Clariom D application in breast cancer

The TAILORx clinical trial showed that up to 70% of patients with breast cancer with estrogen receptor (ER+) do not benefit from chemotherapy. However, these patients continue to develop recurrences when treated with endocrine therapy alone. The addition of CDK4/5 inhibitors to endocrine therapy results in significant (8-12 months) delay in progression of disease. There is critical need to understand the mechanisms of resistance and development of novel targets/therapies for prevention of endocrine resistance and recurrences of ER+ breast cancer. We have recently demonstrated that Epithelial Splicing Regulatory Protein 1 (ESRP1), an epithelial cell-specific RNA binding protein and splicing factor, is associated with poor prognosis and endocrine resistance in human ER+ breast tumors (Gökmen-Polar, EMBO Reports 2019). We documented a novel role for ESRP1 in altering metabolic pathways by altering both gene expression and alternative splicing events. ESRP1 silencing further impacted cell/tumor growth and metabolism significantly. CDK4/CDK6 inhibitor effectively decreased the expression of proliferation pathway significantly, but did not block the metabolic alterations. Here we seek to investigate associated molecular mechanisms by performing RNA Binding Protein (RBP) immunoprecipitation followed by Clariom D transcriptomics microarray (RIP-Chip) to identify mRNA binding partners of ESRP1. RIP-Chip analysis identified PHGDH, a key metabolic enzyme, as a binding partner of ESRP1 in ER+ breast cancer. Binding was further validated using RNA electrophoretic shift assay (REMSA) against 5’ UTR probes and RIP-RT-qPCR. Further functional characterization in vitro showed that ESRP1 posttranscriptional regulates PHGDH by affecting its mRNA stability. Seahorse metabolic analyses of the PHGDH knockdown cells significantly reduced oxygen consumption rate (P<0.04) mimicking the effects of ESRP1 knockdown. In conclusion, this study documents a non-mutational pathway in mRNA and protein down regulation and altered cellular metabolism. We anticipate that the insights garnered from these studies will enable a more precise molecular understanding of metabolic pathways of endocrine resistance and facilitate discovery of new therapeutic targets for treatment of endocrine resistance.