No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
CAMSAP3 negatively regulates lung cancer cell invasion and angiogenesis through nucleolin/HIF-1α mRNA complex stabilization
Abstract
Aims:
Cancer metastasis is a major cause of lung cancer-related mortality, so identification of related molecular mechanisms is of interest. Calmodulin-regulated spectrin-associated protein 3 (CAMSAP3) has been implicated in lung cancer malignancies; however, its role in metastatic processes, including invasion and angiogenesis, is largely unknown.
Main method:
The clinical relevance of CAMSAP3 expression in lung cancer was evaluated. The relevance of CAMSAP3 expression to in vitro cell invasion and angiogenesis was assessed in human lung cancer cells and endothelial cells, respectively. The molecular mechanism was identified by qRT-PCR, immunoprecipitation, mass spectrometry, and RNA immunoprecipitation. The in vivo metastatic and angiogenic activities of lung cancer cells were assessed.
Key findings:
Low CAMSAP3 expression was found in malignant lung tissues and strongly correlated with a poor prognosis in lung adenocarcinoma (LUAD). CAMSAP3-knockout NSCLC exhibited high invasive ability, and CAMSAP3 knockout induced HUVEC proliferation and tube formation; these effects were significantly attenuated by reintroduction of exogenous wild-type CAMSAP3. Mechanistically, in the absence of CAMSAP3, the expression of hypoxia-inducible factor-1α (HIF-1α) was upregulated, which increased the levels of downstream HIF-1α targets such as vascular endothelial growth factor A (VEGFA) and matrix metalloproteinases (MMPs) 2 and 9. Proteomic analysis revealed that nucleolin (NCL) bound to CAMSAP3 to regulate HIF-1α mRNA stabilization. In addition, CAMSAP3-knockout lung cancer cells displayed highly aggressive behavior in metastasis and angiogenesis in vivo.
Significance:
This study reveals that CAMSAP3 plays a negative regulatory role in lung cancer cell metastatic behavior both in vitro and in vivo through NCL/HIF-1α mRNA complex stabilization.
... Discover Oncology (2025) 16:3 | https://doi.org/10.1007/s12672-024-01723-5 of solid tumors [17]. Particularly under hypoxic conditions, the expression of HIF-1α significantly increases, thereby influencing the survival and growth of tumor cells through the regulation of relevant gene expression [14][15][16]. Studies indicate that HIF-1α is connected to tumor development by promoting the expression of Vascular endothelial growth factor B (VEGF-B) and angiogenesis [18,19]. ...
... Hypoxia-inducible factor 1 alpha (HIF-1α) is a critical transcription factor that plays a pivotal role in the tumor microenvironment [12,13]. HIF-1α is involved in regulating cellular metabolism, proliferation, and apoptosis, among other biological processes [14][15][16]. Tumor angiogenesis plays a critical role in the growth and treatment response Supplementary Information The online version contains supplementary material available at https:// doi. org/ 10. 1007/ s12672-024-01723-5. ...
Ovarian cancer is a common malignant tumor in women, exhibiting a certain sensitivity to chemotherapy drugs like gemcitabine (GEM). This study, through the analysis of ovarian cancer single-cell RNA sequencing (scRNA-seq) data and transcriptome data post-GEM treatment, identifies the pivotal role of hypoxia-inducible factor 1 alpha (HIF-1α) in regulating the treatment process. The results reveal that HIF-1α modulates the expression of VEGF-B, thereby inhibiting the fibroblast growth factor 2 (FGF2)/FGFR1 signaling pathway and impacting tumor formation. In vitro experiments validate the mechanistic role of HIF-1α in GEM treatment, demonstrating that overexpression of HIF-1α reverses the drug's effects on ovarian cancer cells while silencing fibroblast growth factor receptor 1 (FGFR1) can restore treatment efficacy. These findings provide essential molecular targets and a theoretical foundation for the development of novel treatment strategies for ovarian cancer in the future.
Supplementary Information
The online version contains supplementary material available at 10.1007/s12672-024-01723-5.
... Recent research bySeephan et al. (2023) has shown that reduced CAMSAP3 expression correlated with poor outcomes in LAUD patients, and its decrease enhanced invasive and angiogenic capabilities in NSCLC cells.Restoration of CAMSAP3 levels has been observed to counter these effects. At the molecular level, the absence of CAMSAP3 increases the activity of hypoxia-inducible factor 1-alpha (HIF-1α) and its downstream targets, including VEGFA and MMPs 2 and 9, vital in promoting metastasis and angiogenesis. ...
... This process is partly due to the interaction between NCL and CAMSAP3, which governs the stability and expression of HIF-1α mRNA. This interaction highlights the importance of the NCL/CAMSAP3 relationship in regulating aggressive metastatic and angiogenic tendencies in lung cancer cells, establishing CAMSAP3 as a key suppressor in lung cancer progression by influencing the NCL/HIF-1α mRNA complex.61 5.5 | MMP7MMP7 plays a nuanced yet pivotal role in lung cancer progression by targeting NCL at Asp255. ...
In addressing the challenges of lung cancer, attention has increasingly turned to molecular diagnostics and targeted therapies, with nucleolin (NCL) assuming a pivotal role, especially in non‐small cell lung cancer. The aberrant activity and cellular distribution of NCL act as crucial biomarkers for early detection and treatment monitoring, showing a strong correlation with disease progression and patient prognosis. Elevated NCL levels signal advanced disease and poorer outcomes, underscoring its significance in evaluating disease severity and therapeutic response. Strategies targeting the molecular interactions of NCL have spurred innovative approaches to inhibit tumour growth, overcome drug resistance and improve treatment efficacy. These advancements are paving the way for personalized therapies tailored to the unique molecular profiles of patients' tumours. Consequently, NCL stands at the forefront of lung cancer management, symbolizing the move towards more precise and individualized oncology care, and marking substantial progress in therapeutic development.
... CAMSAP3 can regulate microtubule stability and played a key role in the apical-to-basal polarity of microtubules in oriented epithelial cells [10]. Knockout of CAMSAP3 promoted invasion and malignant progression of lung cancer and the expression of HIF-1α [11]. The activation of HIF-1α was one of the main initiating factors of the interaction between macrophage and tumor cells [9]. ...
The clinical heterogeneity of early-stage endometrial cancer (EC) is worthy of further study to identify high-quality prognostic markers and their potential role in aggressive tumor behavior. Mutation of TP53 was considered as an important primary triage in modified molecular typing for EC, it still cannot precisely predict the prognosis of EC. After proteomic analysis of cancer and para-cancerous tissues from 24 early-stage endometrioid EC patients with different survival outcomes, 13 differentially expressed proteins were screen out while 2 proteins enriched in p53 signaling pathway were further identified by single-cell transcriptome (scRNA-seq). Interestingly, tumor necrosis factor type-1 receptor-associated protein (TRAP1) and calmodulin-regulated spectrin-associated protein family member 3 (CAMSAP3) were found to be significantly downregulated in the specific cell cluster. Expectedly, the signature genes of TRAP1low/CAMSAP3low cluster included classical oncogenes. Moreover, close cellular interactions were observed between myeloid cells and the TRAP1low/CAMSAP3low cluster after systematically elucidating their relationship with tumor microenvironment (TME). The expression of TRAP1 and CAMSAP3 was verified by immunohistochemistry. Thus, a novel prediction model combining TRAP1, CAMSAP3 and TP53 was construct by multi-omics. Compared with the area under the curve, it demonstrated a significantly improvemrnt in the diagnostic efficacy in EC patients from TCGA bank. In conclusion, this work improved the current knowledge regarding the prognosis of early-stage EC through proteomics and scRNA-seq. These findings may lead to improvements in precise risk stratification of early-stage EC patients.
Lung cancer is one of the most lethal tumors in the world with a 5-year overall survival rate of less than 20%, mainly including lung adenocarcinoma (LUAD). Tumor microenvironment (TME) has become a new research focus in the treatment of lung cancer. The TME is heterogeneous in composition and consists of cellular components, growth factors, proteases, and extracellular matrix. The various cellular components exert a different role in apoptosis, metastasis, or proliferation of lung cancer cells through different pathways, thus contributing to the treatment of adenocarcinoma and potentially facilitating novel therapeutic methods. This review summarizes the research progress on different cellular components with cell–cell interactions in the TME of LUAD, along with their corresponding drug candidates, suggesting that targeting cellular components in the TME of LUAD holds great promise for future theraputic development.
The major cause of death in cancer patients is strongly associated with metastasis. While much remains to be understood, microtubule-associated proteins (MAPs) have shed light on metastatic progression’s molecular mechanisms. In this review article, we focus on the role of MAPs in cancer aggressiveness, particularly cancer metastasis activity. Increasing evidence has shown that a growing number of MAP member proteins might be fundamental regulators involved in altering microtubule dynamics, contributing to cancer migration, invasion, and epithelial-to-mesenchymal transition. MAP types have been established according to their microtubule-binding site and function in microtubule-dependent activities. We highlight that altered MAP expression was commonly found in many cancer types and related to cancer progression based on available evidence. Furthermore, we discuss and integrate the relevance of MAPs and related molecular signaling pathways in cancer metastasis. Our review provides a comprehensive understanding of MAP function on microtubules. It elucidates how MAPs regulate cancer progression, preferentially in metastasis, providing substantial scientific information on MAPs as potential therapeutic targets and prognostic markers for cancer management.
Molecular oxygen (O2) is essential for most biological reactions in mammalian cells. When the intracellular oxygen content decreases, it is called hypoxia. The process of hypoxia is linked to several biological processes, including pathogenic microbe infection, metabolic adaptation, cancer, acute and chronic diseases, and other stress responses. The mechanism underlying cells respond to oxygen changes to mediate subsequent signal response is the central question during hypoxia. Hypoxia-inducible factors (HIFs) sense hypoxia to regulate the expressions of a series of downstream genes expression, which participate in multiple processes including cell metabolism, cell growth/death, cell proliferation, glycolysis, immune response, microbe infection, tumorigenesis, and metastasis. Importantly, hypoxia signaling also interacts with other cellular pathways, such as phosphoinositide 3-kinase (PI3K)- mammalian target of rapamycin (mTOR) signaling, nuclear factor kappa-B (NF-κB) pathway, extracellular signal-regulated kinases (ERK) signaling, and endoplasmic reticulum (ER) stress. This paper systematically reviews the mechanisms of hypoxia signaling activation, the control of HIF signaling, and the function of HIF signaling in human health and diseases. In addition, the therapeutic targets involved in HIF signaling to balance health and diseases are summarized and highlighted, which would provide novel strategies for the design and development of therapeutic drugs.
Background
Cellular senescence is an aging-related process found in cancer cells that contributes to irreversible growth arrest and tumor aggressiveness. Recently, calmodulin-regulated spectrin-associated protein 3 (CAMSAP3), a minus-end microtubule-stabilizing protein, has received increasing attention in cancer cell biology. However, the biological role of CAMSAP3 on senescence in human lung cancer remains incompletely understood.
Methods
The function of CAMSAP3 on the regulation of cellular senescence-associated phenotypes in human non-small cell lung cancer H460 cells were determined in CAMSAP3 deletion (H460/C3ko) cells. The effects of CAMSAP3 on cell proliferation were investigated using MTT and colony formation assays. The cell cycle activity was evaluated by flow cytometry and the senescence-associated phenotypes were observed by SA-β-Gal staining. Quantitative RT-PCR and westen blot were used to evaluate the expression of cell cycle and senescence markers. Moreover, the interaction of CAMSAP3-ERK1/2 and possible partner protein was quantified using immunoprecipitation/mass spectrometry and immunofluorescence. Lastly, an xenograft model were performed.
Results
CAMSAP3 knockout promotes lung cancer cell senescence-associated phenotypes and induces G1 cell cycle arrest. Mechanistic investigation revealed that phosphorylated ERK (p-ERK) was markedly downregulated in CAMSAP3-deleted cells, suppressing cyclin D1 expression levels, and full-length CAMSAP3 abrogated these phenotypes. Proteomic analysis demonstrated that vimentin, an intermediate filament protein, is required as a scaffold for CAMSAP3-modulating ERK signaling. Furthermore, an in vivo tumor xenograft experiment showed that tumor initiation is potentially delayed in CAMSAP3 knockout tumors with the downregulation of p-ERK and cyclin D1, resulting in a senescence-like phenotype.
Conclusion
This study is the first to report an intriguing role of CAMSAP3 in lung carcinoma cell senescence-associated phenotypes via the modulation of p-ERK/cyclin D1 signaling.
Metastasis of bladder cancer is a complex process and has been associated with poor clinical outcomes. However, the mechanisms of bladder cancer metastasis remain largely unknown. The present study found that the long noncoding RNA lnc00892 was significantly downregulated in bladder cancer tissues, with low lnc00892 expression associated with poor prognosis of bladder cancer patients. Lnc00892 significantly inhibited the migration, invasion, and metastasis of bladder cancer cells in vitro and in vivo. In-depth analysis showed that RhoA/C acted downstream of lnc00892 to inhibit bladder cancer metastasis. Mechanistically, lnc00892 reduces nucleolin gene transcription by competitively binding the promoter of nucleolin with c-Jun, thereby inhibiting nucleolin-mediated stabilization of RhoA/RhoC mRNA. Taken together, these findings provide novel insights into understanding the mechanisms of bladder cancer metastasis and suggest that lnc00892 can serve as a potential therapeutic target in patients with invasive bladder cancer.
Simple Summary
Breast cancer is one of the most common cancers affecting women today. Astrocyte Elevated Gene-1 (AEG-1) is elevated in breast cancer patients and is associated with metastasis and poor prognosis. However, the mechanisms by which AEG-1 promotes breast cancer are not fully understood. This report focuses on a novel AEG-1 interacting protein, nucleolin (NCL), which we identified via mass spectrometry-based interactome profiling. We found NCL to be important for the oncogenic function of AEG-1 in breast cancer. Further, c-Met was identified as a novel mediator of the oncogenic function of the AEG-1-NCL complex. Collectively, our study suggests that targeting the AEG-1-NCL protein complex could be an effective therapeutic approach for the treatment of some breast cancers.
Abstract
Breast cancer is one of the most common malignant diseases worldwide. Astrocyte elevated gene-1 (AEG-1) is upregulated in breast cancer and regulates breast cancer cell proliferation and invasion. However, the molecular mechanisms by which AEG-1 promotes breast cancer have yet to be fully elucidated. In order to delineate the function of AEG-1 in breast cancer development, we mapped the AEG-1 interactome via affinity purification followed by LC-MS/MS. We identified nucleolin (NCL) as a novel AEG-1 interacting protein, and co-immunoprecipitation experiments validated the interaction between AEG-1 and NCL in breast cancer cells. The silencing of NCL markedly reduced not only migration/invasion, but also the proliferation induced by the ectopic expression of AEG-1. Further, we found that the ectopic expression of AEG-1 induced the tyrosine phosphorylation of c-Met, and NCL knockdown markedly reduced this AEG-1 mediated phosphorylation. Taken together, our report identifies NCL as a novel mediator of the oncogenic function of AEG-1, and suggests that c-Met could be associated with the oncogenic function of the AEG-1-NCL complex in the context of breast cancer.
This article provides an update on the global cancer burden using the GLOBOCAN 2020 estimates of cancer incidence and mortality produced by the International Agency for Research on Cancer. Worldwide, an estimated 19.3 million new cancer cases (18.1 million excluding nonmelanoma skin cancer) and almost 10.0 million cancer deaths (9.9 million excluding nonmelanoma skin cancer) occurred in 2020. Female breast cancer has surpassed lung cancer as the most commonly diagnosed cancer, with an estimated 2.3 million new cases (11.7%), followed by lung (11.4%), colorectal (10.0 %), prostate (7.3%), and stomach (5.6%) cancers. Lung cancer remained the leading cause of cancer death, with an estimated 1.8 million deaths (18%), followed by colorectal (9.4%), liver (8.3%), stomach (7.7%), and female breast (6.9%) cancers. Overall incidence was from 2‐fold to 3‐fold higher in transitioned versus transitioning countries for both sexes, whereas mortality varied <2‐fold for men and little for women. Death rates for female breast and cervical cancers, however, were considerably higher in transitioning versus transitioned countries (15.0 vs 12.8 per 100,000 and 12.4 vs 5.2 per 100,000, respectively). The global cancer burden is expected to be 28.4 million cases in 2040, a 47% rise from 2020, with a larger increase in transitioning (64% to 95%) versus transitioned (32% to 56%) countries due to demographic changes, although this may be further exacerbated by increasing risk factors associated with globalization and a growing economy. Efforts to build a sustainable infrastructure for the dissemination of cancer prevention measures and provision of cancer care in transitioning countries is critical for global cancer control.
Background
Under physiological conditions, CXCL12 modulates cell proliferation, survival, angiogenesis, and migration mainly through CXCR4. Interestingly, the newly discovered receptor CXCR7 for CXCL12 is highly expressed in many tumor cells as well as tumor-associated blood vessels, although the level of CXCR7 in normal cells is low. Recently, many studies have suggested that CXCR7 promotes cell growth and metastasis in more than 20 human malignancies, among which lung cancer is the leading cause of cancer-associated deaths worldwide. Thus, the mechanism of CXCR7 in the progression of lung cancer is urgently needed.
Methods
First, we explored CXCR4 and CXCR7 expression in human lung cancer specimens and cell lines by immunohistochemistry, western blot and flow cytometry. Then, we chose the human lung adenocarcinoma cell line A549 that stably overexpressed CXCR7 through the way of lentivirus-mediated transduction. Next, “wound healing” assay and transwell assay were applied to compare the cell migration and invasion ability, and stripe assay was used to evaluate the cell polarization. Last, our team established a mouse xenograft model of human lung cancer and monitored tumor proliferation and metastasis by firefly luciferase bioluminescence imaging in SCID/Beige mice.
Results
In clinical lung cancer samples, CXCR7 expression was almost not detected in normal tissue but upregulated in lung tumor tissue, whereas, CXCR4 was highly expressed in both normal and tumor tissues. Furthermore, overexpression of CXCR7 enhanced A549 cell migration and polarization in vitro. Besides, mouse xenograft model of human lung cancer showed that CXCR7 promoted primary lung tumor’s growth and metastasis to the second organ, such as liver or bone marrow in SCID/Beige mice in vivo.
Conclusions
This study describes the multiple functions of CXCR7 in lung cancer. Thus, these results suggest that CXCR7 may be a malignancy marker and may provide a novel target for anticancer therapy.
Tumor vascularization occurs through several distinct biological processes, which not only vary between tumor type and anatomic location, but also occur simultaneously within the same cancer tissue. These processes are orchestrated by a range of secreted factors and signaling pathways and can involve participation of non-endothelial cells, such as progenitors or cancer stem cells. Anti-angiogenic therapies using either antibodies or tyrosine kinase inhibitors have been approved to treat several types of cancer. However, the benefit of treatment has so far been modest, some patients not responding at all and others acquiring resistance. It is becoming increasingly clear that blocking tumors from accessing the circulation is not an easy task to accomplish. Tumor vessel functionality and gene expression often differ vastly when comparing different cancer subtypes, and vessel phenotype can be markedly heterogeneous within a single tumor. Here, we summarize the current understanding of cellular and molecular mechanisms involved in tumor angiogenesis and discuss challenges and opportunities associated with vascular targeting.
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide and is characterized by a fast‐paced growth. Like other solid tumors, the HNSCC growth rate results in the development of hypoxic regions identified by the expression of hypoxia‐inducible factor 1α (HIF‐1α). Interestingly, clinical data have shown that pharmacological induction of intratumoral hypoxia caused an unexpected rise in tumor metastasis and the accumulation of cancer stem cells (CSCs). However, little is known on the molecular circuitries involved in the presence of intratumoral hypoxia and the augmented population of CSCs. Here we explore the impact of hypoxia on the behavior of HNSCC and define that the controlling function of phosphatase and tensin homolog (PTEN) over HIF‐1α expression and CSC accumulation are de‐regulated during hypoxic events. Our findings indicate that hypoxic niches are poised to accumulate CSCs in a molecular process driven by the loss of PTEN activity. Furthermore, our data suggest that targeted therapies aiming at the PTEN/PI3K signaling may constitute an effective strategy to counteract the development of intratumoral hypoxia and the accumulation of CSCs.—Nascimento‐Filho, C. H. V., Webber, L. P., Borgato, G. B., Goloni‐Bertollo, E. M., Squarize, C. H., Castilho, R. M. Hypoxic niches are endowed with a protumorigenic mechanism that supersedes the protective function of PTEN. FASEB J. 33, 13435–13449 (2019). www.fasebj.org
Endothelial cells play a critical role in the process of angiogenesis during skin wound healing. The migration and proliferation of endothelial cells are processes that are initiated by the hypoxic microenvironment in a wound, but the underlying mechanisms remain largely unknown. Here, we identified a novel role for microtubule-associated protein 4 (MAP4) in angiogenesis. We firstly demonstrated that MAP4 phosphorylation was induced in hypoxic endothelial cells; the increase in MAP4 phosphorylation enhanced the migration and proliferation of endothelial cells. We also found that hypoxia (2% O2) activated p38/mitogen-activated protein kinase (MAPK) signaling, and we identified p38/MAPK as an upstream regulator of MAP4 phosphorylation in endothelial cells. Moreover, we showed that the promigration and proproliferation effects of MAP4 phosphorylation were attributed to its role in microtubule dynamics. These results indicated that MAP4 phosphorylation induced by p38/MAPK signaling promotes angiogenesis by inducing the proliferation and migration of endothelial cells cultured under hypoxic conditions via microtubule dynamics regulation. These findings provide new insights into the potential mechanisms underlying the initiation of the migration and proliferation of endothelial cells.
Globally, gastric cancer is the fifth most common malignancy, with high rates of incidence and mortality. The high mortality rate and poor prognosis of gastric cancer are closely associated with its profound invasiveness, high incidence of metastasis, rapid proliferation, and high rate of recurrence. Previous studies have confirmed that stathmin (STMN) has an important role in the occurrence, development and prognosis of gastric cancer. However, the detailed mechanisms by which STMN affects these processes remain unclear. The aim of the present study was to determine how STMN promotes invasion, migration and proliferation in gastric cancer tumor cells. The results of immunohistochemistry indicated that STMN is overexpressedinstomachneoplasmtissues,andthatitisassociated with migration, invasion, proliferation and anti‑apoptotic states of gastric cancer cells. The secretory proteins of gastric cancer cells with or without STMN knockdown were further analyzed using the isobaric tags for relative and absolute quantitation method to identify differentially expressed proteins verified by reverse transcription‑quantitative polymerase chain reaction and western blot analysis. Inhibition of STMN decreases the levels of clusterin, cystatin C and matrix metalloproteinases, followed by inhibiting the protein kinase B and signal transducer and activation of transcription activation. These findings suggest that STMN could be a promising therapeutic target for gastric cancer.
Despite many efforts to develop hormone therapy and chemotherapy, no effective strategy to suppress prostate cancer metastasis has been established because the metastasis is not well understood. We here investigate a role of CBP/p300-interacting transactivator with E/D-rich carboxy-terminal domain-2 (CITED2) in prostate cancer metastasis. CITED2 is highly expressed in metastatic prostate cancer, and its expression is correlated with poor survival. The CITED2 gene is highly activated by ETS-related gene that is overexpressed due to chromosomal translocation. CITED2 acts as a molecular chaperone to guide PRMT5 and p300 to nucleolin, thereby activating nucleolin. Informatics and experimental data suggest that the CITED2-nucleolin axis is involved in prostate cancer metastasis. This axis stimulates cell migration through the epithelial-mesenchymal transition and promotes cancer metastasis in a xenograft mouse model. Our results suggest that CITED2 plays a metastasis-promoting role in prostate cancer and thus could be a target for preventing prostate cancer metastasis.
Hypoxia is a common feature of solid tumours that promotes invasion and metastatic dissemination. Invadopodia are actin-rich membrane protrusions that direct extracellular matrix proteolysis and facilitate tumour cell invasion. Here, we show that CSRP2, an invadopodial actin bundling protein, is upregulated by hypoxia in various breast cancer cell lines, as well as in pre-clinical and clinical breast tumour specimens. We functionally characterized two hypoxia responsive elements within the proximal promoter of CSRP2 gene which are targeted by hypoxia-inducible factor-1 (HIF-1) and required for
promoter transactivation in response to hypoxia. Remarkably, CSRP2 knockdown significantly inhibits hypoxia-stimulated invadopodium formation, ECM degradation and invasion in MDA-MB-231 cells, while CSRP2 forced expression was sufficient to enhance the invasive capacity of HIF-1α-depleted cells under hypoxia. In MCF-7 cells, CSRP2 upregulation was required for hypoxia-induced formation of invadopodium precursors that were unable to promote ECM degradation. Collectively, our data support
that CSRP2 is a novel and direct cytoskeletal target of HIF-1 which facilitates hypoxia-induced breast cancer cell invasion by promoting invadopodia formation.
The relationship between the expression levels of matrix metalloproteinase-2 (MMP-2) and MMP-9 and breast cancer prognosis was studied. Two breast cancer cell lines (MDA-MB-231 and MCF-7) and one human normal breast cell line (HS578Bst) were investigated. Fluorescence real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blotting were used to detect cellular mRNA and protein MMP-2 and MMP-9 expression levels. Breast cancer tissue samples from 80 patients and tumor-adjacent normal tissue samples from 40 patients were collected, and MMP-2 and MMP-9 expression in these samples were examined using immunohistochemistry (IHC). The relationship of MMP-2 and MMP-9 expression levels with breast cancer patient clinicopathological parameters and prognosis was analyzed. RT-PCR and western blot results showed that MMP-2 and MMP-9 mRNA and protein expression levels were significantly higher in MDA-MB-231 and MCF-7 cells than in HS578Bst cells. A high expression of MMP-2 and MMP-9 was found in 83.75% (67/80) and 78.75% (63/80) of breast cancer tissue samples, respectively. MMP-2 and MMP-9 expression in breast cancer tissues were significantly different from that in tumor-adjacent normal tissues (p<0.01). MMP-2 and MMP-9 expression levels in breast cancer tissues were correlated with lymph node metastasis and tumor staging. Single factor survival analysis showed that MMP-2 and MMP-9 were factors influencing breast cancer prognosis. MMP-2 and MMP-9 are highly expressed in breast cancer tissues and are closely related to lymph node metastasis and tumor staging. MMP-2 and MMP-9 can be used as reference indices for guiding breast cancer prognosis and treatment.
Circulating tumor cells (CTCs) represent the key step of cancer cell dissemination. The alteration of CTCs correlates with the treatment outcome and prognosis. To enrich and identify CTCs from billions of blood cells renders a very challenging task, which triggers development of several methods, including lysis of RBC plus negative or positive enrichment using antibodies, and filter membrane or spiral microfluidics to capture CTCs. To compare the advantages of different enrichment methods for CTCs, we utilized the 4T1 breast cancer cells transfected with both green fluorescent protein (GFP) and luciferase to trace CTCs in the experimental lung metastasis model. Three methods were used to detect CTCs at the same time: bioluminescence assay, smearing method, and membrane filter method. The in vivo alive mouse imaging was used to dynamically monitor the growth of lung metastases. The sensitivity and accuracy of three detection methods were compared side-by-side. Our results showed that 1) the sensitivity of bioluminescence assay was the highest, but there was no information of CTC morphology; 2) the smearing method and membrane filter method could observe the detail of CTC morphology, such as in single or in cluster, while their sensitivity was lower than bioluminescence assay; 3) A dynamic observation at a 7-day intervals, the lung metastatic cancer grew at a log speed, while CTCs were increased at a low speed. This might be due to the activated immune cells eliminating the CTCs at a speed much faster than CTCs were generated. This comparison of three CTC detection methods in mouse model suggests that bioluminescence assay could be used in quantitative study of the effect of certain agent on the suppression of CTCs, while GFP-based morphological assays could be used to study the dissemination mechanism of CTCs. The combination of both bioluminescence assay and GFP-based assay would generate more information for quantity and quality of CTCs.
Tremendous amount of RNA sequencing data have been produced by large consortium projects such as TCGA and GTEx, creating new opportunities for data mining and deeper understanding of gene functions. While certain existing web servers are valuable and widely used, many expression analysis functions needed by experimental biologists are still not adequately addressed by these tools. We introduce GEPIA (Gene Expression Profiling Interactive Analysis), a web-based tool to deliver fast and customizable functionalities based on TCGA and GTEx data. GEPIA provides key interactive and customizable functions including differential expression analysis, profiling plotting, correlation analysis, patient survival analysis, similar gene detection and dimensionality reduction analysis. The comprehensive expression analyses with simple clicking through GEPIA greatly facilitate data mining in wide research areas, scientific discussion and the therapeutic discovery process. GEPIA fills in the gap between cancer genomics big data and the delivery of integrated information to end users, thus helping unleash the value of the current data resources. GEPIA is available at http://gepia.cancer-pku.cn/.
Vol. 210, No. 5, May 2013. Pages 951–968.
The authors regret that in their original paper, the RNU6 loading control panels shown in Fig. 1 (E and F) were incorrect as a result of an error in figure presentation during the final production process. The conclusions of the experiments shown in these panels are not affected. The corrected figure appears below. The online HTML and PDF versions of this article have been corrected. The error only remains in the print version.
• Download figure
• Open in new tab
• Download powerpoint
Studies have found that hypoxia is the most common feature in all of solid tumor progression, thus it has become a central issue in tumor physiology and cancer treatment. Hypoxia-inducible factor-1α (HIF-1α) could make the tumor produce adaptive biological response to hypoxia and become more aggressive. In this paper, we used enzyme linked immune sorbent assay to detect the plasma level of HIF-1α in patients with NSCLC and healthy volunteers. The results indicated that the 5-year survival rate of patients with squamous cell carcinomas is negatively correlated with the plasma level of HIF-1α and the 5-year survival rate of patients with low level of HIF-1α is higher than those with high level of HIF-1α. The plasma level of HIF-1α in patients with NSCLC is significantly higher than healthy volunteers. There is no significant correlation between the plasma level of HIF-1α and clinical features of NSCLC patients. In a word, there is no connection between the plasma level of HIF-1α and the clinical features of NSCLC patients as well as their prognosis. In stratified analysis, the plasma level of HIF-1α in patients with squamous cell carcinoma is associated with regional lymph node status.
Hypoxia-inducible factors (HIFs) regulate the transcription of genes that mediate the response to hypoxia. HIFs are constantly expressed and degraded under normoxia, but stabilized under hypoxia. HIFs have been widely studied in physiological and pathological conditions and have been shown to contribute to the pathogenesis of various vascular diseases. In clinical settings, the HIF pathway has been studied for its role in inhibiting carcinogenesis. HIFs might also play a protective role in the pathology of ischemic diseases. Clinical trials of therapeutic angiogenesis after the administration of a single growth factor have yielded unsatisfactory or controversial results, possibly because the coordinated activity of different HIF-induced factors is necessary to induce mature vessel formation. Thus, manipulation of HIF activity to simultaneously induce a spectrum of angiogenic factors offers a superior strategy for therapeutic angiogenesis. Because HIF-2α plays an essential role in vascular remodeling, manipulation of HIF-2α is a promising approach to the treatment of ischemic diseases caused by arterial obstruction, where insufficient development of collateral vessels impedes effective therapy. eIF3e/INT6 interacts specifically with HIF-2α and induces the proteasome inhibitor-sensitive degradation of HIF-2α, independent of hypoxia and VHL. Treatment with eIF3e/INT6 siRNA stabilizes HIF-2α activity even under normoxic conditions and induces the expression of several angiogenic factors, at levels sufficient to produce functional arteries and veins in vivo. We have demonstrated that administration of eIF3e/INT6 siRNA to ischemic limbs or cold-injured brains reduces ischemic damage in animal models. This review summarizes the current understanding of the relationship between HIFs and vascular diseases. We also discuss novel oxygen-independent regulatory proteins that bind HIF-α and the implications of a new method for therapeutic angiogenesis using HIF stabilizers.
Matrix metalloproteinases (MMPs) and adamalysin-like metalloproteinase with thrombospondin motifs (ADAMTSs) belong to the metzincin superfamily of metalloproteinases and they play key roles in extracellular matrix catabolism, activation and inactivation of cytokines, chemokines, growth factors, and other proteinases at the cell surface and within the extracellular matrix. Their activities are tightly regulated in a number of ways, such as transcriptional regulation, proteolytic activation and interaction with tissue inhibitors of metalloproteinases (TIMPs). Here, we highlight recent studies that have illustrated novel mechanisms regulating the extracellular activity of these enzymes. These include allosteric activation of metalloproteinases by molecules that bind outside the active site, modulation of location and activity by interaction with cell surface and extracellular matrix molecules, and endocytic clearance from the extracellular milieu by low-density lipoprotein receptor-related protein 1 (LRP1).
Copyright © 2015. Published by Elsevier B.V.
Abstract Nucleolin is a pleiotropic protein involved in a variety of cellular processes. Although multipolar spindle formation has been observed after nucleolin depletion, the roles of nucleolin in centrosome regulation and functions have not been addressed. Here we report using immunofluorescence and biochemically purified centrosomes that nucleolin co-localized only with one of the centrioles during interphase which was further identified as the mature centriole. Upon nucleolin depletion, cells exhibited an amplification of immature centriole markers surrounded by irregular pericentrin staining; these structures were exempt from maturation markers and unable to nucleate microtubules. Furthermore, the microtubule network was disorganized in these cells, exhibiting frequent non-centrosomal microtubules. At the mature centriole a reduced kinetics in the centrosomal microtubule nucleation phase was observed in live silenced cells, as well as a perturbation of microtubule anchoring. Immunoprecipitation experiments showed that nucleolin belongs to protein complexes containing two key centrosomal proteins, γ-tubulin and ninein, involved in microtubule nucleation and anchoring steps. Altogether, our study uncovered a new role for nucleolin in restricting microtubule nucleation and anchoring at centrosomes in interphase cells.
Hypoxia-inducible factor-1α (HIF-1α) plays an important role in tumour progression and metastasis through activation of many target genes that are especially involved in pivotal aspects of cancer biology. However, the prognostic role of HIF-1α has been controversial in primary patients with lung cancer. This meta-analysis was performed to systematically evaluate whether HIF-1α expression is associated with the clinical outcomes in lung cancer patients.
We retrieved relevant articles from Cochrane library, PubMed, EMbase, CNKI, CBM, VIP and Wan Fang Databases from inception to May 2012. Studies were selected using specific inclusion and exclusion criteria. A systematic review and meta-analysis was performed on the association between HIF-1α expression and clinical outcomes in lung cancer patients. All analyses were performed using the Revman 5.1 software.
A total of 30 studies were identified as eligible for the systematic review and meta-analysis. The expression of HIF-1α was significantly higher than those in normal lung tissue; and III‒IV stage, lymph node metastasis, poorly differentiation, squamous cell carcinoma and small cell lung cancer (SCLC) were significantly higher than those in I‒II stage, no lymph node metastasis, well differentiation, adenocarcinomas and non small cell lung cancer (NSCLC), respectively (odds ratio (OR) = 19.00, 95% confidence interval (CI):12.12-29.78, p <0.00001; OR = 0.23, 95% CI:0.14-0.36, p <0.00001; OR = 3.72, 95% CI:2.38-5.80, p <0.00001; OR = 0.47, 95% CI:0.31-0.70, p <0.00002, OR = 0.24, 95% CI:0.07-0.77, p = 0.02; OR = 0.78, 95% CI:0.63-0.98, p = 0.03). VEGF and CA IX positive expression in HIF-1α positive tumour tissues were significantly higher than those in HIF-1α negative tumour tissues, respectively (OR = 3.23, 95% CI: 1.90-5.46, p <0.0001; OR = 3.84, 95% CI: 2.10-7.03, p <0.0001). The positive HIF-1α tumour tissues of patients had lower 5-year survival rates (OR = 0.13, 95% CI: 0.03-0.47, p = 0.002) and overall survival (relative risk (RR) = 1.68, 95% CI: 1.12-2.50, p = 0.01).
HIF-1α is related to a differing degree of lung cancer cell, lymph node metastasis, post-operative survival time and histology (NSCLC vs. SCLC, adenocarcinomas vs. squamous cell carcinoma). HIF-1 α , which combines other proteins, such as vascular endothelial growth factor (VEGF) or CA IX, might serve as important parameters in evaluating biological behaviour and prognosis of lung cancer; it will be of benefit to clinical treatment and prognostic evaluation.
Numerous studies have described the altered expression and the causal role of microRNAs (miRNAs) in human cancer. However, to date, efforts to modulate miRNA levels for therapeutic purposes have been challenging to implement. Here we find that nucleolin (NCL), a major nucleolar protein, posttranscriptionally regulates the expression of a specific subset of miRNAs, including miR-21, miR-221, miR-222, and miR-103, that are causally involved in breast cancer initiation, progression, and drug resistance. We also show that NCL is commonly overexpressed in human breast tumors and that its expression correlates with that of NCL-dependent miRNAs. Finally, inhibition of NCL using guanosine-rich aptamers reduces the levels of NCL-dependent miRNAs and their target genes, thus reducing breast cancer cell aggressiveness both in vitro and in vivo. These findings illuminate a path to novel therapeutic approaches based on NCL-targeting aptamers for the modulation of miRNA expression in the treatment of breast cancer.
Background The authors consider whether differences in stage at diagnosis could explain the variation in lung cancer survival between six developed countries in 2004–2007.
Methods Routinely collected population-based data were obtained on all adults (15–99 years) diagnosed with lung cancer in 2004–2007 and registered in regional and national cancer registries in Australia, Canada, Denmark, Norway, Sweden and the UK. Stage data for 57 352 patients were consolidated from various classification systems. Flexible parametric hazard models on the log cumulative scale were used to estimate net survival at 1 year and the excess hazard up to 18 months after diagnosis.
Results Age-standardised 1-year net survival from non-small cell lung cancer ranged from 30% (UK) to 46% (Sweden). Patients in the UK and Denmark had lower survival than elsewhere, partly because of a more adverse stage distribution. However, there were also wide international differences in stage-specific survival. Net survival from TNM stage I non-small cell lung cancer was 16% lower in the UK than in Sweden, and for TNM stage IV disease survival was 10% lower. Similar patterns were found for small cell lung cancer.
Conclusions There are comparability issues when using population-based data but, even given these constraints, this study shows that, while differences in stage at diagnosis explain some of the international variation in overall lung cancer survival, wide disparities in stage-specific survival exist, suggesting that other factors are also important such as differences in treatment. Stage should be included in international cancer survival studies and the comparability of population-based data should be improved.
The mRNA of hif-1α is considered as being constitutively and ubiquitously expressed, regardless of the level of oxygen tension. However many
recent reports have showed that hif-1α mRNA could be regulated by natural antisense transcripts, potential microRNAs, and low O2. In this study, it was found that a deficiency of JNK2 expression reduced HIF-1α protein induction in response to nickel
treatment resulting from the impaired expression of hif-1α mRNA. Both the promoter luciferase assay and mRNA degradation assay clearly showed that depletion of JNK2 affected stability
of hif-1α mRNA, rather than regulated its transcription. In addition, nucleolin, a classic histone chaperone, was demonstrated to
physically bind to hif-1α mRNA and maintain its stability. Further investigation indicated that JNK2 regulated nucleolin expression and might in turn
stabilize hif-1α mRNA. Collectively, we provided one more piece of evidence for the oncogenic role of JNK2 and nucleolin in regulating the
cancer microenvironments by controlling HIF-1α expression.
β-Arrestins 1 and 2 are multifunctional adaptor proteins originally discovered for their role in desensitizing seven-transmembrane receptor signaling via the heterotrimeric guanine nucleotide-binding proteins. Recently identified roles of β-arrestins include regulation of cancer cell chemotaxis and proliferation. Herein, we report that β-arrestin1 expression regulates breast tumor colonization in nude mice and cancer cell viability during hypoxia. β-Arrestin1 robustly interacts with nuclear hypoxia-induced factor-1α (HIF-1α) that is stabilized during hypoxia and potentiates HIF-1-dependent transcription of the angiogenic factor vascular endothelial growth factor-A (VEGF-A). Increased expression of β-arrestin1 in human breast cancer (infiltrating ductal carcinoma or IDC and metastatic IDC) correlates with increased levels of VEGF-A. While the anti-angiogenic drug thalidomide inhibits HIF-1-dependent VEGF transcription in breast carcinoma cells, it does not prevent HIF-1α stabilization, but leads to aberrant localization of HIF-1α to the perinuclear compartments and surprisingly stimulates nuclear export of β-arrestin1. Additionally, imatinib mesylate that inhibits release of VEGF induces nuclear export of β-arrestin1-HIF-1α complexes. Our findings suggest that β-arrestin1 regulates nuclear signaling during hypoxia to promote survival of breast cancer cells via VEGF signaling and that drugs that induce its translocation from the nucleus to the cytoplasm could be useful in anti-angiogenic and breast cancer therapies.
Background & Aims
Pancreatic ductal adenocarcinomas (PDACs) are hypovascular, resulting in the upregulation of hypoxia inducible factor 1 alpha (HIF1A), which promotes survival of cells under low-oxygen conditions. We studied the roles of HIF1A in development of pancreatic tumors in mice.
Methods
We performed studies with KrasLSL-G12D/+;Trp53LSL-R172H/+; Pdx1-Cre (KPC) mice, KPC mice with labeled pancreatic epithelial cells (EKPC), and EKPC mice with pancreas-specific depletion of HIF1A. Pancreatic and other tissues were collected and analyzed by histology, and immunohistochemistry. Cancer cells were cultured from PDACs from mice and analyzed in cell migration and invasion assays and by immunoblots, real-time PCR, and liquid chromatography–mass spectrometry. We performed studies with the human pancreatic cancer cell lines PATU-8988T, BxPC-3, PANC-1 and MiaPACA-2, which have no or low metastatic activity, and PATU-8988S, AsPC-1, SUIT-2 and Capan-1, which have high-metastatic activity. Expression of genes was knocked down in primary cancer cells and pancreatic cancer cell lines using small hairpin RNAs; cells were injected intravenously into immune-competent and NOD/SCID mice and lung metastases were quantified. We compared levels of mRNAs in pancreatic tumors and normal pancreas in the Cancer Genome Atlas.
Results
EKPC mice with pancreas-specific deletion of HIF1A developed more advanced pancreatic neoplasias and PDACs with more invasion and metastasis, and had significantly shorter survival times, than EKPC mice. Pancreatic cancer cells from these tumors had higher invasive and metastatic activity in culture than cells from tumors of EKPC mice. HIF1A-knockout pancreatic cancer cells had increased expression of protein phosphatase 1 regulatory inhibitor subunit 1B (PPP1R1B). There was an inverse correlation between levels of HIF1A and PPP1R1B in human PDAC tumors; higher expression of PPP1R1B correlated with shorter survival times of patients. Metastatic human pancreatic cancer cell lines had increased levels of PPP1R1B and lower levels of HIF1A compared with non-metastatic cancer cell lines; knockdown of PPP1R1B significantly reduced the ability of pancreatic cancer cells to form lung metastases in mice. PPP1R1B promoted degradation of p53 by stabilizing phosphorylation of MDM2 at Ser166.
Conclusions
HIF1A can act a tumor suppressor by preventing expression of PPP1R1B and subsequent degradation of the p53 protein in pancreatic cancer cells. Loss of HIF1A from pancreatic cancer cells increases their invasive and metastatic activity.
Microtubules are cytoskeletal elements known as drivers of directed cell migration, vesicle and organelle trafficking, and mitosis. In this review, we discuss new research in the lens that has shed light into further roles for stable microtubules in the process of development and morphogenesis. In the lens, as well as other systems, distinct roles for characteristically dynamic microtubules and stabilized populations are coming to light. Understanding the mechanisms of microtubule stabilization and the associated microtubule post-translational modifications is an evolving field of study. Appropriate cellular homeostasis relies on not only one cytoskeletal element, but also rather an interaction between cytoskeletal proteins as well as other cellular regulators. Microtubules are key integrators with actin and intermediate filaments, as well as cell–cell junctional proteins and other cellular regulators including myosin and RhoGTPases to maintain this balance.
Impact statement
The role of microtubules in cellular functioning is constantly expanding. In this review, we examine new and exciting fields of discovery for microtubule’s involvement in morphogenesis, highlight our evolving understanding of differential roles for stabilized versus dynamic subpopulations, and further understanding of microtubules as a cellular integrator.
Metastasis is the primary cause of cancer morbidity and mortality. The process involves a complex interplay between intrinsic tumor cell properties as well as interactions between cancer cells and multiple microenvironments. The outcome is the development of a nearby or distant discontiguous secondary mass. To successfully disseminate, metastatic cells acquire properties in addition to those necessary to become neoplastic. Heterogeneity in mechanisms involved, routes of dissemination, redundancy of molecular pathways that can be utilized, and the ability to piggyback on the actions of surrounding stromal cells makes defining the hallmarks of metastasis extraordinarily challenging. Nonetheless, this review identifies four distinguishing features that are required: motility and invasion, ability to modulate the secondary site or local microenvironments, plasticity, and ability to colonize secondary tissues. By defining these first principles of metastasis, we provide the means for focusing efforts on the aspects of metastasis that will improve patient outcomes.
Background
A Lung cancer death account for approximately 1 in 5 of all cancer-related deaths and is particularly virulent due to its enhanced metastasis and resistance to chemotherapy. Chrysotobibenzyl has been reported to decrease cell metastasis, according to the results of an anchorage-independent growth assay; however, its underlying mechanism has not been investigated yet.
Purpose
The aim of this study was to investigate the effect of chrysotobibenzyl on lung cancer cell migration and drug sensitization and its mechanism.
Methods
Cell viability, cell proliferation and drug sensitization were determined by MTT assay. Cell migration was analyzed using a wound-healing assay. Transwell migration and invasion were analyzed using Boyden chamber assay. Mechanisms of chrysotobibenzyl against metastasis including cell migration, invasion, and epithelial to mesenchymal transition (EMT) were evaluated by Western blot analysis and immunofluorescence.
Results
Treatment with chrysotobibenzyl was applied at concentrations of 0–50 µM and the results showed non-cytotoxicity in human lung cancer cells (H460, H292, A549, and H23) and other non-cancerous human cells (HCT116, primary DP1 and primary DP2). However, 50 µM of chrysotobibenzyl significantly altered cell proliferation in H292 cells at 48 h. In addition, 1–50 µM of chrysotobibenzyl significantly inhibited H460 and H292 cell migration, invasion, filopodia formation, and decreased EMT in a dose-dependent manner at 48 h, which were correlated with reduced protein levels of integrins β1, β3, and αν, p-FAK, p-AKT, Cdc42, and Cav-1. We also established shRNA-Cav-1-transfected (shCav-1) H460 and H292 cells. shCav-1 transfected cells can decrease cell migration and downregulate the expression of integrins β1, β3, and αν when compared with the control. Moreover, chrysotobibenzyl was shown to suppress EMT indicated by the reduction of EMT markers (Vimentin, Snail, and Slug), and sensitize lung cancer cells to cisplatin-mediated apoptosis.
Conclusion
Treatment with chrysotobibenzyl inhibited lung cancer cell migration via Cav-1, integrins β1, β3, and αν, and EMT suppressions. The downregulation of integrins in response to the compound not only inhibited cell metastasis, but also sensitized lung cancer cells to cisplatin-mediated apoptosis.
Epithelial-to-mesenchymal transition (EMT) plays pivotal roles in a variety of biological processes, including cancer invasion. Although EMT involves alterations of cytoskeletal proteins such as microtubules, the role of microtubules in EMT is not fully understood. Microtubule dynamics are regulated by microtubule-binding proteins, and one such protein is CAMSAP3, which binds the minus-end of microtubules. Here, we show that CAMSAP3 is important to preserve the epithelial phenotypes in lung carcinoma cells. Deletion of CAMSAP3 in human lung carcinoma-derived cell lines showed that CAMSAP3-deficient cells acquired increased mesenchymal features, mostly at the transcriptional level. Analysis of the mechanisms underlying these changes demonstrated that tubulin acetylation was dramatically increased following CAMSAP3 removal, leading to the upregulation of Akt proteins (also known as protein kinase B proteins, hereafter Akt) activity, which is known to promote EMT. These findings suggest that CAMSAP3 functions to protect lung carcinoma cells against EMT by suppressing Akt activity via microtubule regulation and that CAMSAP3 loss promotes EMT in these cells.
This article has an associated First Person interview with the first author of the paper.
Vascular leak, or plasma extravasation, has a number of causes, and may be a serious consequence or symptom of an inflammatory response. This study may ultimately lead to new knowledge concerning the causes of or new ways to inhibit or treat plasma extravasation. It is important that researchers have the proper tools, including the best methods available, for studying plasma extravasation. In this article, we describe a protocol, using the Evans blue dye method, for assessing plasma extravasation in the organs of FVBN mice. This protocol is intentionally simple, to as great a degree as possible, but provides high quality data. Evans blue dye has been chosen primarily because it is easy for the average laboratory to use. We have used this protocol to provide evidence and support for the hypothesis that the enzyme neprilysin may protect the vasculature against plasma extravasation. However, this protocol may be experimentally used and easily adapted for use in other strains of mice or in other species, in many different organs or tissues, for studies which may involve other factors that are important in understanding, preventing, or treating plasma extravasation. This protocol has been extensively optimized and modified from existing protocols, and combines reliability, ease of use, economy, and general availability of materials and equipment, making this protocol superior for the average laboratory to use in quantifying plasma extravasation from organs. © 2018, Journal of Visualized Experiments. All rights reserved.
Important advancements in the treatment of non-small cell lung cancer (NSCLC) have been achieved over the past two decades, increasing our understanding of the disease biology and mechanisms of tumour progression, and advancing early detection and multimodal care. The use of small molecule tyrosine kinase inhibitors and immunotherapy has led to unprecedented survival benefits in selected patients. However, the overall cure and survival rates for NSCLC remain low, particularly in metastatic disease. Therefore, continued research into new drugs and combination therapies is required to expand the clinical benefit to a broader patient population and to improve outcomes in NSCLC.
Although NK cells are recognized as direct antitumor effectors, the ability of NK cells to control cancer-associated inflammation, which facilitates tumor progression, remains unknown. In this study, we demonstrate that NK cells control tumor-promoting inflammation through functional modification of neutrophils. NK cells control the tumor-promoting function of neutrophils through an IFNgamma-dependent mechanism. Tumor progression in an NK cell-depleted host is diminished when the IL17A-neutrophil axis is absent. In NK cell-depleted mice, neutrophils acquire a tumor-promoting phenotype, characterized by up-regulation of VEGF-A expression, which promotes tumor growth and angiogenesis. A VEGFR inhibitor which preferentially suppressed tumor growth in NK cell-depleted mice was dependent on neutrophils. Furthermore, the systemic neutropenia caused by an anti-metabolite treatment showed an anti-cancer effect only in mice lacking NK cells. Thus, NK cells likely control the tumor-promoting and angiogenic function of neutrophils.
Our recent studies demonstrate that X-linked inhibitor of apoptosis protein (XIAP) is essential for regulating colorectal cancer invasion. Here we discovered that RhoGDIβ was a key XIAP downstream effector mediating bladder cancer (BC) invasion in vitro and in vivo. We found that both XIAP and RhoGDIβ expressions were consistently elevated in BCs of N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-treated mice in comparison to bladder tissues from vehicle-treated mice and human BCs in comparison to the paired adjacent normal bladder tissues. Knockdown of XIAP attenuated RhoGDIβ expression and reduced cancer cell invasion, whereas RhoGDIβ expression was attenuated in BBN-treated urothelium of RING-deletion knockin mice. Mechanistically, XIAP stabilized RhoGDIβ mRNA by its positively regulating nucleolin mRNA stability via Erks-dependent manner. Moreover, ectopic expression of GFP-RhoGDIβ in T24T(shXIAP) cells restored its lung metastasis in nude mice. Our results demonstrate that XIAP-regulated Erks/nucleolin/RhoGDIβ axis promoted BC invasion and lung metastasis. This article is protected by copyright. All rights reserved.
Introduction The vascular endothelial growth factor A (VEGF) is the main mediator of angiogenesis. In addition, VEGF contributes to cancer growth and metastasis directly targeting tumor cells. VEGF overexpression and/or high VEGF serum levels have been reported in lung cancer.
Areas covered We searched Pubmed for relevant preclinical studies with the terms “lung cancer” “VEGF” and “in vivo”. We also searched the Clinicaltrials.gov database, the FDA and the EMA websites for the most recent updates on clinical development of anti-VEGF agents.
Expert opinion VEGF plays an important role in sustaining the development and progression of lung cancer and it might represent an attractive target for therapeutic strategies. Nevertheless, clinical trials failed to attend the promising expectations deriving from preclinical studies with anti-VEGF agents. To improve the efficacy of anti-VEGF therapies in lung cancer, potential strategies might be the employment of combinatory therapies with immune checkpoint inhibitors or agents that inhibit signaling pathways and proangiogenic factors activated in response to VEGF blockade, and the identification of novel targets in the VEGF cascade. Finally, the identification of predictive markers might help to select patients who are more likely to respond to anti-angiogenic drugs.
Lung cancer is the most frequent cause of cancer-related deaths worldwide. Every year, 1·8 million people are diagnosed with lung cancer, and 1·6 million people die as a result of the disease. 5-year survival rates vary from 4-17% depending on stage and regional differences. In this Seminar, we discuss existing treatment for patients with lung cancer and the promise of precision medicine, with special emphasis on new targeted therapies. Some subgroups, eg-patients with poor performance status and elderly patients-are not specifically addressed, because these groups require special treatment considerations and no frameworks have been established in terms of new targeted therapies. We discuss prevention and early detection of lung cancer with an emphasis on lung cancer screening. Although we acknowledge the importance of smoking prevention and cessation, this is a large topic beyond the scope of this Seminar.
Significance
Polarization is essential for epithelial cells to exert a variety of functions. Epithelial polarization includes characteristic microtubule array formation. The microtubules are oriented along the apicobasal axis with their minus ends facing apically. The molecules that regulate such epithelial-specific microtubule assembly remain unknown, however. Our study demonstrates that in intestinal epithelial cells, the microtubule minus-end binding protein CAMSAP3 (calmodulin-regulated–spectrin-associated protein 3) tethers noncentrosomal microtubules to the apical cortex, leading to their longitudinal orientation. This mechanism is essential for maintaining epithelial intracellular organization, such as positioning of organelles. Our findings facilitate our understanding of how epithelial cells acquire polarized structures, which are crucial for their physiological functions.
the present retrospective study was performed to evaluate the clinicopathological characteristics associated with distant metastasis from nonsmallcell lung cancer (NSCLC). The records of NSCLC patients with metastasis at the time of diagnosis between 1999 and 2012 were reviewed. Of the consecutive 1,542 NSCLC patients diagnosed during the study period, 729 (47.3%) patients presented with distant metastasis. Among those 729 metastatic NSCLC patients, 250 (34.3%), 234 (32.1%), 207 (28.4%), 122 (16.7%), 98 (13.4%) and 69 (9.5%) had bone, lung, brain, adrenal gland, liver and extrathoracic lymph node metastasis, respectively. In a multivariate analysis using the Cox proportional hazards model, liver and adrenal gland metastases were unfavorable prognostic factors. However, brain and bone metastases were not statistically significant prognostic factors. Using a logistic regression analysis, metastasis to the adrenal glands and the presence of pleural andor pericardial fluid effusion were correlated with a poor performance status. Therefore, when planning the treatment of NSCLC patients, particularly those with liver and adrenal gland metastases, we should take into consideration information regarding these unfavorable organ metastases.
Expression of microtubule‑associated protein 1 light chain 3 (LC3) is correlated with poor prognosis in many human cancers. Hypoxia induces LC3 expression and is an essential characteristic of epithelial ovarian cancer (EOC). The aim of the present study was to elucidate the mechanism by which LC3 facilitates EOC cell migration and invasion under conditions of hypoxia. The effects of LC3B inhibition under hypoxic conditions on migration, invasion, and adhesion in HO8910PM and HO8910 EOC cell lines were investigated. LC3B inhibition was achieved by small‑interfering RNA (siRNA) targeting LC3B or by treatment with 3‑methyladenine (3‑MA). Cell migration, invasion and adhesion and the arrangement of the cytoskeleton were determined by Transwell migration assays and rhodamine phalloidin staining. Western blot analysis was performed to evaluate the expression level of LC3B and the expression and activity of ras homolog gene family member A (RhoA). Increased LC3B expression was associated with HO8910PM and HO8910 cell migration and invasion promoted under hypoxic conditions. LC3B siRNA and 3‑MA treatment each attenuated hypoxia‑induced LC3B expression, along with migration and invasion, and this was associated with a decrease in RhoA expression and disorganization of the actin cytoskeleton. LC3B may promote the migration and invasion of EOC cells by affecting the cytoskeleton via the RhoA pathway. In addition, LC3B may be a marker of tumor hypoxia and/or metastasis in EOC cells.
Nucleolin is a ubiquitously expressed protein and participates in many important biological processes, such as cell cycle regulation and ribosomal biogenesis. The activity of nucleolin is regulated by intracellular localization and post-translational modifications (PTMs), including phosphorylation, methylation and ADP-ribosylation. Small ubiquitin-like modifier (SUMO) is a category of lately verified forms of PTMs, and exerts various effects on the target proteins. In the studies reported here, we discovered SUMOylational modification of human nucleolin protein at K294, which facilitated the mRNA binding property of nucleolin by maintaining its nucleus localization. In response to arsenic exposure, nucleolin-SUMO was induced and promoted its binding with gadd45a mRNA, which increased gadd45a mRNA stability and protein expression, subsequently causing GADD45α-mediated cell death. On the other hand, ectopic expression of MnSOD attenuated arsenite-generated superoxide radical level, abrogated nucleolin-SUMO, and in turn inhibited arsenite-induced apoptosis by reducing GADD45α expression. Collectively, our results for the first time demonstrated that nucleolin-SUMO at K294R played a critical role in its nucleus sequestration and gadd45a mRNA binding activity. This novel biological function of nucleolin was distinct from its conventional role as proto-oncogene. Therefore, our findings here not only discovered a new modification of nucleolin protein and its novel functional paradigm in mRNA metabolism, but also expanded our understanding regarding the dichotomous roles of nucleolin in term of cancer development, which were dependent on multiple intracellular conditions, and consequently the appropriate regulations of its modifications, including SUMOylation.
Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
Unlabelled:
Aberrant activation of the Wnt/β-catenin signaling pathway is a critical event in advanced prostate cancer, but the genetic alterations that activate the Wnt signaling pathway in many other cancers are rarely observed in prostate cancer. Other molecular mechanisms that regulate the Wnt signaling pathway in prostate cancer remain to be identified. Here, it is demonstrated that KIF3a, a subunit of kinesin-II motor protein, functions as an agonist of the Wnt signaling pathway in prostate cancer. KIF3a is upregulated in the majority of human prostate cancer cell lines and primary tumor biopsies. The expression levels of KIF3a correlate with a higher Gleason score, tumor-node-metastasis stage, and metastatic status of prostate cancer. Moreover, exogenous expression of KIF3a promoted cell growth in the benign prostate cells, whereas silencing KIF3a in cancer cells decreased cell proliferation, anchorage-independent cell growth, and cell migration/invasion. Mechanistically, KIF3a increases CK1-dependent DVL2 phosphorylation and β-catenin activation in prostate cancer cells, leading to transactivation of the Wnt-signaling target genes such as cyclin D1, HEF1, and MMP9. These findings support the notion that upregulation of KIF3a is causal of aberrant activation of Wnt signaling in advanced prostate cancer through the KIF3a-DVL2-β-catenin axis.
Implications:
Inactivation of KIF3a may improve survival of patients with advanced prostate cancer in which Wnt signaling is activated.
Hypoxia-inducible factor 1α (HIF1α) is a transcription factor involved in the growth, invasion and metastasis of malignant tumors. Glycogen synthase kinase 3 beta (GSK3β) is a protein kinase involved in a variety of signaling pathways, such as the Wnt and NF-κB pathways; this kinase can affect tumor progress through the regulation of transcription factor expression and apoptosis. Recent studies showed that GSK3β was involved in the expression of HIF1α. However, the effect of GSK3β on HIF1α expression in osteosarcoma cells remains unknown. To understand the relationship between GSK3β and HIF1α comprehensively, small RNA interference techniques, western blot analyses, quantitative real-time PCR analyses and luciferase assays were used in our study. Experimental data revealed that inhibition of GSK3β could increase HIF1α protein levels and expression of its target genes by increasing the stability of the HIF1α mRNA, not by affecting the HIF1α protein stability, and that this process could be mediated by nucleolin.
The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-DeltaDeltaCr) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-DeltaDeltaCr) method. In addition, we present the derivation and applications of two variations of the 2(-DeltaDeltaCr) method that may be useful in the analysis of real-time, quantitative PCR data. (C) 2001 Elsevier science.
The cBio Cancer Genomics Portal (http://cbioportal.org) is an open-access resource for interactive exploration of multidimensional cancer genomics data sets, currently providing access to data from more than 5,000 tumor samples from 20 cancer studies. The cBio Cancer Genomics Portal significantly lowers the barriers between complex genomic data and cancer researchers who want rapid, intuitive, and high-quality access to molecular profiles and clinical attributes from large-scale cancer genomics projects and empowers researchers to translate these rich data sets into biologic insights and clinical applications.
Cell motility is an essential aspect of metastatic spread of cancer. Microtubule-targeted agents exhibit anti-metastatic properties, the underlying mechanism of which remains understudied. In this study, we have investigated the role of microtubule dynamics in migration of cancer cells using indanocine, a synthetic small molecule inhibitor of tubulin. We found that indanocine, at concentrations that did not visibly affect microtubule organization, suppressed dynamic instability of microtubules and reduced the rate of migration of highly metastatic MDA-MB-231 cells. Indanocine-treated cells were defective in lamellipodium formation and could not develop polarized morphology. The kinetic stabilization of microtubules was associated with a marked increase in their acetylation level and a perturbation in the localization of EB1, a microtubule plus end binding protein. Using standard scratch wound healing assay and immunofluorescence analysis; we found that microtubule acetylation occurred in the direction of migration in vehicle-treated cells, whereas indanocine treatment led to a global acetylation of microtubules. The results together suggested that selective stabilization of microtubules was perturbed in the presence of indanocine that possibly resulted in lack of cell polarization and a concurrent reduction in migration of cells. Moreover, microtubule stabilization by indanocine affected adhesion turnover and impaired the polarized pattern of adhesion sites in cells. Together the results indicated that the regulation of microtubule dynamics is required to coordinate cell polarization as well as adhesion asymmetry and support the hypothesis that the perturbation of microtubule dynamics by tubulin-targeted agents can be exploited to restrict the migration of tumor cells.
Hypoxia-inducible factors (HIFs) mediate adaptive physiological responses to hypoxia. In human cancers that are accessible for O(2) electrode measurements, intratumoral hypoxia is common and severe hypoxia is associated with increased risk of mortality. HIF activity in regions of intratumoral hypoxia mediates angiogenesis, epithelial-mesenchymal transition, stem-cell maintenance, invasion, metastasis, and resistance to radiation therapy and chemotherapy. A growing number of drugs have been identified that inhibit HIF activity by a variety of molecular mechanisms. Because many of these drugs are already FDA-approved for other indications, clinical trials can (and should) be initiated to test the hypothesis that incorporation of HIF inhibitors into current standard-of-care therapy will increase the survival of cancer patients.
Hypoxia is a critical event in solid tumor development, invasion, and metastasis. Cellular adaptation to hypoxic microenvironment is essential for tumor progression and is largely mediated by hypoxia-inducible factor-1α (HIF-1α) through coordinated regulation of hypoxia-responsive genes. In this study, we found that membrane type-2 matrix metalloproteinase (MT2-MMP), one of the matrix metalloproteinase (MMP) family members, was a novel hypoxia-responsive gene and was upregulated by HIF-1α under hypoxia. When cancer cells were subjected to hypoxia (1% O(2) ) treatment, the mRNA and protein levels of MT2-MMP were significantly increased in a time-dependent manner in all three tested cancer cell lines including pancreatic cancer cells (PANC-1), nonsmall cell lung cancer cells (A-549), and cervix cancer cells (HeLa). Further analyses indicated that there were two hypoxia-responsive elements (HREs) in the MT2-MMP promoter, and HRE1 but not HRE2 was essential for MT2-MMP transcriptional activation under hypoxia. HIF-1α specifically and directly bound to MT2-MMP promoter was analyzed by HIF-1α binding/competition and chromatin immunoprecipitation (ChIP) assays. Furthermore, we found that upregulation of MT2-MMP under hypoxia could confer resistance to hypoxia-induced apoptosis and increase invasiveness of cancer cells. These findings provided a new insight into how cancer cells overcome hypoxic stress and trend to survive and invade, demonstrated a new regulatory mechanism of MT2-MMP expression in caner cells, and also revealed that MT2-MMP was a novel hypoxia-responsive gene and was upregulated by HIF-1α under hypoxia. © 2011 Wiley-Liss, Inc.