Biophysical and molecular characterization of HuR inhibitors, CMLD-2 and Dihydrotanshinone I (DHTS), in Triple Negative Breast Cancer (TNBC)

Abstract

In the present study, we explored the oncogenic role of Human Antigen R (HuR), a post-transcriptional regulator implicated in cancer progression, metastasis, and therapy resistance. The present study evaluates the specificities and efficacies of two HuR inhibitors - CMLD2 and DHTS in silico and in vitro, in TNBCs. Apart from in-depth biophysical analysis, the cytotoxic effect of CMLD2 and DHTS was compared on the proliferation of TNBC cell lines [human: MDA-MB-231 & MDA-MB-468] and the 4T1[murine]. Western blotting was performed to access the differential modulation of HuR and its downstream targets i.e., MMP9 and β-catenin along with classical EMT markers viz. epithelial (E-cadherin), and mesenchymal (N-cadherin, and vimentin). Functional assays like wound healing, matrigel droplet invasion, and colony formation were performed and both drugs displayed substantial inhibitory activity on the invasiveness, migratory, and clonogenic potential of TNBC cells. However, at the level of regulation of molecular targets of HuR, CMLD2 shows much better specificity and reproducibility compared to DHTS which was validated using HuR-specific siRNA. In conclusion, both inhibitors successfully diminished the aggressive phenotype (i.e., migration, invasion, and colony formation capabilities) of TNBC cells. However, CMLD2 demonstrated greater specificity in inhibiting HuR and its downstream targets, compared to DHTS.