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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.

High circulating lipids occurring in obese individuals and insulin-resistant patients are considered a contributing factor to type 2 diabetes. Exposure to high lipid concentration is proposed to both protect and damage beta-cells under different circumstances. Here, by feeding mice a high-fat diet (HFD) for 2 weeks to up to 14 months, the study showed that HFD initially causes the beta-cells to expand in population, whereas long-term exposure to HFD is associated with failure of beta-cells and the inability of animals to respond to glucose challenge. To prevent the failure of beta-cells and the development of type 2 diabetes, the molecular mechanisms that underlie this biphasic response of beta-cells to lipid exposure were explored. Using palmitic acid (PA) in cultured beta-cells and islets, the study demonstrated that chronic exposure to lipids leads to reduced viability and inhibition of cell cycle progression concurrent with down-regulation of a pro-growth/survival kinase AKT, independent of glucose. This AKT down-regulation by PA is correlated with the induction of mTOR/S6K activity. Inhibiting mTOR activity with rapamycin induced Raptor and restored AKT activity, allowing beta-cells to gain proliferation capacity that was lost after HFD exposure. In summary, a novel mechanism in which lipid exposure may cause the dipole effects on beta-cell growth was elucidated, where mTOR acts as a lipid sensor. These mechanisms can be novel targets for future therapeutic developments.

An analogue of nicotinamide adenine dinucleotide (NAD+) featuring an azido group at 3′-OH of adenosine moiety is found to possess high specificity for human PARP1-catalyzed protein poly-ADP-ribosylation.

The estrogen-related receptor (ERR) family of orphan nuclear receptors are transcriptional activators for genes involved in mitochondrial bioenergetics and metabolism. The goal of this study was to explore the role of ERRα in lipid metabolism and the potential effect of inhibiting ERRα on the development of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). In the current study, three experimental mouse models: high-fat diet, high-carbohydrate diet, and a genetic model of hepatic insulin resistance where the liver hyperinsulinemia signal is mimicked via hepatic deletion of Pten (phosphatase and tensin homolog deleted on chromosome 10), the negative regulator of the insulin/phosphatidylinositol 3-kinase signaling pathway, were used. A recently developed small-molecule inhibitor for ERRα was used to demonstrate that inhibiting ERRα blocked NAFLD development induced by either high-carbohydrate diet or high-fat diet feeding. ERRα inhibition also diminished lipid accumulation and attenuated NASH development in the Pten null mice. Glycerolipid synthesis was discovered as an additional mechanism for ERRα-regulated NAFLD/NASH development and glycerophosphate acyltransferase 4 was identified as a novel transcriptional target of ERRα. In summary, these results establish ERRα as a major transcriptional regulator of lipid biosynthesis in addition to its characterized primary function as a regulator for mitochondrial function. This study recognizes ERRα as a potential target for NAFLD/NASH treatment and elucidates novel signaling pathways regulated by ERRα.

Hepatic glucose metabolism signaling downstream of insulin can diverge to multiple pathways including AKT. Genetic studies suggest that AKT is necessary for insulin to suppress gluconeogenesis. To specifically address the role of AKT2, the dominant liver isoform of AKT in the regulation of gluconeogenesis genes, we generated hepatocytes lacking AKT2 (Akt2−/−). We found that, in the absence of insulin signal, AKT2 is required for maintaining the basal level expression of phosphoenolpyruvate carboxyl kinase (PEPCK) and to a lesser extent G6Pase, two key rate-limiting enzymes for gluconeogenesis that support glucose excursion due to pyruvate loading. We further showed that this function of AKT2 is mediated by the phosphorylation of cyclic AMP response element binding (CREB). Phosphorylation of CREB by AKT2 is needed for CREB to induce the expression of PEPCK and likely represents a priming event for unstimulated cells to poise to receive glucagon and other signals. The inhibition of gluconeogenesis by insulin is also dependent on the reduced FOXO1 transcriptional activity at the promoter of PEPCK. When insulin signal is absent, this activity appears to be inhibited by AKT2 in manner that is independent of its phosphorylation by AKT. Together, this action of AKT2 on FOXO1 and CREB to maintain basal gluconeogenesis activity may provide fine-tuning for insulin and glucocorticoid/glucagon to regulate gluconeogenesis in a timely manner to meet metabolic needs.

Liver malignancies consist of hepatocellular carcinoma (HCC) with the highest occurrence, intrahepatic cholangiocarcinoma (iCCA), and serval rare subtypes, which is the third lethal cause among all cancer types worldwide. PTEN is a well-known tumor suppressor gene, liver-specific loss of PTEN leads to the development of liver tumors from tumor-initiating cells (TICs). A mouse model that specifically mutant PTEN in hepatocytes (PM mice, PTENloxP/loxP; Alb-Cre+) has been used to mimic the natural progression of liver malignancy and study the mechanism of liver tumorigenesis. AKT, also known as protein kinase B, is a downstream kinase that is negatively regulated by PTEN. PTEN loss will unequivocally result in AKT phosphorylation and activation of the AKT pathway. In this study, we explored the role of AKT2, the most abundant liver isoform of AKT in the PTEN loss-driven liver malignancy by generating a new double mutant mouse model (DM mice, PTENloxP/loxP; AKT2loxP/loxP; Alb-Cre+). Our data demonstrated that only PM mice developed tumors starting from a 6-month age. A moderate reactive duct/oval cell accumulation phenotype is observed in the PM livers with Von Meyenbury complex (VMC) formation. And both HCC and iCCA phenotypes are observed following steatosis development in PM mice. AKT2 loss arrested tumor development at the pre-malignant stage. The DM mice also developed VMC condition with minimum steatosis starting from 9-month age and some of them manifest an advanced stage called polycystic liver disease. No tumors are observed in these mice up to 16 months of age. Our preliminary data showed that the deletion of AKT2 attenuated the accumulation of TICs marked by Sox9 suggesting a potential role of SOX9 in the regulation of PTEN-driven tumorigenesis. In summary, our result shows that AKT2 is a determining factor in PTEN loss-induced liver malignancy. Citation Format: Qi Tang, LIna He, Chien-Yu Chen, Shefali Chopra, Bangyan L. Stiles. AKT2 as the determining factor for PTEN loss-induced liver malignancy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2583.

Liver carcinoma is the 6th most prevalent cancer worldwide in 2020. Moreover, it is the 3rd leading cause of cancer related deaths. In addition to the genomic and transcriptomic heterogeneity of liver tumor cells which is recognized as a major driver in liver cancer progression, the liver immune system is also fundamental to liver carcinogenesis and presents a promising target for therapy. The liver immune response is orchestrated by cytokines and chemokines. Recent studies suggest that chemokines not only recruit immune cells but also regulate various liver functions. In partial hepatectomy, CXCL2 has been shown to promote hepatocyte proliferation. CXCL1, 2, 5 and 8 can induce endothelial cells chemotaxis to promote angiogenesis through binding to CXCR2. These diverse functions suggest that chemokines could play multifaceted roles in liver cancer development. However, chemokines that are commonly associated with liver cancer is still unknown.We analyzed HCC patient data from the GEO database, and we categorized the datasets based on HCC etiologies including HBV, HCV, alcoholic and NASH. We identified CXCL5 as the only chemokine consistently upregulated in HCC with different etiologies compared to healthy or cirrhotic livers. Immunohistochemistry (IHC) analysis reveals that CXCL5 was produced by immune cells but not tumor cells in human HCC tissues. To further study HCC associated CXCL5 expression, the liver-specific Pten deletion mouse model (PM mice) that recapitulates NAFLD-NASH-HCC progression was used. A gradual increase of hepatic CXCL5 expression is observed during HCC development, reaching nearly 100-fold upregulation of CXCL5 mRNA expression in 12-month-old PM mice livers carrying tumors. Examination of liver immune cell populations showed that macrophages were significantly enriched in Pten deleted livers bearing tumors than wild type livers without tumors. Flow cytometry and IHC analysis further identified Kupffer cells (KCs), the liver resident macrophages as the source of CXCL5 in tumor bearing livers using these mice. Since increased LPS is a prominent feature in most chronic liver diseases, we isolated and treated mouse KCs with LPS and found that LPS treatment robustly increased CXCL5 expression by nearly 20-fold. Interestingly, neither murine macrophage cell lines nor primary peritoneal macrophages displayed induced CXCL5 expression in response to LPS. These data suggest that induction of CXCL5 in KCs is likely a unique function of the KCs but not of other macrophages. To explore the function of CXCL5 in HCC development, we treated mouse hepatocytes and HCC cells with CXCL5 and showed that CXCL5 induces the proliferation of these cells. This effect is further blocked by the inhibition of CXCR2, the receptor of CXCL5, demonstrating the specificity for CXCL5 mediated effects. Together we show here for the first time that CXCL5 expression is a unique property of Kupffer cells and the induction of CXCL5 promotes HCC progression. Citation Format: Taojian Tu, Handan Hong, Lina He, Mario Alba, Curtis T. Okamoto, Bangyan L. Stiles. Kupffer cells secrete CXCL5 to promote liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1350.

Among various protein posttranslational modifiers, poly-ADP-ribose polymerase 1 (PARP1) is a key player for regulating numerous cellular processes and events through enzymatic attachments of target proteins with ADP-ribose units donated by nicotinamide adenine dinucleotide (NAD

Extracellular vesicles (EV) containing microRNAs (miRNAs) have tremendous potential as biomarkers for the early detection of disease. Here, we present a simple and rapid PCR-free integrated microfluidics platform capable of absolute quantification (, Zeinab Ramashani, Chenguang Zhang et al. developed a microfluidic device that allows for quantification of extracellular vesicle (EV) microRNA from plasma. The platform does not require EV extraction, so it is able to process samples in as little as 30 minutes and requires much smaller volumes of sample than competing platforms.

Protein poly-ADP-ribosylation (PARylation) is a heterogeneous and dynamic posttranslational modification regulated by various writers, readers, and erasers. It participates in a variety of biological events and is involved in many human diseases. Currently, tools and technologies have yet to be developed for unambiguously defining readers and erasers of individual PARylated proteins or cognate PARylated proteins for known readers and erasers. Here, we report the generation of a bifunctional nicotinamide adenine dinucleotide (NAD(+)) characterized by diazirine-modified adenine and clickable ribose. By serving as an excellent substrate for poly-ADP-ribose polymerase 1 (PARP1)-catalyzed PARylation, the generated bifunctional NAD(+) enables photocrosslinking and enrichment of PARylation-dependent interacting proteins for proteomic identification. This bifunctional NAD(+) provides an important tool for mapping cellular interaction networks centered on protein PARylation, which are essential for elucidating the roles of PARylation-based signals or activities in physiological and pathophysiological processes.

SOX9 (Sex-determining region Y Box 9) is a well-characterized transcription factor that is a marker for progenitor cells in various tissues. In the liver, cells delineated by SOX9 are responsible for regenerating liver parenchyma when cell proliferation is impaired following chronic injury. However, whether these SOX9+ cells play a role in liver carcinogenesis has not been fully understood, although high SOX9 expression has been linked to poor survival outcome in liver cancer patients. To address this question, we developed a liver cancer mouse model (PtenloxP/loxP; Sox9-CreERT+; R26RYFP) where tumor suppressor Pten (phosphatase and tensin homolog deleted on chromosome ten) is deleted in SOX9+ cells following tamoxifen injection. In this paper, we employ lineage-tracing to demonstrate the tumorigenicity potential of the Pten-, SOX9+ cells. We show that these cells are capable of giving rise to mixed-lineage tumors that manifest features of both hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (CCA). Our results suggest that PTEN loss induces the transformation of SOX9+ cells. We further show that to activate these transformed SOX9+ cells, the presence of liver injury is crucial. Liver injury, induced by hepatotoxin 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or high-fat diet (HFD), substantially increases tumor incidence and accelerates liver carcinogenesis from SOX9+ cells in Pten null mice but not in control mice. We further examine the mechanisms underlying tumor formation in this model to show that concurrent with the induction of niche signal (i.e., Wnt signaling), liver injury significantly stimulates the expansion of tumor-initiating cells (TICs). Together, these data show that (1) SOX9+ cells have the potential to become TICs following the primary transformation (i.e. Pten deletion) and that (2) liver injury is necessary for promoting the activation and proliferation of transformed SOX9+ cells, resulting in the genesis of mixed-lineage liver tumors.

High circulating lipids occurring in obese individuals and insulin resistant patients are considered a contributing factor to Type 2 Diabetes (T2D). Exposure to high lipids initially causes the beta-cells to expand in population. Long-term exposure to high lipids however is associated with failure of beta-cells and the development of T2D. To prevent the failure of beta-cells and development of Type 2 Diabetes, this study focuses on understanding the molecular mechanisms that underlie this biphasic response of beta-cells to lipid exposure. Using palmitic acid (PA) in cultured beta-cells and islets, we demonstrated that chronic exposure to lipids leads to reduced viability and inhibition of cell cycle progression concurrent with downregulation of a pro-growth/survival kinase AKT, independent of glucose. This AKT downregulation by PA treatment is correlated with a consistent induction of mTOR/S6K activity concurrent with AKT downregulation. Inhibiting mTOR activity restores AKT activity and allows beta-cells to gain proliferation capacity that are lost after high fat diet exposure. In summary, we elucidated a novel mechanism for which lipid exposure may cause the dipole effects on beta-cell growth, where mTOR acts as a lipid sensor. These mechanisms can be novel targets for future therapeutic developments.

Isoforms of protein kinase B (also known as AKT) play important roles in mediating insulin and growth factor signals. Previous studies have suggested that the AKT2 isoform is critical for insulin-regulated glucose metabolism, while the role of the AKT1 isoform remains less clear. This study focuses on the effects of AKT1 on the adaptive response of pancreatic β cells. Using a mouse model with inducible β-cell-specific deletion of the Akt1 gene (βA1KO mice), we showed that AKT1 is involved in high-fat-diet (HFD)-induced growth and survival of β cells but is unnecessary for them to maintain a population in the absence of metabolic stress. When unchallenged, βA1KO mice presented the same metabolic profile and β-cell phenotype as the control mice with an intact Akt1 gene. When metabolic stress was induced by HFD, β cells in control mice with intact Akt1 proliferated as a compensatory mechanism for metabolic overload. Similar effects were not observed in βA1KO mice. We further demonstrated that AKT1 protein deficiency caused endoplasmic reticulum (ER) stress and potentiated β cells to undergo apoptosis. Our results revealed that AKT1 protein loss led to the induction of eukaryotic initiation factor 2 α subunit (eIF2α) signaling and ER stress markers under normal-chow-fed conditions, indicating chronic low-level ER stress. Together, these data established a role for AKT1 as a growth and survival factor for adaptive β-cell response and suggest that ER stress induction is responsible for this effect of AKT1.

The orphan nuclear receptors estrogen-related receptors (ERRs) bind to the estrogen-related receptor response element (ERRE) to regulate transcriptional programs in cellular metabolism and cancer cell growth. In this study, we evaluated the potential for a pyrrole-imidazole polyamide to block ERRα binding to ERREs to inhibit gene expression. We demonstrated that the ERRE-targeted polyamide 1 blocked the binding of ERRα to the consensus ERRE and reduced the transcriptional activity of ERRα in cell culture. We further showed that inhibiting ERRα transcriptional activity with polyamide 1 led to reduced mitochondrial oxygen consumption, a primary biological effect regulated by ERRα. Finally, our data demonstrated that polyamide 1 is an inhibitor for cancer cell growth.

Liver cancer is hallmarked with chronic inflammation resulting from underlying liver diseases such as liver steatosis. Steatotic liver injury recruits inflammatory cells and establishes the tumor immune environment that can promote the development of liver cancer. Previously, we showed that macrophages produce growth factors to promote liver cancer growth in a genetic model where steatosis and liver tumors were induced by the deletion of tumor suppressor Pten (phosphatase and tensin homologue deleted on chromosome 10). In this model, we showed that attenuating steatosis and depletion of macrophages leads to inhibition of liver cancer development. To investigate the recruitment and functions of immune cells in liver cancer, we analyzed the expression profiles of these tumors as well as tumors from other mouse models together with human liver samples. Our analysis of the inflammatory cytokines identified one chemokine that was commonly upregulated in all tumor model and attenuated in non-tumor models. CXCL5 is a member of the neutrophil-activating chemokines. In the Pten deleted liver tumors, CXCL5 is significantly upregulated whereas its expression is inhibited when steatosis is attenuated via caloric restriction or deletion of a metabolic kinase, Akt2. We explored the cell types that may produce CXCL5. To our surprise, the upregulation of CXCL5 is concurrent with increases in macrophage but not neutrophil populations. We further observed significant upregulation of CXCL5 mRNA expression in Kupffer cells, the liver resident macrophages isolated from the Pten deleted mice. We explored the hypothesis that macrophages secrets CXCL5 to establish the tumor environment and promote tumor growth. Our data showed that CXCL5 treatment increased mouse hepatocytes and human HepG2 cells proliferation. This effect is blocked by inhibition of CXCR2, the receptor for CXCL5. Additionally, we explored the mechanisms of CXCL5 upregulation in Kupffer cells and discovered that lipopolysaccharide (LPS) induces the expression of CXCL5 by nearly 20-fold in Kupffer cells. This is in contrast with the lack of induction in murine macrophage cell lines and primary peritoneal macrophages. This unique response of Kupffer cells suggests that the source of CXCL5 in the Pten deletion mouse model is likely Kupffer cells. This data is collaborated by bioinformatic analysis of datasets where steatotic liver injury is induced by diet feeding. Furthermore, analysis of patient proteomic data detected CXCL5 as the primary chemokine that is induced in tumors among other neutrophil-activating peptides. In summary, our data identified CXCL5 as a novel chemokine produced by Kupffer cells that plays key roles in steatosis driven liver cancer development. Chronic liver diseases such as steatohepatitis can establish tumorigenesis environment through accumulation of LPS stimulated CXCL5 release from Kupffer cells. Citation Format: Bangyan L. Stiles, Taojian Tu, Lina He, Mario Alba. Steatosis promote liver cancer development by inducing chemokine production from Kupffer cells [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P048.

Obesity confers an independent risk for carcinogenesis. In the liver, steatosis often proceeds cancer formation; however, the mechanisms by which steatosis promotes carcinogenesis is unknown. We hypothesize that steatosis alters the microenvironment to promote proliferation of tumor initiating cells (TICs) and carcinogenesis. We used several liver cancer models to address the mechanisms underlying the role of obesity in cancer and verified these findings in patient populations. Using bioinformatics analysis and verified by biochemical assays, we identified that hepatosteatosis resulting from either Pten deletion or transgenic expression of HCV core/NS5A proteins, promotes the activation of Wnt/β-catenin. We verified that high fat diet lipid accumulation is also capable of inducing Wnt/β-catenin. Caloric restriction inhibits hepatosteatosis, reduces Wnt/β-catenin activation and blocks the expansion of TICs leading to complete inhibition of tumorigenesis without affecting the phosphatase and tensin homologue deleted on chromosome 10 (PTEN) loss regulated protein kinase B (AKT) activation. Pharmacological inhibition or loss of the Wnt/β-catenin signal represses TIC growth in vitro, and decreases the accumulation of TICs in vivo. In human liver cancers, ontology analysis of gene set enrichment analysis (GSEA)-defined Wnt signature genes indicates that Wnt signaling is significantly induced in tumor samples compared with healthy livers. Indeed, Wnt signature genes predict 90% of tumors in a cohort of 558 patient samples. Selective depletion of macrophages leads to reduction of Wnt and suppresses tumor development, suggesting infiltrating macrophages as a key source for steatosis-induced Wnt expression. These data established Wnt/β-catenin as a novel signal produced by infiltrating macrophages induced by steatosis that promotes growth of tumor progenitor cells, underlying the increased risk of liver tumor development in obese individuals.

Liver kinase B 1 (LKB1 or STK11) and phosphatase and tensin homologue deleted on chromosome 10 (PTEN) are two tumor suppressors that regulate the mammalian target of rapamycin signaling pathway. Deletion studies show that loss of either Lkb1 (Lkb+/–) or Pten (PtenloxP/loxP; Alb-Cre+) leads to liver injury and development of hepatocarcinoma. In this study, we investigated the crosstalk of LKB1 and PTEN loss during tumorigenesis and liver development. We show that haplo-insufficiency of Lkb1 in the liver leads to advanced tumor development in Pten-null mice (PtenloxP/loxP; LkbloxP/+; Alb-Cre+). Our analysis shows that LKB1 and PTEN interact with each other in their regulation of fatty acid synthase as well as p21 expression. The combined loss of LKB1 and PTEN (PtenloxP/loxP; LkbloxP/loxP; Alb-Cre+) also leads to the inability to form zonal structures in the liver. The lack of metabolic zonal structures is consistent with the inability of the livers to store glycogen as well as elevated plasma bilirubin and alanine aminotransferase, indicative of liver dysfunction. These structural and functional defects are associated with cytoplasm distribution of a canalicular membrane protein multidrug resistant protein 2, which is responsible for clearing bilirubin. This observed regulation of multidrug resistant protein 2 by LKB1 likely contributes to the lack of cellular polarity and the early lethality phenotype associated with the homozygous loss of Lkb1 alone or in combination with Pten. Finally, Pten deletion does not rescue the precocious ductal plate formation reported for Lkb1-deleted livers. Conclusion: Our study dissected the functional and molecular crosstalk of PTEN and LKB1 and elucidated key molecular targets for such interactions. (Hepatology Communications 2017;1:153-167)

Translocator protein (TSPO, 18kDa) levels increase in parallel with the evolution of simple steatosis (SS) to nonalcoholic steatohepatitis (NASH) in non-alcoholic fatty liver disease (NAFLD). However, TSPO function in SS and NASH is unknown. Loss of TSPO in hepatocytes in vitro downregulated acetyl-CoA acetyltransferase 2 (ACAT2) and increased free cholesterol (FC). FC accumulation induced endoplasmic reticulum stress via IRE1A and PERK/ATF4/CHOP pathways and autophagy. TSPO deficiency activated cellular adaptive antioxidant protection; this adaptation was lost upon excessive FC accumulation. 19-Atriol, a TSPO ligand, blocked cholesterol binding and recapitulated many of the alterations seen in TSPO-deficient cells. These data suggest that TSPO deficiency accelerated the progression of SS. In NASH, however, loss of TSPO ameliorated liver fibrosis through downregulation of bile acid synthesis by reducing CYP7A1 and CYP27A1 levels and increasing farnesoid X receptor expression. These studies indicate a dynamic and complex role for TSPO in the evolution of NAFLD.

Summary Translocator protein (TSPO, 18 kDa) levels increase in parallel with the evolution of simple steatosis (SS) to nonalcoholic steatohepatitis (NASH) in nonalcoholic fatty liver disease (NAFLD). However, TSPO function in SS and NASH is unknown. Loss of TSPO in hepatocytes in vitro downregulated acetyl-CoA acetyltransferase 2 and increased free cholesterol (FC). FC accumulation induced endoplasmic reticulum stress via IRE1A and protein kinase RNA-like ER kinase/ATF4/CCAAT-enhancer-binding protein homologous protein pathways and autophagy. TSPO deficiency activated cellular adaptive antioxidant protection; this adaptation was lost upon excessive FC accumulation. A TSPO ligand 19-Atriol blocked cholesterol binding and recapitulated many of the alterations seen in TSPO-deficient cells. These data suggest that TSPO deficiency accelerated the progression of SS. In NASH, however, loss of TSPO ameliorated liver fibrosis through downregulation of bile acid synthesis by reducing CYP7A1 and CYP27A1 levels and increasing farnesoid X receptor expression. These studies indicate a dynamic and complex role for TSPO in the evolution of NAFLD., Graphical abstract, Highlights • TSPO expression levels correlate with the progression of NAFLD • TSPO deficiency inhibits ACAT2 leading to FC accumulation • Loss of TSPO in hepatocytes leads to FC accumulation that promotes simple steatosis • Loss of TSPO attenuates liver fibrosis via downregulation of bile acid production, Molecular biology; Cell biology; Metabolomics

Insulin resistance–as observed in aging, diabetes, obesity, and other pathophysiological situations, affects brain function, for insulin signaling is responsible for neuronal glucose transport and control of energy homeostasis and is involved in the regulation of neuronal growth and synaptic plasticity. This study investigates brain metabolism and function in a liver-specific Phosphatase and Tensin Homologue (Pten) knockout mouse model (Liver-PtenKO), a negative regulator of insulin signaling. The Liver-PtenKO mouse model showed an increased flux of glucose into the liver–thus resulting in an overall hypoglycemic and hypoinsulinemic state–and significantly lower hepatic production of the ketone body beta-hydroxybutyrate (as compared with age-matched control mice). The Liver-PtenKO mice exhibited increased brain glucose uptake, improved rate of glycolysis and flux of metabolites in the TCA cycle, and improved synaptic plasticity in the hippocampus. Brain slices from both control- and Liver-PtenKO mice responded to the addition of insulin (in terms of pAKT/AKT levels), thereby neglecting an insulin resistance scenario. This study underscores the significance of insulin signaling in brain bioenergetics and function and helps recognize deficits in diseases associated with insulin resistance.

Macroautophagy, hereafter autophagy, is a catabolic process by which cells remove protein aggregates and damaged organelles for recycling. This process is important for maintaining cellular homeost...

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) encodes a 403-amino acid protein with an amino-terminal domain that shares sequence homology with the actin-binding protein tensin and the putative tyrosine-protein phosphatase auxilin. Crystal structure analysis of PTEN has revealed a C2 domain that binds to phospholipids in membranes and a phosphatase domain that displays dual-specific activity toward both tyrosine (Y), serine (S)/threonine (T), as well as lipid substrates in vitro. Characterized primarily as a lipid phosphatase, PTEN plays important roles in multiple cellular processes including cell growth/survival as well as metabolism.

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.

PTEN is a critical tumor suppressor whose dysregulation leads to metabolic disease and cancer. How these diseases are linked at a molecular level is poorly understood. Maf1 is a novel PTEN target that connects PTEN's ability to repress intracellular lipid accumulation with its tumor suppressor function. Maf1 represses the expression of rRNAs and tRNAs to restrain biosynthetic capacity and oncogenic transformation. Recent studies demonstrate that Maf1 also controls intracellular lipid accumulation. In animal models, dysregulation of RNA polymerase I- and III-dependent transcription, and subsequent upregulation of rRNAs and tRNAs, leads to altered lipid metabolism and storage. Together these results identify unexpected connections between RNA and lipid metabolism that may help explain the strong epidemiological association between obesity and cancer.

Autophagy is a catabolic process that functions in recycling and degrading cellular proteins, and is also induced as an adaptive response to the increased metabolic demand upon nutrient starvation. However, the prosurvival role of autophagy in response to metabolic stress due to deprivation of glutamine, the most abundant nutrient for mammalian cells, is not well understood. Here, we demonstrated that when extracellular glutamine was withdrawn, autophagy provided cells with sub-mM concentrations of glutamine, which played a critical role in fostering cell metabolism. Moreover, we uncovered a previously unknown connection between metabolic responses to ATG5 deficiency and glutamine deprivation, and revealed that WT and atg5 (-/-) MEFs utilized both common and distinct metabolic pathways over time during glutamine deprivation. Although the early response of WT MEFs to glutamine deficiency was similar in many respects to the baseline metabolism of atg5 (-/-) MEFs, there was a concomitant decrease in the levels of essential amino acids and branched chain amino acid catabolites in WT MEFs after 6 h of glutamine withdrawal that distinguished them from the atg5 (-/-) MEFs. Metabolomic profiling, oxygen consumption and pathway focused quantitative RT-PCR analyses revealed that autophagy and glutamine utilization were reciprocally regulated to couple metabolic and transcriptional reprogramming. These findings provide key insights into the critical prosurvival role of autophagy in maintaining mitochondrial oxidative phosphorylation and cell growth during metabolic stress caused by glutamine deprivation.

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.)

Background & Aims Intraductal papillary mucinous neoplasias (IPMNs) are precancerous cystic lesions that can develop into pancreatic ductal adenocarcinomas (PDACs). These large macroscopic lesions are frequently detected during medical imaging, but it is unclear how they form or progress to PDAC. We aimed to identify cells that form IPMNs and mutations that promote IPMN development and progression. Methods We generated mice with disruption of Pten specifically in ductal cells ( Sox9CreER T2 ; Pten flox/flox ; R26R YFP or Pten ΔDuct/ΔDuct mice) and used Pten ΔDuct/+ and Pten +/+ mice as controls. We also generated Kras G12D ; Pten ΔDuct/ΔDuct and Kras G12D ; Pten ΔDuct/+ mice. Pancreata were collected when mice were 28 weeks to 14.5 months old and analyzed by histology, immunohistochemistry, and electron microscopy. We performed multiplexed droplet digital polymerase chain reaction to detect spontaneous Kras mutations in Pten ΔDuct/ΔDuct mice and study the effects of Ras pathway activation on initiation and progression of IPMNs. We obtained 2 pancreatic sections from a patient with an invasive pancreatobiliary IPMN and analyzed the regions with and without the invasive IPMN (control tissue) by immunohistochemistry. Results Mice with ductal cell–specific disruption of Pten but not control mice developed sporadic, macroscopic, intraductal papillary lesions with histologic and molecular features of human IPMNs. Pten ΔDuct/ΔDuct mice developed IPMNs of several subtypes. In Pten ΔDuct/ΔDuct mice, 31.5% of IPMNs became invasive; invasion was associated with spontaneous mutations in Kras . Kras G12D ; Pten ΔDuct/ΔDuct mice all developed invasive IPMNs within 1 month. In Kras G12D ; Pten ΔDuct/+ mice, 70% developed IPMN, predominately of the pancreatobiliary subtype, and 63.3% developed PDAC. In all models, IPMNs and PDAC expressed the duct-specific lineage tracing marker yellow fluorescent protein. In immunohistochemical analyses, we found that the invasive human pancreatobiliary IPMN tissue had lower levels of PTEN and increased levels of phosphorylated (activated) ERK compared with healthy pancreatic tissue. Conclusions In analyses of mice with ductal cell–specific disruption of Pten , with or without activated Kras, we found evidence for a ductal cell origin of IPMNs. We also showed that PTEN loss and activated Kras have synergistic effects in promoting development of IPMN and progression to PDAC.

Since its discovery in 1997, phosphatase and tensin homologue deleted on chromosome 10 (PTEN) has become one of the most important molecules in tumor biology. Mutations, deletions or dysregulation of PTEN is found in many human tumors. Recent studies have extended the reach of PTEN to include diabetes and neurological diseases such as Parkinson's and autism. In this review, we summarize the traditionally characterized function of PTEN as the lipid phosphatase that dephosphorylates PI-3,4,5-P(3), and several other newly discovered functions. The inhibition of the phosphatidylinositol-3-kinase (PI3K)/AKT signaling pathway may account for most of PTEN's tumor suppressing function. However, other growth inhibiting functions of PTEN may not involve this pathway. PTEN can also inhibit growth through its protein phosphatase activity and in ways not related to its enzymatic activity at all. We survey the many functions and biochemical interactions of PTEN in cytoplasm, the nucleus and throughout the cell in this paper.

With their unique ability to differentiate into all cell types, embryonic stem (ES) cells hold great therapeutic promise. To improve the efficiency of embryoid body (EB)-mediated ES cell differentiation, we studied murine EBs on the basis of their size and found that EBs with an intermediate size (diameter 100–300 μm) are the most proliferative, hold the greatest differentiation potential, and have the lowest rate of cell death. In an attempt to promote the formation of this subpopulation, we surveyed several biocompatible substrates with different surface chemical parameters and identified a strong correlation between hydrophobicity and EB development. Using self-assembled monolayers of various lengths of alkanethiolates on gold substrates, we directly tested this correlation and found that surfaces that exhibit increasing hydrophobicity enrich for the intermediate-size EBs. When this approach was applied to the human ES cell system, similar phenomena were observed. Our data demonstrate that hydrophobic surfaces serve as a platform to deliver uniform EB populations and may significantly improve the efficiency of ES cell differentiation.

Background Hepatic 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. Results The 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. Conclusions Wnt/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.

Alcoholic liver disease is a significant contributor to global liver failure. In murine models, chronic ethanol consumption dysregulates PTEN/Akt signaling. Hepatospecific deletion of phosphatase and tensin homolog deleted on chromosome 10 (PTENLKO) mice possess constitutive activation of Akt(s) and increased de novo lipogenesis resulting in increased hepatocellular steatosis. This makes PTENLKO a viable model to examine the effects of ethanol in an environment of preexisting steatosis. The aim of this study was to determine the impact of chronic ethanol consumption and the absence of PTEN (PTENLKO) compared to Alb-Cre control mice (PTENf/f) on hepatocellular damage as evidenced by changes in lipid accumulation, protein carbonylation and alanine amino transferase (ALT). In the control PTENf/f animals, ethanol significantly increased ALT, liver triglycerides and steatosis. In contrast, chronic ethanol consumption in PTENLKO mice decreased hepatocellular damage when compared to PTENLKO pair-fed controls. Consumption of ethanol elevated protein carbonylation in PTENf/f animals but had no effect in PTENLKO animals. In PTENLKO mice, overall hepatic mRNA expression of genes that contribute to GSH homeostasis as well as reduced glutathione (GSH) and oxidized glutathione (GSSG) concentrations were significantly elevated compared to respective PTENf/f counterparts. These data indicate that during conditions of constitutive Akt activation and steatosis, increased GSH homeostasis assists in mitigation of ethanol-dependent induction of oxidative stress and hepatocellular damage. Furthermore, data herein suggest a divergence in EtOH-induced hepatocellular damage and increases in steatosis due to polyunsaturated fatty acids downstream of PTEN.

In injury conditions, myofibroblasts are induced to lay down matrix proteins and support the repair process. In this study, we investigated the role of myofibroblasts, particularly stellate cells, in the growth and regeneration of pancreatic β cells.We used both in vitro and in vivo approaches to address whether stellate cells may promote the growth of β cells.Our experiments demonstrated that activated stellate cells support the proliferation of β cells in vitro. In vivo, mesenchymals surrounding the pancreatic islets are activated (induced to proliferate) in the islet regeneration model of Pten null mice. These mesenchymals display markers of pancreatic stellate cells, such as desmin and to a lesser extent, smooth muscle actin α. We have shown previously that targeted β-cell deletion of Pten lead to a significant increase in total islet mass. This phenotype was accompanied by an increase in peri-islet mitotic activity, particularly in islets injured by streptozotocin, a β cell-specific toxin.Together with the in vitro observations, our data, here, suggest that that these mesenchymal cells may support the regeneration of the islets. Identifying how the communication occurs may provide clinically relevant mechanism for inducing β-cell regeneration.

Cholangiocellular carcinoma (CC), the second most common primary liver cancer, is associated with a poor prognosis. It has been shown that CCs harbor alterations of a number of tumor-suppressor genes and oncogenes, yet key regulators for tumorigenesis remain unknown. Here we have generated a mouse model that develops CC with high penetrance using liver-specific targeted disruption of tumor suppressors SMAD4 and PTEN. In the absence of SMAD4 and PTEN, hyperplastic foci emerge exclusively from bile ducts of mutant mice at 2 months of age and continue to grow, leading to tumor formation in all animals at 4-7 months of age. We show that CC formation follows a multistep progression of histopathological changes that are associated with significant alterations, including increased levels of phosphorylated AKT, FOXO1, GSK-3beta, mTOR, and ERK and increased nuclear levels of cyclin D1. We further demonstrate that SMAD4 and PTEN regulate each other through a novel feedback mechanism to maintain an expression balance and synergistically repress CC formation. Finally, our analysis of human CC detected PTEN inactivation in a majority of p-AKT-positive CCs, while about half also lost SMAD4 expression. These findings elucidate the relationship between SMAD4 and PTEN and extend our understanding of CC formation.

In adipose tissue, insulin controls glucose and lipid metabolism through the intracellular mediators phosphatidylinositol 3-kinase and serine-threonine kinase AKT. Phosphatase and a tensin homolog deleted from chromosome 10 (PTEN), a negative regulator of the phosphatidylinositol 3-kinase/AKT pathway, is hypothesized to inhibit the metabolic effects of insulin. Here we report the generation of mice lacking PTEN in adipose tissue. Loss of Pten results in improved systemic glucose tolerance and insulin sensitivity, associated with decreased fasting insulin levels, increased recruitment of the glucose transporter isoform 4 to the cell surface in adipose tissue, and decreased serum resistin levels. Mutant animals also exhibit increased insulin signaling and AMP kinase activity in the liver. Pten mutant mice are resistant to developing streptozotocin-induced diabetes. Adipose-specific Pten deletion, however, does not alter adiposity or plasma fatty acids. Our results demonstrate that in vivo PTEN is a potent negative regulator of insulin signaling and insulin sensitivity in adipose tissue. Furthermore, PTEN may be a promising target for nutritional and/or pharmacological interventions aimed at reversing insulin resistance.

High-fat diet (HFD)-induced obesity is associated with insulin resistance, which may affect brain synaptic plasticity through impairment of insulin-sensitive processes underlying neuronal survival, learning, and memory. The experimental model consisted of 3 month-old C57BL/6J mice fed either a normal chow diet (control group) or a HFD (60% of calorie from fat; HFD group) for 12 weeks. This model was characterized as a function of time in terms of body weight, fasting blood glucose and insulin levels, HOMA-IR values, and plasma triglycerides. IRS-1/Akt pathway was assessed in primary hepatocytes and brain homogenates. The effect of HFD in brain was assessed by electrophysiology, input/output responses and long-term potentiation. HFD-fed mice exhibited a significant increase in body weight, higher fasting glucose- and insulin levels in plasma, lower glucose tolerance, and higher HOMA-IR values. In liver, HFD elicited (a) a significant decrease of insulin receptor substrate (IRS-1) phosphorylation on Tyr608 and increase of Ser307 phosphorylation, indicative of IRS-1 inactivation; (b) these changes were accompanied by inflammatory responses in terms of increases in the expression of NFκB and iNOS and activation of the MAP kinases p38 and JNK; (c) primary hepatocytes from mice fed a HFD showed decreased cellular oxygen consumption rates (indicative of mitochondrial functional impairment); this can be ascribed partly to a decreased expression of PGC1α and mitochondrial biogenesis. In brain, HFD feeding elicited (a) an inactivation of the IRS-1 and, consequentially, (b) a decreased expression and plasma membrane localization of the insulin-sensitive neuronal glucose transporters GLUT3/GLUT4; (c) a suppression of the ERK/CREB pathway, and (d) a substantial decrease in long-term potentiation in the CA1 region of hippocampus (indicative of impaired synaptic plasticity). It may be surmised that 12 weeks fed with HFD induce a systemic insulin resistance that impacts profoundly on brain activity, i.e., synaptic plasticity.

PTEN is mutated at high frequency in many primary human cancers and several familial cancer predisposition disorders. Activation of AKT is a common event in tumors in which the PTEN gene has been inactivated. We previously showed that deletion of the murine Pten gene in embryonic stem (ES) cells led to increased phosphatidylinositol triphosphate (PIP(3)) accumulation, enhanced entry into S phase, and better cell survival. Since PIP(3) controls multiple signaling molecules, it was not clear to what degree the observed phenotypes were due to deregulated AKT activity. In this study, we mutated Akt-1 in Pten(-/-) ES cells to directly assess the role of AKT-1 in PTEN-controlled cellular processes, such as cell proliferation, cell survival, and tumorigenesis in nude mice. We showed that AKT-1 is one of the major downstream effectors of PTEN in ES cells and that activation of AKT-1 is required for both the cell survival and cell proliferation phenotypes observed in Pten(-/-) ES cells. Deletion of Akt-1 partially reverses the aggressive growth of Pten(-/-) ES cells in vivo, suggesting that AKT-1 plays an essential role in PTEN-controlled tumorigenesis.

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., Author Summary Obesity is a strong risk factor for human cancers, yet the biological basis for this is unclear. In addition to aberrant growth, abnormal lipid synthesis is a hallmark of cancer cells. Our results have identified a novel role for Maf1 in suppressing both lipid biogenesis and tumor formation. Maf1 elicits these biological responses through its ability to repress genes that that synthesize lipids and regulate biosynthetic capacity. Maf1 amounts are regulated through a critical cellular pathway involving PTEN/PI3K/Akt/FoxO1, which is deregulated in many human cancers. Our results support the idea that deregulation of this pathway in cancer cells results in decreases in cellular Maf1, resulting in both abnormal growth and lipid synthesis. Thus, Maf1 represents a novel link between lipid metabolism and oncogenic transformation providing a new molecular basis for the strong association between obesity and cancer.

Tumors from patients with high-grade aggressive prostate cancer (PCa) exhibit increased expression of monoamine oxidase A (MAOA), a mitochondrial enzyme that degrades monoamine neurotransmitters and dietary amines. Despite the association between MAOA and aggressive PCa, it is unclear how MAOA promotes PCa progression. Here, we found that MAOA functions to induce epithelial-to-mesenchymal transition (EMT) and stabilize the transcription factor HIF1α, which mediates hypoxia through an elevation of ROS, thus enhancing growth, invasiveness, and metastasis of PCa cells. Knockdown and overexpression of MAOA in human PCa cell lines indicated that MAOA induces EMT through activation of VEGF and its coreceptor neuropilin-1. MAOA-dependent activation of neuropilin-1 promoted AKT/FOXO1/TWIST1 signaling, allowing FOXO1 binding at the TWIST1 promoter. Importantly, the MAOA-dependent HIF1α/VEGF-A/FOXO1/TWIST1 pathway was activated in high-grade PCa specimens, and knockdown of MAOA reduced or even eliminated prostate tumor growth and metastasis in PCa xenograft mouse models. Pharmacological inhibition of MAOA activity also reduced PCa xenograft growth in mice. Moreover, high MAOA expression in PCa tissues correlated with worse clinical outcomes in PCa patients. These findings collectively characterize the contribution of MAOA in PCa pathogenesis and suggest that MAOA has potential as a therapeutic target in PCa.