Abstract
Notch dysregulation has been implicated in numerous tumors, including triple-negative breast cancer (TNBC), which is the breast cancer subtype with the worst clinical outcome. However, the importance of individual receptors in TNBC and their specific mechanism of action remain to be elucidated, even if recent findings suggested a specific role of activated-Notch3 in a subset of TNBCs. Epidermal growth factor receptor (EGFR) is overexpressed in TNBCs but the use of anti-EGFR agents (including tyrosine kinase inhibitors, TKIs) has not been approved for the treatment of these patients, as clinical trials have shown disappointing results. Resistance to EGFR blockers is commonly reported. Here we show that Notch3-specific inhibition increases TNBC sensitivity to the TKI-gefitinib in TNBC-resistant cells. Mechanistically, we demonstrate that Notch3 is able to regulate the activated EGFR membrane localization into lipid rafts microdomains, as Notch3 inhibition, such as rafts depletion, induces the EGFR internalization and its intracellular arrest, without involving receptor degradation. Interestingly, these events are associated with the EGFR tyrosine dephosphorylation at Y1173 residue (but not at Y1068) by the protein tyrosine phosphatase H1 (PTPH1), thus suggesting its possible involvement in the observed Notch3-dependent TNBC sensitivity response to gefitinib. Consistent with this notion, a nuclear localization defect of phospho-EGFR is observed after combined blockade of EGFR and Notch3, which results in a decreased TNBC cell survival. Notably, we observed a significant correlation between EGFR and NOTCH3 expression levels by in silico gene expression and immunohistochemical analysis of human TNBC primary samples. Our findings strongly suggest that combined therapies of TKI-gefitinib with Notch3-specific suppression may be exploited as a drug combination advantage in TNBC treatment.
Highlights
Triple-negative breast cancer (TNBC), which lacks estrogen receptor (ER), progesterone receptor, and human epidermal growth factor 2 receptor (HER2), accounts for about 15–20% of breast cancers and represents the most aggressive breast cancer (BC) subtype[1]
epidermal growth factor receptor (EGFR) crosstalk in TNBC context, we first performed an in silico analysis of the NOTCH3 and EGFR gene expression levels in two cohorts of TNBC patients, collectively consisting of 777 individuals[23,24,25,26] (Fig. 1a)
Almost all TNBC cells expressed activated Notch[1] and/or Notch[3] protein (N3IC), confirming the hyperactivation of Notch signaling observed in this BC subtype[14], mainly involving the upregulation of N3IC expression, as it appears at undetectable levels in MCF10A, a normal immortalized mammary epithelial cell line
Summary
Triple-negative breast cancer (TNBC), which lacks estrogen receptor (ER), progesterone receptor, and human epidermal growth factor 2 receptor (HER2), accounts for about 15–20% of breast cancers and represents the most aggressive breast cancer (BC) subtype[1]. TNBC-bearing patients better respond to current chemotherapy than do non-TNBC ones, patients with TNBC experience a more rapid relapse evolving as metastatic disease. For this reason, this BC subtype suffers from the poorest prognosis[1]. No EGFR inhibitory therapies are currently approved for BC treatment, including TNBC, as results from clinical trials are disappointing[4]. This limited clinical activity is often due to the existence of compensatory pathways that confer resistance to EGFR inhibition, allowing continued cancer cell growth and survival[5,6,7]
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