Protein Kinase C as a Therapeutic Target (2006)

The recognition of protein kinase C (PKC) as the long sought-after receptor for the tumor-promoting phorbol esters established the potential role of PKC in carcinogenesis and as a potentially important target for cancer therapeutics (1, 2). PKC is a family of serine/threonine kinases that regulate a variety of cell functions including proliferation, gene expression, cell cycle, differentiation, cytoskeletal organization cell migration, and apoptosis (3). The PKC family was the first recognized receptor of diacylglycerol. Aberrant PKC activation can lead to diseases of cellular dysregulation such as cancer and diabetes. The existence of multiple isozymes of PKC raised the question of whether each PKC isozyme has a specific function. The PKC family includes isozymes [α, βI, βII, γ, δ, ε, 𝛉, η, λ/ι (mouse/human), and ζ] which are involved in signal transduction from membrane receptors to the nucleus (Fig. 1; ref. 4). Differences in PKC isozyme protein structure and substrate preferences have allowed the family to be divided into three groups (2). First, the conventional PKC isozymes (α, βI, βII, and γ) are calcium-dependent and are phospholipid- and diacylglycerol-activated kinases. Second, the novel isozymes (δ, ε, μ, and 𝛉) are calcium-insensitive, phospholipid-dependent, and diacylglycerol-dependent. Third, the atypical PKC isozymes (η and λ/ι) are both calcium- and diacylglycerol-insensitive enzymes. The activation mechanism of the PKC isozyme family is clearly different among the three subgroups: conventional, novel, and atypical PKC, but whether or not each isozyme in a subgroup has a specific function or activation mechanism has not been clarified. The activation and degradation of the PKC isozymes is controlled spatially and temporally (5). PKC is a single polypeptide with four conserved regions and four variable regions having a COOH-terminal catalytic domain and a NH2-terminal regulatory domain (2). The multiple functions of PKC in signal transduction are regulated by targeting PKC to specific intracellular compartments. Several PKC isozymes may be capable of phosphorylating the same substrate. The constitutive, lipid-dependent protein kinase activity of purified PKC delayed the realization that PKC phosphorylation plays a fundamental role in their catalytic activities. PKC phosphorylation in vivo is well documented and is important in the maturation of the enzyme to a fully functional form localized correctly in the cell.The identification of physiologically relevant substrates of the 11 known PKC isozymes is of obvious importance for a complete understanding of the mechanisms by which this family of serine/threonine protein kinases relays information from intracellular stimuli to biological responses. Many proteins have been identified as PKC substrates both in vitro and in vivo. Interestingly, however, knowledge of PKC substrates is not currently matched by the detailed understanding of PKC regulation (2–4, 6). PKC isoforms are widely distributed in tissues, although some isoforms are selectively expressed. PKCγ is selectively expressed in the central nervous system and spinal cord, PKC𝛉 is expressed by skeletal muscle and hematopoietic cells, and PKCβ is expressed in pancreatic islet cells, monocytes, brain, and retinal tissue (2).Many kinases display overlapping substrate specificities in vitro and can functionally compensate for each other in single-gene knockout experiments. Early investigations regarding the role of PKC isoforms in various intracellular signaling events were based on a linear paradigm (6). Recent studies show that signaling molecules aggregate to form multiprotein complexes, a phenomenon that seems to hold a number of various types of molecules in close proximity. The formation of these complexes may facilitate signal transduction and allow the modulation of biological functions within the cell. PKC isozymes do not function in isolation but exist in complexes with other signaling molecules.PKCβI, PKCε, and PKCγ are targets in phorbol ester–mediated tumor promotion/progression, whereas PKCδ is tumor-suppressive (Fig. 1). Disulfide forms of thiols have oxidative regulatory effects on PKC isozymes and PKC S-thiolation by disulfiram induces differential regulatory effects on PKC isozymes that correlate with the cancer-preventive activity of disulfiram (7, 8). Overexpression of PKCδ stimulates apoptosis in a wide variety of cell types through a mechanism that is incompletely understood (9). PKCδ-deficient cells are impaired in their response to DNA damage–induced apoptosis, suggesting that PKCδ is required for an apoptotic response to stress. Using adenoviral vectors, Santiago-Walker et al. (9) found a modest increase in PKCδ activity but PKCα or PKCε activity was not able to selectively stimulate quiescent cells to initiate G(1) phase cell cycle progression. The PKCδ-infected cells arrest in S phase and proceed to caspase-dependent apoptotic cell death. Further delineating the PKC isozymes, Gustafson et al. (10) showed that atypical PKCι is required for Bcr-Abl-mediated resistance of human K562 chronic myelogenous leukemia cells to paclitaxel-induced apoptosis. When Bcr-Abl was expressed in Bcr-Abl-negative HL-60 promyelocytic leukemia cells, the expression of PKCβI, PKCβII, and PKCι was induced whereas expression of PKCδ was decreased. Thus, Bcr-Abl-mediated transformation involves transcriptional activation of the PKCι gene leading to Bcr-Abl-mediated chemoresistance.Understanding the mechanisms by which PKC contribute to malignancy remains a challenge (11). One model critically implicates conventional PKCβII (diacylglycerol-responsive) and atypical PKCλ/ι (diacylglycerol-unresponsive) in cancer cell transformation and invasion. PKCλ/ι is markedly up-regulated in human colon and lung tumors, in an in vivo model of chemically induced colon carcinogenesis, and in the expression of activated PKCλ/ι-promoted hypersensitivity of mice to a carcinogen.The PKC family is important in normal keratinocyte biology and in skin diseases, especially skin cancer (12). Keratinocyte apoptosis induced by UV radiation is a major protective mechanism from photocarcinogenesis. The constitutively active catalytic domain of PKCδ is an apoptotic effector generated by caspase-3 cleavage of full-length PKCδ in response to a wide variety of apoptotic stimuli including UV radiation (13). The activated PKCδ catalytic domain triggers the redistribution and activation of Bax that can directly induce cytochrome c release. PKCδ also activates upstream components of the death effector pathway to insure apoptosis. The cleavage and activation of PKCδ are critical components of UV-induced apoptosis in human keratinocytes. Inactivation of PKCδ can promote the survival of keratinocytes exposed to UV radiation (14). Recently, Aziz et al. (15) showed that PKCδ overexpression in transgenic mice failed to suppress the induction of squamous cell carcinoma developed by repeated exposure to UV radiation.Transgenic mice that overexpress PKCε protein in basal epidermal cells and cells of the hair follicle develop papilloma-independent metastatic squamous cell carcinoma elicited by 7,12-dimethylbenz(a)anthracene-initiation and 12-O-tetradecanoylphorbol-13-acetate promotion. Chronic exposure of PKCε transgenic mice to UV radiation indicates that PKCε signals tumor necrosis factor-α release, which is linked to photosensitivity in these mice (16). The overexpression of PKCε in the epidermis of mice may lead to the induction of specific cytokines that perturb normal hematopoiesis in bone marrow, resulting in a granulocytic skew toward neutrophils and eosinophils (17, 18). Both the transgenic overexpression of PKCε in mouse epidermis and human squamous cell carcinoma indicate that high expression of PKCε in the epidermis may lead to a microenvironment suitable for the development of squamous cell carcinoma by a paracrine mechanism.PKC activity is higher in actively proliferating colonic epithelial cells than in their quiescent counterparts, suggesting a role for PKC in proliferation. Colonic epithelial cells express multiple PKC isozymes that are differentially modulated during colon carcinogenesis, suggesting a direct connection between PKC and colon carcinogenesis (10, 11).PKCδ expression is decreased in azoxymethane-induced rat and sporadic human colon tumors (19). Human CaCo-2 gastric cancer cells stably transfected with PKCδ showed that increased expression of PKCδ induced cellular differentiation and limited survival. Elevated expression of PKCβII is an early event in colon carcinogenesis. Transgenic expression of PKCβII in mouse colon led to hyperproliferation and increased susceptibility to colon carcinogenesis that correlated with decreased expression of transforming growth factor-β type II receptor. Yu et al. (20) showed that transfection with PKCβII induced the expression of cyclooxygenase-2 in rat intestinal epithelial cells in culture and in transgenic PKCβII mice. Transgenic mice expressing PKCβII in colonic epithelium show that high expression of PKCβII is both necessary and sufficient to confer susceptibility to azoxymethane-induced colon carcinogenesis (21). PKCβII regulated its own expression in rat intestinal epithelial cells in culture and in animals through a mitogen-activated protein/extracellular signal-regulated kinase kinase–dependent signaling pathway. In the colon epithelium, elevated expression of PKCβII seems to be procarcinogenic, acting through both cyclooxygenase-2 and transforming growth factor-β signaling pathways.PKCι is implicated as a critical downstream effector of oncogenic Ras in the colonic epithelium (11). Using the synthetic ether lipid, 1-O-hexadecyl-2-O-methyl-rac-glycerol, which inhibits PKC activation by competing with the natural PKC activator, diacylglycerol, Liou et al. (22) showed that human tumor cell lines with oncogenic p21ras mutations were much more sensitive to PKC inhibition by 1-O-hexadecyl-2-O-methyl-rac-glycerol than tumor cell lines with normal K-ras alleles. In transgenic mice expressing constitutively active PKCι in the colon epithelium, PKCι activity was required for oncogenic Ras- and carcinogen-mediated colon carcinogenesis. PKC isoforms may act in concert to promote malignancy in the colon because transgenic expression of PKCβII in mouse colon induces hyperproliferation and increased susceptibility to colon cancer through pathways that include the activation of Ras, PKCι/Rac1, and Mek leading to invasion in intestinal epithelial cells (23). PKC phosphorylation promotes rapid dissociation of K-Ras from the plasma membrane and association with intracellular membranes, including the outer membrane of mitochondria, in which phosphor-K-Ras interacts with Bcl-XL (24). Interestingly, gastric adenocarcinoma SNU-1 cells respond to dietary phytoalexin resveratrol by loss of membrane-associated PKCδ and an increase in cytosolic PKCα prior to the up-regulation of antiproliferative and proapoptotic signals (25).PKCα and PKCδ promote apoptosis in androgen-dependent prostate cancer cells (26). Stewart and O'Brian (27) found 12-O-tetradecanoylphorbol-13-acetate–induced PKCα activation in epidermal growth factor receptor transactivation and signaling to Erk1/2 activation in PC-3 cells. The PKC-selective kinase inhibitors, GF109203X and Go6983, blocked phorbol ester–induced epidermal growth factor receptor transactivation, indicating a requirement for PKC, and supporting PKCα inhibition as a therapeutic approach for prostate cancer.PKCε expression in LNCaP cells was sufficient to confer a significant resistance to phorbol ester induction of Bax oligomerization suggesting the association of PKCε with Bax and the neutralization of apoptotic mitochondrial death-signaling pathways (28). Failure of hormonal therapy frequently involves an outgrowth of PKCε-positive cells in recurrent prostate cancer. Wu et al. (29) found a functional interplay among the integrin receptors, PKCε and protein kinase B/Akt, in recurrent CWR-R1 prostate cancer, suggesting that sustained activation of these β1 integrins induced an androgen independent–like behavior in these cells.Atypical PKCι is critical in the establishment and maintenance of epithelial cell polarity and is genomically amplified and overexpressed in serous epithelial ovarian cancers (30). PKCι is markedly increased and mislocalized in all serous ovarian cancers. In nonserous ovarian cancers, increased PKCι is associated with decreased overall survival. Thus, PKCι may be an oncogene in ovarian cancer regulating epithelial cell polarity and proliferation. Compared with normal tissues, endometrial cancers also exhibit alterations in the expression profile or in the activities of the PKC family, and aberrant levels or activation of specific PKC isoforms are postulated to contribute to endometrial neoplasia and transformation. Lahn et al. (31) found that PKCα increased in breast cancers with low or negative estrogen receptor levels. In breast cancer cells, the indolocarbazole PKC inhibitor, Go6976, abrogated S and G2 cell cycle arrest (32). In p53-mutated human SKOV3 ovarian cancer xenograft tumors, the administration of Go6976 and caffeine was able to restore the response to cisplatin (33).Atypical PKCι is highly expressed in human non–small cell lung carcinoma (NSCLC) lines, whereas the closely related PKC isozyme, PKCζ, is undetectable in these cells (34). PKCι is a critical lung cancer gene that activates a Rac1 → Pak → Mek1,2 → Erk1,2 signaling pathway required for transformed growth. PKCι may be an oncogene in NSCLC because: (a) PKCι is overexpressed in most NSCLCs, (b) PKCι expression predicts poor survival in patients with NSCLC, (c) the PKCι gene is frequently amplified in NSCLC lines and primary NSCLC, (d) gene amplification drives PKCι expression in NSCLC lines and primary tumors, and (e) PKCι disruption signaling with a dominant-negative PKCι allele blocks the transformed growth of human NSCLC cells harboring PKCι gene amplification (34, 35). PKCι is implicated in carcinogen-induced survival of malignant cells (36). Stallings-Mann et al. (37) found that aurothioglucose and aurothiomalate were inhibitors of PKCι-Par6 interactions and slowed the growth of human A549 NSCLC xenograft tumors.Clark et al. (38) found PKCδ to be antiapoptotic in NSCLC cells. Ding et al. (39) identified PKCε as critically involved in drug-resistance in lung cancer cells by preventing cell from undergoing apoptosis. PKCα and PKCε work cooperatively in lung cancer cellular response to various stimuli (40). In cocultures of normal pulmonary fibroblasts and NSCLC cells, PKCα and PKCε, but not PKCδ, were critical to stromelysin-3 (MMP-11) expression, indicating the involvement of both classical and novel PKC in the regulation of stromelysin-3 expression (41).An antisense agent designed to inhibit the production of PKCα has undergone clinical trials in NSCLCs (42). In preclinical studies, the antisense oligonucleotide, LY900003 (ISIS3521), inhibited PKCα mRNA and protein expression and had antitumor activity. In early clinical studies, LY900003 had single-agent activity and was promising in combination, particularly in patients with NSCLC, which led to a phase III clinical trial of LY900003 in combination with either cisplatin and gemcitabine or carboplatin and paclitaxel. However, in the phase III setting, LY900003 did not add significantly to the chemotherapy regimens.In rat hippocampal H19-7 cells and rat pheochromocytoma PC12 cells, the extracellular signal-regulated kinase activation by the neurogenic agents, fibroblast growth factor and nerve growth factor, is PKCδ-dependent, whereas extracellular signal-regulated kinase activation in response to epidermal growth factor is PKCδ-independent. Inhibition of PKCδ by antisense or rottlerin, blocked neurite outgrowth induced by fibroblast growth factor and nerve growth factor in PC12 cells and by activated Raf in H19-7 cells, indicating the involvement of PKCδ in the neurogenic effects of fibroblast growth factor, nerve growth factor, and Raf. Epidermal growth factor activates mitogen-activated protein kinase through a PKCζ-dependent pathway involving phosphatidylinositol-3-kinase and PDK1 in H19-7 cells (43). PKC regulates the activation of Raf-1 signaling from growth factors by phosphorylating the Raf kinase inhibitory protein, thereby relieving the inhibition of the Raf/mitogen-activated protein kinase signaling cascade (44).Phorbol 12-myristate 13-acetate–stimulated activation of PKCε in U-251 glioblastoma cells resulted in the activation of both Akt and the mammalian target of rapamycin signaling pathways as well as an increase in cell proliferation. Increased PKCη expression correlated with increased phorbol 12-myristate 13-acetate–induced proliferation in U-1242 MG cells (45). In the glioblastoma cell lines (U-1242 MG and U-87 MG), phorbol 12-myristate 13-acetate–induced epidermal growth factor receptor phosphorylation was blocked by bisindolylmaleimide, a PKC inhibitor, and rottlerin, a PKCδ-specific inhibitor (46). Thus, phorbol 12-myristate 13-acetate transactivates the epidermal growth factor receptor and increases cell proliferation by activating the PKCδ/c-Src pathway in glioblastomas.PKC is directly involved in modulating apoptosis hematopoietic neoplasms. PKCδ, PKCι, PKCμ, and PKCζ expression was detected in multiple myeloma cell lines (U266, RPMI-8226, and K620; ref. 47). Using immunohistochemistry, PKCδ expression was confirmed in plasma cells from 11 patients. Alkan et al. (48) investigated PKC isoform expressions in chronic lymphocytic leukemia cells from seven patients and found the expression of PKCβ, PKCγ, PKCδ, and PKCζ by Western blot in all of the PKC isoforms including βII, ε, and to be in the and PKCδ seems to have some in the brain, is more and downstream pathways from PKCα and PKCδ are in Many factors related to proliferation and invasion have been with however, the pathways downstream from PKCδ form a Several PKC isoforms have been identified in malignant however, active role in the has been to PKC have been toward the or the levels of isoform can be for and synthetic PKC of with the same for PKC as phorbol esters were the of as a inhibitor the domain of the with were inhibitors of PKC and The of the of inhibitors such as and the of the enzyme The identified the PKC inhibitor, was to inhibit the growth of tumor types in vitro and in vivo has undergone phase and II clinical trials and in combination The was identified as a PKC inhibitor but has been found to inhibit both kinase and serine/threonine kinases is a radiation both in cell culture and human tumor xenograft studies through a pathway In was because of the inhibition of the activities of the cells was more toward cell chronic lymphocytic leukemia than toward normal cells to human NSCLC cells to DNA by Akt kinase activation and induced apoptosis in human multiple myeloma cells is a inhibitor of and of the protein, growth factor receptor as well as kinase activities remains sensitive to In exposure of human leukemia cells to with resulted in inhibition of cells expressing or the exposure of human myelogenous leukemia cells with to or inhibitor, and resulted in increased cell with either agent and increased cell with myelogenous leukemia cells expressing normal has undergone phase clinical trials as a PKC inhibitor and as a kinase single agent studies showed that can be by studies of in combination with of in tumors, in combination with and or gemcitabine and cisplatin in NSCLC, established that be in these phase II trials of and with in leukemia or tumors have a in the kinase domain which resistance to has been identified is a inhibitor of PKCβ PKCβII mRNA levels in cells were significantly by with the on PKCβ activity to inhibited the proliferation of growth factor human cells. When human cell lung carcinoma cells were exposed to for a differential on the malignant cells human cell was culture studies indicate that exposure to can growth inhibition and apoptosis in human multiple myeloma cells, cell and cell for of resulted in markedly decreased growth to of the whereas decreased growth to the of for of fibroblast growth factor resulted in decreased growth to of the fibroblast growth factor When mice human tumor were with for the number of was decreased to to of the in animals (Fig. some tumors to as an in was blocked as in the The tumor growth did not correlate with (Fig. In most tumor the tumor growth by as a single agent was not sufficient to single agent activity in the However, the combination high activity. plasma levels in mice the human and cell or with were on and the of levels were between the and groups to levels in the groups to increase the however, of plasma levels in mice were significantly decreased and with and were in human glioblastoma tumor growth as the the tumor was and as the the tumor was the was on to and was on to 11 tumor In this tumor with tumor growth of and of and (Fig. of the combination of with resulted in a tumor growth of mice with had a survival of of the survival a survival of (Fig. of to mice the tumor resulted in a survival of The combination of with or survival of and in the of was a from to on to tumor growth of to in the tumor (Fig. by resulted in a tumor growth of with from to tumor survival of to with for (Fig. by resulted in survival of to with for clinical studies for were in from patients with showed decreased PKC activity vivo with phorbol ester a potential in a cell II studies have been in patients with cell and in patients with recurrent Several patients with multiple cell of although the tumor response was low In patients with was well and to have promising antitumor activity in a significant of highly patients In early combination studies, has and potentially with and gemcitabine and cisplatin phase III trial of in with in glioblastoma has been phase III trial has been in be as a maintenance therapy in patients with cell have first therapy to among the kinases with inhibitors, the PKC family is the most expressed and involved in cellular the clinical trials to the of kinases by these and therapeutics