The Effects and Underlying Mechanisms of Hepatitis B Virus X Gene Mutants on the Development of Hepatocellular Carcinoma

Background: We aimed to elucidate the mechanism by which hepatitis B virus X (HBx) gene mutations increase the risk of hepatocellular carcinoma (HCC) and identify novel therapeutic targets.Methods: Wild-type and four HBx mutants (M1, A1762T/G1764A; M2, T1674G+T1753C+A1762T/G1764A; M3, C1653T+T1674G+A1762T/G1764A; Ct-HBx, carboxylic acid-terminal truncated HBx) were delivered into the livers of fumarylacetoacetate hydrolase-deficient mice by using the Sleeping Beauty (SB) transposon system, respectively. Seven liver tissues and seven tumor tissues of the SB mouse models were subjected to HBV-capture sequencing. Three liver tissues from WT-HBx mice, three tumor tissues from M3-HBx mice, and three tumor tissues from Ct-HBx mice were subjected to cDNA microarray analysis. HeLa cells stably expressing WT-HBx and the four HBx mutants were also subjected to cDNA microarray assay.Results: The incidence of HCC was higher in the mice injected with M3-HBx or Ct-HBx. M3-HBx had a stronger capacity of upregulating inflammatory cytokines than other HBx variants. HBV-capture sequencing showed that the HBx fragments were mainly integrated into intergenic and intron regions. No significant difference was observed in the number of insertion sites between tumors and liver tissues. Ectopic expression of the HBx mutants, especially M3-HBx and Ct-HBx, significantly increased cell proliferation and the S phase proportion of HepG2 and HeLa cells, compared to WT-HBx. Liver tissues of the SB mice and the transfected cells were subjected to cDNA microarray analysis. Plasminogen activator inhibitor-1 (PAI1) and cell division cycle 20 (CDC20) were identified as novel effectors. M3-HBx and Ct-HBx significantly upregulated the expression of PAI1 and CDC20 in HepG2 and HeLa cells as well as the livers of the SB mice. PAI1 silencing attenuated the effect of M3-HBx and Ct-HBx on the growth of HepG2 cells and greatly decreased the growth of HeLa cells with Ct-HBx. Conclusion: HBx C1653T+T1674G+A1762T/G1764A mutant and Ct-HBx promote carcinogenesis via upregulating PAI1 and CDC20. PAI1, an important player bridging the HBx mutants and HCC, should be a promising candidate as a predictive and prognostic biomarker and therapeutic target in HBV-related HCC.

Interferon-induced protein with tetratricopeptide repeats 3 may be a key factor in primary biliary cholangitis

Accumulating studies suggest that senescent biliary epithelial cells (BECs) produce senescence-associated secretory phenotypes (SASPs) and play various roles in the pathogenesis of primary biliary cholangitis (PBC) and other cholangiopathies. We examined comprehensive profiles of senescent BECs and its contribution to the pathogenesis of PBC taking advantage of microarray analysis. cDNA microarray analysis revealed that 1841 genes including CCL2, IFIT3, CPQ were commonly up-regulated in senescent BECs cultured in serum depleted media or media with glycochenodeoxycholic acid. Knockdown of IFIT3 significantly suppressed cellular senescence (p < 0.01) and significantly increased apoptosis (p < 0.01) in BECs treated with serum depletion or glycochenodeoxycholic acid. Significantly increased expression of IFIT3 was seen in senescent BECs in small bile ducts showing cholangitis and in ductular reactions in PBC, compared to control livers (p < 0.01). An inadequate response to UDCA was inversely correlated to the increased expression of IFIT3 in small bile duct in PBC (p < 0.05). In conclusion, the expression of various genes related to immunity and inflammation including SASPs were increased in senescent BECs. Upregulated IFIT3 in senescent BECs may be associated with the pathogenesis of PBC and may be a possible therapeutic target in PBC.

Acrolein Contributes to Human Colorectal Tumorigenesis Through Activation of RAS/MAPK Pathway

Background. Colorectal cancer (CRC) is one of the most well-known malignancies with high prevalence and poor 5-year survival. Previous studies have demonstrated that intake of food rich in fat, and with low fiber content (as known as high-fat diet, HFD) is capable of increasing the odds of developing CRC. Acrolein, an IARC group 2A carcinogen, can be formed by thermal treatment of animal and vegetable fats, carbohydrates and amino acids through Maillard reaction. Also, acrolein has been shown to be produced from microbial glycerol metabolism in human gut. Consequently, humans are at risk of acrolein exposure through consumption of foods rich in fat. However, whether acrolein contributes to HFD-induced CRC tumorigenesis remains elusive.Methods. The effect of acrolein in oncogenic transformation was analyzed using NIH/3T3 cells with xenograft tumorigenesis mice models. Furthermore, cDNA microarray analysis with Ingenuity Pathway Analysis (IPA) was performed in acrolein-transformed NIH/3T3 cells. Finally, acrolein-induced DNA damages (Acr-dG) were analyzed in tumor tissues and normal epithelial of CRC patients using immunohistochemical analysis. The levels of Acr-dG adducts were associated with tumor characteristics and CRC patients’ survival using Chi-Square analysis and Kaplan Meier survival analysis, respectively.Results. In this present study, we found that acrolein induced oncogenic transformation including faster cell cycling, proliferation, soft agar formation, sphere formation, cell migration in NIH/3T3 cells. Using xenograft tumorigenicity assays, the acrolein-transformed NIH3T3 clone formed tumors whereas no tumors were observed in mice inoculated with NIH3T3 parental cells. In addition, RAS/MAPK pathway contributing to colon tumorigenesis was activated in NIH/3T3 Acr-clone using cDNA microarray analysis with IPA. Finally, Acr-dG adducts were higher in CRC tumor tissues compared to normal epithelial cells in CRC patients. Intriguingly, CRC patients with higher Acr-dG adducts have better prognosis. Conclusions. Taken together, this is the first study to demonstrate that acrolein is important in oncogenic transformation through activating RAS/MAPK signaling pathway contributing to colon tumorigenesis. Thus, acrolein might be a novel target for early detection, prevention and treatment of tumors in the future.

SUN-LB134 Androgen Receptor Phosphorylated at Serine 815 in Mouse and Human Prostates

Androgen receptor (AR) regulates male sexual development and maintenance. AR forms a homodimer in the cytoplasm and monomerizes following hormonal activation, translocating to the nucleus in Cos-1 cells (Shizu et al. Scientific reports. 2019). Utilizing Ser815 of AR, the conserved phosphorylation residue within the ligand binding domains of steroid hormone receptors (NR3C), whether and how this phosphorylation regulates AR functions was investigated. While, like AR WT, a phosphomimic AR S815D mutant formed a homodimer in the cytoplasm, unlike the WT, this mutant remained as a homodimer in the cytoplasm even after hormone treatment. Apparently, Ser815 phosphorylation disabled AR’s capability to monomerize and nuclear translocate in Cos-1 cells. A phospho-Ser815 peptide antibody was used to detect phosphorylation of endogenous AR in mouse as well as human prostates. Immunohistochemistry showed phosphorylation present in both the cytoplasm and nucleus. Mouse prostates were cell fractionated in cell membrane, mitochondria, endoplasmic reticulum (ER) and cytosolic fractions for subsequent Western blot analysis. While AR was found in all of these fractions, phosphorylated AR was only detected in the ER and cytosolic fractions. A cDNA microarray analysis of PC-3 cells with ectopic expression of AR S815D suggested that phosphorylated AR may regulate ER stress.

Tumor-Stroma Crosstalk Enhances REG3A Expressions that Drive the Progression of Hepatocellular Carcinoma

Background: Crosstalk between tumors and their microenvironment plays a crucial role in the progression of hepatocellular carcinoma (HCC). However, there is little existing information about the key signaling molecule that modulates tumor-stroma crosstalk. Methods: Complementary DNA (cDNA) microarray analysis was performed to identify the key molecule in tumor-stroma crosstalk. Subcutaneous xenograft in vivo murine model, immunoblotting, immunofluorescence, and real-time polymerase chain reaction using HCC cells and tissues were performed. Results: The key molecule, regenerating gene protein-3A (REG3A), was most significantly enhanced when coculturing HCC cells and activated human hepatic stellate cells (HSCs) (+8.2 log) compared with monoculturing HCC cells using cDNA microarray analysis. Downregulation of REG3A using small interfering RNA significantly decreased the proliferation of HSC-cocultured HCC cells in vitro and in vivo, and enhanced deoxycholic acid-induced HCC cell apoptosis. Crosstalk-induced REG3A upregulation was modulated by platelet-derived growth factor ββ (PDGF-ββ) in p42/44-dependent manner. REG3A mRNA levels in human HCC tissues were upregulated 1.8-fold compared with non-tumor tissues and positively correlated with PDGF-ββ levels. Conclusions: REG3A/p42/44 pathway/PDGF-ββ signaling plays a significant role in hepatocarcinogenesis via tumor-stroma crosstalk. Targeting REG3A is a potential novel therapeutic target for the management of HCCs by inhibiting crosstalk between HCC cells and HSCs.

Role of TLR4 (Toll-Like Receptor 4) in N1/N2 Neutrophil Programming After Stroke

Background and Purpose— After stroke, the population of infiltrated neutrophils in the brain is heterogeneous, including a population of alternative neutrophils (N2) that express M2 phenotype markers. We explored the role of TLR4 (toll-like receptor 4) on neutrophil infiltration and polarization in this setting. Methods— Focal cerebral ischemia was induced by occlusion of the middle cerebral artery occlusion in TLR4-KO and WT (wild type) mice. Infarct size was measured by Nissl staining and magnetic resonance imaging. Leukocyte infiltration was quantified 48 hours after middle cerebral artery occlusion by immunofluorescence and flow cytometry. To elucidate mechanisms underlying TLR4-mediated N2 phenotype, a cDNA microarray analysis was performed in neutrophils isolated from blood 48 hours after stroke in WT and TLR4-KO mice. Results— As demonstrated previously, TLR4-deficient mice presented lesser infarct volumes than WT mice. TLR4-deficient mice showed higher density of infiltrated neutrophils 48 hours after stroke compared with WT mice, concomitantly to neuroprotection. Furthermore, cytometric and stereological analyses revealed an increased number of N2 neutrophils (YM1 + cells) into the ischemic core in TLR4-deficient mice, suggesting a protective effect of this neutrophil subset that was corroborated by depleting peripheral neutrophils or using mice with TLR4 genetically ablated in the myeloid lineage. Finally, cDNA microarray analysis in neutrophils, confirmed by quantitative polymerase chain reaction, showed that TLR4 modulates several pathways associated with ischemia-induced inflammation, migration of neutrophils into the parenchyma, and their functional priming, which might explain the opposite effect on outcome of the different neutrophil subsets. Conclusions— TLR4 deficiency increased the levels of alternative neutrophils (N2)—an effect associated with neuroprotection after stroke—supporting that modulation of neutrophil polarization is a major target of TLR4 and highlighting the crucial role of TLR4 at the peripheral level after stroke. Visual Overview— An online visual overview is available for this article.

Investigation of aberrant DNA methylation in association with cisplatin-resistant hepatoblastoma.

258 Background: Cisplatin (CDDP) is a key-drug of mainstream treatment for hepatoblastoma (HB), but 22.3% of HB patients have resistance to CDDP, and their prognoses are poor. We investigated methylation status of HB patients and identified CDDP resistant candidate genes in HB. Methods: First, we performed a genome-wide methylation array analysis of 11 resected HB tumors. These cases included six cases of RECIST (Response Evaluation Criteria In Solid Tumors) CR or PR (S group) and five cases of SD or PD (R group). We analyzed methylation status of these patients, comparing with clinical course, and selected significant differentially methylated genes. Next, We made CDDP-resistant HepG2 cells by repeated CDDP treatments for a year, then compared expression levels between the CDDP-resistant cells and original cells, using cDNA microarray analysis. We selected CDDP resistant candidate genes from these data. Results: In methylation array analysis data, unsupervised hierarchical clustering analysis showed that methylation profiles were separable into 2 groups in accord with clinical S group and R group. In principal component analysis, the S group also showed a different methylation pattern to the R group. We compared survival rates using Kaplan-Meier method, and results showed that the R group had poorer prognosis significantly. The R group showed 336 hypermethylated genes and 106 hypomethylated genes around the transcriptional start site compared with S group significantly. And in cDNA microarray analysis, 1271 genes were downregulated and 1571 genes were upregulated significantly in CDDP-resistant cells. From among these genes, we selected 15 suppressor and 8 promoter candidate genes that could be related to CDDP-resistance. Conclusions: We identified methylation status associated with CDDP resistance and poor prognosis, and 23 candidates genes in HB. Further refining might lead us to find out the pathogenesis of chemoresistance or new target of therapies and biomarkers in HB.