Shrikanth A. G. Reddy

The PI 3-kinase/Akt signaling pathway is activated due to aberrant Pten expression and targets transcription factors NF-κB and c-Myc in pancreatic cancer cells

The persistent activation of signaling cascades results in dramatic consequences that include loss of cellular growth control and neoplastic transformation. We show here that phosphoinositide 3-kinase (PI 3-kinase) and its mediator Akt were constitutively activated in pancreatic cancer and that this might be due to the aberrant expression of their natural antagonist MMAC/PTEN. Indeed, our results show that MMAC/PTEN expression was either lost or significantly reduced in five of eight cell lines and in twelve of seventeen tumor specimens examined. That the poor expression of MMAC/PTEN in pancreatic cancer cells could be due to promoter methylation was indicated by methylation-specific PCR analysis. Our studies also indicated that PI 3-kinase targeted two important transcription factors in pancreatic cancer cells. The ability of constitutively activated NF-κB to induce gene expression and the stabilization of c-MYC protein by decreased phosphorylation of Thr58 were both dependent on PI 3-kinase activity. When pancreatic cancer cells were treated with a peptide antagonist of NF-κB nuclear translocation, or stably transfected with a dominant-negative mutant of MYC, their proliferation was markedly inhibited. Taken together, these data indicate that the aberrant expression of MMAC/PTEN contributes to the activation of the PI 3-kinase/Akt pathway and its transcription factor mediators in pancreatic cancer.

Phosphatidylinositol 3-Kinase in Interleukin 1 Signaling PHYSICAL INTERACTION WITH THE INTERLEUKIN 1 RECEPTOR AND REQUIREMENT IN NFκB AND AP-1 ACTIVATION

The signaling mechanisms utilized by the proinflammatory cytokine interleukin-1 (IL-1) to activate the transcription factors NFκB and activator protein-1 (AP-1) are poorly defined. We present evidence here that IL-1 not only stimulates a dramatic increase in phosphatidylinositol 3-kinase (PI 3-kinase) activity but also induces the physical interaction of its type I receptor with the p85 regulatory subunit of PI 3-kinase. Furthermore, two PI 3-kinase-specific inhibitors, wortmannin and a dominant-negative mutant of the p85 subunit, inhibited IL-1-induced activation of both NFκB and AP-1. Transient transfection experiments indicated that whereas overexpression of PI 3-kinase may be sufficient to induce AP-1 and increase nuclear c-Fos protein levels, PI 3-kinase may need to cooperate with other IL-1-inducible signals to fully activate NFκB-dependent gene expression. In this regard, cotransfection studies suggested that PI 3-kinase may functionally interact with the recently-identified IL-1-receptor-associated kinase to activate NFκB. Our results thus indicate that PI 3-kinase is a novel signal transducer in IL-1 signaling and that it may differentially mediate the activation of NFκB and AP-1.

Curcumin is a non-competitive and selective inhibitor of phosphorylase kinase

Recently, we reported that curcumin (diferuloylmethane) inhibits the growth of several different kinds of tumor cells. In order to investigate the mechanism of this inhibition, we examined the effects of curcumin on different protein kinases: highly purified protein kinase A (PkA), protein kinase C (PkC), protamine kinase (cPK), phosphorylase kinase (PhK), autophosphorylation‐activated protein kinase (AK) and pp60c‐src tyrosine kinase. While all kinases tested were inhibited by curcumin, only PhK was completely inhibited at relatively lower concentrations. At around 0.1 mM curcumin, PhK, pp60c‐src, PkC, PkA, AK, and cPK were inhibited by 98%, 40%, 15%, 10%, 1%, and 0.5%, respectively. Lineweaver‐Burk plot analysis indicated that curcumin is a non‐competitive inhibitor of PhK with a K i of 0.075 mM. Overall, our results indicate that curcumin is a potent and selective inhibitor of phosphorylase kinase, a key regulatory enzyme involved in the metabolism of glycogen. This has important implications for the anti‐proliferative effects of curcumin.

Phosphatidylinositol 3-Kinase as a Mediator of TNF-Induced NF-κB Activation

The activation of transcription factor NF-κB by TNF involves the stimulation of a novel signaling cascade. In this paper we show that phosphatidylinositol 3-kinase (PI 3-kinase) may play a pivotal role in TNF-mediated activation of NF-κB-dependent genes. Consistent with its involvement in TNF signaling, PI 3-kinase activities in HepG2 and U937 cells can be stimulated by TNF in a rapid but transient manner through a mechanism that may involve its association with the insulin receptor substrate-1. A dominant-negative mutant of the p85 regulatory subunit of PI 3-kinase, which is a potent inhibitor of PI 3-kinase signaling, effectively blocked the TNF-induced expression of an NF-κB-dependent reporter gene. Although PI 3-kinase may be required for NF-κB activation, overexpression of its p110 catalytic subunit alone was unable to induce an NF-κB/chloramphenicol acetyltransferase (CAT) reporter gene. However, when TNF was added to p110-overexpressing cells, there was a synergistic activation of the NF-κB/CAT reporter, suggesting that other TNF-inducible signals may cooperate with PI 3-kinase to activate NF-κB. Consistent with its role in NF-κB activation, inhibition of PI 3-kinase activity by wortmannin or LY294002 greatly potentiated TNF-induced apoptosis. This TNF/wortmannin-induced apoptosis was markedly prevented in cells overexpressing Rel A. Taken together, our results indicate that a PI 3-kinase-regulated step in TNF-signaling is critical for the expression of NF-κB-dependent genes.

Inhibition of the mammalian target of rapamycin (mTOR) in advanced pancreatic cancer: results of two phase II studies

BackgroundThe phosphoinositide 3-kinase (PI3K)/Akt pathway is constitutively activated in pancreatic cancer and the mammalian target of rapamycin (mTOR) kinase is an important mediator for its signaling. Our recent in vitro studies suggest that prolonged exposure of pancreatic cancer cells to mTOR inhibitors can promote insulin receptor substrate-PI3K interactions and paradoxically increase Akt phosphorylation and cyclin D1 expression in pancreatic cancer cells (negative feedback loop). The addition of erlotinib to rapamycin can down-regulate rapamycin-stimulated Akt and results in synergistic antitumor activity with erlotinib in preclinical tumor models.MethodsTwo studies prospectively enrolled adult patients with advanced pancreatic cancer, Eastern Cooperative Oncology Group performance status 0-1, adequate hematologic, hepatic and renal parameters and measurable disease. In Study A, temsirolimus was administered intravenously at 25 mg weekly. In Study B, everolimus was administered orally at 30 mg weekly and erlotinib was administered at 150 mg daily. The primary endpoint in both studies was overall survival at 6 months. Secondary endpoints included time to progression, progression-free survival, overall survival, response rate, safety and toxicity. Pretreatment tumor biopsies were analyzed by immunofluorescence and laser scanning cytometry for the expression of pmTOR/mTOR, pAkt/Akt, pErk/Erk, pS6, p4EBP-1 and PTEN.ResultsFive patients enrolled in Study A; Two patients died within a month (rapid disease progression and hemorrhagic stroke, respectively). One patient developed dehydration and another developed asthenia. Sixteen patients enrolled in Study B.: 12 males, all ECOG PS = 1. Median cycles = 1 (range 1-2). Grade 4 toxicity: hyponatremia (n = 1), Grade 3: diarrhea (n = 1), cholangitis (n = 3), hyperglycemia (n = 1), fatigue (n = 1). Grade 2: pneumonia (n = 2), dehydration (n = 2), nausea (n = 2), neutropenia (n = 1), mucositis (n = 2) & rash (n = 2). Four patients were hospitalized. Progressive disease occurred in 15 and 1 was non-evaluable. Pretreatment biopsies revealed a higher pAkt/Akt ratio in tumor specimens that in nonmalignant pancreatic tissue. No such trends were noted for the other biomarkers.ConclusionsNeither study with mTOR inhibitors demonstrated objective responses or disease stability. The negative feedback loop resulting from mTOR inhibition may account for the disease progression and toxicity noted in these studies. Future strategies should aim for a broader targeting of the PI3K pathway in pancreatic cancer.Trial RegistrationTrial registration: Study A: NCT 0075647. Study B: NCT00640978

Alleviating the Suppression of Glycogen Synthase Kinase-3β by Akt Leads to the Phosphorylation of cAMP-response Element-binding Protein and Its Transactivation in Intact Cell Nuclei

Glycogen synthase kinase-3β (GSK-3β) activity is suppressed when it becomes phosphorylated on serine 9 by protein kinase B (Akt). To determine how GSK-3β activity opposes Akt function we used various methods to alleviate GSK-3β suppression in prostate carcinoma cells. In some experiments, LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (a kinase involved in activating Akt) and tumor necrosis factor-α (TNF-α) were used to activate GSK-3β. In other experiments mutant forms of GSK-3β, GSK-3βΔ9 (a constitutively active deletion mutant of GSK-3β) and GSK-3βY216F (an inactive point mutant of GSK-3β) were used to alter GSK-3β activity. LY294002, TNF-α, and overexpression of wild-type GSK-3β or of GSK-3βΔ9, but not GSK-3βY216F, alleviated the suppression of GSK-3β activity in prostate carcinoma cells and enhanced the turnover of β-catenin. Forced expression of wild-type GSK-3β or of GSK-3βΔ9, but not GSK-3βY216F, suppressed cell growth and showed that the phosphorylation status of GSK-3β can affect its intracellular distribution. When transcription factors activator protein-1 and cyclic AMP-response element (CRE)-binding protein were analyzed as targets of GSK-3β activity, overexpression of wild-type GSK-3β suppressed AP1-mediated transcription and activated CRE-mediated transcription. Overexpression of GSK-3βΔ9 caused an (80-fold) increase in CRE-mediated transcription, which was further amplified (up to 130-fold) by combining GSK-3βΔ9 overexpression with the suppression of Jun activity. This study also demonstrated for the first time that expression of constitutively active GSK-3βΔ9 results in the phosphorylation of CRE-binding protein on serine 129 and enhancement of CRE-mediated transcription in intact cell nuclei.

Tocotrienols inhibit AKT and ERK activation and suppress pancreatic cancer cell proliferation by suppressing the ErbB2 pathway.

Tocotrienols are members of the vitamin E family but, unlike tocopherols, possess an unsaturated isoprenoid side chain that confers superior anti-cancer properties. The ability of tocotrienols to selectively inhibit the HMG-CoA reductase pathway through posttranslational degradation and to suppress the activity of transcription factor NF-κB could be the basis for some of these properties. Our studies indicate that γ- and δ-tocotrienols have potent antiproliferative activity in pancreatic cancer cells (Panc-28, MIA PaCa-2, Panc-1, and BxPC-3). Indeed both tocotrienols induced cell death (>50%) by the MTT cell viability assay in all four pancreatic cancer cell lines. We also examined the effects of the tocotrienols on the AKT and the Ras/Raf/MEK/ERK signaling pathways by Western blotting analysis. γ- and δ-tocotrienol treatment of cells reduced the activation of ERK MAP kinase and that of its downstream mediator RSK (ribosomal protein S6 kinase) in addition to suppressing the activation of protein kinase AKT. Suppression of activation of AKT by γ-tocotrienol led to downregulation of p-GSK-3β and upregulation accompanied by nuclear translocation of Foxo3. These effects were mediated by the downregulation of Her2/ErbB2 at the messenger level. Tocotrienols but not tocopherols were able to induce the observed effects. Our results suggest that the tocotrienol isoforms of vitamin E can induce apoptosis in pancreatic cancer cells through the suppression of vital cell survival and proliferative signaling pathways such as those mediated by the PI3-kinase/AKT and ERK/MAP kinases via downregulation of Her2/ErbB2 expression. The molecular components for this mechanism are not completely elucidated and need further investigation.

Insulin Receptor Substrate Is a Mediator of Phosphoinositide 3-Kinase Activation in Quiescent Pancreatic Cancer Cells

Phosphoinositide 3-kinase (PI3K) is activated in pancreatic cancer cells and plays a central role in their proliferation, survival, and drug resistance. Although the mechanism is unclear, PI3K activation in these cells could be due to physical interaction between its regulatory subunit (p85) and specific tyrosine kinases or their mediators. Consistent with this possibility, PI3K was precipitated with anti-phosphotyrosine antibodies and Akt phosphorylation was blocked by the tyrosine kinase inhibitors SU6656 and PD158780 in quiescent pancreatic cancer cells. Pull-down assays with a fusion protein (GST-p85NC-SH2), and coimmunoprecipitation studies, indicated that the insulin receptor substrate (IRS), and not the epidermal growth factor and insulin-like growth factor receptors or the Src tyrosine kinase, was physically associated with PI3K in these cells. Our data also indicated that SU6656 and PD158780 inhibited Akt activation in pancreatic cancer cells by interfering with the ability of IRS-1 to recruit PI3K. Furthermore, IRS-1 was phosphorylated on a p85-binding site (Y(612)), and IRS-specific small interfering RNA potently inhibited activation of PI3K and Akt in transfected cells. Taken together, these observations indicate that IRS is a mediator of PI3K activation in quiescent pancreatic cancer cells.

Purification and Characterization of the Serum Amyloid A3 Enhancer Factor

Serum amyloid A (SAA) is a major acute-phase protein synthesized and secreted mainly by the liver. In response to acute inflammation, its expression may be induced up to 1000-fold, primarily as a result of a 200-fold increase in the rate ofSAA gene transcription. We showed previously that cytokine-induced transcription of the SAA3 gene promoter requires a transcriptional enhancer that contains three functional elements: two CCAAT/enhancer-binding protein (C/EBP)-binding sites and a third site that interacts with a constitutively expressed transcription factor, SAA3 enhancer factor (SEF). Each of these binding sites as well as cooperation among their binding factors is necessary for maximum transcription activation by inflammatory cytokines. Deletion or site-specific mutations in the SEF-binding site drastically reduced SAA3 promoter activity, strongly suggesting that SEF is important in SAA3 promoter function. To further elucidate its role in the regulation of the SAA3 gene, we purified SEF from HeLa nuclear extracts to near homogeneity by using conventional liquid chromatography and DNA affinity chromatography. Ultraviolet cross-linking and Southwestern experiments indicated that SEF consisted of a single polypeptide with an apparent molecular mass of 65 kDa. Protein sequencing and antibody supershift experiments identified SEF as transcription factor LBP-1c/CP2/LSF. Cotransfection of SEF expression vector with SAA3-luciferase reporter resulted in approximately a 5-fold increase in luciferase activity. Interestingly, interleukin-1 treatment of SEF-transfected cells caused dramatic synergistic activation (31-fold) of the SAA3 promoter. In addition to its role in regulating SAA3 gene expression, we provide evidence that SEF could also bind in a sequence-specific manner to the promoters of the α2-macroglobulin and Aα-fibrinogen genes and to an intronic enhancer of the human Wilm’s tumor 1 gene, suggesting a functional role in the regulation of these genes.

PTEN down regulates AP-1 and targets c-fos in human glioma cells Via PI3-kinase/Akt pathway

The continual activation of signaling cascades results in dramatic consequences that include loss of cellular growth control and neoplastic transformation. We show here that phosphoinositide 3-kinase and its mediator Akt was constitutively activated in glioma and that this might be due to the aberrant expression of their natural antagonist PTEN. The PTEN (phosphatase and tensin homologue deleted on chromosome ten) tumor suppressor gene modulates cell growth and survival through mechanisms that are incompletely understood. In this study, we investigated the possibility that PTEN mediates its effects through modulation of transcription factor AP-1, which is in part due to decrease in c-fos expression which was dependent on PI3kinase activity. Consistent with a reduction in the c-fos levels, an AP-1 dependent reporter gene was poorly induced in the PTEN expressing cell lines. In contrast to its effect on c-fos, PTEN did not affect the expression of c-Jun and other fos family members. We also show that the effect of PTEN on c-fos expression was due to its ability to antagonize PI3-kinase and could be mimicked by the expression of dominant negative Akt mutant. Taken together, these data indicate that the aberrant expression of PTEN contributes to the activation of the PI3kinase/Akt pathway and its transcription factor mediators in glioma. We conclude that the ectopic expression of PTEN down regulates the proliferation of glioma cells through the suppression of AP-1 and that this target might be essential for its central role in the growth and survival of glioma cancer cells.