RESUMO
BACKGROUND: Epithelial-to-mesenchymal transition (EMT) is associated with downregulated E-cadherin and frequently with decreased proliferation. Proliferation may be restored in secondary metastases by mesenchymal-to-epithelial transition (MET). We tested whether E-cadherin maintains epithelial proliferation in MDA-MB-468 breast cancer cells, facilitating metastatic colonization in severe combined immunodeficiency (SCID) mice. METHODS: EMT/MET markers were assessed in xenograft tumors by immunohistochemistry. Stable E-cadherin manipulation was effected by transfection and verified by Western blotting, immunocytochemistry, and quantitative polymerase chain reaction (qPCR). Effects of E-cadherin manipulation on proliferation and chemomigration were assessed in vitro by performing sulforhodamine B assays and Transwell assays, respectively. Invasion was assessed by Matrigel outgrowth; growth in vivo was assessed in SCID mice; and EMT status was assessed by qPCR. Hypoxic response of E-cadherin knockdown cell lines was assessed by qPCR after hypoxic culture. Repeated measures analysis of variance (ANOVA), one- and two-way ANOVA with posttests, and paired Student's t tests were performed to determine significance (p < 0.05). RESULTS: EMT occurred at the necrotic interface of MDA-MB-468 xenografts in regions of hypoxia. Extratumoral deposits (vascular and lymphatic inclusions, local and axillary nodes, and lung metastases) strongly expressed E-cadherin. MDA-MB-468 cells overexpressing E-cadherin were more proliferative and less migratory in vitro, whereas E-cadherin knockdown (short hairpin CDH1 [shCDH1]) cells were more migratory and invasive, less proliferative, and took longer to form tumors. shCDH1-MDA-MB-468 xenografts did not contain the hypoxia-induced necrotic areas observed in wild-type (WT) and shSCR-MDA-MB-468 tumors, but they did not exhibit an impaired hypoxic response in vitro. Although vimentin expression was not stimulated by E-cadherin knockdown in 2D or 3D cultures, xenografts of these cells were globally vimentin-positive rather than exhibiting regional EMT, and they expressed higher SNA1 than their in vitro counterparts. E-cadherin suppression caused a trend toward reduced lung metastasis, whereas E-cadherin overexpression resulted in the reverse trend, consistent with the increased proliferation rate and predominantly epithelial phenotype of MDA-MB-468 cells outside the primary xenograft. This was also originally observed in WT xenografts. Furthermore, we found that patients with breast cancer that expressed E-cadherin were more likely to have metastases. CONCLUSIONS: E-cadherin expression promotes growth of primary breast tumors and conceivably the formation of metastases, supporting a role for MET in metastasis. E-cadherin needs to be reevaluated as a tumor suppressor.
Assuntos
Neoplasias da Mama/genética , Caderinas/genética , Proliferação de Células/genética , Transição Epitelial-Mesenquimal/genética , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Células Epiteliais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Camundongos , Metástase Neoplásica , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Recent evidence has implicated the transmembrane co-receptor neuropilin-1 (NRP1) in cancer progression. Primarily known as a regulator of neuronal guidance and angiogenesis, NRP1 is also expressed in multiple human malignancies, where it promotes tumor angiogenesis. However, non-angiogenic roles of NRP1 in tumor progression remain poorly characterized. In this study, we define NRP1 as an androgen-repressed gene whose expression is elevated during the adaptation of prostate tumors to androgen-targeted therapies (ATTs), and subsequent progression to metastatic castration-resistant prostate cancer (mCRPC). Using short hairpin RNA (shRNA)-mediated suppression of NRP1, we demonstrate that NRP1 regulates the mesenchymal phenotype of mCRPC cell models and the invasive and metastatic dissemination of tumor cells in vivo. In patients, immunohistochemical staining of tissue microarrays and mRNA expression analyses revealed a positive association between NRP1 expression and increasing Gleason grade, pathological T score, positive lymph node status and primary therapy failure. Furthermore, multivariate analysis of several large clinical prostate cancer (PCa) cohorts identified NRP1 expression at radical prostatectomy as an independent prognostic biomarker of biochemical recurrence after radiation therapy, metastasis and cancer-specific mortality. This study identifies NRP1 for the first time as a novel androgen-suppressed gene upregulated during the adaptive response of prostate tumors to ATTs and a prognostic biomarker of clinical metastasis and lethal PCa.
Assuntos
Neuropilina-1/genética , Neuropilina-1/metabolismo , Neoplasias de Próstata Resistentes à Castração/mortalidade , Neoplasias da Próstata/tratamento farmacológico , Regulação para Cima , Antagonistas de Androgênios/uso terapêutico , Linhagem Celular Tumoral , Progressão da Doença , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Gradação de Tumores , Metástase Neoplásica , Neoplasias da Próstata/genética , Neoplasias da Próstata/mortalidade , Análise de SobrevidaRESUMO
BACKGROUND: Epidermal homeostasis is maintained through the balance between keratinocyte proliferation, differentiation and desquamation; however, human skin equivalent (HSE) models are known to differentiate excessively. In native tissue, proteases such as kallikrein-related peptidase (KLK) 5 and KLK7 cleave the extracellular components of corneodesmosomes; proteins corneodesmosin, desmocollin 1 and desmoglein 1, loosening the cellular connections and enabling desquamation. The actions of KLK7 are tightly controlled by protease inhibitors, skin-derived antileucoproteinase (SKALP) and lymphoepithelial Kazal-type-related inhibitor (LEKTI), which also inhibits KLK5, localizing protease activity to the stratum corneum. OBJECTIVES: To investigate the mechanisms that inhibit the desquamation cascade in HSE models. METHODS: Human skin tissue and HSE models were investigated using gene microarray, real-time polymerase chain reaction (PCR), immunohistochemistry and Western blot analysis to examine key components of the desquamation pathway. To elucidate proteolytic activity in HSEs and native skin, in situ and gel zymography was performed. RESULTS: Histological analysis indicated that HSE models form a well-organized epidermis, yet develop an excessively thick and compact stratum corneum. Gene microarray analysis revealed that the desquamation cascade was dysregulated in HSE models and this was confirmed using real-time PCR and immunohistochemistry. Immunohistochemistry and Western blot indicated overexpression of LEKTI and SKALP in HSEs. Although KLK7 was also highly expressed in HSEs, zymography indicated that protease activation and activity was lower than in native skin. CONCLUSIONS: These findings demonstrate that stratum corneum thickening is due to inhibited KLK5 and KLK7 activation and a subsequent lack of corneodesmosome degradation in the HSE model epidermis.
Assuntos
Epiderme/patologia , Calicreínas/metabolismo , Queratinócitos/efeitos dos fármacos , Adulto , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Desmossomos/genética , Células Epidérmicas , Epiderme/metabolismo , Expressão Gênica , Genoma Humano/genética , Humanos , Queratinócitos/metabolismo , Antígeno Ki-67/metabolismo , Proteínas de Membrana/metabolismo , Análise em Microsséries/métodos , Modelos Biológicos , Proteínas/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
MicroRNA-375 (miR-375) is frequently elevated in prostate tumors and cell-free fractions of patient blood, but its role in genesis and progression of prostate cancer is poorly understood. In this study, we demonstrated that miR-375 is inversely correlated with epithelial-mesenchymal transition signatures (EMT) in clinical samples and can drive mesenchymal-epithelial transition (MET) in model systems. Indeed, miR-375 potently inhibited invasion and migration of multiple prostate cancer lines. The transcription factor YAP1 was found to be a direct target of miR-375 in prostate cancer. Knockdown of YAP1 phenocopied miR-375 overexpression, and overexpression of YAP1 rescued anti-invasive effects mediated by miR-375. Furthermore, transcription of the miR-375 gene was shown to be directly repressed by the EMT transcription factor, ZEB1. Analysis of multiple patient cohorts provided evidence for this ZEB1-miR-375-YAP1 regulatory circuit in clinical samples. Despite its anti-invasive and anti-EMT capacities, plasma miR-375 was found to be correlated with circulating tumor cells in men with metastatic disease. Collectively, this study provides new insight into the function of miR-375 in prostate cancer, and more broadly identifies a novel pathway controlling epithelial plasticity and tumor cell invasion in this disease.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Fosfoproteínas/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Transdução de Sinais , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Regiões 3' não Traduzidas , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Biomarcadores , Linhagem Celular Tumoral , Epitélio/metabolismo , Epitélio/patologia , Expressão Gênica , Humanos , Masculino , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologia , Fenótipo , Fosfoproteínas/metabolismo , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Interferência de RNA , Fatores de Transcrição , Proteínas de Sinalização YAP , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genéticaRESUMO
Metastatic competence is contingent upon the aberrant activation of a latent embryonic program, known as the epithelial-mesenchymal transition (EMT), which bestows stem cell properties as well as migratory and invasive capabilities upon differentiated tumor cells. We recently identified the transcription factor FOXC2 as a downstream effector of multiple EMT programs, independent of the EMT-inducing stimulus, and as a key player linking EMT, stem cell traits and metastatic competence in breast cancer. As such, FOXC2 could serve as a potential therapeutic target to attenuate metastasis. However, as FOXC2 is a transcription factor, it is difficult to target by conventional means such as small-molecule inhibitors. Herein, we identify the serine/threonine-specific kinase p38 as a druggable upstream regulator of FOXC2 stability and function that elicits phosphorylation of FOXC2 at serine 367 (S367). Using an orthotopic syngeneic mouse tumor model, we make the striking observation that inhibition of p38-FOXC2 signaling selectively attenuates metastasis without impacting primary tumor growth. In this model, circulating tumor cell numbers are significantly reduced in mice treated with the p38 inhibitor SB203580, relative to vehicle-treated counterparts. Accordingly, genetic or pharmacological inhibition of p38 decreases FOXC2 protein levels, reverts the EMT phenotype and compromises stem cell attributes in vitro. We also identify the EMT-regulator ZEB1-known to directly repress E-cadherin/CDH1-as a downstream target of FOXC2, critically dependent on its activation by p38. Consistent with the notion that activation of the p38-FOXC2 signaling axis represents a critical juncture in the acquisition of metastatic competence, the phosphomimetic FOXC2(S367E) mutant is refractory to p38 inhibition both in vitro and in vivo, whereas the non-phosphorylatable FOXC2(S367A) mutant fails to elicit EMT and upregulate ZEB1. Collectively, our data demonstrate that FOXC2 regulates EMT, stem cell traits, ZEB1 expression and metastasis in a p38-dependent manner, and attest to the potential utility of p38 inhibitors as antimetastatic agents.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Fatores de Transcrição Forkhead/metabolismo , Serina/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Antineoplásicos/farmacologia , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal/genética , Feminino , Xenoenxertos , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Metástase Neoplásica , Células-Tronco Neoplásicas/metabolismo , Fenótipo , Fosforilação , Ligação Proteica , RNA Interferente Pequeno/genéticaRESUMO
Hematogenous metastases are rarely present at diagnosis of ovarian clear cell carcinoma (OCC). Instead dissemination of these tumors is characteristically via direct extension of the primary tumor into nearby organs and the spread of exfoliated tumor cells throughout the peritoneum, initially via the peritoneal fluid, and later via ascites that accumulates as a result of disruption of the lymphatic system. The molecular mechanisms orchestrating these processes are uncertain. In particular, the signaling pathways used by malignant cells to survive the stresses of anchorage-free growth in peritoneal fluid and ascites, and to colonize remote sites, are poorly defined. We demonstrate that the transmembrane glycoprotein CUB-domain-containing protein 1 (CDCP1) has important and inhibitable roles in these processes. In vitro assays indicate that CDCP1 mediates formation and survival of OCC spheroids, as well as cell migration and chemoresistance. Disruption of CDCP1 via silencing and antibody-mediated inhibition markedly reduce the ability of TOV21G OCC cells to form intraperitoneal tumors and induce accumulation of ascites in mice. Mechanistically our data suggest that CDCP1 effects are mediated via a novel mechanism of protein kinase B (Akt) activation. Immunohistochemical analysis also suggested that CDCP1 is functionally important in OCC, with its expression elevated in 90% of 198 OCC tumors and increased CDCP1 expression correlating with poor patient disease-free and overall survival. This analysis also showed that CDCP1 is largely restricted to the surface of malignant cells where it is accessible to therapeutic antibodies. Importantly, antibody-mediated blockade of CDCP1 in vivo significantly increased the anti-tumor efficacy of carboplatin, the chemotherapy most commonly used to treat OCC. In summary, our data indicate that CDCP1 is important in the progression of OCC and that targeting pathways mediated by this protein may be useful for the management of OCC, potentially in combination with chemotherapies and agents targeting the Akt pathway.
Assuntos
Adenocarcinoma de Células Claras/mortalidade , Adenocarcinoma de Células Claras/patologia , Antígenos CD/metabolismo , Moléculas de Adesão Celular/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/patologia , Adenocarcinoma de Células Claras/metabolismo , Animais , Antígenos CD/análise , Antígenos CD/genética , Antígenos de Neoplasias , Carboplatina/farmacologia , Moléculas de Adesão Celular/análise , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral/efeitos dos fármacos , Movimento Celular , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Estimativa de Kaplan-Meier , Camundongos Endogâmicos NOD , Proteínas de Neoplasias/análise , Proteínas de Neoplasias/genética , Neoplasias Ovarianas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Transition between epithelial and mesenchymal states is a feature of both normal development and tumor progression. We report that expression of chloride channel accessory protein hCLCA2 is a characteristic of epithelial differentiation in the immortalized MCF10A and HMLE models, while induction of epithelial-to-mesenchymal transition by cell dilution, TGFß or mesenchymal transcription factors sharply reduces hCLCA2 levels. Attenuation of hCLCA2 expression by lentiviral small hairpin RNA caused cell overgrowth and focus formation, enhanced migration and invasion, and increased mammosphere formation in methylcellulose. These changes were accompanied by downregulation of E-cadherin and upregulation of mesenchymal markers such as vimentin and fibronectin. Moreover, hCLCA2 expression is greatly downregulated in breast cancer cells with a mesenchymal or claudin-low profile. These observations suggest that loss of hCLCA2 may promote metastasis. We find that higher-than-median expression of hCLCA2 is associated with a one-third lower rate of metastasis over an 18-year period among breast cancer patients compared with lower-than-median (n=344, unfiltered for subtype). Thus, hCLCA2 is required for epithelial differentiation, and its loss during tumor progression contributes to metastasis. Overexpression of hCLCA2 has been reported to inhibit cell proliferation and is accompanied by increases in chloride current at the plasma membrane and reduced intracellular pH (pHi). We found that knockdown cells have sharply reduced chloride current and higher pHi, both characteristics of tumor cells. These results suggest a mechanism for the effects on differentiation. Loss of hCLCA2 may allow escape from pHi homeostatic mechanisms, permitting the higher intracellular and lower extracellular pH that are characteristic of aggressive tumor cells.