Figure S7 shows: (A) Histograms show the fold increasein Annexin V positive cells following treatment with LY2090314 3μM for 72 hours.(B) AsPC1, Panc1 and MDA-Panc28 cell lines were treated as in (A). Cell extracts were subjected to immunoblot with the indicated antibodies. (C) AsPC1, Panc1 and MDA- Panc28 cell lines were treated with LY2090314 3μM for 72 hours and then either treated or not with nab-paclitaxel 0.02ng/ml for additional 24 hours. Cell extracts were subjected to immunoblot with the indicated antibodies. (D) Normal human pancreatic duct epithelial cells HPDE were pretreated with LY2090314 3μM or dimethyl sulfoxide (DMSO) as control for 72h, and subsequently treated with equitoxic increasing doses of nab- paclitaxel and washed-out after 24h. Sulforhodamine B (SRB) assay was used to obtain relative estimates of viable cell number. Means and SD are shown. Curves were fitted by nonlinear regression analysis.
ARTICLE ABSTRACT
YAP and TAZ are central determinants of malignancy; however, their functions remain still undruggable. We identified TGFβ-activated kinase 1 (TAK1) as a central hub integrating the most relevant signals sustaining pancreatic cancer aggressiveness and chemoresistance. Glycogen synthase kinase (GSK)3 is known to stabilize TAK1, and its inhibition causes a reduction in TAK1 levels. Here, we hypothesized that TAK1 could sustain YAP/TAZ program, and thus, modulation of TAK1 expression through the inhibition of GSK3 could impair YAP/TAZ functions in pancreatic cancer.Differentially expressed transcripts between pancreatic cancer cells expressing scramble or TAK1-specific shRNA were annotated for functional interrelatedness by ingenuity pathway analysis. TAK1 expression was modulated by using different GSK3 inhibitors, including LY2090314. In vivo activity of LY2090314 alone or in combination with nab-paclitaxel was evaluated in an orthotopic nude mouse model.Differential gene expression profiling revealed significant association of TAK1 expression with HIPPO and ubiquitination pathways. We measured a significant downregulation of YAP/TAZ and their regulated genes in shTAK1 cells. TAK1 prevented YAP/TAZ proteasomal degradation in a kinase independent manner, through a complex with TRAF6, thereby fostering their K63-ubiquitination versus K48-ubiquitination. Pharmacologic modulation of TAK1 by using GSK3 inhibitors significantly decreased YAP/TAZ levels and suppressed their target genes and oncogenic functions. In vivo, LY2090314 plus nab-paclitaxel significantly prolonged mice survival duration.Our study demonstrates a unique role for TAK1 in controlling YAP/TAZ in pancreatic cancer. LY2090314 is a novel agent that warrants further clinical development in combination with nab-paclitaxel for the treatment of pancreatic cancer.
