Introduction: Fibrotic and malignant pancreatic ductal adenocarcinoma (PDAC) tumors are often resistant to chemotherapy, and remain a leading cause of morbidity and mortality. Epithelial–mesenchymal transition (EMT), a process by which epithelial cells lose their cell polarity and adhesion to become migratory and invasive mesenchymal stem cells, contributes to chemoresistance in PDAC. Likewise, PDAC tumors benefit from autophagy, a self-degradation pathway that confers a survival benefit against metabolic stress. Over-activation of EMT and autophagy has been linked to increased drug resistance in PDAC. Both these processes are largely mediated by TGFβ1. Recently, we demonstrated the role of neuropilin-1 (NRP-1) in TGFβ1-dependent endothelial-mesenchymal transition and fibrosis in PDAC. However, the potential mechanisms linking NRP-1 with EMT and autophagy-mediated chemoresistance remain unexplored. We hypothesized that NRP-1 blockade will enhance chemotherapeutic effect, by suppressing EMT and autophagy in PDAC.
Methods: Human PDAC cells (BxPC-3) or serially gemcitabine exposed chemoresistant (CR-BxPC-3) cells were transfected with siNRP-1 or scramble using DharmaFECT-2. For EMT and chemotherapy studies, transfected cells were treated with TGFβ1 (5ng/mL, 24h), followed by gemcitabine (1μM) for 48h. RNA (24h) and protein (48h) was isolated using TRIzolTM and RIPA, respectively. EMT markers were evaluated by RT-PCR and immunoblotting/immunostaining. For autophagy studies, cells were serum starved post NRP-1 silencing and protein was isolated. For autophagy inhibition, cells were treated with bafilomycin-A1 (100nM) for 24h post NRP-1 silencing and protein was isolated. Autophagy flux (LC3-II/I) and P62 were measured by immunoblotting/immunostaining. Cell viability was evaluated by MTT assay.
Results: SiNRP-1 treatment in BxPC-3 and CR-BxPC-3 cells resulted in NRP-1 knockdown and promoted cell-death after chemotherapy. TGFβ1-stimulated BxPC-3 cells demonstrated a morphological change consistent with EMT that was revoked by siNRP-1. Also, siNRP-1 maintained epithelial cellular morphology via gain of epithelial and loss of mesenchymal markers. Remarkably, CR-BxPC-3 cells exhibited enhanced EMT features. SiNRP-1 inhibited EMT and promoted cell-death by overcoming gemcitabine resistance in CR-BxPC-3 cells, possibly through reduced ERK signaling. Moreover, siNRP-1 inhibited autophagy (decreased LC3-II/I ratio and increased P62 levels) following starvation or chemotherapy stress, and enhanced cell-death after chemotherapy and autophagy inhibition in BxPC-3 cells.
Conclusions: Our findings define a previously undetermined role of NRP-1 in regulating autophagy and TGFβ1-induced EMT in PDAC, suggesting that NRP-1 may represent a novel therapeutic target to overcome chemoresistance through reduced EMT and autophagy.
Citation Format: Pratiek N. Matkar, Krishna K. Singh, Gerald Prud’homme, David Hedley, Howard Leong-Poi. Neuropilin-1 blockade chemosensitizes pancreatic cancer cells via dual inhibition of epithelial-mesenchymal transition and autophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1198. doi:10.1158/1538-7445.AM2017-1198