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Video 3. N+/E- cells 3D reconstruction animation shows localization of ENT1 (red) and DAPI (blue)

Epithelial–mesenchymal transition (EMT) in cancer cells drives cancer chemoresistance, yet the molecular events of EMT that underpin the acquisition of chemoresistance are poorly understood. Here, we demonstrate a loss of gemcitabine chemosensitivity facilitated by human equilibrative nucleoside transporter 1 (ENT1) during EMT in pancreatic cancer and identify that cadherin switching from the epithelial (E) to neuronal (N) type, a hallmark of EMT, contributes to this loss. Our findings demonstrate that N-cadherin decreases ENT1 expression, membrane localization, and gemcitabine transport, while E-cadherin augments each of these. Besides E- and N-cadherin, another epithelial cell adhesion molecule, EpCAM, played a more prominent role in determining ENT1 membrane localization. Forced expression of EpCAM opposed cadherin switching with restored ENT1 expression, membrane localization, and gemcitabine transport in EMT-committed pancreatic cancer cells. In gemcitabine-treated mice, EpCAM-positive tumors had high ENT1 expression and reduced metastasis, whereas tumors with N-cadherin expression resisted gemcitabine treatment and formed extensive secondary metastatic nodules. Tissue microarray profiling and multiplexed IHC analysis of pancreatic cancer patient-derived primary tumors revealed EpCAM and ENT1 cell surface coexpression is favored, and ENT1 plasma membrane expression positively predicted median overall survival times in patients treated with adjuvant gemcitabine. Together, our findings identify ENT1 as an inadvertent target of EMT signaling mediated by cadherin switching and provide a mechanism by which mesenchymal pancreatic cancer cells evade gemcitabine therapy during EMT.

Video 2. Animation displays 3D reconstruction of PANC-1 cells grown in monolayer immunostained for ENT1 (red) and DAPI (blue)

Video 4. E+/N- cells animation displays 3D reconstruction of ENT1 (red) and DAPI (blue)

Video 5. PANC-1 cells stably overexpressing EpCAM are shown in this animation of a 3D reconstruction of compiled z-stack images. ENT1 (red), EpCAM (green), DAPI (blue).

Video 1. PANC-1 cells stably overexpressing E-cadherin are shown in this animation of a 3D reconstruction of compiled z-stack images. ENT1 (red), E-cadherin (green), DAPI (blue).

Epithelial-mesenchymal transition (EMT) in cancer cells drives cancer chemoresistance, yet the molecular events of EMT that underpin the acquisition of chemoresistance are poorly understood. Here, we demonstrate a significant loss of gemcitabine chemosensitivity facilitated by human equilibrative nucleoside transporter 1 (ENT1) during EMT in pancreatic cancer and identify that cadherin switching from the epithelial (E) to neuronal (N) type, a hallmark of EMT, contributes to this loss. Our findings demonstrate that N-cadherin decreases ENT1 expression, basolateral membrane localization and gemcitabine transport, while E-cadherin augments each of these. Besides E- and N-cadherin, another epithelial cell adhesion molecule, EpCAM, played a more prominent role in determining ENT1 membrane localization. Forced expression of EpCAM opposed cadherin switching with restored ENT1 expression, basolateral localization and gemcitabine transport in EMT-committed pancreatic cancer cells. In gemcitabine-treated mice, EpCAM positive tumors had high ENT1 expression and reduced metastasis, whereas tumors with N-cadherin expression resisted gemcitabine treatment and formed extensive secondary metastatic nodules. Tissue microarray profiling and multiplexed immunohistochemical analysis of pancreatic cancer patient-derived primary tumors revealed EpCAM and ENT1 cell surface co-expression is favored, and ENT1 plasma membrane expression positively predicted median overall survival times in patients treated with adjuvant gemcitabine. Together, our findings identify ENT1 as an inadvertent target of EMT signaling mediated by cadherin switching and provide a mechanism by which mesenchymal pancreatic cancer cells evade gemcitabine therapy during EMT. Citation Format: Brenna Weadick, Debasis Nayak, Avinash K. Persaud, Sau Wai Hung, Radhika Raj, Wei Chen, Junan Li, Terence M. Williams, Rajgopal Govindarajan. EMT-induced gemcitabine resistance in pancreatic cancer involves the functional loss of equilibrative nucleoside transporter 1 [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-062.