GSK-3? promotes oncogenic KRAS function in pancreatic cancer via TAK1-TAB stabilization and regulation of noncanonical NF-?B.
ABSTRACT: Mutations in KRAS drive the oncogenic phenotype in a variety of tumors of epithelial origin. The NF-?B transcription factor pathway is important for oncogenic RAS to transform cells and to drive tumorigenesis in animal models. Recently, TGF-?-activated kinase 1 (TAK1), an upstream regulator of I?B kinase (IKK), which controls canonical NF-?B signaling, was shown to be important for chemoresistance in pancreatic cancer and for regulating KRAS-mutant colorectal cancer cell growth and survival. Here, we show that mutant KRAS upregulates glycogen synthase kinase 3? (GSK-3?), leading to its interaction with TAK1 to stabilize the TAK1-TAB complex to promote IKK activity. In addition, GSK-3? is required for promoting critical noncanonical NF-?B 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-3? as a key downstream effector of oncogenic KRAS via its ability to coordinately regulate distinct NF-?B signaling pathways.
Project description:Mutations in KRAS occur in a variety of tumors of epithelial origin, driving the oncogenic phenotype.The NF-kB 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-kB, was shown to be important for chemoresistance in pancreatic cancer and for regulating KRAS+ colorectal cancer cell growth and survival. Here we show that GSK-3alpha is upregulated by KRAS leading to interaction with TAK1 to stabilize the TAK1/TAB complex to promote IKK activity. Additionally, GSK-3alpha is required for promoting critical non-canonical NF-kB signaling in pancreatic cancer cells. Pharmacologic inhibition of GSK-3 suppresses growth of human pancreatic tumor explants, consistent with loss of expression of genes such as c-myc and TERT. These data identify GSK-3alpha as a key downstream effector of oncogenic RAS via its ability to coordinately regulate distinct NF-kB signaling pathways GSK-3 inhibition at 2 and 8 hours
Project description:Constitutive nuclear factor kappaB (NF-kappaB) activation is among the many deregulated signaling pathways that are proposed to drive pancreatic cancer cell growth and survival. Recent reports suggest that glycogen synthase kinase-3beta (GSK-3beta) plays a key role in maintaining basal NF-kappaB target gene expression and cell survival in pancreatic cancer cell lines. However, the mechanism by which GSK-3beta facilitates constitutive NF-kappaB signaling in pancreatic cancer remains unclear. In this report, we analyze the contributions of both GSK-3 isoforms (GSK-3alpha and GSK-3beta) in regulating NF-kappaB activation and cell proliferation in pancreatic cancer cell lines (Panc-1 and MiaPaCa-2). We show that GSK-3 isoforms are differentially required to maintain basal NF-kappaB DNA binding activity, transcriptional activity, and cell proliferation in Panc-1 and MiaPaCa-2 cells. Our data also indicate that IkappaB kinase (IKK) subunits are not equally required to regulate pancreatic cancer-associated NF-kappaB activity and cell growth. Importantly, we provide the first evidence that GSK-3 maintains constitutive NF-kappaB signaling in pancreatic cancer by regulating IKK activity. These data provide new insight into GSK-3-dependent NF-kappaB regulation and further establish GSK-3 and IKK as potential therapeutic targets for pancreatic cancer.
Project description:SRY (sex determining region Y)-box 9 (SOX9) is required for oncogenic Kras-mediated acinar-to-ductal metaplasia (ADM), pancreatic intraepithelial neoplasias (PanINs) and ultimately pancreatic ductal adenocarcinoma (PDAC). However, how oncogenic Kras affects SOX9 activity is not yet understood, and SOX9-associated genes in PDAC are also unknown at all. Here, we investigated the mechanistic link between SOX9 and oncogenic Kras, studied biological function of SOX9, and identified SOX9-related genes and their clinical significance in patients with PDAC. Our studies reveal that oncogenic Kras induces SOX9 mRNA and protein expression as well as phosphorylated SOX9 expression in human pancreatic ductal progenitor cells (HPNE) and pancreatic ductal cells (HPDE). Moreover, oncogenic Kras promoted nuclear translocation and transcriptional activity of SOX9 in these cells. TAK1/I?B?/NF-?B pathway contributed to induction of SOX9 by oncogenic Kras, and SOX9 in turn enhanced NF-?B activation. SOX9 promoted the proliferation of HPNE and PDAC cells, and correlated with minichromosome maintenance complex components (MCMs) and mediator of DNA damage checkpoint 1 (MDC1) expression. The overexpressive MDC1 was associated with less perineural and lymph node invasion of tumors and early TNM-stage of patients. Our results indicate that oncogenic Kras induces constitutive activation of SOX9 in HPNE and HPDE cells, and Kras/TAK1/I?B?/NF-?B pathway and a positive feedback between SOX9 and NF-?B are involved in this inducing process. SOX9 accelerates proliferation of cells and affects MCMs and MDC1 expression. MDC1 is associated negatively with invasion and metastasis of PDAC.
Project description:OBJECTIVES:The ability of tumor cells to drive angiogenesis is an important cancer hallmark that positively correlates with metastatic potential and poor prognosis. Therefore, targeting angiogenesis is a rational therapeutic approach and dissecting proangiogenic pathways is important, particularly for malignancies driven by oncogenic KRAS, which are widespread and lack effective targeted therapies. Based on published studies showing that oncogenic RAS promotes angiogenesis by upregulating the proangiogenic NF-?B target genes IL-8 and VEGF, that NF-?B activation by KRAS requires the IKK? kinase, and that targeting IKK? reduces KRAS-induced lung tumor growth in vivo, but has limited effects on cell growth in vitro, we hypothesized that IKK? targeting would reduce lung tumor growth by inhibiting KRAS-induced angiogenesis. MATERIALS AND METHODS:To test this hypothesis, we targeted IKK? in KRAS-mutant lung cancer cell lines either by siRNA-mediated transfection or by treatment with Compound A (CmpdA), a highly specific IKK? inhibitor, and used in vitro and in vivo assays to evaluate angiogenesis. RESULTS AND CONCLUSIONS:Both pharmacological and siRNA-mediated IKK? targeting in lung cells reduced expression and secretion of NF-?B-regulated proangiogenic factors IL-8 and VEGF. Moreover, conditioned media from IKK?-targeted lung cells reduced human umbilical vein endothelial cell (HUVEC) migration, invasion and tube formation in vitro. Furthermore, siRNA-mediated IKK? inhibition reduced xenograft tumor growth and vascularity in vivo. Finally, IKK? inhibition also affects endothelial cell function in a cancer-independent manner, as IKK? inhibition reduced pathological retinal angiogenesis in a mouse model of oxygen-induced retinopathy. Taken together, these results provide a novel mechanistic understanding of how the IKK? pathway affects human lung tumorigenesis, indicating that IKK? promotes KRAS-induced angiogenesis both by cancer cell-intrinsic and cancer cell-independent mechanisms, which strongly suggests IKK? inhibition as a promising antiangiogenic approach to be explored for KRAS-induced lung cancer therapy.
Project description:Resibufogenin (RB), one of the major active compounds of the traditional Chinese medicine Chansu, has received considerable attention for its potency in cancer therapy. However, the anticancer effects and the underlying mechanisms of RB on pancreatic cancer remain elusive. Here, we found that RB inhibited the viability and induces caspase-dependent apoptosis in human pancreatic cancer cells Panc-1 and Aspc. Resibufogenin-induced apoptosis was through inhibition of constitutive nuclear factor-?B (NF-?B) activity and its target genes' expression, which was caused by downregulation of transforming growth factor-?-activated kinase 1 (TAK1) levels and suppression of I?B kinase activity in Panc-1 and Aspc cells. This induction of TAK1-mediated NF-?B inactivation by RB was associated with increased glycogen synthase kinase-3 (GSK-3) phosphorylation and subsequent suppression of its activity. Moreover, RB-induced GSK-3 phosphorylation/inactivation acted through activation of protein kinase C but not Akt. Finally, RB suppressed human pancreatic tumor xenograft growth in athymic nude mice. Thus, our findings reveal a novel mechanism by which RB suppresses TAK1-mediated NF-?B activity through protein kinase C-dependent inhibition of GSK-3. Our findings provide a rationale for the potential application of RB in pancreatic cancer therapy.
Project description:The conversion of transforming growth factor beta (TGF-beta) from a tumor suppressor to a tumor promoter occurs frequently during mammary tumorigenesis, yet the molecular mechanisms underlying this phenomenon remain undefined. We show herein that TGF-beta repressed nuclear factor-kappaB (NF-kappaB) activity in normal NMuMG cells, but activated this transcription factor in their malignant counterparts, 4T1 cells, by inducing assembly of TGF-beta-activated kinase 1 (TAK1)-binding protein 1 (TAB1):I kappaB kinase beta (IKK beta) complexes, which led to the stimulation of a TAK1:IKK beta:p65 pathway. TAB1:IKK beta complexes could only be detected in NMuMG cells following their induction of epithelial-mesenchymal transition (EMT), which, on TGF-beta treatment, activated NF-kappaB. Expression of a truncated TAB1 mutant [i.e., TAB1(411)] reduced basal and TGF-beta-mediated NF-kappaB activation in NMuMG cells driven to undergo EMT by TGF-beta and in 4T1 cells stimulated by TGF-beta. TAB1(411) expression also inhibited TGF-beta-stimulated tumor necrosis factor-alpha and cyclooxygenase-2 expression in 4T1 cells. Additionally, the ability of human MCF10A-CA1a breast cancer cells to undergo invasion in response to TGF-beta absolutely required the activities of TAK1 and NF-kappaB. Moreover, small interfering RNA-mediated TAK1 deficiency restored the cytostatic activity of TGF-beta in MCF10A-CA1a cells. Finally, expression of truncated TAB1(411) dramatically reduced the growth of 4T1 breast cancers in syngeneic BALB/c, as well as in nude mice, suggesting a potentially important role of NF-kappaB in regulating innate immunity by TGF-beta. Collectively, our findings have defined a novel TAB1:TAK1:IKK beta:NF-kappaB signaling axis that forms aberrantly in breast cancer cells and, consequently, enables oncogenic signaling by TGF-beta.
Project description:Activating mutations in KRAS are prevalent in cancer, but therapies targeted to oncogenic RAS have been ineffective to date. These results argue that targeting downstream effectors of RAS will be an alternative route for blocking RAS-driven oncogenic pathways. We and others have shown that oncogenic RAS activates the NF-?B transcription factor pathway and that KRAS-induced lung tumorigenesis is suppressed by expression of a degradation-resistant form of the I?B? inhibitor or by genetic deletion of IKK? or the RELA/p65 subunit of NF-?B. Here, genetic and pharmacological approaches were utilized to inactivate IKK in human primary lung epithelial cells transformed by KRAS, as well as KRAS mutant lung cancer cell lines. Administration of the highly specific IKK? inhibitor Compound A (CmpdA) led to NF-?B inhibition in different KRAS mutant lung cells and siRNA-mediated knockdown of IKK? or IKK? reduced activity of the NF-?B canonical pathway. Next, we determined that both IKK? and IKK? contribute to oncogenic properties of KRAS mutant lung cells, particularly when p53 activity is disrupted. Based on these results, CmpdA was tested for potential therapeutic intervention in the Kras-induced lung cancer mouse model (LSL-Kras (G12D)) combined with loss of p53 (LSL-Kras (G12D)/p53 (fl/fl)). CmpdA treatment was well tolerated and mice treated with this IKK? inhibitor presented smaller and lower grade tumors than mice treated with placebo. Additionally, IKK? inhibition reduced inflammation and angiogenesis. These results support the concept of targeting IKK as a therapeutic approach for oncogenic RAS-driven tumors with altered p53 activity.
Project description:Responses to transforming growth factor beta and multiple cytokines involve activation of transforming growth factor beta-activated kinase-1 (TAK1) kinase, which activates kinases IkappaB kinase (IKK) and MKK3/6, leading to the parallel activation of NF-kappaB and p38 MAPK. Activation of TAK1 by autophosphorylation is known to involve three different TAK1-binding proteins (TABs). Here we report a protein phosphatase subunit known as type 2A phosphatase-interacting protein (TIP) that also acts as a TAB because it co-precipitates with and directly binds to TAK1, enhances TAK1 autophosphorylation at unique sites, and promotes TAK1 phosphorylation of IKKbeta and signaling to NF-kappaB. Mass spectrometry demonstrated that co-expression of TAB4 protein significantly increased phosphorylation of four sites in TAK1, in a linker region between the kinase and TAB2/3 binding domains, and two sites in TAB1. Recombinant GST-TAB4 bound in an overlay assay directly to inactive TAK1 and activated TAK1 but not TAK1 phosphorylated in the linker sites, suggesting a bind and release mechanism. In kinase assays using TAK1 immune complexes, added GST-TAB4 selectively stimulated IKK phosphorylation. TAB4 co-precipitated polyubiquitinated proteins dependent on a Phe-Pro motif that was required to enhance phosphorylation of TAK1. TAB4 mutated at Phe-Pro dominantly interfered with IL-1beta activation of NF-kappaB involving IKK-dependent but not p38 MAPK-dependent signaling. The results show that TAB4 binds TAK1 and polyubiquitin chains to promote specific sites of phosphorylation in TAK1-TAB1, which activates IKK signaling to NF-kappaB.
Project description:Tumor cells carrying KRAS mutations activate the NF-?B pathway by different mechanisms, which contribute to the acquisition of essential cancer properties. The BRAF kinase, a downstream mediator of KRAS, is also mutated in a subset of colorectal cancers (CRC), which predicts bad prognosis and therapy resistance. However, nothing is known on whether NF-?B participates of BRAF-mediated tumorigenesis. We here found that in CRC cells, mutant BRAF does not trigger canonical or alternative NF-?B signaling but induces p45-IKK? activation. Moreover, IKK? activity is required for BRAF-induced transformation and to support BRAF-dependent transcription in CRC cells. Activation of p45-IKK? downstream of BRAF requires the TAK1 kinase, and is associated to the endosomal compartment. Inhibition of endosomal V-ATPase abolished p45-IKK? phosphorylation, and induced apoptosis of BRAF mutated CRC cells. Pharmacologic inhibition of endosome acidification reduced the in vivo growth of tumors carrying mutant BRAF, and abrogated the metastatic capacity of a primary human CRC tumor with acquired resistance to standard chemotherapy in an orthotopic xenograft model. 18 samples were analyzed: HT29 controls (n=3); HT28 BRAF inhibited (n=3), WiDr controls (n=3); WiDr BRAF inhibited (n=3); WiDr transduced with control shRNA (n=3) and WiDr transduced with shRNA against IKKalpha (n=3)