Protein kinase C (PKC) isozymes have long been implicated in carcinogenesis. However, little is known about the functional significance of these enzymes in human cancer. We recently showed that the atypical PKC (aPKC) isozyme PKCiota is overexpressed in human non-small cell lung cancer (NSCLC) cells and that PKCiota plays a critical role in the transformed growth of the human lung adenocarcinoma A549 cell line in vitro and tumorigenicity in vivo. Here we provide compelling evidence that PKCiota is an oncogene in NSCLC based on the following criteria: (a) aPKCiota is overexpressed in the vast majority of primary NSCLC tumors; (b) tumor PKCiota expression levels predict poor survival in patients with NSCLC; (c) the PKCiota gene is frequently amplified in established NSCLC cell lines and primary NSCLC tumors; (d) gene amplification drives PKCiota expression in NSCLC cell lines and primary NSCLC tumors; and (e) disruption of PKCiota signaling with a dominant negative PKCiota allele blocks the transformed growth of human NSCLC cells harboring PKCiota gene amplification. Taken together, our data provide conclusive evidence that PKCiota is required for the transformed growth of NSCLC cells and that the PKCiota gene is a target for tumor-specific genetic alteration by amplification. Interestingly, PKCiota expression predicts poor survival in NSCLC patients independent of tumor stage. Therefore, PKCiota expression profiling may be useful in identifying early-stage NSCLC patients at elevated risk of relapse. Our functional data indicate that PKCiota is an attractive target for development of novel, mechanism-based therapeutics to treat NSCLC.

Atypical protein kinase C (aPKC) isozymes function in epithelial cell polarity, proliferation, and survival and have been implicated in cellular transformation. However, the role of these enzymes in human cancer is largely unexplored. Here, we report that aPKC is highly expressed in human non-small cell lung cancer cell lines, whereas the closely related aPKC isozyme PKC is undetectable in these cells. Disruption of PKC signaling reveals that PKC is dispensable for adherent growth of non-small cell lung cancer cells but is required for transformed growth in soft agar in vitro and for tumorigenicity in vivo. Molecular dissection of signaling downstream of PKC demonstrates that Rac1 is a critical molecular target for PKC-dependent transformation, whereas PKC is not necessary for NFB activation in vitro or in vivo. Expression of the PB1 domain of PKC (PKC-(1-113)) blocks PKC-dependent Rac1 activity and inhibits cellular transformation indicating a role for this domain in the transforming activity of PKC. Taken together, our data demonstrate that PKC is a critical lung cancer gene that activates a Rac13 Pak3 Mek1,23 Erk1,2 signaling pathway required for transformed growth. Our data indicate that PKC may be an attractive molecular target for mechanism-based therapies for treatment of lung cancer.

We recently showed that atypical protein kinase CI (PKCI) is required for transformed growth of human non-small-cell lung cancer (NSCLC) cells by activating Rac1. Genetic disruption of PKCI signaling blocks Rac1 activity and transformed growth, indicating that PKCI is a viable target for development of novel therapeutics for NSCLC. Here, we designed and implemented a novel fluorescence resonance energy transfer-based assay to identify inhibitors of oncogenic PKCI signaling. This assay was used to identify compounds that disrupt the interaction between PKCI and its downstream effector Par6, which links PKCI to Rac1. We identified aurothioglucose (ATG), a gold compound used clinically to treat rheumatoid arthritis, and the related compound, aurothiomalate (ATM), as potent inhibitors of PKCI-Par6 interactions in vitro (IC 50 f1 Mmol/L). ATG blocks PKCIdependent signaling to Rac1 and inhibits transformed growth of NSCLC cells. ATG-mediated inhibition of transformation is relieved by expression of constitutively active Rac1, consistent with a mechanism at the level of the interaction between PKCI and Par6. ATG inhibits A549 cell tumor growth in nude mice, showing efficacy against NSCLC in a relevant preclinical model. Our data show the utility of targeting protein-protein interactions involving PKCI for antitumor drug development and provide proof of concept that chemical disruption of PKCI signaling can be an effective treatment for NSCLC. ATG and ATM will be useful reagents for studying PKCI function in transformation and represent promising new agents for the clinical treatment of NSCLC. (Cancer Res 2006; 66(3): 1767-74)

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.

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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.

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.

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