Abstract 712: TNFα-induced MUC4 elicits trastuzumab resistance in ErbB-2-positive breast cancer
Authors: Mercogliano María F. , De Martino Mara , Venturutti Leandro , Rivas Martín A. , Inurrigarro Gloria , Frahm Isabel , Proietti Cecilia J. , Izzo Franco , Elizalde Patricia V. , Schillaci Roxana 
Published: August 2015

Abstract
ErbB-2 is a transmembrane tyrosine kinase receptor overexpressed/amplified in ∼15% of breast cancer patients and its overexpression correlates with poor prognosis. These patients are treated with trastuzumab (T), an anti-ErbB-2 monoclonal antibody, but only 40-60% of them respond when used in combination with chemotherapy. This lack of response is due to de novo or acquired resistance to T. In previous studies we have demonstrated that tumor necrosis factor alpha (TNF) transactivates ErbB-2, and subsequently activates Akt and NF-κB pathways in T-sensitive cell lines, which leads to an increase in cyclin D1 levels and proliferation, even in the presence of T.
In this work we explored the role and the underlying mechanism of TNF in T resistance. In JIMT-1 and KPL-4, two de novo T-resistant cell lines, targeting TNF with etanercept (E, TNFR2-human Fc IgG fusion protein) in presence or absence of T did not affect cell growth. Remarkably, combined administration of T plus E decreased in vitro proliferation of both cell lines. Moreover, we observed a reduction in Akt and p65 NF-κB phosphorylation in the combined treatment, measured by Western Blot (WB). Similar results were obtained in vivo: tumor growth inhibition of the E+T group was &gt;50% for JIMT-1 and &gt;70% for KPL-4 vs. control, E or T groups. To explore acquired resistance to T, we engineered the T-sensitive BT-474 cell line to stably overexpress TNF (T2). Our results show that T2 xenografts are resistant to T administration while control tumors expressing empty vector (C2) dramatically regressed under the same treatment. Histopathological studies shed some light to understand the mechanism of TNF-induced T resistance by demonstrating that T2 tumors showed mucinous foci. It has been reported that one of the mechanisms of T resistance in JIMT-1 is due to mucin 4 (MUC4) expression, a transmembrane glycoprotein that masks the ErbB-2 epitope recognized by T. Evaluation of MUC4 by immunohistochemistry (IHC) showed a more intense MUC4 staining in T2 than in C2 tumors, which was confirmed by WB of cell extracts. Treatment with E and E+T in JIMT-1 and KPL-4 tumors showed weak to null staining for MUC4, in contrast to the strong staining obtained in IgG or T-treated tumors by IHC. Interestingly, in vitro treatment of JIMT-1 and KPL-4 cells with E, induced an increase in T binding measured by flow cytometry. In addition, T2 cells exhibited reduced binding of T and reduced antibody-dependent cellular cytotoxicity compared to C2 cells, which was reversed by MUC4 knockdown. Finally, p65 NF-κB knockdown in T2 cells impaired TNF-induced MUC4 expression.
These results indicate that TNF plays an important role in T resistance stimulating MUC4 expression through activation of NF-κB pathway. Our data suggest that both TNF and MUC4 could be used as biomarkers of resistance to T. Furthermore, blockage of TNF could be a promising therapy for ErbB-2-positive breast cancer patients with acquired or de novo T resistance.
Citation Format: María F. Mercogliano, Mara De Martino, Leandro Venturutti, Martín A. Rivas, Gloria Inurrigarro, Isabel Frahm, Cecilia J. Proietti, Franco Izzo, Patricia V. Elizalde, Roxana Schillaci. TNFα-induced MUC4 elicits trastuzumab resistance in ErbB-2-positive breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 712. doi:10.1158/1538-7445.AM2015-712