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.