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J. Nair-Menon

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    MINI 09 - Drug Resistance (ID 107)

    • Event: WCLC 2015
    • Type: Mini Oral
    • Track: Biology, Pathology, and Molecular Testing
    • Presentations: 1
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      MINI09.13 - Neuropilin-2 Promotes Acquired Resistance to EGFR-TKI Associated with the Epithelial–Mesenchymal Transition in Lung Cancer (ID 1271)

      16:45 - 18:15  |  Author(s): J. Nair-Menon

      • Abstract
      • Slides

      Lung cancer accounts for one-fifth of cancer deaths worldwide with invasion, metastases and drug resistance representing major causes of mortality and barriers to cure. While lung cancers with activating mutations in the EGF receptor (EGFR) are susceptible to tyrosine kinase inhibitors (TKI), such as erlotinib and gefitinib, the efficacy of these agents is limited by the inevitable development of resistance. The epithelial-mesenchymal transition (EMT), by which epithelial cells acquire a mesenchymal and invasive phenotype, is one mechanism promoting EGFR-TKI resistance, including resistance to 3[rd] generation T790M-specific inhibitors. However, the molecular connections between EMT and resistance are not well understood. Here we report that upregulation of Neuropilin-2 (NRP2) is crucial for development of EGFR-TKI resistance associated with the EMT. NRP2 is a cell surface receptor for SEMA3F, a secreted semaphorin with tumor suppressor activity that is down-regulated during EMT. NRP1 and NRP2 are also co-receptors and signaling enhancers for several growth-promoting ligands such as VEGF, HGF and FGF. We previously reported that NRP2 was induced by TGFβ as part of an EMT response in lung cancers and that NRP2 knockdown suppressed the EMT phenotype, including local tumor invasion in a subcutaneous xenograft model.

      Immunohistochemistry (IHC) was performed for NRP2 on patient biopsies, before and after development of gefitinib resistance. EGFR mutant NSCLC cell lines, transfected with control or NRP2-specific shRNAs, were selected for gefitinib/erlotinib resistance in vitro, using progressively increasing concentrations or continuous exposure to IC~50~ levels of EGFR TKIs. Western blot analysis confirmed changes in NRP2 expression along with selected markers of EMT. MTS viability assays determined drug sensitivity while migration and invasion were assessed using Boyden chambers. Growth as spheroids was assessed in 1% methylcellulose medium in low-adherence plates.

      Increased NRP2 was observed in lung tumor biopsies following acquisition of EMT-associated gefitinib-resistance, and in HCC4006-ER cells, which acquired a stable erlotinib-resistant EMT phenotype. In vitro, using multiple EGFR mutant cell lines, NRP2 knockdown blocked acquired gefitinib-resistance, arising both spontaneously following growth in IC~50~ concentrations or after exposure to TGFβ. Of interest, spontaneously-resistant cells exhibited increased migration similar to cells stimulated with TGFβ. NRP2 knockdown also blocked tumorsphere formation, which has been associated with stem-cell characteristics and drug resistance.

      Collectively, our results demonstrate that NRP2 is a mediator of acquired EGFR-TKI resistance. The results also suggest that NRP2 blocking antibodies could be useful for enhancing the duration of response to EGFR inhibitors, including those targeting the T790M mutation.

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