Dual Src kinase/pretubulin inhibitor KX-01, sensitizes ERα-negative breast cancers to tamoxifen through ERα reexpression

Unlike breast cancer that is positive for estrogen receptor-a (ERa), there are no targeted therapies for triple-negative breast cancer (TNBC). ERa is silenced in TNBC through epigenetic changes including DNA methylation and histone acetylation. Restoring ERa expression in TNBC may sensitize patients to endocrine therapy. Expression of c-Src and ERa are inversely correlated in breast cancer suggesting that c-Src inhibition may lead to reexpression of ERa in TNBC. KX-01 is a peptide substrate–targeted Src/pretubulin inhibitor in clinical trials for solid tumors. KX-01 (1 mg/kg body weight-twice daily) inhibited growth of tamoxifen-resistant MDA-MB-231 and MDA-MB-157 TNBC xenografts in nude mice that was correlated with Src kinase inhibition. KX-01 also increased ERa mRNA and protein, as well as increased the ERa targets progesterone receptor (PR), pS2 (TFF1), cyclin D1 (CCND1), and c-myc (MYC) in MDA-MB-231 and MDA-MB-468, but not MDA-MB-157 xenografts. MDA-MB-231 and MDA-MB-468 tumors exhibited reduction in mesenchymal markers (vimentin, b-catenin) and increase in epithelial marker (E-cadherin) suggesting mesenchymal-to-epithelial transition (MET). KX-01 sensitized MDA-MB-231 and MDA-MB-468 tumors to tamoxifen growth inhibition and tamoxifen repression of the ERa targets pS2, cyclin D1, and c-myc. Chromatin immunoprecipitation (ChIP) of the ERa promoter in KX-01–treated tumors demonstrated enrichment of active transcription marks (acetyl-H3, acetyl-H3Lys9), dissociation of HDAC1, and recruitment of RNA polymerase II. Methylation-specific PCR and bisulfite sequencing demonstrated no alteration in ERa promoter methylation by KX-01. These data demonstrate that in addition to Src kinase inhibition, peptidomimetic KX-01 restores ERa expression in TNBC through changes in histone acetylation that sensitize tumors to tamoxifen.