Biomarker Discovery for TNBC and CRC Tumor Sensitivity to Novel CDK8 Inhibitors

Abstract

BackgroundPrevious work done by the Russu lab developed a novel piperazinylpyrimidine compound, Q12, which acts as a competitive inhibitor for the ATP binding pocket in cyclin‐dependent kinase 8 (CDK8). Q12 was tested for its ability to selectively inhibit the growth of various tumor cell lines within the NCI‐60 cell line panel. Those results revealed that HCT‐116, a colorectal cancer cell line, and MDA‐MB‐468, a triple negative breast cancer (TNBC) cell line, showed the highest sensitivity to Q12 with GI50 values of 0.27M and 0.06M respectively. Q12 was studied in the context of these two cell lines through in vitro analysis. We hypothesize that Q12 can be used as a molecular probe for biomarker discovery in tumor types that may benefit from clinical CDK8 inhibition.Materials and methodsWe performed in vitro analysis of the cancer cell lines previously mentioned: MDA‐MB‐468 and HCT‐116. A MUSE Cell Analyzer was used to perform cell viability and apoptosis assays. Western blot analysis was performed to test Q12’s effect on various proteins including: β‐catenin, STATs, and E2F1. Genetic knockdown experiments of E2F1 were performed using siRNA transfection in the MDA‐MB‐468 cell line and results were obtained through western blot analysis and the MUSE Cell Analyzer. Conditioned media and co‐treatment of Q12 and cryptotanshinone (CPT) was performed to test the regulation of STAT3 phosphorylation.ResultsOur results showed that Q12 reduced cellular viability and p‐STAT1(Ser727) expression as well as induced apoptosis across both cell lines. For HCT‐116, we found that Q12 reduced ‐catenin, and p‐STAT3(Tyr 705) levels. However, the MDA‐MB‐468 cells showed no observable decrease in ‐catenin levels and a significant increase in p‐STAT3(Tyr 705) levels. The conditioned media experiments showed upregulation of p‐STAT3(Tyr 705) protein in the MDA‐MB‐468 cells. Co‐treatment with Q12 and CPT showed retention of cell viability even after Q12 treatment in MDA‐MB‐468 cells. Treatment of MDA‐MB‐468 cells with Q12 also revealed upregulation of total E2F1 protein expression. siRNA knockdown of E2F1 revealed a retention of cell viability in MDA‐MB‐468 cells after treatment with Q12.ConclusionOur results showed that, while both HCT‐116 and MDA‐MB‐468 are both sensitive to CDK8 inhibition, the mechanism through which Q12 induces apoptosis may stem from different biochemical pathways. HCT‐116 harbors a ‐catenin stabilizing mutation which contributes to its cellular proliferation. The results of our in vitro studies on the colon cancer cell lines are consistent with the literature indicating wnt/catenin signaling may be the primary biochemical pathway involved in HCT‐116 cell proliferation and likely associated with its sensitivity to Q12. MDA‐MB‐468 cells, on the other hand, harbor a deletion of the RB1 tumor suppressor gene which, under normal conditions, inhibits the E2F1 transcription factor. Our results indicated that interactions between E2F1, STAT3 and the RB biochemical pathway may be more significantly associated with Q12 sensitivity in the MDA‐MB‐468 cell line. There is also the possibility of autocrine/paracrine apoptosis induction from secreted cytokines which can be tested through an IL Family ELISA assay in both HCT‐116 and MDA‐MB‐468 cells.