Akt Cell Survival Research Articles

Novel Role of Raft-Associated Smoothened (SMO) in AKT Signal Regulation in Diffuse Large B Cell Lymphoma

Constitutive PI3K/AKT activation is relevant to multiple aspects of tumor growth and survival in numerous cancers including diffuse large B cell lymphoma (DLBCL). For example, PTEN loss is one of the mechanisms leading to constitutive PI3K/AKT activation in a subset of DLBCL. Smoothened (SMO) is a seven transmembrane spanning and Frizzled-class G-protein coupled receptor that functions as a Hedgehog (Hh) signal transducer. SMO is overexpressed in DLBCL cell lines and tumors. While canonical Hh signaling culminates in the activation of GLI transcription factors and is best understood in the context of cilia, "noncanonical" Hh signaling does not involve GLI transcriptional activity and remains less well characterized. Here, we found that SMO is not only an integral component of lipid rafts but also plays an unexpected central role in the organization of raft microdomains (specialized glycolipid-enriched microdomains known to serve as a highly dynamic signaling platform for cell surface receptors and signaling proteins) and in the sorting of lipid raft-associated proteins. To address whether SMO co-localizes to lipid rafts in the context of DLBCL, HBL1 cells were engineered to stably overexpress a C-terminal SMO-mCherry-fusion protein and were incubated with FITC-Cholera toxin. Within 15 min, a large fraction of SMO-mCherry was co-localized with FITC-Cholera toxin in lipid raft clusters. We also performed immunostaining of endogenous SMO and CD59 in HBL1 cells. CD59 is a glycol-phosphatidyl inositol-anchored lipid raft protein. Immunostaining of live HBL1 cells revealed the colocalization of SMO and CD59 on the extracellular surface of HBL1 cells. No colocalization was found between SMO and the transferrin receptor (TF-R), a plasma membrane protein not associated with lipid rafts. Finally, immunoblotting analysis of detergent-free fractionation further corroborated SMO as a bona fide component of lipid rafts. We then tried to explore the functional relevance of SMO association to the lipid compartment. First, we examined the effects of SMO stable knockdown in DLBCL. We observed a marked decrease in the expression of raft-associated receptors and signaling proteins (e.g. IGFR1, EGFR, IRS1) while caveolin and flotillin, two functional components of lipid rafts, remained unaltered in their levels and distribution. Although only a portion of the overall pool of AKT and pAKT were localized to lipid rafts, SMO loss significantly reduced raft-localized total AKT and pAKT (T308/S473). Consistent with the well-established role of AKT in cell survival, SMO silencing also resulted in reduced DLBCL cell viability. To evaluate whether SMO regulated IGF1R expression at the transcriptional level, we analyzed the mRNA levels of GLI1, the immediate transcriptional target of canonical SMO signaling, and IGF1R in SMO-/- MEFs. Even if GLI1 transcript levels were reduced, consistent with the established mode of GLI1 regulation by SMO through the GLI2 transcription factor, IGF1R mRNA levels remained unchanged. Using GLI1-/- MEFs, we could confirm that the effect of SMO on surface receptors was independent of canonical Hh signaling. We also determined that the rate of IGFR degradation was comparable in the presence and absence of SMO. Total IGF1R receptor levels at steady state represent the balance of total protein synthesis and the fraction of existing receptors that is directed towards lysosomal degradation. To test whether the absence of SMO results in increased degradation, we inhibited lysosomal function with chloroquine (CHLQ). CHLQ had a far more pronounced impact on restoring IGF1R protein levels in SMO deficient cells than it did in control cells. These last results suggested that in the absence of SMO, a larger fraction of this receptor is directed towards lysosomal degradation, and thus resulting in lower steady state levels. In summary, our data confirm that SMO is localized to raft microdomains in lymphoma cells and play a novel role in the sorting of surface proteins for degradation or recycling. In particular, SMO increases the levels of raft resident receptors and facilitates the assembly of an AKT activating machinery to enhance lymphoma cell survival. This novel role of SMO in signal regulation at the level of lipid rafts has broad implications for cancer biology. Disclosures Vega: National Cancer Institute, national Institutes of Health: Other: Grant Funding-R01CA222918.

Abstract 5163: Angiocrine induction of colorectal cancer cell HER3 activation and cell survival is independent of CRC mutational status

Abstract INTRODUCTION: Colorectal cancer (CRC) is the second-leading cause of cancer-related death in the United States. A better understanding of the regulation of CRC cell survival pathways is necessary for developing new therapeutic strategies for patients with metastatic CRC (mCRC). We have previously found that endothelial cells (ECs) from the liver, the most common site of CRC metastases, secrete soluble factors in their conditioned medium (CM) that, in turn, increase cell growth and chemoresistance of CRC cells. We also determined that EC-induced CRC cell survival is mediated, in part, by HER3/ERRB3 signaling. OBJECTIVES: In the current study we sought to determine if alterations in driver mutations mediate the role of liver ECs in activating HER3 signaling and promoting cell survival in CRC cells. METHODS: Primary ECs from non-malignant liver parenchyma were isolated as previously described (Wang et al., Mol Cancer Res., 2018). CRC cell lines with distinct molecular profiles (MSI status, and wildtype or mutant KRAS, BRAF, and PIK3CA) were incubated with either their own CM (control) or CM from ECs. The effects of CM on CRC cell growth and response to 5-fluorouracil (5-FU) was determined by MTT and colony formation assays. The effect of angiocrine signaling on chemoresistance was also determined by fluorescence-activated cell sorting (FACS) for apoptosis after treating CRC cells with 5-FU, either in control CM or EC CM. HER3 blockade was accomplished by 1) siRNA knockdown or 2) a HER3 antibody. RESULTS: CM from liver ECs significantly increased cell growth and chemoresistance in CRC cells regardless of the gene alteration profiles in in vitro assays. We confirmed that EC CM activated the HER3-AKT signaling pathway in CRC cells. Furthermore, we found that HER3 blockade attenuated EC CM-induced AKT activation and cell survival in CRC cells independent of the cell mutational profile. CONCLUSIONS: Our results demonstrate that HER3 mediates liver EC-induced cell growth and chemoresistance in CRC cells independent of the driver mutational profiles of the cells. The genetic background of CRC cells is not a mediator of EC CM-induced HER3 activation and cell survival. These findings suggest that effects of HER3 targeting of tumor cells in patients with mCRC should be independent of MSI status, or RAS, BRAF, or PIK3CA mutational status. Citation Format: Rui Wang, Rajat Bhattacharya, Fan Fan, Xiangcang Ye, Lee M. Ellis. Angiocrine induction of colorectal cancer cell HER3 activation and cell survival is independent of CRC mutational status [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5163.

Abstract 4198: mTORC2 directs breast morphogenesis through Rictor-dependent PKCα/Rac1 signaling independent of Akt

Abstract Akt phosphorylation is a major driver of cell survival, motility, and proliferation in development and disease, causing increased interest in upstream regulators of Akt like mTOR complex 2 (mTORC2). We used genetic disruption of Rictor to impair mTORC2 activity in mouse mammary epithelia, which decreased Akt phosphorylation, ductal length, secondary branching, cell motility, and cell survival. These effects were recapitulated with a pharmacological dual inhibitor of mTORC1/mTORC2, but not upon genetic disruption of mTORC1 function via Raptor knock-out. Surprisingly, Akt re-activation was insufficient to rescue survival, branching, or motility of mTORC2-impaired mammary epithelial cells (MECs). However, activation of the mTORC2 substrate protein kinase C (PKC)-α fully rescued branching, invasion, and survival of MECs after genetic or pharmacological mTORC2 inhibition. PKCα-mediated signaling through the small GTPase Rac1 was necessary for mTORC2-dependent mammary morphogenesis, revealing a novel role for Rictor/mTORC2 in survival and motility of untransformed MECs through an Akt-independent, PKCα/Rac1-dependent mechanism. Ongoing analyses of mammary specific Rictor-deletion in genetically engineered mouse models of breast cancer support a key role for mTORC2 in tumor progression and metastasis. Citation Format: Dana M. Brantley-Sieders, Meghan M. Morrison, Donna J. Hicks, Rebecca S. Cook. mTORC2 directs breast morphogenesis through Rictor-dependent PKCα/Rac1 signaling independent of Akt. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4198. doi:10.1158/1538-7445.AM2015-4198

Abstract B28: Rictor/mTORC2 drives formation, progression, and therapeutic resistance of HER2-amplified breast cancers

Abstract Although HER2-targeted therapies substantially improve the outcome for patients with HER2-amplified breast cancer, resistance to HER2 targeting is frequently observed. HER2-amplified tumors depend on the HER2-mediated activation of the phosphatidylinositol-3kinase (PI3K)/Akt pathway, which becomes reactivated in many therapeutically resistant HER2-amplified tumors. We investigated the mTOR complexes mTORC1 and mTORC2 as potential therapeutic targets in HER2-amplified breast cancers, based on their roles as activators (mTORC2) or effectors (mTORC1) of Akt. We found that genomic gains in Rictor, encoding a necessary mTORC2 cofactor, correlated with poor survival in breast cancer patients, while genomic gains in Raptor, an mTORC1 cofactor, did not. Rictor protein levels were increased in invasive breast cancers as compared to ductal carcinoma in situ (DCIS). Conditional Rictor gene ablation decreased growth, cell survival and invasion of human HER2-amplified breast cancer cells by blocking Akt signaling. Treatment of HER2 positive cells with the HER2/EGFR inhibitor Lapatinib caused Rictor upregulation. However, Rictor knockdown improved cell killing in Lapatinib-treated cells and impaired Akt signaling and cell survival in Lapatinib-resistant HER2 positive breast cancer cells. Together, these results demonstrate the pivotal role of mTORC2 in HER2-amplified breast cancers and support efforts to develop mTORC2-targeted therapies. Citation Format: Meghan M. Morrison, Bayley A. Jones, Donna J. Hicks, Violeta Sanchez, Valeria M. Estrada, Michelle M. Williams, Dana Brantley-Sieders, Rebecca S. Cook. Rictor/mTORC2 drives formation, progression, and therapeutic resistance of HER2-amplified breast cancers. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B28.

Abstract B49: mTORC2 directs breast morphogenesis through Rictor-dependent PKCα/Rac1 signaling independent of Akt

Abstract Akt phosphorylation is a major driver of cell survival, motility, and proliferation in development and disease, causing increased interest in upstream regulators of Akt like mTOR complex 2 (mTORC2). We used genetic disruption of Rictor to impair mTORC2 activity in mouse mammary epithelia, which decreased Akt phosphorylation, ductal length, secondary branching, cell motility, and cell survival. These effects were recapitulated with a pharmacological dual inhibitor of mTORC1/mTORC2, but not upon genetic disruption of mTORC1 function via Raptor knock-out. Surprisingly, Akt re-activation was insufficient to rescue survival, branching, or motility of mTORC2-impaired mammary epithelial cells (MECs). However, activation of the mTORC2 substrate protein kinase C (PKC)-α fully rescued branching, invasion, and survival of MECs after genetic or pharmacological mTORC2 inhibition. PKCα-mediated signaling through the small GTPase Rac1 was necessary for mTORC2-dependent mammary morphogenesis, revealing a novel role for Rictor/mTORC2 in survival and motility of untransformed MECs through an Akt-independent, PKCα/Rac1-dependent mechanism. Citation Format: Meghan M. Morrison, Christian D. Young, Shan Wang, Violeta M. Sanchez, Cook S. Rebecca, Donna J. Hicks, Dana M. Brantley-Sieders. mTORC2 directs breast morphogenesis through Rictor-dependent PKCα/Rac1 signaling independent of Akt. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B49.

ErbB3 plays a key role in the early phase of establishment of resistance to BRAF and/or MEK inhibitors

Background A major issue in the management of cancer is the development of drug resistance. In metastatic melanoma bearing V600 mutations in the BRAF oncogene, all patients undergo disease relapse after combination therapy with BRAF and MEK inhibitors. Hence, understanding the mechanisms at the basis of development of resistance is fundamental to the discovery of new therapeutic approaches. In our group we have spent the last years to identify mechanisms of early adaptation of BRAF mutated melanoma to BRAF and or MEK inhibitors. We have recently shown that the ErbB3 receptor is involved in the activation of an early feedback survival loop upon cell exposure to BRAF and/or MEK inhibitors. Upregulation of pErbB3, due to enhanced production of its ligand neuregulin-1 (HRG), causes increased AKT phosphorylation and cell survival. Furthermore, we demonstrated that activation of the ErbB3/AKT axis is abrogated by cotreatment with anti-ErbB3 mAbs previously generated in our laboratory.

Abstract 594: Dual regulation of cell death by Akt kinase inhibitor Mk-2206 in colorectal cancer.

Abstract Background: Colorectal cancer (CRC) is the second highest cause of cancer related deaths in the United States. Dissemination of CRC cells to distant organs (liver and lungs) often leads to treatment failure. Thus, the identification of novel strategies for the treatment of metastases is a pressing need in this type of cancer. Several reports have suggested the development of inappropriate cell survival signaling for various steps in the metastatic process and have noted the particular importance of aberrant cell survival to successful colonization at metastatic sites. We have earlier reported that Akt inactivation is associated with survivin and XIAP downregulation and increased cell death. Given the widespread role of Akt in cell survival and association of survivin and XIAP with CRC progression and metastasis, as well as other types of cancer, we hypothesize that Akt phosphorylation may be key to the aberrant survival mechanism permitting metastatic growth at distal organ sites. Thus, inactivation of Akt activity might be an important therapeutic target in CRC metastasis. Methods: Mk-2206 is a potent allosteric kinase inhibitor of Akt (provided by Merck). It inhibits the translocation of Akt to plasma membrane to prevent its phosphorylation and activation. IGF1R dependent CRC cell lines were treated with MK-2206 to study its anti-tumor effects in vitro and in vivo. Results: Mk-2206 promotes anti-tumor activity in the IGF1R dependent CRC cell lines and increases cell death both in vitro and in vivo. Mk-2206 inhibits Akt phosphorylation at S473 and T308 leading to decreased protein expression of XIAP and Survivin. Interestingly, Mk-2206 treatment upregulated apoptosis inducing factor (AIF) and its translocation from mitochondria to the nucleus and a concomitant inactivation of phospho-Ezrin at Y354 and T567.Ezrin is a cytoskeleton organizing protein implicated in the metastatic process of several other types of cancer in addition to CRC. RNAi studies were performed to confirm the role of AIF and Ezrin in Mk-2206 mediated cell death. We are currently analyzing the anti-metastatic effects of MK-2206 in an orthotopic mouse model of human CRC cells. Conclusion: In this work we have demonstrated the effectiveness of Mk-2206 as a pharmacological agent for targeting CRC cells. We have identified dual mechanisms by which Mk-2206-mediated Akt inactivation promotes cell death.The first mechanism involved caspase-independent AIF mediated DNA fragmentation; while the second involved caspase-dependent Akt-Ezrin-XIAP mediated cell death. Citation Format: Ekta Agarwal, Anathbandhu Chaudhuri, Michael G. Brattain, Sanjib Chowdhury. Dual regulation of cell death by Akt kinase inhibitor Mk-2206 in colorectal cancer. [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 594. doi:10.1158/1538-7445.AM2013-594 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 838: Distinct effects of S6K homologues on breast cancer cell survival.

Abstract The 40S ribosomal protein S6 kinase (S6K) acts downstream of the mammalian target of rapamycin (mTOR), which is an important target for cancer therapy. mTOR inhibitors are, however, of limited success, partly due to the activation of Akt caused by persistent inhibition of mTOR/p70 S6 kinase or S6K1 via a negative feedback loop. S6K exists as two homologs, S6K1 and S6K2. While both homologs are overexpressed in breast cancer, little is known about the function of S6K2. The objective of the present study is to determine whether S6K homologs perform redundant or distinct functions with respect to Akt activation and breast cancer cell survival. Silencing of S6K1 inhibited whereas knockdown of S6K2 potentiated cell death by various apoptotic stimuli, including tumor necrosis factor-alpha (TNF) and TNF-Related Apoptosis Inducing Ligand (TRAIL). In contrast to S6K1, depletion of S6K2 decreased Akt phosphorylation in MCF-7 and T47D cells. Ectopic expression of constitutively-active Akt in MCF-7 cells restored cell survival in S6K2-depleted cells. We have previously shown that activation of Akt induces downregulation of Bid via p53. Knockdown of S6K2 caused an increase in p53 and silencing of Bid blunted the ability of S6K2 deficiency to enhance TNF-induced apoptosis. Thus, our study demonstrates for the first time that the two homologs of S6K have distinct effects on Akt activation and cell survival and that S6K2 depletion enhanced cell death via the proapoptotic Bcl-2 family member Bid. Thus, targeting S6K2 may be an effective therapeutic strategy to treat cancers. (This work was supported by the grant CA71727 from the NCI). Citation Format: Savitha Sridharan, Alakananda Basu. Distinct effects of S6K homologues on breast cancer cell survival. [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 838. doi:10.1158/1538-7445.AM2013-838

Vorinostat, an HDAC inhibitor attenuates epidermoid squamous cell carcinoma growth by dampening mTOR signaling pathway in a human xenograft murine model

Histone deacetylase (HDAC) inhibitors are potent anticancer agents and show efficacy against various human neoplasms. Vorinostat is a potent HDAC inhibitor and has shown potential to inhibit growth of human xenograft tumors. However, its effect on the growth of skin neoplasm remains undefined. In this study, we show that vorinostat (2μM) reduced expression of HDAC1, 2, 3, and 7 in epidermoid carcinoma A431 cells. Consistently, it increased acetylation of histone H3 and p53. Vorinostat (100mg/kg body weight, IP) treatment reduced human xenograft tumor growth in highly immunosuppressed nu/nu mice. Histologically, the vorinostat-treated tumor showed features of well-differentiation with large necrotic areas. Based on proliferating cell nuclear antigen (PCNA) staining and expression of cyclins D1, D2, E, and A, vorinostat seems to impair proliferation by down-regulating the expression of these proteins. However, it also induced apoptosis. The mechanism by which vorinostat blocks proliferation and makes tumor cells prone to apoptosis, involved inhibition of mTOR signaling which was accompanied by reduction in cell survival AKT and extracellular-signal regulated kinase (ERK) signaling pathways. Our data provide a novel mechanism-based therapeutic intervention for cutaneous squamous cell carcinoma (SCC). Vorinostat may be utilized to cure skin neoplasms in organ transplant recipient (OTR). These patients have high morbidity and surgical removal of these lesions which frequently develop in these patients, is difficult.

Abstract B19: Cigarette smoke promotes human airway epithelial migration through temporal coordination of p120-catenin-dependent and -independent pathways

Abstract Background: Cigarette smoking has been linked to almost all major types of cancer. Emerging evidence suggests that smoking initiates transformed cell growth and migration by disrupting cell-cell interactions in the polarized mucosal epithelium. Together with other adherens junction proteins, p120-catenin (p120ctn) maintains cell-cell adhesion through its direct interaction with E-cadherin. Mislocalization and/or loss of p120ctn have been reported in all lung cancer subtypes and are related to poor prognosis. Our purpose was to investigate the initiating (intact p120ctn) and late (loss of 120ctn) events of smoke-promoted lung carcinogenesis using primary human bronchial epithelial (HBE) cells. Methods: Wide-type and p120ctn-knockdown HBE cells exposed to cigarette smoke-conditioned medium were used to mimic the early and late progressive stages of lung cancer. Signaling cascades and cell migration under the two conditions were investigated. Results: Src and Jnk activation by smoke were delayed in p120ctn-knockdown cells and inhibition of migration through the chemical blockade of EGFR, Src, and/or Jnk was dependent on p120ctn. In contrast, chemical inhibition of signaling via Akt (cell survival) or Erk (cell proliferation) was sufficient to block smoke-induced migration independent of p120ctn. Also independent of p120ctn was smoke's effect on the RhoGTPase, Rac1. Rac1 knockdown abolished dephosphorylation of actin binding protein, cofilin, in the presence and absence of p120ctn, indicating its essential role in smoke-induced cell migration. Conclusions: These studies provide unprecedented insight regarding the multiple and interdependent molecular strategies employed by smoke to escape tightly regulated cellular processes such as cell migration, proliferation, and survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B19.

Abstract 5248: Neuroblastoma tumors with different ALK mutations are sensitive to Perifosine

Abstract Background: Neuroblastoma (NB) is the most common extracranial solid tumor of children. Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase predominantly expressed in the developing nervous system. Recently, mutated ALK has been identified in association with familial and sporadic neuroblastoma (NB). Patients whose tumors contain amplification, mutation or express increased levels of activated ALK have a poor prognosis. Downstream targets of ALK include the STAT and Akt pathways. Perifoisine is an Akt inhibitor that has been reported to inhibit tumor growth in adult pre-clinical models. The aim of this study was to assess whether ALK mutations affect the response of NB cells to perifosine. Methods: We selected 4 NB cell lines with different ALK mutation status (SY5Y-ALK F1174L mutation; KCNR-ALK R1275Q mutation, AS and NGP with wild-type (wt) ALK. The effect of perifosine on Akt phosphorylation and cell survival was evaluated in these cell lines. In vivo studies were also performed in nude mice by subcutaneous injection of NB cells with or without ALK mutation. The tumor growth and phosphorylated Akt levels in tumor tissues were studied. Results: In vitro, treatment with perifosine induced decreases in Akt phosphorylation in all the 4 cell lines. SY5Y-ALK F1174L which is reported to be sensitive to ALK inhibitor TAE684, and KCNR-ALK R1275Q, AS-ALK wt and NGP-ALK wt which are reported to be resistant to ALK inhibitor TAE684, all responded to perifosine treatment. In vivo, perifosine treatment delayed or inhibited growth of tumors containing mutant or wild-type ALK. The phosphorylated Akt levels were lower in tumors treated with perifosine compared to tumors treated with control reagent. Conclusions: These data indicate that Perifosine is able to control NB tumor cell growth despite the mutational status of ALK. Perifosine may be a candidate treatment for NB tumors not only for those with wt ALK but especially for those that carry mutations that are resistant to ALK inhibitor treatment. 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 5248.

Differential regulation of TNF receptors by vagal nerve stimulation protects heart against acute ischemic injury

Vagal nerve stimulation (VS) has been reported to improve the survival after both acute and chronic myocardial infarction through the release of neurotransmitter ACh. However, the precise mechanism behind its beneficial effect is still unknown. In this study, we demonstrate the upregulation of tumor necrosis factor-alpha (TNF-α) and its cell survival TNF receptor-2 (TNFR2) as the mechanism behind VS induced myocardial protection.We investigated the effects of efferent VS on myocardial ischemic injury with in vivo and in vitro mouse models. In in vivo hearts VS significantly increased the expression of TNF-α both at the messenger and protein level after 3-hours of myocardial ischemia. In the in vitro studies ACh treatment before hypoxia, induced a significant upregulation of TNF-α compared to the untreated cardiomyocytes. Immunofluorescence analysis confirmed the synthesis of TNF-α by cardiomyocytes both in vivo and in vitro. VS also significantly reduced the myocardial infarct size (23.9±5.7% vs. 56±1.9%) and activated the cell survival Akt cascade system. Further, ACh upregulated the cell survival TNFR2 expression, while downregulating the cell destructive TNF receptor 1 (TNFR1) expression. These results were confirmed using the TNF receptors deficient mice, where the VS mediated protection was lost both in vivo and in vitro in TNFR2 (TNFR2-/-) and TNF receptors double knock out (TNFR1-/-2-/-) mice.VS and ACh protects the heart against acute ischemia or hypoxic injury by differentially regulating the TNF receptor subtypes.

Oleanane triterpenoid CDDO-Me inhibits growth and induces apoptosis in prostate cancer cells through a ROS-dependent mechanism

CDDO-Me, a synthetic triterpenoid derived from oleanolic acid, is a promising anticancer agent that has shown strong activity against a wide variety of cancer types in vitro and in vivo. We have previously shown that CDDO-Me induces apoptosis in prostate cancer cells irrespective of their hormonal status. To further understand the proapoptotic mechanism of CDDO-Me, we investigated the role of reactive oxygen species (ROS) in mediating the apoptosis inducing activity of CDDO-Me in LNCaP and PC-3 prostate cancer cell lines. Here, we show that CDDO-Me induces ROS generation from both nonmitochondrial and mitochondrial sources, which is associated with the induction of apoptosis as characterized by increased annexin V-binding, cleavage of PARP-1 and procaspases-3, -8, -9, loss of mitochondrial membrane potential and release of cytochrome c. In addition, CDDO-Me inhibited cell survival Akt, NF-κB and mTOR signaling proteins. The inhibition of ROS generation by N-acetylcysteine (NAC) or by overexpression of antioxidant enzymes glutathione peroxidase (GPx) and superoxide dismutase-1 (SOD-1) prevented CDDO-Me-induced apoptosis. Pretreatment with NAC blocked annexin V-binding, cleavage of PARP-1 and procaspases-3, -8, -9, loss of mitochondrial membrane potential and release of cytochrome c by CDDO-Me. NAC also prevented the inhibition of constitutively active Akt, NF-κB and mTOR by CDDO-Me. Together, these data indicate that ROS plays an essential role in the induction of apoptosis by CDDO-Me in prostate cancer cells.

AKT inhibitor, GSK690693, induces growth inhibition and apoptosis in acute lymphoblastic leukemia cell lines

AbstractThe PI3K/AKT signaling is activated in various hematologic malignancies. We evaluated the effect of a novel, pan-AKT kinase inhibitor, GSK690693, on the proliferation of 112 cell lines representing different hematologic neoplasia. Fifty-five percent of all cell lines tested were sensitive to AKT inhibitor (EC50 < 1 μM), with acute lymphoblastic leukemia (ALL), non-Hodgkin lymphoma, and Burkitt lymphoma showing 89%, 73%, and 67% sensitivity to GSK690693, respectively. The antiproliferative effect was selective for the malignant cells, as GSK690693 did not inhibit the proliferation of normal human CD4+ peripheral T lymphocytes as well as mouse thymocytes. Phosphorylation of downstream substrates of AKT was reduced in both sensitive and insensitive cell lines on treatment with GSK690693, suggesting that the cause of resistance was not related to the lack of AKT kinase inhibition. Consistent with the role of AKT in cell survival, GSK690693 also induced apoptosis in sensitive ALL cell lines. Overall, our data provide direct evidence for the role of AKT signaling in various hematologic malignancies, especially ALL and some lymphomas.

Abstract 5364: Vitamin B1 Analogue Benfotiamine Supplementation Prevents Diabetic Cardiomyopathy through Activation of eNOS/Akt Signaling Pathway

Cardiomyopathy (DCM) is increasingly recognized as contributing to mortality and morbidity of diabetic patients. Importantly, initial signs of cardiac dysfunction are detected in 75% of young patients with well controlled diabetes, suggesting the necessity for early prevention. Vitamin B1 is defective in diabetics and supplementation with vitamin B1 analogue Benfotiamine (BFT) has been demonstrated to be protective against experimental diabetic limb microangiopathy, nephropathy and retinopathy. Here, we tested whether supplementation of BFT could exhibit its protective effects against DCM. Streptozocin-induced CD1 diabetic (DM) mice were fed with BFT (70mg/kg/day) or vehicle starting from 2wks after glycosuria documentation. Cardiac function was monitored every month using Vevo high resolution echocardiography. 12wks after DM induction, the animals given vehicle started developing the signs of diastolic and systolic dysfunction which was more significant at 20wks of DM. However, BFT (without insulin supplement) markedly suppressed the DM induced left ventricular cavity dilatation (LV end-diastolic volume; 61±4 vs 74±5μl, p&lt;0.001), improved fractional shortening (40±4 vs 26±4%, P&lt;0.001) and ejection fraction (72±5 vs 54±4%, p&lt;0.001) and shifted pressure volume loop leftward compared with the vehicle treated animals. BFT also improved indexes of systolic and diastolic function (dp/dt max 5886±400 vs 4730±356mmHg/s and dp/dt min 3963±282 vs 3145±284mmHg/s, P&lt;0.05 for both comparison). Further, BFT benefited cardiac perfusion (4.5±0.4 vs 3.8±0.1ml/min/gm of tissue, P&lt;0.01, using fluorescent microspheres), which was supported by increased microvasculature (4332±321 vs 3654±263 capillaries/mm 2 ). Moreover, BFT (150μM) reduced the high glucose (30mM) induced apoptosis of cultured adult cardiomyocytes by 21±4% (P&lt;0.05) through activation of the cell survival Akt and eNOS pathway, suggesting its direct effect on cardiomyocytes. Altogether, these results demonstrate that BFT supplementation protects the heart from DM induced cardiac dilatation and dysfunction through activation of prosurvival mechanisms and preservation of myocardial perfusion. Thus, BFT merits attention for potential application in clinical practice.

AKT1, AKT2 and AKT3-dependent cell survival is cell line-specific and knockdown of all three isoforms selectively induces apoptosis in 20 human tumor cell lines

AKT is a key serine/threonine kinase in the PTEN/PI3K/AKT pathway1 and activation of AKT is often observed in human cancers. To explore the role of AKT in cell survival in different tumor cells, we tested 20 human tumor cell lines for response to knockdown of AKT by small interference RNA (siRNA) and/or a kinase-dead mutant AKT. siRNA-mediated knockdown of all three AKT isoforms in tumor cell lines led to a reduction of phosphorylation of AKT substrates. Knockdown of AKT resulted in apoptosis in 6 out of 11 tumor cells with activated AKT. In contrast, knockdown of AKT induced apoptosis in 3 out of 9 cell lines with a low level of active AKT. The responsiveness of the cells to knockdown of AKT was not affected by mutational status of p53 but appeared correlated with overexpression of HER2. To assess the role of individual AKT isoforms, five of the cell lines responsive to knockdown of AKT were further characterized. In ZR-75 cells, AKT1 is the predominant isoform responsible for cell proliferation and survival. Conversely, in IGROV1 cells, AKT2 plays a major role in cell proliferation, but no single isoform is essential for cell survival. Thus, the relative importance of the AKT isoforms is cell linespecific. Our data suggest that inhibiting all three AKT isoforms is necessary to elicit maximal apoptotic response in tumor cells, and the level of activated AKT is a favorable but not always reliable biomarker for pre-selection of responsive tumor cell lines to AKT inhibitors.

Marked suppression of tumor growth by FTY720 in a rat liver tumor model: the significance of down-regulation of cell survival Akt pathway.

We aim to investigate the anticancer effect of a novel immunomodulator FTY720 on a rat orthotopic liver tumor model. A buffalo rat orthotopic liver tumor model was established by injection of a buffalo hepatoma cell line MH7777 into the right portal vein. FTY720 was administered by intraperitoneal injection starting at 10 days after tumor cell injection at a dosage of 5 mg/kg/day. FTY720 markedly suppressed tumor growth and inhibited tumor progression by selective induction of apoptosis of tumor cells via down-regulation of phospho-Akt(ser473) and up-regulation of cleaved caspase-3, together with decrease of focal adhesion kinase. Moreover, the proliferation index of tumor cells was significantly reduced to 15.92+/-5.03% by FTY720 compared with that of 42.92+/-4.47% in the control group (p<0.001). In addition, we confirmed that FTY720 caused no effect on infiltrated lymphocyte in tumor tissue. We conclude that FTY720 is an effective anticancer agent for liver tumor in a rat model without affecting the immune system of the host.

Regulation of AKT in Richardson’s ground squirrels during hibernation

Hibernation is a winter survival strategy for many small mammals metabolic rate falls by >95%, core body temperature can approach 0°C, and all physiological functions are suppressed. Energy savings of ~90% are achieved compared with the costs of remaining euthermic (37°C) over the winter. The protein kinase Akt (or PKB) plays a central role in coordinating growth and survival responses in cells and we hypothesized that regulation of Akt would be critical in hibernation. Kinetic properties of muscle Akt were compared in euthermic vs hibernating states; total and phosphorylated Akt protein was also quantified by Western blotting. Total Akt protein did not change in hibernation but phospho-Akt (the active form) and measured Akt activity decreased significantly by 40% and 60%, respectively, compared with euthermic controls. S0.5 values for Akt peptide fell by 28% during hibernation whereas S0.5 ATP increased by 330%. Low temperature assay (10°C) strongly affected S0.5 ATP of euthermic Akt (a 350% increase). Akt activation energy (Ea) and sensitivity to denaturation by urea did not change between euthermia and hibernation. DEAE Sephadex chromatography showed three peaks (isozymes) of Akt in euthermic muscle but only two peaks during hibernation. The results document differential regulation of Akt during hibernation and suggest a key role for Akt in cell survival during torpor. Funded by NSERC Canada.

Retracted: Fat-Derived Hormone Adiponectin Combined with FTY720 Significantly Improves Small-for-Size Fatty Liver Graft Survival

Owing to the discrepancy between organ donation and the demand for liver transplantation, expanding the liver donor pool is of vital importance. However, marginal liver grafts, such as small-for-size and/or fatty grafts, were associated with primary graft nonfunction or poor function. Therefore, novel combination therapies to rescue small-for-size fatty liver grafts should be investigated. In this study, we applied a combination therapy using a fat-derived hormone adiponectin (anti-steatosis) plus immunomodulator FTY720 (anti-inflammatory) in a rat liver transplantation model using small-for-size fatty liver grafts, and investigated the underlying protective mechanism such as anti-steatosis, intra-graft energy metabolism, hepatic microcirculatory changes, cell signaling cascades for survival, apoptosis and inflammation. The current study demonstrated that even a single treatment of adiponectin or FTY720 improved the 7-day graft survival from 0% to 62.5% (p = 0.001). The combination therapy significantly increased the 7-day graft survival rate to 100% by remarkable attenuation of graft steatosis and acute phase inflammatory response, significant activation of cell survival Akt pathway and maintenance of intra-graft adenosine triphosphate metabolism and improvement of hepatic microcirculation. In conclusion, the fat-derived hormone adiponectin combined with FTY720 might be a novel combination drug therapy for prevention of small-for-size fatty liver graft injury.

FTY720 Attenuates Hepatic Ischemia-Reperfusion Injury in Normal and Cirrhotic Livers

Hepatic ischemia-reperfusion injury is an inevitable consequence during liver surgery. The outcome is particularly poor in cirrhotic livers, which are more prone to hepatic ischemia-reperfusion injury. We aim to study whether FTY720 could attenuate hepatic ischemia-reperfusion injury both in normal and in cirrhotic livers. We applied a 70% liver-ischemia (60 min) model in rats with normal or cirrhotic livers. FTY720 was given 20 min before ischemia and 10 min before reperfusion (1 mg/kg, i.v.). Liver tissues and blood were sampled at 20 min, 60 min, 90 min, 6 h and 24 h after reperfusion for detection of MAPK-Egr-1, Akt pathways and caspase cascade. Hepatic ultrastructure and apoptosis were also compared. FTY720 significantly improved liver function in the rats with normal and cirrhotic livers. Akt pathway was activated at 6 and 24 h after reperfusion. FTY720 significantly down-regulated Egr-1, ET-1, iNOS and MIP-2 accompanied with up-regulation of A20, IL-10, HO-1 and Hsp70. MAPK (Raf-MEK-Erk) pathway was down-regulated. Hepatic ultrastructure was well maintained and fewer apoptotic liver cells were found in the FTY720 groups. In conclusion, FTY720 attenuates ischemia-reperfusion injury in both normal and cirrhotic livers by activation of cell survival Akt signaling and down-regulation of Egr-1 via Raf-MEK-Erk pathway.