NOTCH and PTP4A3 alterations emerge as novel predictive biomarkers and potential therapeutic targets in pleural mesothelioma

Background: Previous studies showed opposite effects of NOTCH1 and NOTCH2 on mesothelioma cell survival under hypoxia. Mechanisms underlying these effects are not still clear and this pathway plays a key role in angiogenesis and cancer stem cells (CSCs) self-renewal processes. Purpose: In this study we evaluated whether NOTCH1, NOTCH2 copy number alterations (CNAs) might predict prognosis of patients with pleural mesothelioma (PM) and if the modulation of this pathway might target CSCs, potentiating pemetrexed activity, also in hypoxic conditions. Methods: Recurrent CNAs were determined by high-resolution whole-genome sequencing from paraffin-embedded samples of a “discovery cohort” (26 patients treated with pemetrexed-based chemotherapy). Prognostic CNAs were validated by PCR gene copy-number and expression analyses in the “discovery” and in two independent “validation” cohorts of pemetrexed-treated and untreated patients (N = 45 and N = 40). Functional analyses of emerging genes were performed through siRNA in different subpopulation of PM cells, growing under hypoxia. Results: A copy number gain of NOTCH2 was observed in 50% of patients with progressive disease and its overexpression correlated with a worse prognosis in both pemetrexed-treated and untreated-patients’ cohorts. Conversely, losses of PTP4A3 correlated with clinical benefit, while patients with overexpression of both NOTCH2 and PTP4A3 had the worse prognosis. Moreover, NOTCH2 silencing through siRNA in vitro reduced migration, enhancing apoptosis of PM cells, while the PTP4A3 inhibitor BR-1 overcame pemetrexed resistance in PM cells characterized by high NOTCH2/PTP4A3 expression. Conclusions: NOTCH2 and PTP4A3 alterations are associated with clinical outcomes in pemetrexed-treated PM patients. The inhibition of NOTCH pathway may be exploited to eradicate CSCs and improve patients’ survival.