Dysregulation and oncogenic activities of ubiquitin specific peptidase 2a in the pathogenesis of hepatocellular carcinoma.

Ubiquitin specific peptidase 2a (USP2a) plays critical roles in protein degradation and other cellular activities. Currently, our understanding on USP2a dysregulation in subjects with hepatocellular carcinoma (HCC) and its roles in HCC pathogenesis is limited. In this study, we found that USP2a mRNA and protein levels were significantly upregulated in HCC tumors from both human and mice. USP2a overexpression in HepG2 and Huh 7 cells significantly increased cell proliferation while inhibition of USP2a activity by chemical inhibitor or stable knockout of USP2 by CRISPR markedly reduced cell proliferation. In addition, USP2a overexpression significantly augmented the resistance while knockout of USP2a markedly increased the susceptibility of HepG2 cells to bile acid-induced apoptosis and necrosis. Consistent with the oncogenic activities detected in vitro, overexpression of USP2a promoted de novo HCC development in mice with significantly increased tumor occurrence rates, tumor sizes and liver/body ratios. Further investigations with unbiased co-immunoprecipitation (Co-IP)-coupled proteomic analysis and Western blot identified novel USP2a target proteins involved in cell proliferation, apoptosis, and tumorigenesis. Analysis of those USP2a target proteins revealed that USP2a's oncogenic activities are mediated through multiple pathways, including modulating protein folding and assembling through regulating protein chaperones/co-chaperones HSPA1A, DNAJA1 and TCP1, promoting DNA replication and transcription through regulating RUVBL1, PCNA and TARDBP, and altering mitochondrial apoptotic pathway through regulating VDAC2. Indeed, those newly identified USP2a target proteins were markedly dysregulated in HCC tumors. In summary, USP2a was upregulated in HCC subjects and acted as an oncogene in the pathogenesis of HCC through multiple downstream pathways. The findings provided molecular and pathogenesis bases for developing interventions to treat HCC by targeting USP2a or its downstream pathways.

Dysregulation and activities of ubiquitin specific peptidase 2b in the pathogenesis of hepatocellular carcinoma.

Ubiquitin specific peptidase-2 (USP2) plays important roles in a myriad of cellular activities through deubiquitinating target proteins and its implications in various diseases, especially cancers, are starting to emerge. Our current understanding on USP2 expression in subjects with hepatocellular carcinoma (HCC) and its roles in the pathogenesis of HCC is limited. In this study, we found that USP2 protein and mRNA levels were significantly dysregulated in HCC tumor (HCC-T) when compared to adjacent non-tumor (HCC-NT) or normal liver tissues from both human and mouse HCC model. Among the USP2 isoforms, USP2b was the predominant isoform in the normal liver and markedly down-regulated in HCC-T tissues in both human and mice. Data from overexpression, chemical inhibition and knockout studies consistently demonstrated that USP2b promoted cell proliferation, colony formation and wound healing in HepG2 and Huh 7 cells. On the other hand, USP2b exhibited proapoptotic and pronecrtotic activities through enhancing bile acid-induced apoptosis and necrosis in both HepG2 and Huh 7 cells. Unbiased proteomic analysis of USP2-knockout (KO) and parental HepG2 cells resulted in identification of USP2-regulated downstream target proteins involved in cell proliferation, apoptosis, and tumorigenesis, including serine/threonine kinase 4 (STK4), epidermal growth factor receptor (EGFR), dipeptidyl peptidase 4 (DPP4) and fatty acid binding protein 1 (FABP1). In conclusion, USP2b expression was dysregulated in subjects with HCC and contributed to the pathogenesis of HCC by promoting cell proliferation and exerting proapoptotic and pronecrotic activities. The findings provide the molecular basis for developing therapies for HCC through modulating USP2b expression or activities.

Dysregulation of bile acids increases the risk for preterm birth in pregnant women.

Preterm birth (PTB) is the leading cause of perinatal mortality and newborn complications. Bile acids are recognized as signaling molecules regulating a myriad of cellular and metabolic activities but have not been etiologically linked to PTB. In this study, a hospital-based cohort study with 36,755 pregnant women is conducted. We find that serum total bile acid levels directly correlate with the PTB rates regardless of the characteristics of the subjects and etiologies of liver disorders. Consistent with the findings from pregnant women, PTB is successfully reproduced in mice with liver injuries and dysregulated bile acids. More importantly, bile acids dose-dependently induce PTB with minimal hepatotoxicity. Furthermore, restoring bile acid homeostasis by farnesoid X receptor activation markedly reduces PTB and dramatically improves newborn survival rates. The findings thus establish an etiologic link between bile acids and PTB, and open an avenue for developing etiology-based therapies to prevent or delay PTB.

Correction: Aspartate ß-hydroxylase expression promotes a malignant pancreatic cellular phenotype.

[This corrects the article DOI: 10.18632/oncotarget.2840.].

Dysregulation of Δ4-3-oxosteroid 5ß-reductase in diabetic patients: Implications and mechanisms.

Aldo-keto reductase family 1 member D1 (AKR1D1) is a Δ4-3-oxosteroid 5ß-reductase required for bile acid synthesis and steroid hormone metabolism. Both bile acids and steroid hormones, especially glucocorticoids, play important roles in regulating body metabolism and energy expenditure. Currently, our understanding on AKR1D1 regulation and its roles in metabolic diseases is limited. We found that AKR1D1 expression was markedly repressed in diabetic patients. Consistent with repressed AKR1D1 expression, hepatic bile acids were significantly reduced in diabetic patients. Mechanistic studies showed that activation of peroxisome proliferator-activated receptor-α (PPARα) transcriptionally down-regulated AKR1D1 expression in vitro in HepG2 cells and in vivo in mice. Consistently, PPARα signaling was enhanced in diabetic patients. In summary, dysregulation of AKR1D1 disrupted bile acid and steroid hormone homeostasis, which may contribute to the pathogenesis of diabetes. Restoring bile acid and steroid hormone homeostasis by modulating AKR1D1 expression may represent a new approach to develop therapies for diabetes.

Differential Feedback Regulation of Δ4-3-Oxosteroid 5ß-Reductase Expression by Bile Acids.

Δ4-3-oxosteroid 5ß-reductase is member D1 of the aldo-keto reductase family 1 (AKR1D1), which catalyzes 5ß-reduction of molecules with a 3-oxo-4-ene structure. Bile acid intermediates and most of the steroid hormones carry the 3-oxo-4-ene structure. Therefore, AKR1D1 plays critical roles in both bile acid synthesis and steroid hormone metabolism. Currently our understanding on transcriptional regulation of AKR1D1 under physiological and pathological conditions is very limited. In this study, we investigated the regulatory effects of primary bile acids, chenodeoxycholic acid (CDCA) and cholic acid (CA), on AKR1D1 expression. The expression levels of AKR1D1 mRNA and protein in vitro and in vivo following bile acid treatments were determined by real-time PCR and Western blotting. We found that CDCA markedly repressed AKR1D1 expression in vitro in human hepatoma HepG2 cells and in vivo in mice. On the contrary, CA significantly upregulated AKR1D1 expression in HepG2 cells and in mice. Further mechanistic investigations revealed that the farnesoid x receptor (FXR) signaling pathway was not involved in regulating AKR1D1 by bile acids. Instead, CDCA and CA regulated AKR1D1 through the mitogen-activated protein kinases/c-Jun N-terminal kinases (MAPK/JNK) signaling pathway. Inhibition of the MAPK/JNK pathway effectively abolished CDCA and CA-mediated regulation of AKR1D1. It was thus determined that AKR1D1 expression was regulated by CDCA and CA through modulating the MAPK/JNK signaling pathway. In conclusion, AKR1D1 expression was differentially regulated by primary bile acids through negative and positive feedback mechanisms. The findings indicated that both bile acid concentrations and compositions play important roles in regulating AKR1D1 expression, and consequently bile acid synthesis and steroid hormone metabolism.

Liver receptor homolog-1 (LRH-1): a potential therapeutic target for cancer.

Liver receptor homolog-1 (LRH-1) is a nuclear receptor involved in various biological processes. This nuclear receptor has critical functions in embryonic development as well as in adult homeostasis. Although the physiological functions of LRH-1 in normal breast, pancreas, and intestine have been widely investigated, the dysregulation that occurs during pathological conditions is not well understood. LRH-1 has been implicated in pancreatic, breast, and gastrointestinal cancer, where it exerts its effect of initiation and progression by promoting cell proliferation and metastasis. In addition to mechanistic studies, LRH-1 agonists and antagonists are being explored. Identification and development of endogenous and synthetic ligands has been pursued using computational-based structural analysis. Through ligand identification and a thorough understanding of the pathological roles of LRH-1, new therapeutic avenues for cancer treatment based upon LRH-1 may be a desirable focus for further research.

Estrogen and Estrogen Receptor-α-Mediated Transrepression of Bile Salt Export Pump.

Among diseases unique to pregnancy, intrahepatic cholestasis of pregnancy is the most prevalent disorder with elevated serum bile acid levels. We have previously shown that estrogen 17ß-estradiol (E2) transrepresses bile salt export pump (BSEP) through an interaction between estrogen receptor (ER)-α and farnesoid X receptor (FXR) and transrepression of BSEP by E2/ERα is an etiological contributing factor to intrahepatic cholestasis of pregnancy. Currently the mechanistic insights into such transrepression are not fully understood. In this study, the dynamics of coregulator recruitment to BSEP promoter after FXR activation and E2 treatment were established with quantitative chromatin immunoprecipitation assays. Coactivator peroxisome proliferator-activated receptor-γ coactivator-1 was predominantly recruited to the BSEP promoter upon FXR activation, and its recruitment was decreased by E2 treatment. Meanwhile, recruitment of nuclear receptor corepressor was markedly increased upon E2 treatment. Functional evaluation of ERα and ERß chimeras revealed that domains AC of ERα are the determinants for ERα-specific transrepression on BSEP. Further studies with various truncated ERα proteins identified the domains in ERα responsible for ligand-dependent and ligand-independent transrepression. Truncated ERα-AD exhibited potent ligand-independent transrepressive activity, whereas ERα-CF was fully capable of transrepressing BSEP ligand dependently in vitro in Huh 7 cells and in vivo in mice. Both ERα-AD and ERα-CF proteins were associated with FXR in the coimmunoprecipitation assays. In conclusion, E2 repressed BSEP expression through diminishing peroxisome proliferator-activated receptor-γ coactivator-1 recruitment with a concurrent increase in nuclear receptor corepressor recruitment to the BSEP promoter. Domains AD and CF in ERα mediated ligand-independent and ligand-dependent transrepression on BSEP, respectively, through interacting with FXR.

Aspartate ß-Hydroxylase expression promotes a malignant pancreatic cellular phenotype.

Pancreatic cancer (PC) is one of the leading causes of cancer related deaths due to aggressive progression and metastatic spread. Aspartate ß-hydroxylase (ASPH), a cell surface protein that catalyzes the hydroxylation of epidermal growth factor (EGF)-like repeats in Notch receptors and ligands, is highly overexpressed in PC. ASPH upregulation confers a malignant phenotype characterized by enhanced cell proliferation, migration, invasion and colony formation in vitro as well as PC tumor growth in vivo. The transforming properties of ASPH depend on enzymatic activity. ASPH links PC growth factor signaling cascades to Notch activation. A small molecule inhibitor of ß-hydroxylase activity was developed and found to reduce PC growth by downregulating the Notch signaling pathway. These findings demonstrate the critical involvement of ASPH in PC growth and progression, provide new insight into the molecular mechanisms leading to tumor development and growth and have important therapeutic implications.

Anti-bacterial glycosyl triazoles - Identification of an N-acetylglucosamine derivative with bacteriostatic activity against Bacillus.

N-acetylglucosaminidases (GlcNAcases) play an important role in the remodeling and recycling of bacterial peptidoglycan. Inhibitors of bacterial GlcNAcases can serve as antibacterial agents and provide an opportunity for the development of new antibiotics. We report the synthesis of triazole derivatives of N-acetylglucosamine using a copper promoted azide-alkyne coupling reaction between 1-azido-N-acetylglucosamine and a small library of terminal alkynes prepared via the Ugi reaction. These compounds were evaluated for their ability to inhibit the growth of bacteria. Two compounds that show bacteriostatic activity against Bacillus were identified, with MIC values of approximately 60 µM in both cases. Bacillus subtilis cultured in the presence of sub-MIC amounts of the glycosyl triazole inhibitors exhibit an elongated phenotype characteristic of impaired cell division. This represents the first report of inhibitors of bacterial cell wall GlcNAcases that demonstrate inhibition of cell growth in whole cell assays.

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.

Ang II-AT1R increases cell migration through PI3K/AKT and NF-κB pathways in breast cancer.

Angiotensin II (Ang II), a biologically active peptide of the renin-angiotensin system (RAS), plays an important role in promoting cell migration via Angiotensin II type 1 receptor (AT1R). In this study, we examined the mechanisms by which Ang II affected cell migration in AT1R-positive MDA-MB-231 human breast cancer cells. Ang II increased cell migration and expression of matrix metalloproteinase (MMP)-2,-9 in a dose-dependent manner. Ang II-mediated cell migration was reduced by specific blocking of MMP-2 and MMP-9, as well as with pretreatment with inhibitors of AT1R, phosphatidylinositol 3-kinase (PI3K), Akt, and NF-κB. Similarly, Ang II-mediated expression of MMP-2,-9 was downregulated by pretreatment with inhibitors of AT1R and PI3K. In addition, Ang II treatment significantly induced phosphorylation of PI3K, Akt, and resulted in increased NF-κB activity. These findings suggest that Ang II activates the AT1R/PI3K/Akt pathway, which further activates IKKα/ß and NF-κB, resulting in enhanced expression of MMP-2,-9 and migration in human breast cancer cells. Therefore, targeting Ang II/AT1R/PI3K/Akt/NF-κB signaling could be a novel anti-metastatic therapy for breast cancer.

Serum Helicobacter pylori NapA antibody as a potential biomarker for gastric cancer.

Helicobacter pylori (H. pylori) infection is strongly associated with gastric cancer. However, only a minority of infected individuals ever develop gastric cancer. This risk stratification may be in part due to differences among strains. The relationship between neutrophil-activating protein (NapA) and gastric cancer is unclear. The purpose of this study is to evaluate the significance of NapA as a biomarker in gastric cancer. We used enzyme linked immunosorbent assay (ELISA) to determine the status of H. pylori infection. Indirect ELISA method was used for detection of NapA antibody titer in the serum of H. pylori infected individuals. Unconditional logistic regressions were adopted to analyze the variables and determine the association of NapA and gastric cancer. The results of study indicated serum H. pylori NapA antibody level were associated with a reduced risk for development of gastric cancer. It may be used in conjugation with other indicators for gastric cancer detection.

The effects of fungal volatile organic compounds on bone marrow stromal cells.

Evidence has shown that individuals exposed to indoor toxic molds for extended periods of time have elevated risk of developing numerous respiratory illnesses. It is not clear at the cellular level what impact mold exposure has on the immune system. Herein, we show that 2 fungal volatiles (E)-2-octenal and oct-1-en-3-ol have cytotoxic effects on murine bone marrow stromal cells. To further analyze alterations to the cell, we evaluated the impact these volatile organic compounds have on membrane composition and hence fluidity. Both (E)-2-octenal and oct-1-en-3-ol exposure caused a shift to unsaturated fatty acids and lower cholesterol levels in the membrane. This indicates that the volatile organic compounds under investigation increased membrane fluidity. These vast changes to the cell membrane are known to contribute to the breakdown of normal cell function and possibly lead to death. Since bone marrow stromal cells are vital for the appropriate development and activation of immune cells, this study provides the foundation for understanding the mechanism at a cellular level for how mold exposure can lead to immune-related disease conditions.

Serum antibody against Helicobacter pylori FlaA and risk of gastric cancer.

BACKGROUND: Helicobacter pylori (H. pylori) infection is a major risk factor for gastric cancer (GC); however, only a minority of infected individuals develops GC. We aim to assess the association between serostatus of antibody against H. pylori flagellin A (FlaA) and risk of GC and to evaluate the value of serum FlaA antibody as a novel screening biomarker for GC risk. METHODS: A hospital-based case-control study including 232 cases and 264 controls was conducted. Logistic regression was adopted to analyze the association between the serostatus of FlaA antibody and risk of GC. Serum FlaA antibody was measured by an enzyme-linked immunosorbent assay (ELISA). Receiver operating characteristic (ROC) curve was used to evaluate the screening efficacy and to identify a cutoff point of serum FlaA antibody level. RESULTS: Helicobacter pylori infection was associated with an increased risk of GC (p = .007). A positive association between serum FlaA antibody and GC risk was observed in overall subjects and H. pylori-positive subjects (OR [95% CI]: 6.8 [4.3-10.7] and 6.9 [3.6-13.4], respectively; p < .001). The seropositivity of FlaA antibody was strongly related to GC risk in a dose-dependent manner (p for trend < .001). The optimal cutoff value (OD) was 0.1403, providing a sensitivity of 74.1% and a specificity of 64.4%. The area under the ROC curve (AUC) was 0.74 in overall subjects and 0.73 in H. pylori-positive subjects, respectively. CONCLUSIONS: FlaA was an independent risk factor for H. pylori-related GC. Serum FlaA antibody may serve as a novel noninvasive biomarker for early detection of GC.