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.

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.

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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)

SUMMARY The guanine nucleotide exchange factor (GEF) Epithelial Cell Transforming sequence 2 (Ect2) has been implicated in cancer. However, it is not clear how Ect2 causes transformation, and whether Ect2 is necessary for tumorigenesis in vivo. Here, we demonstrate that nuclear Ect2 GEF activity is required for Kras-Trp53 lung tumorigenesis in vivo, and that Ect2-mediated transformation requires Ect2-dependent ribosomal DNA (rDNA) transcription. Ect2 activates rRNA synthesis by binding the nucleolar transcription factor Upstream Binding Factor 1 (UBF1) on rDNA promoters, and recruiting Rac1 and its downstream effector nucleophosmin (NPM) to rDNA. Protein kinase Cι (PKCι)-mediated Ect2 phosphorylation stimulates Ect2-dependent rDNA transcription. Thus, Ect2 regulates rRNA synthesis through a PKCι-Ect2-Rac1-NPM signaling axis that is required for lung tumorigenesis.

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We recently identified the gold compound aurothiomalate (ATM) as a potent inhibitor of the Phox and Bem1p (PB1)-PB1 domain interaction between protein kinase C (PKC) and the adaptor molecule Par6. ATM also blocks oncogenic PKC signaling and the transformed growth of human lung cancer cells. Here we demonstrate that ATM is a highly selective inhibitor of PB1-PB1 domain interactions between PKC and the two adaptors Par6 and p62. ATM has no appreciable inhibitory effect on other PB1-PB1 domain interactions, including p62-p62, p62-NBR1, and MEKK3-MEK5 interactions. ATM can form thio-gold adducts with cysteine residues on target proteins. Interestingly, PKC (and PKC) contains a unique cysteine residue, Cys-69, within its PB1 domain that is not present in other PB1 domain containing proteins. Cys-69 resides within the OPR, PC, and AID motif of PKC at the binding interface between PKC and Par6 where it interacts with Arg-28 on Par6. Molecular modeling predicts formation of a cysteinyl-aurothiomalate adduct at Cys-69 that protrudes into the binding cleft normally occupied by Par6, providing a plausible structural explanation for ATM inhibition. Mutation of Cys-69 of PKC to isoleucine or valine, residues frequently found at this position in other PB1 domains, has little or no effect on the affinity of PKC for Par6 but confers resistance to ATM-mediated inhibition of Par6 binding. Expression of the PKC C69I mutant in human non-small cell lung cancer cells confers resistance to the inhibitory effects of ATM on transformed growth. We conclude that ATM inhibits cellular transformation by selectively targeting Cys-69 within the PB1 domain of PKC.