RESUMEN
Epithelial-mesenchymal transition (EMT) programs are essential in promoting breast cancer invasion, systemic dissemination and in arousing proliferative programs in breast cancer micrometastases, a reaction that is partially dependent on focal adhesion kinase (FAK). Many functions of FAK are shared by its homolog, protein tyrosine kinase 2 (Pyk2), raising the question as to whether Pyk2 also participates in driving the metastatic outgrowth of disseminated breast cancer cells. In addressing this question, we observed Pyk2 expression to be (i) significantly upregulated in recurrent human breast cancers; (ii) differentially expressed across clonal isolates of human MDA-MB-231 breast cancer cells in a manner predictive for metastatic outgrowth, but not for invasiveness; and (iii) dramatically elevated in ex vivo cultures of breast cancer cells isolated from metastatic lesions as compared with cells that produced the primary tumor. We further show that metastatic human and murine breast cancer cells robustly upregulate their expression of Pyk2 during EMT programs stimulated by transforming growth factor-ß (TGF-ß). Genetic and pharmacological inhibition of Pyk2 demonstrated that the activity of this protein tyrosine kinase was dispensable for the ability of breast cancer cells to undergo invasion in response to TGF-ß, and to form orthotopic mammary tumors in mice. In stark contrast, Pyk2-deficiency prevented TGF-ß from stimulating the growth of breast cancer cells in 3D-organotypic cultures that recapitulated pulmonary microenvironments, as well as inhibited the metastatic outgrowth of disseminated breast cancer cells in the lungs of mice. Mechanistically, Pyk2 expression was inversely related to that of E-cadherin, such that elevated Pyk2 levels stabilized ß1 integrin expression necessary to initiate the metastatic outgrowth of breast cancer cells. Thus, we have delineated novel functions for Pyk2 in mediating distinct elements of the EMT program and metastatic cascade regulated by TGF-ß, particularly the initiation of secondary tumor outgrowth by disseminated cells.
Asunto(s)
Neoplasias de la Mama/enzimología , Transición Epitelial-Mesenquimal/fisiología , Quinasa 2 de Adhesión Focal/biosíntesis , Neoplasias Mamarias Experimentales/enzimología , Factor de Crecimiento Transformador beta/farmacología , Animales , Biomarcadores de Tumor/biosíntesis , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Transición Epitelial-Mesenquimal/efectos de los fármacos , Femenino , Humanos , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/secundario , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Transducción de Señal/efectos de los fármacos , Proteína Smad4/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
Transforming growth factor-ß (TGF-ß) and epidermal growth factor (EGF) have critical roles in regulating the metastasis of aggressive breast cancers, yet the impact of epithelial-mesenchymal transition (EMT) induced by TGF-ß in altering the response of breast cancer cells to EGF remains unknown. We show in this study that murine metastatic 4T1 breast cancer cells formed compact and dense spheroids when cultured under three-dimensional (3D) conditions, which was in sharp contrast to the branching phenotypes exhibited by their nonmetastatic counterparts. Using the human MCF10A series, we show that epithelial-type and nonmetastatic breast cancer cells were unable to invade to EGF, whereas their mesenchymal-type and metastatic counterparts readily invaded to EGF. Furthermore, EMT induced by TGF-ß was sufficient to manifest dense spheroid morphologies, a phenotype that increased primary tumor exit and invasion to EGF. Post-EMT invasion to EGF was dependent on increased activation of EGF receptor (EGFR) and p38 mitogen-activated protein kinase, all of which could be abrogated either by pharmacologic (PF-562271) or by genetic (shRNA) targeting of focal adhesion kinase (FAK). Mechanistically, EMT induced by TGF-ß increased cell-surface levels of EGFR and prevented its physical interaction with E-cadherin, leading instead to the formation of oncogenic signaling complexes with TßR-II. Elevated EGFR expression was sufficient to transform normal mammary epithelial cells, and to progress their 3D morphology from that of hollow acini to branched structures characteristic of nonmetastatic breast cancer cells. Importantly, we show that TGF-ß stimulation of EMT enabled this EGFR-driven breast cancer model to abandon their inherent branching architecture and form large, undifferentiated masses that were hyperinvasive to EGF and showed increased pulmonary tumor growth upon tail vein injection. Finally, chemotherapeutic targeting of FAK was sufficient to revert the aggressive behaviors of these structures. Collectively, this investigation has identified a novel EMT-based approach to neutralize the oncogenic activities of EGF and TGF-ß in aggressive and invasive forms of breast cancer.
Asunto(s)
Neoplasias de la Mama/metabolismo , Carcinoma/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Transición Epitelial-Mesenquimal , Neoplasias Mamarias Experimentales/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Cadherinas/metabolismo , Carcinoma/tratamiento farmacológico , Carcinoma/secundario , Línea Celular Tumoral , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/metabolismo , Femenino , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Humanos , Indoles/farmacología , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/patología , Ratones , ARN Interferente Pequeño/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Sulfonamidas/farmacología , Factor de Crecimiento Transformador beta/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Expression of the chemokine receptor CXCR4 has been linked with increased metastasis and decreased clinical prognosis in breast cancer. The current paradigm dictates that CXCR4 fosters carcinoma cell metastasis along a chemotactic gradient to organs expressing the ligand CXCL12. The present study asked if alterations in autocrine CXCR4 signaling via dysregulation of CXCL12 in mammary carcinoma cells modulated their metastatic potential. While CXCR4 was consistently detected, expression of CXCL12 characteristic of human mammary epithelium was silenced by promoter hypermethylation in breast cancer cell lines and primary mammary tumors. Stable re-expression of functional CXCL12 in ligand null cells increased orthotopic primary tumor growth in the mammary fat-pad model of tumorigenesis. Those data parallel increased carcinoma cell proliferation measured in vitro with little-to-no-impact on apoptosis. Moreover, re-expression of autocrine CXCL12 markedly reduced metastatic lung invasion assessed using in vivo bioluminescence imaging following tail vein injection. Consistent with those data, decreased metastasis reflected diminished intracellular calcium signaling and chemotactic migration in response to exogenous CXCL12 independent of changes in CXCR4 expression. Together these data suggest that an elevated migratory signaling response to ectopic CXCL12 contributes to the metastatic potential of CXCR4-expressing mammary carcinoma cells, subsequent to epigenetic silencing of autocrine CXCL12.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Quimiocina CXCL12/antagonistas & inhibidores , Quimiocina CXCL12/genética , Epigénesis Genética/fisiología , Silenciador del Gen/fisiología , Neoplasias Pulmonares/secundario , Animales , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Quimiocina CXCL12/fisiología , Metilación de ADN , Femenino , Células HT29 , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/prevención & control , Ratones , Ratones SCID , Invasividad Neoplásica , Regiones Promotoras Genéticas/fisiología , Receptores CXCR4/fisiología , Células U937RESUMEN
Cellular metastasis is the most detrimental step in carcinoma disease progression, yet the mechanisms that regulate this process are poorly understood. CXCL12 and its receptor CXCR4 are co-expressed in several tissues and cell types throughout the body and play essential roles in development. Disruption of either gene causes embryonic lethality due to similar defects. Post-natally, CXCL12 signaling has a wide range of effects on CXCR4-expressing cells, including the directed migration of leukocytes, lymphocytes and hematopoietic stem cells. Recently, this signaling axis has also been described as an important regulator of directed carcinoma cell metastasis. We show herein that while CXCR4 expression remains consistent, constitutive colonic epithelial expression of CXCL12 is silenced by DNA hypermethylation in primary colorectal carcinomas as well as colorectal carcinoma-derived cell lines. Inhibition of DNA methyltransferase (Dnmt) enzymes with 5-aza-2'-deoxycytidine or genetic ablation of both Dnmt1 and Dnmt3b prevented promoter methylation and restored CXCL12 expression. Re-expression of functional, endogenous CXCL12 in colorectal carcinoma cells dramatically reduced metastatic tumor formation in mice, as well as foci formation in soft agar. Decreased metastasis was correlated with increased caspase activity in cells re-expressing CXCL12. These data constitute the unique observation that silencing CXCL12 within colonic carcinoma cells greatly enhances their metastatic potential.