Chronic ethanol-mediated hepatocyte apoptosis links to decreased TET1 and 5-hydroxymethylcytosine formation.

The 5-hydroxymethylcytosine (5hmc) is a newly identified epigenetic modification thought to be regulated by the TET family of proteins. Little information is available about how ethanol consumption may modulate 5hmC formation and alcoholic liver disease (ALD) progression. A rat ALD model was used to study 5hmC in relationship to hepatocyte apoptosis. Human ALD liver samples were also used to validate these findings. It was found that chronic ethanol feeding significantly reduced 5hmC formation in a rat ALD model. There were no significant changes in TET2 and TET3 between the control- and ethanol-fed animals. In contrast, methylcytosine dioxygenase TET1 (TET1) expression was substantially reduced in the ethanol-fed rats and was accompanied by increased hepatocyte apoptosis. Similarly, knockdown of TET1 in human hepatocyte-like cells also significantly promoted apoptosis. Down-regulation of TET1 resulted in elevated expression of the DNA damage marker, suggesting a role for 5hmc in hepatocyte DNA damage as well. Mechanistic studies revealed that inhibition of TET1 promoted apoptotic gene expression. Similarly, targeting TET1 activity by removing cosubstrate promoted apoptosis and DNA damage. Furthermore, treatment with 5-azacitidine significantly mimics these effects, suggesting that chronic ethanol consumption promotes hepatocyte apoptosis and DNA damage by diminishing TET1-mediated 5hmC formation and DNA methylation. In summary, the current study provides a novel molecular insight that TET1-mediated 5hmC is involved in hepatocyte apoptosis in ALD progression.-Ji, C., Nagaoka, K., Zou, J., Casulli, S., Lu, S., Cao, K. Y., Zhang, H., Iwagami, Y., Carlson, R. I., Brooks, K., Lawrence, J., Mueller, W., Wands, J. R., Huang, C.-K. Chronic ethanol-mediated hepatocyte apoptosis links to decreased TET1 and 5-hydroxymethylcytosine formation.

Aspartate ß-hydroxylase modulates cellular senescence through glycogen synthase kinase 3ß in hepatocellular carcinoma.

UNLABELLED: Aspartate ß-hydroxylase (ASPH) is an enzyme overexpressed in human hepatocellular carcinoma (HCC) tumors that participates in the malignant transformation process. We determined if ASPH was a therapeutic target by exerting effects on cellular senescence to retard HCC progression. ASPH knockdown or knockout was achieved by short hairpin RNAs or the CRISPR/Cas9 system, respectively, whereas enzymatic inhibition was rendered by a potent second-generation small molecule inhibitor of ASPH. Alterations of cell proliferation, colony formation, and cellular senescence were evaluated in human HCC cell lines. The potential mechanisms for activating cellular senescence were explored using murine subcutaneous and orthotopic xenograft models. Inhibition of ASPH expression and enzymatic activity significantly reduced cell proliferation and colony formation but induced tumor cell senescence. Following inhibition of ASPH activity, phosphorylation of glycogen synthase kinase 3ß and p16 expression were increased to promote senescence, whereas cyclin D1 and proliferating cell nuclear antigen were decreased to reduce cell proliferation. The mechanisms involved demonstrate that ASPH binds to glycogen synthase kinase 3ß and inhibits its subsequent interactions with protein kinase B and p38 upstream kinases as shown by coimmunoprecipitation. In vivo experiments demonstrated that small molecule inhibitor treatment of HCC bearing mice resulted in significant dose-dependent reduced tumor growth, induced phosphorylation of glycogen synthase kinase 3ß, enhanced p16 expression in tumor cells, and promoted cellular senescence. CONCLUSIONS: We have identified a new mechanism that promotes HCC growth and progression by modulating senescence of tumor cells; these findings suggest that ASPH enzymatic activity is a novel therapeutic target for HCC.

Tumor progression-related transmembrane protein aspartate-ß-hydroxylase is a target for immunotherapy of hepatocellular carcinoma.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) has a poor survival rate due to recurrent intrahepatic metastases and lack of effective adjuvant therapy. Aspartate-ß-hydroxylase (ASPH) is an attractive cellular target since it is a highly conserved transmembrane protein overexpressed in both murine and human HCC tumors, and promotes a malignant phenotype as characterized by enhanced tumor cell migration and invasion. METHODS: Dendritic cells (DCs), expanded and isolated from the spleen, were incubated with a cytokine cocktail to optimize IL-12 secretion and co-stimulatory molecule expression, then subsequently loaded with ASPH protein for immunization. Mice were injected with syngeneic BNL HCC tumor cells followed by subcutaneous inoculation with 5-10×10(5) ASPH loaded DCs using a prophylactic and therapeutic experimental approach. Tumor infiltrating lymphocytes (TILs) were characterized, and their role in producing anti-tumor effects determined. The immunogenicity of ASPH protein with respect to activating antigen specific CD4+ T cells derived from human peripheral blood mononuclear cells (PBMCs) was also explored. RESULTS: We found that immunotherapy with ASPH-loaded DCs suppressed and delayed established HCC and tumor growth when administered prophylactically. Ex-vivo re-stimulation experiments and in vivo depletion studies demonstrated that both CD4+ and CD8+ cells contributed to anti-tumor effects. Using PBMCs derived from healthy volunteers and HCC patients, we showed that ASPH stimulation led to significant development of antigen-specific CD4+ T-cells. CONCLUSIONS: Immunization with ASPH-loaded DCs has substantial anti-tumor effects which could reduce the risk of HCC recurrence.

Bio-nanoparticle based therapeutic vaccine induces immunogenic response against triple negative breast cancer.

Triple negative breast cancer (TNBC) is more aggressive and has a poorer prognosis than other sub-types of breast tumors. This study elucidates how aspartate beta-hydroxylase (ASPH) network promotes drug resistance, and immunotherapy targeting ASPH may improve the efficacy of Doxorubicin (DOX) therapy. An orthotopic model of breast cancer generated by 4T1 cells in immunocompetent mice was used to explore efficacy of immunotherapy in combination with DOX chemotherapy. We evaluated mRNA and protein expression in cultured tumor cells and tissue, as well as assessed cell proliferation, apoptosis, soluble factors/cytokine production, immune cell population diversity and function. We observed that ASPH expression enables TNBC cells to exhibit primary resistance to DOX induced single-/double-strand breaks (SSB/DSB) and enhanced proliferation and survival. Specific bio-nanoparticle based therapeutic vaccine (BNP-TV) promoted ASPH uptake by and maturation of DCs. This BNP-TV combined with DOX induces immunogenic cell death (ICD) in orthotopic xenograft tumors and significantly suppressed primary mammary tumor growth and distant multi-organ metastases. Immunogenic cell death induced by BNP-TV targeting ASPH combined with DOX provides opportunities to treat a highly resistant and metastatic form of breast cancer.

Anti-tumor activity of antibody drug conjugate targeting aspartate-ß-hydroxylase in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive malignancy with very limited treatment options. Antibody drug conjugates (ADCs) are promising cytotoxic agents capable of highly selective delivery. Aspartate-ß-hydroxylase (ASPH) is a type II transmembrane protein highly expressed in PDACs (97.1%) but not normal pancreas. We investigated anti-tumor effects of an ADC guided by a human monoclonal antibody (SNS-622) against ASPH in human PDAC cell lines and derived subcutaneous (s.c.) xenograft as well as a patient-derived xenograft (PDX) murine model with spontaneous pulmonary metastasis. The cytotoxic effects exhibited by several candidate payloads linked to SNS-622 antibody targeting ASPH+ PDACs were analyzed. After i.v. administration of SNS-622-emtansine (DM1) ADC, the primary PDAC tumor growth and progression (number and size of pulmonary metastases) were determined. The PDAC cell lines, s.c. and PDX tumors treated with ADC were tested for cell proliferation, cytotoxicity and apoptosis by MTS and immunohistochemistry (IHC) assays. SNS-622-DM1 construct has demonstrated optimal anti-tumor effects in vitro. In the PDX model of human PDAC, SNS-622-DM1 ADC exerted substantially inhibitory effects on tumor growth and pulmonary metastasis through attenuating proliferation and promoting apoptosis.

Aspartate beta-hydroxylase promotes cholangiocarcinoma progression by modulating RB1 phosphorylation.

Cholangiocarcinoma (CCA) is a highly lethal and aggressive disease. Recently, IDH1/2 mutations have been identified in approximately 20% of CCAs which suggests an involvement of 2-oxoglutarate (2-OG) -dependent dioxygenases in oncogenesis. We investigated if the 2-OG dependent dioxygenase, aspartate beta-hydroxylase (ASPH) was important in tumor development and growth. Immunoassays were used to clarify how ASPH modulates CCA progression by promoting phosphorylation of the retinoblastoma protein (RB1). A xenograft model was employed to determine the role of ASPH on CCA growth. Knockdown of ASPH expression inhibited CCA development and growth by reducing RB1 phosphorylation. Expression of ASPH promoted direct protein interaction between RB1, cyclin-dependent kinases, and cyclins. Treatment with 2-OG-dependent dioxygenase and ASPH inhibitors suppressed the interaction between RB1 and CDK4 as well as RB1 phosphorylation. Knockdown of ASPH expression inhibited CCA progression and RB1 phosphorylation in vivo and they were found to be highly expressed in human CCAs. Knockdown of ASPH expression altered CCA development by modulating RB1 phosphorylation, as one of the major factors regulating the growth of these tumors.

Role of aspartyl-(asparaginyl) beta-hydroxylase in placental implantation: Relevance to early pregnancy loss.

Aspartyl-(asparaginyl) beta-hydroxylase (AAH) is a type 2 transmembrane protein with catalytic activity that hydroxylates epidermal growth factor-like domains of proteins that have a functional role in cell motility and invasion. Extravillous cytotrophoblasts (CTB) are motile and invasive unpolarized epithelial cells that mediate early implantation through interaction with the endometrium. This study characterizes the potential role of AAH in CTB implantation using human placentas from (1) terminated pregnancies (n = 11), (2) normal term deliveries (n = 21), (3) spontaneous abortuses (n = 21), and (4) small-for-gestational-age (SGA) term deliveries (n = 21). The SGA cases all had established clinical histories of intrauterine growth restriction or preeclampsia. Formalin-fixed, paraffin-embedded sections of placenta were immunostained using the 15C7 monoclonal antibody generated to recombinant AAH. In addition, snap-frozen or RNAlater-preserved specimens (Ambion, Austin, TX) were used for RNA analysis of AAH expression by real-time quantitative reverse transcriptase-polymerase chain reaction and protein analysis by Western blotting. The immunohistochemical staining studies demonstrated AAH expression in amniocytes, villous CTB, syncytiotrophoblast, extravillous CTB, decidua, and endometrial glands at all gestational ages and in all 4 groups. Higher levels of AAH immunoreactivity were observed in extravillous CTB compared with villous CTB. Immunohistochemical staining and RNA analysis demonstrated abundant AAH expression in placental trophoblastic cells as well as in decidua and endometrial glands, with reduced expression in spontaneous abortion and SGA, suggesting that AAH may serve as a biomarker of impaired implantation. The high levels of AAH in decidua and endometrial glands suggest a role for this molecule in "receptivity" of endometrium.

Lambda phage-based vaccine induces antitumor immunity in hepatocellular carcinoma.

BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) is a difficult to treat tumor with a poor prognosis. Aspartate ß-hydroxylase (ASPH) is a highly conserved enzyme overexpressed on the cell surface of both murine and human HCC cells. METHODS: We evaluated therapeutic effects of nanoparticle lambda (λ) phage vaccine constructs against ASPH expressing murine liver tumors. Mice were immunized before and after subcutaneous implantation of a syngeneic BNL HCC cell line. Antitumor actively was assessed by generation of antigen specific cellular immune responses and the identification of tumor infiltrating lymphocytes. RESULTS: Prophylactic and therapeutic immunization significantly delayed HCC growth and progression. ASPH-antigen specific CD4+ and CD8+ lymphocytes were identified in the spleen of tumor bearing mice and cytotoxicity was directed against ASPH expressing BNL HCC cells. Furthermore, vaccination generated antigen specific Th1 and Th2 cytokine secretion by immune cells. There was widespread necrosis with infiltration of CD3+ and CD8+ T cells in HCC tumors of λ phage vaccinated mice compared to controls. Moreover, further confirmation of anti-tumor effects on ASPH expressing tumor cell growth were obtained in another murine syngeneic vaccine model with pulmonary metastases. CONCLUSIONS: These observations suggest that ASPH may serve as a highly antigenic target for immunotherapy.

Human aspartyl (asparaginyl) beta-hydroxylase monoclonal antibodies: potential biomarkers for pancreatic carcinoma.

INTRODUCTION: Pancreatic adenocarcinoma is among the top 10 leading causes of death due to cancer in the United States. The lack of reliable and sensitive biomarkers for this disease makes it difficult to render an early diagnosis. AIMS: To evaluate carcinoma-associated monoclonal antibodies (MoAbs), including AF-20, SF-25, and FB-50, for their binding specificity to pancreatic adenocarcinoma relative to normal pancreatic tissue. In addition, binding of the Th9 MoAb to human Reg 1 protein was studied because of its potential role in cell growth. METHODOLOGY: Adjacent histologic sections were immunostained with each of the MoAbs and graded on a scale of 0 to 4+, corresponding to the relative distribution and intensity of immunoreactivity within the tumor and normal adjacent tissue. RESULTS: Intense levels (grade 3 or 4) of FB50 immunoreactivity were detected in 19 of 19 tumors but not in normal adjacent pancreatic tissue. In addition, increased levels of FB50 immunoreactivity were detected in at least 75% of the tumor cells in 18 of the 19 cases. SF-25 immunoreactivity similarly distinguished pancreatic adenocarcinoma from normal pancreas in 14 of 19 cases. In contrast, AF20 immunoreactivity was detected in 6 of 19 pancreatic adenocarcinomas, and for the most part, the labeling was focal and of a low level. TH9 immunoreactivity was detected in 5 of 19 tumors but also in normal as well as inflamed adjacent pancreatic tissue. CONCLUSION: These results suggest that the FB50 and SF25 MoAbs represent excellent potential biomarkers of pancreatic adenocarcinoma and could be configured in an immunoassay for detecting pancreatic adenocarcinoma cells in biologic fluids.

LRH1 promotes pancreatic cancer metastasis.

The transcriptional factor liver receptor homolog 1 (LRH1) regulates pancreatic development, and may participate in pancreatic oncogenesis through activation of growth factor signaling transduction cascades. We measured transcriptional activity of ß-catenin in response to LRH1 stimulation by a Topflash reporter assay. The pancreatic cancer (PC) phenotype was then characterized by cell migration, wound healing, invasion, and sphere formation in vitro, as well as tumor formation and distant metastatic spread in vivo. We compared results between vector control and LRH1-overexpressing stable PC cell lines. In addition, tumor burden, angiogenesis, histologic characteristics, and hepatic spread were assessed in orthotopic and experimental liver metastatic murine models. Expression of downstream LRH1 related genes was evaluated by Western blot and immunohistochemistry in PC cell lines and human tumor specimens. Specific inhibition of LRH1 expression and function was accomplished by shRNAs "knockdown" experiments. It was found that LRH1 enhanced transcriptional activity of ß-catenin and the expression of downstream target genes (c-Myc, MMP2/9), as well as promoted migration, wound healing, invasion, and sphere formation of PC cell lines. Specific inhibition of LRH1 by shRNAs reduced cell migration, invasion, sphere formation and expression of c-Myc and MMP2/9 target genes. Mice injected with LRH1 overexpressing stable PC cells developed tumors with increased size and exhibited striking hepatic metastatic spread. More important, LRH1 was overexpressed in PC tumors compared to adjacent normal pancreas. Our findings demonstrate that LRH1 overexpression is associated with increased PC growth and metastatic spread, indicating that LRH1-targeted therapy could inhibit tumor progression.

LRH1 as a driving factor in pancreatic cancer growth.

Liver receptor homolog 1 (LRH1), directs the development and differentiation of embryonic pancreas, and is overexpressed in pancreatic cancer (PC). We hypothesized that LRH1 promotes PC growth. Cell proliferation and tumorigenicity in nude mice were compared between empty vector-transfected (control) and stable LRH1-overexpressed PC cell lines. The subsequent tumor burden, vasculature development, and histologic features were evaluated. LRH1 overexpression enhanced the expression of downstream target genes (cyclin D1/E1) and stimulated cell proliferation in PC cell lines. LRH1 upregulated cyclin E1 truncated T1/T2 isoforms expression which may occur through ERα-calpain1 signaling. Compared with the control, LRH1 overexpressing stable cells generated tumors with increased weight, proliferation index and enhanced angiogenesis. Cyclin D1/E1 and calpain1 were overexpressed in human PC tumors compared to adjacent normal pancreas. These observations demonstrate that LRH1 promotes PC growth and angiogenesis, suggesting that LRH1 is a driving factor in tumorigenesis and may serve as a potential therapeutic target.

Rat strain differences in susceptibility to alcohol-induced chronic liver injury and hepatic insulin resistance.

The finding of more severe steatohepatitis in alcohol fed Long Evans (LE) compared with Sprague Dawley (SD) and Fisher 344 (FS) rats prompted us to determine whether host factors related to alcohol metabolism, inflammation, and insulin/IGF signaling predict proneness to alcohol-mediated liver injury. Adult FS, SD, and LE rats were fed liquid diets containing 0% or 37% (calories) ethanol for 8 weeks. Among controls, LE rats had significantly higher ALT and reduced GAPDH relative to SD and FS rats. Among ethanol-fed rats, despite similar blood alcohol levels, LE rats had more pronounced steatohepatitis and fibrosis, higher levels of ALT, DNA damage, pro-inflammatory cytokines, ADH, ALDH, catalase, GFAP, desmin, and collagen expression, and reduced insulin receptor binding relative to FS rats. Ethanol-exposed SD rats had intermediate degrees of steatohepatitis, increased ALT, ADH and profibrogenesis gene expression, and suppressed insulin receptor binding and GAPDH expression, while pro-inflammatory cytokines were similarly increased as in LE rats. Ethanol feeding in FS rats only reduced IL-6, ALDH1-3, CYP2E1, and GAPDH expression in liver. In conclusion, susceptibility to chronic steatohepatitis may be driven by factors related to efficiency of ethanol metabolism and degree to which ethanol exposure causes hepatic insulin resistance and cytokine activation.

Ethanol inhibition of aspartyl-asparaginyl-beta-hydroxylase in fetal alcohol spectrum disorder: potential link to the impairments in central nervous system neuronal migration.

Fetal alcohol spectrum disorder (FASD) is caused by prenatal exposure to alcohol and associated with hypoplasia and impaired neuronal migration in the cerebellum. Neuronal survival and motility are stimulated by insulin and insulin-like growth factor (IGF), whose signaling pathways are major targets of ethanol neurotoxicity. To better understand the mechanisms of ethanol-impaired neuronal migration during development, we examined the effects of chronic gestational exposure to ethanol on aspartyl (asparaginyl)-beta-hydroxylase (AAH) expression, because AAH is regulated by insulin/IGF and mediates neuronal motility. Pregnant Long-Evans rats were pair-fed isocaloric liquid diets containing 0, 8, 18, 26, or 37% ethanol by caloric content from gestation day 6 through delivery. Cerebella harvested from postnatal day 1 pups were used to examine AAH expression in tissue, and neuronal motility in Boyden chamber assays. We also used cerebellar neuron cultures to examine the effects of ethanol on insulin/IGF-stimulated AAH expression, and assess the role of GSK-3beta-mediated phosphorylation on AAH protein levels. Chronic gestational exposure to ethanol caused dose-dependent impairments in neuronal migration and corresponding reductions in AAH protein expression in developing cerebella. In addition, prenatal ethanol exposure inhibited insulin and IGF-I-stimulated directional motility in isolated cerebellar granule neurons. Ethanol-treated neuronal cultures (50mMx96h) also had reduced levels of AAH protein. Mechanistically, we showed that AAH protein could be phosphorylated on Ser residues by GSK-3beta, and that chemical inhibition of GSK-3beta and/or global Caspases increases AAH protein in both control- and ethanol-exposed cells. Ethanol-impaired neuronal migration in FASD is associated with reduced AAH expression. Because ethanol increases the activities of both GSK-3beta and Caspases, the inhibitory effect of ethanol on neuronal migration could be mediated by increased GSK-3beta phosphorylation and Caspase degradation of AAH protein.