The role of RhoA GTPases in breast cancer tumorigenesis and metastasis is unclear. Early studies within which mutations in RhoA were designed based on cancer-associated mutations in Ras supported an oncogene role for RhoA. However, recent whole-genome sequencing studies of cancers raised the possibility that RhoA may have a tumor suppression function. Here, using a syngeneic triple negative breast cancer murine model we investigated the physiological effects of reduced RhoA expression on breast cancer tumorigenesis and metastasis. RhoA knockdown had no effect on primary tumor formation and tumor proliferation, concurring with our in vitro findings where reduced RhoA had no effect on breast cancer cell proliferation and clonogenic growth. In contrast, primary tumors with RhoA knockdown efficiently invaded sentinel lymph nodes and significantly metastasized to lungs compared to control tumors. Mechanistically, the current study demonstrated that this is achieved by promoting a pro-tumor microenvironment, with increased cancer-associated fibroblasts and macrophage infiltration, and by modulating the CCL5-CCR5 and CXCL12-CXCR4 chemokine axes in the primary tumor. To our knowledge, this is the first such mechanistic study in breast cancer showing the ability of RhoA to suppress chemokine receptor expression in breast tumor cells. Our work suggests a physiological lung and lymph node metastasis suppressor role for RhoA GTPase in breast cancer.

Raf-1 kinase inhibitor protein was initially discovered as a physiological kinase inhibitor of the MAPK signaling pathway and was later shown to suppress cancer cell invasion and metastasis. Yet, the molecular mechanism through which RKIP executes its effects is not completely defined. RhoA has both a pro- and anti-metastatic cell-context dependent functions. Given that Rho GTPases primarily function on actin cytoskeleton dynamics and cell movement regulation, it is possible that one way RKIP hinders cancer cell invasion/metastasis is by targeting these proteins. Here we show that RKIP inhibits cancer cell invasion and metastasis by stimulating RhoA anti-tumorigenic functions. Mechanistically, RKIP activates RhoA in an Erk2 and GEF-H1 dependent manner to enhance E-cadherin membrane localization and inhibit CCL5 expression.

Recent high-throughput-sequencing of cancer genomes has identified oncogenic mutations in the B-Raf genetic locus as one of the critical events in melanomagenesis. B-Raf encodes a serine/threonine kinase that regulates the MAPK/ERK kinase (MEK) and extracellular signal-regulated kinase (ERK) protein kinase cascade. In normal cells, the activity of B-Raf is tightly regulated and is required for cell growth and survival. B-Raf gain-of-function mutations in melanoma frequently lead to unrestrained growth, enhanced cell invasion and increased viability of cancer cells. Although it is clear that the invasive phenotypes of B-Raf mutated melanoma cells are stringently dependent on B-Raf-MEK-ERK activation, the downstream effector targets that are required for oncogenic B-Raf-mediated melanomagenesis are not well defined. miRNAs have regulatory functions towards the expression of genes that are important in carcinogenesis. We observed that miR-10b expression correlates with the presence of the oncogenic B-Raf ( B-Raf V600E ) mutation in melanoma cells. While expression of miR-10b enhances anchorage-independent growth of B-Raf wild-type melanoma cells, miR-10b silencing decreases B-Raf V600E cancer cell invasion in vitro . Importantly, the expression of miR-10b is required for B-Raf V600E -mediated anchorage independent growth and invasion of melanoma cells in vitro . Taken together our results suggest that miR-10b is an important mediator of oncogenic B-Raf V600E activity in melanoma.

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26Recent high-throughput-sequencing of cancer genomes has identified oncogenic 27 mutations in the BRaf genetic locus as one of the critical events in melanomagenesis. 28 BRaf encodes a serine/threonine kinase that regulates the MAPK/ERK kinase (MEK) 29 and extracellular signal-regulated kinase (ERK) protein kinase cascade. In normal cells, 30 the activity of BRaf is tightly regulated and is required for cell growth and survival. BRaf 31 gain-of-function mutations in melanoma frequently lead to unrestrained growth, 32 enhanced cell invasion and increased viability of cancer cells. Although it is clear that 33 the invasive phenotypes of BRaf mutated melanoma cells are stringently dependent on 34 BRaf-MEK-ERK activation, the downstream effector targets that are required for 35 oncogenic BRaf-mediated melanomagenesis are not well defined. miRNAs have 36 regulatory functions towards the expression of genes that are important in 37 carcinogenesis. We observed that miR-10b expression correlates with the presence of 38 the oncogenic BRaf (BRaf V600E ) mutation in melanoma cells. While expression of miR-39 10b enhances anchorage-independent growth of BRaf wild-type melanoma cells, miR-40 10b silencing decreases BRaf V600E cancer cell invasion in vitro. Importantly, the 41 expression of miR-10b is required for BRaf V600E -mediated anchorage-independent 42 growth and invasion of melanoma cells in vitro. Taken together our results suggest that 43 miR-10b is an important mediator of oncogenic BRaf V600E activity in melanoma.44 45 46 3 47 48 49 Melanoma is the most aggressive of all the skin cancers. BRaf is a 50 serine/threonine protein kinase that activates the MEK/ERK-signaling pathway. About 51 25-70% of malignant melanomas harbor gain-of-function mutations in the oncogene 52 BRaf (1). Among several BRaf gain-of-function mutations, BRaf V600E is the most 53 common mutation and accounts for nearly 80% of them (1). Not only does BRaf V600E 54 cause a sustained activation of ERK signaling pathway in melanoma, it is also critical for 55 the malignant process and is one of the few identified driver mutations essential for 56 melanoma proliferation and survival. The transformation of melanocytes to melanoma 57 by BRaf V600E requires activation of the MEK-ERK kinases cascade with multiple 58 downstream components (2-4). The mechanism that integrates the diverse components 59 into a coordinated response to the BRaf V600E mutation remains undefined. 60 61 MicroRNAs (miRNAs) are small, non-protein coding RNA molecules and they 62 regulate gene expression through a combination of translational repression and mRNA 63 destabilization (5). Each miRNA targets ~200 mRNA molecules (6). Because of their 64 pleiotropic potentials, miRNAs are attractive candidates as master regulators of the 65 BRaf V600E oncogenic transformation program. In this study, we identified for the first 66 time, significant positive correlation between BRaf V600E mutation and microRNA-10b 67 (miR-10b) expression. Furthermore, we show that miR-10b is a novel ...

Background: Tumor metastasis suppressors are proteins that impede secondary tumor formation by inhibiting one or more steps of the metastasis cascade without stimulating primary tumor growth. Raf-1 kinase inhibitor protein (RKIP) is a metastasis suppressor that inhibits metastasis in breast, prostate, melanoma and several other types of cancers. The molecular mechanism through which RKIP executes its anti-metastasis effects is not yet completely defined. The objective of the current study is to understand how RKIP inhibits breast cancer cell invasion and metastasis in molecular level. Given its primary function in regulating actin cytoskeleton and cell movements, Rho GTPases were studied as possible downstream effectors of RKIP. These small GTPases belong to the Ras superfamily and consist of nearly 22 members with RhoA and Rac1 as the major players affecting cell motility. Rho GTPases oscillate between a GTP-bound active form and a GDP-bound inactive form. The GDP-GTP cycle is regulated by GEFs that facilitate the exchange of bound GDP with GTP, and by GAPs that stimulate the Rho GTPase activity. Aberrantly activated GEFs are reported to drive tumorigenesis, while activated GAPs are assumed to inhibit the cancer formation and metastasis. Hence, Rho GTPases are widely accepted as oncogenes. Contrary to this notion, several recent studies suggested a possible metastasis inhibitory function for wild-type RhoA in triple negative breast cancer and several other types of cancers. Experimental procedures and results: In this study, several mouse (4T1, 168 FARN) and human (BT20, MDA-MB 231) breast cancer cell lines were used to eliminate possible cell line specific observations. Using in vitro matrigel invasion assay, we showed that RhoA, not Rac1, acts downstream of RKIP and is needed for RKIP-mediated inhibition of breast cancer cell invasion in vitro. GTPase activity pull-down assay results revealed that RKIP specifically increases RhoA activation, thus inhibiting invasiveness of these cells. Similarly, orthotopic mice tumor implantation experiments showed that ectopic expression of dominant negative RhoA in 4T1 cells confers significantly greater proclivity to metastasize in mice. These observations are consistent with RhoA's emerging role as a metastasis suppressor. In BT20 breast cancer cells, RhoA enhanced E-cadherin expression and negatively affected the cell invasiveness. Interestingly, RKIP phenocopied the RhoA effect on E-cadherin and cell invasion, suggesting that RKIP-RhoA axis inhibits breast cancer cell invasiveness by increasing E-cadherin expression. Conclusions: Our results conclude that RKIP specifically increases RhoA activation in breast cancer cells, and this activated RhoA stabilizes E-cadherin and negatively affects the invasiveness of these cells. Citation Format: Gardiyawasam Kalpana, Vu Bach, Clariza Borile, Miranda Yeung, Rafael Garcia-Mata, Kam C. Yeung. RKIP-RhoA axis inhibits breast cancer invasion and metastasis by increasing E-cadherin expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5153.