TNFα-induced MUC4 elicits trastuzumab resistance in ErbB-2-positive breast cancer

MERCOGLIANO, MARÍA FLORENCIA; DE MARTINO, MARA; VENTURUTTI, LEANDRO; RIVAS, MARTÍN A.; INURRIGARRO, GLORIA; FRAHM, ISABEL; IZZO, FRANCO; PROIETTI, CECILIA J.; ELIZALDE, PATRICIA V.; SCHILLACI, ROXANA

Philadelphia

Congreso; AACR 106th Annual Meeting; 2015

American Association for Cancer Research (AACR)

ErbB-2 is a transmembrane tyrosine kinase receptor overexpressed/amplified in ~15% of breast cancer patients and correlates with poor prognosis. These patients receive 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 alpha (TNF) transactivates ErbB-2 and activates Akt and NF-κB pathways in T-sensitive cell lines, which leads to an increase in cyclin D1 levels and subsequent proliferation, even in presence of T. In this work we explored the role of TNF in T resistance and the underlying mechanism. First, we blocked TNF using etanercept (E, TNFR2-human Fc IgG fusion protein) in presence or absence of T, in models of de novo resistance to the monoclonal antibody such as JIMT-1 and KPL-4 cell lines. Neither T or E alone affected cell growth, but combined administration decreased proliferation of both cell lines. Moreover, we observed a reduction in Akt and NF-κB phosphorylation in the combined treatment, measured by Western Blot (WB). We obtained similar results in vivo: tumor growth inhibition of the E+T group was > 50% for JIMT-1 and >70% for KPL-4 vs. control, E or T groups. Second, using the T-sensitive BT-474 cell line, we generated cells that stably overexpress TNF (T2) or control cells expressing empty vector (C2). Our results showed that T2 xenografts in nude mice were resistant to T administration while C2 tumors dramatically regressed under the same treatment. In order to understand the mechanism of TNF induced T resistance, histopathological studies shed some light by demonstrating that T2 tumors showed mucinous foci. It had been reported that one of the mechanisms of T resistance in JIMT-1 was due to mucin 4 (MUC4) expression, a transmembrane glycoprotein that masks the ErbB-2 epitope recognized by T. Then, we evaluated MUC4 by immunohistochemistry (IHC). T2 tumors show a more intense MUC4 staining than C2, which was confirmed by WB of cell extracts. In the de novo resistant tumors, E and E¬+T treated JIMT-1 and KPL-4 tumors, revealed weak to null staining for MUC4 in contrast to the strong staining obtained in IgG or T-treated tumors. When JIMT-1 and KPL-4 cells were treated in vitro with E, we observed an increase in T binding to these cells measured by flow cytometry. In addition, T2 exhibited reduced binding of T and ADCC compared to C2, that was reversed when MUC4 was blocked by siRNA. Finally, by knocking down p65 NF-kB by siRNA in T2 cells, we determined that TNF induces MUC4 expression via NF-κB activation.These results indicate that TNF plays an important role in T resistance stimulating MUC4 expression through activation of NF-κB pathway. Both TNF and MUC4 could be used as biomarkers of resistance to T. Furthermore blocking TNF could be a promising therapy for ErbB-2-positive breast cancer patients with acquired or de novo T resistance.