Therapeutic targeting of HuR using a specific small-molecule inhibitor in triple negative breast cancer

Human Antigen R (HuR/ELAVL1) is an RNA-binding protein that interacts with U/AU-rich elements in target mRNAs, and its aberrant cytoplasmic accumulation have been observed in multiple cancers. HuR modulates the expression of key oncogenic and survival-related transcripts, thereby driving tumor progression, metastasis, and therapeutic resistance. Given this central role, HuR represents a compelling molecular target for cancer therapy. The present study specifically evaluates the potential of CMLD2, a highly selective small-molecule inhibitor of HuR, in the context of Triple-Negative Breast Cancer (TNBC). Comparative assessments were conducted using CMLD2 and DHTS across in silico and in vitro models. Immunohistochemical analysis of 71 TNBC samples revealed HuR overexpression in 66% of cases, along with elevated expression of HuR downstream target MMP9 (56%). Furthermore, in silico analysis established significant association between HuR overexpression and poor patient survival (p = 0.028). Functional assays in TNBC cell lines MDA-MB-231 and MDA-MB-468 demonstrated that both inhibitors suppressed proliferation, invasion, and clonogenicity; however, CMLD2 exhibited markedly higher specificity and consistency in inhibiting HuR function. Mechanistically, CMLD2 treatment downregulated HuR and its downstream effectors MMP9 and beta-catenin, while reversing epithelial-mesenchymal transition (EMT) marker expression (E-cadherin, N-cadherin and Vimentin). Immunofluorescence and cell fractionation confirmed effective cytoplasmic HuR inhibition. Moreover, CMLD2 distinctly suppressed aerobic glycolysis, evidenced by reduced extracellular acidification and downregulation of key glycolytic mediators-PFKP, LDHA, and MCT4-validated at both transcript and protein levels. HuR silencing by siRNA supported the role of HuR in glycolytic regulation and TNBC progression. In vivo , CMLD2 significantly impaired tumor-forming ability in female BALB/c mice. Together, these findings underscore the therapeutic promise of CMLD2 as a specific and potent HuR inhibitor, providing strong experimental rationale for its further development as a targeted intervention in Triple-Negative Breast Cancer.