Abstract
Abstract The EGF receptor (EGFR) ligand epiregulin (EREG) is delivered preferentially to the basolateral cell surface of polarized MDCK cells. Previously, we showed that EREG basolateral trafficking is regulated by a conserved tyrosine residue within a YXXΦ motif (Y156ERV) in its cytoplasmic domain. Interestingly, a Y156A substitution led to apical mistrafficking of EREG and transformation of polarized MDCK cells (PNAS 110: 8960-5, 2013). We have identified an EREG mutation (R147stop) in human tumors that we now report mistrafficks to the apical surface. To test the hypothesis that apical mistrafficking of EREG can be a driver event, we have generated MDCK cells stably expressing inducible (Tet-ON) wild-type and Y156A mutant EREG. In 3D Matrigel cultures, we show that uninduced clones form normal-appearing cysts, regardless of EREG status. Induction of WT or mutant EREG leads to increased cyst diameter. However, only (Y156A)EREG-expressing cells form abnormal cysts with ectopic lumens and inward growth, features of in vivo transformation. Furthermore, we show that ligand processing and EGFR activity is required for EREG mistrafficking-induced ectopic lumen formation as preincubation with a broad-spectrum metalloprotease inhibitor, BB-94, abrogates ectopic lumen formation. We have extended these studies to polarizing human colon cancer cells, HCA-7, which preserve the trafficking fidelity of WT and mutant EREG. Furthermore, using EREG cytoplasmic domain as a bait in a yeast-2-hybrid screen, we have identified APOBEC3C as an interacting protein. APOBECs are a family of cytidine deaminases that have been identified as a major contributor to human cancer mutations. We have subsequently confirmed EREG-APOBEC3C interaction by co-immunoprecipitation. Results from MDCK cells stably expressing differentially inducible EREG (Tet-ON) and APOBEC3C (Cumate-ON) will be presented. Note: This abstract was not presented at the meeting. Citation Format: Bhuminder Singh, Galina Bogatcheva, Robert J. Coffey. A 3D system to elucidate a direct role for epiregulin mistrafficking in epithelial transformation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2143. doi:10.1158/1538-7445.AM2015-2143
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