Role Of Akt Research Articles

Akt1 Stimulates Homologous Recombination Repair of DNA Double-Strand Breaks in a Rad51-Dependent Manner.

Akt1 is known to promote non-homologous end-joining (NHEJ)-mediated DNA double-strand break (DSB) repair by stimulation of DNA-PKcs. In the present study, we investigated the effect of Akt1 on homologous recombination (HR)-dependent repair of radiation-induced DSBs in non-small cell lung cancer (NSCLC) cells A549 and H460. Akt1-knockdown (Akt1-KD) significantly reduced Rad51 protein level, Rad51 foci formation and its colocalization with γH2AX foci after irradiation. Moreover, Akt1-KD decreased clonogenicity after treatment with Mitomycin C and HR repair, as tested by an HR-reporter assay. Double knockdown of Akt1 and Rad51 did not lead to a further decrease in HR compared to the single knockdown of Rad51. Consequently, Akt1-KD significantly increased the number of residual DSBs after irradiation partially independent of the kinase activity of DNA-PKcs. Likewise, the number of residual BRCA1 foci, indicating unsuccessful HR events, also significantly increased in the irradiated cells after Akt1-KD. Together, the results of the study indicate that Akt1 seems to be a regulatory component in the HR repair of DSBs in a Rad51-dependent manner. Thus, based on this novel role of Akt1 in HR and the previously described role of Akt1 in NHEJ, we propose that targeting Akt1 could be an effective approach to selectively improve the killing of tumor cells by DSB-inducing cytotoxic agents, such as ionizing radiation.

Akt3 inhibits adipogenesis and protects from diet-induced obesity via WNK1/SGK1 signaling.

Three Akt isoforms, encoded by 3 separate genes, are expressed in mammals. While the roles of Akt1 and Akt2 in metabolism are well established, it is not yet known whether Akt3 plays a role in metabolic diseases. We now report that Akt3 protects mice from high-fat diet-induced obesity by suppressing an alternative pathway of adipogenesis via with no lysine protein kinase-1 (WNK1) and serum/glucocorticoid-inducible kinase 1 (SGK1). We demonstrate that Akt3 specifically phosphorylates WNK1 at T58 and promotes its degradation via the ubiquitin-proteasome pathway. A lack of Akt3 in adipocytes increases the WNK1 protein level, leading to activation of SGK1. SGK1, in turn, promotes adipogenesis by phosphorylating and inhibiting transcription factor FOXO1 and, subsequently, activating the transcription of PPARγ in adipocytes. Akt3-deficient mice have an increased number of adipocytes and, when fed a high-fat diet, display increased weight gain, white adipose tissue expansion, and impaired glucose homeostasis. Pharmacological blockade of SGK1 in high-fat diet-fed Akt3-deficient mice suppressed adipogenesis, prevented excessive weight gain and adiposity, and ameliorated metabolic parameters. Thus, Akt3/WNK1/SGK1 represents a potentially novel signaling pathway controlling the development of obesity.

Phosphorylated AKT expression in tumor-adjacent normal tissue is associated with poor prognosis in patients with hepatocellular carcinoma.

The AKT pathway serves important roles in tumor cell growth. Its overexpression is associated with poor prognosis in a number of types of cancer; however, the role of AKT in the role of the pathogenesis of hepatocellular carcinoma (HCC) remains unclear. The present study was undertaken to explore the clinical relevance of phosphorylated AKT (p-AKT) in HCC. The level of p-AKT in tumor (TU) and paired adjacent normal liver (AN) tissue from 202 HCC patients was evaluated with immunohistochemistry. The results demonstrated that p-AKT was more highly expressed in TU than in AN tissue. Kaplan-Meier curves and Cox regression revealed that patients with a high expression of p-AKT (AN) exhibited reduced overall and relapse-free survival times; this was not observed at a statistically significant level in p-AKT (TU). Additionally, the high expression of p-AKT (AN) was positively correlated with hepatitis C virus (HCV) infection in HCC patients. These results support the hypothesis that AKT activation is a mechanism of HCV-induced hepatocarcinogenesis, suggesting that AKT can be a therapeutic target for the treatment of recurrent HCC subsequent to surgical resection.

Abstract 4893: AKT1 signaling negatively regulates invasion and metastasis of non-small cell lung cancer cells carrying mutations in KRAS or EGFR

Abstract Signaling driven by the phosphoinositide-3 kinase (PI3K) and AKT/protein kinase B (PKB) pathway plays an important role in cancer development and progression, and thus this pathway is an attractive therapeutic target. Although many different inhibitors targeting either PI3K or AKT have been developed, the therapeutic benefit of these inhibitors in the clinic remains elusive, in part because of the perplexing role of AKT signaling in the regulation of cell motility, invasion and metastasis. AKT activation is known to promote migration, invasion and metastasis; however under certain circumstances and different cellular systems, it also exhibits an inhibitory activity on these cellular processes and the underlying cause of these conflicting results is unclear. Through analysis of the Reverse Phase Protein Array (RPPA) performed on a series of non-small cell lung cancer (NSCLC) brain metastasis models generated via intracardiac injection in mice, we identified AKT1 (one of three AKT family members) as a critical negative regulator of A549 tumor metastasis. AKT1 tyrosine phosphorylation (but not AKT2 or AKT3) inversely correlated with the metastatic potential of A549 cells which carry a KRAS mutation. Using AKT1 siRNAs, we demonstrated that inhibition of AKT1 significantly enhanced migration and invasion of KRAS- or EGFR-mutant NSCLC cells, but not wild type KRAS and EGFR cells. Similarly, the AKT inhibitor MK-2206 also enhanced the migration and invasion of KRAS- or EGFR-mutant NSCLC cells in vitro, and promoted metastasis of KRAS-mutant A549 cells in vivo. The PI3K-AKT pathway is downstream of both EGFR and KRAS, whose constitutive activation caused by mutations likely alter the signal output of PI3K-AKT pathway and thus the activity on cell invasiveness. Therefore, our results provide a sound explanation that under certain genetic backgrounds (as we demonstrated here with KRAS or EGFR mutations) the AKT signaling may switch from being a promoter to an inhibitor of cell migration, invasion and metastasis. This observation may have strong clinical implications for the development of inhibitors of this pathway for cancer treatment. Furthermore, we found that AKT1 blockade induced phosphorylation of Myristoylated alanine-rich C-kinase substrate (MARCKS) and elevation of Laminin subunit gamma-2 (LAMC2), which in turn inhibited AKT phosphorylation and promoted cell invasiveness. In contrast, MARCKS or LAMC2 knockdown abrogated the activities of migration and invasion induced by AKT1 inhibition. This study unravels an anti-metastatic role of AKT1 in NSCLC cells with KRAS or EGFR mutation, and provides mechanistic insights for the understanding of the potentially contrasting roles of AKT signaling in the regulation of cell invasiveness. Citation Format: Guanhua Rao, Mariaelena Pierobon, In-Kyu Kim, Wei-Hsun Hsu, Jianghong Deng, Yong-Wha Moon, Emanuel F. Petricoin, Yu-Wen Zhang, Yisong Wang, Giuseppe Giaccone. AKT1 signaling negatively regulates invasion and metastasis of non-small cell lung cancer cells carrying mutations in KRAS or EGFR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4893. doi:10.1158/1538-7445.AM2017-4893

Abstract 1368: RABL6A, a novel critical regulator of Akt-mTOR signaling in pancreatic neuroendocrine tumor cells

Abstract Introduction: A better molecular understanding of pancreatic neuroendocrine tumors (PNETs) is needed to improve patient diagnosis and treatment. Everolimus (mTOR inhibitor) is a standard-of-care therapy for PNET patients based on aberrant activation of the PI3K/Akt/mTOR kinase pathway in tumors. However, sustained mTOR inhibition paradoxically promotes Akt kinase hyperactivation due to loss of negative feedback regulation and tumors become drug resistant. Our data reveal that RABL6A, a novel oncoprotein amplified in PNETs, is a key regulator of this clinically relevant pathway. Methods: RABL6A and Akt protein levels were manipulated using viral shRNAs in BON1 PNET cells. Transcript levels were assayed by microarray and qRT-PCR, proteins assessed by western blotting, and cell proliferation and survival measured by cell counts, trypan blue exclusion and EdU incorporation. Effect of RABL6A expression on sensitivity to clinically relevant drugs, MK2206 (Akt inhibitor) and everolimus, were tested. Results: Silencing of RABL6A in PNET cells causes G1 and G2/M cell cycle arrest, and pathway analysis of microarray data suggested inactivation of Akt signaling in the arrested cells. Immunoblotting confirmed dramatic loss of Akt phosphorylation at Ser-473 along with impaired phosphorylation and activation of its targets, PRAS40 and FOXO-1/3. Phosphorylation of S6K, a downstream target of Akt-mTOR signaling, was also reduced by RABL6A deficiency. The mechanism by which RABL6A controls Akt-S473 phosphorylation is currently not known although we demonstrated that mTORC2 (the kinase that phosphorylates Akt at Ser473) remains active in RABL6A deficient cells since the phosphorylation of other mTORC2 substrates (SGK1 and PKCα) is unaffected. Given the central role of Akt1 in tumorigenesis, we hypothesized that reinstating its activity may rescue the arrest phenotype caused by RABL6A loss. Restoration of Akt1 in RABL6A-depleted cells partially rescued the G1 phase arrest and induced S phase entry but was insufficient to allow mitosis, suggesting RABL6A regulates other factors required for cell division. Finally, drug response assays showed that RABL6A loss desensitizes PNET cells to Akt and mTOR inhibitors. Conclusion: Our previous work showed RABL6A promotes G1 progression in PNET cells by inactivating Rb1, an established suppressor of PNET pathogenesis. We now show that RABL6A also controls Akt phosphorylation and is essential for Akt-mTOR activation. Thus, RABL6A controls multiple cancer pathways necessary for PNET cell cycle progression and survival. We are testing if RABL6A status in PNETs predicts responsiveness to combination therapies targeting Akt and mTOR. Overall, this work identifies RABL6A as a new essential activator of Akt1-mTOR signaling, suggesting it is a new potential biomarker and target for anticancer therapy in PNET patients. Citation Format: Shaik Amjad Ume Salma, Jussara Hagen, Jacki Reilly, Ryan Sheehy, Nitija Tiwari, Jackson Nteeba, Scott K. Sherman, Thomas M. O'Dorisio, James R. Howe, Andrew M. Bellizzi, Benjamin W. Darbro, Dawn E. Quelle. RABL6A, a novel critical regulator of Akt-mTOR signaling in pancreatic neuroendocrine tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1368. doi:10.1158/1538-7445.AM2017-1368

AKT2 is involved in the IL‑17A‑mediated promotion of differentiation and calcification of murine preosteoblastic MC3T3‑E1 cells.

Interleukin (IL)-17A exhibits pleiotropic biological activities and serves a role in the progression of periodontitis. However, data describing the association between IL-17 and osteogenesis are not conclusive. It was previously demonstrated that RAC-β serine/threonine protein kinase (AKT2)-specific knockdown in MC3T3-E1 cells weakened osteogenic effects. The role of AKT2 in the regulation of IL-17A for osteoblast differentiation and calcification remains unclear. The MTT method was adopted in the present study to assess cell proliferation; cell cycle distribution was analyzed by flow cytometry. Following osteogenic induction treatment, the involvement of phosphatidylinositol 3-kinase (PI3K) and phosphorylated-PI3K was evaluated by western blotting. The effects of IL-17A on osteogenesis-associated markers, including Runt-related transcription factor 2 (Runx-2), alkaline phosphatase (ALP) and osteocalcin (OCN) were evaluated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. An ALP activity assay and Alizarin Red S staining were used to assess the differentiation and calcification functions. AKT2 knockdown inhibited MC3T3-E1 cell proliferation, inducing significantly increased G0/G1 cell counts, and reduced S and G2/M cell numbers. IL-17A exerted no significant effects. The protein levels of p-PI3K, gene expression levels of IL-17A, Runx-2, ALP and OCN, and relative ALP activity and calcification areas were increased in the induction group, and these effects were markedly promoted by treatment with IL-17A. AKT2 knockdown in MC3T3-E1 cells resulted in reduced IL-17A-induced differentiation and calcification, although it was not completely inhibited. The results of the present study suggested that AKT2 signaling was required for MC3T3-E1 cell proliferation. IL-17A promoted osteoblast differentiation and calcification in a partly AKT2-dependent manner in MC3T3-E1 cells in vitro, possibly reflecting compensation by other signaling pathways. The results of the present study may offer novel perspectives to guide the clinical strategy for the prevention and treatment of periodontitis.

The Role of Akt in Chronic Liver Disease and Liver Regeneration

The liver is continuously exposed to diverse insults, which may culminate in pathological processes causing liver disease. An effective therapeutic strategy for chronic liver disease should control the causal factors of the disease and stimulate functional liver regeneration. Preclinical studies have shown that interventions aimed at maintaining Akt activity in a dysfunctional liver meet most of the criteria. Although the central function of Akt is cell survival, other cellular aspects such as glucose uptake, glycogen synthesis, cell-cycle progression, and lipid metabolism have been shown to be prominent functions of Akt in the context of hepatic physiology. In this review, the authors describe the benefits of the Akt signaling pathway, emphasizing its importance in coordinating proper cellular growth and differentiation during liver regeneration, hepatic function, and liver disease.

Hederagenin Supplementation Alleviates the Pro-Inflammatory and Apoptotic Response to Alcohol in Rats.

In this study, we determined the effects of hederagenin isolated from Akebia quinata fruit on alcohol-induced hepatotoxicity in rats. Specifically, we investigated the hepatoprotective, anti-inflammatory, and anti-apoptotic effects of hederagenin, as well as the role of AKT and mitogen-activated protein kinase (MAPK) signaling pathways in ethanol-induced liver injury. Experimental animals were randomly divided into three groups: normal (sham), 25% ethanol, and 25% ethanol + hederagenin (50 mg/kg/day). Each group was orally administered the respective treatments once per day for 21 days. Acetaldehyde dehydrogenase-2 mRNA expression was higher and alcohol dehydrogenase mRNA expression was lower in the ethanol + hederagenin group than those in the ethanol group. Pro-inflammatory cytokines, including TNF-α, IL-6, and cyclooxygenase-2, significantly increased in the ethanol group, but these increases were attenuated by hederagenin. Moreover, Western blot analysis showed increased expression of the apoptosis-associated protein, Bcl-2, and decreased expression of Bax and p53 after treatment with hederagenin. Hederagenin treatment attenuated ethanol-induced increases in activated p38 MAPK and increased the levels of phosphorylated AKT and ERK. Hederagenin alleviated ethanol-induced liver damage through anti-inflammatory and anti-apoptotic activities. These results suggest that hederagenin is a potential candidate for preventing alcoholic liver injury.

Abstract B07: Differential roles for AKT isoforms in BRAF mutant melanoma

Abstract Despite recent advances, melanoma remains the deadliest form of skin cancer, and new therapeutic strategies are urgently needed. The MAP kinase (MAPK) and PI3K/AKT pathways are central to both disease progression and chemoresistance in melanoma, with dual targeting approaches yielding promising results that would benefit from increased target specificity and selectivity. The AKT family of serine/threonine kinases comprises three highly homologous and functionally distinct isoforms (AKT1, AKT2, and AKT3) that play unique roles in melanoma but have not yet been leveraged for isoform-specific targeting. Our lab has engineered a unique mouse model of melanoma, in which mutant BRAFV600E is constitutively targeted to mouse melanocytes, resulting in spontaneous malignant disease upon additional loss of tumor suppressor genes, and faithfully recapitulates the human disease. We have identified differential and dynamic activation of AKT isoforms in our mouse melanoma model, which supports the hypothesis that AKT isoforms may play both non-redundant and compensatory roles in melanoma progression. To systematically address this question, we have genetically ablated each AKT isoform individually in melanoma-prone mice in order to elucidate the individual and unique contribution of each isoform to melanoma progression. In parallel, we performed in vitro studies using shRNA mediated conditional knockdown of AKTs in a wide variety of human melanoma cell lines. Our results support an isoform-specific role for AKT1 in promoting melanoma cell proliferation, while AKT2 stimulates cell migration. Further studies utilizing the precision genome editing technology CRISPR/Cas9 allows us to interrogate the effects of AKT isoform specific knockout (KO) on in vivo tumor formation. This work moves toward establishing the contribution of AKT isoforms to tumor progression in melanoma, using both human melanoma cell lines and mouse models, to advance our understanding of the role of AKT in melanomagenesis. With AKT inhibitors in clinical trials for melanoma, our findings could yield novel therapeutic strategies and increase the efficiency of existing therapies to improve treatment options and outcome for this devastating disease. Citation Format: Siobhan K. McRee, Jodie R. Pietruska, Elisabeth Dignan, Philip W. Hinds. Differential roles for AKT isoforms in BRAF mutant melanoma. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr B07.

Inhibition of JNK by cellular stress- and tumor necrosis factor α-induced AKT2 through activation of the NFκB pathway in human epithelial cells.

Previous studies have demonstrated that AKT1 and AKT3 are activated by heat shock and oxidative stress via both phosphatidylinositol 3-kinase-dependent and -independent pathways. However, the activation and role of AKT2 in the stress response have not been fully elucidated. In this study, we show that AKT2 in epithelial cells is activated by UV-C irradiation, heat shock, and hyperosmolarity as well as by tumor necrosis factor α (TNFα) through a phosphatidylinositol 3-kinase-dependent pathway. The activation of AKT2 inhibits UV- and TNFα-induced c-Jun N-terminal kinase (JNK) and p38 activities that have been shown to be required for stress- and TNFα-induced programmed cell death. Moreover, AKT2 interacts with and phosphorylates IκB kinase α. The phosphorylation of IκB kinase α and activation of NFκB mediates AKT2 inhibition of JNK but not p38. Furthermore, phosphatidylinositol 3-kinase inhibitor or dominant negative AKT2 significantly enhances UV- and TNFα-induced apoptosis, whereas expression of constitutively active AKT2 inhibits programmed cell death in response to UV and TNFα stimulation with an accompanying decreased JNK and p38 activity. These results indicate that activated AKT2 protects epithelial cells from stress- and TNFα-induced apoptosis by inhibition of stress kinases and provide the first evidence that AKT inhibits stress kinase JNK through activation of the NFκB pathway.

Cannabis and a lower BMI in psychosis: What is the role of AKT1?

Cannabis use has been associated with favorable outcomes on metabolic risk factors. The cause of this relation is still unknown. In this study we investigated whether this effect is mediated by the AKT1 gene, as activation of the related enzyme by cannabis may cause metabolic changes.Six Single Nucleotide Polymorphisms (SNPs) of the AKT1 gene (rs1130214, rs1130233, rs2494732, rs2498784, rs3730358, and rs3803300) of patients with psychotic disorders (n=623) were related to Body Mass Index (BMI), levels of glycosylated hemoglobin (HBA1c) and total metabolic risk. Next, mediation analysis was performed with BMI as outcome, cannabis as predictor, and AKT1 as mediator.Cannabis use was inversely related to BMI but not with levels of HBA1c and total metabolic risk. Moreover, out of 6 AKT1 SNPs, rs2494732 was associated with cannabis use, but AKT1 did not mediate the effect of cannabis on BMI.In conclusion, cannabis use is likely to be associated with a lower BMI in patients with a psychotic disorder. Moreover, AKT1 risk alleles may increase the incidence of cannabis use in patients with a psychotic disorder, but AKT1 does not appear to mediate the effect of cannabis on BMI.

Tert-butylhydroquinone lowers blood pressure in AngII-induced hypertension in mice via proteasome-PTEN-Akt-eNOS pathway.

Tert-butylhydroquinone (tBHQ), as an antioxidant, has been widely used for many years to prevent oxidization of food products. The aim of this study was to investigate whether tBHQ activates endothelial nitric oxide synthase (eNOS) to prevent endothelial dysfunction and lower blood pressure. The role of Akt in tBHQ-induced eNOS phosphorylation was examined in human umbilical vein endothelial cells (HUVEC) or in mice. tBHQ treatment of HUVEC increased both Akt-Ser473 phosphorylation, accompanied with increased eNOS-Ser1177 phosphorylation and NO release. Mechanically, pharmacologic or genetic inhibition of Akt abolished tBHQ-enhanced NO release and eNOS phosphorylation in HUVEC. Gain-function of PTEN or inhibition of 26S proteasome abolished tBHQ-enhanced Akt phosphorylation in HUVEC. Ex vivo analysis indicated that tBHQ improved Ach-induced endothelium-dependent relaxation in LPC-treated mice aortic arteries, which were abolished by inhibition of Akt or eNOS. In animal study, administration of tBHQ significantly increased eNOS-Ser1177 phosphorylation and acetylcholine-induced vasorelaxation, and lowered AngII-induced hypertension in wildtype mice, but not in mice deficient of Akt or eNOS. In conclusion, tBHQ via proteasome-dependent degradation of PTEN increases Akt phosphorylation, resulting in upregulation of eNOS-derived NO production and consequent improvement of endothelial function in vivo. In this way, tBHQ lowers blood pressure in hypertensive mice.

Abstract 188: Isoform-specific AKT functions in thyroid carcinoma development

Abstract Mammalian cells contain three genes that encode three AKT isoforms (AKT1-3). The three genes have >85% sequence identity, and possess conserved threonine and serine residues that are critical for full activation. All AKT isoforms seem to have identical or similar substrate specificity, and it is not clear whether they possess different functional specificities in vivo. Using conditional, organ-specific isoform deletion in a model of PI3K-dependent neoplastic transformation and progression, we are dissecting the molecular and biological roles of AKT1 and AKT2. Thyroid-specific deletion of Pten (Ptenthy-/− mice) results in thyroid hyperplasia at birth which then slowly progresses to solid nodular lesions and finally to invasive carcinomas. Simultaneous thyroid-specific deletion of Akt1 and/or Akt2 demonstrates that both AKT1 and AKT2 are necessary to confer proliferative ability to Pten−/- thyrocytes in an additive manner. However, with age, (Pten,Akt1)thy-/− mice have a significantly higher incidence of thyroid lesions compared to (Pten,Akt2)thy-/− mice. In addition, deletion of Akt2 is more effective than that of Akt1 to extend survival and reduce lesion aggressiveness in Ptenthy-/−/KrasG12D mice. Transcriptomic and proteomic analyses have revealed that AKT2 loss rescues most of the expression and activation changes induced, in vivo, by Pten deletion, while AKT1 loss has more restricted effects. Futhermore we have identified and are characterizing subsets of genes that are differentially regulated by AKT isoforms. These data point to the existence of dramatically different molecular pathways associated with each AKT isoform in the thyroid, and represent a strong rationale for the development of less toxic, more effective treatments based on isoform-specific inhibitors. Citation Format: Michela Ranieri, Antonio Di Cristofano. Isoform-specific AKT functions in thyroid carcinoma development. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 188.

Abstract 4617: Targeting mTORC1 by overexpression of PRAS40(T246A) in basal keratinocytes suppresses proliferation and migration of keratinocyte stem cells and skin inflammation during tumor promotion

Abstract Considerable evidence indicates a crucial role for Akt and mTORC1 signaling in cancer. Previously, we discovered that the Akt and mTORC1 signaling pathways play an important role in epithelial multistage carcinogenesis using the mouse skin model, especially during the tumor promotion stage. The proline-rich Akt substrate of 40 kDa (PRAS40), a subunit of mTORC1 was originally identified as a novel Akt substrate through Akt-mediated phosphorylation at Thr246 and this phosphorylation is critical for mTORC1 activity. Moreover, PRAS40 has been identified to negatively regulate mTORC1 signaling. In this study, transgenic mice were generated that overexpress a mutant form of PRAS40 [PRAS40(T246A)] using the bovine keratin 5 (BK5) promoter to further examine the importance of mTORC1 signaling in epithelial carcinogenesis. BK5.PRAS40(T246A) transgenic mice did not have a discernable gross skin phenotype. However, these mice were significantly less sensitive to TPA-induced epidermal hyperproliferation and hyperplasia. Overexpression of PRAS40(T246A) in the epidermis led to significant suppression of skin tumor development and significant reductions in tumor weight and tumor size compared to wild-type mice. Immunoprecipitation analysis of epidermal protein lysates from BK5.PRAS40(T246A) mice showed that PRAS40(T246A) remained bound to raptor even after TPA treatment. BK5.PRAS40(T246A) mice also displayed reduced mTORC1 signaling, reduced levels of cell cycle proteins and increased autophagy signaling as well as the skin inflammatory response in the epidermis following treatment with TPA. Notably, TPA-induced proliferation and migration of bulge-region label retaining cells were also significantly inhibited in BK5.PRAS40(T246A) mice. In addition, expression of PRAS40(T246A) in basal keratinocytes significantly inhibited keratinocyte migration both in vivo and in vitro and led to delayed wound healing. Decreased migration and impaired wound healing may be attributed to altered expression of EMT markers as identified in PRAS40(T246A) epidermal cells. The current data using this novel mouse model provides further evidence that mTORC1 specific signaling in keratinocytes contributes significantly to the process of skin tumor promotion by regulating proliferation and migration of keratinocyte stem cells. In addition, the data demonstrate an important role for mTORC1 specific signaling in keratinocytes in regulating skin inflammation. Collectively, using this novel mouse model where mTORC1 signaling is selectively suppressed in keratinocytes has provided further evidence that this signaling pathway regulates several important mechanistic events involved in the process of skin tumor promotion. Research supported by NIH Grants CA129409, CA037111, and American Cancer Research Center and Foundation Citation Format: Okkyung Rho, Jaya Srivastava, Jiyoon Cho, John DiGiovanni. Targeting mTORC1 by overexpression of PRAS40(T246A) in basal keratinocytes suppresses proliferation and migration of keratinocyte stem cells and skin inflammation during tumor promotion. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4617.

Lack of AKT in adipocytes causes severe lipodystrophy.

ObjectiveAdipose depot mass is tightly regulated to maintain energy homeostasis. AKT is a critical kinase in the insulin-signaling cascade that is required for the process of adipogenesis in vitro. However, the role of AKT in the maintenance and/or function of mature adipocytes in vivo had not been examined.MethodsTo study this, we deleted Akt1 and Akt2 in adipocytes of mice using the AdipoQ-Cre driver.ResultsStrikingly, mice lacking adipocyte AKT were severely lipodystrophic, having dramatically reduced gonadal adipose and no discernible subcutaneous or brown adipose tissue. As a result, these mice developed severe insulin resistance accompanied by fatty liver, hepatomegaly and with enlarged islets of Langerhans.ConclusionsThese data reveal the critical role of adipocyte AKT and insulin signaling for maintaining adipose tissue mass.

Inhibition of AKT sensitizes chemoresistant ovarian cancer cells to cisplatin by abrogating S and G2/M arrest

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway is frequently altered in human malignancies and Akt over-expression and/or activation induces malignant transformation and chemoresistance. However, the role of Akt in the mechanisms of chemoresistance remains elusive. Here we reported that cisplatin treatment of chemosensitive, but not resistant, ovarian cancer cells (OVCAs) markedly increased the cell proportion in sub-G1 phase. Cisplatin however caused a significant accumulation of the resistant cells in S and G2/M phases, which was associated with a rapid and sustained checkpoint kinase 1 (Chk1) activation. In contrast, while cisplatin also elicited a rapid activation of Chk1 in sensitive cells, it markedly decreased total ChK1 and phospho-Chk1 contents over 12 h. Over-expression of dominant negative (DN)-AKT alone increased phospho-Chk1 content, and induced G2/M arrest and apoptosis. However, it inhibited Chk1 activation and G2/M arrest with combination of cisplatin treatment, resulting in p53-independent apoptosis. Furthermore, the responses of the chemoresistant cells to cisplatin were attenuated with forced expression of constitutive active AKT2. Chk1 knock-down also facilitated cisplatin-induced apoptosis in chemoresistant cells. Our studies implicate that, in addition to its cell survival and anti-apoptotic actions, Akt might also play an important role in the regulation of G2-M transition, possibly via up-regulation of Chk1 activity and stability. These data provide strong support for the concept that Akt is important in cell cycle regulation in the control of chemosensitivity in OVCAs and offers an alternate regulatory pathway for the development of rationale therapy for cisplatin-resistant ovarian cancer.

Differential metformin dose-dependent effects on cognition in rats: role of Akt.

Epidemiological evidence suggests that individuals with diabetes mellitus are at greater risk of developing Alzheimer's disease, and controversy overwhelms the usefulness of the widely prescribed insulin-sensitizing drug, metformin, on cognition. Through the scopolamine-induced memory deficit model, we investigated metformin influence on cognitive dysfunction and explored underlying mechanisms. Sixty adult male Wistar rats were randomly assigned into 5 groups (12 rats each) to receive either normal saline, scopolamine 1mg/kg intraperitoneally once daily, scopolamine + oral metformin (100mg/kg/day), scopolamine + oral metformin (300mg/kg/day) or scopolamine + oral rivastigmine (0.75mg/kg/day) for 14days. Cognitive behaviours were tested using Morris water maze and passive avoidance tasks. Biochemically, brain oxidative (malondialdehyde) and inflammatory (TNF-α) markers, nitric oxide, Akt, phospho-Akt, phospho-tau and acetyl cholinesterase activity in hippocampal and cortical tissues were assessed. The lower dose of metformin (100mg/kg) ameliorated scopolamine-induced impaired performance in both Morris water maze and passive avoidance tasks, and was associated with significant reduction of inflammation and to a lesser extent oxidative stress versus rivastigmine. Given the role of total Akt in regulation of abnormal tau accumulation and degradation, our finding that metformin 100 decreased the elevated total Akt while increasing its phosphorylated form explains its beneficial modulatory effect on phosphorylated tau in both tissues, and could further clarify its protection against memory impairment. Metformin, only in the average human antidiabetic dose, offers a protective effect against scopolamine-induced cognitive impairment, while no deleterious effect was observed with the higher dose, which may support a bonus effect of metformin in type 2 diabetic patients.

Abstract B39: PI3K therapy-induced adaptive mitochondria reprogramming fuels metastatic traits

Abstract Tumors rewire cell metabolism and autophagy to adapt to environmental stress conditions, including hypoxia, starvation and oxidative stress. Such adaptive reprogramming includes a switch from oxidative phosphorylation to glycolysis, use of glutamine as fuel instead of glucose and increased biosynthesis of lipids and nucleotides. Although these processes promote resistance to apoptosis and support biomass expansion, they are also energetically inefficient, making it unclear how transformed cells accomplish highly energy-demanding tasks, including invasion and metastasis. Scattered evidences suggest that mitochondrial oxidative phosphorylation may be important for cancer traits, including stemmness, tumor repopulation and drug resistance. We recently demonstrated that targeting of the universal cancer node phosphatidylinositol-3 kinase (PI3K) induces extensive bioenergetics and transcriptional reprogramming in tumor cells, paradoxically culminating with preservation of homeostatic mitochondrial bioenergetics, opposing permeability transition and tumor cell death. Here we sought to understand how tumors adapt to molecular therapy as it pertains to tumor progression. Our results show that PI3K inhibitors used in the clinic induce global changes in activation of growth factor receptor kinases, reactivation of Akt and mTOR signaling, and dramatically increased tumor cell invasion. This involves the trafficking of energetically active mitochondria to the cortical cytoskeleton of tumor cells, supporting membrane lamellipodia dynamics, heightened turnover of focal adhesion complexes and increased velocity and distance of random cell migration. Furthermore, a single dose of PI3Ki increased liver metastasis after intrasplenic-injection of tumor cells in mice. Mechanistic studies pinpoint the role of Akt, mTOR, mitochondrial Hsp90s, the autophagy initiating complex ULK1/FIP200 and focal adhesion kinase (FAK) in the adaptive response to PI3Ki. Blocking oxidative phosphorylation by targeting mtHsp90s prevented the repositioning of mitochondria to sites of cell movements, impaired membrane dynamics and suppressed tumor cell invasion. Our results suggest a potential drawback for PI3K therapy, namely the increased risk of metastasis in patients. Targeting mitochondrial reprogramming is feasible, and could provide a novel therapeutic strategy to limit disease dissemination. Note: This abstract was not presented at the conference. Citation Format: M. Cecilia Caino, Dario C. Altieri. PI3K therapy-induced adaptive mitochondria reprogramming fuels metastatic traits. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B39.

Upregulated ATF6 contributes to chronic intermittent hypoxia-afforded protection against myocardial ischemia/reperfusion injury.

In the present study, we investigated the role of activating transcription factor 6 (ATF6) in the mechanism by which chronic intermittent hypoxia (CIH) increases tolerance to myocardial ischemia/reperfusion (I/R). Experiments were conducted using a rat model of I/R injury in vivo and isolated Langendorff-perfused rat hearts ex vivo. The role of Akt in this process was also investigated in vitro using rat myoblast H9c2 cells. Cell viability was measured using a cell counting kit-8 assay. Lactate dehydrogenase (LDH) and creatine kinase cardiac isoenzyme activity were also measured as markers of cellular damage. ATF6, Akt and phosphorylated (p)-Akt expression was analyzed by western blot analysis. RNA interference (RNAi) was used to suppress ATF6 expression. We noted that ATF6 expression in the ventricular myocardium was significantly increased in rats exposed to CIH. Furthermore, we noted that CIH preserved cardiac function after I/R in vivo and improved post-ischemic recovery of myocardial performance in isolated rat hearts. ATF6 and p-Akt expression was upregulated in cultured H9c2 cells exposed to chronic mild hypoxia compared with those cultured under normoxic conditions. Chronic mild hypoxia attenuated subsequent simulated I/R injury in H9c2 cells (48 h), as evidenced by increased cell viability and decreased LDH activity. By contrast, decreased cell viability and increased LDH activity were observed in siRNA-ATF6-transfected H9c2 cells, with a concomitant reduction in p-Akt levels. These results indicated that ATF6 upregulation is involved in the mechanism by which CIH attenuates myocardial I/R injury, possibly through upregulation of p-Akt, which is a key regulator of cardiomyocyte survival.

Overexpression of the rice AKT1 potassium channel affects potassium nutrition and rice drought tolerance.

Potassium (K(+)) is the most important cationic nutrient for all living organisms and has roles in most aspects of plant physiology. To assess the impact of one of the main K(+) uptake components, the K(+) inward rectifying channel AKT1, we characterized both loss of function and overexpression of OsAKT1 in rice. In many conditions, AKT1 expression correlated with K(+) uptake and tissue K(+) levels. No salinity-related growth phenotype was observed for either loss or gain of function mutants. However, a correlation between AKT1 expression and root Na(+) when the external Na/K ratio was high suggests that there may be a role for AKT1 in Na(+) uptake in such conditions. In contrast to findings with Arabidopsis thaliana, we did not detect any change in growth of AKT1 loss of function mutants in the presence of NH4 (+) Nevertheless, NH4 (+)-dependent inhibition was detected during K(+) uptake assays in loss of function and wild type plants, depending on pre-growth conditions. The most prominent result of OsAKT1 overexpression was a reduction in sensitivity to osmotic/drought stress in transgenic plants: the data suggest that AKT1 overexpression improved rice osmotic and drought stress tolerance by increasing tissue levels of K(+), especially in the root.