Subtype-specific overexpression of the Rac-GEF P-REX1 in breast cancer is associated with promoter hypomethylation.

INTRODUCTION: The Rac-GEF P-REX1 is a key mediator of ErbB signaling in breast cancer recently implicated in mammary tumorigenesis and metastatic dissemination. Although P-REX1 is essentially undetectable in normal human mammary epithelial tissue, this Rac-GEF is markedly upregulated in human breast carcinomas, particularly of the luminal subtype. The mechanisms underlying P-REX1 upregulation in breast cancer are unknown. Toward the goal of dissecting the mechanistic basis of P-REX1 overexpression in breast cancer, in this study we focused on the analysis of methylation of the PREX1 gene promoter. METHODS: To determine the methylation status of the PREX1 promoter region, we used bisulfite genomic sequencing and pyrosequencing approaches. Re-expression studies in cell lines were carried out by treatment of breast cancer cells with the demethylating agent 5-aza-2'-deoxycitidine. PREX1 gene methylation in different human breast cancer subtypes was analyzed from the TCGA database. RESULTS: We found that the human PREX1 gene promoter has a CpG island located between -1.2 kb and +1.4 kb, and that DNA methylation in this region inversely correlates with P-REX1 expression in human breast cancer cell lines. A comprehensive analysis of human breast cancer cell lines and tumors revealed significant hypomethylation of the PREX1 promoter in ER-positive, luminal subtype, whereas hypermethylation occurs in basal-like breast cancer. Treatment of normal MCF-10A or basal-like cancer cells, MDA-MB-231 with the demethylating agent 5-aza-2'-deoxycitidine in combination with the histone deacetylase inhibitor trichostatin A restores P-REX1 levels to those observed in luminal breast cancer cell lines, suggesting that aberrant expression of P-REX1 in luminal breast cancer is a consequence of PREX1 promoter demethylation. Unlike PREX1, the pro-metastatic Rho/Rac-GEF, VAV3, is not regulated by methylation. Notably, PREX1 gene promoter hypomethylation is a prognostic marker of poor patient survival. CONCLUSIONS: Our study identified for the first time gene promoter hypomethylation as a distinctive subtype-specific mechanism for controlling the expression of a key regulator of Rac-mediated motility and metastasis in breast cancer.

Predictive Outcomes for HER2-enriched Cancer Using Growth and Metastasis Signatures Driven By SPARC.

Understanding the mechanism of metastatic dissemination is crucial for the rational design of novel therapeutics. The secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein which has been extensively associated with human breast cancer aggressiveness although the underlying mechanisms are still unclear. Here, shRNA-mediated SPARC knockdown greatly reduced primary tumor growth and completely abolished lung colonization of murine 4T1 and LM3 breast malignant cells implanted in syngeneic BALB/c mice. A comprehensive study including global transcriptomic analysis followed by biological validations confirmed that SPARC induces primary tumor growth by enhancing cell cycle and by promoting a COX-2-mediated expansion of myeloid-derived suppressor cells (MDSC). The role of SPARC in metastasis involved a COX-2-independent enhancement of cell disengagement from the primary tumor and adherence to the lungs that fostered metastasis implantation. Interestingly, SPARC-driven gene expression signatures obtained from these murine models predicted the clinical outcome of patients with HER2-enriched breast cancer subtypes. In total, the results reveal that SPARC and its downstream effectors are attractive targets for antimetastatic therapies in breast cancer.Implications: These findings shed light on the prometastatic role of SPARC, a key protein expressed by breast cancer cells and surrounding stroma, with important consequences for disease outcome. Mol Cancer Res; 15(3); 304-16. ©2016 AACR.

SPARC promotes cathepsin B-mediated melanoma invasiveness through a collagen I/α2ß1 integrin axis.

In melanoma, the extracellular protein SPARC (secreted protein acidic and rich in cysteine) is related to tumor progression. Some of the evidence that links SPARC to melanoma progression indicates that SPARC may be involved in the acquisition of mesenchymal traits that favor metastatic dissemination. However, no molecular pathways that link extracellular SPARC to a mesenchymal phenotype have been described. In this study, global protein expression analysis of the melanoma secretome following enforced downregulation of SPARC expression led us to elucidate a new molecular mechanism by which SPARC promotes cathepsin B-mediated melanoma invasiveness using collagen I and α2ß1 integrins as mediators. Interestingly, we also found that the transforming growth factor (TGF)-ß1 contribution to cathepsin B-mediated invasion is highly SPARC dependent. In addition, induction of the E-cadherin to N-cadherin switch by SPARC enabled melanoma cells to transmigrate across an endothelial layer through a mechanism independent to that of enhancing invasion. Finally, SPARC also enhanced the extracellular expression of other proteins involved in epithelial-mesenchymal transformation, such as family with sequence similarity 3, member C/interleukin-like EMT-inducer. Our findings demonstrate a previously unreported molecular pathway for SPARC activity on invasion and support an active role of SPARC in the mesenchymal transformation that contributes to melanoma dissemination.

SPARC endogenous level, rather than fibroblast-produced SPARC or stroma reorganization induced by SPARC, is responsible for melanoma cell growth.

SPARC (secreted protein acidic and rich in cysteine) is a matricellular protein whose overexpression in malignant or tumor-stromal cells is often associated with increased aggressiveness and bad prognosis in a wide range of human cancer types, particularly melanoma. We established the impact that changes in the level of SPARC produced by malignant cells and neighboring stromal cells have on melanoma growth. Melanoma cell growth in monolayer was only slightly affected by changes in SPARC levels. However, melanoma growth in spheroids was strongly inhibited upon SPARC hyperexpression and conversely enhanced when SPARC expression was downregulated. Interestingly, SPARC overexpression in neighboring fibroblasts had no effect on spheroid growth irrespective of SPARC levels expressed by the melanoma cells, themselves. Downregulation of SPARC expression in melanoma cells induced their rejection in vivo through a mechanism mediated exclusively by host polymorphonuclear cells. On the other hand, SPARC hyperexpression enhanced vascular density, collagen deposition, and fibroblast recruitment in the surrounding stroma without affecting melanoma growth. In agreement with the in vitro data, overexpression of SPARC in co-injected fibroblasts did not affect melanoma growth in vivo. All the data indicate that melanoma growth is not subject to regulation by exogenous SPARC, nor by stromal organization, but only by SPARC levels produced by the malignant cells themselves.