Negative Regulator Of Akt Signaling Research Articles

Accelerated plasma-cell differentiation in Bach2-deficient mouse B cells is caused by altered IRF4 functions.

Transcription factors BACH2 and IRF4 are both essential for antibody class-switch recombination (CSR) in activated B lymphocytes, while they oppositely regulate the differentiation of plasma cells (PCs). Here, we investigated how BACH2 and IRF4 interact during CSR and plasma-cell differentiation. We found that BACH2 organizes heterochromatin formation of target gene loci in mouse splenic B cells, including targets of IRF4 activation such as Aicda, an inducer of CSR, and Prdm1, a master plasma-cell regulator. Release of these gene loci from heterochromatin in response to B-cell receptor stimulation was coupled to AKT-mTOR pathway activation. In Bach2-deficient B cells, PC genes' activation depended on IRF4 protein accumulation, without an increase in Irf4 mRNA. Mechanistically, a PU.1-IRF4 heterodimer in activated B cells promoted BACH2 function by inducing gene expression of Bach2 and Pten, a negative regulator of AKT signaling. Elevated AKT activity in Bach2-deficient B cells resulted in IRF4 protein accumulation. Thus, BACH2 and IRF4 mutually modulate the activity of each other, and BACH2 inhibits PC differentiation by both the repression of PC genes and the restriction of IRF4 protein accumulation.

Scaffolding Protein Connector Enhancer of Kinase Suppressor of Ras 1 (CNKSR1) Regulates MAPK Inhibition Responsiveness in Pancreas Cancer via Crosstalk with AKT Signaling.

The CNKSR1 scaffold, identified within an RNAi screen as a novel mediator of resistance to MEK inhibition in pancreas cancer, connects the MAPK pathway and AKT signaling and may be adopted as a biomarker to select patients for combined MEK AKT blockade.

Differential roles of ERRFI1 in EGFR and AKT pathway regulation affect cancer proliferation.

AKT signaling is modulated by a complex network of regulatory proteins and is commonly deregulated in cancer. Here, we present a dual mechanism of AKT regulation by the ERBB receptor feedback inhibitor 1 (ERRFI1). We show that in cells expressing high levels of EGFR, ERRF1 inhibits growth and enhances responses to chemotherapy. This is mediated in part through the negative regulation of AKT signaling by direct ERRFI1‐dependent inhibition of EGFR. In cells expressing low levels of EGFR, ERRFI1 positively modulates AKT signaling by interfering with the interaction of the inactivating phosphatase PHLPP with AKT, thereby promoting cell growth and chemotherapy desensitization. These observations broaden our understanding of chemotherapy response and have important implications for the selection of targeted therapies in a cell context‐dependent manner. EGFR inhibition can only sensitize EGFR‐high cells for chemotherapy, while AKT inhibition increases chemosensitivity in EGFR‐low cells. By understanding these mechanisms, we can take advantage of the cellular context to individualize antineoplastic therapy. Finally, our data also suggest targeting of EFFRI1 in EGFR‐low cancer as a promising therapeutic approach.

ABL2 suppresses FLT3-ITD-induced cell proliferation through negative regulation of AKT signaling.

The type III receptor tyrosine kinase FLT3 is one of the most commonly mutated oncogenes in acute myeloid leukemia (AML). Inhibition of mutated FLT3 in combination with chemotherapy has displayed promising results in clinical trials. However, one of the major obstacles in targeting FLT3 is the development of resistant disease due to secondary mutations in FLT3 that lead to relapse. FLT3 and its oncogenic mutants signal through associating proteins that activate downstream signaling. Thus, targeting proteins that interact with FLT3 and their downstream signaling cascades can be an alternative approach to treat FLT3-dependent AML. We used an SH2 domain array screen to identify novel FLT3 interacting proteins and identified ABL2 as a potent interacting partner of FLT3. To understand the role of ABL2 in FLT3-mediated biological and cellular events, we used the murine pro-B cell line Ba/F3 as a model system. Overexpression of ABL2 in Ba/F3 cells expressing an oncogenic mutant of FLT3 (FLT3-ITD) resulted in partial inhibition of FLT3-ITD-dependent cell proliferation and colony formation. ABL2 expression did not alter the kinase activity of FLT3, its ubiquitination or its stability. However, it partially blocked FLT3-induced AKT phosphorylation without affecting ERK1/2 and p38 activation. Taken together our data suggest that ABL2 acts as negative regulator of signaling downstream of FLT3.

Hyperglycemia and hyperlipidemia blunts the Insulin-Inpp5f negative feedback loop in the diabetic heart.

The leading cause of death in diabetic patients is diabetic cardiomyopathy, in which alteration of Akt signal plays an important role. Inpp5f is recently found to be a negative regulator of Akt signaling, while its expression and function in diabetic heart is largely unknown. In this study, we found that in both the streptozotocin (STZ) and high fat diet (HFD) induced diabetic mouse models, Inpp5f expression was coordinately regulated by insulin, blood glucose and lipid levels. Increased Inpp5f was inversely correlated with the cardiac function. Further studies revealed that Insulin transcriptionally activated Inpp5f in an Sp1 dependent manner, and increased Inpp5f in turn reduced the phosphorylation of Akt, forming a negative feedback loop. The negative feedback plays a protective role under diabetic condition. However, high blood glucose and lipid, which are characteristics of uncontrolled diabetes and type 2 diabetes, increased Inpp5f expression through activation of NF-κB, blunts the protective feedback. Thus, our study has revealed that Inpp5f provides as a negative feedback regulator of insulin signaling and downregulation of Inpp5f in diabetes is cardioprotective. Increased Inpp5f by hyperglycemia and hyperlipidemia is an important mediator of diabetic cardiomyopathy and is a promising therapeutic target for the disease.

Abstract 2103: SOX2 suppresses PTEN expression via miR-19a in esophageal squamous cell carcinoma

Abstract We previously showed that SOX2, a master regulator during embryogenesis, is an amplification target of 3q26.33 in esophageal squamous cell carcinoma (ESCC), and that SOX2 promotes ESCC proliferation in vitro and in vivo via activation of the AKT/mammalian target of rapamycin complex 1 (mTORC1) signaling pathway. However, the mechanisms by which SOX2 promotes AKT/mTORC1 signaling remain to be elucidated. We determined that SOX2 decreased PTEN, a negative regulator of AKT signaling, via microRNA (miRNA) in ESCC cells. SOX2 knockdown increased PTEN and decreased phosphorylated AKT (p-AKT), and PTEN knockdown restored p-AKT decreased by SOX2 knockdown in SOX2 amplified ESCC cells (KYSE70, KYSE140 and TE10 cells), indicating that SOX2 elevates p-AKT via PTEN suppression in these cells. DICER knockdown increased PTEN expression without SOX2 expression change in these cells, suggesting that PTEN expression is regulated by miRNA. Therefore we hypothesized that SOX2 may suppress PTEN expression through miRNA regulation. To examine whether SOX2 modulate the expression of miRNAs, we investigated the expression of cancer-related 84 miRNAs using miRNA PCR arrays, and found that SOX2 elevated miR-17∼92 cluster (miR-17, miR-18a, miR-19a, miR-19b-1, miR-20a, and miR-92) in KYSE70 cells. Quantitative RT-PCR confirmed that the expression of mir-17∼92 cluster is elevated by SOX2 in KYSE70, KYSE140, and TE10 cells. Of these miRNA, transfection of miR-19a mimic suppressed PTEN and conversely miR-19a inhibition elevated PTEN in these cells. Collectively, SOX2 may suppress PTEN expression via miR-19a in ESCC cells. Citation Format: Yasuyuki Gen, Kohichiroh Yasui, Tomoko Kitaichi, Osamu Dohi, Yoshito Ito. SOX2 suppresses PTEN expression via miR-19a in esophageal squamous cell carcinoma. [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 2103. doi:10.1158/1538-7445.AM2015-2103

MicroRNA-155 regulates cell proliferation and invasion by targeting FOXO3a in glioma

microRNAs (miRNAs) are short noncoding RNAs, which modulate the expression of numerous genes by targeting mRNAs. Numerous abnormal miRNA expression patterns are found in various human malignancies, and certain miRNAs act as oncogenes or tumor suppressors. microRNA-155 (miR‑155) may not only function as an oncogene but also as a tumor suppressor in various types of cancer cells, such as melanoma. Although miR-155 has been found to be upregulated in glioma, its role has not yet been eludicated in glioma tumorigenesis. Based on the prediction of the target genes of miR-155, we hypothesized that there is a significant association between miR-155 and FOXO3a, a negative regulator of Akt signaling. In the present study, we found that FOXO3a expression was significantly downregulated and miR-155 was upregulated in a panel of glioma cells and tissue specimens. Furthermore, we demonstrated that miR-155 induced cell proliferation by inhibiting apoptosis and promoted the migration and invasiveness of glioma cells, while miR-155 had no effect on the cell cycle as determined by gain-of-function and loss-of-function experiments. Moreover, we confirmed that miR-155 downregulated the expression of FOXO3a by directly targeting its 3'-UTR. These findings indicate that miR-155 may function as an oncogene by targeting FOXO3a in the development and progression of glioma.

Abstract 846: ROS-mediated activation of AKT induces apoptosis via pVHL in prostate cancer cells .

Abstract Reactive oxygen species (ROS) play a central role in oxidative stress, which leads to the onset of diseases, such as cancer. Furthermore, ROS contributes to the delicate balance between tumor cell survival and death. However, the mechanisms by which tumor cells decide to elicit survival or death signals during oxidative stress are not completely understood. We have previously reported that ROS enhanced tumorigenic functions in prostate cancer cells, such as cell migration and invasion, which depended on CXCR4 and AKT signaling. Here, we report a novel mechanism by which ROS facilitated cell death through activation of AKT. We initially observed that ROS increased expression of phosphorylated AKT (p-AKT) in 22Rv1 human prostate cancer cells. The tumor suppressor PTEN, a negative regulator of AKT signaling, was rendered catalytically inactive through oxidation by ROS, although the expression levels remained consistent. Despite these events, cells still underwent apoptosis. Further investigation into apoptosis revealed that expression of the tumor suppressor pVHL increased, with concomitant decreased expression of HIF1α. Further investigation into pVHL during apoptosis revealed that the tumor suppressor becomes phosphorylated at S111 prior to activity; S111 is within a target motif (RXXS) forphosphorylation by AKT kinase. We then determined that pVHL and p-AKT associated in vitro via co-immunoprecipitation assay. Moreover, knockdown of pVHL by siRNA rescued HIF1α protein expression and rescued cells from apoptosis, which was determined by Annexin-V and cleaved-PARP. Collectively, our study suggests that in the context of oxidative stress, activatedAKT facilitated apoptosis by inducing pVHL function. Moreover, our studies suggest a novel paradigm for AKT-that it may participate in tumor suppressor rather than oncogenesis. Citation Format: Mahandranauth A. Chetram, Kia J. Jones, Christopher Coke, Ayesha Don-Salu-Hewage, Danaya A. Bethea, Cimona V. Hinton. ROS-mediated activation of AKT induces apoptosis via pVHL in prostate cancer cells . [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 846. doi:10.1158/1538-7445.AM2013-846

ROS-mediated activation of AKT induces apoptosis via pVHL in prostate cancer cells

Reactive oxygen species (ROS) play a central role in oxidative stress, which leads to the onset of diseases, such as cancer. Furthermore, ROS contributes to the delicate balance between tumor cell survival and death. However, the mechanisms by which tumor cells decide to elicit survival or death signals during oxidative stress are not completely understood. We have previously reported that ROS enhanced tumorigenic functions in prostate cancer cells, such as transendothelial migration and invasion, which depended on CXCR4 and AKT signaling. Here, we report a novel mechanism by which ROS facilitated cell death through activation of AKT. We initially observed that ROS enhanced the expression of phosphorylated AKT (p-AKT) in 22Rv1 human prostate cancer cells. The tumor suppressor PTEN, a negative regulator of AKT signaling, was rendered catalytically inactive through oxidation by ROS, although the expression levels remained consistent. Despite these events, cells still underwent apoptosis. Further investigation into apoptosis revealed that expression of the tumor suppressor pVHL increased, and contains a target site for p-AKT phosphorylation. pVHL and p-AKT associated in vitro, and knockdown of pVHL rescued HIF1α expression and the cells from apoptosis. Collectively, our study suggests that in the context of oxidative stress, p-AKT facilitated apoptosis by inducing pVHL function.

Induction of IL-4Rα–dependent microRNAs identifies PI3K/Akt signaling as essential for IL-4–driven murine macrophage proliferation in vivo

AbstractMacrophage (MΦ) activation must be tightly controlled to preclude overzealous responses that cause self-damage. MicroRNAs promote classical MΦ activation by blocking antiinflammatory signals and transcription factors but also can prevent excessive TLR signaling. In contrast, the microRNA profile associated with alternatively activated MΦ and their role in regulating wound healing or antihelminthic responses has not been described. By using an in vivo model of alternative activation in which adult Brugia malayi nematodes are implanted surgically in the peritoneal cavity of mice, we identified differential expression of miR-125b-5p, miR-146a-5p, miR-199b-5p, and miR-378-3p in helminth-induced MΦ. In vitro experiments demonstrated that miR-378-3p was specifically induced by IL-4 and revealed the IL-4–receptor/PI3K/Akt-signaling pathway as a target. Chemical inhibition of this pathway showed that intact Akt signaling is an important enhancement factor for alternative activation in vitro and in vivo and is essential for IL-4–driven MΦ proliferation in vivo. Thus, identification of miR-378-3p as an IL-4Rα–induced microRNA led to the discovery that Akt regulates the newly discovered mechanism of IL-4–driven macrophage proliferation. Together, the data suggest that negative regulation of Akt signaling via microRNAs might play a central role in limiting MΦ expansion and alternative activation during type 2 inflammatory settings.

Abstract C40: Recombinant viral protein suppresses metastasis of ovarian adenocarcinoma cells through negative Akt signaling

Abstract Ovarian cancer has a high fatality rate, mainly due to metastatic progression of tumor cells. Advances in the treatment of metastatic ovarian cancer are needed. Here, we studied the mechanism of action of a recombinant viral capsid protein (rVP1) and explored its effect against ovarian tumor growth and metastasis in vivo. We found that rVP1 was cytotoxic to several human ovarian cancer cell lines, namely doxorubicin-resistant SKOV3, cisplatin-resistant OVCAR-3, and TOV-21G. rVP1 was more potent than doxorubicin and cisplatin in inducing apoptosis and inhibiting growth of SKOV3 in vitro. Treatment of SKOV3 or peritoneal adapted SKOV3ip.1 cells with rVP1 at certain concentrations below IC50 resulted mainly in inhibition of cell invasion rather than apoptosis. This inhibitory effect of rVP1 on cell invasion was accompanied with activation of PTEN and GSK-3β as well as downregulation of FAK, Akt and MMP-2. Anti-integrin antibodies or overexpression of constitutively active Akt reversed the cellular effects of rVP1. In vivo studies confirmed that rVP1 attenuated survival and metastasis of SKOV3 cells through the selective regulation of Akt and GSK-3β activity in an orthotopic xenograft mouse model as revealed by bioluminescence imaging of mice and immunohistological analysis. In addition, rVP1 decreased the formation of ascites and metastatic tumor nodules formed by SKOV3 cells in an intraperitoneal xenograft mouse model. These results indicate that negative regulation of Akt signaling by rVP1 may have the potential to suppress ovarian tumor growth and metastasis in vivo. 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 C40.

MiR-190-Mediated Downregulation of PHLPP Contributes to Arsenic-Induced Akt Activation and Carcinogenesis

The role of trivalent arsenic (As(3+)) on the regulation of the recently identified noncoding small RNAs, mainly microRNAs, has not been explored so far. In the present study, we provide evidence showing that As(3+) is a potent inducer for the expression of miR-190 in human bronchial epithelial cells. The induction of miR-190 by As(3+) is concentration dependent and associated with the expression of the host gene of miR-190, talin 2, a gene encoding a high-molecular-weight cytoskeletal protein. The elevated level of miR-190 induced by As(3+) is capable of downregulating the translation of the PH domain leucine-rich repeat protein phosphatase (PHLPP), a negative regulator of Akt signaling. Such a downregulation is occurred through direct interaction of the miR-190 with the 3'-UTR region of the PHLPP mRNA, leading to a diminished PHLPP protein expression and consequently, an enhanced Akt activation and expression of vascular endothelial growth factor, an Akt-regulated protein. Overexpression of miR-190 itself is able to enhance proliferation and malignant transformation of the cells as determined by anchorage-independent growth of the cells in soft agar. Accordingly, the data presented suggest that induction of miR-190 is one of the key mechanisms in As(3+)-induced carcinogenesis.

MiR-155, a Modulator of FOXO3a Protein Expression, Is Underexpressed and Cannot Be Upregulated by Stimulation of HOZOT, a Line of Multifunctional Treg

MicroRNAs (miRNAs) play important roles in regulating post-transcriptional gene repression in a variety of immunological processes. In particular, much attention has been focused on their roles in regulatory T (Treg) cells which are crucial for maintaining peripheral tolerance and controlling T cell responses. Recently, we established a novel type of human Treg cell line, termed HOZOT, multifunctional cells exhibiting a CD4+CD8+ phenotype. In this study, we performed miRNA profiling to identify signature miRNAs of HOZOT, and therein identified miR-155. Although miR-155 has also been characterized as a signature miRNA for FOXP3+ natural Treg (nTreg) cells, it was expressed quite differently in HOZOT cells. Under both stimulatory and non-stimulatory conditions, miR-155 expression remained at low levels in HOZOT, while its expression in nTreg and conventional T cells remarkably increased after stimulation. We next searched candidate target genes of miR-155 through bioinformatics, and identified FOXO3a, a negative regulator of Akt signaling, as a miR-155 target gene. Further studies by gain- and loss-of-function experiments supported a role for miR-155 in the regulation of FOXO3a protein expression in conventional T and HOZOT cells.

A novel activation-induced suicidal degradation mechanism for Akt by selenium

Selenium has been associated with an anti-cancer effect via the modulation of Akt. In order to investigate whether selenium modulates Akt by hitherto unidentified molecular mechanisms, we examined the effect of selenium on the stability and activity of Akt. Selenium induced destabilization of Akt which is coupled to its own enzyme activation. Mutation of T308 and S473 of Akt to alanine as well as the inhibition or depletion of upstream kinases for Akt activation blocked Akt degradation. These features of Akt degradation are reminiscent of the 'activation-induced suicidal degradation' mechanism. PTEN was also required for Akt destabilization as Akt activation alone was unable to elicit Akt degradation in the absence of PTEN. Conversely, PTEN introduction in PTEN-null prostate cancer cells restored the ability to degrade Akt upon selenium treatment. Collectively, selenium seems to achieve ultimate negative regulation of Akt signaling by destabilizing the protein, and this regulation mechanism might provide a paradigm for the anti-cancer activity of selenium.