Fields, Alan P.

Rac1b mediates the matrix metalloproteinase–induced epithelial-mesenchymal transition in lung adenocarcinoma and is a potential therapeutic target in early-stage lung cancer.

Abstract Background: Alveolar rhabdomyosarcoma (ARMS) is an aggressive pediatric cancer showing skeletal muscle differentiation. New therapeutic targets are required to improve the dismal cure rates for patients with metastatic ARMS. Protein Kinase C iota (PRKCI) has been shown to play an important role in many cancers but little to date is known of this kinase's role in rhabdomyosarcoma. Materials and Methods: Human samples were obtained from the Pediatric Cooperative Human Tissue Network (Columbus, OH). Mouse samples were obtained from a Myf6Cre, Pax3: Fkhr, p53 conditional mouse model of ARMS and a Pax7CreER, Ptch1, p53 conditional model of embryonal rhabdomyosarcoma (ERMS). Expression of PKCι in tumors was evaluated by quantitative RT-PCR, western blotting and immunohistochemistry. To elucidate the functional role of PKCι in ARMS, we performed invasion assays, anchorage-independent colony formation assays and in vitro growth inhibition assays using primary cell lines obtained from mouse ARMS tumors, treating either with the PKCι specific inhibitor Aurothiomalate (ATM) or by PKCι RNA interference. Results: Over-expression of PKCι at the mRNA and protein level was confirmed in human and mouse ARMS and ERMS. Immunoblotting confirmed that PKCι and its downstream targets, mitogen-activated protein kinase and nuclear factor-kappa B were expressed and highly activated in primary cell cultures. Inhibition of PKCι by RNA interference resulted in a dramatic decrease in cell viability and colony formation. Treatment of primary cell culture using ATM resulted in decreased cell viability (IC50: 54.3 – 256.9 uM) and colony formation, and increased G2/M blockade. Co-treatment with ATM and vincristine, a microtubule inhibitor currently used in rhabdomyosarcoma treatment regimens, resulted in a Combination Index of 0.572 – 1.017, suggesting cooperativity between these agents. Conclusion: These results suggest that PKCι has an important function in proliferation of ARMS and that the PKCι specific inhibitor ATM shows activity in ARMS at clinically-feasible drug concentrations. Furthermore, these results suggest that combination therapy with vincristine and ATM should be investigated further in ARMS. Ken Kikuchi and Anuradha Soundararajan contributed this presentation equally. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1043. doi:10.1158/1538-7445.AM2011-1043

Abstract Motivation: KRAS is commonly mutated in a variety of cancers including lung cancer. The KRAS gene is frequently mutated at codons 12 and 13 in lung adenocarcinomas in patients with a history of smoking. Tumors harboring an activating KRAS mutation are aggressive and are often resistant to available therapies. In the present study, we set out to identify pathways that are specifically altered in lung cancer patients whose tumors harbor a KRAS mutation using next generation sequencing technology. Methods: We performed 50nt paired-end RNA-sequencing in 15 lung adenocarcinoma samples (8 and 7 samples with and without KRAS mutation). Reads were aligned to genome and exon junctions using Illumina's alignment tool ELAND_RNA. CASAVA and Genome Studio data analysis software were used to obtain read counts for genes, exons and exon junctions. We applied a variety of computational methods (Casper R package, Bowtie, TopHat) and softwares (Partek, R statistical software, JMP, Ingenuity Pathway Anlaysis, DAVID) to carry out our analyses. Results: Differential gene expression and splicing analysis were performed between the two groups. We identified 115 genes that were differentially expressed and 112 genes that consist of splicing variants with at least 2 fold changes and p-value < 0.01. We randomly selected 6/15 samples and performed real-time qRT-PCR for 6 differentially expressed genes. Significant correlations were observed ranging from 0.44 to 0.83, when qRT-PCR results were compared with RNA-sequencing expression data. Pathway analysis with 115 differentially expressed genes and 112 splicing variants revealed that the most significant pathway is composed entirely of NFκB focus genes, suggesting that there is a direct connection between oncogenic KRAS and activation of the NFκB signaling pathway. Conclusions: NFκB has been implicated in KRas-mediated formation of early stage lung adenomas in the LSL-Kas mouse model. Our data indicate that this connection also occurs in human lung cancer and establish NFκB activation as a key manifestation of KRAS mutation in human lung cancer. Hence drugs targeting NFκB and NFκB related genes may potentially be helpful for the treatment of patients with oncogenic KRAS mutations. (Supported in part by grants from the 26.2 with Donna Foundation and the Florida Department of Health Bankhead/Coley program.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4975. doi:10.1158/1538-7445.AM2011-4975

Abstract 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 Cι (PKCι) is required for the oncogenic Ras–mediated transformed growth of 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 overexpressed 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 show 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. Cancer Res; 70(5); 2064–74

Abstract Protein kinase Cι (PKCι) is an oncogene required for maintenance of the transformed phenotype of non–small cell lung cancer 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 to undergo Kras-mediated expansion and morphologic transformation in vitro and in vivo. Furthermore, the small molecule PKCι inhibitor aurothiomalate inhibits Kras-mediated bronchioalveolar stem cell 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 antitumor 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. [Cancer Res 2009;69(19):7603–11]

Abstract Purpose: 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. Experimental Design: 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. Results: 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. Conclusion: 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.

Abstract 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. [Cancer Res 2009;69(4):1643–50]

Abstract We previously showed that elevated expression of either protein kinase CβII (PKCβII) or PKCι/λ enhances colon carcinogenesis in mice. Here, we use novel bitransgenic mice to determine the relative importance of PKCβII and PKCι/λ in colon carcinogenesis in two complimentary models of colon cancer in vivo. Bitransgenic mice overexpressing 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 with nontransgenic 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 knockout of PKCλ significantly suppresses intestinal tumor formation in Apcmin/+ mice. Our data show that PKCβII and PKCι/λ serve distinct, nonoverlapping 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. [Cancer Res 2009;69(2):656–62]

Abstract The antirheumatoid 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 nmol/L to >100 μmol/L. 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. 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 antitumor 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. [Cancer Res 2008;68(14):5888–95]

PKC (protein kinase C) isoenzymes are key signalling components involved in the regulation of normal cell proliferation, differentiation, polarity and survival. The aberrant regulation of PKC isoenzymes has been implicated in the development of many human diseases including cancer [Fields and Gustafson (2003) Methods Mol. Biol. 233, 519–537]. To date, however, only one PKC isoenzyme, the aPKC [atypical PKCι (protein kinase Cι)], has been identified as a human oncogene [Regala, Weems, Jamieson, Khoor, Edell, Lohse and Fields (2005) Cancer Res. 65, 8905–8911]. PKCι has also proven to be a useful prognostic marker and legitimate target for the development of novel pharmacological agents for the treatment of cancer. The PKCι gene resides at chromosome 3q26 and is a frequent target of tumour-specific gene amplification in multiple forms of human cancer. PKCι gene amplification in turn drives PKCι overexpression in these cancers. Genetic disruption of PKCι expression blocks multiple aspects of the transformed phenotype of human cancer cells including transformed growth in soft agar, invasion through Matrigel and growth of subcutaneous tumours in nude mice. Genetic dissection of oncogenic PKCι signalling mechanisms demonstrates that PKCι drives transformed growth by activating a PKCι → Rac1 → PAK (p21-activated kinase) → MEK [MAPK (mitogen-activated protein kinase) 1,2/ERK (extracellular-signal-regulated kinase) kinase] 1,2 signalling pathway [Regala, Weems, Jamieson, Copland, Thompson and Fields (2005) J. Biol. Chem. 280, 31109–31115]. The transforming activity of PKCι requires the N-terminal PB1 (Phox-Bem1) domain of PKCι, which serves to couple PKCι with downstream effector molecules. Hence, there exists a strong rationale for developing novel cancer therapeutics that target the PB1 domain of PKCι and thereby disrupt its interactions with effector molecules. Using a novel high-throughput drug screen, we identified compounds that can disrupt PB1–PB1 domain interactions between PKCι and the adaptor molecule Par6 [Stallings-Mann, Jamieson, Regala, Weems, Murray and Fields (2006) Cancer Res. 66, 1767–1774]. Our screen identified the gold compounds ATG (aurothioglucose) and ATM (aurothiomalate) as specific inhibitors of the PB1–PB1 domain interaction between PKCι and Par6 that exhibit anti-tumour activity against NSCLC (non-small-cell lung cancer) both in vitro and in vivo. Structural analysis, site-directed mutagenesis and modelling indicate that ATM specifically targets the PB1 domain of PKCι to mediate its anti-tumour activity [Erdogan, Lamark, Stallings-Mann, Lee, Pellechia, Thompson, Johansen and Fields (2006) J. Biol. Chem. 281, 28450–28459]. Taken together, our recent work demonstrates that PKCι signalling is required for transformed growth of human tumours and is an attractive target for development of mechanism-based cancer therapies. ATM is currently in Phase I clinical trials for the treatment of NSCLC.