RESUMO
Cell plasticity is a crucial hallmark leading to cancer metastasis. Upregulation of Rho/ROCK pathway drives actomyosin contractility, protrusive forces, and contributes to the occurrence of highly invasive amoeboid cells in tumors. Cancer stem cells are similarly associated with metastasis, but how these populations arise in tumors is not fully understood. Here, we show that the novel oncogene RASSF1C drives mesenchymal-to-amoeboid transition and stem cell attributes in breast cancer cells. Mechanistically, RASSF1C activates Rho/ROCK via SRC-mediated RhoGDI inhibition, resulting in generation of actomyosin contractility. Moreover, we demonstrate that RASSF1C-induced amoeboid cells display increased expression of cancer stem-like markers such as CD133, ALDH1, and Nanog, and are accompanied by higher invasive potential in vitro and in vivo. Further, RASSF1C-induced amoeboid cells employ extracellular vesicles to transfer the invasive phenotype to target cells and tissue. Importantly, the underlying RASSF1C-driven biological processes concur to explain clinical data: namely, methylation of the RASSF1C promoter correlates with better survival in early-stage breast cancer patients. Therefore, we propose the use of RASSF1 gene promoter methylation status as a biomarker for patient stratification.
Assuntos
Neoplasias da Mama/genética , Vesículas Extracelulares/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteínas Supressoras de Tumor/genética , Proteína rhoA de Ligação ao GTP/genética , Quinases da Família src/genética , Antígeno AC133/genética , Antígeno AC133/metabolismo , Família Aldeído Desidrogenase 1/genética , Família Aldeído Desidrogenase 1/metabolismo , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Ilhas de CpG , Metilação de DNA , Vesículas Extracelulares/química , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Camundongos , Camundongos SCID , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Células-Tronco Neoplásicas/patologia , Transdução de Sinais , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Análise de Sobrevida , Proteínas Supressoras de Tumor/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína rhoA de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismoRESUMO
RASSF1A promoter methylation has been correlated with tumor dedifferentiation and aggressive oncogenic behavior. Nevertheless, the underlying mechanism of RASSF1A-dependent tumor dedifferentiation remains elusive. Here, we show that RASSF1A directly uncouples the NOTCH-HES1 axis, a key suppressor of differentiation. Interestingly, the crosstalk of RASSF1A with HES1 occurs independently from the signaling route connecting RASSF1A with the Hippo pathway. At the molecular level, we demonstrate that RASSF1A acts as a scaffold essential for the SUMO-targeted E3 ligase SNURF/RNF4 to target HES1 for degradation. The reciprocal relationship between RASSF1A and HES1 is evident across a wide range of human tumors, highlighting the clinical significance of the identified pathway. We show that HES1 upregulation in a RASSF1A-depleted environment renders cells non-responsive to the downstream effects of γ-secretase inhibitors (GSIs) which restrict signaling at the level of the NOTCH receptor. Taken together, we report a mechanism through which RASSF1A exerts autonomous regulation of the critical Notch effector HES1, thus classifying RASSF1A expression as an integral determinant of the clinical effectiveness of Notch inhibitors.
Assuntos
Receptores Notch , Transdução de Sinais , Fatores de Transcrição HES-1 , Proteínas Supressoras de Tumor , Humanos , Proteínas Nucleares/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Fatores de Transcrição HES-1/genética , Fatores de Transcrição HES-1/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Epigenetic inactivation of the Hippo pathway scaffold RASSF1A is associated with poor prognosis in a wide range of sporadic human cancers. Loss of expression reduces tumor suppressor activity and promotes genomic instability, but how this pleiotropic biomarker is regulated at the protein level is unknown. Here we show that TGF-ß is the physiological signal that stimulates RASSF1A degradation by the ubiquitin-proteasome pathway. In response to TGF-ß, RASSF1A is recruited to TGF-ß receptor I and targeted for degradation by the co-recruited E3 ubiquitin ligase ITCH. RASSF1A degradation is necessary to permit Hippo pathway effector YAP1 association with SMADs and subsequent nuclear translocation of receptor-activated SMAD2. We find that RASSF1A expression regulates TGF-ß-induced YAP1/SMAD2 interaction and leads to SMAD2 cytoplasmic retention and inefficient transcription of TGF-ß targets genes. Moreover, RASSF1A limits TGF-ß induced invasion, offering a new framework on how RASSF1A affects YAP1 transcriptional output and elicits its tumor-suppressive function.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias Pulmonares/metabolismo , Fosfoproteínas/metabolismo , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Transporte Ativo do Núcleo Celular , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Movimento Celular , Metilação de DNA , Epigênese Genética , Feminino , Regulação Neoplásica da Expressão Gênica , Via de Sinalização Hippo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Invasividade Neoplásica , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise , Interferência de RNA , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Fatores de Transcrição , Transcrição Gênica , Transfecção , Fator de Crescimento Transformador beta1/farmacologia , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Sinalização YAPRESUMO
Nuclear actin participates in many essential cellular processes including gene transcription, chromatin remodelling and mRNA processing. Actin shuttles into and out the nucleus through the action of dedicated transport receptors importin-9 and exportin-6, but how this transport is regulated remains unclear. Here, we show that RASSF1A is a novel regulator of actin nucleocytoplasmic trafficking and is required for the active maintenance of nuclear actin levels through supporting binding of exportin-6 (XPO6) to RAN GTPase. RASSF1A (Ras association domain family 1 isoform A) is a tumour suppressor gene frequently silenced by promoter hypermethylation in all major solid cancers. Specifically, we demonstrate that endogenous RASSF1A localises to the nuclear envelope (NE) and is required for nucleocytoplasmic actin transport and the concomitant regulation of myocardin-related transcription factor A (MRTF-A), a co-activator of the transcription factor serum response factor (SRF). The RASSF1A/RAN/XPO6/nuclear actin pathway is aberrant in cancer cells where RASSF1A expression is lost and correlates with reduced MRTF-A/SRF activity leading to cell adhesion defects. Taken together, we have identified a previously unknown mechanism by which the nuclear actin pool is regulated and uncovered a previously unknown link of RASSF1A and MRTF-A/SRF in tumour suppression.
Assuntos
Actinas/metabolismo , Neoplasias da Mama/genética , Neoplasias Hepáticas/genética , Membrana Nuclear/metabolismo , Fator de Resposta Sérica/genética , Proteínas Supressoras de Tumor/metabolismo , Transporte Biológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Citoplasma/metabolismo , Metilação de DNA , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Carioferinas/metabolismo , Neoplasias Hepáticas/metabolismo , Prognóstico , Fator de Resposta Sérica/metabolismo , Transativadores/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
Lung cancer remains the leading cause of cancer-related death due to poor treatment responses and resistance arising from tumour heterogeneity. Here, we show that adverse prognosis associated with epigenetic silencing of the tumour suppressor RASSF1A is due to increased deposition of extracellular matrix (ECM), tumour stiffness and metastatic dissemination in vitro and in vivo. We find that lung cancer cells with RASSF1A promoter methylation display constitutive nuclear YAP1 accumulation and expression of prolyl 4-hydroxylase alpha-2 (P4HA2) which increases collagen deposition. Furthermore, we identify that elevated collagen creates a stiff ECM which in turn triggers cancer stem-like programming and metastatic dissemination in vivo. Re-expression of RASSF1A or inhibition of P4HA2 activity reverses these effects and increases markers of lung differentiation (TTF-1 and Mucin 5B). Our study identifies RASSF1A as a clinical biomarker associated with mechanical properties of ECM which increases the levels of cancer stemness and risk of metastatic progression in lung adenocarcinoma. Moreover, we highlight P4HA2 as a potential target for uncoupling ECM signals that support cancer stemness.
Assuntos
Adenocarcinoma de Pulmão/patologia , Metilação de DNA , Neoplasias Pulmonares/patologia , Células-Tronco Neoplásicas/metabolismo , Proteínas Supressoras de Tumor/genética , Regulação para Cima , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Animais , Linhagem Celular Tumoral , Progressão da Doença , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Metástase Neoplásica , Transplante de Neoplasias , Prolil Hidroxilases/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Proteínas de Sinalização YAPRESUMO
BACKGROUND: Although there is extensive evidence for the amoeboid invasiveness of cancer cells in vitro, much less is known about the role of amoeboid invasiveness in metastasis and the importance of Rho/ROCK/MLC signaling in this process. RESULTS: We analyzed the dependence of amoeboid invasiveness of rat and chicken sarcoma cells and the metastatic activity of chicken cells on individual elements of the Rho/ROCK/MLC pathway. In both animal models, inhibition of Rho, ROCK or MLC resulted in greatly decreased cell invasiveness in vitro, while inhibition of extracellular proteases using a broad spectrum inhibitor did not have a significant effect. The inhibition of both Rho activity and MLC phosphorylation by dominant negative mutants led to a decreased capability of chicken sarcoma cells to metastasize. Moreover, the overexpression of RhoA in non-metastatic chicken cells resulted in the rescue of both invasiveness and metastatic capability. Rho and ROCK, unlike MLC, appeared to be directly involved in the maintenance of the amoeboid phenotype, as their inhibition resulted in the amoeboid-mesenchymal transition in analyzed cell lines. CONCLUSION: Taken together, these results suggest that protease-independent invasion controlled by elements of the Rho/ROCK/MLC pathway can be frequently exploited by metastatic sarcoma cells.
Assuntos
Cadeias Leves de Miosina/metabolismo , Sarcoma/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular , Galinhas , Invasividade Neoplásica , Ratos , Sarcoma/patologia , Transdução de SinaisRESUMO
Tumor cell invasion is the most critical step of metastasis. Determination of the mode of invasion within the particular tumor is critical for effective cancer treatment. Protease-independent amoeboid mode of invasion has been described in carcinoma cells and more recently in sarcoma cells on treatment with protease inhibitors. To analyze invasive behavior, we compared highly metastatic sarcoma cells with parental nonmetastatic cells. The metastatic cells exhibited a functional up-regulation of Rho/ROCK signaling and, similarly to carcinoma cells, an amoeboid mode of invasion. Using confocal and traction force microscopy, we showed that an up-regulation of Rho/ROCK signaling leads to increased cytoskeletal dynamics, myosin light chain localization, and increased tractions at the leading edge of the cells and that all of these contributed to increased cell invasiveness in a three-dimensional collagen matrix. We conclude that cells of mesenchymal origin can use the amoeboid nonmesenchymal mode of invasion as their primary invading mechanism and show the dependence of ROCK-mediated amoeboid mode of invasion on the increased capacity of cells to generate force.
Assuntos
Colágeno/metabolismo , Citoesqueleto/patologia , Sarcoma/metabolismo , Sarcoma/patologia , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Animais , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Células Cultivadas , Citoesqueleto/metabolismo , Quinases Lim/genética , Quinases Lim/metabolismo , Magnetismo , Metaloproteinase 2 da Matriz/metabolismo , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Microscopia de Fluorescência , Cadeias Leves de Miosina/metabolismo , Invasividade Neoplásica , Fosforilação , Análise Serial de Proteínas , Ratos , Regulação para Cima , Proteínas rho de Ligação ao GTP/genética , Quinases Associadas a rho/genéticaRESUMO
Cancer-associated fibroblasts (CAFs) are an emerging target for cancer therapy as they promote tumour growth and metastatic potential. However, CAF targeting is complicated by the lack of knowledge-based strategies aiming to selectively eliminate these cells. There is a growing body of evidence suggesting that a pro-inflammatory microenvironment (e.g. ROS and cytokines) promotes CAF formation during tumorigenesis, although the exact mechanisms involved remain unclear. In this study, we reveal that a prolonged pro-inflammatory stimulation causes a de facto deficiency in base excision repair, generating unrepaired DNA strand breaks and thereby triggering an ATF4-dependent reprogramming of normal fibroblasts into CAF-like cells. Based on the phenotype of in vitro-generated CAFs, we demonstrate that midostaurin, a clinically relevant compound, selectively eliminates CAF-like cells deficient in base excision repair and prevents their stimulatory role in cancer cell growth and migration.
RESUMO
Tumor cells gain metastatic capacity through a Golgi phosphoprotein 3-dependent (GOLPH3-dependent) Golgi membrane dispersal process that drives the budding and transport of secretory vesicles. Whether Golgi dispersal underlies the pro-metastatic vesicular trafficking that is associated with epithelial-to-mesenchymal transition (EMT) remains unclear. Here, we have shown that, rather than causing Golgi dispersal, EMT led to the formation of compact Golgi organelles with improved ribbon linking and cisternal stacking. Ectopic expression of the EMT-activating transcription factor ZEB1 stimulated Golgi compaction and relieved microRNA-mediated repression of the Golgi scaffolding protein PAQR11. Depletion of PAQR11 dispersed Golgi organelles and impaired anterograde vesicle transport to the plasma membrane as well as retrograde vesicle tethering to the Golgi. The N-terminal scaffolding domain of PAQR11 was associated with key regulators of Golgi compaction and vesicle transport in pull-down assays and was required to reconstitute Golgi compaction in PAQR11-deficient tumor cells. Finally, high PAQR11 levels were correlated with EMT and shorter survival in human cancers, and PAQR11 was found to be essential for tumor cell migration and metastasis in EMT-driven lung adenocarcinoma models. We conclude that EMT initiates a PAQR11-mediated Golgi compaction process that drives metastasis.
Assuntos
Adenocarcinoma/metabolismo , Movimento Celular , Transição Epitelial-Mesenquimal , Complexo de Golgi/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Progesterona/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patologia , Linhagem Celular Tumoral , Deleção de Genes , Complexo de Golgi/genética , Complexo de Golgi/patologia , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Metástase Neoplásica , Proteínas de Neoplasias/genética , Domínios Proteicos , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Receptores de Progesterona/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismoRESUMO
UNLABELLED: Intratumoral collagen cross-links heighten stromal stiffness and stimulate tumor cell invasion, but it is unclear how collagen cross-linking is regulated in epithelial tumors. To address this question, we used Kras(LA1) mice, which develop lung adenocarcinomas from somatic activation of a Kras(G12D) allele. The lung tumors in Kras(LA1) mice were highly fibrotic and contained cancer-associated fibroblasts (CAF) that produced collagen and generated stiffness in collagen gels. In xenograft tumors generated by injection of wild-type mice with lung adenocarcinoma cells alone or in combination with CAFs, the total concentration of collagen cross-links was the same in tumors generated with or without CAFs, but coinjected tumors had higher hydroxylysine aldehyde-derived collagen cross-links (HLCC) and lower lysine-aldehyde-derived collagen cross-links (LCCs). Therefore, we postulated that an LCC-to-HLCC switch induced by CAFs promotes the migratory and invasive properties of lung adenocarcinoma cells. To test this hypothesis, we created coculture models in which CAFs are positioned interstitially or peripherally in tumor cell aggregates, mimicking distinct spatial orientations of CAFs in human lung cancer. In both contexts, CAFs enhanced the invasive properties of tumor cells in three-dimensional (3D) collagen gels. Tumor cell aggregates that attached to CAF networks on a Matrigel surface dissociated and migrated on the networks. Lysyl hydroxylase 2 (PLOD2/LH2), which drives HLCC formation, was expressed in CAFs, and LH2 depletion abrogated the ability of CAFs to promote tumor cell invasion and migration. IMPLICATIONS: CAFs induce a collagen cross-link switch in tumor stroma to influence the invasive properties of tumor cells.
Assuntos
Adenocarcinoma/patologia , Colágeno/metabolismo , Fibroblastos/patologia , Neoplasias Pulmonares/patologia , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/metabolismo , Células Tumorais Cultivadas/patologia , Adenocarcinoma/genética , Adenocarcinoma de Pulmão , Animais , Movimento Celular , Células Cultivadas , Técnicas de Cocultura , Fibroblastos/metabolismo , Neoplasias Pulmonares/genética , Camundongos , Neoplasias Experimentais , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
Tumor progression to invasive carcinoma is associated with activation of SRC family kinase (SRC, YES, FYN) activity and loss of cellular cohesion. The hippo pathway-regulated cofactor YAP1 supports the tumorigenicity of RAS mutations but requires both inactivation of hippo signaling and YES-mediated phosphorylation of YAP1 for oncogenic activity. Exactly how SRC kinases are activated and hippo signaling is lost in sporadic human malignancies remains unknown. Here, we provide evidence that hippo-mediated inhibition of YAP1 is lost upon promoter methylation of the RAS effector and hippo kinase scaffold RASSF1A. We find that RASSF1A promoter methylation reduces YAP phospho-S127, which derepresses YAP1, and actively supports YAP1 activation by switching RASSF1 transcription to the independently transcribed RASSF1C isoform that promotes Tyr kinase activity. Using affinity proteomics, proximity ligation, and real-time molecular visualization, we find that RASSF1C targets SRC/YES to epithelial cell-cell junctions and promotes tyrosine phosphorylation of E-cadherin, ß-catenin, and YAP1. RASSF1A restricts SRC activity, preventing motility, invasion, and tumorigenesis in vitro and in vivo, with epigenetic inactivation correlating with increased inhibitory pY527-SRC in breast tumors. These data imply that distinct RASSF1 isoforms have opposing functions, which provide a biomarker for YAP1 activation and explain correlations of RASSF1 methylation with advanced invasive disease in humans. The ablation of epithelial integrity together with subsequent YAP1 nuclear localization allows transcriptional activation of ß-catenin/TBX-YAP/TEAD target genes, including Myc, and an invasive phenotype. These findings define gene transcript switching as a tumor suppressor mechanism under epigenetic control.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Transformação Celular Neoplásica , Regulação Neoplásica da Expressão Gênica , Fosfoproteínas/genética , Transdução de Sinais , Proteínas Supressoras de Tumor/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Fosfoproteínas/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Fatores de Transcrição , Ativação Transcricional , Proteínas Supressoras de Tumor/metabolismo , Proteínas de Sinalização YAPRESUMO
Epithelial tumor metastasis is preceded by an accumulation of collagen cross-links that heighten stromal stiffness and stimulate the invasive properties of tumor cells. However, the biochemical nature of collagen cross-links in cancer is still unclear. Here, we postulated that epithelial tumorigenesis is accompanied by changes in the biochemical type of collagen cross-links. Utilizing resected human lung cancer tissues and a p21CIP1/WAF1-deficient, K-rasG12D-expressing murine metastatic lung cancer model, we showed that, relative to normal lung tissues, tumor stroma contains higher levels of hydroxylysine aldehyde-derived collagen cross-links (HLCCs) and lower levels of lysine aldehyde-derived cross-links (LCCs), which are the predominant types of collagen cross-links in skeletal tissues and soft tissues, respectively. Gain- and loss-of-function studies in tumor cells showed that lysyl hydroxylase 2 (LH2), which hydroxylates telopeptidyl lysine residues on collagen, shifted the tumor stroma toward a high-HLCC, low-LCC state, increased tumor stiffness, and enhanced tumor cell invasion and metastasis. Together, our data indicate that LH2 enhances the metastatic properties of tumor cells and functions as a regulatory switch that controls the relative abundance of biochemically distinct types of collagen cross-links in the tumor stroma.
Assuntos
Adenocarcinoma/enzimologia , Carcinoma de Células Escamosas/enzimologia , Colágeno/metabolismo , Neoplasias Pulmonares/enzimologia , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/fisiologia , Adenocarcinoma/mortalidade , Adenocarcinoma/secundário , Animais , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/secundário , Linhagem Celular Tumoral , Células Cultivadas , Indução Enzimática , Matriz Extracelular/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Camundongos da Linhagem 129 , Camundongos Transgênicos , Transplante de Neoplasias , Regiões Promotoras Genéticas , Fator de Transcrição STAT3/metabolismo , Microambiente Tumoral , Regulação para CimaRESUMO
The aim of this study was to analyze the potential role of NG2 chondroitin sulfate proteoglycan in amoeboid morphology and invasiveness of cancer cells. In the highly metastatic amoeboid cell lines A3 and A375M2, siRNA-mediated down-regulation of NG2 induced an amoeboid-mesenchymal transition associated with decreased invasiveness in 3D collagen and inactivation of the GTPase Rho. Conversely, the expression of NG2 in mesenchymal sarcoma K2 cells as well as in A375M2 cells resulted in an enhanced amoeboid phenotype associated with increased invasiveness and elevated Rho-GTP levels. Remarkably, the amoeboid-mesenchymal transition in A375M2 cells triggered by NG2 down-regulation was associated with increased extracellular matrix-degrading ability, although this was not sufficient to compensate for the decreased invasive capability caused by down-regulated Rho/ROCK signaling. Conversely, in K2 cells with overexpression of NG2, the ability to degrade the extracellular matrix was greatly reduced. Taken together, we suggest that NG2-mediated activation of Rho leading to effective amoeboid invasiveness is a possible mechanism through which NG2 could contribute to tumor cell invasion and metastasis.
Assuntos
Movimento Celular , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias/metabolismo , Neoplasias/ultraestrutura , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Linhagem Celular Tumoral , Proteoglicanas de Sulfatos de Condroitina/genética , Colágeno/química , Regulação para Baixo , Matriz Extracelular/metabolismo , Humanos , Proteínas de Membrana/genética , Conformação Molecular , Invasividade Neoplásica , Neoplasias/patologia , RNA Interferente Pequeno , Ratos , Regulação para Cima , Quinases Associadas a rho/metabolismoRESUMO
The newly established breast cancer cell line G3S1, derived from EM-G3 breast cancer progenitors, was analyzed for functional changes related to neoplastic progression manifested by elevated invasiveness and enhanced capability to degrade gelatin. Degradation of gelatin and invasiveness of G3S1 cells was found to be dependent on the activity of matrix proteinases and actin cytoskeletal dynamics. Therefore, the expression and activity of these proteases was compared in G3S1 and EM-G3 cells. Despite enhanced capability of G3S1 cells to degrade gelatin, these cells exhibited lower levels of secreted extracellular matrix degrading proteases than parental EM-G3 cells. However, the expression of membrane-bound MT1-MMP was strongly elevated in G3S1 cells. While the degradation of gelatin was associated with invadopodia-like structures in both EM-G3 and G3S1 cells, the cytoskeletal remodeling dynamics was greatly elevated in G3S1 cells, suggesting that upregulation of MT1-MMP, together with elevation of cytoskeletal remodeling dynamics can effectively cause elevated invasiveness and enhanced matrix degrading capability in G3S1 cells.
Assuntos
Actinas/metabolismo , Neoplasias da Mama/enzimologia , Movimento Celular , Citoesqueleto/enzimologia , Metaloproteinase 14 da Matriz/metabolismo , Células-Tronco Neoplásicas/enzimologia , Aprotinina/farmacologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/patologia , Dipeptídeos/farmacologia , Progressão da Doença , Feminino , Gelatina/metabolismo , Humanos , Leucina/análogos & derivados , Leucina/farmacologia , Toxinas Marinhas/farmacologia , Inibidores de Metaloproteinases de Matriz , Metaloproteinases da Matriz Secretadas/metabolismo , Invasividade Neoplásica , Células-Tronco Neoplásicas/patologia , Inibidores de Proteases/farmacologia , Pseudópodes/enzimologia , Regulação para CimaRESUMO
G3S1 cells are a new line derived from EM-G3 breast cancer cells by chronic nutritional stress and treatments with 12-O-tetradecanoylphorbol-13-acetate. These cells are capable of growing in standard medium. G3S1 cells exhibited elevated invasiveness in Matrigel invasion chambers as compared with parental EM-G3 cells. Elevated invasiveness of G3S1 cells was accompanied by higher incidence of myzitiras morphotype (sucker-like) and newly observed vthela morphotype (leech-like) both inducible in Hanks' Balanced Salt Solution test. Time-lapse phase contrast microscopy showed a capacity of G3S1 cells to form lobopodial protrusions already 20 min after seeding on gelatin. These protrusions could make contact with the dish and possibly produce the vthela shape. The possible relationship of mysitiras and vthela morphotypes to an increase in malignant potential marked by enhanced invasiveness was thus indicated.