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Supplemental Methods from Downregulating Neuropilin-2 Triggers a Novel Mechanism Enabling EGFR-Dependent Resistance to Oncogene-Targeted Therapies

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posted on 2023-03-31, 01:21 authored by Sabrina Rizzolio, Chiara Battistini, Gabriella Cagnoni, Maria Apicella, Viviana Vella, Silvia Giordano, Luca Tamagnone

This document describes Materials and Methods applied for siRNA, shRNA, and cDNA transfer in mammalian cells, by means of transfection or lentiviral-mediated cell transduction. Moreover, supplementary information are provided about genomic DNA and RNA isolation and Real-time quantitative PCR analysis, as well as gene promoter reporter assays. Finally, cell surface protein-biotinylation and Western blotting methods are described.

Funding

AIRC

FPRC

Fondazione Veronesi

Italian Association for Cancer Research

Fondazione Piemontese per la Ricerca sul Cancro - ONLUS

FPRC-ONLUS

History

ARTICLE ABSTRACT

Neuropilins are a class of cell surface proteins implicated in cell migration and angiogenesis, with aberrant expression in human tumors. Here, we show that the expression of Neuropilin-2 (NRP2) controls EGFR protein levels, thereby impinging on intracellular signaling, viability, and response to targeted therapies of oncogene-addicted cells. Notably, increased NRP2 expression in EGFR-addicted tumor cells led to downregulation of EGFR protein and tumor cell growth inhibition. NRP2 also blunted upregulation of an EGFR "rescue" pathway induced by targeted therapy in Met-addicted carcinoma cells. Cancer cells acquiring resistance to MET oncogene-targeted drugs invariably underwent NRP2 loss, a step required for EGFR upregulation. Mechanistic investigations revealed that NRP2 loss activated NFkB and upregulated the EGFR-associated protein KIAA1199/CEMIP, which is known to oppose the degradation of activated EGFR kinase. Notably, KIAA1199 silencing in oncogene-addicted tumor cells improved therapeutic responses and counteracted acquired drug resistance. Our findings define NRP2 as the pivotal switch of a novel broad-acting and actionable pathway controlling EGFR signaling, and driving resistance to therapies targeting oncogene-addiction.Significance: These important findings identify the cell surface molecule Nrp2 as the pivotal switch of a novel, actionable pathway driving EGFR upregulation and resistance to oncogene- targeted therapies. Cancer Res; 78(4); 1058–68. ©2017 AACR.