Co‐expression of Zeb‐1 and K17 provides new insights into the role of EMT in pancreatic cancer progression

Abstract

INTRODUCTIONDespite improvements in screening and therapeutic options, pancreatic ductal adenocarcinoma (PDAC) has the third‐worst mortality rate, highlighting the need for better diagnostic and therapeutic approaches. Epithelial‐mesenchymal transition (EMT) has been conferred with a greater likelihood of metastasis in multiple cancers, specifically identified through markers like Zeb‐1 and Twist‐1. Zeb‐1 (Zinc Finger E‐Box Binding Homeobox 1) was shown to repress E‐cadherin expression, consequently inducing EMT.Our lab has previously identified K17 (Keratin 17) as an independent prognostic biomarker of poor outcomes in PDAC patients. While increasing evidence has suggested that EMT drives pancreatic cancer migration and metastasis, there have not been any well‐established connections between K17 expression and EMT. This study aims to provide new insights into the role of K17 in EMT by evaluating the co‐expression of K17 and Zeb‐1 in human PDAC biopsies. We hypothesized that tumor cells co‐express K17 and Zeb‐1 as a hallmark of EMT in PDAC. In addition, our study aims to establish a novel method of scoring Zeb‐1 expression in stromal fibroblasts via immunofluorescence to remove variability in the stromal constituency.METHODSPDAC tissues obtained from patients who underwent surgical resection between 2013 and 2017 at Stony Brook University (n=20) were selected for serial sectioning. Double immunofluorescence and single immunohistochemical staining were achieved by incubation with Zeb‐1 and K17 antibodies followed by incubation of fluorescent or biotin conjugated secondary antibodies, respectively. Histologic examination was performed using immunofluorescence staining along with H&E and immunohistochemical staining (IHC) to determine concordance.RESULTS● IHC for Zeb‐1 was detected predominantly in the nuclei of centroacinar cells, benign proliferative ducts, and stromal fibroblasts. Focal nuclear staining in tumor epithelial was also detected in some cases.● High levels of K17 and but decreased expression of Zeb‐1 were detected in diffusely infiltrative cells with morphologic features of EMT.● Both high Zeb‐1 and high K17 protein expression were associated with poor survival of PDAC patients.● Using confocal immunofluorescence microscopy, we detected co‐localization of Zeb‐1 and K17 in PDAC.● We quantified Zeb‐1 protein expression in PDAC samples from 20 patients and confirmed significantly higher expression in the tumor‐associated stroma of high‐grade tumors compared to stromal fibroblasts in areas devoid of tumor.CONCLUSIONOur results provide evidence that K17 is upregulated in tumor epithelial cells that show morphologic immunohistochemical properties of EMT. The loss of Zeb‐1 in diffusely infiltrative, K17‐positive tumor cells suggests that EMT upregulates K17, driving tumor aggression. Our results suggest that K17 may enhance PDAC aggression by mediating EMT. Hence, novel therapeutic agents targeting K17 may improve clinical care for this biologically distinct patient subgroup.