Abstract 2738: Wnt11 regulates migration and invasion in pancreatic ductal adenocarcinoma

Abstract

Abstract Canonical Wnt/β-catenin signaling is strongly associated with cancer development and metastasis, but non-canonical Wnt signaling and its role in pancreatic ductal adenocarcinoma (PDAC) metastasis is not well understood. Wnt11 is implicated in cancer cell motility through non-canonical Wnt signaling pathways, however, previously described mechanisms are diverse and tissue specific. We investigated Wnt11 expression and its effects on PDAC cell motility/invasion and correlated these with gene expression signatures generated through single cell RNA-sequencing of PDAC tumors. Expression of Wnt11 was confirmed by RT-qPCR in PDAC cell lines derived from genetically engineered mouse models (KPC) and PDAC patient derived xenografts. Wnt11 was overexpressed in three PDAC cell lines following transfection with Wnt11 cDNA cloned into a pcDNA3.1 backbone and confirmed by RT-qPCR and western blot. siRNAs targeting Wnt11 were used to silence Wnt11 in PDAC cell lines relative to non-targeting controls and confirmed by RT-qPCR. Transwell migration/invasion assays were performed in triplicate with matched MTT proliferation assays in 3 PDAC cell lines with stable Wnt11 overexpression and 3 PDAC cell lines following Wnt11 siRNA knockdown. For single cell transcriptomic analysis, single cell suspensions were generated from 14 unique patient PDAC tumors. 10x GemCode microfluidics technology was used to isolate single cells into gel-bead in emulsion (GEM) units and were individually barcoded, lysed and reverse-transcribed. Barcoded cDNA libraries were then amplified and underwent next-generation sequencing. PDAC cell subpopulations with >4-fold levels of Wnt11 expression relative to all sequenced cells were identified and compared to the remaining tumor cell population to develop differentially expressed gene profiles. Pathways analysis of this gene set was performed using ENRICHR. Single cell transcriptomic analysis of PDAC cells derived from 14 different PDAC patients confirmed strong Wnt11 expression in subpopulations of PDAC cells. Wnt11 overexpression resulted in a 3.2-fold increase in migration and invasion (p<0.001) and RNAi-mediated depletion of Wnt11 resulted in a 6.6-fold reduction in migration and invasion (p<0.001). In PDAC cell populations with increased (>4-fold) Wnt11 expression, ERICHR analysis using the NCI pathway interaction database revealed significant enrichment in β-integrin signaling (p<0.001, z = -1.16). Wnt11 is strongly expressed in subpopulations of human PDAC cells and significantly enhances PDAC cell motility and invasion. Ongoing tail vein injection experiments with Wnt11-null PDAC cell lines and Wnt11 wildtype controls will test the in vivo role of Wnt11 in PDAC metastasis. β-integrin signaling is associated with Wnt11 expression in human PDAC tumors and may be involved in PDAC cell motility and invasion. Citation Format: Tara Hughes, Xinqun Li, Bingbing Dai, Jenying Deng, Christian Siangco, Kaberi Das, Shanshan Bai, Min Hu, Emi Sei, Tapsi Seth, Nicholas Navin, Michael Kim. Wnt11 regulates migration and invasion in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2738.