Abstract 682: PKCá mediates FOXC2 transcriptional repression of p120-catenin in breast cancer

Background: Protein kinase C alpha (PKCa) has been studied as a predictive biomarker for breast cancer aggressiveness and resistance to therapy. In this study, we identified a novel signaling pathway regulated by PKCa in breast cancer cells that involves FOXC2 and p120-catenin (p120), a prominent member of adherens junctions (AJs). We report her that PKCa causes dissolution of AJs, a mechanism which contributes to cancer cells becoming more migratory and invasive, two key features of metastasizing cells. Interestingly, we found that this mechanism is relevant in both estrogen receptor (ER)-positive, endocrine resistant as well as triple negative breast cancer (TNBC) (lack of ER, progesterone receptor, and HER2/neu amplification). Methods: ER+, endocrine resistant (T47D/PKCa and T47D:A18-TAM1) and TNBC cell lines (HCC1143 and HCC1937) were cultured according to previous publication and ATCC guidelines. Migration and invasion assays were done in Transwell® polycarbonate membranes (Corning). Promoter activity of p120 was measured by luciferase assay using p120 reporter construct obtained from Dr. Fariborz Mortazavi (Division of Hematology/Oncology, UCLA). Chromatin immunoprecipitation (ChIP) to assess binding of FOXC2 on p120 promoter was done using ChIP-grade FOXC2 antibody (Abcam). Lipofectamine®2000 (Invitrogen) was used for transfection of PKCa (Sigma) and FOXC2 (IDT) siRNAs Results and Conclusions: We found that FOXC2 is a downstream target of PKCa; activation of PKCa by a phorbol ester significantly up-regulated FOXC2 mRNA and knockdown of PKCa by siRNA reduced expression of FOXC2 at both the mRNA and protein level. Immunofluorescence staining showed that depletion of PKCa rescued E-cadherin, secondary to the recovery of p120 expression at the AJ. We demonstrated by ChIP that FOXC2 binds to the promoter region and represses transcription of p120, indicating a novel finding that FOXC2 is a transcriptional repressor of p120 in breast cancer. Therefore, through FOXC2, PKCa can negatively regulate the AJ complex integrity. Correspondingly, knockdown of either PKCa or FOXC2 led to a reduction in the migration and invasion, concomitant with an increase of p120 at the AJ. Data obtained from the Cancer Genome Atlas (TCGA) indicates that high co-expression of PKCa and FOXC2 along with low expression level of p120 are characteristic of TNBC patients, confirming the relevance of this signaling axis in clinical samples. In conclusion, we have identified a new mechanism for migration and invasion in breast cancer cells: in the presence of PKCa, transcriptional repression of p120 by FOXC2 results in down-regulation of E-cadherin and dissociation of the AJs. Notably, we demonstrated that this mechanism is relevant in two distinct molecular subtypes of breast cancer: ER+, endocrine resistant and TNBC. Targeting the PKCa-FOXC2-p120 axis has the potential to reduce the metastatic capacity of these cancer cells. Citation Format: Thao N. Pham. PKCa mediates FOXC2 transcriptional repression of p120-catenin in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 682.