CCN6 regulates IGF2BP2and HMGA2 signaling in metaplastic carcinomas of the breast.

Abstract

Metaplastic breast carcinomas are an aggressive subtype of triple-negative breast cancer (TNBC) in which part or all of the adenocarcinoma transforms into a non-glandular component (e.g., spindled, squamous, or heterologous). We discovered that mammary-specific Ccn6/Wisp3 knockout mice develop mammarycarcinomas with spindle and squamous differentiation that share upregulation of the oncofetal proteins IGF2BP2 (IMP2) and HMGA2 with human metaplastic carcinomas. Here, we investigated the functionalrelationship between CCN6,IGF2BP2, and HMGA2 proteinsin vitro and in vivo, andtheir expression in human tissue samples. MMTV-cre;Ccn6fl/fl tumors and spindle TNBC cell lines were treated with recombinant CCN6 protein or vehicle. IGF2BP2 was downregulated using shRNAsinHME cells with stable CCN6 shRNA knockdown, and subjected to invasion and adhesion assays.Thirty-one humanmetaplastic carcinomaswere arrayed in a tissue microarray (TMA) and immunostained for CCN6, IGF2BP2, and HMGA2. CCN6 regulates IGF2BP2 and HMGA2 protein expression in MMTV-cre;Ccn6fl/fltumors, in MDA-MB-231 and - 468, and in HME cells. CCN6 recombinant protein reduced IGF2BP2 and HMGA2 proteinexpression, and decreased growth of MMTV-cre;Ccn6fl/fl tumors in vivo. IGF2BP2 shRNA knockdown was sufficient to reverse the invasive abilities conferred by CCN6 knockdown in HME cells. Analyses of the TCGA Breast Cancer Cohort (n = 1238) showed that IGF2BP2 and HMGA2 are significantly upregulated in metaplastic carcinoma compared to other breast cancersubtypes. In clinical samples, low CCN6 is frequent in tumorswithhigh IGF2BP2/HMGA2 with spindle and squamous differentiation. These data shed light into the pathogenesis of metaplastic carcinoma and demonstrate a novel CCN6/IGF2BP2/HMGA2 oncogenic pathway with biomarker andtherapeutic implications.