Liver carcinoma is the 6th most prevalent cancer worldwide in 2020. Moreover, it is the 3rd leadi... more 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.
bioRxiv (Cold Spring Harbor Laboratory), Jun 22, 2023
SUMMARYsnoRNAs are a large family of noncoding (nc)RNAs present across eukaryotes and archaea. Wh... more SUMMARYsnoRNAs are a large family of noncoding (nc)RNAs present across eukaryotes and archaea. While a subset of them guide 2’-O- methylation (Nm) and pseudouridylation (Ψ) of rRNAs and snRNAs, targets of most snoRNAs remain unknown. Here we used PARIS2 to map snoRNA targets, revealing an extensive and conserved snoRNA-tRNA interaction network. Using optimized denatured RiboMeth-seq (dRMS), we discovered snoRNA-guided Nm sites in ncRNAs, including tRNAs. Loss of snoRNAs and their associated 2’-O-methyltransferase FBL reduced tRNA modifications and increased fragmentation. CRISPR knockout of the D97/D133 family of snoRNAs reduced the activity and levels of several target tRNAs, including elongator (e)Met-CAU, leading to codon-biased transcriptome and translatome in human cells. The codon-biased gene expression tipped the balance between the dichotomous cellular states of proliferation and differentiation, and skewed germ layer potential of mouse embryonic stem cells. Together, we discovered a snoRNA-guided tRNA modification mechanism controlling codon-biased gene expression and cellular states.
Among various protein posttranslational modifiers, poly-ADP-ribose polymerase 1 (PARP1) is a key ... more 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 +). Human PARP1 is involved in the pathogenesis and progression of many diseases. PARP1 inhibitors have received approvals for cancer treatment. Despite these successes, our understanding about PARP1 remains limited, partially due to the presence of various ADP-ribosylation reactions catalyzed by other PARPs and their overlapped cellular functions. Here we report a synthetic NAD + featuring an adenosyl 3 0-azido substitution. Acting as an ADP-ribose donor with high activity and specificity for human PARP1, this compound enables labelling and profiling of possible protein substrates of endogenous PARP1. It provides a unique and valuable tool for studying PARP1 in biology and pathology and may shed light on the development of PARP isoformspecific modulators.
Insulin resistance-as observed in aging, diabetes, obesity, and other pathophysiological situatio... more 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.
Protein poly-ADP-ribosylation (PARylation) is a heterogeneous and dynamic posttranslational modif... more 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.
The a-subunit of eukaryotic initiation factor 2 (eIF2a) is a key translation regulator that plays... more The a-subunit of eukaryotic initiation factor 2 (eIF2a) is a key translation regulator that plays an important role in cellular stress responses. In the present study, we investigated how eIF2a 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 eIF2a 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 H 2 O 2-induced cellular toxicity. We showed that the messenger level of CReP (constitutive repressor of eIF2a phosphorylation), a constitutive phosphatase of eIF2a, was downregulated in Pten-null hepatocytes, providing a possible mechanism through which PTEN/AKT pathway regulates eIF2a 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-eIF2a 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/eIF2a pathway. Mol Cancer Res; 9(12); 1708-17. Ó2011 AACR.
Members of estrogen-related receptors (ERRs) are orphan nuclear receptors (NRs) that play primary... more Members of estrogen-related receptors (ERRs) are orphan nuclear receptors (NRs) that play primary roles in mitochondrial biogenesis and bioenergetics. The ERRs regulate a range of cellular functions, including oxidative phosphorylation (OXPHOS) as well as glucose and lipid metabolism. ERRs are considered important targets for the treatment of metabolic diseases, particularly type II diabetes (T2D), insulin resistance (IR) and obesity. In this review, we will overview the transcriptional network regulated by the members of ERR transcriptional factors and elaborate on the regulation of ERR via its binding to PGC-1α, the primary co-activator of ERR as well as post-translational regulation of ERRs by upstream kinase signals. Recent development in ERR's cellular function has identified lipid metabolism/lipogenesis as a process that ERR regulates, and this function significantly impacts metabolic syndrome. Here, we will focus on their roles in lipid metabolic regulation and discuss the in vivo functions of ERRs in the development of non-alcoholic fatty liver disease (NAFLD), a comorbid metabolic syndrome concurrent with T2D, IR as well as obesity. Finally, we will explore ERRs as potential therapeutic targets by discussing the ligands that serve as antagonist/agonists for ERRs as well as efforts that target DNA binding of ERR as a transcriptional factor.
Estrogen-related receptors (ERRs) are orphan nuclear receptors identified based on their high seq... more Estrogen-related receptors (ERRs) are orphan nuclear receptors identified based on their high sequence similarity to estrogen receptors (ERs), but ERRs do not have a known endogenous ligand. ERRs play a primary role in regulating the transcription of genes involved in mitochondrial and lipid metabolism and are abundantly expressed across tissues. Because of ERR’s role in metabolism, it is suggested that they may also play a role in tumor metabolism, where dysfunction in lipid metabolism promotes tumor cell growth. The ERR subfamily is comprised of three isoforms: ERRα, ERRβ, and ERRγ. Studies targeting the ERR isoforms found that the absence of ERRα presents obesity and insulin resistance with an increase in bone mass, deletion of ERRβ causes placental abnormalities and embryonic lethality, and deletion of ERRγ leads to mitochondrial dysfunction. Together, these studies strongly suggest that ERRs, particularly ERR α and γ, function primarily as metabolic regulators, with ERRα being the predominant isoform expressed in the liver. In addition, the mechanisms leading to lipid accumulation vary under different feeding conditions. Previous studies from our lab showed that PTEN and PI3K/AKT signaling regulates ERRa expression and its function. Furthermore, we demonstrated that inhibiting ERRα blocks liver steatosis and steatohepatitis developed in a mouse model where loss of tumor suppressor PTEN drives both steatosis and cancer development. In addition, we found that ERR-PA, a small molecule inhibitor for ERR, attenuated cancer cell growth and proliferation in both mouse hepatocytes and human cancer cell lines. Here, we report transcriptome network regulated by ERRα under different metabolic conditions and further explored its regulation by the PI3K/AKT pathway. A better understanding of the role ERRα plays in physiology will allow further development of ERR-PA as a potential therapy for liver steatohepatitis, which progresses to end stage cirrhosis and ultimately liver cancer. Citation Format: Brittney A. Hua, Chien-yu Chen, Yang Li, Lina He, Bangyan Stiles, Ielyzaveta Slarve. Investigating the transcriptional regulation by estrogen-related receptor alpha (ERRα) under different metabolic conditions. [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 4847.
4566 Hepatocellular carcinoma (HCC) is the 5th most common cause of cancer related mortality worl... more 4566 Hepatocellular carcinoma (HCC) is the 5th most common cause of cancer related mortality worldwide. The disease progression for HCC is biphasic. The first stage consists of lipid accumulation in the liver, which progresses to the final stage characterized by tumor development. A number of studies have demonstrated a high correlation between PTEN loss and HCC development. PTEN (phosphatase and tensin homologue deleted on chromosome ten) is a negative regulator of the insulin signaling pathway. Hepatic insulin signaling plays a central role in glucose and lipid metabolism. The actions of insulin are mediated via cell surface receptors which in the presence of growth factor activate the phosphatidylinositol-3 kinase (PI3K) pathway. PI3K generates phosphatidylinositol, second messengers which can activate a number of downstream targets including the serine/threonine kinase (AKT). AKT regulates a number of key metabolic proteins including the mammalian target of rapamycin (mTOR) and AMP-activated kinase (AMPK). The AMPK pathway is an evolutionarily conserved sensor of cellular energy status. Activation of this pathway (via phosphorylation by upstream kinase LKB1) promotes ATP production and attenuation of ATP consuming biosynthetic processes. To better understand the molecular interactions between the PTEN and AMPK energy signaling pathways we have crossed PtenloxP/loxP;Alb-Cre+ mice with Lkb1loxP/loxP mice to obtain PtenloxP/loxP ; Lkb1loxP/loxP ;Cre+ mice, which are deficient in both PTEN and LKB1. Liver specific Pten null animals exhibit bile duct hyperplasia and develop liver cancer of both cholangiocyte and hepatocyte origin by 12 months of age. Because Pten deletion is consequential to decreased activation of the AMPK pathway via the actions of AKT, we hypothesize that concomitant deletion of Pten and Lkb1 will lead to advanced tumorigenesis observed in Pten null mice alone. Mice which harbor liver specific deletions of both of Pten and Lkb1 demonstrate lethality at weaning age (21-30 days). Phenotypically, double mutant mice weigh roughly one third as much as wildtype cohorts. In addition systemic glucose levels exhibited by double mutant mice in the fed state are a third to half that observed in control littermates. Therefore, we hypothesize that concurrent deletion of Ptenand Lkb1 in the liver induces hypoglycemia. Due to the observed lethality of homozygous mice, Lkb1loxP/+;PtenloxP/loxP;Alb-Cre+ mice will be used to acertain that Pten deletion and a bypass of AMPK energy sensing pathway may act synergistically to regulate the progression of HCC.
Liver malignancies consist of hepatocellular carcinoma (HCC) with the highest occurrence, intrahe... more 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.
The amounts of the intracellular glycosylation, O-GlcNAc modification, are increased in essential... more The amounts of the intracellular glycosylation, O-GlcNAc modification, are increased in essentially all tumors when compared to healthy tissue, and lowering O-GlcNAcylation levels results in reduced tumorigenesis and increased cancer cell death. Therefore, the pharmacological reduction of O-GlcNAc may represent a therapeutic vulnerability. The most direct approach to this goal is the inhibition of O-GlcNAc transferase (OGT), the enzyme that directly adds the modification to proteins. However, despite some recent success, this enzyme has proven difficult to inhibit. An alternative strategy involves starving OGT of its sugar substrate UDP-GlcNAc by targeting enzymes of the hexosamine biosynthetic pathway (HBP). Here, we explore the potential of the rate determining enzyme of this pathway, glutamine fructose-6-phosphate amidotransferase (GFAT). We first show that CRISPR-mediated knockout of GFAT results in inhibition of cancer cell growth in vitro and a xenograft model that correlates with O-GlcNAcylation levels. We then demonstrate that pharmacological inhibition of GFAT sensitizes a small panel of cancer cells to undergo apoptosis in response to diamide-induced oxidative stress. Finally, we find that GFAT expression and O-GlcNAc levels are increased in a spontaneous mouse model of liver cancer. Together these experiments support the further development of inhibitors of the HBP as an indirect approach to lowering O-GlcNAcylation levels in cancer.
Liver cancer is one of the most common malignant tumors. It is reported to be the third most leth... more Liver cancer is one of the most common malignant tumors. It is reported to be the third most lethal malignancy worldwide. Recent studies including our own identified CD133+ cell population as the tumor initiating cells for liver cancer. Tumor suppressor PTEN (phosphatase and tensin homologue deleted on chromosome ten) is aberrantly expressed in liver cancers. Liver specific Pten (Pm) null mice develop liver cancer following an extensive phase of chronic lipid accumulation and demonstrate escalating levels of hepatic injury markers from 6-12M, prior to hepatic progenitor cell proliferation. Concomitantly, expression of mRNA levels for Wnt ligands and receptors also increased progressively. Wnt/β-Catenin signaling pathway has various roles in regulating embryonic development and tumorigenesis. In Pten null liver progenitor cell line and tissues; we observed high levels of βcatenin, a downstream target component of the Wnt signaling pathway compared to control cells. In this study, we ...
Liver carcinoma is the 6th most prevalent cancer worldwide in 2020. Moreover, it is the 3rd leadi... more 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.
bioRxiv (Cold Spring Harbor Laboratory), Jun 22, 2023
SUMMARYsnoRNAs are a large family of noncoding (nc)RNAs present across eukaryotes and archaea. Wh... more SUMMARYsnoRNAs are a large family of noncoding (nc)RNAs present across eukaryotes and archaea. While a subset of them guide 2’-O- methylation (Nm) and pseudouridylation (Ψ) of rRNAs and snRNAs, targets of most snoRNAs remain unknown. Here we used PARIS2 to map snoRNA targets, revealing an extensive and conserved snoRNA-tRNA interaction network. Using optimized denatured RiboMeth-seq (dRMS), we discovered snoRNA-guided Nm sites in ncRNAs, including tRNAs. Loss of snoRNAs and their associated 2’-O-methyltransferase FBL reduced tRNA modifications and increased fragmentation. CRISPR knockout of the D97/D133 family of snoRNAs reduced the activity and levels of several target tRNAs, including elongator (e)Met-CAU, leading to codon-biased transcriptome and translatome in human cells. The codon-biased gene expression tipped the balance between the dichotomous cellular states of proliferation and differentiation, and skewed germ layer potential of mouse embryonic stem cells. Together, we discovered a snoRNA-guided tRNA modification mechanism controlling codon-biased gene expression and cellular states.
Among various protein posttranslational modifiers, poly-ADP-ribose polymerase 1 (PARP1) is a key ... more 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 +). Human PARP1 is involved in the pathogenesis and progression of many diseases. PARP1 inhibitors have received approvals for cancer treatment. Despite these successes, our understanding about PARP1 remains limited, partially due to the presence of various ADP-ribosylation reactions catalyzed by other PARPs and their overlapped cellular functions. Here we report a synthetic NAD + featuring an adenosyl 3 0-azido substitution. Acting as an ADP-ribose donor with high activity and specificity for human PARP1, this compound enables labelling and profiling of possible protein substrates of endogenous PARP1. It provides a unique and valuable tool for studying PARP1 in biology and pathology and may shed light on the development of PARP isoformspecific modulators.
Insulin resistance-as observed in aging, diabetes, obesity, and other pathophysiological situatio... more 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.
Protein poly-ADP-ribosylation (PARylation) is a heterogeneous and dynamic posttranslational modif... more 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.
The a-subunit of eukaryotic initiation factor 2 (eIF2a) is a key translation regulator that plays... more The a-subunit of eukaryotic initiation factor 2 (eIF2a) is a key translation regulator that plays an important role in cellular stress responses. In the present study, we investigated how eIF2a 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 eIF2a 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 H 2 O 2-induced cellular toxicity. We showed that the messenger level of CReP (constitutive repressor of eIF2a phosphorylation), a constitutive phosphatase of eIF2a, was downregulated in Pten-null hepatocytes, providing a possible mechanism through which PTEN/AKT pathway regulates eIF2a 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-eIF2a 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/eIF2a pathway. Mol Cancer Res; 9(12); 1708-17. Ó2011 AACR.
Members of estrogen-related receptors (ERRs) are orphan nuclear receptors (NRs) that play primary... more Members of estrogen-related receptors (ERRs) are orphan nuclear receptors (NRs) that play primary roles in mitochondrial biogenesis and bioenergetics. The ERRs regulate a range of cellular functions, including oxidative phosphorylation (OXPHOS) as well as glucose and lipid metabolism. ERRs are considered important targets for the treatment of metabolic diseases, particularly type II diabetes (T2D), insulin resistance (IR) and obesity. In this review, we will overview the transcriptional network regulated by the members of ERR transcriptional factors and elaborate on the regulation of ERR via its binding to PGC-1α, the primary co-activator of ERR as well as post-translational regulation of ERRs by upstream kinase signals. Recent development in ERR's cellular function has identified lipid metabolism/lipogenesis as a process that ERR regulates, and this function significantly impacts metabolic syndrome. Here, we will focus on their roles in lipid metabolic regulation and discuss the in vivo functions of ERRs in the development of non-alcoholic fatty liver disease (NAFLD), a comorbid metabolic syndrome concurrent with T2D, IR as well as obesity. Finally, we will explore ERRs as potential therapeutic targets by discussing the ligands that serve as antagonist/agonists for ERRs as well as efforts that target DNA binding of ERR as a transcriptional factor.
Estrogen-related receptors (ERRs) are orphan nuclear receptors identified based on their high seq... more Estrogen-related receptors (ERRs) are orphan nuclear receptors identified based on their high sequence similarity to estrogen receptors (ERs), but ERRs do not have a known endogenous ligand. ERRs play a primary role in regulating the transcription of genes involved in mitochondrial and lipid metabolism and are abundantly expressed across tissues. Because of ERR’s role in metabolism, it is suggested that they may also play a role in tumor metabolism, where dysfunction in lipid metabolism promotes tumor cell growth. The ERR subfamily is comprised of three isoforms: ERRα, ERRβ, and ERRγ. Studies targeting the ERR isoforms found that the absence of ERRα presents obesity and insulin resistance with an increase in bone mass, deletion of ERRβ causes placental abnormalities and embryonic lethality, and deletion of ERRγ leads to mitochondrial dysfunction. Together, these studies strongly suggest that ERRs, particularly ERR α and γ, function primarily as metabolic regulators, with ERRα being the predominant isoform expressed in the liver. In addition, the mechanisms leading to lipid accumulation vary under different feeding conditions. Previous studies from our lab showed that PTEN and PI3K/AKT signaling regulates ERRa expression and its function. Furthermore, we demonstrated that inhibiting ERRα blocks liver steatosis and steatohepatitis developed in a mouse model where loss of tumor suppressor PTEN drives both steatosis and cancer development. In addition, we found that ERR-PA, a small molecule inhibitor for ERR, attenuated cancer cell growth and proliferation in both mouse hepatocytes and human cancer cell lines. Here, we report transcriptome network regulated by ERRα under different metabolic conditions and further explored its regulation by the PI3K/AKT pathway. A better understanding of the role ERRα plays in physiology will allow further development of ERR-PA as a potential therapy for liver steatohepatitis, which progresses to end stage cirrhosis and ultimately liver cancer. Citation Format: Brittney A. Hua, Chien-yu Chen, Yang Li, Lina He, Bangyan Stiles, Ielyzaveta Slarve. Investigating the transcriptional regulation by estrogen-related receptor alpha (ERRα) under different metabolic conditions. [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 4847.
4566 Hepatocellular carcinoma (HCC) is the 5th most common cause of cancer related mortality worl... more 4566 Hepatocellular carcinoma (HCC) is the 5th most common cause of cancer related mortality worldwide. The disease progression for HCC is biphasic. The first stage consists of lipid accumulation in the liver, which progresses to the final stage characterized by tumor development. A number of studies have demonstrated a high correlation between PTEN loss and HCC development. PTEN (phosphatase and tensin homologue deleted on chromosome ten) is a negative regulator of the insulin signaling pathway. Hepatic insulin signaling plays a central role in glucose and lipid metabolism. The actions of insulin are mediated via cell surface receptors which in the presence of growth factor activate the phosphatidylinositol-3 kinase (PI3K) pathway. PI3K generates phosphatidylinositol, second messengers which can activate a number of downstream targets including the serine/threonine kinase (AKT). AKT regulates a number of key metabolic proteins including the mammalian target of rapamycin (mTOR) and AMP-activated kinase (AMPK). The AMPK pathway is an evolutionarily conserved sensor of cellular energy status. Activation of this pathway (via phosphorylation by upstream kinase LKB1) promotes ATP production and attenuation of ATP consuming biosynthetic processes. To better understand the molecular interactions between the PTEN and AMPK energy signaling pathways we have crossed PtenloxP/loxP;Alb-Cre+ mice with Lkb1loxP/loxP mice to obtain PtenloxP/loxP ; Lkb1loxP/loxP ;Cre+ mice, which are deficient in both PTEN and LKB1. Liver specific Pten null animals exhibit bile duct hyperplasia and develop liver cancer of both cholangiocyte and hepatocyte origin by 12 months of age. Because Pten deletion is consequential to decreased activation of the AMPK pathway via the actions of AKT, we hypothesize that concomitant deletion of Pten and Lkb1 will lead to advanced tumorigenesis observed in Pten null mice alone. Mice which harbor liver specific deletions of both of Pten and Lkb1 demonstrate lethality at weaning age (21-30 days). Phenotypically, double mutant mice weigh roughly one third as much as wildtype cohorts. In addition systemic glucose levels exhibited by double mutant mice in the fed state are a third to half that observed in control littermates. Therefore, we hypothesize that concurrent deletion of Ptenand Lkb1 in the liver induces hypoglycemia. Due to the observed lethality of homozygous mice, Lkb1loxP/+;PtenloxP/loxP;Alb-Cre+ mice will be used to acertain that Pten deletion and a bypass of AMPK energy sensing pathway may act synergistically to regulate the progression of HCC.
Liver malignancies consist of hepatocellular carcinoma (HCC) with the highest occurrence, intrahe... more 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.
The amounts of the intracellular glycosylation, O-GlcNAc modification, are increased in essential... more The amounts of the intracellular glycosylation, O-GlcNAc modification, are increased in essentially all tumors when compared to healthy tissue, and lowering O-GlcNAcylation levels results in reduced tumorigenesis and increased cancer cell death. Therefore, the pharmacological reduction of O-GlcNAc may represent a therapeutic vulnerability. The most direct approach to this goal is the inhibition of O-GlcNAc transferase (OGT), the enzyme that directly adds the modification to proteins. However, despite some recent success, this enzyme has proven difficult to inhibit. An alternative strategy involves starving OGT of its sugar substrate UDP-GlcNAc by targeting enzymes of the hexosamine biosynthetic pathway (HBP). Here, we explore the potential of the rate determining enzyme of this pathway, glutamine fructose-6-phosphate amidotransferase (GFAT). We first show that CRISPR-mediated knockout of GFAT results in inhibition of cancer cell growth in vitro and a xenograft model that correlates with O-GlcNAcylation levels. We then demonstrate that pharmacological inhibition of GFAT sensitizes a small panel of cancer cells to undergo apoptosis in response to diamide-induced oxidative stress. Finally, we find that GFAT expression and O-GlcNAc levels are increased in a spontaneous mouse model of liver cancer. Together these experiments support the further development of inhibitors of the HBP as an indirect approach to lowering O-GlcNAcylation levels in cancer.
Liver cancer is one of the most common malignant tumors. It is reported to be the third most leth... more Liver cancer is one of the most common malignant tumors. It is reported to be the third most lethal malignancy worldwide. Recent studies including our own identified CD133+ cell population as the tumor initiating cells for liver cancer. Tumor suppressor PTEN (phosphatase and tensin homologue deleted on chromosome ten) is aberrantly expressed in liver cancers. Liver specific Pten (Pm) null mice develop liver cancer following an extensive phase of chronic lipid accumulation and demonstrate escalating levels of hepatic injury markers from 6-12M, prior to hepatic progenitor cell proliferation. Concomitantly, expression of mRNA levels for Wnt ligands and receptors also increased progressively. Wnt/β-Catenin signaling pathway has various roles in regulating embryonic development and tumorigenesis. In Pten null liver progenitor cell line and tissues; we observed high levels of βcatenin, a downstream target component of the Wnt signaling pathway compared to control cells. In this study, we ...
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