Alan P. Fields

Atypical Protein Kinase Cι Is an Oncogene in Human Non–Small Cell Lung Cancer

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 ι Protects Human Leukemia Cells against Drug-induced Apoptosis

Protein kinase C (PKC) isozymes play distinct roles in cellular function. In human K562 leukemia cells, PKC α is important for cellular differentiation and PKC βIIis required for proliferation. In this report, we assess the role of the atypical PKC isoform PKC ι in K562 leukemia cell physiology. K562 cells were stably transfected with expression plasmids containing the cDNA for human PKC ι in sense or antisense orientation to increase or decrease cellular PKC ι levels, respectively. Overexpression or inhibition of expression of PKC ι had no significant effect on the proliferative capacity of K562 cells nor their sensitivity to phorbol myristate acetate-induced cytostasis and megakaryocytic differentiation, suggesting that PKC ι does not play a critical role in these processes. Rather, PKC ι serves to protect K562 cells against drug-induced apoptosis. K562 cells, which are resistant to most apoptotic agents, undergo apoptosis when treated with the protein phosphatase inhibitor okadaic acid (OA). Overexpression of PKC ι leads to increased resistance to OA-induced apoptosis whereas inhibition of PKC ι expression sensitizes cells to OA-induced apoptosis. Overexpression of the related atypical PKC ζ has no protective effect, demonstrating that the effect is isotype-specific. PKC ι also protects K562 cells against taxol-induced apoptosis, indicating that it plays a general protective role against apoptotic stimuli. These data support a role for PKC ι in leukemia cell survival.

A novel small-molecule inhibitor of protein kinase Cι blocks transformed growth of non-small-cell lung cancer cells

We recently showed that atypical protein kinase Cι (PKCι) is required for transformed growth of human non–small-cell lung cancer (NSCLC) cells by activating Rac1. Genetic disruption of PKCι signaling blocks Rac1 activity and transformed growth, indicating that PKCι 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 PKCι signaling. This assay was used to identify compounds that disrupt the interaction between PKCι and its downstream effector Par6, which links PKCι 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 PKCι-Par6 interactions in vitro (IC50 ∼1 μmol/L). ATG blocks PKCι-dependent 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 PKCι 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 PKCι for antitumor drug development and provide proof of concept that chemical disruption of PKCι signaling can be an effective treatment for NSCLC. ATG and ATM will be useful reagents for studying PKCι function in transformation and represent promising new agents for the clinical treatment of NSCLC. (Cancer Res 2006; 66(3): 1767-74)

A Role for Nuclear Phosphatidylinositol-specific Phospholipase C in the G2/M Phase Transition

Protein kinase C (PKC) is activated at the nucleus during the G2 phase of cell cycle, where it is required for mitosis. However, the mechanisms controlling cell cycle-dependent activation of nuclear PKC are not known. We now report that nuclear levels of the major physiologic PKC activator diacylglycerol (DAG) fluctuate during cell cycle. Specifically, nuclear DAG levels in G2/M phase cells are 2.5–3-fold higher than in G1 phase cells. In synchronized cells, nuclear DAG levels rise to a peak coincident with the G2/M phase transition and return to basal levels in G1 phase cells. This increase in DAG level is sufficient to stimulate βIIPKC-mediated phosphorylation of its mitotic nuclear envelope substrate lamin B in vitro. Isolated nuclei from G2 phase cells contain an active phospholipase activity capable of generating DAG in vitro. Nuclear phospholipase activity is inhibited by the selective phosphatidylinositol-specific phospholipase C (PI-PLC) inhibitor 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine and neomycin sulfate, but not by the phosphatidylcholine-PLC selective inhibitor D609 or inhibitors of phospholipase D-mediated DAG generation. Treatment of synchronized cells with 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine leads to decreased nuclear PI-PLC activity and cell cycle blockade in the G2 phase, suggesting a role for nuclear PI-PLC in the G2/M phase transition. Our data are consistent with the hypothesis that nuclear PI-PLC generates DAG to activate nuclear βII PKC, whose activity is required for mitosis.

Identification of Protein Phosphatase 1 as a Mitotic Lamin Phosphatase

At the onset of mitosis, the nuclear lamins are hyperphosphorylated leading to nuclear lamina disassembly, a process required for nuclear envelope breakdown and entry into mitosis. Multiple lamin kinases have been identified, including protein kinase C, that mediate mitotic lamin phosphorylation and mitotic nuclear lamina disassembly. Conversely, lamin dephosphorylation is required for nuclear lamina reassembly at the completion of mitosis. However, the protein phosphatase(s) responsible for the removal of mitotic phosphates from the lamins is unknown. In this study, we use human lamin B phosphorylated at mitosis-specific sites as a substrate to identify and characterize a lamin phosphatase activity from mitotic human cells. Several lines of evidence demonstrate that the mitotic lamin phosphatase corresponds to type 1 protein phosphatase (PP1). First, mitotic lamin phosphatase activity is inhibited by high nanomolar concentrations of okadaic acid and the specific PP1 peptide inhibitor, inhibitor-2. Second, mitotic lamin phosphatase activity cofractionates with PP1 after ion exchange chromatography. Third, microcystin-agarose depletes mitotic extracts of both PP1 and lamin phosphatase activity. Our results demonstrate that PP1 is the major mitotic lamin phosphatase responsible for removal of mitotic phosphates from lamin B, a process required for nuclear lamina reassembly.

Role of cyclooxygenase 2 in protein kinase C βII-mediated colon carcinogenesis

Elevated expression of protein kinase C βII (PKCβII) is an early promotive event in colon carcinogenesis (Gokmen-Polar, Y., Murray, N. R., Velasco, M. A., Gatalica, Z., and Fields, A. P. (2001) Cancer Res. 61, 1375–1381). Expression of PKCβII in the colon of transgenic mice leads to hyperproliferation and increased susceptibility to colon carcinogenesis due, at least in part, to repression of transforming growth factor beta type II receptor (TGF-βRII) expression (Murray, N. R., Davidson, L. A., Chapkin, R. S., Gustafson, W. C., Schattenberg, D. G., and Fields, A. P. (1999)J. Cell Biol., 145, 699–711). Here we report that PKCβII induces the expression of cyclooxygenase type 2 (Cox-2) in rat intestinal epithelial (RIE) cells in vitro and in transgenic PKCβII mice in vivo. Cox-2 mRNA increases more than 10-fold with corresponding increases in Cox-2 protein and PGE2 production in RIE/PKCβII cells. PKCβII activates the Cox-2 promoter by 2- to 3-fold and stabilizes Cox-2 mRNA by at least 4-fold. The selective Cox-2 inhibitor Celecoxib restores expression of TGF-βRII both in vitro and in vivo and restores TGFβ-mediated transcription in RIE/PKCβII cells. Likewise, the ω-3 fatty acid eicosapentaenoic acid (EPA), which inhibits PKCβII activity and colon carcinogenesis, causes inhibition of Cox-2 protein expression, re-expression of TGF-βRII, and restoration of TGF-β1-mediated transcription in RIE/PKCβII cells. Our data demonstrate that PKCβII promotes colon cancer, at least in part, through induction of Cox-2, suppression of TGF-β signaling, and establishment of a TGF-β-resistant, hyperproliferative state in the colonic epithelium. Our data define a procarcinogenic PKCβII → Cox-2 → TGF-β signaling axis within the colonic epithelium, and provide a molecular mechanism by which dietary ω-3 fatty acids and nonsteroidal antiinflammatory agents such as Celecoxib suppress colon carcinogenesis.

Protein kinase calpha regulates human monocyte O-2 production and low density lipoprotein lipid oxidation.

Our previous studies have shown that human native low density lipoprotein (LDL) can be oxidized by activated human monocytes. In this process, both activation of protein kinase C (PKC) and induction of superoxide anion (O·̄2) production are required. PKC is a family of isoenzymes, and the functional roles of individual PKC isoenzymes are believed to differ based on subcellular location and distinct responses to regulatory signals. We have shown that the PKC isoenzyme that is required for both monocyte O·̄2 production and oxidation of LDL is a member of the conventional PKC group of PKC isoenzymes (Li, Q., and Cathcart, M. K. (1994) J. Biol. Chem. 269, 17508–17515). The conventional PKC group includes PKCα, PKCβI, PKCβII, and PKCγ. With the exception of PKCγ, each of these isoenzymes was detected in human monocytes. In these studies, we investigated the requirement for select PKC isoenzymes in the process of monocyte-mediated LDL lipid oxidation. Our data indicate that PKC activity was rapidly induced upon monocyte activation with the majority of the activity residing in the membrane/particulate fraction. This enhanced PKC activity was sustained for up to 24 h after activation. PKCα, PKCβI, and PKCβII protein levels were induced upon monocyte activation, and PKCα and PKCβII substantially shifted their location from the cytosol to the particulate/membrane fraction. To distinguish between these isoenzymes for regulating monocyte O·̄2 production and LDL oxidation, PKCα or PKCβ isoenzyme-specific antisense oligonucleotides were used to selectively suppress isoenzyme expression. We found that suppression of PKCα expression inhibited both monocyte-mediated O·̄2 production and LDL lipid oxidation by activated human monocytes. In contrast, inhibition of PKCβ expression (including both PKCβI and PKCβII) did not affect O·̄2 production or LDL lipid oxidation. Further studies demonstrated that the respiratory burst oxidase responsible for O·̄2 production remained functionally intact in monocytes with depressed levels of PKCα because O·̄2 production could be restored by treating the monocytes with arachidonic acid. Taken together, our data reveal that PKCα, and not PKCβI or PKCβII, is the predominant isoenzyme required for O·̄2 production and maximal oxidation of LDL by activated human monocytes.

Ect2 links the PKCι-Par6α Complex to Rac1 Activation and Cellular 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 epithelial cell transforming sequence 2 (Ect2), a guanine nucleotide exchange factor for Rho family GTPases, is coordinately amplified and overexpressed with PKCι in NSCLC tumors. RNA interference-mediated knockdown 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. RNA interference-mediated knockdown 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.

Atypical Protein Kinase Cι Is Required for Bronchioalveolar Stem Cell Expansion and Lung Tumorigenesis

Protein kinase Ciota (PKCiota) is an oncogene required for maintenance of the transformed phenotype of non-small cell lung cancer cells. However, the role of PKCiota in lung tumor development has not been investigated. To address this question, we established a mouse model in which oncogenic Kras(G12D) is activated by Cre-mediated recombination in the lung with or without simultaneous genetic loss of the mouse PKCiota 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 PKCiota 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 PKCiota as a therapeutic target and for the clinical use of aurothiomalate for lung cancer treatment.

Atypical Protein Kinase Cι Expression and Aurothiomalate Sensitivity in Human Lung Cancer Cells

The antirheumatoid agent aurothiomalate (ATM) is a potent inhibitor of oncogenic PKC iota. ATM inhibits non-small lung cancer (NSCLC) growth by binding PKC iota and blocking activation of a PKC iota-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 IC(50)s ranging from approximately 300 nmol/L to >100 micromol/L. ATM sensitivity correlates positively with expression of PKC iota and Par6, but not with the PKC iota binding protein p62, or the proposed targets of ATM in rheumatoid arthritis (RA), thioredoxin reductase 1 or 2. PKC iota expression profiling revealed that a significant subset of primary NSCLC tumors express PKC iota 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 PKC iota and Par6. Our results indicate that PKC iota expression profiling will be useful in identifying lung cancer patients most likely to respond to ATM therapy in an ongoing clinical trial.