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posted on 2023-03-31, 19:51 authored by María F. Mercogliano, Mara De Martino, Leandro Venturutti, Martín A. Rivas, Cecilia J. Proietti, Gloria Inurrigarro, Isabel Frahm, Daniel H. Allemand, Ernesto Gil Deza, Sandra Ares, Felipe G. Gercovich, Pablo Guzmán, Juan C. Roa, Patricia V. Elizalde, Roxana Schillaci Supplementary Figure S2. Determination and scoring of MUC4 and TNFα expression in breast cancer biopsies by IHC. A, tissue microarrays were used to establish a score of MUC4 by IHC analysis as described by Workman et al (17). Score 0 represents no stain to less than 30% of cells stained faintly, 1+ greater than 30% of cells stained with light to moderate intensity, 2+ greater than 50% of cells stained moderately, 3+ intense staining of majority of the epithelial population. The yellow arrow in the inset of MUC4 (score 0) shows positive staining in endothelial cells, used as an internal control. B, TNFα score was equivalent to that described for MUC4. For both proteins scores of 2+ and 3+ were considered as positive expression.
Funding
IDB/PICT
National Cancer Institute (Argentina)
Alberto J. Roemmers Foundation
Productos Roche S.A.Q e I, Argentina
CONICET
ANPCyT
History
ARTICLE ABSTRACT
Purpose: Although trastuzumab administration improved the outcome of HER2-positive breast cancer patients, resistance events hamper its clinical benefits. We demonstrated that TNFα stimulation in vitro induces trastuzumab resistance in HER2-positive breast cancer cell lines. Here, we explored the mechanism of TNFα-induced trastuzumab resistance and the therapeutic strategies to overcome it.Experimental Design: Trastuzumab-sensitive breast cancer cells, genetically engineered to stably overexpress TNFα, and de novo trastuzumab-resistant tumors, were used to evaluate trastuzumab response and TNFα-blocking antibodies effectiveness respectively. Immunohistochemistry and antibody-dependent cell cytotoxicity (ADCC), together with siRNA strategy, were used to explore TNFα influence on the expression and function of its downstream target, mucin 4 (MUC4). The clinical relevance of MUC4 expression was studied in a cohort of 78 HER2-positive breast cancer patients treated with adjuvant trastuzumab.Results: TNFα overexpression turned trastuzumab-sensitive cells and tumors into resistant ones. Histopathologic findings revealed mucin foci in TNFα-producing tumors. TNFα induced upregulation of MUC4 that reduced trastuzumab binding to its epitope and impaired ADCC. Silencing MUC4 enhanced trastuzumab binding, increased ADCC, and overcame trastuzumab and trastuzumab-emtansine antiproliferative effects in TNFα-overexpressing cells. Accordingly, administration of TNFα-blocking antibodies downregulated MUC4 and sensitized de novo trastuzumab-resistant breast cancer cells and tumors to trastuzumab. In HER2-positive breast cancer samples, MUC4 expression was found to be an independent predictor of poor disease-free survival (P = 0.008).Conclusions: We identified TNFα-induced MUC4 expression as a novel trastuzumab resistance mechanism. We propose MUC4 expression as a predictive biomarker of trastuzumab efficacy and a guide to combination therapy of TNFα-blocking antibodies with trastuzumab. Clin Cancer Res; 23(3); 636–48. ©2016 AACR.
