We report that two oncogenes co-amplified on chromosome 3q26, PRKCI and SOX2, cooperate to drive a stem-like phenotype in lung squamous cell carcinoma (LSCC). PKCι phosphorylates SOX2, a master transcriptional regulator of stemness, and recruits it to the promoter of Hedgehog Acyl Transferase (HHAT), which catalyzes the rate-limiting step in Hh ligand production. PKCι-mediated SOX2 phosphorylation is required for HHAT promoter occupancy, HHAT expression, and maintenance of a stem-like phenotype. Primary LSCC tumors coordinately overexpress PKCι, SOX2, and HHAT, and require PKCι-SOX2-HHAT signaling to maintain a stem-like phenotype. Thus, PKCι and SOX2 are genetically, biochemically and functionally linked in LSCC, and together they drive tumorigenesis by establishing a cell autonomous Hh signaling axis.
Protein Kinase Cι; SOX2; 3q26 amplification; Hedgehog signaling; proliferation; transformed growth; LSCC tumor initiation; clonal expansion
Protein kinase Cι (PKCι) is an oncogene in lung and ovarian cancers. PKCι is an attractive therapeutic target for treatment of lung cancer, particularly those whose tumors express elevated PKCι. However, it is unknown whether PKCι is a viable therapeutic target in the ovary, and virtually nothing is known about the mechanism by which PKCι drives ovarian tumorigenesis. Here, we demonstrate that PKCι maintains a tumor-initiating cell (TIC) phenotype that drives ovarian tumorigenesis. A highly tumorigenic population of cells from human ovarian cancer cell lines exhibit properties of cancer stem-like TICs including self-renewal, clonal expansion, expression of stem-related genes, enhanced transformed growth in vitro, and aggressive tumor-initiating activity in vivo. Genetic disruption of PKCι inhibits the proliferation, clonal expansion, anchorage-independent growth and enhanced tumorigenic properties of ovarian TICs. Biochemical analysis demonstrates that PKCι acts through its oncogenic partner Ect2 to activate a Mek-Erk signaling axis that drives the ovarian TIC phenotype. Genomic analysis reveals that PRKCI and ECT2 are coordinately amplified and overexpressed in the majority of primary ovarian serous tumors, and these tumors exhibit evidence of an active PKCι-Ect2 signaling axis in vivo. Finally, we demonstrate that auranofin is a potent and selective inhibitor of oncogenic PKCι signaling that inhibits the tumorigenic properties of ovarian TIC cells in vitro and in vivo. Our data demonstrate that PKCι is required for a TIC phenotype in ovarian cancer, and that auranofin is an attractive therapeutic strategy to target deadly ovarian TICs in ovarian cancer patients.
Protein kinase C iota (PKCι) is overexpressed in non-small cell lung (NSCLC), ovarian and pancreatic cancers where it plays a critical role in oncogenesis. The gold compound aurothiomalate (ATM) has been shown to inhibit PKCι signaling and exhibits potent anti-tumor activity in preclinical models. We sought to determine the maximum tolerated dose (MTD) of ATM.
We conducted a phase I dose escalation trial of ATM in patients with NSCLC, ovarian or pancreatic cancer. Patients received ATM IM weekly for three cycles (cycle duration 4 weeks) at 25 mg, 50 mg or 75 mg in a 3+3 design. The dose was not escalated for individual patients. Blood samples were analyzed for elemental gold levels. Patients were evaluated every four weeks for toxicity and every eight weeks for response.
Fifteen patients were enrolled in this study. Six patients were treated at 25 mg, 7 patients at 50 mg, and 2 at 75 mg. There was 1 dose limiting toxicity at 25 mg (hypokalemia), one at 50 mg (urinary tract infection), and none at 75 mg. There were 3 grade 3 hematologic toxicities. The recommended MTD of ATM is 50 mg. Patients received treatment for a median of 2 cycles (range 1-3). There appeared to be a dose-related accumulation of steady-state plasma concentrations of gold consistent with linear pharmacokinetics.
In summary, this phase I study was successful in identifying ATM 50 mg IM weekly as the MTD. Future clinical investigations targeting PKCι are currently in progress.
protein kinase C iota; aurothiomalate; non-small cell lung cancer; ovarian cancer; pancreatic cancer
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
Human colorectal cancer (CRC) cells are resistant to the anti-proliferative effect of transforming growth factor-β (TGF-β), suggesting that disruption of TGF-β signaling plays an important role in colorectal carcinogenesis. Ecotropic virus integration site-1 (Evi-1) oncoprotein represses TGF-β signaling by interacting with Smads, but its role in CRC has not been established. The purpose of this study is to determine whether Evi-1 plays role(s) in CRCs and to characterize Evi-1 transcript(s) in CRCs. Evi-1 was overexpressed in 53% of human CRC samples, 100% of colon adenoma samples, and 100% of human colon cancer cell lines tested. Using 5′ RACE, we cloned a novel Evi-1 transcript (Evi-1e) from a human CRC tissue and found that this novel transcript was expressed at a higher level in CRC tissues than in normal tissues and was the major Evi-1 transcript in CRCs. Transient Evi-1 transfection inhibited TGF-β-induced transcriptional activity and reversed the growth inhibitory effect of TGF-β in MC-26 mouse colon cancer cells. In conclusion, we have identified overexpression of Evi-1 oncoprotein as a novel mechanism by which a subset of human CRCs may escape TGF-β regulation. We have also identified a novel Evi-1 transcript, Evi-1e, as the major Evi-1 transcript expressed in human CRCs.
colorectal cancer; ecotropic virus integration site-1; transforming growth factor-β; Smad proteins; rapid amplification of cDNA ends; growth inhibition
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.
Oncogenic K-ras mutations are frequently observed in colon cancers and contribute to transformed growth. Oncogenic K-ras is detected in aberrant crypt foci (ACF), precancerous colonic lesions, demonstrating that acquisition of a K-ras mutation is an early event in colon carcinogenesis. Here, we investigate the role of oncogenic K-ras in neoplastic initiation and progression. Transgenic mice in which an oncogenic K-rasG12D allele is activated in the colonic epithelium by sporadic recombination (K-rasLA2 mice) develop spontaneous ACF that are morphologically indistinguishable from those induced by the colon carcinogen azoxymethane (AOM). Similar neoplastic changes involving the entire colon are induced in transgenic mice constitutively expressing K-rasG12D throughout the colon (LSL-K-rasG12D/Villin-Cre mice). However, the biochemistry and fate of K-ras-induced lesions differ depending upon their location within the colon in these mice. In the proximal colon, K-rasG12D induces increased expression of procarcinogenic protein kinase CβII (PKCβII), activation of the MEK/ERK signaling axis and increased epithelial cell proliferation. In contrast, in the distal colon, K-rasG12D inhibits expression of procarcinogenic PKCβII and induces apoptosis. Treatment of K-rasLA2 mice with AOM leads to neoplastic progression of small ACF to large, dysplastic microadenomas in the proximal, but not the distal colon. Thus, oncogenic K-ras functions differently in the proximal and distal colon of mice, inducing ACF capable of neoplastic progression in the proximal colon, and ACF with little or no potential for progression in the distal colon. Our data indicate that acquisition of a K-ras mutation is an initiating neoplastic event in proximal colon cancer development in mice.
K-ras; colon; carcinogenesis; dysplasia; PKCβII; ACF; progression
We evaluated RS5444, a thiazolidinedione high affinity PPARγ agonist, for the ability to inhibit colon carcinogenesis in azoxymethane (AOM)-treated mice. In our initial experiment, mice were treated with RS5444 during AOM treatment, and the drug was withdrawn 12 weeks after the last injection of AOM. RS5444 significantly inhibited aberrant crypt focus formation under these circumstances. Furthermore, exposure to RS5444 during the course of AOM treatment effectively blocked colon tumor formation after withdrawal of the agonist. PPARγ expression and nuclear localization were reduced in adenomas. RS5444 did not inhibit DNA synthesis in tumor cells, suggesting that PPARγ activity was impaired in adenomas. To test this hypothesis, pre-existing adenomas were treated with RS5444 for 16 weeks. We observed a slight, albeit not statistically significant, reduction in tumor incidence in RS5444-treated mice. However, histological examination revealed that tumors from RS5444-treated mice were of significantly lower grade, as evaluated by the extent of dysplasia. Furthermore, carcinoma in situ was observed in about one-third of control tumors, but was never observed in RS5444-treated tumors. We conclude that RS5444 inhibits both initiation and progression of colon tumors in the AOM model of sporadic colon carcinogenesis.
PPARγ; colon carcinogenesis; colon cancer chemoprevention; azoxymethane-induced colon tumors; nuclear receptors
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
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.
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
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.
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
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
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
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
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
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
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
Matrix metalloproteinases (Mmps) stimulate tumor invasion and metastasis by degrading the extracellular matrix. Here we reveal an unexpected role for Mmp10 (stromelysin 2) in the maintenance and tumorigenicity of mouse lung cancer stem-like cells (CSC). Mmp10 is highly expressed in oncosphere cultures enriched in CSCs and RNAi-mediated knockdown of Mmp10 leads to a loss of stem cell marker gene expression and inhibition of oncosphere growth, clonal expansion, and transformed growth in vitro. Interestingly, clonal expansion of Mmp10 deficient oncospheres can be restored by addition of exogenous Mmp10 protein to the culture medium, demonstrating a direct role for Mmp10 in the proliferation of these cells. Oncospheres exhibit enhanced tumor-initiating and metastatic activity when injected orthotopically into syngeneic mice, whereas Mmp10-deficient cultures show a severe defect in tumor initiation. Conversely, oncospheres implanted into syngeneic non-transgenic or Mmp10−/− mice show no significant difference in tumor initiation, growth or metastasis, demonstrating the importance of Mmp10 produced by cancer cells rather than the tumor microenvironment in lung tumor initiation and maintenance. Analysis of gene expression data from human cancers reveals a strong positive correlation between tumor Mmp10 expression and metastatic behavior in many human tumor types. Thus, Mmp10 is required for maintenance of a highly tumorigenic, cancer-initiating, metastatic stem-like cell population in lung cancer. Our data demonstrate for the first time that Mmp10 is a critical lung cancer stem cell gene and novel therapeutic target for lung cancer stem cells.
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.
KRAS mutations are highly prevalent in non-small cell lung cancer (NSCLC), and tumors harboring these mutations tend to be aggressive and resistant to chemotherapy. We used next-generation sequencing technology to identify pathways that are specifically altered in lung tumors harboring a KRAS mutation. Paired-end RNA-sequencing of 15 primary lung adenocarcinoma tumors (8 harboring mutant KRAS and 7 with wild-type KRAS) were performed. Sequences were mapped to the human genome, and genomic features, including differentially expressed genes, alternate splicing isoforms and single nucleotide variants, were determined for tumors with and without KRAS mutation using a variety of computational methods. Network analysis was carried out on genes showing differential expression (374 genes), alternate splicing (259 genes), and SNV-related changes (65 genes) in NSCLC tumors harboring a KRAS mutation. Genes exhibiting two or more connections from the lung adenocarcinoma network were used to carry out integrated pathway analysis. The most significant signaling pathways identified through this analysis were the NFκB, ERK1/2, and AKT pathways. A 27 gene mutant KRAS-specific sub network was extracted based on gene–gene connections from the integrated network, and interrogated for druggable targets. Our results confirm previous evidence that mutant KRAS tumors exhibit activated NFκB, ERK1/2, and AKT pathways and may be preferentially sensitive to target therapeutics toward these pathways. In addition, our analysis indicates novel, previously unappreciated links between mutant KRAS and the TNFR and PPARγ signaling pathways, suggesting that targeted PPARγ antagonists and TNFR inhibitors may be useful therapeutic strategies for treatment of mutant KRAS lung tumors. Our study is the first to integrate genomic features from RNA-Seq data from NSCLC and to define a first draft genomic landscape model that is unique to tumors with oncogenic KRAS mutations.
transcriptome sequencing; RNA-Seq; KRAS mutation; NSCLC; bioinformatics; network analysis; data integration and computational methods
Matrix metalloproteinase 10 (MMP-10; stromelysin 2) is a member of a large family of structurally related matrix metalloproteinases, many of which have been implicated in tumor progression, invasion and metastasis. We recently identified Mmp10 as a gene that is highly induced in tumor-initiating lung bronchioalveolar stem cells (BASCs) upon activation of oncogenic Kras in a mouse model of lung adenocarcinoma. However, the potential role of Mmp10 in lung tumorigenesis has not been addressed. Here, we demonstrate that Mmp10 is overexpressed in lung tumors induced by either the smoke carcinogen urethane or oncogenic Kras. In addition, we report a significant reduction in lung tumor number and size after urethane exposure or genetic activation of oncogenic Kras in Mmp10 null (Mmp10−/−) mice. This inhibitory effect is reflected in a defect in the ability of Mmp10-deficient BASCs to expand and undergo transformation in response to urethane or oncogenic Kras in vivo and in vitro, demonstrating a role for Mmp10 in the tumor-initiating activity of Kras-transformed lung stem cells. To determine the potential relevance of MMP10 in human cancer we analyzed Mmp10 expression in publicly-available gene expression profiles of human cancers. Our analysis reveals that MMP10 is highly overexpressed in human lung tumors. Gene set enhancement analysis (GSEA) demonstrates that elevated MMP10 expression correlates with both cancer stem cell and tumor metastasis genomic signatures in human lung cancer. Finally, Mmp10 is elevated in many human tumor types suggesting a widespread role for Mmp10 in human malignancy. We conclude that Mmp10 plays an important role in lung tumor initiation via maintenance of a highly tumorigenic, cancer-initiating, stem-like cell population, and that Mmp10 expression is associated with stem-like, highly metastatic genotypes in human lung cancers. These results indicate that Mmp10 may represent a novel therapeutic approach to target lung cancer stem cells.