Pancreatic ductal adenocarcinoma (PDAC) is an exceptionally lethal cancer that is characterized by an abundant desmoplastic stroma containing numerous tumor-associated fibroblasts (TAFs). TAFs are believed to contribute to PDAC's rapid progression and its resistance to therapy via multiple mechanisms, including remodeling of the extracellular matrix and secretion of motility-promoting cytokines and growth factors. We recently showed that one isoform of the actin-binding protein palladin is highly expressed in TAFs within the stroma of PDAC patient specimens. To investigate palladin's functional role in this cell type, we obtained a line of immortalized human pancreatic TAFs, and generated a sub-line of palladin knockdown TAFs using lentiviral expression of shRNA. The palladin knockdown TAFs were characterized in a mixed-cell, mouse xenograft model, and the results showed that palladin plays a critical role in the metastasis-promoting activity of TAFs. Recent studies in smooth muscle cells yielded new insights into palladin's potential molecular function in TAFs, as these studies have shown that palladin is detected in the nucleus of smooth muscle cells, where it regulates gene expression through interactions with Myocardin-Related Transcription Factor. As TAFs and smooth muscle cells share similar patterns of gene expression, we set out to test the hypothesis that palladin functions as a transcriptional co-regulator also in TAFs. To address this question, we first investigated palladin's subcellular localization in cultured TAFs using immunofluorescence staining and cellular fractionation combined with immunoblot analysis. The results demonstrated that endogenous palladin is detected in both the stress fibers and the nucleus of TAFs. To ask if palladin plays a role in regulating gene expression in TAFs, total RNA from replicate cultures of knockdown and control cells was isolated and subjected to transcriptomic analysis (RNAseq). The results showed that the levels of expression of over 100 genes were significantly altered when palladin was knocked down in TAFs. Pathway analysis revealed that palladin regulates the expression of multiple genes involved in focal adhesion assembly and cell-matrix interactions, including the matrix metalloproteinases MMP1 and MMP3, and also secreted cytokines that regulate cell motility and epithelial-to-mesenchymal transition, including bone morphogenetic protein 4 (BMP4) and its secreted antagonist Gremlin-1. These novel results define an unexpected role for palladin within the nuclear compartment, and suggest that palladin may regulate multiple TAF behaviors that contribute to tumor progression, including matrix remodeling and cytokine secretion. Future studies will be aimed at investigating the functional roles of palladin-regulated genes in the biology and behavior of TAFs.
Citation Format: Meredith Owen, Michael Kerber, Silvia Goicoechea, Rosa F. Hwang, Hong Jin Kim, Carol A. Otey. The actin-binding protein palladin localizes to the nucleus of pancreatic tumor-associated fibroblasts and regulates gene expression. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B46.