Abstract 4555: Identification of bone morphogenetic protein 2 splice variant in pancreatic cancer reveals cross-talk between endocytic and autophagy pathways

The inherent heterogeneity in pancreatic cancer (PC) confers complex transcription patterns that require a subset approach to identifying novel gene targets. Thus, we performed meta-analysis on 11 COPA (Cancer Outlier Profile Analysis) transformed datasets (www.oncomine.org) depicting the expression profiles of 416 pancreatic tumors. This analysis revealed Bone Morphogenetic Protein 2 (BMP-2), a pivotal regulator of cellular proliferation, motility, & differentiation, to be increased in a subset of PC tumors. Canonical BMP-2 signaling is mediated via type 1 & type 2 serine/threonine kinase receptors, which upon ligand binding, phosphorylate SMAD proteins leading to initiation or attenuation of gene expression programs. Receptor mediated endocytosis has been shown to be an integral regulator of BMP signaling intensity and duration whereby endocytic internalization of activated receptors leads to their subsequent inactivation and recycling back to the plasma membrane or degradation via the lysosomal pathway. Our investigation of BMP-2 in PC has revealed a novel splice variant (BMP-2 SV), which due to exclusion of its signal peptide bearing exon, escapes the secretory pathway and is endowed with an intracellular pattern of expression. Furthermore, this BMP-2 isoform undergoes preferential degradation via the autophagy lysosomal pathway (ALP). Endosomal and autophagosomal trafficking has been previously shown to converge, through endosome and autophagosome vesicular fusion, to form a hybrid structure described as an amphisome. We hypothesize that endocytosed bone morphogenetic receptors and autophagocytosed BMP-2 SV converge in the amphisome thereby promoting signal transduction. Indeed, immunofluorescence analysis has revealed colocalization of the exogenous BMP-2 SV with components of both the endocytic and autophagic pathways. Furthermore, overexpression of BMP-2 SV in HEK-293 and pancreatic cancer cell lines was performed, which led to a corresponding increased Smad 1/5/8 phosphorylation. In contrast, biochemical inhibition of receptor mediated endocytosis with PitStop ® 2 or disruption of microtubule-dependent vesicular transport with vinblastine was found to diminish the BMP-SV dependent increase in Smad 1/5/8 phosphorylation. Likewise, increasing autophagy through Rapamycin treatment led to a corresponding increase in Smad 1/5/8 phosphorylation. Altogether, our results suggest a novel amphisome-mediated mechanism of BMP-2 signaling due to convergence of endocytosed receptors and autophagocytosed BMP-2 SV. Consequently, this intracellular mode of signaling may endow pancreatic tumor cells with a cell-autonomous means of driving its own growth and metastasis. Citation Format: Eric Cruz, Surinder K. Batra. Identification of bone morphogenetic protein 2 splice variant in pancreatic cancer reveals cross-talk between endocytic and autophagy pathways. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4555.