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Exosomes from Nischarin-Expressing Cells Reduce Breast Cancer Cell Motility and Tumor Growth
Abstract
Exosomes are small extracellular microvesicles that are secreted by cells when intracellular multivesicular bodies fuse with the plasma membrane. We have previously demonstrated that Nischarin inhibits focal adhesion formation, cell migration, and invasion, leading to reduced activation of focal adhesion kinase. In this study, we propose that the tumor suppressor Nischarin regulates the release of exosomes. When cocultured on exosomes from Nischarin-positive cells, breast cancer cells exhibited reduced survival, migration, adhesion, and spreading. The same cocultures formed xenograft tumors of significantly reduced volume following injection into mice. Exosomes secreted by Nischarin-expressing tumors inhibited tumor growth. Expression of only one allele of Nischarin increased secretion of exosomes, and Rab14 activity modulated exosome secretions and cell growth. Taken together, this study reveals a novel role for Nischarin in preventing cancer cell motility, which contributes to our understanding of exosome biology.
Significance
Regulation of Nischarin-mediated exosome secretion by Rab14 seems to play an important role in controlling tumor growth and migration.
See related commentary by McAndrews and Kalluri, p. 2099
Supplementary resources (5)
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... Nischarin can also inhibit tumor cell growth in cancer by induction of exosome secretion. Nischarin expressing exosomes suppressed the cancer cell motility and tumor growth in a breast caner mouse model [59]. Finally, Nischarin knockout-PyMT animal model data showed that deletion of Nischarin enhances tumor growth and metastasis [60]. ...
... Regulates Rac signaling cascade at multiple levels Actin filament organization (Rac signaling) Regulates focal adhesion proteins Induces exosome secretion [59]. ...
Integrins are cell adhesion receptors, which are typically transmembrane glycoproteins that connect to the extracellular matrix (ECM). The function of integrins regulated by biochemical events within the cells. Understanding the mechanisms of cell growth by integrins is important in elucidating their effects on tumor progression. One of the major events in integrin signaling is integrin binding to extracellular ligands. Another event is distant signaling that gathers chemical signals from outside of the cell and transmit the signals upon cell adhesion to the inside of the cell. In normal breast tissue, integrins function as checkpoints to monitor effects on cell proliferation, while in cancer tissue these functions altered. The combination of tumor microenvironment and its associated components determines the cell fate. Hypoxia can increase the expression of several integrins. The exosomal integrins promote the growth of metastatic cells. Expression of certain integrins is associated with increased metastasis and decreased prognosis in cancers. In addition, integrin-binding proteins promote invasion and metastasis in breast cancer. Targeting specific integrins and integrin-binding proteins may provide new therapeutic approaches for breast cancer therapies. This review will examine the current knowledge of integrins’ role in breast cancer.
... Interestingly, when SCID mice xenografted with PFN2-overexpressing SCLC cells were treated with exosomes originating from PFN2-knockdown SCLC cells, their tumors were smaller than those in the untreated mice (Supplementary Figure 4), implying that PFN2 may be a valuable therapeutic target for SCLC in clinical practice. Because cancer cell migration and invasion are prerequisites for metastasis, PFN2 OE significantly promoted proliferation, migration, and invasion, demonstrating a novel mechanism underlying PFN2 mediated tumor metastasis [25]. ...
... Exosomes, which are membrane microvesicles secreted by cells, are important carriers of various bioactive molecules and are well-known promoters of the invasion and migration of cancer cells [25][26][27][28][29][30][31]. An increasing number of studies have revealed that cancerderived exosomes could influence cancer growth, progression, and metastasis in a lymphatic and vascular manner [32,33]. ...
Small cell lung cancer (SCLC) is highly aggressive and prone to hypervascular metastases. Recently, we found profilin 2 (PFN2) expression in SCLC but not in normal tissues. Furthermore, PFN2 expression had been shown to promote angiogenesis through exosomes. However, it remains unclear whether PFN2 contributes to the progression and metastasis of SCLC through angiogenesis. We report here that overexpression (OE) of PFN2 increased, whereas its knockdown (KD) decreased the proliferation, migration, and invasion of SCLC cell H446. The exosomes from OE-H446 (SCLC-OE-exo) exhibited similar effects on H446 properties. Culturing of endothelial cells (ECs) in SCLC-OE conditioned medium (CM) or SCLC-OE-exo increased the migration and tube formation ability of ECs, whereas SCLC-KD-CM and SCLC-KD-exo had inhibitory effects. Interestingly, both SCLC- and EC-derived exosomes were internalized in H446 more rapidly than in ECs. More importantly, OE-PFN2 dramatically elevated SCLC growth and vasculature formation as well as lung metastasis in tumor xenograft models. Finally, we found that PFN2 activated Smad2/3 in H446 and pERK in ECs, respectively. Taken together, our study revealed the role of PFN2 in SCLC development and metastasis, as well as tumor angiogenesis through exosomes, providing a new molecular target for SCLC treatment.
... Nischarin prevents cell migration and invasion by altering the expression of key focal adhesion proteins (17). Additionally, Nischarin regulates cell motility via exosomes; when co-cultured with exosomes from Nischarin-positive cells, the survival ability, migration ability and adhesion of breast cancer cells are decreased (18). In Prkdc scid mice xenograft tumor models, exosomes secreted by Nischarin-positive tumor cells inhibit tumor growth (18). ...
... Additionally, Nischarin regulates cell motility via exosomes; when co-cultured with exosomes from Nischarin-positive cells, the survival ability, migration ability and adhesion of breast cancer cells are decreased (18). In Prkdc scid mice xenograft tumor models, exosomes secreted by Nischarin-positive tumor cells inhibit tumor growth (18). ...
Nischarin is an integrin-binding protein, which is well known as a novel tumor suppressor. In breast cancer, Nischarin serves a critical role in breast cancer cell migration and invasion. However, the molecular mechanism underlying the role of Nischarin remains unclear. Recent findings have demonstrated that epithelial-mesenchymal transition (EMT) increases the capacity of cell migration and invasion. As a member of the integrin family, it was hypothesized that Nischarin may regulate cellular processes via various signaling pathways associated with the EMT process. The present study detected the mRNA levels of EMT regulators via reverse transcription-quantitative PCR and related protein levels via western blotting in breast cancer cells, following NISCH-overexpression and -knockdown. The results demonstrated that Nischarin inhibits cell proliferation, migration and invasion in breast cancer cells. Furthermore, when the NISCH gene was overexpressed, the relative mRNA level of E-cadherin was increased, while the relative mRNA levels of several transcription factors, such as Snail, ZEB1, N-cadherin, Slug, Twist1 and vimentin, decreased. When NISCH was silenced, these results were reversed. The present results demonstrated that Nischarin suppresses cell migration and invasion via inhibiting the EMT process.
... The metastasis suppressor cd82 is known to reverse EMT process in cancer cells by inhibiting the TGF-β1/Smad andWnt/β-catenin pathways [57]. The tumor suppressor gene Nisch drives transcriptional up-regulation of E-cadherin and, contextually, downregulation of several mesenchymal genes, including N-cadherin in cancer cells [58] and was shown to be delivered through EV conferring traits of reduced migration, adhesion, and spreading in target cancer cells [59]. Some of the mRNAs preferentially expressed in Eo33-EV are involved in negative regulation of cellular activities. ...
Immune cell-derived extracellular vesicles (EV) affect tumor progression and hold promise for therapeutic applications. Eosinophils are major effectors in Th2-related pathologies recently implied in cancer. Here, we evaluated the anti-tumor activities of eosinophil-derived EV following activation with the alarmin IL-33. We demonstrate that IL-33-activated mouse and human eosinophils produce higher quantities of EV with respect to eosinophils stimulated with IL-5. Following incorporation of EV from IL-33-activated eosinophils (Eo33-EV), but not EV from IL-5-treated eosinophils (Eo5-EV), mouse and human tumor cells increased the expression of cyclin-dependent kinase inhibitor (CDKI)-related genes resulting in cell cycle arrest in G0/G1, reduced proliferation and inhibited tumor spheroid formation. Moreover, tumor cells incorporating Eo33-EV acquired an epithelial-like phenotype characterized by E-Cadherin up-regulation, N-Cadherin downregulation, reduced cell elongation and migratory extent in vitro, and impaired capacity to metastasize to lungs when injected in syngeneic mice. RNA sequencing revealed distinct mRNA signatures in Eo33-EV and Eo5-EV with increased presence of tumor suppressor genes and enrichment in pathways related to epithelial phenotypes and negative regulation of cellular processes in Eo33-EV compared to Eo5-EV. Our studies underscore novel IL-33-stimulated anticancer activities of eosinophils through EV-mediated reprogramming of tumor cells opening perspectives on the use of eosinophil-derived EV in cancer therapy.
Supplementary Information
The online version contains supplementary material available at 10.1186/s13046-024-03129-1.
... Nischarin was so far known as a novel tumor suppressor gene whose downregulation promotes tumorigenesis [13], tumor progression [3,6,18], and poor survival in breast, ovarian and lung cancer patients [3,5,7,18]. It was reported that exogenous expression of NISCH could suppress breast cancer cell survival and motility in vitro and growth in vivo [7,18,69,70]. NISCH was found to be downregulated in breast and ovarian cancer tissues compared to the healthy counterparts and was found to be a marker of better prognosis [3,6,18]. Surprisingly, although NISCH expression was downregulated in melanoma tissue compared to the uninvolved skin, our group found that it was a favorable prognostic marker only in female melanoma patients, but not in males [19]. ...
Nischarin was reported to be a tumor suppressor that plays a critical role in breast cancer initiation and progression, and a positive prognostic marker in breast, ovarian and lung cancers. Our group has found that nischarin had positive prognostic value in female melanoma patients, but negative in males. This opened up a question whether nischarin has tumor type-specific and sex-dependent roles in cancer progression. In this study, we systematically examined in the public databases the prognostic value of nischarin in solid tumors, regulation of its expression and associated signaling pathways. We also tested the effects of a nischarin agonist rilmenidine on cancer cell viability in vitro. Nischarin expression was decreased in tumors compared to the respective healthy tissues, most commonly due to the deletions of the nischarin gene and promoter methylation. Unlike in healthy tissues where it was located in the cytoplasm and at the membrane, in tumor tissues nischarin could also be observed in the nuclei, implying that nuclear translocation may also account for its cancer-specific role. Surprisingly, in several cancer types high nischarin expression was a negative prognostic marker. Gene set enrichment analysis showed that in tumors in which high nischarin expression was a negative prognostic marker, signaling pathways that regulate stemness were enriched. In concordance with the findings that nischarin expression was negatively associated with pathways that control cancer growth and progression, nischarin agonist rilmenidine decreased the viability of cancer cells in vitro. Taken together, our study lays a ground for functional studies of nischarin in a context-dependent manner and, given that nischarin has several clinically approved agonists, provides rationale for their repurposing, at least in tumors in which nischarin is predicted to be a positive prognostic marker.
... In BC xenografts, sulfisoxazole, an oral antibiotic with anti-tumor and antimetastatic properties, interferes with endothelin receptor A and decreases exosome release, ultimately inhibiting progression and metastasis in BC cells [235]. Additionally, the tumor suppressor nischarin has been found to regulate early metastatic events in BC, with further research demonstrating its novel role in preventing BC cell motility and tumor growth by regulating Rab14 activity and secreting exosomes that control tumor malignancy [236,237]. Another study suggested that antisense non-coding mitochondrial RNA could be a novel target for BC therapy, with exosomes derived from knockdown cells reducing tumorigenic properties and inhibiting the development of BC metastatic niches [238]. ...
Breast cancer (BC) continues to be a significant global challenge due to drug resistance and severe side effects. The increasing prevalence is alarming, requiring new therapeutic approaches to address these challenges. At this point, Extracellular vesicles (EVs), specifically small endosome-released nanometer-sized EVs (SEVs) or exosomes, have been explored by literature as potential theranostics. Therefore, this review aims to highlight the therapeutic potential of exosomes in BC, focusing on their advantages in drug delivery and their ability to mitigate metastasis. Following the review, we identified exosomes' potential in combination therapies, serving as miRNA carriers and contributing to improved anti-tumor effects. This is evident in clinical trials investigating exosomes in BC, which have shown their ability to boost chemotherapy efficacy by delivering drugs like paclitaxel (PTX) and doxorubicin (DOX). However, the translation of EVs into BC therapy is hindered by various challenges. These challenges include the heterogeneity of EVs, the selection of the appropriate parent cell, the loading procedures, and determining the optimal administration routes. Despite the promising therapeutic potential of EVs, these obstacles must be addressed to realize their benefits in BC treatment.
... 36 There were reports showing RAB14 protein expression was positively correlated with increased tumor size, 37 and its activity could regulate exosome secretions and cell growth. 38 Protein transport protein Sec31A (SEC31A) is a core component of the COPII coat complex, which is involved in the formation of transport vesicles from the endoplasmic reticulum (ER), contributing to the efficient transport of proteins from the ER to the Golgi apparatus. This gene is essential for maintaining proper protein trafficking and secretion within the cell. ...
Endometrial cancer (EC) is a frequently diagnosed gynecologic cancer. Identifying reliable prognostic genes for predicting EC onset is crucial for reducing patient morbidity and mortality. Here, a comprehensive strategy with transcriptomic and proteomic data was performed to measure EC’s characteristics. Based on the publicly available RNA-seq data, death-associated protein kinase 3, recombination signal-binding protein for the immunoglobulin kappa J region, and myosin light chain 9 were screened out as potential biomarkers that affect the EC patients’ prognosis. A linear model was further constructed by multivariate Cox regression for the prediction of the risk of being malignant. From further integrative analysis, exosomes were found to have a highly enriched role that might participate in EC occurrence. The findings were validated by qRT-polymerase chain reaction (PCR) and western blotting. Collectively, we constructed a prognostic-gene-based model for EC prediction and found that exosomes participate in EC incidents, revealing significantly promising support for the diagnosis of EC.
... Recently, immunotherapy provided the unprecedented opportunities to malignancies effective treatment and tumor immunology research has been the fastest growing area in cancer, including BC26 . Exosomes are a kind of nanosized vesicles involved in varieties of cellular functions 14 , containing several proteins and nucleic acids including lncRNAs and miRNAs27,28 . LncRNAs are a wide heterogeneous group defined as transcripts longer than 200 bp in length and ...
Breast cancer (BC) is one of the most frequent malignancies among women worldwide. Accumulating evidence indicates that long non-coding RNA (lncRNA) may affect BC progression. Exosomes, a class of small membrane vesicles, have been reported to promote tumor progression through transporting proteins, mRNAs, lncRNAs and some other small molecules. However, the interaction between exosome-related lncRNAs and the microenvironment of malignancies is unclear. Hence, we proceeded to investigate the relationship between exosome-related lncRNAs and BC microenvironment. 121 exosome-associated genes were extracted from ExoBCD database. Then, the Pearson analysis was used to screened out the exosome-related lncRNAs. After that, 15 exosome-related differentially expressed lncRNAs were identified by the correlation with BC prognosis. According to the sum of the expression of these 15 lncRNAs, extracted from The Cancer Genome Atlas, and the regression coefficients, an exosome-related lncRNAs signature was developed by using Cox regression analysis. With the median risk score of the training set, the patients in training and validation sets were separated to low-risk group and high-risk group. Subsequently, the lncRNA–mRNA co-expression network was constructed. The distinct enrichment pathways were compared among the different risk groups by using the R package clusterProfiler. The ESTIMATE method and ssGESA database were adopted to study the ESTIMATE Score and immune cell infiltration. Eventually, the expression of immune checkpoint associated genes, microsatellite instable and the immunophenoscore were further analyzed between different risk groups. Different risk groups exhibited different prognosis, with lower survival rate in the high-risk group. The differentially expressed genes between the different risk groups were enriched in biological processes pathways as well as immune responses. BC patients in high-risk group were identified with lower scores of ESTIMATE scores. Subsequently, we noticed that the infiltrating levels of aDCs, B cells, CD8+ T cells, iDCs, DCs, Neutrophils, macrophages, NK cells, pDCs, Tfh, T helper cells, TIL and Tregs were obvious elevated with the decreased risk score in training and validation cohorts. And some immune signatures were significantly activated with the decreased risk score in both cohorts. Eventually, the exosome-associated lncRNAs risk model was demonstrated to accurately predict immunotherapy response in patients with BC. The results of our study suggest that exosome-related lncRNAs risk model has close relationship with prognosis and immune cells infiltration in BC patients. These findings could make a great contribution to improving BC immunotherapy.
... Exosomes can be secreted by all kinds of cells to produce the extracellular vesicles with diameters of 30 to 200 nm, which transfer diverse biological molecules such as lipids, proteins and nucleic acids with specific functions [5][6][7]. The function of exosomes tightly depends on cell types from which they are derived. ...
Many evidences show that exosomes play an important role in cancer development, invasion and metastasis. This study is based on the need to explore exosomal protein that promote breast cancer metastasis. We found that tyrosine kinase EphA2 was enriched in Triple-negative breast cancer -derived exosomes and it could disrupt the endothelial monolayer barrier through downregulating tight junction proteins of endothelial cells. These mechanisms were confirmed by in vivo experiments. After periodical injection of exosomal EphA2 into mice caudal vein, we found increased vascular permeability and breast cancer metastases in distant organs, and this phenomenon decreased dramatically after exosomal EphA2 knockdown. This study provides a new mechanism of exosome promoting breast cancer metastasis and suggests a new therapeutic target for the prevention and treatment of breast cancer metastasis.
... It has been demonstrated that tumor-derived sEVs contain proteins and nucleic acids cargo molecules that promoted BC cells proliferation. Nischarin is an integrin a5b1-binding protein, the work by Maziveyi et al. found that sEVs enriched with Nischarin-positive cells promoted in situ BC cells migration and tumor growth in vivo (68). Another recent study in BC was done by Li et al. showed that sEVs-derived miR-1246 is highly expressed in metastatic BC (MDA-MB-231) cells compared to non-metastatic or non-malignant BC cells, and that it promoted BC cells proliferation by targeting cell cycle protein-G2 (CCNG2) (69). ...
Breast cancer (BC) is the most common malignancy and the leading cause of cancer-related deaths in women worldwide. Currently, patients’ survival remains a challenge in BC due to the lack of effective targeted therapies and the difficult condition of patients with higher aggressiveness, metastasis and drug resistance. Small extracellular vesicles (sEVs), which are nanoscale vesicles with lipid bilayer envelopes released by various cell types in physiological and pathological conditions, play an important role in biological information transfer between cells. There is growing evidence that BC cell-derived sEVs may contribute to the establishment of a favorable microenvironment that supports cancer cells proliferation, invasion and metastasis. Moreover, sEVs provide a versatile platform not only for the diagnosis but also as a delivery vehicle for drugs. This review provides an overview of current new developments regarding the involvement of sEVs in BC pathogenesis, including tumor proliferation, invasion, metastasis, immune evasion, and drug resistance. In addition, sEVs act as messenger carriers carrying a variety of biomolecules such as proteins, nucleic acids, lipids and metabolites, making them as potential liquid biopsy biomarkers for BC diagnosis and prognosis. We also described the clinical applications of BC derived sEVs associated MiRs in the diagnosis and treatment of BC along with ongoing clinical trials which will assist future scientific endeavors in a more organized direction.
... e expression of NISCH is closely related to tumorigenesis, progression, and poor prognosis in cancers, including breast, lungs, and ovarian cancers [22,24,37,38]. A series of publications reported that NISCH was a tumor-suppressive Journal of Oncology factor in breast cancer, and NISCH-expressing cells delivered exosomes that suppress breast cancer cell growth and mobility [21,37,39,40]. In ovarian cancer, promoter hypermethylation-induced NISCH silence-promoted cell cycle progression through the focal adhesion kinase (FAK) signal [22]. ...
Background. MicroRNAs (miRNAs) have emerged as crucial regulators in various cancers. However, the potential role of miR-2355-5p in head and neck squamous cell carcinoma (HNSC) remains unclear. Methods. Bioinformatics methods were implemented to find the candidate target gene of miR-2355-5p. Quantitative real-time PCR was performed to detect RNA expression levels of miR-2355-5p and NISCH, while western blot was carried out for the detection of protein levels of NISCH and cell cycle-related biomarkers. CCK-8, EdU staining, and flow cytometry were employed to measure cell proliferation and cell cycle progression. Dual-luciferase assay and RNA pulldown were conducted to verify the binding relationship between miR-2355-5p and NISCH. Results. The expression levels of miR-2355-5p and NISCH were, respectively, higher and lower in HNSC tissues than those in normal adjacent tissues. The transfection of the miR-2355-5p inhibitor suppressed cell proliferation by arresting the cells at the G1/S transition. The results of luciferase activity and RNA pulldown assays indicated that miR-2355-5p directly targeted the NISCH 3′-untranslated region. Furthermore, the effects of miR-2355-5p inhibition on cell proliferation were reversed after treatment with siRNA against NISCH. Conclusion. In summary, our findings indicate that miR-2355-5p promotes cell cycle progression in HNSC by targeting NISCH. Hence, targeting miR-2355-5p could be a promising therapeutic strategy for the treatment of HNSC
... Exosomes derived from tumors promote angiogenesis and coagulation, regulate the immune system, reshape the surrounding parenchyma, and jointly support tumor progression (8,9). These exosomes participate in many cellular functions and are considered important cellular communication connectors as they contain various proteins and nucleic acids, including miRNAs and lncRNAs (10,11). ...
Background:
Breast cancer (BC) is a common tumor that seriously affects women's physical/mental health and even life. BC invasion and metastasis are still the main causes of mortality in BC patients. Exosomal long non-coding RNAs (exo-lncRNA) play an important role in cell communication and can help to understand better the physiological and pathological conditions that result from BC. This study investigates new potential targets and functions of the expression profiles of exo-lncRNAs in BC patients through high-throughput screening and bioinformatics.
Methods:
Samples were collected from two BC patients and one healthy subject. The serum exosomal RNAs were subsequently purified, and a library was established for quality inspection and sequencing. The resultant data was compared with the reference data to obtain the differential expression of exo-lncRNAs, and predict the target genes. To obtain the final results, Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were used to annotate the function and pathway of the differentially expressed genes.
Results:
After a comprehensive comparison of the BC patients and healthy subjects, we discovered five up-regulated exo-lncRNAs and six down-regulated exo-lncRNAs of interest. Combining our results with a literature review and screening, we found that VIM-AS1, SNHG8, and ELDR play a role in the progression of BC, with VIM-AS1 predicting 35 target miRNAs; SNHG8 predicting 12 target miRNAs, and ELDR predicting 24 target miRNAs. Target prediction considered that the target gene of VIM-AS1 was VIM and that the target gene of SNHG8 was PRSS12. GO enrichment analysis showed that VIM mainly played a role in cell processes, biological regulation, metabolic regulation, and molecular adhesion, while PRSS12 was enriched through cell metabolism, catalytic activity, and hydrolase activity. KEGG pathway enrichment results also indicated how the VIM protein functions in cancer development through the viral infection signaling pathway and miRNA signaling pathway.
Conclusions:
There is a significant difference in the expression profiles of serum exo-lncRNAs between BC patients and healthy individuals. This may be closely related to BC's occurrence, development, and metastasis, and therefore provides a theoretical basis for more in-depth studies into exo-lncRNA.
... When corruption becomes systemic, multiple individuals can work together in complementary roles and collude to garner resources and subvert the signaling environment. Cancer cells, both within and among tumors, can "collude" by exchanging information, such as RNA, DNA, and proteins, through exosomes and other mechanisms (Li et al., 2006;Hough et al., 2017;Maziveyi et al., 2019). Although far from fully understood, the proteins, metabolites, and nucleic acids delivered in this way are thought to facilitate survival, differentiation, and proliferation, promote angiogenesis and wound healing, contribute to metastasis and migration, and reprogram metabolic profiles of receiving cells (Kalluri and LeBleu, 2020). ...
All biological systems depend on signals for coordination: signals which pass information among agents that run the gamut from cells to organisms. However, their very importance makes signals vulnerable to subversion. How can a receiver know whether a signal is honest or deceptive? In other words, are signals necessarily a reliable indicator of agent quality or need? By drawing parallels to ecological phenomena ranging from begging by nestlings to social insects, we investigate the role of signal degradation in cancer. We thus think of cancer as a form of corruption, in which cells command huge resource investment through relatively cheap signals, just as relatively small bribes can leverage large profits. We discuss various mechanisms which prevent deceptive signaling in the natural world and within tissues. We show how cancers evolve ways to escape these controls and relate these back to evasion mechanisms in ecology. We next introduce two related concepts, co-option and collusion, and show how they play critical roles in ecology and cancer. Drawing on public policy, we propose new approaches to view treatment based on taxation, changing the incentive structure, and the recognition of corrupted signaling networks.
... Various of small GTPases have been implicated in exosome biogenesis and secretion. Specifically, Ras-related (Ral) proteins (Hyenne et al., 2015), Rab7, Rab27a (Dorayappan et al., 2018), and RhoA (Li et al., 2012) contribute to the formation of MVBs; Rab31 drives MVB formation but suppresses their degradation simultaneously (Wei et al., 2021); Rab14 positively modulates the MVB diameter and number (Maziveyi et al., 2019). Additionally, Cdc42 and Rac1 promote MVB maturation (Kajimoto et al., 2018); and Rab7 mediates the transportation of MVBs (Sun et al., 2020). ...
Membrane trafficking is critical for cellular homeostasis, which is mainly carried out by small GTPases, a class of proteins functioning in vesicle budding, transport, tethering and fusion processes. The accurate and organized membrane trafficking relies on the proper regulation of small GTPases, which involves the conversion between GTP- and GDP-bound small GTPases mediated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Emerging evidence indicates that post-translational modifications (PTMs) of small GTPases, especially ubiquitination, play an important role in the spatio-temporal regulation of small GTPases, and the dysregulation of small GTPase ubiquitination can result in multiple human diseases. In this review, we introduce small GTPases-mediated membrane trafficking pathways and the biological processes of ubiquitination-dependent regulation of small GTPases, including the regulation of small GTPase stability, activity and localization. We then discuss the dysregulation of small GTPase ubiquitination and the associated human membrane trafficking-related diseases, focusing on the neurological diseases and infections. An in-depth understanding of the molecular mechanisms by which ubiquitination regulates small GTPases can provide novel insights into the membrane trafficking process, which knowledge is valuable for the development of more effective and specific therapeutics for membrane trafficking-related human diseases.
... Exosomes derived from cancer cells encapsulate various kinds of tumor-specific molecules, such as nucleic acid and proteins (7,8). Therefore, they can reflect biological characteristics of parental cells and interact with adjacent or distant cells to mediate information exchange (9)(10)(11)(12). Recently, a number of studies have revealed that long noncoding RNAs (lncRNAs) are enriched and more stable in exosome, playing the crucial role in tumorigenesis and tumor development. ...
Background
Exosomes derived from cancer cells encapsulate various kinds of tumor-specific molecules and thus can interact with adjacent or distant cells to mediate information exchange. Long non-coding RNAs (lncRNAs) in exosomes have the potential as diagnostic and prognostic biomarkers in different types of cancers. The current study was aimed to identify circulating exosomal lncRNAs for the diagnosis of colorectal cancer (CRC).
Methods
Exosomes were isolated from the serum by ultracentrifugation and verified by transmission electron microscope (TEM), qNano, and immunoblotting. Exosomal lncRNAs FOXD2-AS1, NRIR, and XLOC_009459 were selected by lncRNA microarray and validated by qPCR in 203 CRC patients and 201 healthy donors. The receiver operating characteristic curve (ROC) was used to assess the diagnostic efficiency of serum exosomal lncRNAs.
Results
Exosomal FOXD2-AS1, NRIR, and XLOC_009459 (TCONS_00020073) levels were significantly upregulated in 203 CRC patients and 80 early-stage CRC patients compared to 201 healthy donors, possessing the area under the curve (AUC) of 0.728, 0.660, and 0.682 for CRC, as well as 0.743, 0.660, and 0.689 for early-stage CRC, respectively. Notably, their combination demonstrated the markedly elevated AUC of 0.736 for CRC and 0.758 for early-stage CRC, indicating their potential as diagnostic biomarkers for CRC.
Conclusions
Our data suggested that exosomal lncRNAs FOXD2-AS1, NRIR, and XLOC_009459 act as the promising biomarkers for the diagnostics of CRC and early-stage CRC.
... La combinaison la plus efficace dans notre étude, en thérapeutique comme en prophylactique, est celle du composé IP2 avec le SLP MCA205 KB-1. L'épitope ciblé est le nonapeptide TNQDFIQRL produit à partir d'un exon du gène codant pour la nischarine (NISCH602-608) Maziveyi et al., 2019). Il est présenté à un niveau basal par les cellules MCA205 mais il est enrichi plus de cent fois à la surface cellulaire après traitement avec IP2. ...
Le succès des vaccins thérapeutiques contre le cancer repose sur l’établissement d’une réponse cellulaire adaptative efficace dirigée contre les antigènes présentés par les cellules tumorales dans les molécules du CMH. Les antigènes ciblés sont des antigènes associés à la tumeur (TAAs) ou des néoantigènes spécifiques de la tumeur (TSAs) qui peuvent être produits à partir des séquences exoniques du génome mais également des séquences présumées non-codantes par des évènements de traduction non-canoniques. Les Pioneer Translation Products (PTPs) sont par exemple produits par un événement de traduction non-conventionnelle se déroulant dans le compartiment nucléaire sur des ARNm pré-matures avant l’épissage des introns. Nous avons mis en évidence les propriétés immunomodulatrices de l’inhibiteur de l’épissage isoginkgetine et de son dérivé hydrosoluble et non toxique, IP2. Le composé IP2 augmente la présentation des peptides dérivés des PTPs in-vitro et exerce une activité antitumorale dépendante du système immunitaire in-vivo. Nous avons identifié par spectrométrie de masse les TAAs et les TSAs induits par IP2 à la surface des cellules tumorales. Nous avons trouvé des antigènes dérivant de séquences exoniques mais également d’introns retenus. Nous avons évalué l’immunogénicité de ces peptides ex-vivo avant de tester leurs propriétés antitumorales in-vivo dans le cadre de stratégies de vaccination prophylactique et thérapeutique. Finalement nous proposons une nouvelle immunothérapie antitumorale qui consiste à combiner le composé immunomodulateur IP2 qui augmente la présentation antigénique par les cellules tumorales avec un vaccin peptidique qui stimule la prolifération de LT CD8+ cytotoxiques spécifiques des antigènes tumoraux.
... The exon-derived TL9 epitope from the tumor suppressor nischarin was shown to be immunogenic and to synergize with IP2 in cancer vaccines to inhibit the growth of MCA205 fibrosarcoma. Recent studies highlighted the role of nischarin in the regulation of breast tumorigenesis 56 related to the secretion of exosomes from breast cancer cells 57 . The nischarin-derived TL9 epitope has been identified throughout multiple proteomic studies referenced in the Immune Epitope Database (IEDB) but has never been further exploited so far. ...
The success of cancer immunotherapy relies on the induction of an immunoprotective response targeting tumor antigens (TAs) presented on MHC-I molecules. We demonstrated that the splicing inhibitor isoginkgetin and its water-soluble and non-toxic derivative IP2 act at the production stage of the pioneer translation products (PTPs). We showed that IP2 increases PTP-derived antigen presentation in cancer cells in vitro and impairs tumor growth in vivo. IP2 action is long-lasting and dependent on the CD8⁺ T cell response against TAs. We observed that the antigen repertoire displayed on MHC-I molecules at the surface of MCA205 fibrosarcoma is modified upon treatment with IP2. In particular, IP2 enhances the presentation of an exon-derived epitope from the tumor suppressor nischarin. The combination of IP2 with a peptide vaccine targeting the nischarin-derived epitope showed a synergistic antitumor effect in vivo. These findings identify the spliceosome as a druggable target for the development of epitope-based immunotherapies.
... Exosomes are critical cellular connectors containing a variety of proteins and nucleic acids including miRNAs and lncRNAs, and they participate in many cellular functions [125,126]. Accumulating evidence indicated that signals transmitted by exosomal lncRNAs regulate many types of local and distant recipient cells. Moreover, there are differences in levels and types of exosome-mediated transferring pathways of lncRNAs in normal physiological situations and pathological conditions such as cancer. ...
Breast cancer is the most common cancer, and the second cause of cancer-related deaths (after lung cancer) among women. Developing tumor metastasis and invasion is the most important cause of death in breast cancer patients. Several key factors participate in breast cancer metastasis including long noncoding RNAs (lncRNAs). lncRNAs are a category of cellular RNAs that are longer than 200 nucleotides in length. Accumulating evidence suggests that lncRNAs have the potential to be promising diagnostic, prognostic biomarkers and therapeutic targets in breast cancer. Understanding the role of lncRNAs and their mechanisms of functions might help to further discovery of breast cancer biological characteristics. In this review, we discuss physiological functions, epigenetic regulation, transcriptional regulation of lncRNAs, and their important role in tumor progression and metastasis. Some lncRNAs function as oncogenes and some function as tumor suppressors. Interestingly, recent reports depict that hypomethylation of promoters of lncRNAs play a pivotal role in cancer progression, suggesting the importance of epigenetic regulation. Furthermore, we discuss the role of lncRNAs in exosomes and their function in drug resistance, and therapeutic importance of exosomal lncRNAs in cancer biology. In summary, lncRNAs have a great potential to consider them as novel prognostic biomarkers as well as new therapeutic targets in breast cancer
... Consistent with this, we showed that exosomes prepared from Nischarin ablated tissues promote breast tumor growth. 37 To examine the effect of Nischarin ablation on mammary cancer progression, breast tumor growth in Nc −/− -PyMT and Nc +/+ -PyMT was measured every 3 days after tumor onset. The change in tumor volume (and tumor burden) was significantly higher in Nc −/− -PyMT tumors compared to WT tumors. ...
Previously, our lab discovered the protein Nischarin and uncovered its role in regulating cell migration and invasion via its interactions with several proteins. We subsequently described a role for Nischarin in breast cancer, in which it is frequently underexpressed. To characterize Nischarin's role in breast tumorigenesis and mammary gland development more completely, we deleted a critical region of the Nisch gene (exons 7–10) from the mouse genome and observed the effects. Mammary glands in mutant animals showed delayed terminal end bud formation but did not develop breast tumors spontaneously. Therefore, we interbred the animals with transgenic mice expressing the mouse mammary tumor virus‐polyoma middle T‐antigen (MMTV‐PyMT) oncogene. The MMTV‐PyMT mammary glands lacking Nischarin showed increased hyperplasia compared to wild‐type animal tissues. Furthermore, we observed significantly increased tumor growth and metastasis in Nischarin mutant animals. Surprisingly, Nischarin deletion decreased activity of AMPK and subsequently its downstream effectors. Given this finding, we treated these animals with metformin, which enhances AMPK activity. Here, we show for the first time, metformin activates AMPK signaling and inhibits tumor growth of Nischarin lacking PyMT tumors suggesting a potential use for metformin as a cancer therapeutic, particularly in the case of Nischarin‐deficient breast cancers.
Objective: This study was aimed at exploring a specific open region of chromatin in the peripheral blood mononuclear cells (PBMCs) of patients with breast cancer and evaluating its feasibility as a biomarker for diagnosing and predicting breast cancer prognosis.
Methods: We obtained PBMCs from breast cancer patients and healthy people for the assay for transposase-accessible chromatin (ATAC) sequencing (n = 3) and obtained the GSE27562 chip sequencing data for secondary analyses. Through bioinformatics analysis, we mined the pattern changes for chromatin accessibility in the PBMCs of breast cancer patients.
Results: A total of 1,906 differentially accessible regions (DARs) and 1,632 differentially expressed genes (DEGs) were identified via ATAC sequencing. The upregulated DEGs in the disease group were mainly distributed in the cells, organelles, and cell-intima-related structures and were mainly responsible for biological functions such as cell nitrogen complex metabolism, macromolecular metabolism, and cell communication, in addition to functions such as nucleic acid binding, enzyme binding, hydrolase reaction, and transferase activity. Combined with microarray data analysis, the following set of nine DEGs showed intersection between the ATAC and microarray data: JUN, MSL2, CDC42, TRIB1, SERTAD3, RAB14, RHOB, RAB40B, and PRKDC. HOMER predicted and identified five transcription factors that could potentially bind to these peak sites, namely NFY, Sp 2, GFY, NRF, and ELK 1.
Conclusion: Chromatin accessibility analysis of the PBMCs from patients with early-stage breast cancer underscores its potential as a significant avenue for biomarker discovery in breast cancer diagnostics and treatment. By screening the transcription factors and DEGs related to breast cancer, this study provides a comprehensive theoretical foundation that is expected to guide future clinical applications and therapeutic developments.
Extracellular vesicles (EVs) are nano-sized, membranous structures secreted into the extracellular space. They exhibit diverse sizes, contents, and surface markers and are ubiquitously released from cells under normal and pathological conditions. Human serum is a rich source of these EVs, though their isolation from serum proteins and non-EV lipid particles poses challenges. These vesicles transport various cellular components such as proteins, mRNAs, miRNAs, DNA, and lipids across distances, influencing numerous physiological and pathological events, including those within the tumor microenvironment (TME). Their pivotal roles in cellular communication make EVs promising candidates for therapeutic agents, drug delivery systems, and disease biomarkers. Especially in cancer diagnostics, EV detection can pave the way for early identification and offers potential as diagnostic biomarkers. Moreover, various EV subtypes are emerging as targeted drug delivery tools, highlighting their potential clinical significance. The need for non-invasive biomarkers to monitor biological processes for diagnostic and therapeutic purposes remains unfulfilled. Tapping into the unique composition of EVs could unlock advanced diagnostic and therapeutic avenues in the future. In this review, we discuss in detail the roles of EVs across various conditions, including cancers (encompassing head and neck, lung, gastric, breast, and hepatocellular carcinoma), neurodegenerative disorders, diabetes, viral infections, autoimmune and renal diseases, emphasizing the potential advancements in molecular diagnostics and drug delivery.
Extracellular vesicles (EVs) are key mediators of cell-to-cell communication. Their content reflects the state of diseased cells representing a window into disease progression. Collagen-VI Related Muscular Dystrophy (COL6-RD) is a multi-systemic disease involving different cell types. The role of EVs in this disease has not been explored. We compared by quantitative proteomics the protein cargo of EVs released from fibroblasts from patients with COL6-RD and controls. Isolated EVs contained a significant proportion of the most frequently reported proteins in EVs according to Exocarta and Vesiclepedia. We identified 67 differentially abundant proteins associated with vesicle transport and exocytosis, actin remodelling and the cytoskeleton, hemostasis and oxidative stress. Treatment of control fibroblasts with EVs from either patient or healthy fibroblasts altered significantly the motility of cells on a cell migration assay highlighting the functional relevance of EVs. In parallel, we analysed the secretome from the same cells and found a distinctly different set of 48 differentially abundant proteins related to extracellular matrix organisation and remodelling, growth factor response, RNA metabolism and the proteasome. The EVs and secretome sets of proteins only shared two identifiers indicating that the sorting of proteins towards EVs or the secretory pathway is tightly regulated for different functions.
The limitations of traditional cancer treatments are driving the creation and development of new nanomedicines. At present, with the rapid increase of research on nanomedicine in the field of cancer, there is a lack of intuitive analysis of the development trend, main authors and research hotspots of nanomedicine in the field of cancer, as well as detailed elaboration of possible research hotspots. In this review, data collected from the Web of Science Core Collection database between January 1st, 2000, and December 31st, 2021, were subjected to a bibliometric analysis. The co-authorship, co-citation, and co-occurrence of countries, institutions, authors, literature, and keywords in this subject were examined using VOSviewer, Citespace, and a well-known online bibliometrics platform. We collected 19,654 published papers, China produced the most publications (36.654%, 7204), followed by the United States (29.594%, 5777), and India (7.780%, 1529). An interesting fact is that, despite China having more publications than the United States, the United States still dominates this field, having the highest H-index and the most citations. Acs Nano, Nano Letters, and Biomaterials are the top three academic publications that publish articles on nanomedicine for cancer out of a total of 7580 academic journals. The most significant increases were shown for the keywords "cancer nanomedicine", "tumor microenvironment", "nanoparticles", "prodrug", "targeted nanomedicine", "combination", and "cancer immunotherapy" indicating the promising area of research. Meanwhile, the development prospects and challenges of nanomedicine in cancer are also discussed and provided some solutions to the major obstacles.
Extracellular vesicles (EVs) are lipid-based nanosized particles that convey biological material from donor to recipient cells. EVs play key roles in glioblastoma progression, as Glioblastoma Stem-like Cells (GSCs) releases pro-oncogenic, pro-angiogenic, and pro-inflammatory EVs. However, the molecular basis of EV release remains poorly understood. Here, we report the identification of the pseudokinase MLKL, a crucial effector of cell death by necroptosis, as a regulator of the constitutive secretion of EVs in GSCs. We find that genetic, protein, and pharmacological targeting of MLKL alters intracellular trafficking and EV release, and reduces GSC expansion. Nevertheless, this function ascribed to MLKL appears independent of its role during necroptosis. In vivo, pharmacological inhibition of MLKL reduces the tumor burden and the level of plasmatic EVs. This work highlights the necroptosis-independent role of MLKL in vesicle release and suggests that interfering with EV is a promising therapeutic option to sensitize glioblastoma cells.
Increasing evidences show that unmodified extracellular vesicles (EVs) derived from various cells can effectively inhibit the malignant progression of different types of tumors by delivering the bioactive molecules. Therefore, EVs are expected to be developed as emerging anticancer drugs. Meanwhile, unmodified EVs as an advanced and promising nanocarrier that is frequently used in targeted delivery therapeutic cargos and personalized reagents for the treatment and diagnosis of cancer. To improve the efficacy of EV-based treatments, researchers are trying to engineering EVs as an emerging nanomedicine translational therapy platform through biological, physical and chemical approaches, which can be broaden and altered to enhance their therapeutic capability. EVs loaded with therapeutic components such as tumor suppressor drugs, siRNAs, proteins, peptides, and conjugates exhibit significantly enhanced anti-tumor effects. Moreover, the design and preparation of tumor-targeted modified EVs greatly enhance the specificity and effectiveness of tumor therapy, and these strategies are expected to become novel ideas for tumor precision medicine. This review will focus on reviewing the latest research progress of functionalized EVs, clarifying the superior biological functions and powerful therapeutic potential of EVs, for researchers to explore new design concepts based on EVs and build next-generation nanomedicine therapeutic platforms.
Background: Breast cancer (BC) is one of the most frequent malignancies among women worldwide. Accumulating evidence indicates that long non-coding RNA (lncRNA) may affect BC progression. Exosomes, a class of small membrane vesicles, have been reported to promote tumor progression through transporting proteins, mRNAs, lncRNAs and so on. However, the interaction between exosome-related lncRNAs and the microenvironment of malignancies is unclear. Hence, we proceeded to investigate the relationship between exosome-related lncRNAs and BC microenvironment.
Method: 121 exosome-associated genes were extracted from ExoBCD database. Then, the Pearson analysis was used to screened out the exosome-related lncRNAs. After that, 15 exosome-related differentially expressed lncRNAs were identified by the correlation with BC prognosis. According to the sum of the expression of these 15 lncRNAs, extracted from The Cancer Genome Atlas (TCGA), and the regression coefficients, an exosome-related lncRNAs signature was developed by using Cox regression analysis. With the median risk score of the training set, the patients in training and validation sets were separated to low-risk group and high-risk group. Subsequently, the lncRNA–mRNA co-expression network was constructed. The distinct enrichment pathways were compared among the different risk groups by using the R package clusterProfiler. The ESTIMATE method and ssGESA database were adopted to study the ESTIMATE Score and immune cell infiltration. Eventually, the expression of immune checkpoint associated genes, microsatellite instable (MSI) and the immunophenoscore (IPS) were further analyzed between different risk groups.
Results: Different risk groups exhibited different prognosis, with lower survival rate in the high-risk group. The differentially expressed genes (DEGs) between the different risk groups were enriched in biological processes pathways as well as immune responses. BC patients in high-risk group were identified with lower scores of ESTIMATE scores. Subsequently, we noticed that the infiltrating levels of aDCs, B cells, CD8+ T cells, iDCs, DCs, Neutrophils, macrophages, NK cells, pDCs, Tfh, T helper cells, TIL and Tregs were obvious elevated with the decreased risk score in training and validation cohorts. And some immune signatures were significantly activated with the decreased risk score in both cohorts. Eventually, the exosome-associated lncRNAs risk model was demonstrated to accurately predict immunotherapy response in patients with BC.
Conclusion: The results of our study suggest that exosome-related lncRNAs risk model has close relationship with prognosis and immune cells infiltration in BC patients. These findings could make a great contribution to improving BC immunotherapy.
Cancer is one of the leading causes of death worldwide, and the factors responsible for its progression need to be elucidated. Exosomes are structures with an average size of 100 nm that can transport proteins, lipids, and nucleic acids. This review focuses on the role of exosomes in cancer progression and therapy. We discuss how exosomes are able to modulate components of the tumor microenvironment and influence proliferation and migration rates of cancer cells. We also highlight that, depending on their cargo, exosomes can suppress or promote tumor cell progression and can enhance or reduce cancer cell response to radio- and chemo-therapies. In addition, we describe how exosomes can trigger chronic inflammation and lead to immune evasion and tumor progression by focusing on their ability to transfer non-coding RNAs between cells and modulate other molecular signaling pathways such as PTEN and PI3K/Akt in cancer. Subsequently, we discuss the use of exosomes as carriers of anti-tumor agents and genetic tools to control cancer progression. We then discuss the role of tumor-derived exosomes in carcinogenesis. Finally, we devote a section to the study of exosomes as diagnostic and prognostic tools in clinical courses that is important for the treatment of cancer patients. This review provides a comprehensive understanding of the role of exosomes in cancer therapy, focusing on their therapeutic value in cancer progression and remodeling of the tumor microenvironment.
Graphical Abstract
Nischarin has been demonstrated to have tumor suppressor functions. In this review, we comprehensively discuss up to date information about Nischarin. In addition, this paper aims to report the prognostic value, clinical relevance, and biological significance of the Nischarin gene (NISCH) in breast cancer (BCa) patients using the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) and The Cancer Genome Atlas (TCGA) datasets. We evaluated NISCH gene expression and its correlation to patient survival, baseline expression, and expression variation based on age groups, tumor stage, tumor size, tumor grade, and lymph node status in different subtypes of BCa. Since NISCH has been extensively reported to inhibit EMT and cancer cell migration, we also checked for the correlation between NISCH and EMT genes in addition to the correlation between NISCH and cell migration genes. Our results indicate that NISCH is a tumor suppressor that plays a critical role in BCa initiation, progression, and tumor development. We find that there is a higher level of NISCH expression in normal breast tissues compared to breast cancer tissues. Also, aggressive subtypes of breast cancers, such as the triple negative/basal category, have decreased levels of NISCH as the disease progresses. Finally, we report that NISCH is inversely correlated with many EMT and cancer cell migration genes in BCa. Interestingly, we identified a significant negative correlation between NISCH expression and its methylation in breast cancer patients. Overall, the goal of this report is to establish a strong clinical basis for further investigation into the cellular, molecular, and physiological roles of NISCH in BCa. Ultimately, NISCH gene expression might be clinically harnessed as a biomarker or predictor of invasiveness and metastasis in BCa. Oncogene; https://doi.
Breast cancer (BC) is reported to be the leading cause of mortality in females worldwide. At the beginning of the year 2021, about 7.8 million women were diagnosed with BC in past 5 years. High prevalence and poor neoadjuvant chemotherapeutic efficacy has motivated the scientists around the globe to investigate alternative management strategies. In recent years, there has been an exponential rise in the scientific studies reporting the role of tumor derived exosomes (TDEs) in the BC pathophysiology and management. TDEs play an important role in the intercellular communication and transportation of biomolecules. This manuscript reviews the role of exosomes in the BC pathophysiology, diagnosis, and therapy. Role of TDEs in the mechanistic pathways of BC metastasis, immunosuppression, migration, dormancy and chemo-resistance is extensively reviewed. We have also highlighted the epigenetic modulations orchestrated by exosomal miRNAs and long noncoding RNAs (lnc RNAs) in the BC environment. Liquid biopsies analyzing blood circulating exosomes for early and accurate detection of the BC have been discussed. Characterization of exosomes, strategies to use them in BC chemotherapy, BC immunotherapy and potential challenges that will present themselves in translating exosomes based technologies to market are discussed.
Breast cancer (BC) develops from breast tissue and is the most common aggressive malignant tumor in women worldwide. Although advanced treatment strategies have been applied and reduced current mortality rates, BC control remains unsatisfactory. It is essential to elucidate the underlying molecular mechanisms to assist clinical options. Exosomes are a type of extracellular vesicles and mediate cellular communications by delivering various biomolecules (oncogenes, oncomiRs, proteins, and even pharmacological compounds). These bioactive molecules can be transferred to change the transcriptome of target cells and influence tumor-related signaling pathways. Extensive studies have implicated exosomes in BC biology, including therapeutic resistance and the surrounding microenvironment. This review focuses on discussing the functions of exosomes in tumor treatment resistance, invasion and metastasis of BC. Moreover, we will also summarize multiple interactions between exosomes and the BC tumor microenvironment. Finally, we propose promising clinical applications of exosomes in BC.
Exosomes are natural lipid bilayer nanovesicles of the cell-endosomal compartment which serve as an emerging therapeutic target of various cancers and novel agents for cancer diagnosis. Exosomes are well-known as cell-cell communicators via transferring biological active messages between cells. Since exosomes derived from tumor cells contain oncogenic, metastatic, and angiogenic components of their parent tumor cells and resemble their characteristics, several studies considered exosomes as a novel cancer therapeutic target in the tumor microenvironment along with their application in cancer diagnosis. Evidence indicated that exosomes could potentiate the progression and metastasis of several cancers such as melanoma and breast and liver cancers. So, mediating exosome secretion or content by chemicals and gene therapy approaches would propose a new strategy for restraining cancer progression. Owing to the existence of exosomes in all bodily fluids including urine, saliva, plasma, and cerebrospinal fluids and their stability against cellular degradation on the one hand and therefore their easy, accessible, and noninvasive sources of sampling on the other hand, exosomes are also considered as valuable cancer biomarkers. Recent studies showed that nucleic acid composition of exosomes such as microRNAs and exosomal surface proteins could be utilized as cancer biomarkers. Usage of exosomes in cancer diagnosis and prognosis has not only attracted immense attention from researchers but also entered the production pipelines of industrial companies. For example, ExoDx Prostate (Intelliscore) manufactured by ExosomeDx Company is a simple exosomal urine-based prostate cancer test that can discriminate against high-grade prostate cancer cells.
Nanotechnology is playing a very vital role in the medical field. Nanoparticles (NPs) are utilized in the field of nanomedicine which partakes in diagnosis and therapy of different diseases including cancer. The interesting distinguishing features of NPs, for example, are their tall exterior to volume proportion which empowers them to stalemate, absorb, and convey small biomolecule like RNA, DNA, proteins, drugs, and dissimilar atoms to embattled site and consequently improves the adequacy of therapeutic agents. In this chapter, common principles of drug target to malignant growth, intracellular mechanism, NP-based detailing in market, and ongoing application in medication as diagnostic and therapeutic aspects are examined based on bibliographic information, as reported in research article on nanotechnology. These features offer different methodologies of tumor nanotechnology in the progression of cancer therapy.
Exosomes are a type of nano-scale biofilm-enclosed vesicles involved in intercellular communications, which are actively secreted by almost all eukaryotes and have been suggested to play an important role in various biological and physiological processes. Since exosomes carry biological contents informative of the original cells and can be easily harvested from body fluids, they are regarded as an emerging and promising biomarker bank for liquid biopsy. Conventional exosome detection methods such as transmission electron microscopy, nanoparticle tracking analysis, and Western blotting are cumbersomely conducted with partial information and limited in sensitivity and specificity. Thus new analytical approaches are urgently needed to facilitate exosome-based in vitro early diagnosis of diseases. Herein, we summarized recent advances in the development of immunoassay-based technologies for rapid exosome detection which have sparked huge interest in the last few years. A brief introduction of exosomes and general principles of immunoassays was first presented. We then described in some detail the studies on four major types of immunoassay technologies incorporated with specific configurations for exosome quantification as well as surface protein profiling, including single immunosensors, immunosensor arrays, immunoassays integrated with microfluidics, and paper-based immunoassays. Challenges and future research perspectives were also discussed in the field of exosome immunoassay detection.
Background:
Exosomes are involved in intercellular communication via specialized molecular cargo, such as microRNAs (miRNAs). However, the mechanisms underlying exosomal miR-19b-1-5p in bladder cancer remain largely unknown, thus, we aim to investigate the effect of exosomal miR-19b-1-5p on bladder cancer with the involvement of non-receptor protein tyrosine kinase Arg (ABL2).
Methods:
miR-19b-1-5p and ABL2 expression were tested in bladder cancer. miR-19b-1-5p inhibition/elevation assays were conducted to determine its role in bladder cancer. Exosomes were extracted from bone marrow mesenchymal stem cells (BMSCs). Exosomes and T24 cells were co-cultured to verify their function in biological characteristics of bladder cancer cells.
Results:
miR-19b-1-5p was down-regulated while ABL2 was upregulated in bladder cancer. Exosomal miR-19b-1-5p suppressed malignant behaviors of bladder cancer cells, and also inhibited tumor growth in vivo. Up-regulated ABL2 mitigated the effects of miR-19b-1-5p up-regulation on bladder cancer cells.
Conclusion:
BMSCs-derived exosomal miR-19b-1-5p suppresses bladder cancer growth via decreasing ABL2.
Exosomes, as cell-cell communicators with an endosomal origin, are involved in the progression of various diseases. RAB5A, a member of the small Rab GTPases family, which is well known as a key regulator of cellular endocytosis, is expected to be involved in exosome secretion. Here, we found the impact of RAB5A on exosome secretion from human hepatocellular carcinoma cell line using a rapid yet reliable bioinformatics approach followed by experimental analysis. Initially, RAB5A and exosome secretion-related genes were gathered from bioinformatics tools, namely, CTD, COREMINE, and GeneMANIA; and published papers. Protein-protein interaction (PPI) was then constructed by the Search Tool for Retrieval of Interacting Genes (STRING) database. Among them, several genes with different combined scores were validated by the real-time quantitative polymerase chain reaction (RT-qPCR) in stable RAB5A knockdown cells. Thereafter, to validate the bioinformatics results functionally, the impact of RAB5A knockdown on exosome secretion was evaluated. Bioinformatics analysis showed that RAB5A interacts with 37 genes involved in exosome secretion regulatory pathways. Validation by RT-qPCR confirmed the association of RAB5A with candidate interacted genes and interestingly showed that even medium to low combined scores of the STRING database could be experimentally valid. Moreover, the functional analysis demonstrated that the stable silencing of RAB5A could experimentally decrease exosome secretion. In conclusion, we suggest RAB5A as a regulator of exosome secretion based on our bioinformatics approach and experimental analysis. Also, we propose the usage of PPI-derived from the STRING database regardless of their combined scores in advanced bioinformatics analysis.
Objective:
To investigate the effects of miR-671-3p on the proliferation and invasion of breast cancer cells and explore the possible mechanism.
Methods:
We examined the expressions of miR-671-3p in human normal epithelial cells (MCF-10A) and breast cancer cell lines (MCF-7, MDA-MB-231, and SK-BR3) using RT-PCR. The effects of transfection with a miR-671-3p mimic or inhibitor on the proliferation, migration and invasion of MCF-7 cells were evaluated using CCK-8 assay and Transwell chamber assay. The target gene of miR-671-3p was predicated with Targetscan and validated by a dual luciferase reporter system and Western blotting.
Results:
The expression of miR-671-3p was significantly lower in breast cancer cells than in normal breast epithelial cells. Compared with negative control group, MCF-7 cells with miR-671-3p overexpression exhibited significantly reduced proliferation and invasion, whereas inhibition of miR-671-3p obviously promoted the cell proliferation and invasion. Luciferase reporter assay demonstrated that DEPTOR was the target gene of miR-671-3p, and miR-671-3p overexpression caused significant down-regulation of the protein expression of DEPTOR.
Conclusions:
MiR-671-3p suppresses the proliferation and invasion of breast cancer cell line MCF-7 by directly targeting DEPTOR protein.
With the development of oncology and bioanalytical chemistry, liquid biopsy emerges as their fruit and manages to bring perspective chance of healthcare. To date, several typical biomarkers (i.e circulating tumor cells, extracellular vesicles, circulating nuclei acids, etc) have been well-established as promising targets of liquid biopsy, and numerous methods, of which optical and electrochemical methods occupy in majority, have been proposed for the idealized detection of these targets. However, the advancements in this field are massive from cancer biology and analytical technology. Therefore, it is necessary to review these advancements from both medical and chemical sides. Here in this paper, our group, of which academic background from medicine and chemistry, aim to provide some comprehensive and in-depth summarization on contribution of optical and electrochemical methods to liquid biopsy, considering both the view of oncologist and analytical chemists. Besides, we will discuss the remaining challenges and future trend of this field in the end. We hope this review can be inspirational to researcher in this field and give helpful introduction to people from various research area.
The intercellular exchange of exosomes may play a regulatory function in tumor progression and metastasis. Maziveyi and colleagues demonstrated that Nischarin regulated the secretion of exosomes from breast cancer cells. Loss of Nischarin expression increased exosome production and promoted tumor cell growth and migration, supporting that Nischarin can influence the behavior of surrounding cancer cells. This study identified a novel function of the tumor suppressor Nischarin in exosome biology and cancer progression.
See related article by Maziveyi et al., p. 2152
Background
During metastasis, tumor cells move through the tracks of extracellular matrix (ECM). Focal adhesions (FAs) are the protein complexes that link the cell cytoskeleton to the ECM and their presence is necessary for cell attachment. The tumor suppressor Nischarin interacts with a number of signaling proteins such as Integrin α5, PAK1, LIMK1, LKB1, and Rac1 to prevent cancer cell migration. Although previous findings have shown that Nischarin exerts this migratory inhibition by interacting with other proteins, the effects of these interactions on the entire FA machinery are unknown. MethodsRT-PCR, Western Blotting, invadopodia assays, and immunofluorescence were used to examine FA gene expression and determine whether Nischarin affects cell attachment, as well as the proteins that regulate it. ResultsOur data show that Nischarin prevents cell migration and invasion by altering the expression of key focal adhesion proteins. Furthermore, we have found that Nischarin-expressing cells have reduced ability to attach the ECM, which in turn leads to a decrease in invadopodia-mediated matrix degradation. Conclusions
These experiments demonstrate an important role of Nischarin in regulating cell attachment, which adds to our understanding of the early events of the metastatic process in breast cancer.
Nischarin (Nisch) is a key protein functioning as a molecular scaffold and thereby hosting interactions with several protein partners. To explore the physiological importance of Nisch, here we generated Nisch loss-of-function mutant mice and analyzed their metabolic phenotype. Nisch mutant embryos exhibited delayed development, characterized by small size and attenuated weight gain. We uncovered the reason for this phenotype by showing that Nisch binds to and inhibits the activity of AMP-activated protein kinase (AMPK), which regulates energy homeostasis by suppressing anabolic and activating catabolic processes. The Nisch mutations enhanced AMPK activation and inhibited mechanistic target of rapamycin (mTOR) signaling in mouse embryonic fibroblasts (MEFs) as well as in muscle and liver tissues of mutant mice. Nisch-mutant mice also exhibited increased rates of glucose oxidation with increased energy expenditure, despite reduced overall food intake. Moreover, the Nisch-mutant mice had reduced expression of liver markers of gluconeogenesis associated with increased glucose tolerance. As a result, these mice displayed decreased growth and body weight. Taken together, our results indicate that Nisch is an important AMPK inhibitor and a critical regulator of energy homeostasis, including lipid and glucose metabolism.
Type 1 diabetes mellitus (T1DM) results from an autoimmune attack against the insulin-producing ß cells which leads to chronic hyperglycemia. Exosomes are lipid vesicles derived from cellular multivesicular bodies that are enriched in specific miRNAs, potentially providing a disease-specific diagnostic signature. To assess the value of exosome miRNAs as biomarkers for T1DM, miRNA expression in plasma-derived exosomes was measured. Nanoparticle tracking analysis and transmission electron microscopy confirmed the presence of plasma-derived exosomes (EXOs) isolated by differential centrifugation. Total RNA extracted from plasma-derived EXOs of 12 T1DM and 12 control subjects was hybridized onto Nanostring human v2 miRNA microarray array and expression data were analyzed on nSolver analysis software. We found 7 different miRNAs (1 up-regulated and 6 down-regulated), that were differentially expressed in T1DM. The selected candidate miRNAs were validated by qRT-PCR analysis of cohorts of 24 T1DM and 24 control subjects. Most of the deregulated miRNAs are involved in progression of T1DM. These findings highlight the potential of EXOs miRNA profiling in the diagnosis as well as new insights into the molecular mechanisms involved in T1DM.
Paxilllin is a multifunctional and multidomain focal adhesion adapter protein which serves an important scaffolding role at focal adhesions by recruiting structural and signaling molecules involved in cell movement and migration, when phosphorylated on specific Tyr and Ser residues. Upon integrin engagement with extracellular matrix, paxillin is phosphorylated at Tyr31, Tyr118, Ser188, and Ser190, activating numerous signaling cascades which promote cell migration, indicating that the regulation of adhesion dynamics is under the control of a complex display of signaling mechanisms. Among them, paxillin disassembly from focal adhesions induced by extracellular regulated kinase (ERK)-mediated phosphorylation of serines 106, 231, and 290 as well as the binding of the phosphatase PEST to paxillin have been shown to play a key role in cell migration. Paxillin also coordinates the spatiotemporal activation of signaling molecules, including Cdc42, Rac1, and RhoA GTPases, by recruiting GEFs, GAPs, and GITs to focal adhesions. As a major participant in the regulation of cell movement, paxillin plays distinct roles in specific tissues and developmental stages and is involved in immune response, epithelial morphogenesis, and embryonic development. Importantly, paxillin is also an essential player in pathological conditions including oxidative stress, inflammation, endothelial cell barrier dysfunction, and cancer development and metastasis.
Electronic supplementary material
The online version of this article (doi:10.1186/s13045-017-0418-y) contains supplementary material, which is available to authorized users.
Various in vitro and in vivo studies have linked mesenchymal stem cells (MSCs) with cancer, but little is known about the effect of MSCs on tumor progression. The present study aimed to analyze the role of the MSCs from different tissues, consisting of human bone marrow, adipose and the umbilical cord tissues, and the heterogeneity of tumors in tumor progression. By collecting the culture supernatants of MSCs as MSC-conditioned media (CMs), the present study found that MSC-CM produces no significant effect on the proliferation of MDA-MB-231 and A549 tumor cells. The migration of MDA-MB-231 cells was enhanced upon incubation with MSC-CM, while that of A549 cells was inhibited. Furthermore, the phosphorylation of insulin receptors (IRs) was upregulated in MSC-CM-treated MDA-MB-231 cells, while in MSC-CM-treated A549 cells, the phosphorylation of human epidermal growth factor receptor 3 (Her3) was downregulated. Taken together, the findings suggest that the phosphorylation of IR and Her3 may contribute to the discrepant effects of MSC-CM on the migration of the 2 cell lines.
Extracellular matrix adhesion is required for normal epithelial cell survival, nutrient uptake and metabolism. This requirement can be overcome by oncogene activation. Interestingly, inhibition of PI3K/mTOR leads to apoptosis of matrix-detached, but not matrix-attached cancer cells, suggesting that matrix-attached cells use alternate mechanisms to maintain nutrient supplies. Here we demonstrate that under conditions of dietary restriction or growth factor starvation, where PI3K/mTOR signalling is decreased, matrix-attached human mammary epithelial cells upregulate and internalize β4-integrin along with its matrix substrate, laminin. Endocytosed laminin localizes to lysosomes, results in increased intracellular levels of essential amino acids and enhanced mTORC1 signalling, preventing cell death. Moreover, we show that starved human fibroblasts secrete matrix proteins that maintain the growth of starved mammary epithelial cells contingent upon epithelial cell β4-integrin expression. Our study identifies a crosstalk between stromal fibroblasts and epithelial cells under starvation that could be exploited therapeutically to target tumours resistant to PI3K/mTOR inhibition.
Packed with biological information, extracellular vesicles (EVs) offer exciting promise for biomarker discovery and applications in therapeutics and non-invasive diagnostics. Currently, our understanding of EV contents is confined by the limited cells from which vesicles have been characterized utilizing the same enrichment method. Using sixty cell lines from the National Cancer Institute (NCI-60), here we provide the largest proteomic profile of EVs in a single study, identifying 6,071 proteins with 213 common to all isolates. Proteins included established EV markers, and vesicular trafficking proteins such as Rab GTPases and tetraspanins. Differentially-expressed proteins offer potential for cancer diagnosis and prognosis. Network analysis of vesicle quantity and proteomes identified EV components associated with vesicle secretion, including CD81, CD63, syntenin-1, VAMP3, Rab GTPases, and integrins. Integration of vesicle proteomes with whole-cell molecular profiles revealed similarities, suggesting EVs provide a reliable reflection of their progenitor cell content, and are therefore excellent indicators of disease.
Exosomes are small extracellular vesicles that carry heterogeneous cargo, including RNA, between cells. Increasing evidence suggests that exosomes are important mediators of intercellular communication and biomarkers of disease. Despite this, the variability of exosomal RNA between individuals has not been well quantified. To assess this variability, we sequenced the small RNA of cells and exosomes from a 17-member family. Across individuals, we show that selective export of miRNAs occurs not only at the level of specific transcripts, but that a cluster of 74 mature miRNAs on chromosome 14q32 is massively exported in exosomes while mostly absent from cells. We also observe more inter-individual variability between exosomal samples than between cellular ones and identify four miRNA expression quantitative trait loci (eQTLs) shared between cells and exosomes. Our findings indicate that genomically co-located miRNAs can be exported together and highlight the variability in exosomal miRNA levels between individuals as relevant for exosome use as diagnostics.
Cell migration is regulated by adhesion to the extracellular matrix (ECM) through integrins and activation of small RhoGTPases, such as RhoA and Rac1, resulting in changes to actomyosin organization. During invasion, epithelial-derived tumor cells switch from laminin-enriched basal membrane to collagen and fibronectin-enriched connective tissue. How this switch affects the tumor migration is still unclear. We tested the hypothesis that ECM dictates the invasiveness of Oral Squamous Cell Carcinoma (OSCC). We analyzed the migratory properties of two OSCC lines, a low invasive cell line with high e-cadherin levels (Linv/HE-cad) or a highly invasive cell line with low e-cadherin levels (Hinv/LE-cad), plated on different ECM components. Compared to laminin, fibronectin induced non-directional collective migration and decreased RhoA activity in Linv/HE-cad OSCC. For Hinv/LE-cad OSCC, fibronectin increased Rac1 activity and induced smaller adhesions, resulting in a fast single cell migration in both 2D and 3D environments. Consistent with these observations, human OSCC biopsies exhibited similar changes in cell-ECM adhesion distribution at the invasive front of the tumor, where cells encounter fibronectin. Our results indicate that ECM composition might induce a switch from collective to single cell migration according to tumor invasiveness due to changes in cell-ECM adhesion and the resulting signaling pathways that alter actomyosin organization.
The epithelial to mesenchymal transition (EMT) is a biological process in which a non-motile epithelial cell changes to a mesenchymal phenotype with invasive capacities. This phenomenon has been well documented in multiple biological processes including embryogenesis, fibrosis, tumor progression and metastasis. The hallmark of EMT is the loss of epithelial surface markers, most notably E-cadherin, and the acquisition of mesenchymal markers including vimentin and N-cadherin. The downregulation of E-cadherin during EMT can be mediated by its transcriptional repression through the binding of EMT transcription factors (EMT-TFs) such as SNAIL, SLUG and TWIST to E-boxes present in the E-cadherin promoter. Additionally, EMT-TFs can also cooperate with several enzymes to repress the expression of E-cadherin and regulate EMT at the epigenetic and post- translational level. In this review, we will focus on epigenetic and post- translational modifications that are important in EMT. In addition, we will provide an overview of the various therapeutic approaches currently being investigated to undermine EMT and hence, the metastatic progression of cancer as well.
Podoplanin (PDPN) is a transmembrane glycoprotein that plays crucial roles in embryonic development, the immune response, and malignant progression. Here, we report that cells ectopically or endogenously expressing PDPN release extracellular vesicles (EVs) that contain PDPN mRNA and protein. PDPN incorporates into membrane shed microvesicles (MVs) and endosomal-derived exosomes (EXOs), where it was found to colocalize with the canonical EV marker CD63 by immunoelectron microscopy. We have previously found that expression of PDPN in MDCK cells induces an epithelial-mesenchymal transition (EMT). Proteomic profiling of MDCK-PDPN cells compared to control cells shows that PDPN-induced EMT is associated with upregulation of oncogenic proteins and diminished expression of tumor suppressors. Proteomic analysis of exosomes reveals that MDCK-PDPN EXOs were enriched in protein cargos involved in cell adhesion, cytoskeletal remodeling, signal transduction and, importantly, intracellular trafficking and EV biogenesis. Indeed, expression of PDPN in MDCK cells stimulated both EXO and MV production, while knockdown of endogenous PDPN in human HN5 squamous carcinoma cells reduced EXO production and inhibited tumorigenesis. EXOs released from MDCK-PDPN and control cells both stimulated in vitro angiogenesis, but only EXOs containing PDPN were shown to promote lymphatic vessel formation. This effect was mediated by PDPN on the surface of EXOs, as demonstrated by a neutralizing specific monoclonal antibody. These results contribute to our understanding of PDPN-induced EMT in association to tumor progression, and suggest an important role for PDPN in EV biogenesis and/or release and for PDPN-EXOs in modulating lymphangiogenesis.
Exosomes regulate cell behavior by binding to and delivering their cargo to target cells; however, the mechanisms mediating exosome-cell interactions are poorly understood. Heparan sul-fates on target cell surfaces can act as receptors for exosome uptake, but the ligand for heparan sulfate on exosomes has not been identified. Using exosomes isolated from myeloma cell lines and from myeloma patients, we identify exosomal fibronectin as a key heparan sulfate-binding ligand and mediator of exosome-cell interactions. We discovered that heparan sulfate plays a dual role in exosome-cell interaction; heparan sulfate on exosomes captures fibronectin, and on target cells it acts as a receptor for fibronectin. Removal of heparan sulfate from the exosome surface releases fibronectin and dramatically inhibits exosome-target cell interaction. Antibody specific for the Hep-II heparin-binding domain of fibronectin blocks exo-some interaction with tumor cells or with marrow stromal cells. Regarding exosome function, fibronectin-mediated binding of exosomes to myeloma cells activated p38 and pERK signaling and expression of downstream target genes DKK1 and MMP-9, two molecules that promote myeloma progression. Antibody against fibronectin inhibited the ability of myeloma-derived exosomes to stimulate endothelial cell invasion. Heparin or hep-arin mimetics including Roneparstat, a modified heparin in phase I trials in myeloma patients, significantly inhibited exo-some-cell interactions. These studies provide the first evidence that fibronectin binding to heparan sulfate mediates exosome-cell interactions, revealing a fundamental mechanism important for exosome-mediated cross-talk within tumor microenvi-ronments. Moreover, these results imply that therapeutic disruption of fibronectin-heparan sulfate interactions will negatively impact myeloma tumor growth and progression.
Exosomes regulate cell behavior by binding to and delivering their cargo to target cells, however the mechanisms mediating
exosome-cell interactions are poorly understood. Heparan sulfates on target cell surfaces can act as receptors for exosome
uptake, but the ligand for heparan sulfate on exosomes has not been identified. Using exosomes isolated from myeloma cell
lines and from myeloma patients, we identify exosomal fibronectin as a key heparan sulfate-binding ligand and mediator of
exosome-cell interactions. We discovered that heparan sulfate plays a dual role in exosome-cell interaction; heparan sulfate
on exosomes captures fibronectin and on target cells it acts as a receptor for fibronectin. Removal of heparan sulfate from
the exosome surface releases fibronectin and dramatically inhibits exosome-target cell interaction. Antibody specific for
the Hep II heparin-binding domain of fibronectin blocks exosome interaction with tumor cells or with marrow stromal cells.
Regarding exosome function, fibronectin-mediated binding of exosomes to myeloma cells activated p38 and pERK signaling and
expression of downstream target genes DKK1 and MMP-9, two molecules that promote myeloma progression. Antibody against fibronectin
inhibited the ability of myeloma-derived exosomes to stimulate endothelial cell invasion. Heparin or heparin mimetics including
Roneparstat, a modified heparin in phase I trials in myeloma patients, significantly inhibited exosome-cell interactions.
These studies provide the first evidence that fibronectin binding to heparan sulfate mediates exosome-cell interactions revealing
a fundamental mechanism important for exosome-mediated crosstalk within tumor microenvironments. Moreover, these results imply
that therapeutic disruption of fibronectin-heparan sulfate interactions will negatively impact myeloma tumor growth and progression.
In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.
Rab GTPases recruit effector proteins, via their GTP-dependent switch regions, to distinct sub-cellular compartments. Rab11 and Rab25 are closely related small GTPases that bind to common effectors termed the Rab11-Family of Interacting Proteins (FIPs). The FIPs are organized into two subclasses (class I and class II) based on sequence and domain organization, and both subclasses contain a highly conserved Rab-binding domain (RBD) at their C-termini. Yeast two-hybrid and biochemical studies have revealed that the more distantly related Rab14 also interacts with class I FIPs. Here, we perform detailed structural, thermodynamic and cellular analyses of the interactions between Rab14 and one of the class I FIPs - the Rab Coupling Protein (RCP) - to clarify the molecular aspects of the interaction. We find that Rab14 indeed binds to RCP, albeit with reduced affinity relative to conventional Rab11:FIP and Rab25:FIP complexes. However, in vivo, Rab11 recruits RCP onto biological membranes. Furthermore, biophysical analyses reveal a non-canonical 1:2 stoichiometry between Rab14:RCP in dilute solutions, in contrast to Rab11/25 complexes. The structure of Rab14:RCP reveals that Rab14 interacts with the canonical RBD, and also provides insight into the unusual properties of the complex. Finally, we show that both the Rab Coupling Protein and Rab14 function in neuritogenesis.
Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
Directional cell movement through tissues is critical for multiple biological processes and requires maintenance of polarity in the face of complex environmental cues. Here we use intravital imaging to demonstrate that secretion of exosomes from late endosomes is required for directionally persistent and efficient in vivo movement of cancer cells. Inhibiting exosome secretion or biogenesis leads to defective tumour cell migration associated with increased formation of unstable protrusions and excessive directional switching. In vitro rescue experiments with purified exosomes and matrix coating identify adhesion assembly as a critical exosome function that promotes efficient cell motility. Live-cell imaging reveals that exosome secretion directly precedes and promotes adhesion assembly. Fibronectin is found to be a critical motility-promoting cargo whose sorting into exosomes depends on binding to integrins. We propose that autocrine secretion of exosomes powerfully promotes directionally persistent and effective cell motility by reinforcing otherwise transient polarization states and promoting adhesion assembly.
We propose a microfluidic system that generates nanovesicles (NVs) by slicing living cell membrane with microfabricated 500 nm-thick silicon nitride (SixNy) blades. Living cells were sliced by the blades while flowing through microchannels lined with the blades. Plasma membrane fragments sliced from the cells self-assembled into spherical NVs of ∼100-300 nm in diameter. During self-assembly, the plasma membrane fragments enveloped exogenous materials (here, polystyrene latex beads) from the buffer solution. About 30% of beads were encapsulated in NVs, and the generated NVs delivered the encapsulated beads across the plasma membrane of recipient cells, but bare beads could not penetrate the plasma membrane of recipient cells. This result implicates that the NVs generated using the method in this study can encapsulate and deliver exogenous materials to recipient cells, whereas exosomes secreted by cells can deliver only endogenous cellular materials.
Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.
Flow cytometry is a powerful method, which is widely used for high-throughput quantitative and qualitative analysis of cells. However, its straightforward applicability for extracellular vesicles (EVs) and mainly exosomes is hampered by several challenges, reflecting mostly the small size of these vesicles (exosomes: ~80-200 nm, microvesicles: ~200-1,000 nm), their polydispersity, and low refractive index. The current best and most widely used protocol for beads-free flow cytometry of exosomes uses ultracentrifugation (UC) coupled with floatation in sucrose gradient for their isolation, labeling with lipophilic dye PKH67 and antibodies, and an optimized version of commercial high-end cytometer for analysis. However, this approach requires an experienced flow cytometer operator capable of manual hardware adjustments and calibration of the cytometer. Here, we provide a novel and fast approach for quantification and characterization of both exosomes and microvesicles isolated from cell culture media as well as from more complex human samples (ascites of ovarian cancer patients) suitable for multiuser labs by using a flow cytometer especially designed for small particles, which can be used without adjustments prior to data acquisition. EVs can be fluorescently labeled with protein-(Carboxyfluoresceinsuccinimidyl ester, CFSE) and/or lipid- (FM) specific dyes, without the necessity of removing the unbound fluorescent dye by UC, which further facilitates and speeds up the characterization of microvesicles and exosomes using flow cytometry. In addition, double labeling with protein- and lipid-specific dyes enables separation of EVs from common contaminants of EV preparations, such as protein aggregates or micelles formed by unbound lipophilic styryl dyes, thus not leading to overestimation of EV numbers. Moreover, our protocol is compatible with antibody labeling using fluorescently conjugated primary antibodies. The presented methodology opens the possibility for routine quantification and characterization of EVs from various sources. Finally, it has the potential to bring a desired level of control into routine experiments and non-specialized labs, thanks to its simple bead-based standardization.
An integrative cell migration model incorporating focal adhesion (FA) dynamics, cytoskeleton and nucleus remodeling, actin motor activity, and lamellipodia protrusion is developed for predicting cell spreading and migration behaviors. This work is motivated by two experimental works: (1) cell migration on 2-D substrates under various fibronectin concentrations and (2) cell spreading on 2-D micropatterned geometries. These works suggest (1) cell migration speed takes a maximum at a particular ligand density (∼1140 molecules/µm) and (2) that strong traction forces at the corners of the patterns may exist due to combined effects exerted by actin stress fibers (SFs). The integrative model of this paper successfully reproduced these experimental results and indicates the mechanism of cell migration and spreading. In this paper, the mechanical structure of the cell is modeled as having two elastic membranes: an outer cell membrane and an inner nuclear membrane. The two elastic membranes are connected by SFs, which are extended from focal adhesions on the cortical surface to the nuclear membrane. In addition, the model also includes ventral SFs bridging two focal adhesions on the cell surface. The cell deforms and gains traction as transmembrane integrins distributed over the outer cell membrane bond to ligands on the ECM surface, activate SFs, and form focal adhesions. The relationship between the cell migration speed and fibronectin concentration agrees with existing experimental data for Chinese hamster ovary (CHO) cell migrations on fibronectin coated surfaces. In addition, the integrated model is validated by showing persistent high stress concentrations at sharp geometrically patterned edges. This model will be used as a predictive model to assist in design and data processing of upcoming microfluidic cell migration assays.
The intracellular pathogenic bacterium Salmonella enterica serovar typhimurium (Salmonella) relies on acidification of the Salmonella-containing vacuole (SCV) for survival inside host cells. The transport and fusion of membrane-bound compartments in a cell is regulated by small GTPases, including Rac and members of the Rab GTPase family, and their effector proteins. However, the role of these components in survival of intracellular pathogens is not completely understood. Here, we identify Nischarin as a novel dual effector that can interact with members of Rac and Rab GTPase (Rab4, Rab14 and Rab9) families at different endosomal compartments. Nischarin interacts with GTP-bound Rab14 and PI(3)P to direct the maturation of early endosomes to Rab9/CD63-containing late endosomes. Nischarin is recruited to the SCV in a Rab14-dependent manner and enhances acidification of the SCV. Depletion of Nischarin or the Nischarin binding partners-Rac1, Rab14 and Rab9 GTPases-reduced the intracellular growth of Salmonella. Thus, interaction of Nischarin with GTPases may regulate maturation and subsequent acidification of vacuoles produced after phagocytosis of pathogens.
Nischarin is a protein known to inhibit breast cancer cell motility by regulating the signaling of the Rho GTPase family. However, little is known about its location and function in the nervous system. The aim of the present study was to investigate the regional and cellular expression and functions of Nischarin in the adult rodent brain. As assessed by real-time PCR, Western blot analysis and immunostaining, we found that Nischarin was widely distributed throughout the brain, with a higher expression in the cerebral cortex and hippocampus. Double-labeling showed that Nischarin was expressed in neurons and was mainly located in the perinuclear region and F-actin-rich protrusions. The expression pattern of Nischarin in the brain was thought to be closely associated with its function. This was verified by our findings from cell migration assays that Nischarin regulated neuronal migration. These results provide a preliminary survey of the distribution of Nischarin in different regions and cell types in the rat brain. This might help to elucidate its physiological roles, and to evaluate its potential clinical implications.
Nischarin (encoded by NISCH), an α5 integrin-binding protein, has been identified as a regulator of breast cancer cell invasion. We hypothesized that it might be a tumor suppressor and were interested in its regulation.
We examined nischarin expression in approximately 300 human breast cancer and normal tissues using quantitative polymerase chain reaction and immunohistochemistry. Loss of heterozygosity analysis was performed by examining three microsatellite markers located near the NISCH locus in normal and tumor tissues. We generated derivatives of MDA-MB-231 human metastatic breast cancer cells that overexpressed nischarin and measured tumor growth from these cells as xenografts in mice; metastasis by these cells after tail vein injection; and α5 integrin expression, Rac, and focal adhesion kinase (FAK) signaling using western blotting. We also generated clones of MCF-7 human breast cancer cells in which nischarin expression was silenced and measured tumor growth in mouse xenograft models (n = 5 for all mouse experiments). P values were from two-sided Student t tests in pairwise comparisons.
Normal human breast tissue samples had statistically significantly higher expression of nischarin mRNA compared with tumor tissue samples (mean level in normal breast = 50.7 [arbitrary units], in breast tumor = 16.49 [arbitrary units], difference = 34.21, 95% confidence interval [CI] = 11.63 to 56.79, P = .003), and loss of heterozygosity was associated with loss of nischarin expression. MDA-MB-231 cells in which nischarin was overexpressed had statistically significantly reduced tumor growth and metastasis compared with parental MDA-MB-231 cells (mean volume at day 40, control vs nischarin-expressing tumors, 1977 vs 42.27 mm(3), difference = 1935 mm(3), 95% CI = 395 to 3475 mm(3), P = .025). Moreover, MCF-7 tumor xenografts in which nischarin expression was silenced grew statistically significantly faster than parental cells (mean volume at day 63, tumors with scrambled short hairpin RNA [shRNA] vs with nischarin shRNA, 224 vs 1262 mm(3), difference = 1038 mm(3), 95% CI = 899.6 to 1176 mm(3), P < .001). Overexpression of nischarin was associated with decreased α5 integrin expression, FAK phosphorylation, and Rac activation.
Nischarin may be a novel tumor suppressor that limits breast cancer progression by regulating α5 integrin expression and subsequently α5 integrin-, FAK-, and Rac-mediated signaling.
Paxillin is a multi-domain scaffold protein that localizes to the intracellular surface of sites of cell adhesion to the extracellular matrix. Through the interactions of its multiple protein-binding modules, many of which are regulated by phosphorylation, paxillin serves as a platform for the recruitment of numerous regulatory and structural proteins that together control the dynamic changes in cell adhesion, cytoskeletal reorganization and gene expression that are necessary for cell migration and survival. In particular, paxillin plays a central role in coordinating the spatial and temporal action of the Rho family of small GTPases, which regulate the actin cytoskeleton, by recruiting an array of GTPase activator, suppressor and effector proteins to cell adhesions. When paxillin was first described 18 years ago, the amazing complexity of cell-adhesion organization, dynamics and signaling was yet to be realized. Herein we highlight our current understanding of how the multiple protein interactions of paxillin contribute to the coordination of cell-adhesion function.
Fibronectin expression and distribution were examined in cancer tissues from 19 patients with cancer of the head and neck regions. Samples taken before and after irradiation of approximately 10 Gy, 20 Gy or 30 Gy were analyzed by the avidin-biotin-horseradish peroxidase method using mouse monoclonal antibodies against human fibronectin. The results were correlated with the patient's prognosis after radiation therapy. No remarkable changes in the fibronectin expression or distribution were found between tissue specimens taken before and after each dose of irradiation. The prognosis, however, varied according to the degree of expression and the distribution pattern of fibronectin. Seven patients in which the cancer tissue was encircled by a thick fibronectin network are still alive without recurrence 4.5-6 years after treatment, whereas 6 patients in which fibronectin was only faintly expressed or focally distributed died or developed recurrence soon after treatment. The present findings demonstrate that fibronectin expression and distribution in cancer tissue are intimately related to the patient's prognosis, and that the analysis of these two parameters is applicable as a predictive assay in radiotherapy of cancer of the head and neck regions.
The PTEN/MMAC1/TEP (PTEN) tumor suppressor gene at 10q23.3 is mutated in multiple types of sporadic tumors including breast cancers and also in the germline of patients with the Cowden's breast cancer predisposition syndrome. The PTEN gene encodes a multifunctional phosphatase capable of dephosphorylating the same sites in membrane phosphatidylinositols phosphorylated by phosphatidylinositol 3'-kinase (PI3K). We demonstrate herein that loss of PTEN function in breast cancer cells results in an increase in basal levels of phosphorylation of multiple components of the P13K signaling cascade as well as an increase in duration of ligand-induced signaling through the P13K cascade. These alterations are reversed by wild-type but not phosphatase inactive PTEN. In the presence of high concentrations of serum, enforced expression of PTEN induces a predominant G1 arrest consistent with the capacity of PTEN to evoke increases in the expression of the p27Kip1 cyclin dependent kinase inhibitor. In the presence of low concentrations of serum, enforced PTEN expression results in a marked increase in cellular apoptosis, a finding which is consistent with the capacity of PTEN to alter the phosphorylation, and presumably function, of the AKT, BAD, p70S6 kinase and GSK3 alpha apoptosis regulators. Under anchorage-independent conditions, PTEN also induces anoikis, a form of apoptosis that occurs when cells are dissociated from the extracellular matrix, which is enhanced in conjunction with low serum culture conditions. Together, these data suggest that PTEN effects on the PI3K signaling cascade are influenced by the cell stimulatory context, and that depending on the exposure to growth factors and other exogenous stimuli such as integrin ligation, PTEN can induce cell cycle arrest, apoptosis or anoikis in breast cancer cells.
Integrins have been implicated in key cellular functions, including cytoskeletal organization, motility, growth, survival, and control of gene expression. The plethora of integrin alpha and beta subunits suggests that individual integrins have unique biological roles, implying specific molecular connections between integrins and intracellular signaling or regulatory pathways. Here, we have used a yeast two-hybrid screen to identify a novel protein, termed Nischarin, that binds preferentially to the cytoplasmic domain of the integrin alpha5 subunit, inhibits cell motility, and alters actin filament organization. Nischarin is primarily a cytosolic protein, but clearly associates with alpha5beta1, as demonstrated by coimmunoprecipitation. Overexpression of Nischarin markedly reduces alpha5beta1-dependent cell migration in several cell types. Rat embryo fibroblasts transfected with Nischarin constructs have "basket-like" networks of peripheral actin filaments, rather than typical stress fibers. These observations suggest that Nischarin might affect signaling to the cytoskeleton regulated by Rho-family GTPases. In support of this, Nischarin expression reverses the effect of Rac on lamellipodia formation and selectively inhibits Rac-mediated activation of the c-fos promoter. Thus, Nischarin may play a negative role in cell migration by antagonizing the actions of Rac on cytoskeletal organization and cell movement.
Suppression of tropomyosins (TMs), a family of actin-binding, microfilament-associated proteins, is a prominent feature of many transformed cells. Yet it is unclear whether downregulation of TMs occur in human tumors. We have investigated the expression of tropomyosin-1 (TM1) in human breast carcinoma tissues by in situ hybridization and immunofluorescence. TM1 mRNA and protein are readily detectable in normal mammary tissue. In contrast, TM1 expression is abolished in the primary human breast tumors. Expression of other TM isoforms, however, is variable among the tumors. The consistent and profound downregulation of TM1 suggests that TM1 may be a novel and useful biomarker of mammary neoplasms. These data also support the hypothesis that suppression of TM1 expression during the malignant conversion of mammary epithelium as a contributing factor of breast cancer. In support of this hypothesis, we show that the ability to suppress malignant growth properties of breast cancer cells is specific to TM1 isoform. Investigations into the mechanisms of TM1-induced tumor suppression reveal that TM1 induces anoikis (detachment induced apoptosis) in breast cancer cells. Downregulation of TM1 in breast tumors may destabilize microfilament architecture and confer resistance to anoikis, which facilitates survival of neoplastic cells outside the normal microenvironment and promote malignant growth.
Integrin-extracellular matrix interactions activate signaling cascades such as mitogen activated protein kinases (MAPK). Integrin binding to extracellular matrix increases tyrosine phosphorylation of focal adhesion kinase (FAK). Inhibition of FAK activity by expression of its carboxyl terminus decreases cell motility, and cells from FAK deficient mice also show reduced migration. Paxillin is a focal adhesion protein which is also phosphorylated on tyrosine. FAK recruitment of paxillin to the cell membrane correlates with Shc phosphorylation and activation of MAPK. Decreased FAK expression inhibits papilloma formation in a mouse skin carcinogenesis model. We previously demonstrated that MAPK activation was required for growth factor induced in vitro migration and invasion by human squamous cell carcinoma (SCC) lines.
Adapter protein recruitment to integrin subunits was examined by co-immunoprecipitation in SCC cells attached to type IV collagen or plastic. Stable clones overexpressing FAK or paxillin were created using the lipofection technique. Modified Boyden chambers were used for invasion assays.
In the present study, we showed that FAK and paxillin but not Shc are recruited to the beta1 integrin cytoplasmic domain following attachment of SCC cells to type IV collagen. Overexpression of either FAK or paxillin stimulated cancer cell migration on type IV collagen and invasion through reconstituted basement membrane which was dependent on MAPK activity.
We concluded that recruitment of focal adhesion kinase and paxillin to beta1 integrin promoted cancer cell migration via the mitogen activated protein kinase pathway.
Exosomes, molecular cargos secreted by almost all mammalian cells, are considered as promising biomarkers to identify many diseases including cancers. However, the small size of exosomes (30-200 nm) poses serious challenges on their isolation from the complex media containing a variety of extracellular vesicles (EVs) of different sizes, especially in small sample volumes. Here we present a viscoelasticity-based microfluidic system to directly separate exosomes from cell culture media or serum in a continuous, size-dependent, and label-free manner. Using a small amount of biocompatible polymer as the additive into the media to control the viscoelastic forces exerted on EVs, we are able to achieve a high separation purity (> 90 %) and recovery (> 80 %) of exosomes. The proposed technique may serve as a versatile platform to facilitate exosome analyses in diverse biochemical applications.
A critical step in metastases formation is cancer-cell invasion through tissue. During invasion, cells change morphology and apply forces to their surroundings. We have previously shown that single, metastatic breast-cancer cells will mechanically indent a synthetic, impenetrable polyacrylamide gel with physiological-stiffness in attempted invasion; benign breast cells do not indent the gels. In solid tumors, e.g., breast cancers, metastases occur predominantly by collective cell-invasion. Thus, here we evaluate the effects of cell proximity on mechanical invasiveness, specifically through changes in gel indention. Gel indentation is induced by 56, 33 and 2% (in >1000 cells), respectively, of adjacent high metastatic potential (MP), low MP and benign breast cells, being double the amounts observed in single, well-separated cells. Single cells exhibited a distribution of indentation depths below 10 µm, while adjacent cells also showed a second peak of deeper indentations. The second peak included 65% of indenting high MP cells as compared to 15% in the low MP cells, illustrating the difference in their invasiveness. Thus, proximity of the metastatic cells enhances their mechanical ability to invade, demonstrating why collective cancer-cell migration is likely more efficient. This could potentially provide a rapid, quantitative approach to identify metastatic cells, and to determine their metastatic potential.
Nischarin, a novel integrin binding protein, has been demonstrated its negative effects on cell migration and invasion. However, the biological role of Nischarin in breast cancer has not been fully elucidated yet. Our study aimed to analyze the association between Nischarin expression and clinical features of breast cancer patients, and further investigate the role of Nischarin in breast cancer cells apoptosis, migration and invasion. Results showed that Nischarin expression was significantly lower in breast cancer tissues (37.8%, 23/67) than in normal tissues (61.8%, 21/34; P < 0.05), and the expression of Nischarin significantly negatively correlated with estrogen receptor status. Similarly, Nischarin expression was highest in normal breast cell line HBL-100 while triple-negative breast cancer cell line MDA-MB-231 had the lowest expression of Nischarin. Further experiments demonstrated that overexpression of Nischarin may induce apoptosis, and inhibit cell migration and invasion. The present data confirmed that Nishcharin might be a novel tumor suppressor and plays an important role in breast cancer cell apoptosis and metastasis, which can be used as a potential therapeutic target for breast cancer treatment.
Elevated expression and aberrant activation of Ras have been implicated in breast cancer aggressiveness. H-Ras, but not N-Ras, induces breast cell invasion. A crucial link between lipid rafts and H-Ras function has been suggested. The present study sought to identify the lipid raft protein(s) responsible for H-Ras-induced tumorigenicity and invasiveness of breast cancer. We conducted a comparative proteomic analysis of lipid raft proteins from invasive MCF10A human breast epithelial cells engineered to express active H-Ras and non-invasive cells expressing active N-Ras. Here, we identified a lipid raft protein flotillin-1 as an important regulator of H-Ras activation and breast cell invasion. Flotillin-1 was required for epidermal growth factor-induced activation of H-Ras, but not that of N-Ras, in MDA-MB-231 triple-negative breast cancer (TNBC) cells. Flotillin-1 knockdown inhibited the invasiveness of MDA-MB-231 and Hs578T TNBC cells in vitro and in vivo. In xenograft mouse tumor models of these TNBC cell lines, we showed that flotillin-1 played a critical role in tumor growth. Using human breast cancer samples, we provided clinical evidence for the metastatic potential of flotillin-1. Membrane staining of flotillin-1 was positively correlated with metastatic spread (p=0.013) and inversely correlated with patient disease-free survival rates (p=0.005). Expression of flotillin-1 was associated with H-Ras in breast cancer, especially in TNBC (p<0.001). Our findings provide insight into the molecular basis of Ras isoform-specific interplay with flotillin-1, leading to tumorigenicity and aggressiveness of breast cancer. This article is protected by copyright. All rights reserved.
Tumor suppressor genes regulate cell growth and prevent spontaneous proliferation that could lead to aberrant tissue function. Deletions and mutations of these genes typically lead to progression through the cell-cycle checkpoints, as well as increased cell migration. Studies of these proteins are important as they may provide potential treatments for breast cancers. In this review, we discuss a comprehensive overview on Nischarin, a novel protein discovered by our laboratory. Nischarin, or imidazoline receptor antisera-selected protein, is a protein involved in a vast number of cellular processes, including neuronal protection and hypotension. The NISCH promoter experiences hypermethylation in several cancers, whereas some highly aggressive breast cancer cells exhibit genomic loss of the NISCH locus. Furthermore, we discuss data illustrating a novel role of Nischarin as a tumor suppressor in breast cancer. Analysis of this new paradigm may shed light on various clinical questions. Finally, the therapeutic potential of Nischarin is discussed. Cancer Res; 75(20); 1-8. ©2015 AACR.
The mammalian brain is composed of an outer layer of gray matter, consisting of cell bodies, dendrites, and unmyelinated axons, and an inner core of white matter, consisting primarily of myelinated axons. Recent evidence suggests that microstructural differences between gray and white matter play an important role during neurodevelopment. While brain tissue as a whole is rheologically well characterized, the individual features of gray and white matter remain poorly understood. Here we quantify the mechanical properties of gray and white matter using a robust, reliable, and repeatable method, flat-punch indentation. To systematically characterize gray and white matter moduli for varying indenter diameters, loading rates, holding times, post-mortem times, and locations we performed a series of n=192 indentation tests. We found that indenting thick, intact coronal slices eliminates the common challenges associated with small specimens: it naturally minimizes boundary effects, dehydration, swelling, and structural degradation. When kept intact and hydrated, brain slices maintained their mechanical characteristics with standard deviations as low as 5% throughout the entire testing period of five days post mortem. White matter, with an average modulus of 1.895kPa±0.592kPa, was on average 39% stiffer than gray matter, p<0.01, with an average modulus of 1.389kPa±0.289kPa, and displayed larger regional variations. It was also more viscous than gray matter and responded less rapidly to mechanical loading. Understanding the rheological differences between gray and white matter may have direct implications on diagnosing and understanding the mechanical environment in neurodevelopment and neurological disorders.
Copyright © 2015 Elsevier Ltd. All rights reserved.
NISCH encodes the imidazoline receptor Nischarin and is a known tumor suppressor in many human malignancies; however, its roles in ovarian cancer are still largely unknown. Here, we aim to investigate the biological functions of NISCH in ovarian cancer. We found that NISCH was significantly downregulated, which correlated considerably with advanced tumor stage, poor differentiation, lymph node metastasis, and the serous/mucinous subtypes in a panel of ovarian cancer tissues. Moreover, NISCH gene silencing was mainly the product of promoter hypermethylation, which could be reversed by treatment with 5-aza-dC. In vitro, NISCH overexpression suppressed cell proliferation and colony formation by hindering cell cycle progression; whereas, the opposite was observed in NISCH knockdown counterparts. In vivo, abundant NISCH expression hindered the growth of HO8910 xenografts, while NISCH knockdown accelerated the growth of SKOV3 xenografts. In addition, NISCH significantly attenuated cell invasion by inhibiting the phosphorylation of FAK and ERK, which could be neutralized by PF-562271 (a FAK/Pyk2 inhibitor). Accordingly, NISCH knockdown xenografts exhibited increased peritoneal/pelvic metastases that were not present in counterparts treated with PF-562271. Furthermore, NISCH expression in primary ovarian cancer cells predicted a cellular resistance to PF-562271. In conclusion, we showed that NISCH was frequently silenced by promoter hypermethylation in human ovarian cancer. NISCH manipulated cellular proliferation and invasion through arresting cell cycle and inhibiting the FAK signal. Our findings revealed the biological functions of NISCH in ovarian cancer, and might be useful for treating patients with aberrant expression of NISCH.
Copyright © 2015, American Association for Cancer Research.
Epithelial cells require attachment to the extracellular matrix (ECM) for survival. However, during tumour progression and metastasis, cancerous epithelial cells must adapt to and survive in the absence of ECM. During the past 20 years, several cellular changes, including anoikis, have been shown to regulate cell viability when cells become detached from the ECM. In this Opinion article, we review in detail how cancer cells can overcome or take advantage of these specific processes. Gaining a better understanding of how cancer cells survive during detachment from the ECM will be instrumental in designing chemotherapeutic strategies that aim to eliminate ECM-detached metastatic cells.
Fibronectin (FN) has raised an interest in cancer research due to its role in tumor progression. The aim of this study was to investigate the expression and clinical relevance of FN in breast cancer as well as to explore its relationship with the expression of Matrix Metalloproteases (MMPs) and their inhibitors (TIMPs).
An immunohistochemical study was performed in 110 breast cancer patients using tissue arrays and specific antibodies against FN, MMP-7, 9, 11, TIMP-1 and 2. The results indicate that FN expression was related to tumour size, histological grade, and with MMP-9 expression. Tumours with high FN expression by tumour cells were significantly associated with higher probability of metastasis, poor overall survival, and with expression of MMP-7, 9, 11, TIMP-1 and 2 by mononuclear inflammatory cells (MICs). In addition, the association between FN expression by cancer cells and MMP-11 by MICs, was strongly associated with distant metastasis development.
Breast carcinomas with distant metastasis have frequently tumour cells expressing intracellular FN. There is a strong association between FN expression by tumour cells and MMPs or TIMPs expression by stromal MICs, which may represent a possible crosstalk of prognostic relevance in breast cancer. This article is protected by copyright. All rights reserved.
Anoikis is a programmed cell death induced upon cell detachment from extracellular matrix, behaving as a critical mechanism in preventing adherent-independent cell growth and attachment to an inappropriate matrix, thus avoiding colonizing of distant organs. As anchorage-independent growth and epithelial-mesenchymal transition, two features associated with anoikis resistance, are vital steps during cancer progression and metastatic colonization, the ability of cancer cells to resist anoikis has now attracted main attention from the scientific community. Cancer cells develop anoikis resistance due to several mechanisms, including change in integrins repertoire allowing them to grow in different niches, activation of a plethora of inside-out pro-survival signals as over-activation of receptors due to sustained autocrine loops, oncogenes activation, growth factor receptors overexpression, or mutation/upregulation of keys enzymes involved in integrins or growth factor receptors signaling. In addition, tumor microenvironment has also been acknowledged to contribute to anoikis resistance of bystander cancer cells, by modulating matrix stiffness, enhancing oxidative stress, producing pro-survival soluble factors, triggering epithelial-mesenchymal transition and self-renewal ability, as well as leading to metabolic deregulations of cancer cells. All these events help cancer cells to inhibit the apoptosis machinery and sustain pro-survival signals after detachment, counteracting anoikis and constituting promising targets for anti-metastatic pharmacological therapy. This article is part of a Special Issue entitled: Cell Death Pathways.
The method we introduced in 1992 for measuring hardness and elastic modulus by instrumented indentation techniques has widely been adopted and used in the characterization of small-scale mechanical behavior. Since its original development, the method has undergone numerous refinements and changes brought about by improvements to testing equipment and techniques as well as from advances in our understanding of the mechanics of elastic–plastic contact. Here, we review our current understanding of the mechanics governing elastic–plastic indentation as they pertain to load and depth-sensing indentation testing of monolithic materials and provide an update of how we now implement the method to make the most accurate mechanical property measurements. The limitations of the method are also discussed.
The present study investigated the effects of adhesion on the elastic modulus determined from nanoindentation curves for soft polydimethylsiloxane (PDMS) elastomers with five different crosslink concentrations. Indentation load-displacement curves were obtained for samples of all concentrations at four different peak loads. All load-displacement curves were nearly linear, resulting in load independent contact stiffnesses (p < 0.003) for the range of loads tested. As a result, elastic modulus calculated from nanoindentation curves with the Hertz contact model exhibited significant differences (p < 0.004) both at different peak loads for a single PDNIS concentration and between different PDMS concentrations at a single peak load (p < 0.001). The differences for different peak loads were attributed to the presence of substantial adhesive forces at the tip-sample interface. By taking these adhesive interactions into account with the Johnson, Kendall, Roberts (JKR) contact model, the differences in elastic modulus at different peak loads could be reconciled. Significant differences (p < 0.001) in moduli between different PDMS concentrations were still present. The results highlight the importance of considering adhesive forces in nanoindentation analyses of low modulus polymeric materials. (c) 2006 Elsevier B.V. All rights reserved.
Biallelic inactivation of LKB1, a serine/threonine kinase, has been detected in 30% of lung adenocarcinomas, and inhibition
of breast tumor growth has been demonstrated. We have identified the tumor suppressor, Nischarin, as a novel binding partner
of LKB1. Our mapping analysis shows that the N terminus of Nischarin interacts with amino acids 44–436 of LKB1. Time lapse
microscopy and Transwell migration data show that the absence of both Nischarin and LKB1 from an invasive breast cancer cell
line (MDA-MB-231) enhances migration as measured by increased distance and speed of migrating cells. Our data suggest that
this is a result of elevated PAK1 and LIMK1 phosphorylation. Moreover, the absence of Nischarin and LKB1 increased tumor growth
in vivo. Consistent with this, the percentage of S phase cells was increased, as demonstrated by flow cytometry and enhanced cyclin
D1. The absence of Nischarin and LKB1 also led to a dramatic increase in the formation of lung metastases. Our studies, for
the first time, demonstrate functional interaction between LKB1 and Nischarin to inhibit cell migration and breast tumor progression.
Mechanistically, we show that these two proteins together regulate PAK-LIMK-Cofilin and cyclin D1/CDK4 pathways.
To facilitate a rapid response to environmental change, cells use scaffolding
— or adaptor — proteins to recruit key components of their signal-transduction
machinery to specific subcellular locations. Paxillin is a multi-domain adaptor
found at the interface between the plasma membrane and the actin cytoskeleton.
Here it provides a platform for the integration and processing of adhesion-
and growth factor-related signals.
The chemokine, stromal cell-derived factor-1 alpha (SDF-1 alpha) and its receptor CXCR-4 (fusin, LESTR) are thought to be involved in the trafficking of hematopoietic progenitors and stem cells, as suggested by the chemotactic effect of SDF-1 alpha on these cells. Gene inactivation studies have shown that both SDF-1 alpha and CXCR-4 are essential for B lymphopoiesis. Migration of leukemic cells may also be dependent on SDF-1 alpha and CXCR-4. Fibronectin (FN) is a component of the extracellular matrix (ECM), and one of the natural supports for cell movement in their bone hematopoietic environment. In the present study, we examined the influence of FN on the chemotactic effect of SDF-1 alpha and on the CXCR-4 expression and function on human precursor-B acute lymphoblastic leukemia (pre-B ALL) cells at sequential stages of development. Fourteen children with pre-B ALL were studied. Their immunophenotypes belonged to the first three stages of B cell differentiation. Despite relatively high levels of CXCR-4 expression at all stages, the responsiveness to SDF-1 alpha, measured as the percentage of migrating cells in the transwell culture system, varied with patients and seems to be less significant for pre-B3 (and pre-B1) than for pre-B2. There was no correlation (r = 0.2) between the SDF-1 alpha induced migration (range: 2.5-39%) and the cell surface density of CXCR-4 (range: 46.5-97.5%). The extracellular matrix protein FN, either coated on the filter (for more than 18 hours) or in soluble form, enhanced the SDF-1 alpha induced migration of pre-B ALL respectively (2 fold and 1.6 fold) without influencing CXCR-4 expression in short term cultures. Therefore, we analyzed the expression of the FN receptors, VLA-4 (CD49d) and VLA-5 (CD49e), by direct immunofluorescence, on these leukemic cells. VLA-4 was strongly expressed in all stages of pre-B ALL (range: 77-97%) while VLA-5 expression was more variable (range: 14-94%), but no correlation with the FN-dependent increased SDF-1 alpha chemotactic effect was noted. In conclusion, the migratory behavior of pre-B leukemic cells in response to SDF-1 alpha partly depends upon the stage of differentiation, and partly upon unexplained patient variability. Our results suggest that several molecules from the extracellular matrix, such as FN, may be implicated in this phenomenon.
Paxillin is a focal adhesion-associated, phosphotyrosine-containing protein that may play a role in several signaling pathways. Paxillin contains a number of motifs that mediate protein-protein interactions, including LD motifs, LIM domains, an SH3 domain-binding site and SH2 domain-binding sites. These motifs serve as docking sites for cytoskeletal proteins, tyrosine kinases, serine/threonine kinases, GTPase activating proteins and other adaptor proteins that recruit additional enzymes into complex with paxillin. Thus paxillin itself serves as a docking protein to recruit signaling molecules to a specific cellular compartment, the focal adhesions, and/or to recruit specific combinations of signaling molecules into a complex to coordinate downstream signaling. The biological function of paxillin coordinated signaling is likely to regulate cell spreading and motility.
Exosomes are small membrane vesicles of endocytic origin that are secreted by most cells in culture. Interest in exosomes has intensified after their recent description in antigen-presenting cells and the observation that they can stimulate immune responses in vivo. In the past few years, several groups have reported the secretion of exosomes by various cell types, and have discussed their potential biological functions. Here, we describe the physical properties that define exosomes as a specific population of secreted vesicles, we summarize their biological effects, particularly on the immune system, and we discuss the potential roles that secreted vesicles could have as intercellular messengers.