RESUMO
Aggressive cancers, such as triple-negative breast cancer (TNBC), are mostly fatal because of their potential to metastasize to distant organs. Cancer cells acquire various abilities to metastasize, including resistance to anoikis, an apoptotic cell death induced by loss of anchorage to the extracellular matrix. Transcriptional coactivator with PDZ binding motif (TAZ) and Yes-associated protein (YAP), the downstream effectors of the Hippo pathway, regulate cell- and tissue-level architectures by responding to mechanical microenvironments of cells, including the cell-extracellular matrix interaction. The Hippo pathway is frequently disrupted in cancer cells, and TAZ and YAP are irrelevantly activated, potentially resulting in anchorage-independent survival/proliferation of cancer cells and metastatic progression. The study aims to investigate the roles of TAZ and YAP in anoikis resistance in basal-like (BL) TNBC cells, which comprise a major subtype (>70%) of TNBC. We found that TAZ and YAP had nonredundant roles in anchorage-independent cancer cell survival or anoikis resistance. Particularly, TAZ was indispensable for anoikis resistance in BL-TNBC cells but not for survival of non-transformed mammary epithelial cells (MECs). In contrast, YAP, a paralog of TAZ, was indispensable for survival of both non-transformed MECs and cancer cells. Therefore, TAZ might be a preferable therapeutic target against dissemination of aggressive cancer cells without killing normal cells. Interestingly, TAZ was abnormally stabilized in BL-TNBC cells under non-adherent conditions, which promoted anoikis resistance. Furthermore, OTUB1, a deubiquitinating enzyme, was responsible for the stabilization of TAZ in detached BL-TNBC cells. Importantly, simultaneous high expression of TAZ and OTUB1 was associated with poor prognosis in BC. Thus, OTUB1 has emerged as a potentially druggable target. Successful inhibition of OTUB1 enzymatic activity is expected to downregulate TAZ and eventually prevents metastasis of aggressive cancers, such as BL-TNBC.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Neoplasias de Mama Triplo Negativas , Humanos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Anoikis/fisiologia , Neoplasias de Mama Triplo Negativas/patologia , Proteínas de Sinalização YAP , Enzimas Desubiquitinantes/metabolismo , Microambiente TumoralRESUMO
Colorectal cancer (CRC) is a deadly malignancy and therapeutic approaches for CRC are evolving every day. Anoikis is a key mechanism for programmed cell death of cancer cells that undergo anchorage-independent growth at a different matrix than the one which is expected. Yet, anoikis is a less studied mechanism of cell death in comparison to other mechanisms such as apoptosis. Relating to this, resistance to anoikis among cancer cells remains critical for improved metastasis and survival in a new environment evading anoikis. Since CRC cells have the ability to metastasize from proximal sites to secondary organs such as liver and promote cancer in those distant sites, a clear knowledge of the mechanisms essential for anchorage-independent growth and subsequent metastasis is necessary to counteract CRC progression and spread. Therefore, the identification of novel drug candidates and studying the roles of anoikis in assisting CRC therapy using such drugs can prevent anchorage-independent cancer cell growth. Additionally, the identification of novel biomarkers or therapeutic targets seems essential for implementing superior therapy, impeding relapse among malignant cells and improving the survival rate of clinical patients. As there are no reviews published on this topic till date, anoikis as a mechanism of cell death and its therapeutic roles in CRC are discussed in this review. In addition, several molecules were identified as therapeutic targets for CRC.
Assuntos
Anoikis , Neoplasias Colorretais , Humanos , Anoikis/fisiologia , Neoplasias Colorretais/patologia , Linhagem Celular TumoralRESUMO
Anoikis resistance is a prerequisite for circulating tumor cells to survive. However, the mechanism underlying anoikis resistance is poorly understood. In the current study, the effect of TNF receptor-associated factor 6 (TRAF6)-induced NF-kB activation on anoikis susceptibility in tumor cells was evaluated. Differential TRAF6-binding proteins in anoikis-sensitive versus anoikis-resistant tumor cells were screened by LC/MS-MS analysis. The effects of TRAF6-binding proteins on the stability of TRAF6, the activation of NF-kB signaling and anoikis susceptibility in tumor cells were detected. We found that the loss of TRAF6 expression is an important molecular event linked to anoikis. X-linked inhibitor of apoptosis protein (XIAP), an E3 ligase, can bind, ubiquitinate, and degrade TRAF6 and may lead to inactivation of NF-κB signaling and anoikis sensitivity. High expression of prohibitin 1 (PHB1) competes with XIAP for binding to TRAF6 and confers anoikis resistance to tumor cells. PHB1 and TRAF6 knockdown eliminated tumor cells from the circulation in vivo. Significant correlations between elevated PHB1 and TRAF6 expression and distant metastasis were observed in patients with oral cancer. Collectively, we elucidated a novel mechanism governing anoikis. Our data also indicated that TRAF6 and PHB1 are potential therapeutic targets for tumor cells disseminating in the circulation. IMPLICATIONS: Our data implicate that PHB1 competes with XIAP for binding to TRAF6 and confers anoikis resistance to tumor cells.
Assuntos
NF-kappa B , Fator 6 Associado a Receptor de TNF , Humanos , NF-kappa B/metabolismo , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Anoikis/fisiologia , Ligação Competitiva , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismoRESUMO
Circulating tumor cells (CTCs) acquire enhanced anti-anoikis abilities after experiencing flow shear stress in the circulatory system. Our previous study demonstrated that low shear stress (LSS) promotes anoikis resistance of human breast carcinoma cells via caveolin-1 (Cav-1)-dependent extrinsic and intrinsic apoptotic pathways. However, the underlying mechanism how LSS enhanced Cav-1 expression in suspended cancer cells remains unclear. Herein, we found that LSS induced redox signaling was involved in the regulation of Cav-1 level and anoikis resistance in suspension cultured cancer cells. Exposure of human breast carcinoma MDA-MB-231 cells to LSS (2 dyn/cm2) markedly induced ROS and â¢NO generation, which promoted the cell viability and reduced the cancer cell apoptosis. Furthermore, ROS and â¢NO scavenging inhibited the upregulation of Cav-1 by interfering ubiquitination, and suppressed the anoikis resistance of suspended tumor cells. These findings provide new insight into the mechanism by which LSS-stimulated ROS and â¢NO generation increases Cav-1 stabilization in suspended cancer cells through inhibition of ubiquitination and proteasomal degradation, which could be a potential target for therapy of metastatic tumors.
Assuntos
Neoplasias da Mama , Caveolina 1 , Feminino , Humanos , Anoikis/fisiologia , Neoplasias da Mama/genética , Caveolina 1/genética , Caveolina 1/metabolismo , Células MDA-MB-231 , Óxido Nítrico/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
During metastasis cancer cells must adapt to survive loss of anchorage and evade anoikis. An important pro-survival adaptation is the ability of metastatic tumor cells to increase their antioxidant capacity and restore cellular redox balance. Although much is known about the transcriptional regulation of antioxidant enzymes in response to stress, how cells acutely adapt to alter antioxidant enzyme levels is less well understood. Using ovarian cancer cells as a model, we demonstrate that an increase in mitochondrial superoxide dismutase SOD2 protein expression is a very early event initiated in response to detachment, an important step during metastasis that has been associated with increased oxidative stress. SOD2 protein synthesis is rapidly induced within 0.5-2 h of matrix detachment, and polyribosome profiling demonstrates an increase in the number of ribosomes bound to SOD2 mRNA, indicating an increase in SOD2 mRNA translation in response to anchorage-independence. Mechanistically, we find that anchorage-independence induces cytosolic accumulation of the RNA binding protein HuR/ELAVL1 and promotes HuR binding to SOD2 mRNA. Using HuR siRNA-mediated knockdown, we show that the presence of HuR is necessary for the increase in SOD2 mRNA association with the heavy polyribosome fraction and consequent nascent SOD2 protein synthesis in anchorage-independence. Cellular detachment also activates the stress-response mitogen-activated kinase p38, which is necessary for HuR-SOD2 mRNA interactions and induction of SOD2 protein output. These findings illustrate a novel translational regulatory mechanism of SOD2 by which ovarian cancer cells rapidly increase their mitochondrial antioxidant capacity as an acute stress response to anchorage-independence.
Assuntos
Antioxidantes , Adesão Celular , Proteína Semelhante a ELAV 1 , Superóxido Dismutase , Anoikis/fisiologia , Antioxidantes/metabolismo , Adesão Celular/genética , Adesão Celular/fisiologia , Proteína Semelhante a ELAV 1/metabolismo , Feminino , Humanos , Neoplasias Ovarianas/genética , RNA Mensageiro/genética , Superóxido Dismutase/biossíntese , Superóxido Dismutase/genéticaRESUMO
Mechanical stress is known to fuel several hallmarks of cancer, ranging from genome instability to uncontrolled proliferation or invasion. Cancer cells are constantly challenged by mechanical stresses not only in the primary tumour but also during metastasis. However, this latter has seldom been studied with regards to mechanobiology, in particular resistance to anoikis, a cell death programme triggered by loss of cell adhesion. Here, we show in vitro that migrating breast cancer cells develop resistance to anoikis following their passage through microporous membranes mimicking confined migration (CM), a mechanical constriction that cancer cells encounter during metastasis. This CM-induced resistance was mediated by Inhibitory of Apoptosis Proteins, and sensitivity to anoikis could be restored after their inhibition using second mitochondria-derived activator of caspase (SMAC) mimetics. Anoikis-resistant mechanically stressed cancer cells displayed enhanced cell motility and evasion from natural killer cell-mediated immune surveillance, as well as a marked advantage to form lung metastatic lesions in mice. Our findings reveal that CM increases the metastatic potential of breast cancer cells.
Assuntos
Anoikis , Neoplasias da Mama , Animais , Anoikis/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Feminino , Humanos , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Transdução de SinaisRESUMO
Peritoneal metastasis is a common mode of spread of ovarian cancer. Despite therapeutic advances, some patients have intractable peritoneal metastasis. Therefore, in-depth characterization of the molecular mechanism of peritoneal metastasis is a key imperative. Angiopoietin-like protein 2 (ANGPTL2) is an inflammatory factor which activates NF-κB signaling and plays an important role in the pathogenesis of various inflammatory diseases including cancers, such as lung and breast cancer. In this study, we examined the role of ANGPTL2 in ovarian cancer peritoneal metastasis. We observed no difference of cell proliferation between ANGPTL2-expressing and control cells. In the mouse intraperitoneal xenograft model, formation of peritoneal metastasis by ANGPTL2-expressing cells was significantly decreased compared to control. In the in vitro analysis, the expressions of integrin α5ß1, α6, and ß4, but not those of αvß3, α3, α4, and ß1, were significantly decreased in ANGPTL2-expressing cells compared to control cells. ANGPTL2-expressing cells showed significantly inhibited adherence to laminin compared to control. In addition, we observed upregulation of anoikis (a form of programmed cell death occurring under an anchorage-independent condition) and significant decrease in the expression of Bcl-2 in ANGPTL2-expressing cells as compared to control cells. These results suggest that ANGPTL2 expression in ovarian cancer cells represses peritoneal metastasis by suppressing anoikis resistance.
Assuntos
Proteínas Semelhantes a Angiopoietina/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Neoplasias Peritoneais/metabolismo , Neoplasias Peritoneais/secundário , Proteína 2 Semelhante a Angiopoietina , Animais , Anoikis/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Feminino , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transdução de SinaisRESUMO
The loss of cell-matrix interactions induces apoptosis, known as anoikis. For successful distant metastasis, circulating tumor cells (CTCs) that have lost matrix attachment need to acquire anoikis resistance in order to survive. Cell aggregate formation confers anoikis resistance, and CTC clusters are more highly metastatic compared to single cells; however, the molecular mechanisms underlying this aggregation are not well understood. In this study, we demonstrated that cell detachment increased cell aggregation and upregulated fibronectin (FN) levels in lung and breast cancer cells, but not in their normal counterparts. FN knockdown decreased cell aggregation and increased anoikis. In addition, cell detachment induced cell-cell adhesion proteins, including E-cadherin, desmoglein-2, desmocollin-2/3, and plakoglobin. Interestingly, FN knockdown decreased the levels of desmoglein-2, desmocollin-2/3, and plakoglobin, but not E-cadherin, suggesting the involvement of desmosomal junction in cell aggregation. Accordingly, knockdown of desmoglein-2, desmocollin-2, or plakoglobin reduced cell aggregation and increased cell sensitivity to anoikis. Previously, we reported that NADPH oxidase 4 (Nox4) upregulation is important for anoikis resistance. Nox4 inhibition by siRNA or apocynin decreased cell aggregation and increased anoikis with the downregulation of FN, and, consequently, decreased desmoglein-2, desmocollin-2/3, or plakoglobin. The coexpression of Nox4 and FN was found to be significant in lung and breast cancer patients, based on cBioPortal data. In vivo mouse lung metastasis model showed that FN knockdown suppressed lung metastasis and thus enhanced survival. FN staining of micro tissue array revealed that FN expression was positive for human lung cancer (61%) and breast cancer (58%) patients. Furthermore, the expression levels of FN, desmoglein-2, desmocollin-2, and plakoglobin were significantly correlated with the poor survival of lung and breast cancer patients, as per the Kaplan-Meier plotter analysis. Altogether, our data suggest that FN upregulation and enhanced desmosomal interactions are critical for cell aggregation and anoikis resistance upon cell detachment.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Fibronectinas/biossíntese , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Células A549 , Animais , Anoikis/fisiologia , Neoplasias da Mama/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Agregação Celular/fisiologia , Linhagem Celular Tumoral , Fibronectinas/genética , Fibronectinas/metabolismo , Xenoenxertos , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/genética , Camundongos , Camundongos Nus , NADPH Oxidase 4/biossíntese , NADPH Oxidase 4/genética , NADPH Oxidase 4/metabolismo , Metástase Neoplásica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise Serial de Tecidos , Regulação para CimaRESUMO
BACKGROUND: The aim of this study was to investigate the role of IL-17A in the cancer microenvironment and the recurrence of triple negative breast cancer (TNBC). METHODS: Using human TNBC cell lines, the role of IL17-A was investigated by knocked down of IL-17A (ΔIL-17A) and by administration of IL-17A into the culture medium. Cell proliferation assays, migration assays, as well as Western blot analysis and real-time PCR, were used to evaluate IL-17A-related signaling. Three types of 4T1 cells were implanted into BALB/c mice, namely wild type (WT), ΔIL-17A, and WT + neutralizing IL-17 antibody (WT + Ab) cells. Tumor weight, necrosis area, and the number of circulating tumor cells (CTCs) were measured. Immunohistochemistry and Western blotting were used to analyze expression of CD34, CD8, and TGF-ß1 as well as anoikis resistance. The Kaplan-Meier's method was used to correlate IL-17A expression and patient outcome, including disease-free survival (DFS) and overall survival (OS). RESULTS: Our results demonstrated that IL-17A was able to stimulate the migratory activity, but not the growth rate, of MDA-MB-231/468 cells. In vivo, for the ΔIL-17A group, there was an increase in necrosis area, a decrease in tumor CD34 expression and a reduction in the number of CTCs. Furthermore, in WT + Ab group, there was a decreased in tumor expression of CD34, fewer CD8 ( +) cells, and fewer CTCs, but an increase in expression of TGF-ß1 expression. Both of the above were compared to the WT group. Knockdown of IL-17A also decreased anoikis resistance in human TNBC and the murine 4T1 cell lines. Kaplan-Meier analysis disclosed a negative correlation between tumor expression of IL-17A and OS in TNBC patients. CONCLUSION: We conclude that IL-17A promotes migratory and angiogenic activity in tumors, enhances anoikis resistance, and modulates the immune landscape of the tumor microenvironment such changes favor cancer metastasis.
Assuntos
Anoikis/fisiologia , Movimento Celular/fisiologia , Interleucina-17/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Microambiente Tumoral/fisiologia , Animais , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologia , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Anoikis is a programmed death of cell induced upon detachment from the extracellular matrix (ECM). Resistance to anoikis is a critical contributor to cancer invasion and metastasis. High frequency of metastatic recurrence is a huge challenge for current therapy of hepatocellular carcinoma (HCC). Our previous study had identified sulfhydryl oxidase 1 (QSOX1) as a suppressor of HCC metastasis. In the present study, we used the anchorage-independent growth condition to mimic the detachment of HCC cells from ECM. We found that QSOX1 was induced in HCC cells under the anchorage-independent growth condition and that could be blocked by endoplasmic reticulum stress (ERS) inhibitor. Overexpression and knockdown of QSOX1 gene were performed on HCC cells. QSOX1 inhibited de novo synthesis of fatty acids (FAs) and cholesterol (ChE) and reduced their content in the detached HCC cells, and thus mediated mitochondrial apoptosis of HCC cells. In conclusion, QSOX1 is induced under detached culture condition via ERS. QSOX1 promotes mitochondrial apoptosis by suppressing the lipid synthesis of HCC cells in detached condition. QSOX1 appears to accelerate anoikis of HCC cells. These findings offer a new insight into how to overcome anoikis resistance of HCC cells and provide a potential target for prevention of HCC metastasis.
Assuntos
Carcinoma Hepatocelular/patologia , Lipídeos/biossíntese , Neoplasias Hepáticas/patologia , Mitocôndrias/patologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Anoikis/fisiologia , Apoptose , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Colesterol/metabolismo , Estresse do Retículo Endoplasmático , Ácidos Graxos/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Ácido Láctico/metabolismo , Neoplasias Hepáticas/metabolismo , Mitocôndrias/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genéticaRESUMO
Acquisition of anoikis resistance is a prerequisite for cancer metastasis and invasion, which are major causes of death from cancer. The molecular mechanisms underlying antianoikis properties in cancer cells are still largely unclear. Here, we describe a protocol for preparation of anoikis-resistant cultured nonmetastatic MCF-7 and metastatic MDA-MB-231 cell lines. The anoikis-resistant cultures were prepared by plating cells in the poly-2-hydroxyethyl methacrylate coated plates and cultured for 24 h. The viability of cells in the cultures was determined using trypan blue staining and annexin V cell death assay, while protein profiles associated with anoikis-resistance in both cells and conditioned media were analyzed by proteomics.
Assuntos
Anoikis/fisiologia , Neoplasias da Mama/metabolismo , Anexina A5/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Feminino , Humanos , Células MCF-7 , Proteômica/métodosRESUMO
BACKGROUND: We previously reported that there were potentially certain correlations between the high expression of SATB1 and the HBV infection in human hepatocellular carcinoma tissues, and SATB1 promoted tumor growth and metastasis in liver cancer. Hepatitis B virus (HBV) infection is internationally recognized as a contributing factor to metastasis in liver cancer. The anoikis prevention of detached malignant cancer cells is the precondition for metastasis. AIMS: Our studies aimed to explore the relationship between HBV infection, SATB1 and liver cancer cell anoikis and their specific regulatory mechanisms in HBV-associated liver cancer. METHODS: HepG2 cell was transiently transfected with pBlue-HBV and seven types of HBV-encoded protein plasmids. Anoikis assay and soft agarose colony formation experiment were analyzed in HepG2.2.15-SATB1 siRNA cells, HBx-overexpressing cells and HepG2-HBx-SATB1 siRNA cells. The inhibitors of signaling molecules were used to treat of HepG2-HBx cells, and then, the SATB1 expression and phosphorylation levels of signaling molecules were evaluated. RESULTS: Our data show that the high expression of SATB1 and enhanced anoikis resistance were observed in HBV stably expressing cell line HepG2.2.15 and high metastatic potential cell line SK-HEP-1. HBV can induce SATB1 expression and suppress anoikis of unattached liver cancer cells. Moreover, SATB1 expression and anoikis resistance were mainly regulated by HBV-encoded viral protein HBx through the activation of ERK and p38 MAPK signaling pathways to promote metastasis of liver cancer. CONCLUSION: These data suggest that the HBV-encoded HBx and SATB1 may play an important role in promoting anoikis resistance and metastasis in HBV-associated liver cancer.
Assuntos
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas de Ligação à Região de Interação com a Matriz/biossíntese , Transativadores/biossíntese , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Anoikis/fisiologia , Carcinoma Hepatocelular/patologia , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Neoplasias Hepáticas/patologia , Proteínas de Ligação à Região de Interação com a Matriz/genética , Transdução de Sinais/fisiologia , Transativadores/genética , Proteínas Virais Reguladoras e AcessóriasRESUMO
Purpose: Cancer stem cells (CSCs) are a subpopulation of cells with the capacity to drive tumor growth. While there is evidence of the existence of CSCs in uveal melanoma (UM), there is no consensus on their defining markers. In this study, we examined putative CSC markers in UM cell lines, primary UM (PUM), and normal choroidal melanocytes (NCM). Methods: Nonadherent sphere assays were used to assess the tumorigenic potential of 15 PUMs, 8 high (M3) and 7 low (D3) metastatic risk. Flow cytometry was used to compare the expression of CSC markers between 10 PUMs and 4 NCMs, as well as in 8 UM cell lines grown under adherent and nonadherent conditions. Based on the data generated and from TCGA analyses, CD166 was investigated in detail, including its effect on cell migration using a tumor transendothelial migration assay. Results: M3 PUM had a greater melanosphere-forming efficiency than D3 PUM. CD166 and Nestin expression was upregulated in PUM compared to NCM by flow cytometry. UM cell lines resistant to anoikis had increased levels of CD271, Nestin, and CD166 compared with adherent cells. TCGA analysis showed that patients with higher CD166 expression had a poorer prognosis: this was supported by a Mel270 CD166high subpopulation that had enhanced migratory capabilities compared with CD166low cells. IHC showed that CD166 is expressed in the cytoplasm and cell membrane of PUM cells. Conclusions: UM contain a population of cells with characteristics of CSCs. In particular, CD166high UM cells appear to represent a subpopulation with enhanced migratory capacity.
Assuntos
Antígenos CD/genética , Biomarcadores Tumorais/genética , Moléculas de Adesão Celular Neuronais/genética , Proteínas Fetais/genética , Melanoma/patologia , Células-Tronco Neoplásicas/patologia , Neoplasias Uveais/patologia , Anoikis/fisiologia , Antígenos CD/metabolismo , Biomarcadores Tumorais/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Linhagem Celular Tumoral , Ensaios de Migração Celular , Movimento Celular/fisiologia , Corioide/metabolismo , Proteínas Fetais/metabolismo , Citometria de Fluxo , Dosagem de Genes , Regulação da Expressão Gênica/fisiologia , Humanos , Imuno-Histoquímica , Melanócitos/metabolismo , Melanoma/genética , Melanoma/metabolismo , Reação em Cadeia da Polimerase Multiplex , Células-Tronco Neoplásicas/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Nestina/genética , Nestina/metabolismo , Receptores de Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/metabolismo , Neoplasias Uveais/genética , Neoplasias Uveais/metabolismoRESUMO
Cancer cells display abnormal redox metabolism. Autophagy, anoikis and reactive oxygen species (ROS) play a regulatory role during metastasis. LC3 is a well-known essential molecule for autophagy. Therefore, we wanted to explore the molecular interplay between autophagy, anoikis, and ROS in relation to LC3B. We observed enhanced LC3B level along with increased expression of p62 and modulation of other autophagy-related molecules (Atg 3, 5, 7, 12, 16L1 and Beclin1) by inducing oxidative-stress in ovarian cancer cells using a ROS-producing pro-oxidant molecule. Surprisingly, enhanced LC3B was unable to induce autophagosome formation rather promoted anoikis. ROS-induced inhibition of autophagosome-formation is possibly due to the instability of autophagy initiator, ULK1 complex. Moreover, such upregulation of LC3B via ROS enhanced several apoptotic molecules. Silencing LC3B reduced these apoptotic molecules and increased when overexpressed, suggesting its role in apoptosis. Furthermore, LC3B-dependent apoptosis was decreased by inhibiting ROS, indicating a possible link between ROS, LC3B, and apoptosis. Additionally, ROS-induced enhanced LC3B promoted detachment-induced cell death (anoikis). This was further reflected by reduced cell adhesion molecules (integrin-ß3 and focal adhesion kinase) and mesenchymal markers (snail and slug). Our in vitro experimental data was further confirmed in primary tumors developed in syngeneic mice, which also showed ROS-mediated LC3B enhancement along with reduced autophagosomes, integrin-ß3 and focal adhesion kinase ultimately leading to the decreased tumor mass. Additionally, primary cells from high-grade serous carcinoma patient's ascites exhibited LC3B enhancement and autophagy inhibition through ROS which provided a clinical relevance of our study. Taken together, this is the first evidence for a non-canonical role of LC3B in promoting anoikis in contrast to autophagy and may, therefore, consider as a potential therapeutic target molecule in ovarian cancer. Taken together, autophagy-inhibition may be an alternative approach to induce apoptosis/anoikis in cancer.
Assuntos
Anoikis/fisiologia , Autofagia/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Neoplasias Ovarianas/metabolismo , Estresse Oxidativo/fisiologia , Animais , Anoikis/genética , Autofagia/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Proliferação de Células/fisiologia , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Feminino , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Estresse Oxidativo/genética , Espécies Reativas de Oxigênio/metabolismoRESUMO
Anoikis is a type of programmed cell death induced by detachment from the extracellular matrix. In cancer cells, anoikis resistance is essential for cancer cell survival in blood circulation and distant metastasis. However, the mechanisms behind anoikis resistance of gastric cancer remain largely unknown. Herein, we demonstrate that NADPH oxidase 4 (NOX4) expression and reactive oxygen species (ROS) generation are upregulated in suspension gastric cell cultures compared with adherent cultures. Silencing of NOX4 decreases ROS generation and downregulates EGFR, sensitizing cells to anoikis. NOX4 overexpression upregulates ROS and EGFR levels and promotes anoikis resistance. NOX4 depletion inhibits gastric cancer survival in blood circulation and attenuates distant metastasis. NOX4 expression is correlated with EGFR expression in patients. In conclusion, induction of NOX4 expression by detachment promotes anoikis resistance of gastric cancer through ROS generation and downstream upregulation of EGFR, which is critical for the metastatic progression of gastric cancer.
Assuntos
Anoikis/fisiologia , Receptores ErbB/metabolismo , NADPH Oxidase 4/metabolismo , Neoplasias Gástricas/metabolismo , Animais , Anoikis/genética , Apoptose/genética , Apoptose/fisiologia , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/genética , Proliferação de Células/fisiologia , Imunofluorescência , Humanos , Imuno-Histoquímica , Lentivirus/genética , Masculino , Camundongos , Camundongos Nus , Microscopia Eletrônica de Varredura , NADPH Oxidase 4/genética , Espécies Reativas de Oxigênio/metabolismo , Neoplasias Gástricas/genéticaRESUMO
Anoikis is a form of apoptosis induced by cell detachment. Integrin inactivation plays a major role in the process but the exact signalling pathway is ill-defined. Here we identify an anoikis pathway using gliotoxin (GT), a virulence factor of the fungus Aspergillus fumigatus, which causes invasive aspergillosis in humans. GT prevents integrin binding to RGD-containing extracellular matrix components by covalently modifying cysteines in the binding pocket. As a consequence, focal adhesion kinase (FAK) is inhibited resulting in dephosphorylation of p190RhoGAP, allowing activation of RhoA. Sequential activation of ROCK, MKK4/MKK7 and JNK then triggers pro-apoptotic phosphorylation of Bim. Cells in suspension or lacking integrin surface expression are insensitive to GT but are sensitised to ROCK-MKK4/MKK7-JNK-dependent anoikis upon attachment to fibronectin or integrin upregulation. The same signalling pathway is triggered by FAK inhibition or inhibiting integrin αV/ß3 with Cilengitide. Thus, GT can target integrins to induce anoikis on lung epithelial cells.
Assuntos
Anoikis/fisiologia , Gliotoxina/metabolismo , Transdução de Sinais/fisiologia , Fatores de Virulência/metabolismo , Amidas , Animais , Anoikis/genética , Linhagem Celular , Citometria de Fluxo , Humanos , Immunoblotting , Imunoprecipitação , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , MAP Quinase Quinase 7/genética , MAP Quinase Quinase 7/metabolismo , Camundongos , Camundongos Knockout , Mutagênese Sítio-Dirigida , Piridinas , Transdução de Sinais/genética , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismoRESUMO
Anoikis resistance is considered to be an essential prerequisite of tumor metastasis and which is an important mechanism in the metastatic process of gastric cancer (GC). Caveolin-1 (CAV-1), a protein component of caveolae, has been reported to regulate several cancer cell behaviors including anoikis resistance. However, the role of CAV-1 in the acquisition of anoikis resistance in GC cells has never been explored. In this study, we investigated the promoting effect of CAV-1 on anchorage-independent growth and anoikis resistance, and the involvement of the related signaling pathways in such process in SGC-7901 cells. The results showed that CAV-1 could promote anchorage-independent growth and anoikis resistance in detached SGC-7901 cells, which was associated with the activation of Src-dependent epidermal growth factor receptor-integrin ß signaling as well as the phosphorylation of PI3K/Akt and MEK/ERK signaling pathways. The data from this study might contribute to the in-depth understanding of the metastatic mechanism for GC.
Assuntos
Anoikis/fisiologia , Caveolina 1/fisiologia , Integrina beta1/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Adesão Celular , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , Metástase Neoplásica , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de SinaisRESUMO
Anoikis is a critical obstacle to cancer metastasis. Colorectal cancer (CRC) exhibits a high rate of metastasis, leading to death, and the mechanisms involved in anoikis resistance are still unclear. We identified that the fatty acid oxidation (FAO) pathway was activated in detached CRC cells. Multiple genes in the FAO pathway, specifically the rate-limiting enzyme CPT1A, were upregulated in CRC cells grown in suspension. Reactive oxygen species elimination mediated by CPT1A in CRC cells was vital to anoikis resistance. In vivo experiments showed that CPT1A-suppressed CRC cells colonized the lung at a much lower rate than normal CRC cells, suggesting that CPT1A-mediated FAO activation increased metastatic capacity. In clinical tissue specimens from CRC patients, elevated expression of CPT1A was observed in metastatic sites compared with primary sites. Our results demonstrate that CPT1A-mediated FAO activation induces CRC cells to resist anoikis, suggesting that CPT1A is an attractive target for treating metastatic CRC.
Assuntos
Anoikis/fisiologia , Carnitina O-Palmitoiltransferase/metabolismo , Neoplasias Colorretais/patologia , Ácidos Graxos/metabolismo , Metástase Neoplásica/patologia , Animais , Células CACO-2 , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Células HCT116 , Células HT29 , Humanos , Metabolismo dos Lipídeos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Oxirredução , Espécies Reativas de Oxigênio/metabolismoRESUMO
Ovarian cancer is the most lethal gynecological cancer, where survival rates have had modest improvement over the last 30 years. Metastasis of cancer cells is a major clinical problem, and patient mortality occurs when ovarian cancer cells spread beyond the confinement of ovaries. Disseminated ovarian cancer cells typically spread within the abdomen, where ascites accumulation aids in their transit. Metastatic ascites contain multicellular spheroids, which promote chemo-resistance and recurrence. However, little is known about the origin and mechanisms through which spheroids arise. Using live-imaging of 3D culture models and animal models, we report that epithelial ovarian cancer (EOC) cells, the most common type of ovarian cancer, can spontaneously detach as either single cells or clusters. We report that clusters are more resistant to anoikis and have a potent survival advantage over single cells. Using in vivo lineage tracing, we found that multicellular spheroids arise preferentially from collective detachment, rather than aggregation in the abdomen. Finally, we report that multicellular spheroids from collective detachment are capable of seeding intra-abdominal metastases that retain intra-tumoral heterogeneity from the primary tumor.
Assuntos
Abdome/patologia , Carcinoma Epitelial do Ovário/patologia , Neoplasias Ovarianas/patologia , Esferoides Celulares/patologia , Anoikis/fisiologia , Ascite/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/fisiologia , Feminino , Humanos , Recidiva Local de Neoplasia/patologiaRESUMO
Anoikis is known as a special type of programmed cell death which occurs in response to loss of correct cell-extracellular matrix (ECM) connections. This process could be as pivotal event in normal development and tissue homeostasis and found as important mechanism in cancer invasiveness and metastasis. The persistent infection with oncoviruses including EBV (Epstein Bar virus), HPV (Human Papillomaviruses), HBV (Hepatitis B virus), KSHV (Human herpesvirus 8), HTLV-1 (Human T-lymphotropic virus-1), and HCV (Hepatitis C virus) accounted as one of main risk factor for cancer progression. Some of them play critical roles in metastasis, especially in anoikis resistance which could contribute to metastasis of tumor cells. The better understanding of effects of oncoviruses on anoikis could contribute to finding of effective therapeutic platforms for treatment of virus-associated cancers. This paper highlighted effects of these oncoviruses on anoikis protection in cancer.