Ccn6 loss cooperates with EZH2 overexpression to induce sarcomatoid differentiation in metaplastic breast carcinoma

Background: Triple negative breast cancers (TNBC) have proven resistant to conventional and immune therapies with 5-year survival rates of 50%. Among the most aggressive of these is metaplastic breast carcinoma (mBrCA), a unique histopathology with spindle or sarcomatoid (chondroid/osseous) components and low immune cell infiltration with poor responses to immunotherapy. We have reported that mammary epithelial cell-specific Ccn6 knockout mice (MMTV-Cre; Ccn6 fl/fl ) develop mBrCAs like human spindle mBrCAs, defining CCN6 as a tumor suppressor for spindle mBrCA . Lately we showed CCN6 antagonizes the pro-invasive effects of Wnt/ beta-catenin/EZH2 signaling. Here, we tested the hypothesis that CCN6 genetically interacts with EZH2 to induce a phenotypic switch from spindle to sarcomatoid cancer cell phenotype and limits CD8 + T cell infiltration. Methods: MDA-MB-231 (TNBC) and Ccn6-KO cells derived from MMTV-Cre;Ccn6 fl/fl (Ccn6-KO) TNBC mouse model were transduced with vector or EZH2 shRNA knockdown (KD). Ccn6-KO EZH2 KD cells were rescued with lentivirus containing EZH2-wild-type (WT). To overexpress EZH2 we infected cancer cells with adenovirus with vector or EZH2-MYC construct. We investigated how Ccn6 loss and EZH2 affect mBrCA sarcomatoid by differentiation assays of the breast cancer cells in chondrogenic and osteogenic specialized media compared to controls. In vivo, to recapitulate the bone microenvironment, we injected Ccn6-KO cells into the tibia of FVB mice and treat them with the EZH2 inhibitor EPZ-6438. Flow cytometry analyses of tumors derived from Ccn6-KOshC, shEZH2 and EZH2-wild type rescue cells and IHC of Ccn6-KO tumors treated with EZH2i were used to measure the percentage of CD8 + gzmb + IFNg + T cells. ChIP-sequencing studies in dominant negative (dn)TCF4 mutant vs. controls were used to study regulation of bivalent genes in Ccn6-KO cells. Results : EZH2 overexpression in Ccn6-KO spindle mBrCA cells induces chondrogenic and osteogenic differentiation in cells grown in specialized media and EZH2 inhibition rescues the sarcomatoid phenotype in vivo. We found that EZH2 shRNA KD or pharmacological inhibition increases CD8+T cell infiltration in Ccn6-KO tumors, which is rescued by EZH2 overexpression. WNT/beta-catenin signaling blockade in Ccn6-KO cells using dnTCF4 downregulates EZH2 and H3K27me3 mark at the promoter regions of 19 bivalent gene targets with roles in development. Conclusion : These data provide a clear link between CCN6 defects and EZH2 overexpression as inducers of sarcomatoid immune cell-low mBrCAs. We demonstrate the effectiveness of pharmacological inhibition of EZH2 methyltransferase activity in reversing these aggressive phenotypes. These data reveal a novel oncogenic cooperation and provide evidence to support the potential therapeutic value of CCN6 restoration, Wnt pathway and EZH2 inhibition as promising strategies for mBrCA patients