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Role of the tumor microenvironment in mediating de novo resistance to drugs and physiological mediators of cell death
Abstract
The emergence of clinical drug resistance continues to be an obstacle for the successful treatment of cancer. Our current understanding of mechanisms associated with drug resistance has been ascertained by investigating drug-resistant models created by exposing a parental population to increasing concentrations of a cytotoxic. These unicellular drug-resistant models have been critical in elucidating drug-resistant mechanism and in some cases have aided in the identification of drug targets. However, these models do not address resistance mechanisms that contribute to de novo drug resistance. We propose that specific niches within the tumor microenvironment may provide a sanctuary for subpopulations of tumors cells that affords a survival advantage following initial drug exposure and may facilitate the acquisition of acquired drug resistance. More specifically, we propose that the bone marrow microenvironment is a sanctuary for hema-topoietic cancers. This review will focus on the bone marrow microenvironment and its role in conferring resistance to cytotoxics and physiological mediators of cell death.
... The microenvironment has emerged as a key player in the development of chemoresistance (16,17,29,30). In fact, we previously reported that TSP-1/CD47 interaction could play a role in cell resistance to Dox (23,24). ...
... Since the last few years, several studies have shown that the tumor microenvironment modulates cell response to chemotherapy and it is clearly now admitted that this ECM-mediated effect contributes to a new form of de novo resistance (14,(33)(34)(35). The Dalton's group was the first to show that fibronectin was able to protect tumor cells from druginduced apoptosis via the b-integrin/PI3-kinase signaling (14,16,17,29,30,33,36). This protective effect has also been described for vitronectin, another ECM protein (37). ...
It is now admitted that in addition to acquired resistance, the tumor microenvironment contributes to the development of chemo-resistance and malignant progression. In a previous study, we showed that Dox induced apoptosis in FTC-133 cells by trigging JNK pathway. This process was accompanied by a decrease of thrombospondin-1 (TSP-1) expression. Moreover, exogenous TSP-1 or its C-terminal-derived peptide interact with receptor CD47 and are able to protect FTC-133 cells against Dox-induced apoptosis. Here, we investigated the involvement of TSP-1/CD47 interaction in a context of acquired multidrug resistance in FTC-133 cells. To that end, we established a Dox-resistant cell line (FTC-133R cells) which developed a resistance against Dox-induced apoptosis. Cell viability was evaluated by Uptiblue assay, nuclear Dox was measured by microspectrofluorimetry, caspase activity was measured by fluorescence of cleaved caspase-3 substrate, gene expression was evaluated by RT-PCR and protein expression was examined by western-blot. Our results showed that FTC-133R overexpressed the P-gp and were 15-fold resistant to Dox. JNK phosphorylation and Dox-induced apoptosis were reduced in FTC-133R cells. Expression of CD47 was increased in FTC-133R cells but TSP-1 expression presented similar levels in two cell lines. VPL restored Dox nuclear uptake and FTC-133R cell sensitivity to apoptosis and induced a decrease in CD47 mRNA expression. Moreover, knockdown of CD47 in FTC-133R cells induced an increase in JNK activation and sensitized FTC-133R cells to Dox. Our data suggest that CD47 is able to contribute to the protection of FTC-133R cells against Dox-induced apoptosis and/or to potentiate the acquired Dox resistance.
... FAs are critical for supporting the cell membrane structure and regulating signal transmission between the cytoskeleton (e.g., actin) and transmembrane receptors (e.g., integrins) during adhesion and migration [16][17][18][19][20][21][22][23][24] . Monitoring the response of FA clusters to drugs is one important mechanism by which the action of pharmaceutical compounds may be evaluated, particularly where approaches that enable characterization to be performed with a small number of cells are especially valuable 22,[25][26][27][28] . During the dynamic assembly and disassembly of a FA, the size of the FA cluster varies and is highly correlated with the level of adhesion engagement and migration speed 13,29 . ...
... Inset: spectra shift before and after the cell attaches to the PC surface been linked to critical cellular processes, including metastasis, apoptosis, and chemotaxis, as well as pathologies of cancers and other diseases 9,29,36,[45][46][47] . As such, monitoring the response of FA clusters to drugs, for example, is an important mechanism by which the action of pharmaceutical compounds may be evaluated 22,[25][26][27][28]36 , and high-throughput approaches that enable the characterization of small cell populations in real time are especially valuable for these applications. Currently available techniques largely make use of fluorescence tagging to mark individual FA proteins, which entails temporal limitations imposed by photobleaching and challenges associated with accurate quantitation and long-term analysis 9,11,48 . ...
We developed a novel label-free imaging approach, named Photonic Resonator Outcoupler Microscopy (PROM) utilizing the reduction of the peak-resonance intensity reflected from a photonic crystal surface. PROM can monitor the variation of focal adhesion areas in live cells dynamically and quantitatively for extended time.
... FAs are critical for supporting the cell membrane structure and regulating signal transmission between the cytoskeleton (e.g., actin) and transmembrane receptors (e.g., integrins) during adhesion and migration [16][17][18][19][20][21][22][23][24] . Monitoring the response of FA clusters to drugs is one important mechanism by which the action of pharmaceutical compounds may be evaluated, particularly where approaches that enable characterization to be performed with a small number of cells are especially valuable 22,[25][26][27][28] . During the dynamic assembly and disassembly of a FA, the size of the FA cluster varies and is highly correlated with the level of adhesion engagement and migration speed 13,29 . ...
... Inset: spectra shift before and after the cell attaches to the PC surface been linked to critical cellular processes, including metastasis, apoptosis, and chemotaxis, as well as pathologies of cancers and other diseases 9,29,36,[45][46][47] . As such, monitoring the response of FA clusters to drugs, for example, is an important mechanism by which the action of pharmaceutical compounds may be evaluated 22,[25][26][27][28]36 , and high-throughput approaches that enable the characterization of small cell populations in real time are especially valuable for these applications. Currently available techniques largely make use of fluorescence tagging to mark individual FA proteins, which entails temporal limitations imposed by photobleaching and challenges associated with accurate quantitation and long-term analysis 9,11,48 . ...
A new imaging technique looks set to provide insights into how biological cells adhere and interact with surfaces. Yue Zhuo and coworkers from the University of Illinois at Urbana-Champaign in the USA have developed a technique called photonic resonator outcoupler microscopy (PROM). In the approach, cells to be studied are attached to the top surface of a photonic crystal biosensor, which is illuminated from below with focused light from a LED. The presence of cellular focal adhesions, a specialized protein located near the cell membrane that dictates how the cell interacts with external surfaces, serves to outcouple evanescent light from the biosensor resulting in a reduction in the sensor’s reflected signal. Measurement of this signal thus makes it possible to image focal adhesions and study their size and dynamic behavior.
... Cysteine-rich protein 61 (Cyr61/CCN1), a member of the CCN (Cyr61/CTGF/NOV) family, plays a pivotal role in maintaining normal bodily physiological functions Cao et al. 2019;Lum et al. 1993;Hazlehurst et al. 2003;Terada et al. 2012). Cyr61 expression becomes abnormal in tumors, and Cyr61 levels are correlated with poor prognosis in tumors (Sabile et al. 2012;Kok et al. 2010;Jiang et al. 2004;Jeong et al. 2014;D'Antonio et al. 2010;Holcik and Korneluk 2001). ...
Purpose
Chemoresistance is a major challenge for acute lymphoblastic leukemia (ALL) treatment. Cysteine-rich protein 61 (Cyr61) plays an important role in drug resistance modulation of tumor cells, and Cyr61 levels are increased in the bone marrow of patients with ALL and contribute to ALL cell survival. However, the effect of Cyr61 on B cell acute lymphoblastic leukemia (B-ALL) cell chemosensitivity and the regulatory mechanisms underlying Cyr61 production in bone marrow remain unknown.
Methods
Nalm-6 and Reh human B-ALL cell lines were used in this study. Cyr61 levels were assessed using quantitative real-time PCR (qRT-PCR), western blot analysis, and enzyme-linked immunosorbent assay. The effect of Cyr61 on B-ALL cell chemosensitivity to daunorubicin (DNR) was evaluated using cell viability and flow cytometry analyses. The regulatory mechanisms of Cyr61 production in bone marrow were examined using qRT-PCR and western blot analysis.
Results
Cyr61 knockdown and overexpression increased and decreased the chemosensitivity of B-ALL cells to DNR, respectively. Cyr61 attenuated chemotherapeutic drug-induced apoptosis by upregulating B cell lymphoma-2. Notably, DNR induced DNA damage response and increased Cyr61 secretion in B-ALL cells through the ataxia telangiectasia mutated (ATM)-dependent nuclear factor kappa B pathway.
Conclusion
DNR induces Cyr61 production in B-ALL cells, and increased Cyr61 levels reduce the chemosensitivity of B-ALL cells. Consequently, targeting Cyr61 or related ATM signaling pathway may present a promising treatment strategy to enhance the chemosensitivity of patients with B-ALL.
... 8. Benner SE, Wahl GM, Von Hoff DD. Double minute chromosomes and homogeneously staining regions in tumors taken directly from patients versus in human tumor cell lines [99,100,101]. The following mechanism are attributed to drug resistance in cancer cells. ...
Bacterial infections are common in the etiology of human diseases owing to the ubiquity of bacteria. Such infections promote the development of periodontal disease, bacterial pneumonia, typhoid, acute gastroenteritis, and diarrhoea in susceptible hosts. In certain hosts, these illnesses may be treated with antibiotics or antimicrobial therapy. But other hosts might not be able to get rid of the bacteria, which would allow them to linger for a long period and raise the carrier's chance of getting cancer over time. In fact, there are ways to reduce the chance of developing cancer, and this thorough analysis elucidates the intricate connection between bacterial infections and the emergence of many cancer forms. The article also discusses the use of antibiotics in the treatment of cancer, the risks associated with their usage, and methods for reducing antibiotic resistance.It is well known that cancers often develop resistance to conventional therapies, leading to an increased prevalence of drug-resistant forms. This research paper provides an overview of current knowledge regarding mechanisms that contribute to or facilitate drug resistance in anticancer drugs.These mechanisms include drug inactivation, alterations in drug targets, drug efux, DNA damage repair, inhibition of cell death, and the epithelial-mesenchymal transition.Finally, the association of bacteria in cancer development as well as in cancer therapy on optimal treatment strategies for existing drug-resistant cancers, preventive measures against drug-resistant cancer formation, and potential future development of novel microbe-based therapeutics research directions.
... In addition, the tumor microenvironment's production of growth factors [17] and cytokines provide additional signals for tumor cell growth and survival [18]. Environment-mediated drug resistance (EM-DR) could be well thought-out as the whole of cell adhesion-mediated drug resistance (CAM-DR) and soluble factor-mediated drug resistance (SM-DR) products including VEGF (vascular endothelial growth factor), bFGF (basic fibroblast growth factor), SDF-1 (stromal cell-derived factor 1), IL-6 (interleukin-6), NO (nitric oxide), IL-3 (interleukin-3), G-CSF (granulocyte colony-stimulating factor), M-CSF (macrophage colony-stimulating factor), GM-CSF (granulocyte-macrophage colony-stimulating factor), TNF superfamily members BAFF (B cell-activating factor of the TNF family) and APRIL (a proliferation-inducing ligand), and many others by the tumor cell interaction [16,19,20]. Cancer drug resistance is multifactorial and not driven exclusively by genetic mechanisms [21,22]. ...
Cancer is one of the deadliest and most heterogeneous diseases. Cancers often develop drug resistance, which can lead to treatment failure or recurrence. Accordingly, anticancer compounds are essential for chemotherapy-resistant cancer cells. Phenolic compounds are of interest in the development of cancer drugs due to their medicinal properties and ability to target different molecular pathways. Gallic acid (GA), as one of the main components of phenol, which is abundantly present in plant compounds such as walnut, sumac, grapes, tea leaves, oak bark, and other plant compounds, has antitumor properties. GA can prevent cancer progression, cell invasion, and metastasis by targeting molecular pathways and is an effective complement to chemotherapy drugs and combating multidrug resistance (MDR). In this review, we discuss various mechanisms related to cancer, the therapeutic potential of GA, the antitumor properties of GA in various cancers, and the targeted delivery of GA with nanocarriers.
... As a consequence, most iCCA patients die within a year after diagnosis 6 . Reciprocal interactions between cancer cells and the tumor microenvironment (TME) play a crucial role in all stages of cancer development, progression and therapeutic resistance [7][8][9][10][11][12][13][14][15][16] . iCCA is often surrounded with a desmoplastic TME as a consequence of chronic inflammation in the bile duct epithelium and nearby tissues 17 . ...
Intrahepatic cholangiocarcinoma (iCCA) arises along the peripheral bile ducts and is often accompanied by a tumor microenvironment (TME) high in extracellular matrices (ECMs). In this study, we aimed to evaluate whether an ECM-rich TME favors iCCA progression. We identified ITGA2, which encodes collagen-binding integrin α2, to be differentially-expressed in iCCA tumors compared with adjacent normal tissues. Elevated ITGA2 is also positively-correlated with its ligand, collagen type I. Increased ITGA2 expression and its role in collagen type I binding was validated in vitro using four iCCA cell lines, compared with a non-cancerous, cholangiocyte cell line. Robust interaction of iCCA cells with collagen type I was abolished by either ITGA2 depletion or integrin α2β1-selective inhibitor treatment. In a phenotypic study, collagen type I significantly enhances clonogenic growth of HuCCA-1 and HuCCT-1 cells by three and sixfold, respectively. Inhibition of integrin α2 expression or its activity significantly blocks collagen type I-induced colony growth in both cell lines. Taken together, our data provide mechanistic evidence that collagen type I promotes growth of iCCA colonies through integrin α2 suggesting that the collagen type I—integrin α2 axis could be a promising target for cancer prevention and a therapeutic opportunity for this cancer.
... VEGF can directly induce activation of VEGFR2, a principal VEGF signalling receptor, which leads to the activation of downstream signalling molecules in a cell proliferation pathway. 32,33 Because the dependence on the signalling pathway in tumour cells may be altered by the microenvironment, 34 It is plausible that certain autocrine growth factors produced by HHV8-unrelated ELBCL cells in vivo and/or paracrine growth factors produced by stroma cells following interaction with tumour cells may contribute to the promotion of tumour proliferation. Malignant ascites serves as a reservoir for a complex mixture of soluble factors and cellular components, which provide a local tumour-promoting microenvironment for tumour cells, especially in ovarian cancer. ...
Primary human herpesvirus 8 (HHV8)‐unrelated effusion large B‐cell lymphoma (ELBCL) is recognized as a new clinical entity, but its pathogenesis and therapeutic strategies remain largely unknown. We have generated two mouse models with profuse lymphomatous effusions that resemble HHV8‐unrelated ELBCL occurring in humans, by grafting the cell lines designated as Pell‐1 and Pell‐2. Using these in vivo models, we evaluated the potential role of vascular endothelial growth factor (VEGF) in the pathogenesis of HHV8‐unrelated ELBCL. Both Pell‐1 and Pell‐2 cells consistently produced very high levels of VEGF in mice, in contrast to in vitro findings of relatively low VEGF production in culture medium by HHV8‐unrelated ELBCL cells, especially Pell‐1 cells. Conversely, returning Pell‐1 cells grown in mice to culture medium markedly suppressed VEGF production to the original in vitro level. These findings suggest that the tumour microenvironment plays a role in the steady production of VEGF. We also found that the interaction between HHV8‐unrelated ELBCL cells and peritoneal mesothelial cells increased the production of VEGF in vitro. Finally, we found that bevacizumab significantly suppressed effusion formation and lymphoma cell growth in both mouse models. These results suggest that bevacizumab is a rational approach to the treatment of HHV8‐unrelated ELBCL.
... The TME plays a significant role in tumorigenesis and cancer progression. The cell types within the TME include bone marrow-derived cells, endothelial cells, cancerassociated fibroblasts (CAFs), and mesenchymal stem cells [12,13,15]. Immune cells may promote cancer initiation by secreting cytokines, which stimulate epithelial proliferation and generate DNA damage. ...
Gastric cancer (GC) is the fourth most common cancer in the world in terms of incidence and second in terms of mortality. Chemotherapy is the main treatment for GC. The greatest challenge and major cause of GC treatment failure is resistance to chemotherapy. As such, research is ongoing into molecular evaluation, investigating mechanisms, and screening therapeutic targets. Several mechanisms related to both the tumor cells and the tumor microenvironment (TME) are involved in resistance to chemotherapy. TME promotes the secretion of various inflammatory cytokines. Recent studies have revealed that inflammatory cytokines affect not only tumor growth, but also chemoresistance. Cytokines in TME can be detected in blood circulation and TME cells. Inflammatory cytokines could serve as potential biomarkers in the assessment of chemoresistance and influence the management of therapeutics in GC. This review presents recent data concerning research on inflammatory cytokines involved in the mechanisms of chemoresistance and provides new clues in GC treatment.
... The interactions among immunosuppressive cells, and together with cancer cells can enhance the tumour growth and stimulate the infiltration of tumour-promoting cells, while limiting the recruitment of anti-tumour cells [57]. The infiltration of immunosuppressive cells promotes tumour growth through various mechanisms such as the inhibition of proliferation of CD8+ T lymphocytes and NK cells, the inhibition of the activity of CTLs, reduction of the secretion of anti-tumour cytokines, stimulation of angiogenesis and metastasis, tumour stiffness, and so on [58]. ...
The interactions and secretions within the tumour have a pivotal role in tumour growth and therapy. Immunosuppressive cells such as regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), tumour-associated macrophages (TAMs), and cancer-associated fibroblasts (CAFs) secrets some substances, which can result in exhaustion of anti-tumour immunity. To stimulate anti-tumour immunity, suppression of the secretion and interactions of immunosuppressive cells, on the other hand, stimulation of proliferation and activation of natural killer (NK) cells and CD8+ T lymphocytes are required. Apigenin is a flavone with anticancer properties. Emerging evidence shows that not only does apigenin modulate cell death pathways in cancer cells but it also can stimulate anti-tumour immune cells to release death signals and suppress the release of tumour-promoting molecules. In this review, we discuss the interactions between apigenin and various cells within the tumour microenvironment (TME). These interactions may enhance anti-tumour immunity to improve the efficiency of anticancer remedies such as immunotherapy.
... Moreover, these events are bolstered by defects in the ability of DTCs to maintain genome integrity that arise in response to aberrant cell cycle checkpoints and DNA repair mechanisms, and to increased rates of proliferation. Additionally, interactions between DTCs and the tumor microenvironment induce unique de novo mechanisms of therapeutic resistance, as cell adhesion networks (e.g., integrins) activate a specialized survival program known as "cell adhesion-mediated" drug resistance that elicit DTC insensitivity to numerous treatment regimens [69,70] . As such, the stromal composition of the metastatic microenvironment creates a natural sanctuary for DTCs to survive therapeutic insults. ...
Although greater than 90% of breast cancer-related mortality can be attributed to metastases, the molecular mechanisms underpinning the dissemination of primary breast tumor cells and their ability to establish malignant lesions in distant tissues remain incompletely understood. Genomic and transcriptomic analyses identified a class of transcripts called long noncoding RNA (lncRNA), which interact both directly and indirectly with key components of gene regulatory networks to alter cell proliferation, invasion, and metastasis. We identified a pro-metastatic lncRNA BMP/OP-Responsive Gene (BORG) whose aberrant expression promotes metastatic relapse by reactivating proliferative programs in dormant disseminated tumor cells (DTCs). BORG expression is broadly and strongly induced by environmental and chemotherapeutic stresses, a transcriptional response that facilitates the survival of DTCs. Transcriptomic reprogramming in response to BORG resulted in robust signaling via survival and viability pathways, as well as decreased activation of cell death pathways. As such, BORG expression acts as a (1) marker capable of predicting which breast cancer patients are predisposed to develop secondary metastatic lesions; and (2) unique therapeutic target to maximize chemosensitivity of DTCs. Here we review the molecular and cellular factors that contribute to the pathophysiological activities of BORG during its regulation of breast cancer metastasis, chemoresistance, and disease recurrence.
... As the second most prevalent cause of cancer-associated mortality in women and men after lung cancer, colon cancer arises due to the conversion of precancerous benign polyps present in the inner lining of the colon, which is responsible for nearly 700,000 deaths and 1.4 million new cases worldwide every year (Jaganathan et al., 2014). Despite the steady decline in incidence rates, and the significant increase in survival rates, potentially due to the increase in cancer screening and improved therapy options, a number of problems remain in the clinical setting, including chemoresistance and severe side effects (Desoize et al., 1998;Hazlehurst et al., 2003). Therefore, it is imperative to investigate and identify novel and safer therapeutic agents with new mechanisms of action against colon carcinoma. ...
Abstract (S)-(-)-N-[2-(3-Hydroxy-2-oxo-2,3-dihydro-1H-indol-3-yl)-ethyl]-acetamide (EA), a new compound isolated from the plant Selaginella pulvinata, has been identified to possess anti-proliferative effects against SK-mel-110 cells in vitro via upregulating the expression of inhibitor of growth family member 4. The present study aimed to investigate whether EA could exert antitumor effects against colon cancer. The results revealed that EA exerted anti-proliferative effects against a broad spectrum of different types of tumor and could induce apoptosis of colon cancer cells via the signal transducer and activator of transcription 1 pathway. It was demonstrated that treating tumor-bearing mice with EA significantly inhibited colon tumor growth in a xenograft tumor model and syngeneic tumor model. Furthermore, EA treatment reshaped the tumor microenvironment by decreasing myeloid-derived suppressor cell accumulation and increasing the tumor infiltrating lymphocytes. The results from the present study suggest that EA could be developed as a potential antitumor drug against colon cancer.
... As a result, the bone marrow niche has also become a key part of the recurrence and MDR of leukemia [4][5][6]. According to recent research, we have known that MDR mediated by the bone marrow microenvironment might stem from the following several mechanisms: i) The influence of cytokines; ii) Changes in adhesion of leukemia cells; iii) The regulation of expression of MDR-related genes; iv) The adjustment of cell cycle and metabolism [7][8][9][10][11][12]. In addition, many factors can also be contributed to the recurrence of leukemia, such as the secretion of cytokines or the changes with certain pathways. ...
Background: Acute myeloid leukemia (AML) is a dangerous type of leukemia. The emergence of multidrug resistance (MDR) and recurrence limits the prognosis and survival of patients. In recent studies, we have known that the bone marrow microenvironment was closely related to the poor prognosis of AML. However, the underlying mechanisms are still far from fully understood. By utilizing the bioinformatics analysis, we screened out integrin B1 (ITGB1) as the hub gene, which is associated with the bone marrow microenvironment mediated changes of AML cells, with expression profile GSE73157 downloaded from the National Center for Biotechnology Information-Gene Expression Omnibus (NCBI-GEO) database. Methods: R studio software was used to screen out candidate hub genes and further visualize the differential expression. R package ‘limma’ was to find out differentially expressed genes (DEGs). Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were conducted by R package “clusterProfiler”. Furthermore, the protein-protein interaction (PPI) network was also performed by the online tool STRING and software Cytoscape. Last but not least, online tools PrognoScan and GEPIA were utilized for the evaluation of the clinical significance of the selected hub gene. P and Cox p-value <0.05 was considered to be statistically significant. Results: ITGB1 was filtrated as the only hub gene in this profile. We found that patients with high expression of ITGB1 had significantly longer overall survival (OS) than those with low expression (COX p-value= 0.016730). Besides, the expression of the ITGB1 gene in AML patients is lower than that in normal people significantly (p-value<0.01). Conclusion: We identified ITGB1 as a key gene in the bone marrow microenvironment mediated poor prognosis in AML. The down-regulated expression of ITGB1 was related to AML patients’ poor outcomes. ITGB1 may be a potential marker for predicting and guiding AML treatment.
... The role of the tumor microenvironment in tumorigenesis (Allinen et al., 2004;Liao et al., 2018;Wang et al., 2017), tumor growth (Lyssiotis and Kimmelman, 2017;Whiteside, 2008), drug resistance (Hazlehurst et al., 2003;Sun, 2016;Trédan et al., 2007), immunosuppression (Arina et al., 2016;Cohen et al., 2017;Wang and DuBois, 2015), and metastasis (Mlecnik et al., 2016;Quail and Joyce, 2013;Yang and Lin, 2017) is becoming increasingly recognized as an important target for therapeutic intervention. Fully elucidating the molecular components that modulate the microenvironment is critical for determining how to eliminate these sanctuaries for cancer cells. ...
Tumor and stromal interactions consist of reciprocal signaling through cytokines, growth factors, direct cell-cell interactions, and extracellular vesicles (EVs). Small EVs (≤200 nm) have been considered critical messengers of cellular communication during tumor development. Here, we demonstrate that gain-of-function (GOF) p53 protein can be packaged into small EVs and transferred to fibroblasts. GOF p53 protein is selectively bound by heat shock protein 90 (HSP90), a chaperone protein, and packaged into small EVs. Inhibition of HSP90 activity blocks packaging of GOF, but not wild-type, p53 in small EVs. GOF p53-containing small EVs result in their conversion to cancer-associated fibroblasts. In vivo studies reveal that GOF p53-containing small EVs can enhance tumor growth and promote fibroblast transformation into a cancer-associated phenotype. These findings provide a better understanding of the complex interactions between cancer and stromal cells and may have therapeutic implications.
... However, drug resistance can often occur within 6 months in BL patients, contributing to poor prognosis (38). Recent studies have revealed that the bone marrow microenvironment, which provides tumour cells with a network of proliferative and survival factors, is one of the most important reasons for drug resistance and relapse (39,40). Specifically, previous studies have found that adhesion to bone marrow stroma is mainly responsible for the development of drugresistance, also known as CAM-DR (41,42). ...
Burkitt lymphoma (BL) is a highly aggressive form of non-Hodgkin's B-cell lymphoma. Currently, multi-agent chemotherapy regimens are being used to significantly improve cure rates and achieve complete remissions in BL patients. However, drug resistance can often occur within 6 months in BL patients, contributing to poor prognosis. Mounting evidence suggests that cell adhesion-mediated drug resistance (CAM-DR), caused by the interaction between the bone marrow microenvironment and tumour cells may play an important role in drug resistance to chemotherapy. However, the molecular mechanism underlying CAM-DR in BL has not been identified yet. In this study, we investigated the molecular mechanism responsible for CAM-DR in BL cells. We also examined the therapeutic targets of CAM-DR in BL cells and found CD49d and CD49e to be the important adhesion molecules involved. However, CD49a, CD49b, CD11a, CD29, CD18, and CD61 were not found to be associated with CAM-DR in BL cells. Furthermore, we clarified that CD49d- and CD49e-mediated CAM-DR could be attributed to an increase in the expression of B cell leukemia-xL (Bcl-xL) and survivin proteins, and a decrease in the expression of Bcl-2 associated X (Bax), Bcl-2 interacting mediator (Bim) and p53 upregulated modulator of apoptosis (PUMA) proteins via nuclear factor kappaB (NF-κB) activation. In addition, bortezomib was found to overcome CAM-DR in BL cells by inhibiting NF-κB. Thus, bortezomib may have potential clinical applications in the treatment of CD49d- and CD49e-mediated CAM-DR in BL patients.
... LFA-1 is one of the most important adhesion molecules that mediate contact between tumor and stromal cells. Furthermore, cell adhesion has been considered as one of the major causes of primary drug resistance 38 . Our results showed that the statins (simvastatin, lovastatin, fluvastatin and rosuvastatin) exerted a cytotoxic effect, while no effect was observed using LFA-1 inhibitors. ...
Chronic lymphocytic leukemia (CLL) is a B lymphoid malignancy highly dependent on the microenvironment. Despite new targeted therapies such as ibrutinib and venetoclax, disease progression and relapse remain an issue. CLL cell interactions with the supportive tissue microenvironment play a critical role in disease pathogenesis. We used a platform for drug discovery based on systems biology and artificial intelligence, to identify drugs targeting key proteins described to have a role in the microenvironment. The selected compounds were screened in CLL cell lines in the presence of stromal cells to mimic the microenvironment and validated the best candidates in primary CLL cells. Our results showed that the commercial drug simvastatin was the most effective and selective out of the tested compounds. Simvastatin decreased CLL cell survival and proliferation as well as cell adhesion. Importantly, this drug enhanced the antitumor effect of venetoclax and ibrutinib. We proposed that systems biology approaches combined with pharmacological screening could help to find new drugs for CLL treatment and to predict new combinations with current therapies. Our results highlight the possibility of repurposing widely used drugs such as statins to target the microenvironment and to improve the efficacy of ibrutinib or venetoclax in CLL cells.
... MM and metastatic prostate cancer progression involves bone marrow disease in 100% and 92% of patients, respectively. [5][6][7][8][9] Herein, we describe the synthesis of cyclized peptidomimetic (Figure 1) of the previously reported linear all Damino acid peptide, HYD1. [10][11][12][13][14] We have developed a molecule with commercial potential to treat MM and have named our lead compound, 10, MTI-101. ...
HYD1 is an all D‐amino acid linear 10‐mer peptide that was discovered by one‐bead‐one‐compound screening. HYD1 has five hydrophobic amino acids flanked by polar amino acids. Alanine scanning studies showed that alternating hydrophobic amino acid residues and N‐ and C‐terminal lysine side chains were contributors to the biological activity of the linear 10‐mer analogs. This observation led us to hypothesize that display of the hydrophobic pentapeptide sequence of HYD1 in a cyclic beta‐hairpin‐like scaffold could lead to better bioavailability and biological activity. An amphipathic pentapeptide sequence was used to form an antiparallel strand and those strands were linked via dipeptide‐like sequences selected to promote β‐turns. Early cyclic analogs were more active but otherwise mimicked the biological activity of the linear HYD1 peptide. The cyclic peptidomimetics were synthesized using standard Fmoc solid phase synthesis to form linear peptides, followed by solution phase or on‐resin cyclization. SAR studies were carried out with an aim to increase the potency of these drug candidates for the killing of multiple myeloma cells in vitro. The solution structures of 1, 5, and 10 were elucidated using NMR spectroscopy. ¹H NMR and 2D TOCSY studies of these peptides revealed a downfield Hα proton chemical shift and 2D NOE spectral analysis consistent with a β‐hairpin‐like structure.
Abstract
... According to recent researches, we have known that MDR mediated by the bone marrow microenvironment might stem from the following several mechanisms: 1. The in uence of cytokines [7,8]; 2. Changes in adhesion of leukemia cells [9,10]; 3. The regulation of expression of MDRrelated genes [11]; 4. The adjustment of cell cycle and metabolism [12]. In addition, many factors can also be contributed to the recurrence of leukemia, such as the secretion of cytokines or the changes with certain pathways. ...
Background
Acute myeloid leukemia (AML) is a dangerous type of leukemia. The emergence of multidrug resistance (MDR) and recurrence limits the prognosis and survival of patients. In recent studies, we have known that the bone marrow microenvironment was closely related to the poor prognosis of AML. However, the underlying mechanisms are still far from fully understood. By utilizing the bioinformatics analysis, we screened out integrin β1 (ITGB1) as the hub gene, which is associated with the bone marrow microenvironment mediated changes of AML cells, with expression profile GSE73157 downloaded from National Center for Biotechnology Information-Gene Expression Omnibus (NCBI-GEO) database.MethodsR studio software was used to screen out candidate hub genes and further visualize the differential expression. R package “limma” was to find out differentially expressed genes (DEGs). Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were conducted by R package “cluster Profiler”. Furthermore, protein-protein interaction (PPI) network was also performed by online tool STRING and software Cytoscape. Last but not least, online tool PrognoScan and GEPIA was utilized for the evaluation of clinical significance of the selected hub gene. P and Cox p value <0.05 was considered to be statistical significance.ResultsITGB1 was filtrated as the only hub gene in this profile. We found that patients with high expression of ITGB1 had significantly longer overall survival (OS) than those with low expression (COX p value= 0.016730). Besides, the expression of the ITGB1 gene in AML patients is lower than that in normal people significantly (p value<0.01).Conclusion
We identified ITGB1 as a key gene in the bone marrow microenvironment mediated poor prognosis in AML. The down-regulated expression of ITGB1 was related to AML patients’ poor outcome. ITGB1 may be a potential marker for predicting and guiding AML treatment.
... These methods induce method-specific changes in response that may hamper or enhance the translation to in vivo conditions. Cultures using stromal cells or in 3D systems modulates response to anti-cancer drugs, through cell adhesion mediated mechanisms or via secreted cytokines and/or vesicles (Hazlehurst et al., 2003;Rashidi and Uy, 2015;Shain and Dalton, 2001). These effects are sample-and drug-specific. ...
New treatment options of acute myeloid leukemia (AML) are rapidly emerging. Pre-clinical models such as ex vivo cultures are extensively used towards the development of novel drugs and to study synergistic drug combinations, as well as to discover biomarkers for both drug response and anti-cancer drug resistance. Although these approaches empower efficient investigation of multiple drugs in a multitude of primary AML samples, their translational value and reproducibility are hampered by the lack of standardized methodologies and by culture system-specific behavior of AML cells and chemotherapeutic drugs. Moreover, distinct research questions require specific methods which rely on specific technical knowledge and skills. To address these aspects, we herein review commonly used culture techniques in light of diverse research questions. In addition, culture-dependent effects on drug resistance towards commonly used drugs in the treatment of AML are summarized including several pitfalls that may arise because of culture technique artifacts. The primary aim of the current review is to provide practical guidelines for ex vivo primary AML culture experimental design.
... Crosstalk between cancerous and stromal cells in an inflammatory-based microenvironment not only affects the aggressive behavior of cancer cell, but also influences the efficacy of anti-cancer drugs [1][2][3][4]. On the other hand, drug impacts are usually assayed in cell culture or immunosuppressed mice models [5], neither of which provides a clear picture of the complicated interactions between tumor cells and their surrounding inflammatory stroma, eventually resulting in inconsistencies in the outcome of clinical trials [6]. ...
Background:
Interaction between microenvironment and breast cancer cells often is not considered at the early stages of drug development leading to failure of many drugs at later clinical stages. Etanercept is a TNF-alpha inhibitor that has been investigated for potential antitumor effect in breast cancer with conflicting results.
Methods:
Secretome data on MDA-MB-231 cancer cell-line were from public repositories and subjected to gene enrichment analyses. Since MDA-MB-231 cells secrete high levels of Granulocyte-Monocyte Colony Stimulating Factor, which activates macrophages to promote tumor growth, the effect of macrophage co-culturing on anticancer efficacy of Etanercept in breast cancer was evaluated using the Boolean network modeling and in vitro experiments including invasion, cell cycle, Annexin PI, and tetrazolium based viability assays and NFKB activity.
Results:
The secretome profile of MDA-MB-231 cells was similar to the expression of genes following treatment of breast cancer cells with TNF-α. Accordingly, inhibition of TNF-α by Etanercept decreased MDA-MB-231 cell survival, induced apoptosis and cell cycle arrest in vitro and inhibited NFKB activation. The inhibitory effect of Etanercept on cell viability, cell cycle progression, invasion and induction of apoptosis decreased following co-culturing of the cancer cells with macrophages. The Boolean network modeling of the changes in the dynamics of intracellular signaling pathways revealed NFKB activation by secretome of macrophages, leading to a decreased efficacy of Etanercept, suggesting NFKB inhibition as an alternative approach to inhibit cancer cell growth in the presence of macrophage crosstalk.
Conclusion:
This study indicates that the effect of Etanercept may be influenced by residing macrophages in tumor microenvironment, and suggests a method to predict the effect of drugs in the presence of stromal cells to guide experimental designs in drug development.
... Anticancer drug resistance is one of the major challenges in the management and treatment of advanced BC which can be caused by intrinsic and acquired factors that alter molecular/signaling pathways leading to poor survival [8]. Among these factors are tumor heterogeneity, genomic instability, self-renewing cancer stem cells (CSC), tumor microenvironment via direct interplay of extracellular matrix, growth factors, cytokines and stromal cells and epigenetic alterations/mutations that lead to metabolic variations (Fig. 2) [9][10][11][12]. ...
The global burden of breast cancer (BC) is increasing significantly. This trend is caused by several factors such as late diagnosis, limited treatment options for certain BC subtypes, drug resistance which all lead to poor clinical outcomes. Recent research has reported the role of epigenetic alterations in the mechanism of BC pathogenesis and its hallmarks include drug resistance and stemness features. The understanding of these modifications and their significance in the management of BC carcinogenesis is challenging and requires further attention. Nevertheless, it promises to provide novel insight needed for utilizing these alterations as potential diagnostic, prognostic markers, predict treatment efficacy, as well as therapeutic agents. This highlights the importance of continuing research development to further advance the existing knowledge on epigenetics and BC carcinogenesis to overcome the current challenges. Hence, this review aims to shed light and discuss the current state of epigenetics research in the diagnosis and management of BC.
... LFA-1 is one of the most important adhesion molecules that mediate contact between tumor and stromal cells. Furthermore, cell adhesion has been considered as one of the major causes of primary drug resistance [40]. Our results showed that the statins (simvastatin, lovastatin, uvastatin and rosuvastatin) exerted a cytotoxic effect, while no effect was observed using LFA-1 inhibitors. ...
Background: Chronic lymphocytic leukemia (CLL) is a B lymphoid malignancy highly dependent on the microenvironment. CLL cell interactions with the supportive tissue microenvironment play a critical role in disease pathogenesis. Despite new targeted therapies such as ibrutinib and venetoclax, disease progression and relapse remain an issue. The present study aimed to identify drugs targeting key proteins described to have a role in the microenvironment.
Methods: We used a platform for drug discovery based on systems biology and artificial intelligence. Bioactive compounds have been screened in silico and selected compounds were evaluated in CLL cell lines in the presence of stromal cells to mimic the microenvironment and validated the best candidates in primary CLL cells.
Results: Our results showed that the commercial drug simvastatin was the most effective and selective out of the tested compounds. Simvastatin decreased CLL cell survival and proliferation as well as cell adhesion. Importantly, this drug enhanced the antitumor effect of venetoclax and ibrutinib.
Conclusions: We proposed that systems biology approaches combined with pharmacology screening could help to find new drugs for CLL treatment and to predict new combinations with current therapies. Our results highlight the possibility of repurposing widely used drugs such as statins to target the microenvironment and to improve the efficacy of ibrutinib or venetoclax in CLL cells.
... As such, the tissue-specific distribution of drug efflux transporters plays a pivotal role in the de novo resistance in carcinomas derived from these tissues, as these channels are inherently poised to protect tumors from pharmacologic insults (103). More importantly, interactions between the tumor microenvironment and the developing carcinoma allow for unique de novo mechanisms of therapeutic resistance that are driven primarily through integrin-based cell adhesion networks that activate cell survival programs coupled to chemoresistance (e.g., cell adhesion-mediated drug resistance; (104,105)). Thus, the stromal composition of metastatic microenvironments can provide disseminated tumor cells a survival sanctuary to resist clinical regimens. ...
Epithelial-mesenchymal transition (EMT) programs play essential functions in normal morphogenesis and organogenesis, including that occurring during mammary gland development and glandular regeneration. Historically, EMT programs were believed to reflect a loss of epithelial gene expression signatures and morphologies that give way to those associated with mesenchymal cells and their enhanced migratory and invasive behaviors. However, accumulating evidence now paints EMT programs as representing a spectrum of phenotypic behaviors that also serve to enhance cell survival, immune tolerance, and perhaps even metastatic dormancy. Equally important, the activation of EMT programs in transformed mammary epithelial cells not only enhances their acquisition of invasive and metastatic behaviors, but also expands their generation of chemoresistant breast cancer stem cells (BCSCs). Importantly, the net-effect of these events results in the appearance of recurrent metastatic lesions that remain refractory to the armamentarium of chemotherapies and targeted therapeutic agents deployed against advanced-stage breast cancers. Here we review the molecular and cellular mechanisms that contribute to the pathophysiology of EMT programs in human breast cancers and how these events impact their "stemness" and acquisition of chemoresistant phenotypes.
... Studies have attached great importance of tumor microenvironment to cancer cell resistance to drugs 6,7 . Of note, tumor microenvironment consists of diverse types of nonmalignant cells, such as mesenchymal stromal cells (MSCs) 8 . ...
Chemotherapy is the first-tier treatment regime for gastric cancer (GC) patients at advance stages. Mesenchymal stem cell (MSC) cam affect drug-resistance of GC cells in tumor microenvironment, but the detailed mechanism remains poorly understood. Present study aimed to investigate the regulation of MSC-induced long non-coding RNA (lncRNA) in GC. Dysregulated lncRNAs in GC were analyzed based on GEO data. Stemness and drug-resistance of GC cells were detected by sphere formation, colony formation, CCK-8, and flow cytometry analyses. MicroRNA (miRNA)-related pathways were analyzed by online KEGG analysis tool DAVID6.8. Molecular interactions were determined by luciferase reporter assay, pulldown, RNA immunoprecipitation (RIP), chromatin immunoprecipitation (ChIP), and co-immunoprecipitation (CoIP). Results revealed that MSC co-culture improved stemness and drug-resistance of GC cells. LncRNA histocompatibility leukocyte antigen complex P5 (HCP5) was induced in GC cells by MSC co-culture, contributing to stemness and drug-resistance. Mechanistically, HCP5 sequestered miR-3619-5p and upregulated PPARG coactivator 1 alpha (PPARGC1A), increasing transcription complex Peroxisome proliferator activated receptor (PPAR) coactivator‐1α (PGC1α)/CEBPB and transcriptionally inducing carnitine palmitoyltransferase 1 (CPT1), which prompted the fatty acid oxidation (FAO) in GC cells. In conclusion, MSC-induced lncRNA HCP5 drove FAO through miR-3619-5p/AMPK/PGC1α/CEBPB axis to promote stemness and chemo-resistance of GC, indicating that targeting HCP5 was a novel approach to enhancing the efficacy of chemotherapy in GC.
... It is also well established that adhesive interactions of MM tumor cells with surrounding microenvironmental cells and extracellular matrix proteins (eg, via integrins) confers resistance to standard-of-care MM agents, including bortezomib. [83][84][85][86] While our analyses revealed that LCRF-0006 treatment did not affect the relative proportion of MSCs, OPs and OBs within long bones, N-cadherin is expressed by numerous cell types within the bone marrow milieu, including MSCs and OBs, and mediates MM plasma cell adhesion to OBs. 47,48,87 As N-cadherin antagonists have been shown to overcome bone marrow stromal cell-mediated resistance to chemotherapeutic agents in chronic myeloid leukemia cells, 88,89 we therefore cannot exclude the possibility that LCRF-0006 treatment may disrupt MM tumor cell interactions with the supportive bone marrow niche in vivo, thereby increasing tumor cell sensitivity to bortezomib. ...
N‐cadherin is a homophilic cell‐cell adhesion molecule that plays a critical role in maintaining vascular stability and modulating endothelial barrier permeability. Pre‐clinical studies have shown that the N‐cadherin antagonist peptide, ADH‐1, increases the permeability of tumour‐associated vasculature thereby increasing anti‐cancer drug delivery to tumours and enhancing tumour response. Small molecule library screens have identified a novel compound, LCRF‐0006, that is a mimetic of the classical cadherin His‐Ala‐Val sequence‐containing region of ADH‐1. Here, we evaluated the vascular permeability‐enhancing and anti‐cancer properties of LCRF‐0006 using in vitro vascular disruption and cell apoptosis assays, and a well‐established pre‐clinical model (C57BL/KaLwRij/5TGM1) of the haematological cancer multiple myeloma (MM). We found that LCRF‐0006 disrupted endothelial cell junctions in a rapid, transient and reversible manner, and increased vascular permeability in vitro and at sites of MM tumour in vivo. Notably, LCRF‐0006 synergistically increased the in vivo anti‐MM tumour response to low dose bortezomib, a frontline anti‐MM agent, leading to regression of disease in 100% of mice. Moreover, LCRF‐0006 and bortezomib synergistically induced 5TGM1 MM tumour cell apoptosis in vitro. Our findings demonstrate the potential clinical utility of LCRF‐0006 to significantly increase bortezomib effectiveness and enhance the depth of tumour response in patients with MM.
... Integrins are an extensive family of glycoproteins expressed by many cell types, including MM cells in which a wide range of integrins (α4, α5, αv, β1, β2, β3, and β7) is detected [2,3]. In particular, α4 β1 (VLA-4) [4][5][6][7] and α4β7 [8] are shown to regulate MM-cell adhesion, migration, homing, invasion as well as drug resistance. ...
Integrins have crucial roles in BM homing, survival, proliferation, or drug resistance of multiple myeloma (MM) cells. Especially, integrin α4β1 (VLA-4) and α4β7 has been reported to have important functions in MM cells, and therefore are potential therapeutic targets. We have recently shown that integrin β7 constitutively adopts the active conformation specifically in MM cells, and found that chimeric antigen receptor (CAR) T cells targeting the activated conformation of integrin β7 is promising for MM. Although the mechanism for the constitutive activation is still being investigated, our results indicate that integrin conformation is different between MM and normal cells and suggest that it may be associated with the pathology of MM.
... In support of this idea, the SCCs displayed the ability to recruit fibroblasts in the TME. Given the ability of CAFs to change the extracellular matrix topology as a main component of tumor progression and anticancer drug resistance (42,43), it was highly likely that the SCCs recruited fibroblasts in the TME to evade tumor dormancy and promote chemoresistance. Indeed, the SCCs appeared to enter the active cell cycle after communicating with fibroblasts. ...
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Quiescent cancer cells are believed to cause cancer progression after chemotherapy through unknown mechanisms. We show here that human non–small cell lung cancer (NSCLC) cell line-derived, quiescent-like, slow-cycling cancer cells (SCC) and residual patient-derived xenograft (PDX) tumors after chemotherapy experience activating transcription factor 6 (ATF6)-mediated upregulation of various cytokines, which acts in a paracrine manner to recruit fibroblasts. Cancer-associated fibroblasts (CAF) underwent transcriptional upregulation of COX2 and type I collagen (Col-I), which subsequently triggered a slow-to-active cycling switch in SCC through prostaglandin E<sub>2</sub> (PGE<sub>2</sub>)- and integrin/Src-mediated signaling pathways, leading to cancer progression. Both antagonism of ATF6 and cotargeting of Src/COX2 effectively suppressed cytokine production and slow-to-active cell cycling transition in SCC, withholding cancer progression. Expression of COX2 and Col-I and activation of Src were observed in patients with NSCLC who progressed while receiving chemotherapy. Public data analysis revealed significant association between COL1A1 and SRC expression and NSCLC relapse. Overall, these findings indicate that a proinflammatory niche created by the interplay between SCC and CAF triggers tumor progression.
Significance:
Cotargeting COX2 and Src may be an effective strategy to prevent cancer progression after chemotherapy.</p
... Given their role in cancer cell survival, it is not surprising that integrins, and therefore the ECM-molecules they interact with, are also strongly involved in regulating resistance to radiotherapy: recent studies reported that β1 integrins are upregulated after radiotherapy and play a role in mediating resistance to radiotherapy (Onoda et al., 1992). Experiments in melanoma and sarcoma cells showed that downregulation of the p53 tumor suppressor gene resulted in increased survival following cell detachment and resistance to apoptosis (Lewis et al., 2002;Hazlehurst et al., 2003). Blocking ECM-induced signaling by targeting either β1 integrins or downstream PI3/AKT signaling enhanced the efficacy of radiotherapy in cultured breast cancer cells and implanted BCa xenografts (Liang et al., 2003;Park et al., 2006Park et al., , 2008. ...
Solid tumors are complex organ-like structures that consist not only of tumor cells but also of vasculature, extracellular matrix (ECM), stromal, and immune cells. Often, this tumor microenvironment (TME) comprises the larger part of the overall tumor mass. Like the other components of the TME, the ECM in solid tumors differs significantly from that in normal organs. Intratumoral signaling, transport mechanisms, metabolisms, oxygenation, and immunogenicity are strongly affected if not controlled by the ECM. Exerting this regulatory control, the ECM does not only influence malignancy and growth of the tumor but also its response toward therapy. Understanding the particularities of the ECM in solid tumor is necessary to develop approaches to interfere with its negative effect. In this review, we will also highlight the current understanding of the physical, cellular, and molecular mechanisms by which the pathological tumor ECM affects the efficiency of radio-, chemo-, and immunotherapy. Finally, we will discuss the various strategies to target and modify the tumor ECM and how they could be utilized to improve response to therapy.
... Although cancer treatments help to reduce or eliminate malignant cells, the current therapies sometimes are not effective and may be accompanied by adverse effects on normal cells and some common issues (Kakde et al. 2011;Reddy et al. 2016). Likewise, the emergence of drug resistant cancer cells, resistance to drug induced apoptosis, high systemic toxicity, complex syntheses (Kibria et al. 2014) and supportive tumor microenvironment are some limitations in chemotherapy (Dandekar et al. 2005;Hazlehurst et al. 2003;Riedl et al. 2011;Van Brussel and Mickisch 2003;Ranganath et al. 2015). It shows the urgent necessity for searching natural compounds that can suppress cancer cells without any detrimental effect (Thakur et al. 2016;Gupta et al. 2007;Satitmanwiwat et al. 2016;Mann 2002). ...
Brevinin-2R (BR-2R), a novel antimicrobial and anti-cancer peptide and two derivatives, BR-C and BR-D, were well designed, synthesized and purified by RP-HPLC (reverse phase high pressure chromatography). Accuracy was confirmed through mass spectrometry. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) assay was carried out to determine cytotoxicity of various concentrations of peptides and their anti-proliferative potential. Results indicate that these compounds have significant anticancer activities in vitro on both MCF-7 (human breast adenocarcinoma) and A549 (human lung carcinoma) cancer cells in a concentration-dependent manner, although showed less cytotoxicity on human red blood cells. Further investigation verified that apoptosis induction, confirmed by Annexin V/PI staining and strong sub G1 cell cycle arrest, was observed in treated MCF-7 and A549 cells. Induction of apoptosis and anti-proliferating activity were closely associated with augmentation of caspase-3/7 activity, surging the apoptotic cells with no change in caspase-8 activity. The growth inhibition of BR-2R was exceedingly stronger than two derivatives; on the other hand, MCF-7 was more susceptible to treatment than A549. Findings prominently suggested development of Brevinin-2R and derivatives as novel promising compounds for cancer therapy, which may conquer drug resistance.
... FAK does not have a specific mutation that can be targeted like BRAF (V600E) (12) nor is it a receptor like HER2 (13) or EGFR (14). Compared to oncogenes that require a mutation to trigger a malignant phenotype, the overexpression of wild-type FAK in tumors relative to the basal expression in surrounding normal tissue is sufficient to drive malignancy within the tumor and TME (15,16). ...
... The ECM-rich phenotype in 3D culture has been especially recognized as protecting the cells from cytotoxic activity and resistance acquisition, especially for chondrosarcoma (CHS), which is notoriously resistant to conventional chemo-and radiotherapy [3,11,12]. CHS, the second most common type of skeletal malignancy after osteosarcoma, with a wide range of clinical behaviors, remains a challenge for oncologists: the overall survival of patients with unresectable disease has not been improved in the last few decades [13]. ...
It has been suggested that chemoresistance of chondrosarcoma (CHS), the cartilage tumor, is caused by the phenotypic microenvironmental features of the tumor tissue, mainly the chondrogenic extracellular matrix (ECM), and hypoxia. We developed and characterized a multicellular tumor spheroid (MCTS) of human chondrosarcoma HEMC-SS cells to gain insight into tumor cell biology and drug response. At Day 7, HEMC-SS spheroids exhibited a homogeneous distribution of proliferative Ki-67 positive cells, whereas in larger spheroids (Day 14 and Day 20), proliferation was mainly localized in the periphery. In the core of larger spheroids, apoptotic cells were evidenced by TUNEL assay, and hypoxia by pimonidazole staining. Interestingly, VEGF excretion, evidenced by ELISA on culture media, was detectable from Day 14 spheroids, and increased as the spheroids grew in size. HEMC-SS spheroids synthesized a chondrogenic extracellular matrix rich in glycosaminoglycans and type-2 collagen. Finally, we investigated the sensitivity of Day 7 and Day 14 chondrosarcoma MCTS to hypoxia-activated prodrug TH-302 and doxorubicin compared with their 2D counterparts. As expected, TH-302 exhibited higher cytotoxic activity on larger hypoxic spheroids (Day 14) than on non-hypoxic spheroids (Day 7), with multicellular resistance index (MCRI) values of 7.7 and 9.1 respectively. For doxorubicin, the larger-sized spheroids exhibited higher drug resistance (MCRI of 5.0 for Day 7 and 18.3 for Day 14 spheroids), possibly due to impeded drug penetration into the deep layer of spheroids, evidenced by its auto-fluorescence property. We have developed a model of human chondrosarcoma MCTS that combines an ECM rich in glycosaminoglycans with a high hypoxic core associated with VEGF excretion. This model could offer a more predictive in vitro chondrosarcoma system for screening drugs targeting tumor cells and their microenvironment.
Background
Cancer drug resistance remains a difficult barrier to effective treatment, necessitating a thorough understanding of its multi-layered mechanism.
Objective
This study aims to comprehensively explore the diverse mechanisms of cancer drug resistance, assess the evolution of resistance detection methods, and identify strategies for overcoming this challenge. The evolution of resistance detection methods and identification strategies for overcoming the challenge.
Methods
A comprehensive literature review was conducted to analyze intrinsic and acquired drug resistance mechanisms, including altered drug efflux, reduced uptake, inactivation, target mutations, signaling pathway changes, apoptotic defects, and cellular plasticity. The evolution of mutation detection techniques, encompassing clinical predictions, experimental approaches, and computational methods, was investigated. Strategies to enhance drug efficacy, modify pharmacokinetics, optimizoptimizee binding modes, and explore alternate protein folding states were examined.
Results
The study comprehensively overviews the intricate mechanisms contributing to cancer drug resistance. It outlines the progression of mutation detection methods and underscores the importance of interdisciplinary approaches. Strategies to overcome drug resistance challenges, such as modulating ATP-binding cassette transporters and developing multidrug resistance inhibitors, are discussed. The study underscores the critical need for continued research to enhance cancer treatment efficacy.
Conclusion
This study provides valuable insights into the complexity of cancer drug resistance mechanisms, highlights evolving detection methods, and offers potential strategies to enhance treatment outcomes.
Resistance to chemotherapy and targeted therapies constitute a common hallmark of most cancers and represent a dominant factor fostering tumor relapse and metastasis. Fibronectin, an abundant extracellular matrix glycoprotein, has long been proposed to play an important role in the pathobiology of cancer. Recent research has unraveled the role of Fibronectin in the onset of chemoresistance against a variety of antineoplastic drugs including DNA-damaging agents, hormone receptor antagonists, tyrosine kinase inhibitors, microtubule destabilizing agents, etc. The current review summarizes the role played by Fibronectin in mediating drug resistance against diverse anticancer drugs. We have also discussed how the aberrant expression of Fibronectin drives the oncogenic signaling pathways ultimately leading to drug resistance through the inhibition of apoptosis, promotion of cancer cell growth and proliferation.
Cisplatin (CDDP)-based chemotherapy is the preferred treatment strategy for advanced stage gastric cancer (GC) patients. Despite the efficacy of chemotherapy, the development of chemoresistance negatively affects the prognosis of GC and the underlying mechanism remains poorly understood. Accumulated evidence suggests that mesenchymal stem cells (MSCs) play important roles in drug resistance. The chemoresistance and stemness of GC cells were observed by colony formation, CCK-8, sphere formation and flow cytometry assays. Cell lines and animal models were utilized to investigate related functions. Western blot, quantitative real-time PCR (qRT-PCR) and co-immunoprecipitation were used to explore related pathways. The results showed that MSCs improved the stemness and chemoresistance of GC cells and accounted for the poor prognosis of GC. Natriuretic peptide receptor A (NPRA) was upregulated in GC cells cocultured with MSCs and knockdown of NPRA reversed the MSC-induced stemness and chemoresistance. At the same time, MSCs could be recruited to GC by NPRA, which formed a loop. In addition, NPRA facilitated stemness and chemoresistance through fatty acid oxidation (FAO). Mechanistically, NPRA protected Mfn2 against protein degradation and promoted its mitochondrial localization, which consequently improved FAO. Furthermore, inhibition of FAO with etomoxir (ETX) attenuated MSC-induced CDDP resistance in vivo. In conclusion, MSC-induced NPRA promoted stemness and chemoresistance by upregulating Mfn2 and improving FAO. These findings help us understand the role of NPRA in the prognosis and chemotherapy of GC. NPRA may be a promising target to overcome chemoresistance.
Despite intensive efforts to develop diagnostic and therapeutic tools, the successful treatment of cancer is still hampered by the obscure boundary between cancerous cells and normal cells, recurrence of the cancer, and the development of drug resistance during chemotherapy. In recent years, sonodynamic therapy (SDT), employing therapeutic ultrasound with sonosensitizers, has attracted attention as a potentially promising approach for cancer therapy. This review describes the current understanding of the mechanisms and the preclinical and clinical efficacy of SDT-based applications in tumors, providing an insight into the therapeutic potential offered by SDT. The limitations and future directions of this emerging treatment are also discussed.
Breast cancer (BC) is mostly observed in women and is responsible for huge mortality in women subjects globally. Due to the continued development of drug resistance and other contributing factors, the scientific community needs to look for new alternatives, and drug repurposing is one of the best opportunities. Here we light upon the drug repurposing with a major focus on breast cancer. BC is a division of cancer known as the leading cause of death of 2.3 million women globally, with 685,000 fatalities. This number is steadily rising, necessitating the development of a treatment that can extend survival time. All available treatments for BC are very costly as well as show side effects. This unfulfilled requirement of the anti-cancer drugs ignited an enthusiasm for drug repositioning, which means finding out the anti-cancer use of already marketed drugs for other complications. With the advancement in proteomics, genomics, and computational approaches, the drug repurposing process hastens. So many drugs are repurposed for the BC, including alkylating agents, antimetabolite, anthracyclines, an aromatase inhibitor, mTOR, and many more. The drug resistance in breast cancer is rising, so reviewing how the challenges in breast cancer can be combated with drug repurposing. This paper provides the updated information on all the repurposed drugs candidates for breast cancer with the molecular mechanism responsible for their anti-tumor activity. Additionally, all the challenges that occur during the repurposing of the drugs are discussed.
Background: The interaction of integrins and growth factor receptors in cells is tightly regulated, ensuring cell survival, proliferation, differentiation, adhesion, and migration. The IR generates reactive oxygen species, which leads to ECM remodeling and cell adhesion through the activation of proteases, soluble cytokines and growth factors. Integrins and adhesion of cells to ECM confer higher resistance to ionizing radiation and cytotoxic drug, a phenomenon known as cell adhesion mediated radiation resistance (CAM-RR) and cell adhesion mediated drug resistance (CAM-DR).Integrins’ involvement in CML progression is well appreciated through its survival, adhesion and migration signaling. The evaluation of global genetic response (in microarray) of ionizing radiation (IR) on integrins expression has not been attempted to specify theirrole and other cell adhesion molecules (CAMs) in CML. In this study, we have compared the microarray based CAMs response in myelogenous leukemia cells onIR exposure.
Results: Results revealed differential regulation of many CAMs, with strongest expression of integrin β2 (CD18), whose role has not been fully appreciated in CML due to low level of expression. However, the synergistic LiCl(GSK3β inhibitor) and IR treatment significantly upregulates CD18 expression leading to enhanced survival, cell adhesion mediated drug/radiation resistance (CAM-DR/RR) and transcription of migration related genes. These effects could be undermined in the presence of CD18 antibody. This may be one of the reasons for CML resistance to radiation therapy and its relapse upon stem cell transplantation.
Conclusion: This study proposes CD18 antagonist administration as an adjuvant in anti-CML therapy and other cancers in which it displays aberrant expression subsuming the contraindication of GSK3β inhibitor. Nevertheless, the CD18 mediated cell adhesion in tumor progression beckons development of improved drug regimensandidentification of diagnostic and prognostic signature for CML.
Secondary plasma cell leukaemia (sPCL) is an aggressive form of multiple myeloma (MM), but the mechanism underlying MM progresses into PCL remains unknown.
Gene expression profiling of MM patients and PCL patients was analysed to identify the molecular differences between the two diseases. Cox survival regression and Kaplan–Meier analysis were performed to illustrate the impact of integrin subunit alpha 6 (ITGA6) on prognosis of MM. Invasion assays were performed to assess whether ITGA6 regulated the progression of MM to PCL.
Gene expression profiling analyses showed that cell metastasis pathways were enriched in PCL and ITGA6 was differentially expressed between PCL and MM. ITGA6 expression was an independent prognostic factor for event-free survival (EFS) and overall survival (OS) of MM patients. Moreover, the stratification ability of the International Staging System (ISS) of MM was improved when including ITGA6 expression. Functional studies uncovered that increased ITGA6 reduced the myeloma cell invasion. Additionally, low expression of ITGA6 resulted from epigenetic downregulating of its anti-sense non-coding RNA, ITGA6-AS1.
Our data reveal that ITGA6 gradually decreases during plasma cell dyscrasias progression and low expression of ITGA6 contributes to myeloma metastasis. Moreover, ITGA6 abundance might help develop MM prognostic stratification.
Introduction
CD38 is expressed by some cells of hematological malignancies and tumor-related immunosuppressive cells, including regulatory T cells, regulatory B cells, and myeloid-derived suppressor cells. CD38 is an effective target in some hematological malignancies such as multiple myeloma (MM). Daratumumab (Dara), a CD38-targeting antibody, can eliminate CD38high immune suppressor cells and is regarded as a standard therapy for MM because of its outstanding clinical efficacy. Other CD38 monospecific antibodies, such as isatuximab, MOR202, and TAK079, showed promising effects in clinical trials.
Area covered
This review examines the expression, function, and targeting of CD38 in MM and its potential to deplete immunosuppressive cells in solid cancers. We summarize the distribution and biological function of CD38 and discuss the application of anti-CD38 drugs in hematological malignancies. We also analyz the role of CD38⁺ immune cells in the tumor microenvironment to encourage additional investigations that target CD38 in solid cancers. PubMed and ClinicalTrials were searched to identify relevant literature from the database inception to April 30, 2020.
Expert opinion
There is convincing evidence that CD38-targeted immunotherapeutics reduce CD38⁺ immune suppressor cells. This result suggests that CD38 can be exploited to treat solid tumors by regulating the immunosuppressive microenvironment.
Ovarian cancer, as one of the most common types of gynecological malignancies, has an increasing rate of incidence worldwide. Despite huge amounts of recent efforts in designing novel therapeutic strategies for complete removal of tumors and increasing overall survival of patients, chemotherapy is still the preferred therapy for ovarian cancer. However, chemotherapy is also challenged by development of drug resistance. Therefore, elucidating the underlying mechanisms of drug reissuance is an urgent need in ovarian cancer. Numerous studies have shown the implication of the Notch signaling pathway in the development of various human malignancies. Therefore, this study will provide a brief overview of the published evidence in support of Notch targeting in reverting multidrug resistance as a safer and novel approach for the improvement of ovarian cancer treatment.
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Extracellular vesicles (EV) from cancer-associated fibroblasts (CAF) are composed of diverse payloads. Although CAFs impact the aggressive characteristics of gastric cancer cells, the contribution of CAF-EV to gastric cancer progression has not been elucidated. Here, we investigated the molecular mechanism of the changes in gastric cancer characteristics induced by CAF-EV. CAF abundance in gastric cancer tissues was associated with poor prognosis of patients with gastric cancer receiving chemotherapy. Moreover, CAF-EV induced tubular network formation and drug resistance of gastric cancer cells in the extracellular matrix (ECM). Comprehensive proteomic analysis of CAF-EV identified that Annexin A6 plays a pivotal role in network formation and drug resistance of gastric cancer cells in the ECM via activation of β1 integrin-focal adhesion kinase (FAK)-YAP. A peritoneal metastasis mouse model revealed that CAF-EV induced drug resistance in peritoneal tumors, and inhibition of FAK or YAP efficiently attenuated gastric cancer drug resistance in vitro and in vivo . These findings demonstrate that drug resistance is conferred by Annexin A6 in CAF-EV and provide a potential avenue for overcoming gastric cancer drug resistance through the inhibition of FAK-YAP signaling in combination with conventional chemotherapeutics.
Significance:
This study elucidates a novel molecular mechanism through which Annexin A6 in CAF-EV activates FAK-YAP by stabilizing β1 integrin at the cell surface of gastric cancer cells and subsequently induces drug resistance.
Graphical Abstract:
http://cancerres.aacrjournals.org/content/canres/80/16/3222/F1.large.jpg.</p
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Pancreatic ductal adenocarcinoma (PDAC) features abundant stromal cells with an excessive extracellular matrix (ECM), termed the desmoplastic reaction. CXCR4 is a cytokine receptor for stromal cell-derived factor-1 (CXCL12) expressed in PDAC, but its roles in PDAC and the characteristic desmoplastic reaction remain unclear. Here, we generated a mouse model of PDAC with conditional knockout of Cxcr4 (KPC-Cxcr4-KO) by crossing Cxcr4 flox mice with Pdx1-Cre;KrasLSL-G12D/+;Trp53LSL-R172H/+ (KPC-Cxcr4-WT) mice to assess the development of pancreatic intraepithelial neoplasia (PanIN) and pancreatic cancers. Tumor cell characteristics of those two types were analyzed in vitro . In addition, CXCR4 expression in human pancreatic cancer specimens was evaluated by IHC staining. In KPC-Cxcr4-KO mice, the number and pathologic grade of PanIN lesions were reduced, but the frequency of pancreatic cancers did not differ from that in KPC-Cxcr4-WT mice. The pancreatic tumor phenotype in KPC-Cxcr4-KO mice was significantly larger and undifferentiated, characterized by abundant vimentin-expressing cancer cells, significantly fewer fibroblasts, and markedly less deposition of ECM. In vitro , KPC-Cxcr4-KO tumor cells exhibited higher proliferative and migratory activity than KPC-Cxcr4-WT tumor cells. Myofibroblasts induced invasion activity in KPC-Cxcr4-WT tumor cells, showing an epithelial–mesenchymal interaction, whereas KPC-Cxcr4-KO tumor cells were unaffected by myofibroblasts, suggesting their unique nature. In human pancreatic cancer, undifferentiated carcinoma did not express CXCR4 and exhibited histologic and IHC features similar to those in KPC-Cxcr4-KO mice. In summary, the CXCL12/CXCR4 axis may play an important role in the desmoplastic reaction in PDAC, and loss of CXCR4 induces phenotype changes in undifferentiated carcinoma without a desmoplastic reaction.
Significance:
The current study uncovers CXCR4 as a key regulator of desmoplastic reaction in PDAC and opens the way for new therapeutic approaches to overcome the chemoresistance in patients with PDAC.</p
Breast cancer is the most common cancer among women and is considered a developed country disease. Moreover, is a heterogenous disease, existing different types and stages of breast cancer development, therefore, better understanding of cancer biology, helps to improve the development of therapies. The conventional treatments accessible after diagnosis, have the main goal of controlling the disease, by improving survival. In more advance stages the aim is to prolong life and symptom palliation care. Surgery, radiation therapy and chemotherapy are the main options available, which must be adapted to each person individually. However, patients are developing resistance to the conventional therapies. This resistance is due to alterations in important regulatory pathways such as PI3K/AKt/mTOR, this pathway contributes to trastuzumab resistance, a reference drug to treat breast cancer.
Therefore, is proposed the repurposing of drugs, instead of developing drugs de novo, for example, to seek new medical treatments within the drugs available, to be used in breast cancer treatment. Providing safe and tolerable treatments to patients, and new insights to efficacy and efficiency of breast cancer treatments. The economic and social burden of cancer is enormous so it must be taken measures to relieve this burden and to ensure continued access to therapies to all patients. In this review we focus on how conventional therapies against breast cancer are leading to resistance, by reviewing those mechanisms and discussing the efficacy of repurposed drugs to fight breast cancer.
Introduction: A major concern that limits the success of cancer chemotherapy is drug resistance (MDR). The drug resistance mechanisms are either host related, or tumor related. The host tumor interacting factors also contribute to MDR. Multifunctional polymeric micelles offer several advantages in circumventing MDR due to their design, selectivity and stability in cancer microenvironment.
Areas covered: The review is broadly divided into two parts the first part covers MDR and its mechanisms. The second part covers multifunctional polymeric micelles in combating MDR through its state-of-the-art design. This part covers various strategies like use of P-gp transporter inhibitors, TPGS, pH & thermo-sensitive and siRNA for selectivity of PMs against multidrug resistant tumors.
Expert opinion: Numerous approaches have been tested using polymeric micelles to overcome MDR tumors. However, these are either limited to only in-vitro investigations and/or preliminary preclinical models and do not investigate the underlying biological mechanism. Hence, there exists an unmet need to perform fundamental research that focuses on studying the underlying mechanism and preclinical/clinical testing of the micellar formulations.
Cancer-associated fibroblasts (CAFs) are a major cellular component of tumor microenvironment in most solid cancers. Altered cellular metabolism is a hallmark of cancer, and much of the published literature has focused on neoplastic cell-autonomous processes for these adaptations. We demonstrate that exosomes secreted by patient-derived CAFs can strikingly reprogram the metabolic machinery following their uptake by cancer cells. We find that CAF-derived exosomes (CDEs) inhibit mitochondrial oxidative phosphorylation, thereby increasing glycolysis and glutamine-dependent reductive carboxylation in cancer cells. Through 13C-labeled isotope labeling experiments we elucidate that exosomes supply amino acids to nutrient-deprived cancer cells in a mechanism similar to macropinocytosis, albeit without the previously described dependence on oncogenic-Kras signaling. Using intra-exosomal metabolomics, we provide compelling evidence that CDEs contain intact metabolites, including amino acids, lipids, and TCA-cycle intermediates that are avidly utilized by cancer cells for central carbon metabolism and promoting tumor growth under nutrient deprivation or nutrient stressed conditions.
Anticancer
drugs resistance is a complex process that arises from altering in the drug
targets. Advances in the DNA microarray, proteomics technology and the
development of targeted therapies provide the new strategies to overcome the
drug resistance. Although a design of the new chemotherapy agents is growing
quickly, effective chemotherapy agent has not been discovered against the
advanced stage of cancer (such as invasion and metastasis). The cancer cell
resistance against the anticancer agents can be due to many factors such as the
individual’s genetic differences, especially in tumoral somatic cells. Also,
the cancer drug resistance is acquired, the drug resistance can be occurred by
different mechanisms, including multi-drug resistance, cell death inhibiting
(apoptosis suppression), altering in the drug metabolism, epigenetic and drug
targets, enhancing DNA repair and gene amplification. In this review, we
outlined the mechanisms of cancer drug resistance and in following, the
treatment failures by common chemotherapy agents in the different type of
cancers.
In MCF-7 human breast carcinoma cells, α5β1 integrin hyperexpression, which was accomplished by transduction of a full-length α5 integrin cDNA, increased by about 50-70% the number of cells, survived during 48-72 h cell treatment with doxorubicin. Up-regulation of α5β1 reduced the level of the apoptogenic p53 protein and p21 cell cycle inhibitor, but enhanced the activity of Akt and mTOR protein kinases. In addition to these findings, we observed a significant decrease in the activity of both isoforms of phosphokinase Erk1/2, which is known to play a key role in cell viability pathways, including pathways alleviating stress damages caused by distinct antitumor drugs. Diminished Erk activity accompanying the rise of drug resistance can be explained by an “atypical” function of this kinase, which, in the cells studied, promotes an enhanced rather than reduced sensitivity to doxorubicin. To verify this suggestion, the effect of a specific Erk inhibitor, PD98059, on the resistance to doxorubicin of control and α5 cDNA-transduced MCF-7 cells was investigated. The data showed that suppression of Erk activity increased the resistance of control cells (transduced with an “empty” vector) to a level higher than that demonstrated by the α5 cDNA-transduced cells. The highest level of resistance was observed in α5β1trancduced cells treated with PD98059. Akt and mTOR kinase inhibitors had little if any effect on doxorubicin resistance of α5 cDNA-transduced MCF-7 cells. The data show for the first time that integrin α5β1 can stimulate drug resistance of tumor cells through a mechanism based on the inhibition of protein kinase Erk. From a more general view, the results of this investigation suggest that signal protein kinases can perform in tumor cells “non-canonical” functions, opposite to those, which are the basis for using kinase inhibitors in targeted cancer therapy. It follows that if a protein kinase is supposed to be used as a target for such therapy, it is important to explore its features in the particular tumor prior to the onset of treatment.
Conventional treatments of gastrointestinal cancers based on surgical resection and chemotherapy are not enough to eradicate potentially relapsing tumor cells and can also impair the immune system functions. Immunotherapies aim to help the body to eradicate cancer and other diseases, by modulating the immune system. They can be performed by active approaches, usually orchestrated by dendritic cell vaccines that present a specific tumor associated antigen to T cells, or passive approaches, which have the T cells as protagonist, and are based on antitumor antibodies, or adoptive cell transfer. T lymphocyte subsets can exhibit different role face to a tumor scenario, varying from an effective cellular antitumor response to a regulatory participation. Although a lot of protocols to combat cancer progression have been proposed, T cell-based immunotherapies in gastrointestinal cancers are still not approved for clinical applications mainly because of their side effects. Nowadays, promising protocols combining two or more approaches, aiming to create an efficient therapy without or with fewer side effects. In this chapter, we made a review about the role of T cells on cancer, especially focusing on gastrointestinal cancer immunotherapeutic methods.
p130Cas (rk ssociated ubstrate) has the structural characteristics of an adapter protein, containing multiple consensus SH2 binding sites, an SH3
domain, and a proline-rich domain. The structure of p130Cas suggests that it may act to provide a framework for protein-protein interactions; however, as yet, its functional role in
cells is unknown. In this report we show that p130Cas is localized to focal adhesions. We demonstrate that p130Cas associates both in vitro and in vivo with pp125FAK (ocal dhesion inase), a kinase implicated in signaling by the integrin family of cell adhesion receptors. p130Cas also associates with pp41/43FRNK (pp125FAK-related, non-kinase), an autonomously expressed form of pp125FAK composed of only the C-terminal noncatalytic domain. We show that the association of p130Cas with pp125Fak and pp41/43FRNK is direct, and is mediated by the binding of the SH3 domain of p130Cas to a proline-rich sequence present in both the C terminus of pp125FAK and in pp41/43FRNK. In agreement with recent studies we show that p130Cas is tyrosine-phosphorylated upon integrin mediated cell adhesion. The association of p130Cas with pp125FAK, a kinase which is activated upon cell adhesion, is likely to be functionally important in integrin mediated signal transduction.
Molluscum contagiosum virus proteins MC159 and MC160 and the equine herpesvirus 2 protein E8 share substantial homology to the death effector domain present in the adaptor molecule Fas-associated death domain protein (FADD) and the initiating death protease FADD-like interleukin-1beta-converting enzyme (FLICE) (caspase-8). FADD and FLICE participate in generating the death signal from both tumor necrosis factor receptor-1 (TNFR-1) and the CD-95 receptor. The flow of death signals from TNFR-1 occurs through the adaptor molecule tumor necrosis factor receptor-associated death domain protein (TRADD) to FADD to FLICE, whereas for CD-95 the receptor directly communicates with FADD and then FLICE. MC159 and E8 inhibited both TNFR-1- and CD-95-induced apoptosis as well as killing mediated by overexpression of the downstream adaptors TRADD and FADD. Neither viral molecule, however, inhibited FLICE-induced killing, consistent with an inhibitory action upstream of the active death protease. These data suggest the existence of a novel strategy employed by viruses to attenuate host immune killing mechanisms. Given that bovine herpesvirus 4 protein E1.1 and Kaposi's sarcoma associated-herpesvirus protein K13 also possess significant homology to the viral inhibitory molecules MC159, MC160, and E8, it may be that this class of proteins is used ubiquitously by viruses to evade host defense.
Integrin ligation initiates intracellular signaling events, among which are the activation of protein tyrosine kinases. The
related adhesion focal tyrosine kinase (RAFTK), also known as PYK2 and CAKβ, is a tyrosine kinase that is homologous to the
focal adhesion kinase (FAK) p125FAK. The structure of RAFTK is similar to p125FAK in that it lacks a transmembrane region, does not contain Src homology 2 or 3 domains, and has a proline-rich region in its
C terminus. Here we report that RAFTK is a target for β1-integrin-mediated tyrosine phosphorylation in both transformed and
normal human B cells. Ligation of the B cell antigen receptor also induced tyrosine phosphorylation of RAFTK. Phosphorylation
of RAFTK following integrin- or B cell antigen receptor-mediated stimulation was decreased by prior treatment of cells with
cytochalasin B, indicating that this process was at least partially cytoskeleton-dependent. One of the tyrosine-phosphorylated
substrates after integrin stimulation in fibroblasts is p130cas, which can associate with p125FAK. RAFTK also interacted constitutively with p130cas in B cells, since p130cas was detected in RAFTK immunoprecipitates. Although the function of RAFTK remains unknown, these data suggest that RAFTK may
have a significant function in integrin-mediated signaling pathways in B cells.
Renewal of the gastrointestinal epithelium involves a coordinated process of terminal differenti- ation and programmed cell death. Integrins have been implicated in the control of apoptotic processes in various cell types. Here we examine the role of integrins in the regulation of apoptosis in gastrointestinal epithelial cells with the use of a rat small intestinal epithelial cell line (RIE1) as a model. Overexpression of the integrin a5 subunit in RIE1 cells conferred protection against several proapoptotic stimuli. In contrast, overexpression of the integrin a2 subunit had no effect on cell survival. The antiapoptotic effect of the a5 subunit was partially retained by a mutated version that had a truncation of the cytoplasmic domain. The antiapoptotic effects of the full- length or truncated a5 subunit were reversed upon treatment with inhibitors of phosphatidylino- sitol 3-kinase (PI-3-kinase), suggesting that the a5b1 integrin might interact with the PI-3-kinase/ Akt survival pathway. When cells overexpressing a5 were allowed to adhere to fibronectin, there was a moderate activation of protein kinase B (PKB)/Akt, whereas no such effect was seen in a2-overexpressing cells adhering to collagen. Furthermore, in cells overexpressing a5 and adher- ing to fibronectin, there was a dramatic enhancement of the ability of growth factors to stimulate PKB/Akt; again, this was not seen in cells overexpressing a2 subunit and adhering to collagen or fibronectin. Expression of a dominant negative version of PKB/Akt in RIE cells blocked to ability of a5 to enhance cell survival. Thus, the a5b1 integrin seems to protect intestinal epithelial cells against proapoptotic stimuli by selectively enhancing the activity of the PI-3-kinase/Akt survival pathway.
The phosphorylation of protein tyrosine kinases (PTKs) on tyrosine residues is a critical regulatory event that modulates catalytic activity and triggers the physical association of PTKs with Src homology 2 (SH2)-containing proteins. The integrin-linked focal adhesion kinase, pp125FAK, exhibits extracellular matrix-dependent phosphorylation on tyrosine and physically associates with two nonreceptor PTKs, pp60src and pp59fyn, via their SH2 domains. Herein, we identify Tyr-397 as the major site of tyrosine phosphorylation on pp125FAK both in vivo and in vitro. Tyrosine 397 is located at the juncture of the N-terminal and catalytic domains, a novel site for PTK autophosphorylation. Mutation of Tyr-397 to a nonphosphorylatable residue dramatically impairs the phosphorylation of pp125FAK on tyrosine in vivo and in vitro. The mutation of Tyr-397 to Phe also inhibits the formation of stable complexes with pp60src in cells expressing Src and FAK397F, suggesting that autophosphorylation of pp125FAK may regulate the association of pp125FAK with Src family kinases in vivo. The identification of Tyr-397 as a major site for FAK autophosphorylation provides one of the first examples of a cellular protein containing a high-affinity binding site for a Src family kinase SH2 domain. This finding has implications for models describing the mechanisms of action of pp125FAK, the regulation of the Src family of PTKs, and signal transduction through the integrins.
Focal adhesion kinase (pp125FAK or FAK) and paxillin colocalize with integrins in structures called focal adhesions. pp125FAK plays an important role in the transmission of integrin-induced cytoplasmic signals. Paxillin has also been implicated in cell signaling by virtue of its association with the protein tyrosine kinases pp60src and Csk (C-terminal Src kinase) as well as with the adapter/oncoprotein p47gag-crk. In this report we show that endogenous pp125FAK and paxillin form a stable complex both in vivo and in vitro and that this interaction is direct, requiring only pp125FAK and paxillin. The paxillin binding site on pp125FAK has been localized to the carboxy-terminal 148 residues of pp125FAK, but appears to be distinct from the previously identified focal adhesion-targeting sequence also present in the carboxy-terminal domain of pp125FAK. The interaction of paxillin and pp125FAK is independent of the adhesion of cells to the extracellular matrix, as the association can be detected in suspension cells as well as those attached to fibronectin.
Cell-matrix interactions have major effects upon phenotypic features such as gene regulation, cytoskeletal structure, differentiation, and aspects of cell growth control. Programmed cell death (apoptosis) is crucial for maintaining appropriate cell number and tissue organization. It was therefore of interest to determine whether cell-matrix interactions affect apoptosis. The present report demonstrates that apoptosis was induced by disruption of the interactions between normal epithelial cells and extracellular matrix. We have termed this phenomenon "anoikis." Overexpression of bcl-2 protected cells against anoikis. Cellular sensitivity to anoikis was apparently regulated: (a) anoikis did not occur in normal fibroblasts; (b) it was abrogated in epithelial cells by transformation with v-Ha-ras, v-src, or treatment with phorbol ester; (c) sensitivity to anoikis was conferred upon HT1080 cells or v-Ha-ras-transformed MDCK cells by reverse-transformation with adenovirus E1a; (d) anoikis in MDCK cells was alleviated by the motility factor, scatter factor. The results suggest that the circumvention of anoikis accompanies the acquisition of anchorage independence or cell motility.
Tumor endothelium is critical for solid tumor growth and is a potential site for anticancer drug action. Within 2 h, etoposide caused marked DNA strand breakage in xenograft tumor-derived endothelial cells (TDECs). Etoposide-induced DNA breakage was inhibited by culturing TDECs on gelatin, type IV collagen, laminin, fibronectin, and the integrin ligand hexapeptide, GRGDSP, but not the inactive peptide, GRADSP. It was also inhibited when TDECs were on surfaces coated with antibodies to alpha 5, beta 1, or beta 3 integrin subunits and by clustering integrins with soluble antibodies. After 8 h with etoposide, TDECs detached from the monolayer, and 50-kb DNA fragments were seen. Fibronectin inhibited both processes. Thus, integrins are survival factors for TDEC that inhibit the genotoxicity of etoposide and may influence the sensitivity of tumors to drugs.
A low proliferating fraction in solid tumors limits the effectiveness of cell cycle-dependent chemotherapeutic agents. To understand the molecular basis of such "kinetic" resistance we cultured tumor cells as multicellular spheroids and examined levels of p27Kip1, a cyclin-dependent kinase inhibitor known to be upregulated by intercellular contact in normal cells. When transferred from monolayer to three-dimensional culture, a consistent upregulation (up to 15-fold) of p27 protein was observed in a panel of mouse and human carcinoma cell lines. Antisense-oligonucleotide-mediated downregulation of p27 in EMT-6 mammary tumor cell spheroids reduced intercellular adhesion, increased cell proliferation, sensitized tumor cells to 4-hydroperoxycyclophosphamide, and restored drug- or radiation-induced cell-cycle perturbations repressed in spheroid culture. Our results implicate p27 as a regulator of drug resistance in solid tumors and suggest that tumor-targeted p27 antagonists may be useful chemosensitizers in conjunction with conventional anticancer therapy.
We provide evidence that a class of integrins combines with the adaptor Shc and thereby with Grb2. Coimmunoprecipitation and mutagenesis experiments indicate that the recruitment of Shc is specified by the extracellular or transmembrane domain of integrin alpha subunit and suggest that this process is mediated by caveolin. Mutagenesis and dominant-negative inhibition studies reveal that Shc is necessary and sufficient for activation of the MAP kinase pathway in response to integrin ligation. Mitogens and Shc-activating integrins cooperate to promote transcription from the Fos serum response element and transit through G1. In contrast, adhesion mediated by integrins not linked to Shc results in cell cycle arrest and apoptosis even in presence of mitogens. These findings indicate that the association of specific integrins with Shc regulates cell survival and cell cycle progression.
Integrin ligation initiates intracellular signaling events, among which are the activation of protein tyrosine kinases. The related adhesion focal tyrosine kinase (RAFTK), also known as PYK2 and CAKbeta, is a tyrosine kinase that is homologous to the focal adhesion kinase (FAK) p125FAK. The structure of RAFTK is similar to p125FAK in that it lacks a transmembrane region, does not contain Src homology 2 or 3 domains, and has a proline-rich region in its C terminus. Here we report that RAFTK is a target for beta1-integrin-mediated tyrosine phosphorylation in both transformed and normal human B cells. Ligation of the B cell antigen receptor also induced tyrosine phosphorylation of RAFTK. Phosphorylation of RAFTK following integrin- or B cell antigen receptor-mediated stimulation was decreased by prior treatment of cells with cytochalasin B, indicating that this process was at least partially cytoskeleton-dependent. One of the tyrosine-phosphorylated substrates after integrin stimulation in fibroblasts is p130cas, which can associate with p125FAK. RAFTK also interacted constitutively with p130cas in B cells, since p130cas was detected in RAFTK immunoprecipitates. Although the function of RAFTK remains unknown, these data suggest that RAFTK may have a significant function in integrin-mediated signaling pathways in B cells.
Integrin-mediated cell adhesion causes activation of MAP kinases and increased tyrosine phosphorylation of focal adhesion kinase (FAK). Autophosphorylation of FAK leads to the binding of SH2-domain proteins including Src-family kinases and the Grb2-Sos complex. Since Grb2-Sos is a key regulator of the Ras signal transduction pathway, one plausible hypothesis has been that integrin-mediated tyrosine phosphorylation of FAK leads to activation of the Ras cascade and ultimately to mitogen activated protein (MAP) kinase activation. Thus, in this scenario FAK would serve as an upstream regulator of MAP kinase activity. However, in this report we present several lines of evidence showing that integrin-mediated MAP kinase activity in fibroblasts is independent of FAK. First, a beta1 integrin subunit deletion mutant affecting the putative FAK binding site supports activation of MAP kinase in adhering fibroblasts but not tyrosine phosphorylation of FAK. Second, fibroblast adhesion to bacterially expressed fragments of fibronectin demonstrates that robust activation of MAP kinase can precede tyrosine phosphorylation of FAK. Finally, we have used FRNK, the noncatalytic COOH-terminal domain of FAK, as a dominant negative inhibitor of FAK autophosphorylation and of tyrosine phosphorylation of focal contacts. Using retroviral infection, we demonstrate that levels of FRNK expression sufficient to completely block FAK tyrosine phosphorylation were without effect on integrin-mediated activation of MAP kinase. These results strongly suggest that integrin-mediated activation of MAP kinase is independent of FAK and indicate the probable existence of at least two distinct integrin signaling pathways in fibroblasts.
The related adhesion focal tyrosine kinase (RAFTK) is tyrosine-phosphorylated following beta1 integrin or B cell antigen receptor stimulation in human B cells. Two substrates that are tyrosine-phosphorylated following integrin ligation in B cells are p130(Cas) and the Cas family member human enhancer of filamentation 1 (HEF1), both of which can associate with RAFTK. In this report we observed that RAFTK was involved in the phosphorylation of these two proteins. While a catalytically active RAFTK was required for both p130(Cas) and HEF1, phosphorylation of p130(Cas), but not of HEF1, was dependent on an intact autophosphorylation site (Tyr402) on RAFTK. To determine if RAFTK phosphorylated p130(Cas) and HEF1 directly or through an intermediate, we assayed the ability of RAFTK and of a Tyr402 mutant to phosphorylate purified HEF1 and p130(Cas) domains. RAFTK was able to phosphorylate the substrate domains of both p130(Cas) and HEF1, but only the C-terminal domain of p130(Cas). Furthermore, Tyr402, which mediates the binding of RAFTK to c-Src kinase, was required for the phosphorylation of the C-terminal domain of p130(Cas). These data suggest that RAFTK itself is sufficient for HEF1 phosphorylation, whereas a cooperation between RAFTK and Src kinases is required for the complete phosphorylation of p130(Cas).
Monoclonal antibodies to very late antigen 4 (VLA-4) recognize the alpha4beta1 integrin receptor. This monoclonal antibody blocks the adhesion between early hematopoietic progenitor cells (CD34-selected cells) and stromal cells when added to cultures of these cells. Addition of the VLA-4 monoclonal antibody to cultures of stromal cells and CD34-selected cells was shown to induce apoptosis of CD34-selected cells in these CD34-selected cell/stromal cell cocultures, as measured by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling method. In contrast to these experiments with early hematopoietic progenitor cells (CD34+), the level of adhesion between more differentiated cells (unfractionated hematopoietic cells) and stromal cells was not significantly altered by addition of the anti-VLA-4 monoclonal antibody. Similarly, the level of apoptosis of unfractionated hematopoietic cells was not significantly increased by the addition of anti-VLA-4 monoclonal antibody to cultures of the latter cells with stromal cells. The binding of the unfractionated cells is less than that of the CD34-selected cells. Given that there is no difference between the alpha4beta1 integrin expression level of the early and late myeloid cells, there may be a difference in the functional state of the integrin between the early and late myeloid cells. We also show that CD34+-selected precursor cells proliferate at a higher rate when these cells are plated on recombinant vascular cell adhesion molecule 1 molecules. These data indicate that the alpha4beta1 integrin receptor (VLA-4) plays a central role in the apoptosis rescue function that results from the anchorage-dependent growth of the CD34-selected early hematopoietic cells on stromal cells. The data suggest that these apoptosis rescue pathways have less significance as the cells mature and become anchorage independent in their growth. These data should assist in the design of systems for the ex vivo proliferation and transduction of early hematopoietic cells for genetic therapy.
Recent studies have demonstrated the importance of E-cadherin, a homophilic cell-cell adhesion molecule, in contact inhibition of growth of normal epithelial cells. Many tumor cells also maintain strong intercellular adhesion, and are growth-inhibited by cell- cell contact, especially when grown in three-dimensional culture. To determine if E-cadherin could mediate contact-dependent growth inhibition of nonadherent EMT/6 mouse mammary carcinoma cells that lack E-cadherin, we transfected these cells with an exogenous E-cadherin expression vector. E-cadherin expression in EMT/6 cells resulted in tighter adhesion of multicellular spheroids and a reduced proliferative fraction in three-dimensional culture. In addition to increased cell-cell adhesion, E-cadherin expression also resulted in dephosphorylation of the retinoblastoma protein, an increase in the level of the cyclin-dependent kinase inhibitor p27(kip1) and a late reduction in cyclin D1 protein. Tightly adherent spheroids also showed increased levels of p27 bound to the cyclin E-cdk2 complex, and a reduction in cyclin E-cdk2 activity. Exposure to E-cadherin-neutralizing antibodies in three-dimensional culture simultaneously prevented adhesion and stimulated proliferation of E-cadherin transfectants as well as a panel of human colon, breast, and lung carcinoma cell lines that express functional E-cadherin. To test the importance of p27 in E-cadherin-dependent growth inhibition, we engineered E-cadherin-positive cells to express inducible p27. By forcing expression of p27 levels similar to those observed in aggregated cells, the stimulatory effect of E-cadherin-neutralizing antibodies on proliferation could be inhibited. This study demonstrates that E-cadherin, classically described as an invasion suppressor, is also a major growth suppressor, and its ability to inhibit proliferation involves upregulation of the cyclin-dependent kinase inhibitor p27.
The focal adhesion kinase (FAK) protein-tyrosine kinase (PTK) links transmembrane integrin receptors to intracellular signaling pathways. We show that expression of the FAK-related PTK, Pyk2, is elevated in fibroblasts isolated from murine fak-/- embryos (FAK-) compared with cells from fak+/+ embryos (FAK+). Pyk2 was localized to perinuclear regions in both FAK+ and FAK- cells. Pyk2 tyrosine phosphorylation was enhanced by fibronectin (FN) stimulation of FAK- but not FAK+ cells. Increased Pyk2 tyrosine phosphorylation paralleled the time-course of Grb2 binding to Shc and activation of ERK2 in FAK- cells. Pyk2 in vitro autophosphorylation activity was not enhanced by FN plating of FAK- cells. However, Pyk2 associated with active Src-family PTKs after FN but not poly-L-lysine replating of the FAK- cells. Overexpression of both wild-type (WT) and kinase-inactive (Ala457), but not the autophosphorylation site mutant (Phe402) Pyk2, enhanced endogenous FN-stimulated c-Src in vitro kinase activity in FAK- cells, but only WT Pyk2 overexpression enhanced FN-stimulated activation of co-transfected ERK2. Interestingly, Pyk2 overexpression only weakly augmented FAK- cell migration to FN whereas transient FAK expression promoted FAK- cell migration to FN efficiently compared with FAK+ cells. Significantly, repression of endogenous Src-family PTK activity by p50(csk) overexpression inhibited FN-stimulated cell spreading, Pyk2 tyrosine phosphorylation, Grb2 binding to Shc, and ERK2 activation in the FAK- but not in FAK+ cells. These studies show that Pyk2 and Src-family PTKs combine to promote FN-stimulated signaling events to ERK2 in the absence of FAK, but that these signaling events are not sufficient to overcome the FAK- cell migration defects.
Integrin-mediated adhesion influences cell survival and may prevent programmed cell death. Little is known about how drug-sensitive tumor cell lines survive initial exposures to cytotoxic drugs and eventually select for drug-resistant populations. Factors that allow for cell survival following acute cytotoxic drug exposure may differ from drug resistance mechanisms selected for by chronic drug exposure. We show here that drug-sensitive 8226 human myeloma cells, demonstrated to express both VLA-4 (alpha4beta1) and VLA-5 (alpha5beta1) integrin fibronectin (FN) receptors, are relatively resistant to the apoptotic effects of doxorubicin and melphalan when pre-adhered to FN and compared with cells grown in suspension. This cell adhesion mediated drug resistance, or CAM-DR, was not due to reduced drug accumulation or upregulation of anti-apoptotic Bcl-2 family members. As determined by flow cytometry, myeloma cell lines selected for drug resistance, with either doxorubicin or melphalan, overexpress VLA-4. Functional assays revealed a significant increase in alpha4-mediated cell adhesion in both drug-resistant variants compared with the drug-sensitive parent line. When removed from selection pressure, drug-resistant cell lines reverted to a drug sensitive and alpha4-low phenotype. Whether VLA-4-mediated FN adhesion offers a survival advantage over VLA-5-mediated adhesion remains to be determined. In conclusion, we have demonstrated that FN-mediated adhesion confers a survival advantage for myeloma cells acutely exposed to cytotoxic drugs by inhibiting drug-induced apoptosis. This finding may explain how some cells survive initial drug exposure and eventually express classical mechanisms of drug resistance such as MDR1 overexpression.
Adherent epithelial cells require interactions with the extracellular matrix for their survival, though the mechanism is ill-defined. In long term cultures of primary mammary epithelial cells, a laminin-rich basement membrane (BM) but not collagen I suppresses apoptosis, indicating that adhesion survival signals are specific in their response (. J. Cell Sci. 109:631-642). We now demonstrate that the signal from BM is mediated by integrins and requires both the alpha6 and beta1 subunits. In addition, a hormonal signal from insulin or insulin-like growth factors, but not hydrocortisone or prolactin, is necessary to suppress mammary cell apoptosis, indicating that BM and soluble factors cooperate in survival signaling. Insulin induced autophosphorylation of its receptor whether mammary cells were cultured on collagen I or BM substrata. However, both the tyrosine phosphorylation of insulin receptor substrate-1 and its association with phosphatidylinositol 3-kinase were enhanced in cells cultured on BM, as was the phosphorylation of the phosphatidylinositol 3-kinase effector, protein kinase B. These results suggest a novel extracellular matrix-dependent restriction point in insulin signaling in mammary epithelial cells. The proximal signal transduction event of insulin receptor phosphorylation is not dependent on extracellular matrix, but the activation of downstream effectors requires adhesion to BM. Since phosphatidylinositol 3-kinase was required for mammary epithelial cell survival, we propose that a possible mechanism for BM-mediated suppression of apoptosis is through its facilitative effects on insulin signaling.
Most transformed cells have lost anchorage and serum dependence for growth and survival. Previously, we established that when serum is absent, fibronectin survival signals transduced by focal adhesion kinase (FAK), suppress p53-regulated apoptosis in primary fibroblasts and endothelial cells (Ilić et al. 1998. J. Cell Biol. 143:547-560). The present goals are to identify survival sequences in FAK and signaling molecules downstream of FAK required for anchorage-dependent survival of primary fibroblasts. We report that binding of the SH3 domain of p130Cas to proline-rich region 1 of FAK is required to support survival of fibroblasts on fibronectin when serum is withdrawn. The FAK-p130Cas complex activates c-Jun NH2-terminal kinase (JNK) via a Ras/Rac1/Pak1/MAPK kinase 4 (MKK4) pathway. Activated (phospho-) JNK colocalizes with FAK in focal adhesions of fibroblasts cultured on fibronectin, which supports their survival, but not in fibroblasts cultured on collagen, which does not. Cells often survive in the absence of extracellular matrix if serum factors are provided. In that case, we confirm work of others that survival signals are transduced by FAK, phosphatidylinositol 3'-kinase (PI3-kinase), and Akt/protein kinase B (PKB). However, when serum is absent, PI3-kinase and Akt/PKB are not involved in the fibronectin-FAK-JNK survival pathway documented herein. Thus, survival signals from extracellular matrix and serum are transduced by FAK via two distinct pathways.
EMT-6 murine mammary tumors were made resistant to cis-diamminedichloroplatinum (II) (CDDP), carboplatin, cyclophosphamide (CTX), or thiotepa in vivo by treatment of tumor-bearing animals with the drug during a 6-month period. In spite of high levels of in vivo resistance, no significant resistance was observed when the cells from these tumors were exposed to the drugs in vitro. The pharmacokinetics of CDDP and CTX were altered in animals bearing the respective resistant tumors. The resistance of all tumor lines except for the EMT-6/thiotepa decreased during 3 to 6 months in vivo passage in the absence of drugs. These results indicate that very high levels of resistance to anticancer drugs can develop through mechanisms that are expressed only in vivo.
The phosphorylation of protein tyrosine kinases (PTKs) on tyrosine residues is a critical regulatory event that modulates catalytic activity and triggers the physical association of PTKs with Src homology 2 (SH2)-containing proteins. The integrin-linked focal adhesion kinase, pp125FAK, exhibits extracellular matrix-dependent phosphorylation on tyrosine and physically associates with two nonreceptor PTKs, pp60src and pp59fyn, via their SH2 domains. Herein, we identify Tyr-397 as the major site of tyrosine phosphorylation on pp125FAK both in vivo and in vitro. Tyrosine 397 is located at the juncture of the N-terminal and catalytic domains, a novel site for PTK autophosphorylation. Mutation of Tyr-397 to a nonphosphorylatable residue dramatically impairs the phosphorylation of pp125FAK on tyrosine in vivo and in vitro. The mutation of Tyr-397 to Phe also inhibits the formation of stable complexes with pp60src in cells expressing Src and FAK397F, suggesting that autophosphorylation of pp125FAK may regulate the association of pp125FAK with Src family kinases in vivo. The identification of Tyr-397 as a major site for FAK autophosphorylation provides one of the first examples of a cellular protein containing a high-affinity binding site for a Src family kinase SH2 domain. This finding has implications for models describing the mechanisms of action of pp125FAK, the regulation of the Src family of PTKs, and signal transduction through the integrins.
Bcl-2 and its homologue Bcl-XL are expressed in a variety of tumors and their expression modulates the sensitivity of tumor cells to a wide spectrum of chemotherapeutic agents and γ-irradiation. In the present report, we generated clones of FL5.12 lymphoid cells with similar levels of Bcl-2 and Bcl-XL using the Flag epitope to determine if these survival proteins could provide equivalent protection when challenged with chemotherapy or γ-irradiation. Using four M-phase specific chemotherapeutic agents, Bcl-XL and Bcl-2 provided similar protection against vincristine and vinblastine whereas Bcl-XL afforded as much as 50% greater cell viability than Bcl-2 against etoposide and teniposide-induced cell death. In addition, Bcl-XL provided significantly greater cell viability than Bcl-2 against methotrexate, fluorouracil, and hydroxyurea, three S-phase specific agents. In apoptosis induced by γ-irradiation and cisplatin, two antitumor treatments that are cell-cycle phase-nonspecific agents, both Bcl-XL and Bcl-2 conferred similar protection against γ-irradiation, but Bcl-XL provided better protection than Bcl-2 against cisplatin. These results indicate that Bcl-XL and Bcl-2 confer a differential ability to protect against chemotherapy-induced cell death, which appears to be dependent on the molecular mechanism targeted by the drug rather than its cell-cycle phase specificity.
Phorbol esters upregulate the functional affinity of beta 1 integrin receptors for fibronectin on human neutrophils and other leukocytes. We investigated the ability of phorbol myristate acetate (PMA) to stimulate the human promyelocytic cell line HL-60 to adhere to fibronectin, either in its undifferentiated state (HL60) or after dimethylsulfoxide-induced differentiation along the granulocytic pathway (dHL60). PMA stimulated little adherence of undifferentiated HL60 to fibronectin or to the 120-kD chymotryptic cell-binding domain (CBD) of fibronectin. In contrast, PMA stimulated dHL60 cells to rapidly adhere to both fibronectin- and to CBD-coated plastic. PMA- stimulated dHL60 adherence to fibronectin was largely mediated by both alpha 4 beta 1 and alpha 5 beta 1, whereas PMA-stimulated dHL60 adherence to CBD was largely mediated by alpha 5 beta 1. There was little contribution from beta 2 integrins to PMA-stimulated dHL60 adherence to fibronectin or CBD. The inability of undifferentiated HL60 to adhere to fibronectin and CBD did not result from lack of expression of alpha 4 beta 1 or alpha 5 beta 1 because HL60 and dHL60 express similar amounts of both alpha 4 beta 1 and alpha 5 beta 1 on their surface. In addition, 1 mmol/L Mn2+ induced similar amounts of alpha 5 beta 1-dependent adherence of both HL60 and dHL60, showing that alpha 5 beta 1 on undifferentiated HL60 is capable of binding to its ligand. These data suggest that activation of protein kinase C cannot functionally upregulate these beta 1 integrins on undifferentiated HL60 cells. The development of PMA-stimulated beta 1-dependent adherence after granulocytic differentiation of HL60 cells suggests that the differentiated HL60 cell is a useful model for investigating functional coupling of protein kinase C to beta 1 integrin in myeloid cells.
Bcl-2 and its homologue Bcl-XL are expressed in a variety of tumors and their expression modulates the sensitivity of tumor cells to a wide spectrum of chemotherapeutic agents and γ-irradiation. In the present report, we generated clones of FL5.12 lymphoid cells with similar levels of Bcl-2 and Bcl-XL using the Flag epitope to determine if these survival proteins could provide equivalent protection when challenged with chemotherapy or γ-irradiation. Using four M-phase specific chemotherapeutic agents, Bcl-XL and Bcl-2 provided similar protection against vincristine and vinblastine whereas Bcl-XL afforded as much as 50% greater cell viability than Bcl-2 against etoposide and teniposide-induced cell death. In addition, Bcl-XL provided significantly greater cell viability than Bcl-2 against methotrexate, fluorouracil, and hydroxyurea, three S-phase specific agents. In apoptosis induced by γ-irradiation and cisplatin, two antitumor treatments that are cell-cycle phase-nonspecific agents, both Bcl-XL and Bcl-2 conferred similar protection against γ-irradiation, but Bcl-XL provided better protection than Bcl-2 against cisplatin. These results indicate that Bcl-XL and Bcl-2 confer a differential ability to protect against chemotherapy-induced cell death, which appears to be dependent on the molecular mechanism targeted by the drug rather than its cell-cycle phase specificity.
Integrin-mediated adhesion influences cell survival and may prevent programmed cell death. Little is known about how drug-sensitive tumor cell lines survive initial exposures to cytotoxic drugs and eventually select for drug-resistant populations. Factors that allow for cell survival following acute cytotoxic drug exposure may differ from drug resistance mechanisms selected for by chronic drug exposure. We show here that drug-sensitive 8226 human myeloma cells, demonstrated to express both VLA-4 (4β1) and VLA-5 (5β1) integrin fibronectin (FN) receptors, are relatively resistant to the apoptotic effects of doxorubicin and melphalan when pre-adhered to FN and compared with cells grown in suspension. This cell adhesion mediated drug resistance, or CAM-DR, was not due to reduced drug accumulation or upregulation of anti-apoptotic Bcl-2 family members. As determined by flow cytometry, myeloma cell lines selected for drug resistance, with either doxorubicin or melphalan, overexpress VLA-4. Functional assays revealed a significant increase in 4-mediated cell adhesion in both drug-resistant variants compared with the drug-sensitive parent line. When removed from selection pressure, drug-resistant cell lines reverted to a drug sensitive and 4-low phenotype. Whether VLA-4–mediated FN adhesion offers a survival advantage over VLA-5–mediated adhesion remains to be determined. In conclusion, we have demonstrated that FN-mediated adhesion confers a survival advantage for myeloma cells acutely exposed to cytotoxic drugs by inhibiting drug-induced apoptosis. This finding may explain how some cells survive initial drug exposure and eventually express classical mechanisms of drug resistance such as MDR1 overexpression.
We have previously reported the presence of monoclonal, tumor-related B lineage cells in the blood of myeloma patients. The cells are continuously differentiating, and the majority are at a very late stage of B cell differentiation into plasma cells, consistent with the hypothesis that they comprise a precursor cell subset responsible for disseminating and possibly for relapse of the disease. The pattern of β1 integrin expression on monoclonal B lineage cells from blood and bone marrow of myeloma patients was evaluated using multiparameter flow cytometry in comparison to normal blood or tissue B cells and malignant B cells from B-CLL, B lymphoma, or plasma cell leukemia. The α4 and β1 chains were found on the majority of normal B cells, usually with a higher expression of α4 compared to β1. α5 was detectable at low density on B cells from lymph node, bone marrow, and lamina propria. The α2 and α6 chains are absent on B cells localized in normal lymphoid tissues as well as on normal blood B cells and in vitro activated B cells. In myeloma, the blood B cells express α2, α5 and α6, suggesting important functional differences between these tumor-related B cells and their normal counterparts. The plasma cells located in myeloma bone marrow express no α2, and almost no α6, although they have variable expression of α4, α5, and β1. Thus the end-stage plasma cells appear to lack receptors that would support a propensity for invasion of basement membranes and exit to extravascular spaces. In contrast, the circulating plasmablasts in a patient with plasma cell leukemia make up a large subset of early plasma cells expressing all integrin receptors analyzed, including α2 and α6. Malignant cells from B-CLL and B lymphoma express only the α4 and β integrins, and some B-CLL have very low levels of α3, but no α2, α5, or α6, suggesting that they may be limited to the vascular spaces and do not extravasate, at least for the stages of disease analyzed here. Our results are therefore consistent with a working hypothesis that invasive capacity in myeloma is to be found within the abnormal monoclonal B cells in peripheral blood, which alone among B cells or plasma cells, and like plasma cell leukemia, express multiple β1 integrins, including the α2 and α6 integrin receptors, necessary for cellular translocation across the endothelial basement membranes and into extravascular spaces. This study raises the possibility that expression of, in particular, the α2 and α6 integrins may underlie the invasiveness of these diseases and offers a new perspective for future avenues of clinical intervention.
Resistance to chemotherapy is a principal problem in the treatment of small
cell lung cancer (SCLC). We show here that SCLC is surrounded by an extensive
stroma of extracellular matrix (ECM) at both primary and metastatic sites.
Adhesion of SCLC cells to ECM enhances tumorigenicity and confers resistance
to chemotherapeutic agents as a result of 1 integrin-stimulated tyrosine
kinase activation suppressing chemotherapy-induced apoptosis. SCLC may create
a specialized microenvironment, and the survival of cells bound to ECM could
explain the partial responses and local recurrence of SCLC often seen clinically
after chemotherapy. Strategies based on blocking 1 integrin-mediated
survival signals may represent a new therapeutic approach to improve the response
to chemotherapy in SCLC.
A homology-based cDNA cloning approach was used to identify a widely expressed protein-tyrosine kinase designated as "focal adhesion kinase" (FadK). The entire mouse FadK amino acid sequence was deduced from cDNA clones, revealing a large (119-kDa) non-membrane-spanning protein-tyrosine kinase that lacks Src-homology SH2 and SH3 domains. Immunostaining of BALB/c 3T3 fibroblasts revealed that FadK is concentrated in focal adhesions. FadK is phosphorylated on tyrosine in growing cultures of BALB/c 3T3 cells but contains little or no phosphotyrosine in cells detached by trypsinization. The tyrosine-phosphorylated state is regained within minutes when the cells are replated onto fibronectin. Activation of FadK may be an important early step in intracellular signal transduction pathways triggered in response to cell interactions with the extracellular matrix.
Chemotherapy remains the mainstay in the treatment of multiple myeloma today. Despite improvements in survival and quality of life that can be attributed to advances in chemotherapeutic treatment, multiple myeloma remains an incurable disease. The development of drug resistance accounts for most treatment failures. Mechanisms of resistance for chemotherapeutic agents occur at the cellular level. Some of these mechanisms are unique for a given individual drug, whereas other mechanisms may confer resistance to a wide variety of agents. Important steps for improving treatment include investigating the resistance mechanisms, developing methods to identify drug-resistant cells, and preventing or circumventing drug resistance once it occurs.
EMT-6 murine mammary tumors were made resistant to cis-diamminedichloroplatinum (II) (CDDP), carboplatin, cyclophosphamide
(CTX), or thiotepa in vivo by treatment of tumor-bearing animals with the drug during a 6-month period. In spite of high levels
of in vivo resistance, no significant resistance was observed when the cells from these tumors were exposed to the drugs in
vitro. The pharmacokinetics of CDDP and CTX were altered in animals bearing the respective resistant tumors. The resistance
of all tumor lines except for the EMT-6/thiotepa decreased during 3 to 6 months in vivo passage in the absence of drugs. These
results indicate that very high levels of resistance to anticancer drugs can develop through mechanisms that are expressed
only in vivo.
Chinese hamster cells in exponential growth as multicellular spheroids were more resistant to radiation damage than were single cells. The survival curve for cells grown and irradiated as spheroids was characterized by a wider shoulder, suggesting enhanced capacity for repair of sublethal damage. This increased repair capacity was developed inherently by cells grown in contact, and was increased further if cells remained in contact during irradiation.
Phorbol esters upregulate the functional affinity of beta 1 integrin receptors for fibronectin on human neutrophils and other leukocytes. We investigated the ability of phorbol myristate acetate (PMA) to stimulate the human promyelocytic cell line HL-60 to adhere to fibronectin, either in its undifferentiated state (HL60) or after dimethylsulfoxide-induced differentiation along the granulocytic pathway (dHL60). PMA stimulated little adherence of undifferentiated HL60 to fibronectin or to the 120-kD chymotryptic cell-binding domain (CBD) of fibronectin. In contrast, PMA stimulated dHL60 cells to rapidly adhere to both fibronectin- and to CBD-coated plastic. PMA-stimulated dHL60 adherence to fibronectin was largely mediated by both alpha 4 beta 1 and alpha 5 beta 1, whereas PMA-stimulated dHL60 adherence to CBD was largely mediated by alpha 5 beta 1. There was little contribution from beta 2 integrins to PMA-stimulated dHL60 adherence to fibronectin or CBD. The inability of undifferentiated HL60 to adhere to fibronectin and CBD did not result from lack of expression of alpha 4 beta 1 or alpha 5 beta 1 because HL60 and dHL60 express similar amounts of both alpha 4 beta 1 and alpha 5 beta 1 on their surface. In addition, 1 mmol/L Mn2+ induced similar amounts of alpha 5 beta 1-dependent adherence of both HL60 and dHL60, showing that alpha 5 beta 1 on undifferentiated HL60 is capable of binding to its ligand. These data suggest that activation of protein kinase C cannot functionally upregulate these beta 1 integrins on undifferentiated HL60 cells. The development of PMA-stimulated beta 1-dependent adherence after granulocytic differentiation of HL60 cells suggests that the differentiated HL60 cell is a useful model for investigating functional coupling of protein kinase C to beta 1 integrin in myeloid cells.
In the germinal center (GC), B cells are either selected to become memory cells or are eliminated through the process of programmed cell death. FDC which are intimately associated with the GC B cells are thought to be important in this selection process. Previously, we have shown that the LFA-1 (CD11a/CD18)-ICAM-1 (CD54) and VLA-4 (CD49d)-VCAM-1 (CD106) adhesion pathways are involved in FDC-B cell interaction. In the present study, we have explored whether these adhesive interactions contribute to the process of B cell selection by studying the effects on apoptosis of GC B cells. Using FDC and B cells derived from human tonsils, we found that only B cells adherent to FDC remain viable: disruption of FDC-B-cell clusters with mAb against LFA-1 alpha (CD11a), VLA-4 (CD49d), ICAM-1 (CD54), or VCAM-1 (CD106) results in apoptosis of the B cells. Furthermore, we found that GC B cells, upon adhesion to plastic-coated purified ICAM-1 (CD54) or VCAM-1 (CD106), show diminished apoptosis. Importantly, we observed that, at low concentration, ICAM-1 (CD54) and VCAM-1 (CD106) act synergistically with anti-IgM, in inhibiting apoptosis. Together, our data strongly suggest that adhesion of B cells via the LFA-1 (CD11a/CD18)-ICAM-1 (CD54) pathway and VLA-4 (CD49d)-VCAM-1 (CD106) pathway contributes to B cell selection.
THE cytoplasmic focal adhesion protein-tyrosine kinase (FAK) localizes with surface integrin receptors at sites where cells attach to the extracellular matrix. Increased FAK tyrosine phosphory-lation occurs upon integrin engagement with fibronectin. Here we show that adhesion of murine NIH3T3 fibroblasts to fibronectin promotes SH2-domain-mediated association of the GRB2 adaptor protein and the c-Src protein-tyrosine kinase (PTK) with FAK in vivo, and also results in activation of mitogen-activated protein kinase (MAPK). In v-Src-transformed NIH3T3, the association of v-Src, GRB2 and Sos with FAK is independent of cell adhesion to fibronectin. The GRB2 SH2 domain binds directly to tyrosine-phosphorylated FAK. Mutation of tyrosine residue 925 of FAK (YENV motif) to phenylalanine blocks GRB2 SH2-domain binding to FAK in vitro
. Our results show that fibronectin binding to integrins on NIH3T3 fibroblasts promotes c-Src and FAK asso-ciation and formation of an integrin-activated signalling complex. Phosphorylation of FAK at Tyr 925 upon fibronectin stimulation creates an SH2-binding site for GRB2 which may link integrin engagement to the activation of the Ras/MAPK signal transduc-tion pathway.
Chronic myelogenous leukemia (CML) is a malignant disease of the hematopoietic stem cell characterized by abnormal circulation and proliferation of malignant progenitors. In contrast to their normal counterparts, CML progenitors adhere poorly to bone marrow stroma or fibronectin (FN). Aside from anchoring progenitors in the marrow microenvironment, beta1 integrin-dependent adhesion of normal progenitors is also associated with inhibition of their proliferation. As the beta1 integrin expression on CML progenitors is normal, we hypothesized that decreased integrin affinity may underlie the abnormal adhesive and proliferative characteristics of CML progenitors. We examined the effect of affinity modulation by the activating antibody 8A2 on the adhesion and proliferation of CML progenitors and the CML cell line, K562. 8A2 induced alpha5Beta1-dependent adhesion of Philadelphia chromosome-positive (Ph+) CD34+/HLA-DR+ cells and K562 cells to FN. Increased adhesion was 8A2- and FN concentration-dependent, time-dependent, and energy-dependent. Further, 8A2-induced adhesion to FN significantly inhibited the proliferation of malignant CML progenitors as well as K562 cells independent of cell differentiation, necrosis, or apoptosis. These studies demonstrate that affinity modulation of the alpha5Beta1 integrin on CML progenitors and K562 cells by 8A2 results in increased adhesion to FN with subsequent decreased proliferation, suggesting that decreased beta1 integrin affinity contributes to the abnormal circulation and proliferation of malignant progenitors in CML.
p130(Cas) (crk associated substrate) has the structural characteristics of an adapter protein, containing multiple consensus SH2 binding sites, an SH3 domain, and a proline-rich domain. The structure of p130(Cas) suggests that it may act to provide a framework for protein-protein interactions; however, as yet, its functional role in cells is unknown. In this report we show that p130(Cas) is localized to focal adhesions. We demonstrate that p130(Cas) associates both in vitro and in vivo with pp125(FAK) (focal adhesion kinase), a kinase implicated in signaling by the integrin family of cell adhesion receptors. p130(Cas) also associates with pp41/43(FRNK) (pp125(FAK)-related, non-kinase), an autonomously expressed form of pp125(FAK) composed of only the C-terminal noncatalytic domain. We show that the association of p130(Cas) with pp125(Fak) and pp41/43(FRNK) is direct, and is mediated by the binding of the SH3 domain of p130(Cas) to a proline-rich sequence present in both the C terminus of pp125(FAK) and in pp41/43(FRNK). In agreement with recent studies we show that p130(Cas) is tyrosine-phosphorylated upon integrin mediated cell adhesion. The association of p130(Cas) with pp125(FAK), a kinase which is activated upon cell adhesion, is likely to be functionally important in integrin mediated signal transduction.
Bcl-2 appears to contribute to neoplasia primarily by promoting cell survival, rather than by stimulating cellular proliferation. Bcl-2, and the related protein Bcl-xL, each suppress apoptosis induced by a wide variety of stimuli in many different cell types. Here we report that suppression of apoptosis by Bcl-2 or Bcl-xL markedly elevates the levels of radiation-induced mutations. This enhanced mutagenesis is the result of an increase in mutation frequency (mutations per survivor) together with a moderate increase in viability. Ectopic expression of either Bcl-2 or Bcl-xL enhances radiation mutagenesis in cells with wtp53. Surprisingly, we found that ectopic expression of Bcl-xL also promotes mutagenesis in p53- cells. These results support the hypothesis that apoptosis plays a crucial role in maintaining genomic integrity by selectively eliminating highly mutated cells from the population.
The neoplastic plasma cells of multiple myeloma differ from normal plasma cells and other B-cell malignancies by an almost exclusive homing to the bone marrow microenvironment which clearly provides the appropriate support, both physical and cytokine, to mediate clonal proliferation and terminal differentiation. Cellular adhesion molecules are involved in the homing of malignant plasma cells to the bone marrow, the production of growth factors and the recirculation of these tumour cells in the advanced stages of disease. Neoplastic plasma cells express H-CAM (CD44), VLA-4 (CD49d/CD29), ICAM-1 (CD54), N-CAM (CD56) and LFA-3 (CD58). In addition VLA-5 (CD49e/CD29) expression seems to be related to cells with less proliferative potential and more potential for paraprotein production. In addition there are fundamental changes in the bone marrow stroma of patients with multiple myeloma including altered composition of the extracellular matrix, increased growth capability of the cellular elements and increased synthesis of interleukin-6 and interleukin-3, which are features postulated to localise and promote growth of the circulating neoplastic progenitors in the bone marrow. However, the evidence to date does not fully explain the inter-relationship of the clonal B cells and the bone marrow stroma in patients with myeloma, including factors which trigger and facilitate the extravasation and recirculation of neoplastic plasma cells as seen in advanced disease.
Adhesion receptors can serve as primary signal transduction molecules that convey information into cells that can affect cell proliferation and differentiation. Since hematopoietic progenitors adhere to marrow stroma and fibronectin via the alpha 4 beta 1 integrin and CD44, we examined the role of these receptors in the transfer of proliferation-regulatory signals to progenitors. Actively proliferating colony-forming cells (CFCs) present in cultured CD34+ cells were incubated with mouse monoclonal antibodies against the alpha 4, beta 1, or CD44 receptors and crosslinking was performed with a secondary goat-anti-mouse antibody. The effect on CFC proliferation was examined with a 3H thymidine suicide assay. Compared with controls (39 to 51% kill), crosslinking the alpha 4 or beta 1 integrins significantly reduced CFC proliferation (12 to 26% kill, p = 0.01), indicating that proliferation-inhibitory signals are transmitted through the VLA-4 integrin. Cytochalasin D, a compound that prevents actin polymerization, prevented not only alpha 4 receptor capping, but also the inhibition of CFC proliferation observed following alpha 4 crosslinking. However, crosslinking of the CD44 receptor with the antibodies Hermes-3 and 50B4, which inhibit adhesion of CFC to fibronectin, failed to cap the CD44 receptor in the majority of CD34+ cells. Furthermore, crosslinking of the CD44 receptor with these antibodies also failed to inhibit proliferation of CFCs. These studies demonstrate that adhesion receptor crosslinking of the alpha 4 beta 1 integrin, together with subsequent changes in F-actin polymerization, negatively regulates hematopoietic progenitor proliferation in a manner independent of the shape change associated with adhesion.
Bcl-2 and its homologue Bcl-XL are expressed in a variety of tumors and their expression modulates the sensitivity of tumor cells to a wide spectrum of chemotherapeutic agents and gamma-irradiation. In the present report, we generated clones of FL5.12 lymphoid cells with similar levels of Bcl-2 and Bcl-XL using the Flag epitope to determine if these survival proteins could provide equivalent protection when challenged with chemotherapy or gamma-irradiation. Using four M-phase specific chemotherapeutic agents, Bcl-XL and Bcl-2 provided similar protection against vincristine and vinblastine whereas Bcl-XL afforded as much as 50% greater cell viability than Bcl-2 against etoposide and teniposide-induced cell death. In addition, Bcl-XL provided significantly greater cell viability than Bcl-2 against methotrexate, fluorouracil, and hydroxyurea, three S-phase specific agents. In apoptosis induced by gamma-irradiation and cisplatin, two antitumor treatments that are cell-cycle phase-nonspecific agents, both Bcl-XL and Bcl-2 conferred similar protection against gamma-irradiation, but Bcl-XL provided better protection than Bcl-2 against cisplatin. These results indicate that Bcl-XL and Bcl-2 confer a differential ability to protect against chemotherapy-induced cell death, which appears to be dependent on the molecular mechanism targeted by the drug rather than its cell-cycle phase specificity.
We have identified and characterized CLARP, a caspase-like apoptosis-regulatory protein. Sequence analysis revealed that human CLARP contains two amino-terminal death effector domains fused to a carboxyl-terminal caspase-like domain. The structure and amino acid sequence of CLARP resemble those of caspase-8, caspase-10, and DCP2, a Drosophila melanogaster protein identified in this study. Unlike caspase-8, caspase-10, and DCP2, however, two important residues predicted to be involved in catalysis were lost in the caspase-like domain of CLARP. Analysis with fluorogenic substrates for caspase activity confirmed that CLARP is catalytically inactive. CLARP was found to interact with caspase-8 but not with FADD/MORT-1, an upstream death effector domain-containing protein of the Fas and tumor necrosis factor receptor 1 signaling pathway. Expression of CLARP induced apoptosis, which was blocked by the viral caspase inhibitor p35, dominant negative mutant caspase-8, and the synthetic caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethylketone (zVAD-fmk). Moreover, CLARP augmented the killing ability of caspase-8 and FADD/MORT-1 in mammalian cells. The human clarp gene maps to 2q33. Thus, CLARP represents a regulator of the upstream caspase-8, which may play a role in apoptosis during tissue development and homeostasis.
The gene for transforming growth factor-beta 1 (TGF-beta 1) was transfected into the murine EMT-6/Parent mammary carcinoma tumor line to form the EMT6/PRK5 beta 1E tumor line. In monolayer culture the EMT-6/PRK5 beta 1E tumor line secretes about 15-times as much TGF-beta 1 into the medium as the EMT-6/Parent line. There was no difference in the response of these two cell lines to 4-hydroperoxycyclophosphamide, cisplatin, melphalan or thiotepa in monolayer culture. When the EMT-6/PRK5 beta 1E cells were grown as a solid tumor in Balb/C mice, plasma levels of TGF-beta 1 were about 5-fold higher than in animals bearing the EMT-6/Parent tumor. The EMT-6/PRK5 beta 1E tumor was markedly resistant to a dosage range of cyclophosphamide, cisplatin, melphalan and thiotepa compared with the EMT-6/Parent tumor. The bone marrow CFU-GM from the animals bearing the EMT-6/PRK5 beta 1E tumor were spared from the cytotoxicity of the drugs compared with the bone marrow CFU-GM from animals bearing the EMT-6/Parent tumor. Administration of decorin, a naturally occurring inhibitor of TGF-beta 1, to animals bearing the EMT-6/PRK5 beta 1E tumor prior to treatment of the animals with the antitumor alkylating agents restored drug sensitivity to the tumor and to the bone marrow CFU-GM. Administration of decorin prior to the antitumor alkylating agents produced very little or no increase in the response of the EMT6/Parent tumor or the bone marrow CFU-GM from those animals. The EMT6/PRK5 beta 1E tumor model allows the effect of secretion of TGF-beta 1 on therapeutic resistance to be assessed directly compared with the EMT-6/Parent tumor. In vivo resistance occurred in the presence of high levels of TGF-beta 1 and was reversed by the TGF-beta 1 inhibitor decorin.
Adhesive interactions are critical for the proliferation, survival and function of all cells. Integrin receptors as the major family of adhesion receptors have been the focus of study for more than a decade. These studies have tremendously enhanced our understanding of the integrin-mediated adhesive interactions and have unraveled novel integrin functions in cell survival mechanisms and in the activation of divergent signaling pathways. The signals from integrin receptors are integrated from those originating from growth factor receptors in order to organize the cytoskeleton, stimulate cell proliferation and rescue cells from matrix detachment-induced programmed cell death. These functions are critical in the regulation of multiple processes such as tissue development, inflammation, angiogenesis, tumor cell growth and metastasis and programmed cell death.
There are two broad categories of drug resistance encountered during cancer chemotherapy, i.e. intrinsic and acquired. They are observed in virtually every type of tumor with every known anticancer chemotherapeutic drug. As such there is an urgent need to develop innovative approaches of preventing or reversing these types of resistance. One strategy to do so is to develop completely new drugs which may be resistance free, such as direct acting angiogenesis inhibitors (T. Boehm, J. Folkman, T. Browder, M.S. O'Reilly, Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance, Nature 390 (1997) 404-407; R.S. Kerbel, Inhibition of tumor angiogenesis as a strategy to circumvent acquired resistance to anti-cancer therapeutic agents, BioEssays 13 (1991) 31-36; R.S. Kerbel, A cancer therapy resistant to resistance, Nature 390 (1997) 335-336). Another is to devise methods which will improve significantly the effectiveness of those conventional drugs already in use, such as adriamycin, cyclophosphamide and taxol. We have directed efforts towards the latter. They depend on the discovery of a new class of chemosensitizers which act as antiadhesive agents rendering solid tumors more susceptible to such conventional cytotoxic therapeutic drugs. Examples of this concept are illustrated with bovine testicular hyaluronidase and a mouse mammary tumor called EMT-6. When this enzyme preparation is used to treat intact multicellular spheroids of the EMT-6 tumor, the spheroids are substantially disaggregated. Dispersed spheroids are more susceptible to the cytotoxic effects of cyclophosphamide than intact spheroids. Moreover, this antiadhesive chemosensitizing effect can actually be reproduced in BALB/c mice when EMT-6 cells are grown intraperitoneally as an ascites tumor (consisting mostly of multicellular aggregates) and the mice are given injections of hyaluronidase and cyclophosphamide. In a similar fashion, the indifference of P-glycoprotein-positive multidrug-resistant EMT-6 spheroids to the P-glycoprotein reversal agent PSC-833 (a cyclosporin A analogue) can be reversed by disaggregation of the intact spheroids by hyaluronidase. This renders the treated cells highly sensitive to a combination of adriamycin and PSC-833 in a manner similar to the striking chemosensitization effects commonly observed in monolayer culture systems. Thus, hyaluronidase has the potential to reverse forms of both intrinsic and acquired drug resistance in solid tumors, such as EMT-6, which are sensitive to its antiadhesive effects.
We have examined the effect of neutralizing TGF-beta antibodies on cisplatin-mediated cytotoxicity against MDA-231 human breast tumor cell spheroids. These tridimensional in vitro systems have been shown to recapitulate the drug sensitivity pattern of tumor cells in vivo. MDA-231 tumor cell spheroids exhibit higher protein levels of the cyclin-dependent kinase (Cdk) inhibitors p21 and p27 and >10-fold lower Cdk2 activity compared to adherent cell monolayers, as well as pRb hypophosphorylation, a predominant G1 population, and a cisplatin 1-h IC50 of approximately 100 microM. Treatment of MDA-231 cells in monolayer with cisplatin for 1 h, subsequently grown as spheroids, increased steady-state TGF-beta1 mRNA levels, secretion of active TGF-beta, cellular Cdk2 activity, pRb phosphorylation, and p21 protein levels, while downregulating p27. Accumulation of cells in G2M and progression into S were noted 48 h after treatment with 100 microM cisplatin. We tested whether drug-induced upregulation of TGF-beta1 and p21, perhaps by preventing cell cycle progression, were protective mechanisms against drug-mediated toxicity by using neutralizing anti-TGF-beta antibodies. Anti-TGF-beta antibodies diminished the induction of p21, enhanced the activation of Cdk2, and facilitated progression into S and G2M following cisplatin treatment. This resulted in a >twofold enhancement of drug-induced DNA fragmentation and a shift in the cisplatin 1-h IC50 from 100 to <10 microM. These data suggest that tumor cell TGF-beta1 may protect from DNA damage and that postchemotherapy administration of TGF-beta inhibitors may facilitate progression beyond G1/S, potentially increasing the efficacy of cytotoxic chemotherapy.
Interleukin 6 (IL-6) is the major survival factor for myeloma tumor cells and induces signaling through the STAT proteins. We report that one STAT family member, Stat3, is constitutively activated in bone marrow mononuclear cells from patients with multiple myeloma and in the IL-6-dependent human myeloma cell line U266. Moreover, U266 cells are inherently resistant to Fas-mediated apoptosis and express high levels of the antiapoptotic protein Bcl-xL. Blocking IL-6 receptor signaling from Janus kinases to the Stat3 protein inhibits Bcl-xL expression and induces apoptosis, demonstrating that Stat3 signaling is essential for the survival of myeloma tumor cells. These findings provide evidence that constitutively activated Stat3 signaling contributes to the pathogenesis of multiple myeloma by preventing apoptosis.
Bcl-2 family members that have only a single Bcl-2 homology domain, BH3, are potent inducers of apoptosis, and some appear to play a critical role in developmentally programmed cell death. We examined the regulation of the proapoptotic activity of the BH3-only protein Bim. In healthy cells, most Bim molecules were bound to LC8 cytoplasmic dynein light chain and thereby sequestered to the microtubule-associated dynein motor complex. Certain apoptotic stimuli disrupted the interaction between LC8 and the dynein motor complex. This freed Bim to translocate together with LC8 to Bcl-2 and to neutralize its antiapoptotic activity. This process did not require caspase activity and therefore constitutes an initiating event in apoptosis signaling.
Detachment of most untransformed adherent cells from the extracellular matrix promotes apoptosis, in a process termed anoikis [1] [2]. The death signalling mechanisms involved in this process are not known, although adhesion or transformation by ras oncogenes have been shown to protect epithelial cells from apoptosis through activation of phosphatidylinositol 3-kinase and protein kinase B (PKB/Akt) [3]. Here we show that detachment-induced apoptosis (anoikis) is blocked by the expression of a dominant-negative form of FAS-associated death domain protein (FADD) in a number of untransformed epithelial cell lines. Because the soluble extracellular domains of the death receptors CD95, DR4 and DR5 failed to block anoikis, we conclude that ligand-dependent activation of these death receptors is not involved in this process. Detachment induced strong activation of caspase 8 and caspase 3. Detachment-induced caspase-8 activation did not require the function of downstream caspases but was blocked by overexpression of the anti-apoptotic proteins Bcl-2 or Bcl-X(L). We propose that caspase-8 activation is the initiating event in anoikis, which is subsequently subject to a positive-feedback loop involving mitochondrial events.
Epigenetic factors (i.e., alterations of gene activity not involving mutations), as well as genetic changes in surviving cancer cells, may play an important role in drug resistance following cancer chemotherapy-a common cause of tumor relapse. Bcl-2 family proteins are central to the regulation of apoptotic cell death and modulate drug sensitivity. We investigated how survival signals in the cellular microenvironment affect the expression, protein conformation, and protein-protein interactions of the Bcl-2 family proteins Bax and Bcl-x(L) and how changes in response to microenvironmental signals alter the response of cancer cells to the drug etoposide.
JLP119 human B-lymphoma cells were treated with etoposide (40 microM) and then cultured in the presence of an activating anti-CD40 antibody, vascular cellular adhesion molecule-1 (VCAM-1)-to activate VLA-4 (alpha4beta1) integrin, and interleukin 4. Cell fate was monitored after etoposide treatment with or without these microenvironmental signals. Bcl-x(L) gene transcription and protein levels of Bcl-x(L) and Bax were measured by northern and western blotting, respectively. Nuclear translocation of transcription factor NF-kappaB was monitored by immunofluorescence and inhibited by (E)-capsaicin. Bax conformation and Bax-Bcl-x(L) interactions were monitored by immunofluorescence and immunoprecipitation, respectively.
Microenvironmental survival signals produced statistically significant reductions in etoposide-induced apoptotic cell death, from 84.6% (95% confidence interval [CI] = 76.7%-92.4%) to 21.3% (95% CI = 19.5%-23.0%); P<.001. Activation of surface protein CD40 increased Bcl-x(L) protein levels via an (E)-capsaicin-inhibitable activation of NF-kappaB; i.e. , (E)-capsaicin restored etoposide sensitivity. Interleukin 4 had no effect on Bcl-x(L) protein levels but accelerated the increase in Bcl-x(L) protein associated with CD40 activation. VCAM-1- and interleukin 4-mediated signals diminished conformational changes in Bax protein and prevented the etoposide-induced disruption of constitutive Bax-Bcl-x(L) binding.
Microenvironmental factors reduce the sensitivity of a B-cell lymphoma to etoposide in vitro by modulating the expression and functions of Bax and Bcl-x(L). This interaction may provide a paradigm for epigenetically induced drug resistance in other tumors.
We evaluated the effect of beta1-integrin receptor engagement on the expression and activity of cell cycle regulatory proteins in CD34(+) cells under conditions that mimic the steady-state marrow microenvironment and in the presence of supraphysiological concentrations of interleukin-3 (IL3) and stem cell factor (SCF). Adhesion of CD34(+) progenitors to fibronectin (FN) was similar whether IL3 or SCF was present or absent. Engagement of beta1-integrins blocked S-phase entry of CD34(+) cells in the absence of IL3 or SCF, whereas addition of 10 ng/mL IL3 or SCF prevented such a block in S-phase entry. In the absence of IL3 or SCF, cyclin-E levels were significantly lower and p27(KIP1) levels significantly higher in FN-adherent than in FN-nonadherent cells, or than in poly-L-lysine (PLL)-adherent or (PLL)-nonadherent cells. Cyclin-dependent-kinase (cdk)-2 activity was decreased and levels of cyclin-E-cdk2 complexes were lower in FN-adherent than in PLL-adherent cells. In contrast, cyclin-E and p27(KIP1) protein levels and cdk2 activity in cells adherent to FN in the presence of IL3 or SCF were similar to those in PLL-adherent and FN-nonadherent or PLL-nonadherent cells. In conclusion, under physiological cytokine conditions, integrin engagement prevents S-phase entrance of CD34(+) cells, which is associated with elevated levels of the contact-dependent cyclin kinase inhibitor p27(KIP1). Supraphysiological concentrations of IL3 or SCF prevent p27(KIP1) elevation and override the integrin-mediated inhibition of entry into S phase.