WNT11 PROMOTES PANCREATIC CANCER CELL MOTILITY AND INVASION
Tara Hughes*1, Xinqun Li1, Bingbing Dai1, Christian Siangco1, Kaberi Das1, Shanshan Bai2, Hu Min2, Tapsi Kumar2, Emi Sei2, Nicholas Navin2, Michael P. Kim1
1Surgical Oncology, MD Anderson Cancer Center, Houston, TX; 2Genetics, MD Anderson Cancer Center, Houston, TX
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, the mechanisms previously described are diverse and tissue specific. We investigated the role of Wnt11 on PDAC cell migration/invasion and identified gene expression signatures associated with Wnt11 expression through single cell transcriptomic analysis of 14 tumors directly isolated from PDAC patients. Expression of Wnt11 was confirmed by RT-qPCR in PDAC cell lines derived from genetically engineered mouse models and PDAC patient derived xenografts. Wnt11 was overexpressed in PDAC cell lines through transfection with Wnt11 cDNA cloned into a pcDNA3.1 backbone and confirmed by RT-qPCR. Wnt11 was silenced using two unique siRNAs along with a non-targeting control and confirmed by RT-qPCR. Transwell migration/invasion assays were performed in 3 PDAC cell lines overexpressing Wnt11 and in 3 cell lines following siRNA-mediated Wnt11 knockdown. For single cell transcriptomic analysis, single cell suspensions were generated from 14 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 tumor cells were selected and compared to remaining tumor cell populations to develop differentially expressed gene profiles. Pathways analysis of derived gene sets was performed using Enrichr. Single cell RNA-sequencing data derived from 14 PDAC patient tumors revealed strong Wnt11 expression in subpopulations of PDAC cells. RT-qPCR confirmed expression of Wnt11 in all tested KPC and human PDAC cell lines. Stable overexpression of Wnt11 resulted in a 3.2-fold increase (3.2 ±1.46; p<0.001) in migration and invasion in three tested PDAC cell lines. RNAi-mediated depletion of Wnt11 resulted in a 6.6 -fold reduction (6.6±3.1; p<0.001) in PDAC cell migration and invasion in three tested cell lines. In PDAC cell populations with increased Wnt11 expression (>4-fold), ERICHR analysis using the NCI pathway interaction database revealed significant enrichment in β-integrin signaling pathways (p<0.001). Wnt11 is strongly expressed in subpopulations of human PDAC cells and significantly increases PDAC cell motility and invasion. Ongoing lung colonization assays with Wnt11-null PDAC cell lines 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.
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