Lina He

The Critical Role of AKT2 in Hepatic Steatosis Induced by PTEN Loss

Insulin signaling in the liver leads to accumulation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3). Deletion of the phosphatase Pten (phosphatase and tensin homologue deleted on chromosome 10) reduces PIP3 levels and leads to fatty liver development. The purpose of this study was to investigate the mechanisms underlying lipogenesis that result from PIP3 accumulation using liver Pten-deletion mice. To explore the role of AKT2, the major liver AKT isoform in steatosis induced by deletion of Pten, we created mice lacking both Pten and Akt2 in hepatocytes and compared the effect of deleting Akt2 and Pten in the double mutants to the Pten deletion mice alone. Hepatic lipid accumulation was significantly reduced in mice lacking both PTEN and AKT2, as compared with Pten mutant mice alone. This effect was due to the role of AKT2 in maintaining expression of genes involved in de novo lipogenesis. We showed that lipid accumulation in the double mutant hepatocytes was partially reversed by expression of constitutive active FOXO1, a transcription factor downstream of AKT not dependent on inhibition of atypical protein kinase C. In summary, this study delineated regulation of lipid metabolism by PI3K signaling pathway by showing that AKT mediates PIP3 accumulation (mimicked by PTEN loss) induced lipid deposition in the liver and provided an important molecular mechanism for insulin-regulated hepatic lipogenesis.

Expansion of CD133‐Expressing Liver Cancer Stem Cells in Liver‐Specific Phosphatase and Tensin Homolog Deleted on Chromosome 10‐Deleted Mice

PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a lipid phosphatase that regulates mitogenic signaling pathways, and deficiency of PTEN results in cell proliferation, survival, and malignancy. Murine liver‐specific Pten deletion models develop liver malignancy by 12 months of age. Using this model, we describe a population of CD133+ liver cancer stem cells isolated during the chronic injury phase of disease progression and before primary carcinoma formation. We performed immunohistochemistry and flow cytometry isolation using livers from 3‐ and 6‐month‐old PtenloxP/loxP; Alb−Cre+ mice (mutants) and controls. CD133+CD45− nonparenchymal (NP) cells were analyzed for gene expression profile and protein levels. Single CD133+CD45− oval cells were isolated for clonal expansion and tumor analysis. Cultured and freshly isolated liver CD133+CD45− and CD133−CD45− NP cells were injected into immune‐deficient and immune‐competent mice. In mutant mice, the NP fraction increased in CD133+CD45− cells in 3‐ and 6‐month‐old Pten‐deleted animals compared with controls. Clone lines expanded from single CD133+CD45− cells demonstrated consistent liver progenitor cell phenotype, with bilineage gene expression of hepatocyte and cholangiocyte markers. CD133+ cells from expanded clone lines formed robust tumors in immune‐deficient and immune‐competent mice. Furthermore, freshly isolated CD133+CD45− NP liver cells from 6‐month‐old mutants formed tumors in vivo, and CD133−CD45− NP cells did not. Consistent with a cancer stem cell phenotype, CD133+ cells demonstrate resistance to chemotherapy agents compared with CD133− cells. CD133+CD45− nonparenchymal cells from chronic injury PtenloxP/loxP; Alb−Cre+ mice represent a bipotent liver progenitor cell population with cancer stem cell phenotype. STEM CELLS 2009;27:290–299

Liver-specific deletion of prohibitin 1 results in spontaneous liver injury, fibrosis, and hepatocellular carcinoma in mice†

Prohibitin 1 (PHB1) is a highly conserved, ubiquitously expressed protein that participates in diverse processes including mitochondrial chaperone, growth and apoptosis. The role of PHB1 in vivo is unclear and whether it is a tumor suppressor is controversial. Mice lacking methionine adenosyltransferase 1A (MAT1A) have reduced PHB1 expression, impaired mitochondrial function, and spontaneously develop hepatocellular carcinoma (HCC). To see if reduced PHB1 expression contributes to the Mat1a knockout (KO) phenotype, we generated liver‐specific Phb1 KO mice. Expression was determined at the messenger RNA and protein levels. PHB1 expression in cells was varied by small interfering RNA or overexpression. At 3 weeks, KO mice exhibit biochemical and histologic liver injury. Immunohistochemistry revealed apoptosis, proliferation, oxidative stress, fibrosis, bile duct epithelial metaplasia, hepatocyte dysplasia, and increased staining for stem cell and preneoplastic markers. Mitochondria are swollen and many have no discernible cristae. Differential gene expression revealed that genes associated with proliferation, malignant transformation, and liver fibrosis are highly up‐regulated. From 20 weeks on, KO mice have multiple liver nodules and from 35 to 46 weeks, 38% have multifocal HCC. PHB1 protein levels were higher in normal human hepatocytes compared to human HCC cell lines Huh‐7 and HepG2. Knockdown of PHB1 in murine nontransformed AML12 cells (normal mouse hepatocyte cell line) raised cyclin D1 expression, increased E2F transcription factor binding to cyclin D1 promoter, and proliferation. The opposite occurred with PHB1 overexpression. Knockdown or overexpression of PHB1 in Huh‐7 cells did not affect proliferation significantly or sensitize cells to sorafenib‐induced apoptosis. Conclusion: Hepatocyte‐specific PHB1 deficiency results in marked liver injury, oxidative stress, and fibrosis with development of HCC by 8 months. These results support PHB1 as a tumor suppressor in hepatocytes. (HEPATOLOGY 2010.)

Expansion of hepatic tumor progenitor cells in Pten-null mice requires liver injury and is reversed by loss of AKT2.

BACKGROUND & AIMS The tumor suppressor PTEN inhibits AKT2 signaling; both are aberrantly expressed in liver tumors. We investigated how PTEN and AKT2 regulate liver carcinogenesis. Loss of PTEN leads to spontaneous development of liver tumors from progenitor cells. We investigated how the loss of PTEN activates liver progenitor cells and induces tumorigenesis. METHODS We studied mice with liver-specific disruptions in Pten and the combination of Pten and Akt2 to investigate mechanisms of liver carcinogenesis. RESULTS PTEN loss leads to hepatic injury and establishes selective pressure for tumor-initiating cells (TICs), which proliferate to form mixed-lineage tumors. The Pten-null mice had increasing levels of hepatic injury before proliferation of hepatic progenitors. Attenuation of hepatic injury by deletion of Akt2 reduced progenitor cell proliferation and delayed tumor development. In Pten/Akt2-null mice given 3,5-diethoxycarbonyl-1,4 dihydrocollidine (DDC), we found that the primary effect of AKT2 loss was attenuation of hepatic injury and not inhibition of progenitor-cell proliferation in response to injury. CONCLUSIONS Liver carcinogenesis in Pten-null mice requires not only the transformation of TICs but selection pressure from hepatic injury and cell death, which activates TICs. Further research is required to elucidate the mechanism for hepatic injury and its relationship with TIC activation.

Maf1 Is a Novel Target of PTEN and PI3K Signaling That Negatively Regulates Oncogenesis and Lipid Metabolism

Maf1 was initially identified as a transcriptional repressor of RNA pol III-transcribed genes, yet little is known about its other potential target genes or its biological function. Here, we show that Maf1 is a key downstream target of PTEN that drives both its tumor suppressor and metabolic functions. Maf1 expression is diminished with loss of PTEN in both mouse models and human cancers. Consistent with its role as a tumor suppressor, Maf1 reduces anchorage-independent growth and tumor formation in mice. PTEN-mediated changes in Maf1 expression are mediated by PTEN acting on PI3K/AKT/FoxO1 signaling, revealing a new pathway that regulates RNA pol III-dependent genes. This regulatory event is biologically relevant as diet-induced PI3K activation reduces Maf1 expression in mouse liver. We further identify lipogenic enzymes as a new class of Maf1-regulated genes whereby Maf1 occupancy at the FASN promoter opposes SREBP1c-mediated transcription activation. Consistent with these findings, Maf1 inhibits intracellular lipid accumulation and increasing Maf1 expression in mouse liver abrogates diet-mediated induction of lipogenic enzymes and triglycerides. Together, these results establish a new biological role for Maf1 as a downstream effector of PTEN/PI3K signaling and reveal that Maf1 is a key element by which this pathway co-regulates lipid metabolism and oncogenesis.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) signaling regulates mitochondrial biogenesis and respiration via estrogen-related receptor α (ERRα).

Background: Aberrant PTEN/PI3K signaling and mitochondrial abnormalities are commonly associated with cancer. Results: PTEN loss and activation of PI3K/protein kinase B up-regulate ERRα, increase mitochondrial mass, and induce a metabolic pattern similar to the “Warburg effect.” Conclusion: PTEN/PI3K signaling controls mitochondrial mass and function by regulating ERRα through the AKT/CREB axis. Significance: This study establishes a novel link between oncogenic signaling and dysregulated mitochondrial metabolism. Mitochondrial abnormalities are associated with cancer development, yet how oncogenic signals affect mitochondrial functions has not been fully understood. In this study, we investigate the relationship between mitochondrial alterations and PI3K/protein kinase B (AKT) signaling activation using hepatocytes and liver tissues as our experimental models. We show here that liver-specific deletion of Pten, which leads to activation of PI3K/AKT, is associated with elevated oxidative stress, increased mitochondrial mass, and augmented respiration accompanied by enhanced glycolysis. Consistent with these observations, estrogen-related receptor α (ERRα), an orphan nuclear receptor known for its role in mitochondrial biogenesis, is up-regulated in the absence of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Our pharmacological and genetic studies show that PI3K/AKT activity regulates the expression of ERRα and mitochondrial biogenesis/respiration. Furthermore, cAMP-response element-binding protein, as a downstream target of AKT, plays a role in the regulation of ERRα, independent of PKA signaling. ERRα regulates reactive oxygen species production, and ERRα knockdown attenuates proliferation and colony-forming potential in Pten-null hepatocytes. Finally, analysis of clinical datasets from liver tissues showed a negative correlation between expressions of ERRα and PTEN in patients with liver cancer. Therefore, this study has established a previously unrecognized link between a growth signal and mitochondrial metabolism.

PTEN CONTROLS β-CELL REGENERATION IN AGED MICE BY REGULATING CELL CYCLE INHIBITOR P16INK4A

Tissue regeneration diminishes with age, concurrent with declining hormone levels including growth factors such as insulin‐like growth factor‐1 (IGF‐1). We investigated the molecular basis for such decline in pancreatic β‐cells where loss of proliferation occurs early in age and is proposed to contribute to the pathogenesis of diabetes. We studied the regeneration capacity of β‐cells in mouse model where PI3K/AKT pathway downstream of insulin/IGF‐1 signaling is upregulated by genetic deletion of Pten (phosphatase and tensin homologue deleted on chromosome 10) specifically in insulin‐producing cells. In this model, PTEN loss prevents the decline in proliferation capacity in aged β‐cells and restores the ability of aged β‐cells to respond to injury‐induced regeneration. Using several animal and cell models where we can manipulate PTEN expression, we found that PTEN blocks cell cycle re‐entry through a novel pathway leading to an increase in p16ink4a, a cell cycle inhibitor characterized for its role in cellular senescence/aging. A downregulation in p16ink4a occurs when PTEN is lost as a result of cyclin D1 induction and the activation of E2F transcription factors. The activation of E2F transcriptional factors leads to methylation of p16ink4a promoter, an event that is mediated by the upregulation of polycomb protein, Ezh2. These analyses establish a novel PTEN/cyclin D1/E2F/Ezh2/p16ink4a signaling network responsible for the aging process and provide specific evidence for a molecular paradigm that explain how decline in growth factor signals such as IGF‐1 (through PTEN/PI3K signaling) may control regeneration and the lack thereof in aging cells.

PI3K/AKT signaling regulates bioenergetics in immortalized hepatocytes

Regulation of cellular bioenergetics by PI3K/AKT signaling was examined in isogenic hepatocyte cell lines lacking the major inhibitor of PI3K/AKT signaling, PTEN (phosphatase and tensin homolog deleted on chromosome 10). PI3K/AKT signaling was manipulated using the activator (IGF-1) and the inhibitor (LY 294002) of the PI3K/AKT pathway. Activation of PI3K/AKT signaling resulted in an enhanced anaerobic glycolysis and mitochondrial respiration. AKT, when phosphorylated and activated, translocated to mitochondria and localized within the membrane structure of mitochondria, where it phosphorylated a number of mitochondrial-resident proteins including the subunits α and β of ATP synthase. Inhibition of GSK3β by either phosphorylation by AKT or lithium chloride resulted in activation of pyruvate dehydrogenase, i.e., a decrease in its phosphorylated form. AKT-dependent phosphorylation of ATP synthase subunits α and β resulted in an increased complex activity. AKT translocation to mitochondria was associated with an increased expression and activity of complex I. These data suggest that the mitochondrial signaling pathway AKT/GSK3β/PDH, AKT-dependent phosphorylation of ATP synthase, and upregulation of mitochondrial complex I expression and activity are involved in the control of mitochondrial bioenergetics by increasing substrate availability and regulating the mitochondrial catalytic/energy-transducing capacity.

Adaptive Basal Phosphorylation of eIF2α Is Responsible for Resistance to Cellular Stress–Induced Cell Death in Pten-Null Hepatocytes

The α-subunit of eukaryotic initiation factor 2 (eIF2α) is a key translation regulator that plays an important role in cellular stress responses. In the present study, we investigated how eIF2α phosphorylation can be regulated by a tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) and how such regulation is used by PTEN-deficient hepatocytes to adapt and cope with oxidative stress. We found that eIF2α was hyperphosphorylated when Pten was deleted, and this process was AKT dependent. Consistent with this finding, we found that the Pten-null cells developed resistance to oxidative glutamate and H2O2-induced cellular toxicity. We showed that the messenger level of CReP (constitutive repressor of eIF2α phosphorylation), a constitutive phosphatase of eIF2α, was downregulated in Pten-null hepatocytes, providing a possible mechanism through which PTEN/AKT pathway regulates eIF2α phosphorylation. Ectopic expression of CReP restored the sensitivity of the Pten mutant hepatocytes to oxidative stress, confirming the functional significance of the downregulated CReP and upregulated phospho-eIF2α in the resistance of Pten mutant hepatocytes to cellular stress. In summary, our study suggested a novel role of PTEN in regulating stress response through modulating the CReP/eIF2α pathway. Mol Cancer Res; 9(12); 1708–17. ©2011 AACR.

Activation of hepatic stellate cell in Pten null liver injury model

BackgroundHepatic fibrosis is a prominent pathological feature associated with chronic liver disease including non-alcoholic hepatosteatosis (NASH), and a precursor for liver cancer development. We previously reported that PTEN loss in the liver, which leads to hyperactivated liver insulin signaling results in NASH development. Here we used the same mouse model to study the progression from steatosis to fibrosis.ResultsThe Pten null livers develop progressive liver fibrosis as indicated by Sirius Red staining and increased expression of collagen I, Timp 1, SMAα, and p75NTR. Consistently, hepatic stellate cells (HSCs) isolated from Pten null livers are readily activated when compared with that from mice with intact PTEN. Deletion of AKT2, the downstream target of PTEN signal, blocked NASH development, and alleviated fibrosis. HSCs from the Pten/Akt2 double null mice are quiescent like those isolated from the control livers. Our analysis shows that the activation of HSCs does not depend on the intrinsic signals regulated by PI3K/AKT, the target of PTEN, but does depend on steatosis and injury to the liver. During the progression of liver fibrosis in the Pten null model, Wnt ligands and signaling receptor are induced, concurrent with the reduction of sFRP5, a Wnt antagonist. We showed that treatment of HSCs with Wnt receptor antagonist blocks the observed morphological changes when HSCs undergo activation in culture. This signal appears to be mediated by β-catenin, as manipulating β-catenin signaling alters marker gene expressions of HSC activation.ConclusionsWnt/β-catenin activation serves as an important mediator for fibrosis development resulting from NASH using a mouse model where NASH is mimicked by PTEN loss.