PKCα Overexpression Research Articles

Activation of protein kinase C-α and Src kinase increases urea transporter A1 α-2, 6 sialylation.

The urea transporter A1 (UT-A1) is a glycosylated protein with two glycoforms: 117 and 97 kD. In diabetes, the increased abundance of the heavily glycosylated 117-kD UT-A1 corresponds to an increase of kidney tubule urea permeability. We previously reported that diabetes not only causes an increase of UT-A1 protein abundance but also, results in UT-A1 glycan changes, including an increase of sialic acid content. Because activation of the diacylglycerol (DAG)-protein kinase C (PKC) pathway is elevated in diabetes and PKC-α regulates UT-A1 urea transport activity, we explored the role of PKC in UT-A1 glycan sialylation. We found that activation of PKC specifically promotes UT-A1 glycan sialylation in both UT-A1-MDCK cells and rat kidney inner medullary collecting duct suspensions, and inhibition of PKC activity blocks high glucose-induced UT-A1 sialylation. Overexpression of PKC-α promoted UT-A1 sialylation and membrane surface expression. Conversely, PKC-α-deficient mice had significantly less sialylated UT-A1 compared with wild-type mice. Furthermore, the effect of PKC-α-induced UT-A1 sialylation was mainly mediated by Src kinase but not Raf-1 kinase. Functionally, increased UT-A1 sialylation corresponded with enhanced urea transport activity. Thus, our results reveal a novel mechanism by which PKC regulates UT-A1 function by increasing glycan sialylation through Src kinase pathways, which may have an important role in preventing the osmotic diuresis caused by glucosuria under diabetic conditions.

Abstract 619: Selective estrogen mimics for the treatment of tamoxifen-resistant breast cancer

Abstract Endocrine-resistant breast cancer, whether de novo or acquired, is a major clinical obstacle. Although recent clinical trials have demonstrated the efficacy of 17β-estradiol (E2) or diethylstilbestrol (DES) following exhaustive use of antiestrogens, E2 treatment is associated with major side effects such as an increased risk of other gynecological cancers, deterring the clinical community from adopting it as a treatment strategy. Our lab has previously shown that Protein kinase C alpha (PKCα) overexpression predicts tamoxifen (TAM) resistance in the clinic (Tonetti DA et al., Br J Cancer. 2003) and may also predict a positive response to an estrogenic therapy (Chisamore MJ et al., Clinical Cancer Research. 2001). Further, the ectopic overexpression of PKCα in T47D breast cancer cells, led to a TAM-resistant, E2-inhibited phenotype in vivo which was accompanied by the translocation of estrogen receptor alpha (ERα) to extranuclear sites (Perez White B et al., Molecular Cancer. 2013). The purpose of this study was to identify novel selective estrogen mimics (SEMs), which could achieve the positive therapeutic effects of E2 treatment in TAM-resistant breast cancers, while minimizing the side effects. In vitro screening identified two SEMs, BTC and TTC-352, which displayed estrogenic activity in breast cancer cell lines. BTC and TTC-352 treatment resulted in significant tumor regression in two xenograft models of TAM-resistant, PKCα-overexpressing breast cancer. Similar to E2, T47D:A18/PKCα tumor regression was accompanied by translocation of ERα to extranuclear sites, possibly defining a mechanism through which these SEMs initiate tumor regression. SEM treatment, however, did not result in growth of parental, TAM-sensitive xenograft tumors. Endometrial thickening, caused by both E2 and TAM, is directly associated with gynecological carcinogenesis and uterine cancer. Interestingly, SEM treatment did not increase uterine weight in mice suggesting negligible hormonal stimulation in gynecological tissues. Both BTC and TTC-352 resulted in regression of two TAM-resistant breast cancer models, while displaying enhanced safety compared to E2 and TAM. These data suggest that further development of SEMs targeted to TAM-resistant breast cancer is a feasible therapeutic strategy. Citation Format: Mary Ellen Molloy, Bethany Perez White, Huiping Zhao, Bradley T. Michalsen, Hitisha K. Patel, Jiong Zhao, Rui Xiong, Marton I. Siklos, Gregory R.J. Thatcher, Debra A. Tonetti. Selective estrogen mimics for the treatment of tamoxifen-resistant breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 619. doi:10.1158/1538-7445.AM2014-619

Pre-ischemic treadmill training alleviates brain damage via GLT-1-mediated signal pathway after ischemic stroke in rats

Physical exercise could play a neuroprotective role in both human and animals. However, the involved signal pathways underlying the neuroprotective effect are still not well established. This study was to investigate the possible signal pathways involved in the neuroprotection of pre-ischemic treadmill training after ischemic stroke. Seventy-two SD rats were randomly assigned into three groups (n=24/group): sham surgery group, middle cerebral artery occlusion (MCAO) group and MCAO with exercise group. Following three weeks of treadmill training exercise, ischemic stroke was induced by occluding the middle cerebral artery (MCA) in rat for 2h, followed by reperfusion. Twenty-four hours after MCAO/reperfusion, 12 rats in each group were evaluated for neurological deficit scores and then sacrificed to measure the infarct volume (n=6) and cerebral edema (n=6). Six rats in each group were sacrificed to measure the expression level of glutamate transporter-1 (GLT-1), protein kinase C-α (PKC-α), Akt, and phosphatidylinositol 3 kinase (PI3K) (n=6). Two hundred and eighty minutes (4.67h) after occlusion, six rats in each group were decapitated to detect the mRNA expression level of metabotropic glutamate receptor 5 (mGluR5) and N-methyl-d-aspartate receptor subunit type 2B (NR2B) (n=6).The results demonstrated that pre-ischemic treadmill training exercise reduced brain infarct volume, cerebral edema and neurological deficits, also decreased the over expression of PKC-α and increased the expression level of GLT-1, Akt and PI3K after ischemic stroke (p<0.05). The over-expression of mGluR5 and NR2B mRNA was also inhibited by pre-ischemic exercise (p<0.05). In summary, exercise preconditioning ameliorated brain damage after ischemic stroke, which might be involved in two signal pathways: PKC-α–GLT-1–Glutamate and PI3K/Akt–GLT-1–Glutamate.

Crosstalk between PKCα and Notch-4 in endocrine-resistant breast cancer cells.

The Notch pathway is functionally important in breast cancer. Notch-1 has been reported to maintain an estrogen-independent phenotype in estrogen receptor α (ERα)+ breast cancer cells. Notch-4 expression correlates with Ki67. Notch-4 also plays a key role in breast cancer stem-like cells. Estrogen-independent breast cancer cell lines have higher Notch activity than estrogen-dependent lines. Protein kinase Cα (PKCα) overexpression is common in endocrine-resistant breast cancers and promotes tamoxifen (TAM)-resistant growth in breast cancer cell lines. We tested whether PKCα overexpression affects Notch activity and whether Notch signaling contributes to endocrine resistance in PKCα-overexpressing breast cancer cells.Analysis of published microarray data from ERα+ breast carcinomas shows that PKCα expression correlates strongly with Notch-4. Real-time reverse transcription PCR and immunohistochemistry on archival specimens confirmed this finding. In a PKCα-overexpressing, TAM-resistant T47D model, PKCα selectively increases Notch-4, but not Notch-1, expression in vitro and in vivo. This effect is mediated by activator protein-1 (AP-1) occupancy of the Notch-4 promoter. Notch-4 knockdown inhibits estrogen-independent growth of PKCα-overexpressing T47D cells, whereas Notch-4IC expression stimulates it. Gene expression profiling shows that multiple genes and pathways associated with endocrine resistance are induced in Notch-4IC- and PKCα-expressing T47D cells. In PKCα-overexpressing T47D xenografts, an orally active γ-secretase inhibitor at clinically relevant doses significantly decreased estrogen-independent tumor growth, alone and in combination with TAM. In conclusion, PKCα overexpression induces Notch-4 through AP-1. Notch-4 promotes estrogen-independent, TAM-resistant growth and activates multiple pathways connected with endocrine resistance and chemoresistance. Notch inhibitors should be clinically evaluated in PKCα- and Notch-4-overexpressing, endocrine-resistant breast cancers.

Nanog expression is negatively regulated by protein kinase C activities in human cancer cell lines

Nanog is a transcription factor that is essential for the maintenance of pluripotency of the embryonic stem cells. Nanog has been shown to be expressed in various kinds of human tumors, suggesting a role in tumorigenesis. In this study, we found that Nanog expression was upregulated by inhibition of protein kinase C (PKC) activity in six human cancer cell lines examined. In a Nanog non-expressing human nasopharyngeal carcinoma cell line, NPC-076, Nanog mRNA level and protein level were both induced and dose-dependently promoted by exposure to PKC inhibitors. Knockdown experiments showed that PKCα and PKCδ were two subtypes exerted most of the effect. The reporter assay showed that Nanog promoter activity was promoted by exposure of the cells to PKC inhibitors and the effect was dependent on the presence of the Octamer-Sox composite element. The involvement of Octamer-Sox composite element was further supported by the observation that silencing of Oct4 and Sox2 in NPC-076 cells attenuated the effects of PKC inhibitors. In Nanog-expressing human embryonal carcinoma cell lines, NT2/D1 and NCCIT, Nanog expression was suppressed by exposure to PKC activator Phorbol-12-myristate-13-acetate (PMA). Further study showed that overexpression of PKCα elicited a repressive effect on Nanog expression in NT2/D1 cells. Consistently, mutation of the Octamer-Sox composite element abolished the suppressive effect by PKC activator. Nanog expression was of cellular significance in that ectopic expression in NPC-076 stimulated cell proliferation and knockdown of the endogenous Nanog expression in NT2/D1-suppressed cell proliferation.

Induction of TRIM22 by IFN-γ Involves JAK and PC-PLC/PKC, but Not MAPKs and pI3K/Akt/mTOR Pathways

Tripartite motif (TRIM) 22 plays an important role in interferons (IFNs)-mediated antiviral activity. We previously demonstrated that interferon regulatory factor-1 (IRF-1) played a central role in IFN-γ-induced TRIM22 expression via binding to a special cis-element named 5' extended IFN-stimulating response element (5'eISRE). In this study, we sought to identify the signaling pathways involved in TRIM22 induction by IFN-γ. By using various pharmacological inhibitors, it was found that the activity of tyrosine kinase and phosphatidylcholine-phospholipase C (PC-PLC), but not phosphatidylinositol-phospholipase C (PI-PLC) and phospholipase D (PLD), was required for IFN-γ-induced TRIM22 expression in HepG2 cells. Tyrosine kinase Janus kinase (JAK), not SRC and PYK2, played an indispensable role in TRIM22 induction. Inhibition of protein kinase C (PKC) activity also significantly attenuated IFN-γ induction of TRIM22. Although treatment with IFN-γ resulted in the stimulation of mitogen-activated protein kinases (MAPKs) (p38, ERK, and JNK) and pI3K/Akt/mTOR pathways in HepG2 cells, the inhibition of their activity did not affect IFN-γ-stimulated TRIM22 expression. Further studies showed that overexpression of JAK1 and PKCα activated TRIM22 promoter activity in a 5'eISRE-dependent manner, and inhibition of not only JAK but also PC-PLC/PKC pathways significantly attenuated IFN-γ-induced IRF-1 expression in HepG2 cells. Taken together, these data indicated that IFN-γ induced TRIM22 expression via activation of JAK and PC-PLC/PKC signaling pathways, which involved the cis-element 5'eISRE and the transactivator IRF-1.

Abstract 554: Differential response to retinoid treatment of normal and transformed murine mammary cells overexpressing protein kinase C (PKC) isoforms.

Abstract Some of the most promising signaling pathways for breast cancer treatment include the PKC family, involved in proliferation and apoptosis, and the retinoid system mainly involved in differentiation. In this work we have overexpressed α or δ PKC isoforms in two murine mammary models, a tumor-derived cell line (LM3) and a normal mammary gland-derived line (NMuMG), in order to analyze whether elevated expression of these kinases alter the sensitivity to retinoid treatment. The overexpression of α or δ PKC isozymes did not induce detectable morphological alterations either in LM3 or in NMuMG cell lines. In the transformed cell context, the overexpression of PKCα increased cell proliferation (population doubling time: 14.8±2.3 h vs 21.2±3.1 h in control cells p&amp;lt;0.05) and motility (wound coverage: 69.3±8.3% vs 41.2±5.1% in control cells p&amp;lt;0.05), alterations that could be associated with an aggressive in vivo tumor behavior. Interestingly, at the same time, PKCα sensitized LM3 cells to the effects of the All-Trans-Retinoic Acid (ATRA) as evidenced by a significant reduction in both parameters. On the contrary, PKCδ overexpression did not affect either LM3 cells in vitro behavior or their response to ATRA. However, through a RARE-luciferase gene reporter assay, we could determine that the overexpression of PKCδ increased the activity of the retinoic acid receptors. In the normal cell context (NMuMG cells), the overexpression of both PKCs did not induce alterations in the proliferative potential (population doubling time: 14.8±0.8 h) or cell motility (wound coverage: 37.8±1.6%). Moreover, the subsequent treatment with ATRA neither affected the above mentioned parameters. Upon othotopic inoculation into syngeneic mice, LM3-PKCα cells formed tumors with higher growth rate and metastatic potential than LM3-vector ones. In vivo treatment with a subcutaneous ATRA pellet reduced both the local tumor growth and the number of spontaneous lung metastases (Md [Range]: 9 [0-27] vs 55 [20-75] for LM3-PKCα treated or not respectively p&amp;lt;0.05). PKCδ overexpression did not affect in vivo LM3 behavior. Our results show that in an already transformed context, PKCα overexpression induced a more aggressive phenotype but also conferred sensitivity to the retinoid treatment. On the other side high PKCδ levels increased the activity of retinoid receptors but did not modulate biological cell responses to this compound. In a normal cell context, both PKC isoforms as well as the retinoid treatment are innocuous thus ensuring the safeness use of retinoids in patients with aggressive breast tumors associated with high PKCα expression. Citation Format: María I. Díaz Bessone, Damian E. Berardi, Stefano M. Cirigliano, Carolina Flumian, Elisa D. Bal de Kier Joffe, Laura B. Todaro, Alejandro J. Urtreger. Differential response to retinoid treatment of normal and transformed murine mammary cells overexpressing protein kinase C (PKC) isoforms. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 554. doi:10.1158/1538-7445.AM2013-554

Clinicopathological correlation and prognostic significance of protein kinase cα overexpression in human gastric carcinoma.

ObjectivesThis study investigated the PKCα protein expression in gastric carcinoma, and correlated it with clinicopathological parameters. The prognostic significance of PKCα protein expression in gastric carcinoma was analyzed.MethodsQuantitative real-time PCR test was applied to compare the PKCα mRNA expression in tumorous and nontumorous tissues of gastric carcinoma in ten randomly selected cases. Then PKCα protein expression was evaluated in 215 cases of gastric carcinoma using immunohistochemical method. The immunoreactivity was scored semiquantitatively as: 0 = absent; 1 = weak; 2 = moderate; and 3 = strong. All cases were further classified into two groups, namely PKCα overexpression group with score 2 or 3, and non-overexpression group with score 0 or 1. The PKCα protein expression was correlated with clinicopathological parameters. Survival analysis was performed to determine the prognostic significance of PKCα protein expression in patients with gastric carcinoma.ResultsPKCα mRNA expression was upregulated in all ten cases of gastric carcinoma via quantitative real-time PCR test. In immunohistochemical study, eighty-eight out of 215 cases (41%) of gastric carcinoma revealed PKCα protein overexpression, which was statistically correlated with age (P = 0.0073), histologic type (P<0.0001), tumor differentiation (P = 0.0110), depth of invasion (P = 0.0003), angiolymphatic invasion (P = 0.0373), pathologic stage (P = 0.0047), and distant metastasis (P = 0.0048). We found no significant difference in overall and disease free survival rates between PKCα overexpression and non-overexpression groups (P = 0.0680 and 0.0587). However, PKCα protein overexpression emerged as a significant independent prognostic factor in multivariate Cox regression analysis (hazard ratio 0.632, P = 0.0415).ConclusionsPKCα protein is upregulated in gastric carcinoma. PKCα protein expression is statistically correlated with age, histologic type, tumor differentiation, depth of invasion, angiolymphatic invasion, pathologic stage, and distant metastasis. The PKCα protein overexpression in patients with gastric carcinoma is a significant independent prognostic factor in multivariate Cox regression analysis.

Protein kinase Cα inhibitor protects against downregulation of claudin-1 during epithelial–mesenchymal transition of pancreatic cancer

Protein kinase Cα (PKCα) is highly expressed in pancreatic cancer. However, the effects of PKCα on Snail and claudin-1, which play crucial roles in epithelial cell polarity during epithelial-mesenchymal transition (EMT), remain unclear. In this study, we investigated the mechanisms of regulation of Snail and claudin-1 via a PKCα signal pathway during EMT in pancreatic cancer cells and in normal human pancreatic duct epithelial cells (HPDEs). By immunostaining, overexpression of PKCα and downregulation of claudin-1 were observed in poorly differentiated human pancreatic cancer tissues and the pancreatic cancer cell line PANC-1. Treatment with the PKCα inhibitor Gö6976 transcriptionally decreased Snail and increased claudin-1 in PANC-1 cells. The PKCα inhibitor prevented upregulation of Snail and downregulation of claudin-1 during EMT induced by transforming growth factor-β1 (TGF-β1) treatment and under hypoxia in PANC-1 cells. The effects of the PKCα inhibitor were in part regulated via an extracellular signal-regulated kinase (ERK) signaling pathway. The PKCα inhibitor also prevented downregulation of the barrier function and fence function during EMT in well-differentiated pancreatic cancer cell line HPAC. In normal HPDEs, the PKCα inhibitor transcriptionally induced not only claudin-1 but also claudin-4, -7 and occludin without a change of Snail. Treatment with the PKCα inhibitor in normal HPDEs prevented downregulation of claudin-1 and occludin by TGF-β1 treatment and enhanced upregulation of claudin-1, -4, -7 and occludin under hypoxia. These findings suggest that PKCα regulates claudin-1 via Snail- and mitogen-activated protein kinase/ERK-dependent pathways during EMT in pancreatic cancer. Thus, PKCα inhibitors may be potential therapeutic agents against the malignancy of human pancreatic cancer cells.

Abstract P6-04-24: Overexpression of protein kinase C alpha differentially activates transcription factors in T47D breast cancer cells in the presence of 17β-estradiol both in the 2D and 3D environments.

Abstract Background: Our lab has previously shown that overexpression of protein kinase C alpha (PKCα) results in a hormone independent, tamoxifen resistant phenotype in the T47D:A18 breast cancer cell line. Moreover, 17β-estradiol (E2) inhibits colony formation of T47D/PKCα in 3D Matrigel and tumor growth in vivo but not in the 2D environment (Zhang et al, Mol. Cancer Res, 2009). Differential transcriptional activation may account for the phenotypic changes observed in T47D/PKCα cells. In this study, we sought to identify transcription factors (TF) differentially activated in T47D:A18/neo versus T47D:A18/PKCα grown in 3D Matrigel in the presence and absence of E2, and compare these TFs with those activated in the 2D environment using a high-throughput screening (HTS) technology. Methods: A system for rapid, non-invasive, large scale, dynamic quantification of TF activity was developed (Weiss et al, PLoS ONE, 2010). Reporter constructs were created containing a TF binding site that preceeds a basal promoter to produce firefly luciferase (Fluc) using a lentiviral backbone. The control construct contained only the basal promoter and was used for normalization. T47D/PKCα and T47D/neo cells were infected with lentiviral reporter constructs. Cells were seeded either on top of Matrigel or without Matrigel in 384 well plates in E2-free media. After 3 days, cells were treated with either E2 (10–9M) or vehicle (0.1% ethanol). Fluc activity was assessed by bioluminescence imaging at 24, 48 and 72 hours following treatment using Xenogen IVIS Spectrum imaging system. Results: Initial findings showed differential transcriptional activities between the parental T47D/neo and T47D/PKCα cell lines at the basal level (without E2 treatment) in the 3D environment. Specifically, TFs involved in tumorigenesis (such as ETS1, STAT5) or the EMT process (such as SNAI1) were more highly activated in T47D/PKCα cells. Furthermore, E2 has a modulating effect on the activities of TFs in the two cell lines over time; observed both in the 2D and 3D environments. In particular, some TFs, such as Oct4, ETS1, and Stat5; were modulated (either induced or suppressed) by E2 only in T47D/PKCα cells. Conclusion: These findings suggest that overexpression of PKCα alters TF activity in T47D breast cancer cells both basally and in response to E2. This HTS technology allowed us to reveal the dynamic regulation of TF activation in PKCα overexpressing T47D breast cancer cells. We are now beginning to understand the mechanism whereby PKCα may mediate differential response to E2 dependent on the microenvironment. This HTS approach is likely to lead to the identification of new therapeutic targets. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-04-24.

Protein kinase Cα protects against multidrug resistance in human colon cancer cells.

Multidrug resistance is the phenomenon by which, after exposure to a single chemotherapeutic agent, cancer cells evade the agent's cytotoxic effects as well as become resistant to several classes of diverse drugs. ATP-binding cassette (ABC) transporters are a family of transporter proteins that contribute to drug resistance via a n ATP - dependent drug efflux pump. P-glycoprotein (P-gp) is a prominent ABC superfamily protein encoded by the mdr gene which has the ability to mediate the cellular extrusion of xenobiotics and anticancer drugs from tumor cells. Exclusively expressed P-gp cells from the human colon cancer HCT15/DOX line showed resistance to doxorubicin while parental HCT15 cells treated with doxorubicin displayed typical signs of apoptosis. In order to verify the hypothesis that expression of MDR is controlled in part, by protein kinase C (PKC), expression patterns of different PKC isoforms were examined in both cell lines. Of the PKC isoforms evaluated, the membrane translocation and expression levels of PKCα were strikingly increased in HCT15/DOX cells. PKCα reversed doxorubicin-induced apoptosis through the scavenging of ROS as well as inhibition of PARP cleavage. In addition, inhibition of PKCα with Go6976, a specific inhibitor of classical PKC, led to reduced MDR expression and increased doxorubicin-induced apoptosis. Knockdown of PKCα by siRNA diminished the protective effects of PKCα for doxorubicin-induced apoptosis. These results suggested that over-expression and activity of PKCα is closely associated with the regulation of the MDR phenotype in human colon cancer HCT15 cells and provided insight into a new strategy for inhibiting doxorubicin resistance in human cancers.

Abstract 172: Utility of retinoid treatment in the reversion of the malignant phenotype of mammary tumors with alterations in the expression of protein kinase C (PKC) isoforms

Abstract Some of the most promising signaling pathways for breast cancer treatment include the PKC family, involved in proliferation and apoptosis, and the retinoid system mainly involved in differentiation. In this work we have developed a murine model of mammary adenocarcinoma-derived cells (LM3) that overexpress α or ≤ PKC isoforms in order to analyze whether elevated levels of these kinases alter cell sensitivity to retinoid treatment (ATRA). LM3 cells are poorly responsive to ATRA. The overexpression of PKCα induced alterations in LM3 in vitro behavior that could be associated with tumor progression. In this sense, PKCα increased proliferation and motility (wound coverage: 69.3±8.3% vs 41.2±5.1% in control cells p&amp;lt;0.05) of the transfected LM3 cells. Interestingly, at the same time PKCα sensitized LM3 cells to ATRA treatment as evidenced by a significant reduction in both parameters. Upon othotopic inoculation into syngeneic mice LM3-PKCα cells formed tumors with higher growth rate and metastatic potential than LM3-vector cells. In vivo treatment with an ATRA pellet reduced both the local tumor growth and the number of spontaneous lung metastases (Md [Range]: 9 [0-27] vs 55 [20-75] for LM3-PKCα treated or not respectively p&amp;lt;0.05). On the contrary PKC ≤ overexpression did not affect LM3 cells behavior either in vitro or in vivo. LM3 cells response to ATRA was not modulated by PKC∈ either. However, through a RARE-luciferase gene reporter assay, we could determine that the overexpression of PKC∈ increased the activity of the retinoic acid receptors. Our results show that PKCα overexpression induced a more aggressive phenotype but also conferred ATRA sensitivity to mammary tumor cells. On the other side high PKC∈ levels increased the activity of retinoid receptors but did not modulate biological cell responses to retinoids. We believe that the use of retinoids may be beneficial in patients with aggressive breast tumors associated with high PKCα expression levels. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 172. doi:1538-7445.AM2012-172

Protein kinase Cδ but not PKCα is involved in insulin‐induced glucose metabolism in hepatocytes

The liver is a major insulin-responsive tissue responsible for glucose regulation. One important mechanism in this phenomenon is insulin-induced glycogen synthesis. Studies in our laboratory have shown that protein kinase Cs delta (PKCδ) and alpha (α) have important roles in insulin-induced glucose transport in skeletal muscle, and that their expression and activity are regulated by insulin. Their importance in glucose regulation in liver cells is unclear. In this study we investigated the possibility that these isoforms are involved in the mediation of insulin-induced glycogen synthesis in hepatocytes. Studies were done on rat hepatocytes in primary culture and on the AML-12 (alpha mouse liver) cell line. Insulin increased activity and tyrosine phosphorylation of PKCδ within 5 min. In contrast, activity and tyrosine phosphorylation of PKCα were not increased by insulin. PKCδ was constitutively associated with IR, and this was increased by insulin stimulation. Suppression of PKCδ expression by transfection with RNAi, or overexpression of kinase dead (dominant negative) PKCδ reduced both the insulin-induced activation of PKB/Akt and the phosphorylation of glycogen synthase kinase 3 (GSK3) and reduced significantly insulin-induced glucose uptake. In addition, treatment of primary rat hepatocytes with rottlerin abrogated insulin-induced increase in glycogen synthesis. Neither overexpression nor inhibition of PKCα appeared to alter activation of PKB, phosphorylation of GSK3 or glucose uptake in response to insulin. We conclude that PKCδ, but not PKCα, plays an essential role in insulin-induced glucose uptake and glycogenesis in hepatocytes.

Iptakalim, a novel ATP-sensitive potassium channel opener, inhibits pulmonary arterial smooth muscle cell proliferation by downregulation of PKC-α

Iptakalim is a new ATP-sensitive potassium (K ATP) channel opener, and it inhibits the proliferation of pulmo-nary arterial smooth muscle cells (PASMCs) and pulmonary vascular remodeling. However, the underlying mech-anism remains unclear. In the present study, we found that iptakalim significantly decreased pulmonary artery pressure, inhibited pulmonary ariery remodeling and PKC-α overexpression in chronic hypoxia in a rat pulmonary hypertension model. Iptakalim reduced hypoxia-iduced expression of PKC-α, and abolished the effect of hypoxia on PASMC proliferation significantly in a dose-dependent manner in vitro. Moreover, these effects were abol-ished by glibenclamide, a selective K ATP channel antagonist. These results indicate that iptakalim inhibits PASMC proliferation and pulmonary vascular remodeling induced by hypoxia through downregulating the expression of PKC-α. Iptakalim can serve as a novel promising treatment for hypoxic pulmonary hypertension.

RTVP-1 expression is regulated by SRF downstream of protein kinase C and contributes to the effect of SRF on glioma cell migration

Gliomas are characterized by increased infiltration into the surrounding normal brain tissue. We recently reported that RTVP-1 is highly expressed in gliomas and plays a role in the migration of these cells, however the regulation of RTVP-1 expression in these cells is not yet described. In this study we examined the role of PKC in the regulation of RTVP-1 expression and found that PMA and overexpression of PKCα and PKCε increased the expression of RTVP-1, whereas PKCδ exerted an opposite effect. Using the MatInspector software, we identified a SRF binding site on the RTVP-1 promoter. Chromatin immunoprecipitation (ChIP) assay revealed that SRF binds to the RTVP-1 promoter in U87 cells, and that this binding was significantly increased in response to serum addition. Moreover, silencing of SRF blocked the induction of RTVP-1 expression in response to serum. We found that overexpression of PKCα and PKCε increased the activity of the RTVP-1 promoter and the binding of SRF to the promoter. In contrast, overexpression of PKCδ blocked the increase in RTVP-1 expression in response to serum and the inhibitory effect of PKCδ was abrogated in cells expressing a SRFT160A mutant. SRF regulated the migration of glioma cells and its effect was partially mediated by RTVP-1. We conclude that RTVP-1 is a PKC-regulated gene and that this regulation is at least partly mediated by SRF. Moreover, RTVP-1 plays a role in the effect of SRF on glioma cell migration.

Inhibitory mechanism of pure curcumin on Wilms’ tumor 1 (WT1) gene expression through the PKCα signaling pathway in leukemic K562 cells

The aim of this study was to investigate the inhibitory mechanism of pure curcumin on WT1 expression in leukemic K562 cells. Pure curcumin suppressed WT1 expression, independent of effects on protein degradation or WT1 mRNA stability. Chromatin immunoprecipitation and reporter gene assays indicate that pure curcumin treatment attenuates WT1 auto-regulation. Interestingly, PKCα inhibition mimicks the repressive effects of pure curcumin in K562 cells. Conversely, myristoylated PKCα over-expression increased WT1 expression and reversed the inhibitory effect of pure curcumin. Our study indicates that pure curcumin attenuates WT1 auto-regulatory function through inhibition of PKCα signaling in K562 cells.

ERK1-independent α-secretase cut of β-amyloid precursor protein via M1 muscarinic receptors and PKCα/ε

The amyloid precursor protein (βAPP) undergoes several proteolytic cleavages. While β- and γ-secretases are responsible for the production of the 40–43 amino-acid long amyloid β peptide (Aβ), the α-secretase cut performed by the disintegrins ADAM10 and ADAM17, occurs in the middle of the Aβ sequence, thereby preventing its formation and leading to the secretion of the large sAPPα neuroprotective fragment. Here we showed that a series of M1 muscarinic receptor agonists dose-dependently stimulated sAPPα secretion without interfering with βAPP subcellular distribution. Carbachol- and PDBu-induced sAPPα secretions were blocked by the general PKC inhibitor GF109203X. We established that HEK293 and rhabdhomyosarcoma cells overexpressing constitutively active (CA) PKCα or PKCε secrete increased amounts of sAPPα while those expressing PKCδ were unable to modify sAPPα recovery. Conversely, the overexpression of PKCα or PKCε dominant negative (DN) constructs abolished PDBU-stimulated sAPPα secretion, whereas DN-PKCδ remained inert. In agreement, PKCα knockout lowered sAPPα recovery in primary cultured fibroblasts. We also demonstrated that the regulated α-secretase processing of βAPP is not controlled by the Extracellular- Regulated Kinase- 1/ MAP- ERK Kinase (ERK1/MEK) cascade and likely does not require ADAM17 phosphorylation on its threonine735 residue. Because the muscarinic-dependent α-secretase-like processing of PrP c is fully dependent on ADAM17 phosphorylation on its threonine735 residue by ERK1, these results indicate that a single extracellular signal triggers ADAM17-dependent regulated cleavages of βAPP and PrP c through distinct signalling cascades. This opens new potential therapeutic strategies aimed, in the context of Alzheimer's disease, at selectively activating ADAM17 towards βAPP without affecting the cleavages of its numerous other substrates.

Opposite Regulation of the Human Apolipoprotein M Gene by Hepatocyte Nuclear Factor 1 and Jun Transcription Factors

HDL is a negative risk factor for atherosclerosis because of its multiple atheroprotective functions. Inflammation converts HDL particles from anti-atherogenic to pro-atherogenic, and this transformation is associated with changes in HDL structure and composition. Apolipoprotein M (apoM) has been recently shown to play a role in the maturation of HDL in plasma and to protect from atherosclerosis. ApoM gene is expressed primarily in the liver and kidney and is down-regulated by pro-inflammatory signals. We now show that the human apoM promoter harbors a dual specificity regulatory element in the proximal region that binds hepatocyte nuclear factor 1 (HNF-1) and members of the AP-1 family of pro-inflammatory transcription factors (c-Jun and JunB). Overexpression of c-Jun or JunB repressed both the basal and the HNF-1-mediated transactivation of the human apoM promoter. Treatment of HepG2 cells with potent inflammation-inducing phorbol esters or overexpression of PKCα was associated with a marked inhibition of apoM gene expression in a c-Jun/JunB-dependent manner. We provide evidence for a novel mechanism of inflammation-induced transcriptional repression that is based on the competition between HNF-1 and Jun proteins for binding to the same regulatory region. A similar mechanism accounts for the down-regulation of the liver-specific apolipoprotein A-II gene by Jun factors. Our studies provide novel insights on the mechanisms that control the expression of liver-specific apolipoprotein genes during inflammation and could affect the maturation and the functionality of HDL particles.

Abstract 2339: Overexpression of PKCα in T47D breast cancer cells induces migration via p120-catenin transcriptional downregulation

Abstract Several factors have been identified that influence epithelial-mesenchymal transition (EMT) in breast cancer cells. We previously reported that overexpression of protein kinase C α (PKCα) in breast cancer predicts tamoxifen resistant secondary tumors (Tonetti, 2003). In T47D breast cancer cells, stable overexpression of PKCα (T47D/PKCα) leads to hormone-independent growth as well as tamoxifen resistance (Chisamore, 2001). In our current study we report that PKCα overexpression increases the metastatic potential of breast cancer cells through induction of a nonclasssical EMT. T47D/PKCα cells are morphologically distinct from T47D/neo cells and do not retain the cuboidal structure of epithelial breast cancer cells. Overexpression of PKCα resulted in significantly induced migratory capabilities in the Boyden chamber assay and PKC activation by phorbol 12-myristate 13 acetate further enhanced T47D/PKCα cell migration. Pharmacological inhibition with the classical PKC inhibitor Go6976 significantly reduced the migratory capacity of T47D/PKCα cells. Transient siRNA-mediated knockdown of PKCα (120 h) significantly reduced both basal and NIH3T3 fibroblast conditioned media-induced T47D/PKCα cell migration. Levels of adherens junction proteins E-cadherin, α-E-catenin, β-catenin and p120-catenin were significantly downregulated in T47D/PKCα cells compared to T47D/neo cells as determined by western blot. Levels of E-cadherin protein were restored by more than 50% after 168 h of PKCα knockdown in T47D/PKCα cells. While SYBR green RT-qPCR revealed that E-cadherin transcripts were significantly higher in T47D/PKCα cells compared to T47D/neo control cells, there was no change in expression of β-catenin or α-E-catenin transcripts. However, p120-catenin transcripts were significantly lower in T47D/PKCα cells compared to T47D/neo cells. Treatment with the proteasomal inhibitor MG132 induces accumulation of E-cadherin but not p120-catenin. Taken together, these data suggest that PKCα may be responsible for EMT in breast cancer cells through upstream signaling that leads to transcriptional inhibition of p120-catenin and subsequent degradation of E-cadherin protein. Further, PKCα expression may not only be predictive of tamoxifen-resistance and hormone-independent growth but also of increased potential for metastasis. 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 2339. doi:10.1158/1538-7445.AM2011-2339

Abstract 2301: E2-induced T47D:A18/PKCα tumor regression is accompanied by translocation of ERα to extranuclear sites in association with caveolin-1

Abstract Background: Acquired resistance to tamoxifen (TAM) is a serious clinical problem in the treatment of breast cancer patients. Our laboratory developed and characterized a TAM-resistant and 17 beta-estradiol (E2) independent preclinical model where PKCα was stably transfected into T47D:A18 cells compared with the E2-dependent T47D:A18/neo cells (Chisamore et al, 2001). We and others have shown that PKCα overexpression is associated with TAM resistance in cell lines and in the clinic (Tonetti et al, 2003, Assender et al, 2007). Additionally the T47D:A18/PKCα model exhibits E2-induced tumor regression in vivo or when grown in matrigel (3D matrix) but not on 2D plastic. Recently we have demonstrated that tumor regression involves participation of estrogen receptor alpha (ERα), Fas/FasL and the extracellular matrix (Zhang et al, 2009). Interestingly the membrane impermeable E2-BSA conjugate mediates growth inhibition of T47D:A18/PKCα colonies in matrigel similar to E2 suggesting a potential role of extranuclear ERα. On the basis of our preliminary data we investigated whether extranuclear ERα signaling pathway might play a critical role in E2-induced tumor regression. Materials and methods: Four micrometer thick sections were prepared from paraffin embedded T47D:A18/PKCα tumor tissues from untreated (control) and E2-treated mice and T47D:A18/neo tumors. Fluorescence tagged secondary antibodies were used for immunofluorescence staining. Co-immunoprecipitation (coIP) was done with control and E2 treatment group tumor extracts. Results: Immunofluorescence images of T47D:A18/PKCα tumor sections from untreated control mice showed that the ERα was mainly localized in the nucleus. Interestingly, treatment with E2 resulted in significant translocation of ERα from the nucleus to the cytoplasm and the plasma membrane of tumor cells. However in T47D:A18/neo tumors the majority of ERα was found in the nucleus suggesting the involvement of ERE mediated classical pathway in tumor growth. Confocal microscopy clearly indicates the colocalization of ERα with caveolin-1 in E2-treated T47D:18/PKCα tumor sections. Furthermore coIP performed with T47D:A18/PKCα tumor extracts revealed greater association of ERα with caveolin-1 in E2-treated tumors compared with control tumors. Discussion: We present evidence that E2-induced regression of our clinically relevant PKCα-overexpressing tumor model is accompanied by translocation of ERα to extranuclear sites in association with caveolin. This result suggests that E2-induced tumor regression is likely to be mediated by a membrane-associated signaling pathway. Therapies that stimulate the extranuclear ERα signaling pathway in PKCα overexpressing tumors may be an attractive approach to treat endocrine resistant tumors. 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 2301. doi:10.1158/1538-7445.AM2011-2301