Mutations in KRAS drive the oncogenic phenotype in a variety of tumors of epithelial origin. The NF-κB transcription factor pathway is important for oncogenic RAS to transform cells and to drive tumorigenesis in animal models. Recently TAK1, an upstream regulator of IKK, which controls canonical NF-κB, was shown to be important for chemoresistance in pancreatic cancer and for regulating KRAS+ colorectal cancer cell growth and survival. Here we show that KRAS+ upregulates GSK-3α leading to its interaction with TAK1 to stabilize the TAK1/TAB complex to promote IKK activity. Additionally, GSK-3α is required for promoting critical non-canonical NF-κB signaling in pancreatic cancer cells. Pharmacologic inhibition of GSK-3 suppresses growth of human pancreatic tumor explants, consistent with the loss of expression of oncogenic genes such as c-myc and TERT. These data identify GSK-3α as a key downstream effector of oncogenic KRAS via its ability to coordinately regulate distinct NF-κB signaling pathways.

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<p>PDF file - 181K, Left: Kras regulates TAK1 levels. HKRAS cells were transiently transfected with non-targeting or Kras siRNA for 72 hours. TAK1 was immunoprecitated from whole cell extracts and immunoblotted for specified antibodies. Right: TAK1 inhibition induces apoptosis. MiaPaCa-2 cells were treated with the TAK1 inhibitor, 5Z-7-oxozaenol at indicated concentrations for 24 hours. Whole cells extracts were immunoblotted with specified antibodies.</p>

<p>PDF file - 63K, GSK-3 inhibition inhibits TAK1 mediated signaling. Panc-1 cells were treated with GSK-3 inhibitor, AR-A014418 (30M) for 24 hours and whole cell lysates immunoblotted for indicated antibodies.</p>

<p>PDF file - 308K, Left: AR-A014418 inhibits GSK-3 activity in a dose dependent mannerPanc-1 cells were treated with increasing concentrations of GSK-3 inhibitor, AR-A014418, like in Fig.1A. Whole cells extracts were prepared and immunoblotted for specified antibodies. p-Glycogen synthase is a downstream substrate of GSK-3 and thus an indicator of its kinase activity. Right: GSK-3 regulates apoptosis in MiaPaCa-2 cells. . Panc-1 and MiaPaCa-2 cells were transiently transfected with indicated siRNAs for 72 hours. Whole cell extracts were immunoblotted for specified antibodies.</p>

<p>PDF file - 3743K, TAK1 inhibition alters cell cycle regulation. Panc-1 cells were treated with TAK1 inhibitor, 5Z-7-oxozaenol or vehicle control, DMSO for 24 hours. Cells were fixed and stained with Propidium Iodide and analyzed by Flow cytometry.</p>

<p>PDF file - 118K, GSK-3 inhibition leads to a decrease in TAK1 protein Panc-1 and MiaPaCa-2 cells were treated with GSK-3 inhibitor, AR-A014418 or vehicle control, DMSO in the presence or absence of MG132 for 24 hours. Whole cell extracts were immunoblotted for indicated antibodies.</p>