Role Of PKCι Research Articles

Prkci Regulates Autophagy and Pancreatic Tumorigenesis in Mice.

Simple SummaryPancreatic cancer is a highly fatal disease that does not respond well to current cancer therapies. We previously showed that protein kinase C iota (PKCι) is highly expressed in pancreatic cancer, and inhibition of PKCι signaling in human pancreatic cell lines blocks tumor growth. In this study, we used a genetic mouse model to investigate the role of PKCι in pancreatic cellular homeostasis and pancreatic tumorigenesis. We found that inhibition of PKCι expression in the mouse pancreas blocked autophagy, altered infiltration of immune cells, and prevented pancreatic cancer formation. Taken together, these findings reveal a promotive role for PKCι in pancreatic cancer development.Protein kinase C iota (PKCι) functions as a bonafide human oncogene in lung and ovarian cancer and is required for KrasG12D-mediated lung cancer initiation and progression. PKCι expression is required for pancreatic cancer cell growth and maintenance of the transformed phenotype; however, nothing is known about the role of PKCι in pancreas development or pancreatic tumorigenesis. In this study, we investigated the effect of pancreas-specific ablation of PKCι expression on pancreatic cellular homeostasis, susceptibility to pancreatitis, and KrasG12D-mediated pancreatic cancer development. Knockout of pancreatic Prkci significantly increased pancreatic immune cell infiltration, acinar cell DNA damage, and apoptosis, but reduced sensitivity to caerulein-induced pancreatitis. Prkci-ablated pancreatic acinar cells exhibited P62 aggregation and a loss of autophagic vesicles. Loss of pancreatic Prkci promoted KrasG12D-mediated pancreatic intraepithelial neoplasia formation but blocked progression to adenocarcinoma, consistent with disruption of autophagy. Our results reveal a novel promotive role for PKCι in pancreatic epithelial cell autophagy and pancreatic cancer progression.

PKC iota promotes cellular proliferation by accelerated G1/S transition via interaction with CDK7 in esophageal squamous cell carcinoma.

Protein kinase C iota (PKCι) has been shown to play an important role in tumorigenesis of many cancers. It was reported that frequent amplification and overexpression of PKCi were correlated with resistance to anoikis in primary esophageal squamous cell carcinomas (ESCC). In this study, we clarified a novel role of PKCι on the cell cycle progression and proliferation in ESCC. Western blot and immunohistochemistry (IHC) analysis showed that the expression of PKCι was higher in ESCC tumor tissues and cell lines. Meanwhile, IHC stain revealed that PKCι was positively correlated with clinical pathologic variables such as tumor size, tumor grade, and tumor invasion, as well as ki67. Immunoprecipitation and immunofluorescence assay revealed that PKCι/CDK7 has the physical interaction and were co-located in the cell nucleus. And this direct interaction could increase the phosphorylation level of CDK7. In vitro studies such as starvation and refeeding assay along with PKCι-shRNA transfection assay demonstrated that PKCι expression promoted proliferation of ESCC cells. And knocking PKCi down by silencing RNA (siRNA) significantly caused cell cycle arrest at G0/G1 phase, decreased rate of colony formation, and alleviated cellular apoptosis. This research provide new insights into PKCi signaling to more deeply understand its cancer-promoting function in ESCC.

Abstract A02: PKCι in pancreatic cancer development: A possible role for autophagy

Abstract Pancreatic cancer is the fourth leading cause of cancer death in the United States with current 5-year survival rates at 6%. Greater than 90% of all pancreatic cancers harbor an activating mutation in codon 12 of the Kras gene (KrasG12D). Protein Kinase C iota (PKCι) has been shown to mediate KrasG12D downstream signaling and acinar-to-ductal metaplasia in an explant model of pancreatic cancer initiation. Additionally, inhibition of PKCι in human pancreatic cancer cells leads to decreased tumor size, angiogenesis, and metastasis in an orthotopic tumor model, suggesting that PKCι plays a role in maintaining the oncogenic phenotype of pancreatic cancer cells. We sought to test the hypothesis that PKCι plays a role development and maintenance of pancreatic cancer using a mouse model of KrasG12D-induced pancreatic cancer. Purpose: To identify the effect of PKCι loss on pancreas function and development of KrasG12D-induced pancreatic cancer. Procedures: Pancreata from mice with pancreas-specific deletion of PKCι, with and without pancreas-specific KrasG12D expression, were analyzed for histological and protein expression alterations. Acinar cells isolated from these transgenic mouse models were subjected to in vitro analyses to determine the role of PKCι in pancreatic cell homeostasis. Results: Mice lacking pancreatic PKCι survived and developed normally. Pancreatic knockout of PKCι expression resulted in a phenotype consistent with a defect in autophagic flux. In vitro analyses of isolated acinar cells supported the hypothesis that PKCι promotes autophagy in pancreatic cells. Autophagy exhibits a dual functionality in pancreatic cancer, acting as a tumor suppressor in normal cells and a tumor promoter in established tumor cells. We sought to determine the effect of PKCι knock-out on both of these cell types in a mouse model of pancreatic cancer. Using the LSL mouse model of K-ras induced pancreatic cancer, we demonstrated that pancreatic loss of PKCι lead to an increase in the number of Pancreatic Intraepithelial (PanIN) lesions formed in young mice. However, these lesions were protected from progressing past the early PanIN1/2 stage such that no mice lacking PKCι developed cancer, even after 1.5 years of age. By contrast, over 50% of mice with wild-type PKCι developed cancer; of those without cancer, many had progressed to the PanIN-3 stage, a stage representative of carcinoma in-situ. These data are similar to that seen in mouse models of pancreatic cancer with genetic inhibition of autophagy. Analysis of PanINs in these mice revealed that those lacking PKCι expressed significantly more P62 protein, a selective autophagy substrate, compared to PanINs with wild-type PKCι, supporting a block in the autophagic flux. These data suggest that PKCι plays an important role in both the initiation and progression of pancreatic cancer, possibly by regulating autophagic flux. Impact: Inhibition of pancreatic PKCι expression prevented progression of PanIN lesions past the early PanIN1/2 stage, resulting in a complete block in cancer development in mouse models of pancreatic cancer. Our preliminary data suggests that PKCι promotes cancer progression by regulating autophagic flux, a process that has been shown to be essential for KrasG12D-mediated pancreatic cancers. Pancreatic cancer is characterized by resistance to current therapies. Novel targets for new and more effective therapies are critical to improve outcomes for patients with this deadly disease. Inhibitors of PKCι are currently in clinical trials for ovarian and lung cancers. Our data supports the feasibility of using PKCι inhibitors for therapy in pancreatic cancer. Citation Format: Kristin S. Inman, Amanda A. Francis, Nicole R. Murray. PKCι in pancreatic cancer development: A possible role for autophagy. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A02.

Atypical PKCι as target for glioblastoma therapy.

Glioblastoma (grade IV astrocytoma) is an aggressive and incurable form of brain tumor. It invariably shows extensive invasion at the time of diagnosis, often involving both hemispheres. Recent studies have given us a very detailed picture of glioblastoma genetics. These paint a picture of a disease with extensive heterogeneity, both between patients and within individual patients. This within patient heterogeneity presents a major challenge in the design of targeted therapies. One approach is to identify targets that are common downstream elements in signaling pathways that are aberrantly activated in glioblastoma. Here we review the evidence that the atypical protein kinase C family member PKCι may fulfill this role. Our current understanding of PKCι activation mechanisms is discussed and related to common genetic changes in glioblastoma. The data showing an essential role for PKCι in multiple aspects of glioblastoma pathology are also reviewed. Finally, data on the role of PKCι in normal brain function are reviewed for insights into potential side effects of PKCι inhibition in the central nervous system.

Protein kinase Cι is a new prognostic factor in gastric cancer.

Protein kinase Cι (PKCι) is an important oncogenic K-ras effector, and its expression is correlated with tumor angiogenesis. The role of PKCι in gastric cancer remains unclear. The aim of this study was to clarify the role of PKCι in gastric cancer. Twenty-eight patients with gastric cancer who underwent gastrectomy were enrolled in this study. The expression of PKCι mRNA was determined, as were the clinicopathological factors. The patients were divided into PKCι high and low expression groups. The 5-year survival rate, ERK mRNA level and VEGF mRNA level were compared between the two groups. The prognostic factors were investigated by a multivariate analysis. High expression of PKCι was observed to be associated with a lack of differentiation, tumor invasion ≥muscularis propria≤, stage III and IV disease and peritoneal dissemination. The 5-year survival rate in the PKCι high group was lower than that in the PKCι low group. The multivariate analysis revealed that a high expression level of PKCι was an independent prognostic factor. The expression levels of ERK and VEGF in the PKCι high group were higher than those in the PKCι low group. Our results indicate that PKCι is correlated with tumor progression and angiogenesis. PKCι may be a new prognostic factor for gastric cancer.

Abstract 7: PKC-ι promotes glioblastoma cell survival by phosphorylating and inhibiting BAD through a phosphotidylinositol 3-kinase pathway

Abstract Robust expression of atypical protein kinase C-iota (PKC-ι) is a hallmark of human glioma, benign and malignant meningiomas [R. Patel, et. al. (2008) Cell Prolif. 41, 122-135]. The phosphoinositide (PI) 3-kinase pathway is frequently found to be over stimulated due to activating mutations in growth factor receptors or loss of function of the tumor suppressor PTEN. Protein kinase C-ι is often activated by the PI 3-kinase pathway and is an essential downstream targeted mediator. We investigated the role of PKC-ι in two human glial tumor derived cell lines T98G and U87MG. PKC-ι directly phosphorylated and inactivated Bad, a pro-apoptotic BH3-only molecule of the Bcl-2 family at three phospho specific residues, Ser-112, Ser-136 and Ser-155. PKC-ι was also observed to co-localize and directly associate with Bad in the cytoplasm of the cells (as shown by Immunofluorescence, Immunoprecipitation, and Western blotting). Furthermore, purified, active PKC-ι induced direct phosphorylation of Bad at all three serine residues subsequently disrupting Bad/Bcl-XL dimerization. Reduction of PKC-ι upon RNA interference exhibited a corresponding reduction in Bad phosphorylation as did Wortmannin and LY294002, known PI3-Kinase inhibitors. Depletion of PKC-ι by siRNA treatment also induced apoptosis with release of cytochrome C, caspase 3 activation and PARP cleavage. These data provide evidence for novel PI3-kinase/PKC-ι/BAD cell survival pathway. 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 7. doi:10.1158/1538-7445.AM2011-7

Abstract 1043: Role of protein kinase Cι in alveolar rhabdomyosarcoma

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 273: Protein kinase C-iota self regulates its expression by regulating the activity of the transcription factor Elk-1

Abstract Protein kinase C-iota (PKC-ι), a member of the atypical PKC family of Ser/Thr kinases has been recognized as an oncogene. PKC-ι manifests its effect by targeting various aspects of cancer cells such as growth, invasion and survival. Bcr-Abl has been shown to regulate the activation of the transcription factor ELK-1 which in turn regulates the expression of PKC-ι. Alternatively, we hypothesize that PKC-ι self regulates its expression by directly regulating the activity of ELK-1. Our preliminary data from immunoprecipitation studies show that PKC-ι directly associates with Elk-1 in neuroblastoma and glioma cells. PKC-ι directly phosphorylated cytoplasmic Elk-1 at Ser 383 in these cells. It has been reported that phosphorylation of cytoplasmic Elk-1 at Ser 383 results in conformational change that promotes the nuclear translocation of Elk-1, increased DNA binding and upregulation of its transcriptional activity. Furthermore, our data from cell cycle analysis showed that the nuclear translocation of Elk-1 coincided with the phosphorylation of cytoplasmic Elk-1 by PKC-ι. Robust phosphorylation of cytoplasmic Elk-1 was observed in the G1/S cell cycle phases, however, as the cells entered the M-phase, no phosphorylation of ELK-1 was observed. Similarly, the cytoplasmic to nuclear ratio of Elk-1 decreased as the cells entered the M-phase suggesting the role of PKC-ι in the promoting the nuclear translocation of Elk-1. Thus, our data supports our hypothesis that PKC-ι self regulates its expression by regulating the transcriptional activity of Elk-1. 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 273. doi:10.1158/1538-7445.AM2011-273

Coordination of glioblastoma cell motility by PKCι

BackgroundGlioblastoma is one of the deadliest forms of cancer, in part because of its highly invasive nature. The tumor suppressor PTEN is frequently mutated in glioblastoma and is known to contribute to the invasive phenotype. However the downstream events that promote invasion are not fully understood. PTEN loss leads to activation of the atypical protein kinase C, PKCι. We have previously shown that PKCι is required for glioblastoma cell invasion, primarily by enhancing cell motility. Here we have used time-lapse videomicroscopy to more precisely define the role of PKCι in glioblastoma.ResultsGlioblastoma cells in which PKCι was either depleted by shRNA or inhibited pharmacologically were unable to coordinate the formation of a single leading edge lamellipod. Instead, some cells generated multiple small, short-lived protrusions while others generated a diffuse leading edge that formed around the entire circumference of the cell. Confocal microscopy showed that this behavior was associated with altered behavior of the cytoskeletal protein Lgl, which is known to be inactivated by PKCι phosphorylation. Lgl in control cells localized to the lamellipod leading edge and did not associate with its binding partner non-muscle myosin II, consistent with it being in an inactive state. In PKCι-depleted cells, Lgl was concentrated at multiple sites at the periphery of the cell and remained in association with non-muscle myosin II. Videomicroscopy also identified a novel role for PKCι in the cell cycle. Cells in which PKCι was either depleted by shRNA or inhibited pharmacologically entered mitosis normally, but showed marked delays in completing mitosis.ConclusionsPKCι promotes glioblastoma motility by coordinating the formation of a single leading edge lamellipod and has a role in remodeling the cytoskeleton at the lamellipod leading edge, promoting the dissociation of Lgl from non-muscle myosin II. In addition PKCι is required for the transition of glioblastoma cells through mitosis. PKCι therefore has a role in both glioblastoma invasion and proliferation, two key aspects in the malignant nature of this disease.

Abstract 3171: Expression of PKC iota in breast cancer

Abstract One in nine (11%) women is expected to develop breast cancer during their lifetime. Abnormal activation of the PI3K signaling cascade is common in breast cancer. The p110α catalytic subunit of PI3K is mutated in 20-40% of breast cancers. The two most common mutations are E545K in the helical domain and H1047R in the kinase domain; these mutations render PI3K constitutively active. PKC iota is a member of atypical class of PKC family serine/threonine kinases and a downstream effector in the PI3K signaling pathway. PKC iota has been implicated in carcinogenesis and oncogenic signaling in lung, colon and ovarian carcinomas but its role in breast cancer progression is unknown. Using immunohistochemistry (IHC), we have evaluated PKCι expression and localization in breast cancer tissue microarrays (TMA). Weak PKCι staining was detected in normal breast tissue. PKCι was over-expressed in subset of breast cancers with no staining in the surrounding stroma. Positive tumor staining was mainly cytoplasmic with nuclear staining in some cases. There was no significant correlation of positive PKCι staining with tumour type. PKCι overexpression was also seen in a subset of ductal carcinoma in situ samples. In vitro, we have also shown that PKCι is over-expressed and has higher levels of phosphorylation in a subset of breast cancer cell lines when compared to mammary epithelial cell lines. Stable cell lines expressing E545K or H1047R mutations were generated in the mammary myoepithelial cell line MCF-10A by retroviral transduction. Using Western blotting, we have shown that these mutations are sufficient to increase PKCι expression and activation. These results suggest a possible mechanism for the PKCι overexpression previously demonstrated by IHC. Two structurally independent PI3K inhibitors, Wortmannin and LY294-002, inhibited PKCι phosphorylation and activation in mutant PI3K expressing cells, confirming that the PKCι activation is PI3K-dependent. These inhibitors did not inhibit the low level of PKCι activation seen in MCF10A cells expressing wild-type PI3K control, suggesting that this activation occurs by a PI3K-independent mechanism. These results demonstrate that PKCι is overexpressed in some breast cancers, and that PI3KCA mutations may drive this overexpression. These results also indicate a potential role for PKCι in breast carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3171.

Abstract 3583: 5-Amino-4-imidazolecarboxamide riboside-potentiated methotrexate uptake in CCRF-CEM cells

Abstract We previously showed that 5-amino-4-imidazolecarboxamide riboside (Z) potentiates methotrexate (MTX) uptake in human leukemia CCRF-CEM cells via the reduced folate carrier (RFC) and also enhances MTX growth inhibitory potency. We have now studied the role of protein kinases (PK) in Z-mediated potentiation of MTX uptake. AMP-activated PK (AMPK), the prototypical target of Z, was not involved because an AMPK activator (D942) alone did not potentiate MTX uptake and an AMPK inhibitor (compound C) did not inhibit Z-mediated potentiation. We then assessed the ability of a PK inhibitor library to inhibit Z-mediated potentiation of [3H]MTX uptake. None of the 80 PK inhibitors at 10 µM affected MTX uptake in the absence of Z. At 10 µM, triciribine (Akt), GF 109203X and Ro 31-8220 (both pan-PKC) demonstrated the greatest inhibition (>78%) of Z potentiation, with IC50 values of 4 µM, 1.8 µM and 1.7 µM, respectively. These data suggest that Akt and/or PKC (classical, c; novel, n; or atypical, a) could be involved in Z potentiation. Further studies with phorbol 12-myristate 13-acetate, which can selectively modulate activity of cPKC and nPKC (but not aPKC), and with PKC isotype-specific inhibitors suggested that cPKC and nPKC are not involved, but aPKC (PKC-ι and/or PKC-ζ) are. Expression of PKC-ι but not PKC-ζ in CCRF-CEM cells indicated PKC-ι to be the most likely candidate. Our PK inhibitor screen would identify inhibitors of Z transport and metabolism, as well as of Z-potentiated RFC function. Our previous findings revealed that Z must enter CCRF-CEM cells via equilibrative nucleoside transporter-1 (ENT-1) and that Z potentiates MTX uptake in a concentration-dependent manner. It was reported by others that PKC-δ and/or -ε enhance nucleoside uptake through ENT-1 in other cell types. To investigate if PKC can regulate transport/metabolism of Z, which could ultimately affect Z potentiation, a modified transport assay and HPLC analysis were used to quantitate intracellular [14C]Z ± pan-PKC inhibitor GF 109203X. GF 109203X decreased cellular Z accumulation by 54 ± 0.7% at 20 min, with an 86 ± 6.5% lower Z monophosphate (ZMP) level. Together with the results above, these data suggest that PKC-ι enhances Z accumulation and ZMP levels, which could potentiate MTX uptake. Further studies are underway investigating the effect of PKC-ι on Z transport alone by measuring the effect of GF 109203X on [14C]Z uptake at 10 sec. In addition, Akt inhibitor, triciribine, also decreased cellular [14C]Z accumulation by 48 ± 7.8%. Current evidence showing a role for PKC-ι and/or Akt in Z-potentiated MTX uptake may be partly or largely due to their effects on Z transport/metabolism, rather than on RFC. Because potentiation of MTX uptake requires Z transport and metabolism and at least 1 potentiating process, direct studies on posttranslational modification of RFC may provide better insight into possible mechanisms of Z-potentiated MTX uptake. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3583.

Protein Kinase Cι Is Required for Pancreatic Cancer Cell Transformed Growth and Tumorigenesis

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 Ciota (PKCiota) is required for the oncogenic Ras-mediated transformed growth of lung cancer and intestinal epithelial cells. However, little is known about the role of PKCiota in pancreatic cancer. In this study, we evaluated the expression of PKCiota in human pancreatic cancer and the requirement for PKCiota for the transformed growth and tumorigenicity of PDAC cells. We find that PKCiota is significantly overexpressed in human pancreatic cancer, and high PKCiota expression correlates with poor patient survival. Inhibition of PKCiota expression blocks PDAC cell transformed growth in vitro and tumorigenicity in vivo. Inhibition of PKCiota expression in pancreatic tumors also significantly reduces tumor angiogenesis and metastasis. Analysis of downstream PKCiota effectors implicates the Rac1-MEK/ERK1/2 signaling axis in PKCiota-mediated transformed growth and cellular invasion. Taken together, our data show a required role for PKCiota in the transformed growth of pancreatic cancer cells and reveal a novel role for PKCiota in pancreatic cancer cell metastasis and angiogenesis in vivo. Our results strongly indicate that PKCiota will be an effective target for pancreatic cancer therapy.

Abstract B223: Protein kinase C iota as a target for glioblastoma therapy

Abstract In spite of advances in surgery, radiation and chemotherapy, glioblastoma is still one of the deadliest forms of cancer. Two key features of the malignant nature of glioblastoma are its abnormal proliferation and its ability to invade both locally and to distant sites within the central nervous system. The phosphoinositide 3-kinase pathway is frequently activated by oncogenic mutations in glioblastoma, leading to activation of multiple downstream signaling molecules including protein kinase C iota (PKCι). Stable suppression of PKCι in glioblastoma cells with a short hairpin RNA caused a significant decrease in the proliferation of glioblastoma cells along with increased actin stress fiber formation and decreased cell motility and invasion. Live cell imaging was used to further assess the role of PKCι in glioblastoma cell motility and proliferation. While control glioblastoma cells form a coordinated leading edge lamellipodia and migrate substantial distances, cells stably depleted of PKCι show a loss of the ability to coordinate the formation of a functional leading edge lamellipodia and instead generate projections from all sides of the cell. These cells are therefore unable to move in a coordinated fashion. In addition live cell imaging showed that while glioblastoma cells round up and initiate mitosis, they are significantly impaired in their ability to complete mitosis. These effects on motility and mitosis were also seen when PKCι activity was inhibited with a myristoylated pseudosubstrate peptide. PKCι is therefore a promising new therapeutic target for glioblastoma. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B223.

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.

Interruption of RNA processing machinery by a small compound, 1-[(4-chlorophenyl)methyl]-1H-indole-3-carboxaldehyde (oncrasin-1)

Protein kinase Ciota (PKCiota) is activated by oncogenic Ras proteins and is required for K-Ras-induced transformation and colonic carcinogenesis in vivo. However, the role of PKCiota in signal transduction and oncogenesis is not clear. We recently identified a small molecule, designated 1-[(4-chlorophenyl)methyl]-1H-indole-3-carboxaldehyde (oncrasin-1), that can selectively kill K-Ras mutant cancer cells and induce abnormal nuclear aggregation of PKCiota in sensitive cells but not in resistant cells. To determine the causes and biological consequences of PKCiota aggregates in the nucleus, we analyzed the effect of oncrasin-1 on proteins involved in DNA repair and RNA processing. Our results showed that oncrasin-1 treatment led to coaggregation of PKCiota and splicing factors into megaspliceosomes but had no obvious effects on the DNA repair molecule Rad51. Moreover, oncrasin-1 treatment suppressed the phosphorylation of the largest subunit of RNA polymerase II and the expression of intronless reporter genes in sensitive cells but not in resistant cells, suggesting that suppression of RNA transcription is a major effect of oncrasin-1 treatment. Studies with cultured cells or with recombinant proteins showed that oncrasin-1 can disrupt the interaction of PKCiota and cyclin-dependent protein kinase 9/cyclin T1 complex, which is known to phosphorylate the largest subunit of RNA polymerase II and is required for RNA transcription. Together, our results suggest that oncrasin-1 suppresses the function of RNA processing machinery and that PKCiota might be involved in the biological function of RNA processing complexes.

Protein Kinase Cι Promotes Nicotine-induced Migration and Invasion of Cancer Cells via Phosphorylation of μ- and m-Calpains

Nicotine is a major component in cigarette smoke that activates the growth-promoting pathways to facilitate the development of lung cancer. However, it is not clear whether nicotine affects cell motility to facilitate tumor metastasis. Here we discovered that nicotine potently induces phosphorylation of both mu- and m-calpains via activation of protein kinase Ciota (PKCiota), which is associated with accelerated migration and invasion of human lung cancer cells. Purified PKCiota directly phosphorylates mu- and m-calpains in vitro. Overexpression of PKCiota results in increased phosphorylation of both mu- and m-calpains in vivo. Nicotine also induces activation of c-Src, which is a known PKCiota upstream kinase. Treatment of cells with the alpha(7) nicotinic acetylcholine receptor inhibitor alpha-bungarotoxin can block nicotine-induced calpain phosphorylation with suppression of calpain activity, wound healing, cell migration, and invasion, indicating that nicotine-induced calpain phosphorylation occurs, at least in part, through a signaling pathway involving the upstream alpha(7) nicotinic acetylcholine receptor. Intriguingly, depletion of PKCiota by RNA interference suppresses nicotine-induced calpain phosphorylation, calpain activity, cell migration, and invasion, indicating that PKCiota is a necessary component in nicotine-mediated cell motility signaling. Importantly, nicotine potently induces secretion of mu- and m-calpains from lung cancer cells into culture medium, which may have potential to cleave substrates in the extracellular matrix. These findings reveal a novel role for PKCiota as a nicotine-activated, physiological calpain kinase that directly phosphorylates and activates calpains, leading to enhanced migration and invasion of human lung cancer cells.

Survival Function of Protein Kinase Cι as a Novel Nitrosamine 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone-activated Bad Kinase

Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is formed by nitrosation of nicotine and has been identified as the most potent carcinogen in cigarette smoke. NNK cannot only induce DNA damage but also promotes the survival of human lung cancer cells. Protein kinase C (PKC)iota is an atypical PKC isoform and plays an important role in cell survival, but the downstream survival substrate(s) is not yet identified. Bad, a proapoptotic BH3-only member of Bcl2 family, is co-expressed with PKCiota in both small cell lung cancer and non-small cell lung cancer cells. We discovered that NNK potently induces multisite Bad phosphorylation at Ser-112, Ser-136, and Ser-155 via activation of PKCiota in association with increased survival of human lung cancer cells. Purified, active PKCiota can directly phosphorylate both endogenous and recombinant Bad at these three sites and disrupt Bad/Bcl-XL binding in vitro. Overexpression of PKCiota results in an enhancement of Bad phosphorylation. NNK also stimulates activation of c-Src, which is a known PKCiota upstream kinase. Treatment of cells with the PKC inhibitor (staurosporine) or a Src-specific inhibitor (PP2) can block NNK-induced Bad phosphorylation and promote apoptotic cell death. The beta-adrenergic receptor inhibitor propranolol blocks both NNK-induced activation of PKCiota and Bad phosphorylation, indicating that NNK-induced Bad phosphorylation occurs at least in part through the upstream beta-adrenergic receptor. Mechanistically, NNK-induced Bad phosphorylation prevents its interaction with Bcl-XL. Because the specific depletion of PKCiota by RNA interference inhibits both NNK-induced Bad phosphorylation and survival, this confirms that PKCiota is a necessary component in NNK-mediated survival signaling. Collectively, these findings reveal a novel role for PKCiota as an NNK-activated physiological Bad kinase that can directly phosphorylate and inactivate this proapoptotic BH3-only protein, which leads to enhanced survival and chemoresistance of human lung cancer cells.

Protein kinase Ciota is required for Ras transformation and colon carcinogenesis in vivo.

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.