Excessive activity of hepatic atypical protein kinase (aPKC) is proposed to play a critical role in mediating lipid and carbohydrate abnormalities in obesity, the metabolic syndrome, and type 2 diabetes mellitus. In previous studies of rodent models of obesity and type 2 diabetes mellitus, adenoviral-mediated expression of kinase-inactive aPKC rapidly reversed or markedly improved most if not all metabolic abnormalities. Here, we examined effects of 2 newly developed small-molecule PKC-ι/λ inhibitors. We used the mouse model of heterozygous muscle-specific knockout of PKC-λ, in which partial deficiency of muscle PKC-λ impairs glucose transport in muscle and thereby causes glucose intolerance and hyperinsulinemia, which, via hepatic aPKC activation, leads to abdominal obesity, hepatosteatosis, hypertriglyceridemia, and hypercholesterolemia. One inhibitor, 1H-imidazole-4-carboxamide, 5-amino-1-[2,3-dihydroxy-4-[(phosphonooxy)methyl]cyclopentyl-[1R-(1a,2b,3b,4a)], binds to the substrate-binding site of PKC-λ/ι, but not other PKCs. The other inhibitor, aurothiomalate, binds to cysteine residues in the PBl-binding domains of aPKC-λ/ι/ζ and inhibits scaffolding. Treatment with either inhibitor for 7 days inhibited aPKC, but not Akt, in liver and concomitantly improved insulin signaling to Akt and aPKC in muscle and adipocytes. Moreover, both inhibitors diminished excessive expression of hepatic, aPKC-dependent lipogenic, proinflammatory, and gluconeogenic factors; and this was accompanied by reversal or marked improvements in hyperglycemia, hyperinsulinemia, abdominal obesity, hepatosteatosis, hypertriglyceridemia, and hypercholesterolemia. Our findings highlight the pathogenetic importance of insulin signaling to hepatic PKC-ι in obesity, the metabolic syndrome, and type 2 diabetes mellitus and suggest that 1H-imidazole-4-carboxamide, 5-amino-1-[2,3-dihydroxy-4-[(phosphonooxy)methyl]cyclopentyl-[1R-(1a,2b,3b,4a)] and aurothiomalate or similar agents that selectively inhibit hepatic aPKC may be useful treatments.
Lung cancer is more deadly than colon, breast, and prostate cancers combined, and treatment improvements have failed to improve prognosis significantly. Here, we identify a critical mediator of lung cancer progression, Rac1b, a tumor-associated protein with cell-transforming properties that are linked to the matrix metalloproteinase (MMP)–induced epithelial-mesenchymal transition (EMT) in lung epithelial cells. We show that expression of mouse Rac1b in lung epithelial cells of transgenic mice stimulated EMT and spontaneous tumor development and that activation of EMT by MMP-induced expression of Rac1b gave rise to lung adenocarcinoma in the transgenic mice through bypassing oncogene-induced senescence. Rac1b is expressed abundantly in stages 1 and 2 of human lung adenocarcinomas and, hence, is an attractive molecular target for the development of new therapies that prevent progression to later-stage lung cancers.
Alveolar rhabdomyosarcoma is an aggressive pediatric cancer exhibiting skeletal muscle differentiation. New therapeutic targets are required to improve the dismal prognosis for invasive or metastatic alveolar rhabdomyosarcoma. Protein kinase C iota (PKCι) has been shown to play an important role in tumorigenesis of many cancers but little is known about its role in rhabdomyosarcoma. Our gene expression studies in human tumor samples revealed overexpression of PRKCI. We confirmed overexpression of PKCι at the mRNA and protein level using our conditional mouse model that authentically recapitulates the progression of rhabdomyosarcoma in humans. Inhibition of Prkci by RNA interference resulted in a dramatic decrease in anchorage-independent colony formation. Interestingly, treatment of primary cell cultures using aurothiomalate (ATM), which is a gold-containing classical anti-rheumatic agent and a PKCι-specific inhibitor, resulted in decreased interaction between PKCι and Par6, decreased Rac1 activity and reduced cell viability at clinically relevant concentrations. Moreover, co-treatment with ATM and vincristine, a microtubule inhibitor currently used in rhabdomyosarcoma treatment regimens, resulted in a combination index (C. I.) of 0.470–0.793 through cooperative accumulation of non-proliferative multinuclear cells in the G2/M phase, indicating that these two drugs synergize. For in vivo tumor growth inhibition studies, ATM demonstrated a trend towards enhanced vincristine sensitivity. Overall, these results suggest that PKCι is functionally important in alveolar rhabdomyosarcoma anchorage-independent growth and tumor cell proliferation and that combination therapy with ATM and microtubule inhibitors holds promise for the treatment of alveolar rhabdomyosarcoma.
Alveolar rhabdomyosarcoma; protein kinase C iota; Rac1; mitosis; aurothiomalate; vincristine
Accumulating evidence demonstrates that PKCι is an oncogene and prognostic marker that is frequently targeted for genetic alteration in many major forms of human cancer. Functional data demonstrate that PKCι is required for the transformed phenotype of NSCLC, pancreatic, ovarian, prostate, colon and brain cancer cells. Future studies will be required to determine whether PKCι is also an oncogene in the many other cancer types that also overexpress PKCι. Studies of PKCι using genetically defined models of tumorigenesis have revealed a critical role for PKCι in multiple stages of tumorigenesis, including tumor initiation, progression and metastasis. Recent studies in a genetic model of lung adenocarcinoma suggest a role for PKCι in transformation of lung cancer stem cells. These studies have important implications for the therapeutic use of aurothiomalate (ATM), a highly selective PKCι signaling inhibitor currently undergoing clinical evaluation. Significant progress has been made in determining the molecular mechanisms by which PKCι drives the transformed phenotype, particularly the central role played by the oncogenic PKCι-Par6 complex in transformed growth and invasion, and of several PKCι-dependent survival pathways in chemo-resistance. Future studies will be required to determine the composition and dynamics of the PKCι-Par6 complex, and the mechanisms by which oncogenic signaling through this complex is regulated. Likewise, a better understanding of the critical downstream effectors of PKCι in various human tumor types holds promise for identifying novel prognostic and surrogate markers of oncogenic PKCι activity that may be clinically useful in ongoing clinical trials of ATM.
tumorigenesis; gene amplification; signal trandsuction; invasion; metastasis; aurothiomalate
Atypical protein kinase Cι (PKCι) is an oncogene in non – small cell lung cancer (NSCLC). Here, we identify four functional gene targets of PKCι in lung adenocarcinoma (LAC), the most prominent form of NSCLC.
Three independent public domain gene expression data sets were interrogated to identify genes coordinately expressed with PKCι in primary LAC tumors. Results were validated by QPCR in an independent set of primary LAC tumors. RNAi-mediated knockdown of PKCι and the target genes was used to determine whether expression of the identified genes was regulated by PKCι, and whether these target genes play a role in anchorage-independent growth and invasion of LAC cells.
Meta-analysis identified seven genes whose expression correlated with PKCι in primary LAC. Subsequent QPCR analysis confirmed coordinate overexpression of four genes (COPB2, ELF3, RFC4, and PLS1) in an independent set of LAC samples. RNAi-mediated knockdown showed that PKCι regulates expression of all four genes in LAC cells, and that the four PKCι target genes play an important role in the anchorage-independent growth and invasion of LAC cells. Meta-analysis of gene expression data sets from lung squamous cell, breast, colon, prostate, and pancreas carcinomas, as well as glioblastoma, revealed that a subset of PKCι target genes, particularly COPB2 and RFC4, correlate with PKCι expression in many tumor types.
Meta-analysis of public gene expression data are useful in identifying novel gene targets of oncogenic PKCι signaling. Our data indicate that both common and cell type – specific signaling mechanisms contribute to PKCι-dependent transformation.
Protein kinase Cι (PKCι) promotes non-small cell lung cancer (NSCLC) by binding to Par6α and activating a Rac1-Pak-Mek1,2-Erk1,2 signaling cascade. The mechanism by which the PKCι-Par6α complex regulates Rac1 is unknown. Here we show that Ect2, a guanine nucleotide exchange factor (GEF) for Rho family GTPases, is coordinately amplified and overexpressed with PKCι in NSCLC tumors. RNAi-mediated knock down of Ect2 inhibits Rac1 activity and blocks transformed growth, invasion and tumorigenicity of NSCLC cells. Expression of constitutively active Rac1 (RacV12) restores transformation to Ect2-deficient cells. Interestingly, the role of Ect2 in transformation is distinct from its well-established role in cytokinesis. In NSCLC cells, Ect2 is mislocalized to the cytoplasm where it binds the PKCι-Par6α complex. RNAi-mediated knock down of either PKCι or Par6α causes Ect2 to redistribute to the nucleus, indicating that the PKCι-Par6α complex regulates the cytoplasmic localization of Ect2. Our data indicate that Ect2 and PKCι are genetically and functionally linked in NSCLC, acting to coordinately drive tumor cell proliferation and invasion through formation of an oncogenic PKCι-Par6α-Ect2 complex.
gene amplification; anchorage-independent growth; invasion; Rac1; Mek-Erk signaling; cytokinesis
We previously demonstrated that elevated expression of either protein kinase CβII (PKCβII) or PKCι/λ enhances colon carcinogenesis in mice. Here we use novel bi-transgenic mice to determine the relative importance of PKCβII and PKCι/λ in colon carcinogenesis in two complimentary models of colon cancer in vivo. Bi-transgenic mice over-expressing PKCβII and constitutively active PKCι (PKCβII/caPKCι) or kinase-deficient, dominant negative PKCι (PKCβII/kdPKCι) in the colon exhibit a similar increase in colon tumor incidence, tumor size and tumor burden in response to azoxymethane (AOM) when compared to non-transgenic littermates. However, PKCβII/kdPKCι mice develop predominantly benign colonic adenomas whereas PKCβII/caPKCι mice develop malignant carcinomas. In contrast, PKCβ deficient (PKCβ−/−) mice fail to develop tumors even in the presence of caPKCι. Our previous data indicated that PKCβII drives tumorigenesis and proliferation by activating β-catenin/Apc signaling. Consistent with this conclusion, genetic deletion of PKCβ has no effect on spontaneous tumorigenesis in APCmin/+ mice. In contrast, tissue-specific knock out of PKCλ significantly suppresses intestinal tumor formation in APCmin/+ mice. Our data demonstrate that PKCβII and PKCι/λ serve distinct, non-overlapping functions in colon carcinogenesis. PKCβII is required for AOM-induced tumorigenesis, but is dispensable for tumor formation in ApcMin/+ mice. PKCι/λ promotes tumor progression in both AOM- and APCmin/+-induced tumorigenesis. Thus PKCβII and PKCι, whose expression is elevated in both rodent and human colon tumors, collaborate to drive colon tumor formation and progression, respectively.
colon carcinogenesis; transgenic mice; β-catenin; proliferation; Adenomatous polyposis coli (Apc); intestinal tumorigenesis
The anti-rheumatoid agent aurothiomalate (ATM) is a potent inhibitor of oncogenic PKCι ATM inhibits non-small lung cancer (NSCLC) growth by binding PKCι and blocking activation of a PKCι-Par6-Rac1-Pak-Mek 1,2-Erk 1,2 signaling pathway. Here, we assessed the growth inhibitory activity of ATM in a panel of human cell lines representing major lung cancer subtypes. ATM inhibited anchorage-independent growth in all lines tested with IC50s ranging from ~300 nM – >100 µM. ATM sensitivity correlates positively with expression of PKCι and Par6, but not with the PKCι binding protein p62, or the proposed targets of ATM in rheumatoid arthritis (RA), thioredoxin reductase 1 or 2 (TrxR1 and TrxR2). PKCι expression profiling revealed that a significant subset of primary NSCLC tumors express PKCι at or above the level associated with ATM sensitivity. ATM sensitivity is not associated with general sensitivity to the cytotoxic agents cis-platin, placitaxel and gemcitabine. ATM inhibits tumorigenicity of both sensitive and insensitive lung cell tumors in vivo at plasma drug concentrations achieved in RA patients undergoing ATM therapy. ATM inhibits Mek/Erk signaling and decreases proliferative index without effecting tumor apoptosis or vascularization in vivo. We conclude that ATM exhibits potent anti-tumor activity against major lung cancer subtypes, particularly tumor cells that express high levels of the ATM target PKCι and Par6. Our results indicate that PKCι expression profiling will be useful in identifying lung cancer patients most likely to respond to ATM therapy in an ongoing clinical trial.
mechanism-based therapy; anchorage-independent growth; tumorigenicity; small cell lung cancer; non-small cell lung cancer
The Protein kinase C (PKC) family of serine/threonine kinases has been the subject of intensive study in the field of cancer since their initial discovery as major cellular receptors for the tumor promoting phorbol esters nearly thirty years ago. However, despite these efforts, the search for a direct genetic link between members of the PKC family and human cancer has yielded only circumstantial evidence that any PKC isozyme is a true cancer gene. This situation changed in the past year with the discovery that atypical protein kinase C iota (PKCι) is a bonafide human oncogene. PKCι is required for the transformed growth of human cancer cells and the PKCι gene is the target of tumor-specific gene amplification in multiple forms of human cancer. PKCι participates in multiple aspects of the transformed phenotype of human cancer cells including transformed growth, invasion and survival. Herein, we review pertinent aspects of atypical PKC structure, function and regulation that relate to the role of these enzymes in oncogenesis. We discuss the evidence that PKCι is a human oncogene, review mechanisms controlling PKCι expression in human cancers, and describe the molecular details of PKCι-mediated oncogenic signaling. We conclude with a discussion of how oncogenic PKCι signaling has been successfully targeted to identify a novel, mechanism-based therapeutic drug currently entering clinical trials for treatment of human lung cancer. Throughout, we identify key unanswered questions and exciting future avenues of investigation regarding this important oncogenic molecule.
Atypical protein kinase C; Par6; Phox/Bem1 domain; cancer signaling; cell polarity; hyperproliferation; invasion and metastasis; mechanism-based therapeutics; aurothiomalate
The integrity of the intestinal epithelium is critical for the absorption and retention of fluid and nutrients. The intestinal epithelium also provides a barrier between the intestinal bacteria and the body's immune surveillance. Therefore, intestinal epithelial barrier function is critically important, and disruption of the intestinal epithelium results in rapid repair of the damaged area.
We evaluated the requirement for protein kinase C iota (PKCι) in intestinal epithelial homeostasis and response to epithelial damage using a well-characterized mouse model of colitis. Mice were analyzed for the clinical, histological and cellular effects of dextran sodium sulfate (DSS) treatment.
Knock out of the mouse PKCι gene (Prkci) in the intestinal epithelium (Prkci KO mice) had no effect on normal colonic homeostasis, however, Prkci KO mice were significantly more sensitive to DSS-induced colitis and death. After withdrawal of DSS, Prkci KO mice exhibited a continued increase in apoptosis, inflammation and damage to the intestinal microvasculature, and a progressive loss of trefoil factor 3 (TFF3) expression, a regulatory peptide important for intestinal wound healing. Knockdown of PKCι expression in HT-29 cells reduced wound healing and TFF3 expression, while addition of exogenous TFF3 restored wound healing in PKCι-depleted cells.
Expression of PKCι in the intestinal epithelium protects against DSS-induced colitis. Our data suggest that PKCι reduces DSS-induced damage by promoting intestinal epithelial wound healing through the control of TFF3 expression.
protein kinase C iota; colitis; wound healing; trefoil factor 3; apoptosis; dextran sodium sulfate; permeability
Protein kinase C ι (PKCι) has been implicated in Ras signaling, however, a role for PKCι in oncogenic Ras-mediated transformation has not been established. Here, we show that PKCι is a critical downstream effector of oncogenic Ras in the colonic epithelium. Transgenic mice expressing constitutively active PKCι in the colon are highly susceptible to carcinogen-induced colon carcinogenesis, whereas mice expressing kinase-deficient PKCι (kdPKCι) are resistant to both carcinogen- and oncogenic Ras-mediated carcinogenesis. Expression of kdPKCι in Ras-transformed rat intestinal epithelial cells blocks oncogenic Ras-mediated activation of Rac1, cellular invasion, and anchorage-independent growth. Constitutively active Rac1 (RacV12) restores invasiveness and anchorage-independent growth in Ras-transformed rat intestinal epithelial cells expressing kdPKCι. Our data demonstrate that PKCι is required for oncogenic Ras- and carcinogen-mediated colon carcinogenesis in vivo and define a procarcinogenic signaling axis consisting of Ras, PKCι, and Rac1.
Rac1; transgenic mice; rat intestinal epithelial cells; cell invasion; soft agar growth
Protein kinase C βII (PKC βII) has been implicated in proliferation of the intestinal epithelium. To investigate PKC βII function in vivo, we generated transgenic mice that overexpress PKC βII in the intestinal epithelium. Transgenic PKC βII mice exhibit hyperproliferation of the colonic epithelium and an increased susceptibility to azoxymethane-induced aberrant crypt foci, preneoplastic lesions in the colon. Furthermore, transgenic PKC βII mice exhibit elevated colonic β-catenin levels and decreased glycogen synthase kinase 3β activity, indicating that PKC βII stimulates the Wnt/adenomatous polyposis coli (APC)/β-catenin proliferative signaling pathway in vivo. These data demonstrate a direct role for PKC βII in colonic epithelial cell proliferation and colon carcinogenesis, possibly through activation of the APC/β-catenin signaling pathway.
protein kinase C; colon carcinogenesis; signal transduction; proliferation; transgenic mice
Pancreatic acinar-to-ductal metaplasia (ADM) is associated with an increased risk of pancreatic cancer and is considered a precursor of pancreatic ductal adenocarcinoma. Transgenic expression of transforming growth factor alpha (TGF-α) or K-rasG12D in mouse pancreatic epithelium induces ADM in vivo. Protein kinase C iota (PKCι) is highly expressed in human pancreatic cancer and is required for the transformed growth and tumorigenesis of pancreatic cancer cells. In this study, PKCι expression was assessed in a mouse model of K-rasG12D-induced pancreatic ADM and pancreatic cancer. The ability of K-rasG12D to induce pancreatic ADM in explant culture, and the requirement for PKCι, was investigated. PKCι is elevated in human and mouse pancreatic ADM and intraepithelial neoplastic lesions in vivo. We demonstrate that K-rasG12D is sufficient to induce pancreatic ADM in explant culture, exhibiting many of the same morphologic and biochemical alterations observed in TGF-α-induced ADM, including a dependence on Notch activation. PKCι is highly expressed in both TGF-α- and K-rasG12D-induced pancreatic ADM and inhibition of PKCι significantly reduces TGF-α- and K-rasG12D-mediated ADM. Inhibition of PKCι suppresses K-rasG12D–induced MMP-7 expression and Notch activation, and exogenous MMP-7 restores K-rasG12D–mediated ADM in PKCι-depleted cells, implicating a K-rasG12D-PKCι-MMP-7 signaling axis that likely induces ADM through Notch activation. Our results indicate that PKCι is an early marker of pancreatic neoplasia and suggest that PKCι is a potential downstream target of K-rasG12D in pancreatic ductal metaplasia in vivo.
Pancreatic cancer is the fourth leading cause of cancer deaths in the United States with an overall 5-year survival rate of <5%. Pancreatic ductal adenocarcinoma (PDAC), the most common form of pancreatic cancer, is highly resistant to conventional chemotherapies underscoring the critical need for new molecular targets for pancreatic cancer chemotherapy. The KRAS proto-oncogene is mutated in >90% of PDAC. Protein kinase C iota (PKCι) is required for oncogenic Ras-mediated transformed growth in lung cancer and intestinal epithelial cells. However, little is known about the role of PKCι in pancreatic cancer. In this study, we evaluated the expression of PKCι in human pancreatic cancer and the requirement for PKCι for the transformed growth and tumorigenicity of PDAC cells. We find that PKCι is significantly over-expressed in human pancreatic cancer and high PKCι expression correlates with poor patient survival. Inhibition of PKCι expression blocks PDAC cell transformed growth in vitro and tumorigenicity in vivo. Inhibition of PKCι expression in pancreatic tumors also significantly reduces tumor angiogenesis and metastasis. Analysis of downstream PKCι effectors implicates the Rac1-MEK/ERK1/2 signaling axis in PKCι-mediated transformed growth and cellular invasion. Taken together, our data demonstrate a required role for PKCι in the transformed growth of pancreatic cancer cells and reveal a novel role for PKCι in pancreatic cancer cell metastasis and angiogenesis in vivo. Our results strongly indicate that PKCι will be an effective target for pancreatic cancer therapy.
pancreatic cancer; transformed growth; invasion; metastasis; protein kinase C iota; Rac1; ERK1/2; VEGF
Protein kinase Cι (PKCι) is an oncogene required for maintenance of the transformed phenotype of non-small cell lung cancer (NSCLC) cells. However, the role of PKCι in lung tumor development has not been investigated. To address this question, we established a mouse model in which oncogenic KrasG12D is activated by Cre-mediated recombination in the lung with or without simultaneous genetic loss of the mouse PKCι gene, Prkci. Genetic loss of Prkci dramatically inhibits Kras-initiated hyperplasia and subsequent lung tumor formation in vivo. This effect correlates with a defect in the ability of Prkci-deficient bronchioalveolar stem cells (BASCs) to undergo Kras-mediated expansion and morphological transformation in vitro and in vivo. Furthermore, the small molecule PKCι inhibitor aurothiomalate inhibits Kras-mediated BASC expansion and lung tumor growth in vivo. Thus, Prkci is required for oncogene-induced expansion and transformation of tumor-initiating lung stem cells. Furthermore, aurothiomalate is an effective anti-tumor agent that targets the tumor-initiating stem cell niche in vivo. These data have important implications for PKCι as a therapeutic target and for the clinical use of aurothiomalate for lung cancer treatment.
lung tumor initiation; aurothiomalate; lung cancer stem cells; Kras transformation; transgenic mice
Ras and Rho small GTPases possessing a C-terminal polybasic region (PBR) are vital signaling proteins whose misregulation can lead to cancer. Signaling by these proteins depends on their ability to bind guanine nucleotides and their prenylation with a geranylgeranyl or farnesyl isoprenoid moiety and subsequent trafficking to cellular membranes. There is little previous evidence that cellular signals can restrain nonprenylated GTPases from entering the prenylation pathway, leading to the general belief that PBR-possessing GTPases are prenylated as soon as they are synthesized. Here, we present evidence that challenges this belief. We demonstrate that insertion of the dominant negative mutation to inhibit GDP/GTP exchange diminishes prenylation of Rap1A and RhoA, enhances prenylation of Rac1, and does not detectably alter prenylation of K-Ras. Our results indicate that the entrance and passage of these small GTPases through the prenylation pathway is regulated by two splice variants of SmgGDS, a protein that has been reported to promote GDP/GTP exchange by PBR-possessing GTPases and to be up-regulated in several forms of cancer. We show that the previously characterized 558-residue SmgGDS splice variant (SmgGDS-558) selectively associates with prenylated small GTPases and facilitates trafficking of Rap1A to the plasma membrane, whereas the less well characterized 607-residue SmgGDS splice variant (SmgGDS-607) associates with nonprenylated GTPases and regulates the entry of Rap1A, RhoA, and Rac1 into the prenylation pathway. These results indicate that guanine nucleotide exchange and interactions with SmgGDS splice variants can regulate the entrance and passage of PBR-possessing small GTPases through the prenylation pathway.
Low Molecular Weight G Proteins; Protein Isoprenylation; Ras; Rho; Trafficking; Rac; Rap; Rap1GDS1; SmgGDS; Polybasic
Rho family GTPases; Rac1; RhoA; Cdc42; Ect2; guanine nucleotide exchange factor (GEF); protein kinase C (PKC); Par6; Mek/Erk; cellular transformation; cellular invasion; lung cancer; oncogene; gene amplification; anchorage-independent growth; tumorigenesis
Colon cancer develops over a period of 10 to 15 years, providing a window of opportunity for chemoprevention and early intervention. However, few molecular targets for effective colon cancer chemoprevention have been characterized and validated. Protein kinase CβII (PKCβII) plays a requisite role in the initiation of colon carcinogenesis in a preclinical mouse model by promoting proliferation and increased β-catenin accumulation. In this study, we test the hypothesis that PKCβII is an effective target for colon cancer chemoprevention using enzastaurin (LY317615), a PKCβ-selective inhibitor, in a mouse model of colon carcinogenesis. We find that enzastaurin potently reduces azoxymethane-induced colon tumor initiation and progression by inhibiting PKCβII-mediated tumor cell proliferation and β-catenin accumulation. Biochemically, enzastaurin reduces expression of the PKCβII- and β-catenin/T-cell factor–regulated genes PKCβII, cyclooxygenase II, and vascular endothelial growth factor, three genes implicated in colon carcinogenesis. Our results show that enzastaurin is an effective chemopreventive agent in a mouse model of sporadic colon cancer that significantly reduces both tumor initiation and progression by inhibiting expression of proproliferative genes. Thus, PKCβII is an important target for colon cancer chemoprevention and the PKCβ-selective inhibitor enzastaurin may represent an effective chemopreventive agent in patients at high risk for colon cancer.
Rit is a novel member of the Ras superfamily of small GTP-binding proteins that regulates signaling pathways controlling cellular fate determination. Constitutively activated mutants of Rit induce terminal differentiation of pheochromocytoma (PC6) cells resulting in a sympathetic neuron-like phenotype characterized by the development of highly-branched neurites. Rit signaling has been found to activate several downstream pathways including MEK/ERK, p38 MAPK, Ral-specific guanine nucleotide exchange factors (GEFs), and Rit associates with the Par6 cell polarity machinery. In this study, a series of Rit effector loop mutants was generated to test the importance of these cellular targets to Rit-mediated neuronal differentiation. We find that Rit-mediated neuritogenesis is dependent upon MEK/ERK MAP kinase signaling but independent of RalGEF activation. In addition, in vivo binding studies identified a novel mechanism of Par6 interaction, suggesting that the cell polarity machinery may serve to spatially restrict Rit signaling.
Neuronal differentiation; PC12 cell; Rit; GTPase; Ras; ERK MAP kinase; Par6
Încreasing evidence demonstrates that protein kinase C βII (PKCβII) promotes colon carcinogenesis. We previously reported that colonic PKCβII is induced during colon carcinogenesis in rodents and humans, and that elevated expression of PKCβII in the colon of transgenic mice enhances colon carcinogenesis. Here, we demonstrate that PKCβII represses transforming growth factor β receptor type II (TGFβRII) expression and reduces sensitivity to TGF-β–mediated growth inhibition in intestinal epithelial cells. Transgenic PKCβII mice exhibit hyperproliferation, enhanced colon carcinogenesis, and marked repression of TGFβRII expression. Chemopreventive dietary ω-3 fatty acids inhibit colonic PKCβII activity in vivo and block PKCβII-mediated hyperproliferation, enhanced carcinogenesis, and repression of TGFβRII expression in the colonic epithelium of transgenic PKCβII mice. These data indicate that dietary ω-3 fatty acids prevent colon cancer, at least in part, through inhibition of colonic PKCβII signaling and restoration of TGF-β responsiveness.
protein kinase C; colon carcinogenesis; ω-3 fatty acids; transforming growth factor β; hyperproliferation
Pancreatic cancer is a very aggressive disease with few therapeutic options. In this study, we investigate the role of protein kinase C zeta (PKCζ) in pancreatic cancer cells. PKCζ has been shown to act as either a tumor suppressor or tumor promoter depending upon the cellular context. We find that PKCζ expression is either maintained or elevated in primary human pancreatic tumors, but is never lost, consistent with PKCζ playing a promotive role in the pancreatic cancer phenotype. Genetic inhibition of PKCζ reduced adherent growth, cell survival and anchorage-independent growth of human pancreatic cancer cells in vitro. Furthermore, PKCζ inhibition reduced orthotopic tumor size in vivo by inhibiting tumor cell proliferation and increasing tumor necrosis. In addition, PKCζ inhibition reduced tumor metastases in vivo, and caused a corresponding reduction in pancreatic cancer cell invasion in vitro. Signal transducer and activator of transcription 3 (STAT3) is often constitutively active in pancreatic cancer, and plays an important role in pancreatic cancer cell survival and metastasis. Interestingly, inhibition of PKCζ significantly reduced constitutive STAT3 activation in pancreatic cancer cells in vitro and in vivo. Pharmacologic inhibition of STAT3 mimicked the phenotype of PKCζ inhibition, and expression of a constitutively active STAT3 construct rescued the transformed phenotype in PKCζ-deficient cells. We conclude that PKCζ is required for pancreatic cancer cell transformed growth and invasion in vitro and tumorigenesis in vivo, and that STAT3 is an important downstream mediator of the pro-carcinogenic effects of PKCζ in pancreatic cancer cells.
Angiogenesis, the recruitment and re-configuration of pre-existing vasculature, is essential for tumor growth and metastasis. Increased tumor vascularization often correlates with poor patient outcomes in a broad spectrum of carcinomas. We identified four jointed box 1 (FJX1) as a candidate regulator of tumor angiogenesis in colorectal cancer. FJX1 mRNA and protein are upregulated in human colorectal tumor epithelium as compared with normal epithelium and colorectal adenomas, and high expression of FJX1 is associated with poor patient prognosis. FJX1 mRNA expression in colorectal cancer tissues is significantly correlated with changes in known angiogenesis genes. Augmented expression of FJX1 in colon cancer cells promotes growth of xenografts in athymic mice and is associated with increased tumor cell proliferation and vascularization. Furthermore, FJX1 null mice develop significantly fewer colonic polyps than wild-type littermates after combined dextran sodium sulfate (DSS) and azoxymethane (AOM) treatment. In vitro, conditioned media from FJX1 expressing cells promoted endothelial cell capillary tube formation in a HIF1-α dependent manner. Taken together our results support the conclusion that FJX1 is a novel regulator of tumor progression, due in part, to its effect on tumor vascularization.
The extracellular matrix of epithelial tumors undergoes structural remodeling during periods of uncontrolled growth, creating regional heterogeneity and torsional stress. How matrix integrity is maintained in the face of dynamic biophysical forces is largely undefined. Here we investigated the role of fibulin-2, a matrix glycoprotein that functions biomechanically as an inter-molecular clasp and thereby facilitates supra-molecular assembly. Fibulin-2 was abundant in the extracellular matrix of human lung adenocarcinomas and was highly expressed in tumor cell lines derived from mice that develop metastatic lung adenocarcinoma from co-expression of mutant K-ras and p53. Loss-of-function experiments in tumor cells revealed that fibulin-2 was required for tumor cells to grow and metastasize in syngeneic mice, a surprising finding given that other intra-tumoral cell types are known to secrete fibulin-2. However, tumor cells grew and metastasized equally well in Fbln2-null and -wild-type littermates, implying that malignant progression was dependent specifically upon tumor cell-derived fibulin-2, which could not be offset by other cellular sources of fibulin-2. Fibulin-2 deficiency impaired the ability of tumor cells to migrate and invade in Boyden chambers, to create a stiff extracellular matrix in mice, to cross-link secreted collagen, and to adhere to collagen. We conclude that fibulin-2 is a driver of malignant progression in lung adenocarcinoma and plays an unexpected role in collagen cross-linking and tumor cell adherence to collagen.
The role of peroxisome proliferator – activated receptor- δ (PPAR δ) gene in colon carcinogenesis remains highly controversial. Here, we established nude mice xenograft model using a human colon cancer cell line KM12C either with PPAR δ silenced or normal. The xenografts in PPAR δ-silenced group grew significantly larger and heavier with less differentiation, promoted cell proliferation, increased expression of vascular endothelial growth factor (VEGF) and similar apoptosis index compared with those of PPAR δ-normal group. After treated with the specific VEGF inhibitor bevacizumab, the capacities of growth and proliferation of xenografts were decreased in both groups while still significantly higher in PPAR δ-silenced group than in PPAR δ-normal group. Administration of PPAR δ agonist significantly decreased VEGF expression in PPAR δ-normal KM12C cells but not in PPAR δ-silenced cells. These findings demonstrate that, knockdown of PPAR δ promotes the growth of colon cancer by inducing less differentiation, accelerating the proliferation and VEGF expression of tumor cells in vivo, and reduces tumor sensitivity to bevacizumab. This study indicates that PPAR δ attenuates colon carcinogenesis.