Table S1: List of qRT-PCR primers; Table. S2: Summary table of mRNA expression for Ly6K and Ly6E in normal and cancer samples in seven comparison; Fig. S1. Expression of Ly6K and Ly6E in human normal and breast cancer tissues; Fig. S2: The knockdown for Ly6K and Ly6E is specific; Fig. S3: Colony assay; Fig. S4: The Ly6K gene is required for intravasation, extravasation and colonization of cancer cells; Fig. S5: Comparison of CD274, CTLA4, IL2RA, LY6E and LY6K gene expression in clinical samples of breast cancer in indicated 6 analysis in three different datasets; Fig. S6: High expression of Ly6K and Ly6E impair NK cell binding to cancer cell; Fig. S7: Indicated cells (MDA-MB-231) were treated with 100ng/ml IFN-g overnight; Fig. S8: Rescue over expression phenotype.
ARTICLE ABSTRACTStem cell antigen Sca-1 is implicated in murine cancer stem cell biology and breast cancer models, but the role of its human homologs Ly6K and Ly6E in breast cancer are not established. Here we report increased expression of Ly6K/E in human breast cancer specimens correlates with poor overall survival, with an additional specific role for Ly6E in poor therapeutic outcomes. Increased expression of Ly6K/E also correlated with increased expression of the immune checkpoint molecules PDL1 and CTLA4, increased tumor-infiltrating T regulatory cells, and decreased natural killer (NK) cell activation. Mechanistically, Ly6K/E was required for TGFβ signaling and proliferation in breast cancer cells, where they contributed to phosphorylation of Smad1/5 and Smad2/3. Furthermore, Ly6K/E promoted cytokine-induced PDL1 expression and activation and binding of NK cells to cancer cells. Finally, we found that Ly6K/E promoted drug resistance and facilitated immune escape in this setting. Overall, our results establish a pivotal role for a Ly6K/E signaling axis involving TGFβ in breast cancer pathophysiology and drug response, and highlight this signaling axis as a compelling realm for therapeutic invention. Cancer Res; 76(11); 3376–86. ©2016 AACR.