Neuropilin-1 upregulation elicits adaptive resistance to oncogene-targeted therapies
Sabrina Rizzolio, … , Silvia Giordano, Luca Tamagnone
Sabrina Rizzolio, … , Silvia Giordano, Luca Tamagnone
Published June 28, 2018
Citation Information: J Clin Invest. 2018;128(9):3976-3990. https://doi.org/10.1172/JCI99257.
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Neuropilin-1 upregulation elicits adaptive resistance to oncogene-targeted therapies

Abstract

Cancer cell dependence on activated oncogenes is therapeutically targeted, but acquired resistance is virtually unavoidable. Here we show that the treatment of addicted melanoma cells with BRAF inhibitors, and of breast cancer cells with HER2-targeted drugs, led to an adaptive rise in neuropilin-1 (NRP1) expression, which is crucial for the onset of acquired resistance to therapy. Moreover, NRP1 levels dictated the efficacy of MET oncogene inhibitors in addicted stomach and lung carcinoma cells. Mechanistically, NRP1 induced a JNK-dependent signaling cascade leading to the upregulation of alternative effector kinases EGFR or IGF1R, which in turn sustained cancer cell growth and mediated acquired resistance to BRAF, HER2, or MET inhibitors. Notably, the combination with NRP1-interfering molecules improved the efficacy of oncogene-targeted drugs and prevented or even reversed the onset of resistance in cancer cells and tumor models. Our study provides the rationale for targeting the NRP1-dependent upregulation of tyrosine kinases, which are responsible for loss of responsiveness to oncogene-targeted therapies.

Authors

Sabrina Rizzolio, Gabriella Cagnoni, Chiara Battistini, Stefano Bonelli, Claudio Isella, Jo A. Van Ginderachter, René Bernards, Federica Di Nicolantonio, Silvia Giordano, Luca Tamagnone

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Figure 1

Adaptive NRP1 neoexpression in BRAF-addicted melanoma cells mediates acquired resistance to targeted therapy.

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Adaptive NRP1 neoexpression in BRAF-addicted melanoma cells mediates acq...
(A) Different melanoma cells were subjected to SOX10 knockdown (KD) by inducible shRNA expression, and SOX10, NRP1, and EGFR mRNA levels were analyzed by qPCR (n = 4). The graph shows log2 fold change variations in SOX10-KD cells versus respective controls. (B) Box plot showing SOX10, NRP1, and EGFR mRNA expression variations in 12 paired melanoma samples (indicated by different symbols) obtained from the same patients before and during treatment with BRAF inhibitors (log2 ratio treated/untreated). (C) qPCR analysis of SOX10, NRP1, and EGFR expression in A375 and SK-MEL-28 melanoma cells that developed acquired resistance to 2 μM PLX-4720 (log2 ratio drug resistant versus parental cells; n = 3). (D) SOX10, NRP1, and EGFR protein expression in the same cells shown in C; vinculin and β-tubulin levels provided loading controls (1 representative experiment of 3 repetitions; duplicate samples were run on parallel gels for staining with different antibodies). (E) The viability of parental or drug-resistant A375 cells was assessed upon NRP1 or EGFR KD with targeted siRNAs (n = 5). (F) A375 parental cells were subjected to NRP1 KD (by targeted shRNAs) before exposure to escalating concentrations of PLX-4720 aimed at establishing drug resistance. NRP1 and EGFR expression was analyzed after 5 weeks of treatment. The statistical analysis compared NRP1-KD samples with respective controls (carrying plkO empty vector) (n = 3). (G) The growth rate of control or NRP1-KD A375, either untreated or exposed to 0.5 μM PLX-4720 for 5 weeks, is scored as the percentage of increase of viable cells across 72 hours (n > 3). (H) The growth rate of control or NRP1-KD A375 cells on therapy with 0.5 μM PLX-4720 for 5 weeks was tracked in culture over 7 days (n > 3). (I) Mice implanted with either mock-transduced or NRP1-KD drug-resistant A375 cells were treated daily with 20 mg/kg PLX-4720 (or vehicle only), and the tumor burden was periodically measured. The statistical analysis compared treated tumors with respective controls (n = 5, per condition). For E, F, G, statistical significance was determined using a Student’s t test with Bonferroni’s correction (*P < 0.01; **P < 0.001; ***P < 0.0001); for H and I, a 2-way ANOVA test with Bonferroni’s correction was used (***P < 0.0001).