MicroRNA-141 enhances anoikis resistance in metastatic progression of ovarian cancer through targeting KLF12/Sp1/survivin axis.

BACKGROUND: Cancer metastasis is determined by the formation of the metastatic niche and the ability of cancer cells to adapt to microenvironmental stresses. Anoikis resistance is a fundamental feature of metastatic cancer cell survival during metastatic cancer progression. However, the mechanisms underlying anoikis resistance in ovarian cancer are still unclear. METHODS: Expressions of miRNA-141 and its downstream targets were evaluated by qPCR, Western blotting, Immunohistochemical (IHC) and in situ hybridization (ISH) assays. The luciferase assays were used to prove KLF12 as the downstream target of miR-141. The cDNA microarray and apoptotic protein arrays were used to identify the targets of miR-141 and KLF12. The competition of KLF12 and Sp1 on survivin promoter was examined by ChIP assay. IHC analysis on ovarian cancer tissue array was used to evaluate the expressions of KLF12 and miR-141 and to show the clinical relevance. The functional studies were performed by in vitro and in vivo tumorigenic assays. RESULTS: Enforced expression of miR-141 promotes, while knockdown of miR-141 expression inhibits, cell proliferation, anchorage-independent capacity, anoikis resistance, tumor growth and peritoneal metastases of ovarian cancer cells. Bioinformatics and functional analysis identified that Kruppel-related zinc finger protein AP-2rep (KLF12) is directly targeted by miR-141. Consistent with this finding, knockdown of KLF12 phenocopied the effects of miR-141 overexpression in ovarian cancer cells. In contrast, restoration of KLF12 in miR-141-expressing cells significantly attenuated anoikis resistance in ovarian cancer cells via interfering with Sp1-mediated survivin transcription, which inhibits the intrinsic apoptotic pathway and is crucial for ovarian cancer cell survival, anoikis resistance and peritoneal metastases. Immunohistochemical (IHC) and in situ hybridization (ISH) assays confirmed that miRNA-141 expression is inversely correlated with KLF12 expression and significantly associated with advanced ovarian cancers accompanied with distal metastases, underscoring the clinical relevance of our findings. CONCLUSIONS: Our data identify a novel signaling axis of miR-141/KLF12/Sp1/survivin in enhancing anoikis resistance and likely serves as a potential therapeutic target for metastatic ovarian cancer.

Genetic variation in STAT4 predicts response to interferon-α therapy for hepatitis B e antigen-positive chronic hepatitis B.

UNLABELLED: Interferon (IFN)-α is a first-line therapy for chronic hepatitis B (CHB) patients but only initiates a response in a minority of patients. A genetic variant, rs7574865 in STAT4, was recently reported to be associated with risk of developing CHB and hepatitis B virus-related hepatocellular carcinoma. We aimed to determine whether this variant is associated with the response to IFNα treatment for hepatitis B e antigen (HBeAg)-positive CHB patients. We studied 466 HBeAg-positive CHB patients who received either IFNα-2b (n = 224) or pegylated IFNα-2a (n = 242) therapy for 48 weeks and were followed for an additional 24 weeks. The rate of sustained virologic response (SVR), defined as HBeAg seroconversion along with hepatitis B virus DNA level <2000 copies/mL at week 72, was compared among patients with different genotypes of rs7574865. After 48 weeks of treatment and 24 weeks off treatment, the SVR rates in the IFNα-2b and pegylated IFNα-2a therapy groups were 30.4% and 28.9%, respectively. Compared to the rs7574865 GT/TT genotype, the GG genotype (a risk factor of CHB and hepatitis B virus-related hepatocellular carcinoma) was significantly associated with a reduced SVR rate in both patients who received IFNα-2b therapy (21.1% versus 37.2%, P = 0.01) and those who received pegylated IFNα-2a therapy (18.0% versus 41.2%, P = 9.74 × 10(-5) ). In joint analysis of the 466 patients, the GG genotype was associated with an approximately half SVR rate compared to the GT/TT genotype (19.3% versus 39.1%, P = 4.15 × 10(-6) ). A multivariate logistic regression model including rs7574865 and clinical variables showed that rs7574865 was the most significant factor for the prediction of SVR. CONCLUSION: STAT4 rs7574865 is a reliable predictor of response to IFNα therapy for HBeAg-positive CHB patients and may be used for optimizing the treatment of CHB.

A missense polymorphism in ATF6 gene is associated with susceptibility to hepatocellular carcinoma probably by altering ATF6 level.

Accumulated evidences indicate that single nucleotide polymorphisms (SNP) are associated with risk of hepatocellular carcinoma (HCC). Activating transcription factor 6 (ATF6) is an important modulator of the unfolded protein response (UPR), which is regarded to be involved in carcinogenesis. So we speculate that SNPs in ATF6 may be associated with susceptibility to HCC. We carried out a two-stage association study in three independent case-control groups in a total of 1,082 chronic hepatitis B (CHB) patients and 816 hepatitis B virus (HBV) related HCC patients in Han Chinese. Four SNPs which can represent all potential functional SNPs with MAF > 0.1 recorded in HapMap database in ATF6 gene were genotyped using TaqMan methods. Functional analyses were conducted to verify the biological significances of the associated SNP. We identified a missense SNP (rs2070150) was significantly associated with susceptibility to HCC (p = 0.008, 0.001 and 0.007 in Beijing_302, Beijing_You'an and Guangxi samples, respectively). This SNP was further validated in four independent groups of major HBV outcomes, indicating it may associate exclusively to HCC. ATF6 mRNA expression was significantly decreased as the disease progressed (p <0.001). Functional analyses show that the protective allele of rs2070150 could significantly increase the expression levels of ATF6 mRNA, as well as ATF6 regulated genes such as GRP78, XBP1 and CHOP. These findings indicate that a common missense SNP in ATF6 may contribute to susceptibility of HCC functionally.

A functional polymorphism in ADAR1 gene affects HBsAg seroclearance both spontaneously and interferon induced.

BACKGROUND & AIMS: Our previous study found that rs4845384 in ADAR1 gene to be associated with HBeAg seroconversion. However, the effect of rs4845384 on HBsAg seroclearance is unknown. To assess the relationship between rs4845384 and HBsAg seroclearance. METHODS: Two independent case-control studies were conducted to test whether rs4845384 in ADAR1 was associated with HBsAg clearance. Reporter gene assays and quantitative PCR experiments were also carried out to verify the functional significances of this polymorphism. RESULTS AND CONCLUSIONS: The rs4845384 polymorphism was associated with HBsAg seroclearance both spontaneously (P = 0.028, OR = 1.36, 95% CI = 1.03-1.78) and interferon induced (P = 0.013, OR = 1.83, 95% CI = 1.13-2.96), in a total of 725 subjects. Luciferase assays showed that pGL3-rs4845384G constructs had higher expression level than pGL3-rs4845384A constructs, especially in HepG2. 2.15 cell line. Quantitative real-time RT-PCR showed that chronic hepatitis B (CHB) patients had lower ADAR1 mRNA level than healthy individuals. The AA carriers of rs4845384 had lower ADAR1 mRNA expression than non-AA carriers. The non-response susceptible allele rs4845384A was functional for regulation of ADAR1 expression, so as to influence HBsAg seroclearance of CHB patients.

Polymorphisms in the VEGFA promoter are associated with susceptibility to hepatocellular carcinoma by altering promoter activity.

Accumulated evidences indicate that single nucleotide polymorphisms (SNP) in angiogenesis and tumorigenesis related genes are associated with risk of hepatocellular carcinoma (HCC). Vascular endothelial growth factor A (VEGFA), one of the most significant mediators of angiogenesis, plays an important role in carcinogenesis and development via promoting tumor growth. We carried out a two-stage association study in 1,838 chronic hepatitis B (CHB) patients and 1,207 hepatitis B virus (HBV) related HCC patients in Han Chinese populations from Beijing, Guangxi and Jiangsu. We systematically screened polymorphisms in the VEGFA gene and examined the association between the SNPs and susceptibility to HCC. Functional analyses were conducted to verify biological significances of associated SNPs. We identified two promoter SNPs (rs833061 and rs1570360) were associated with susceptibility to HCC (rs833061: ptrend = 0.008 in Youan_Beijing samples, ptrend = 0.01 in Guangxi samples, ptrend = 0.01 in Jiangsu samples. rs1570360: ptrend = 0.00003 in Youan_Beijing samples, ptrend = 0.006 in Guangxi samples, ptrend = 0.02 in Jiangsu samples). These two SNPs were further validated in four independent groups of major HBV outcomes, indicating rs833061 and rs1570360 may associate exclusively to HCC. Functional analyses show that CA haplotype constructed by rs833061 and rs1570360 had higher luciferase activity compared with TG haplotype (p < 0.05). A 18 bp insert/del polymorphism was in absolute linkage disequilibrium (LD) with rs833061. The 18 bp insert allele created a Sp1 binding site. We observed higher VEGFA transcription in peripheral blood of HCC patients compared with CHB patients and healthy individuals (p < 0.05). These findings indicate that VEGFA promoter SNPs may contribute to susceptibility of HCC by altering promoter activity.

TMEM25 inhibits monomeric EGFR-mediated STAT3 activation in basal state to suppress triple-negative breast cancer progression.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with poor outcome and lacks of approved targeted therapy. Overexpression of epidermal growth factor receptor (EGFR) is found in more than 50% TNBC and is suggested as a driving force in progression of TNBC; however, targeting EGFR using antibodies to prevent its dimerization and activation shows no significant benefits for TNBC patients. Here we report that EGFR monomer may activate signal transducer activator of transcription-3 (STAT3) in the absence of transmembrane protein TMEM25, whose expression is frequently decreased in human TNBC. Deficiency of TMEM25 allows EGFR monomer to phosphorylate STAT3 independent of ligand binding, and thus enhances basal STAT3 activation to promote TNBC progression in female mice. Moreover, supplying TMEM25 by adeno-associated virus strongly suppresses STAT3 activation and TNBC progression. Hence, our study reveals a role of monomeric-EGFR/STAT3 signaling pathway in TNBC progression and points out a potential targeted therapy for TNBC.

Metagenomic Analyses Reveal Distinct Gut Microbiota Signature for Predicting the Neoadjuvant Chemotherapy Responsiveness in Breast Cancer Patients.

Background: Growing evidence supports the modulatory role of human gut microbiome on neoadjuvant chemotherapy (NAC) efficacy. However, the relationships among the gut microbiome, tumor-infiltrating lymphocytes (TILs), and NAC response for breast cancer (BC) patients remain unclear. We thus proposed this preliminary study to investigate the relationship between gut microbiome and BC patients' responses to NAC treatment as well as underlying mechanisms. Methods: Prior to receiving NAC, the fecal metagenome collected from 23 patients with invasive BC was analyzed. Patients were subsequently assigned to the NAC non-effectual group and the NAC effectual group based on their response to NAC. The peripheral T lymphocyte subset counts were examined by flow cytometry methods. CellMinor analysis was employed to explore the relationship between CD4 mRNA expression and the reaction of tumor cells to NAC drugs. Results: The gut microbiomes of the NAC non-effectual group showed characteristics of low diversity with low abundances, distinct metagenomic composition with decreased butyrate-producing and indolepropionic acid-producing bacteria, and increased potential pathobionts compared with the NAC effectual group. The combination of Coprococcus, Dorea, and uncultured Ruminococcus sp. serves as signature bacteria for distinguishing NAC non-effectual group patients from the NAC effectual group. The absolute numbers of CD4+ and CD8+ TIL infiltration in tumors in the NAC non-effectual group were significantly lower than those in the effectual group. Similar findings were reported for the CD4+ T lymphocytes in the peripheral blood (p's < 0.05). NAC effectual-related signature bacteria were proportional to these patients' CD4+ T lymphocyte counts in peripheral blood and tumors (p's < 0.05). CellMinor analysis showed that the CD4 mRNA expression level dramatically climbed with increased sensitivity of tumor cells to NAC drugs such as cyclophosphamide, cisplatin, and carboplatin (p's < 0.05). Conclusions: The composition of the gut microbial community differs between BC patients for whom NAC is effective to those that are treatment resistant. The modulation of the gut microbiota on host CD4+ T lymphocytes may be one critical mechanism underlying chemosensitivity and NAC pathologic response. Taken together, gut microbiota may serve as a potential biomarker for NAC response, which sheds light on novel intervention targets in the treatment of NAC non-effectual BC patients.

Targeting infected host cells in vivo via responsive azido-sugar mediated metabolic cell labeling followed by click reaction.

The early in vivo diagnosis of infectious disease foci is largely hindered by invasion and concealment of pathogens in host cells, making it difficult for conventional probes to detect and analyze intracellular pathogens. Taking advantage of the excessively produced reactive oxygen species (ROS) within host cells, herein we report the design of thiol-hemiketal blocked N-azidoacetyl galactosamine (Ac3GalNAzSP), an azido unnatural sugar bearing an unprecedent designed ROS-responsive moiety for targeted labelling of infected host cells. Ac3GalNAzSP showed great stability under physiological conditions, specifically released active unnatural sugar in host cells overproducing ROS, metabolically labeled infected host cells with azido groups, and enabled targeting in vivo infection sites by subsequent Click Chemistry reactions, substantiating an unprecedented approach for targeting infected host cells. This technique could be a powerful tool for early in vivo diagnosis and targeted treatment of infectious disease.

Methylation-associated silencing of miR-193a-3p promotes ovarian cancer aggressiveness by targeting GRB7 and MAPK/ERK pathways.

Human growth factor receptor-bound protein-7 (GRB7) is a pivotal mediator involved in receptor tyrosine kinase signaling and governing diverse cellular processes. Aberrant upregulation of GRB7 is frequently associated with the progression of human cancers. However, the molecular mechanisms leading to the upregulation of GRB7 remain largely unknown. Here, we propose that the epigenetic modification of GRB7 at the post-transcriptional level may be a crucial factor leading to GRB7 upregulation in ovarian cancers. Methods: The upstream miRNA regulators were predicted by in silico analysis. Expression of GRB7 was examined by qPCR, immunoblotting and immunohistochemical analyses, while miR-193a-3p levels were evaluated by qPCR and in situ hybridization in ovarian cancer cell lines and clinical tissue arrays. MS-PCR and pyrosequencing analyses were used to assess the methylation status of miR-193a-3p. Stable overexpression or gene knockdown and Tet-on inducible approaches, in combination with in vitro and in vivo tumorigenic assays, were employed to investigate the functions of GRB7 and miR-193a-3p in ovarian cancer cells. Results: Both miR-193a-3p and its isoform, miR-193b-3p, directly targeted the 3' UTR of GRB7. However, only miR-193a-3p showed a significantly inverse correlation with GRB7-upregulated ovarian cancers. Epigenetic studies revealed that methylation-mediated silencing of miR-193a-3p led to a stepwise decrease in miR-193a-3p expression from low to high-grade ovarian cancers. Intriguingly, miR-193a-3p not only modulated GRB7 but also ERBB4, SOS2 and KRAS in the MAPK/ERK signaling pathway to enhance the oncogenic properties of ovarian cancer cells in vitro and in vivo. Conclusion: These findings suggest that epigenetic silencing of miR-193a-3p by DNA hypermethylation is a dynamic process in ovarian cancer progression, and miR-193a-3p may be explored as a promising miRNA replacement therapy in this disease.

Methylation of multiple genes as diagnostic and therapeutic markers in primary head and neck squamous cell carcinoma.

OBJECTIVE: To examine epigenetic events of aberrant promoter methylation as diagnostic markers in primary head and neck squamous cell carcinoma using a novel multigene approach. Promoter methylation-mediated silencing is a hallmark of several established tumor suppressor genes. Changes in DNA methylation have been reported to occur early in carcinogenesis and therefore are potentially important early indicators of existing disease. DESIGN: A multicandidate gene probe panel interrogated DNA for aberrant methylation status in 22 cancer genes using the methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay. Aberrant promoter hypermethylation was confirmed using methylation-specific polymerase chain reaction after bisulfite treatment. SETTING: Primary care medical center. SUBJECTS: We examined fresh-frozen primary head and neck tumor specimens from 28 patients, including 21 late-stage (19 stage IV and 2 stage III) and 7 early-stage (6 stage II and 1 stage I) tumors. RESULTS: Promoter hypermethylation was observed in 14 of the 28 patients (50%). Genes for RARB, APC, and CHFR were most frequently hypermethylated, occurring in 11 (39%) for RARB, 7 (25%) for CHFR, and 6 (21%) for APC. Aberrant methylation of CHFR was solely a stage IV event. Methylation-specific polymerase chain reaction after bisulfite treatment with conventional and real-time polymerase chain reaction confirmed aberrant methylation for RARB and CHFR. CONCLUSIONS: Promoter methylation profiling of primary head and neck squamous cell carcinoma using multiple target genes identified RARB, APC, and CHFR as frequent epigenetic events. The clinical implications of these genes as diagnostic and treatment biomarkers are highly relevant as attractive targets for cancer therapy, given the reversible nature of epigenetic gene silencing.

Replication of genome wide association studies on hepatocellular carcinoma susceptibility loci in a Chinese population.

BACKGROUND: Genome-wide association studies (GWAS) have identified three loci (rs17401966 in KIF1B, rs7574865 in STAT4, rs9275319 in HLA-DQ) as being associated with hepatitis B virus-related hepatocellular carcinoma (HBV-related HCC) in a Chinese population, two loci (rs2596542 in MICA, rs9275572 located between HLA-DQA and HLA-DQB) with hepatitis C virus-related HCC (HCV-related HCC) in a Japanese population. In the present study, we sought to determine whether these SNPs are predictive for HBV-related HCC development in other Chinese population as well. METHOD AND FINDINGS: We genotyped 4 SNPs, rs2596542, rs9275572, rs17401966, rs7574865, in 506 HBV-related HCC patients and 772 chronic hepatitis B (CHB) patients in Han Chinese by TaqMan methods. Odds ratio(OR)and 95% confidence interval (CI) were calculated by logistic regression. In our case-control study, significant association between rs9275572 and HCC were observed (P = 0.02, OR = 0.73, 95% CI = 0.56-0.95). In the further haplotype analysis between rs2596542 at 6p21.33 and rs9275572 at 6p21.3, G-A showed a protective effect on HBV-related HCC occurrence (P<0.001, OR = 0.66, 95% CI = 0.52-0.84). CONCLUSION: These findings provided convincing evidence that rs9275572 significantly associated with HBV-related HCC.

JAK1 gene polymorphisms are associated with the outcomes of hepatitis B virus infection, but not with α interferon therapy response in a Han Chinese population.

AIMS: Janus kinase 1 (JAK1) is a key member in the interferon (IFN) signaling pathway. Recent studies suggested single-nucleotide polymorphisms (SNPs) in IFN pathway genes are associated with outcomes of hepatitis B virus (HBV) infection and response to IFNα therapy. The aim of the study is to investigate whether SNPs in JAK1 were associated with outcomes of HBV infection and response to IFNα therapy. METHODS: We enrolled 395 chronic hepatitis B (CHB) patients and 251 subjects with the inactive carrier state, and 256 CHB patients who received IFNα treatment, with therapy efficacy evaluated. Twelve SNPs: rs310227, rs7531799, rs7546545, rs17127174, rs3790541, rs10493373, rs2780898, rs310247, rs310196, rs2780895, rs4244165, and rs17127024 in JAK1, which could represent all SNPs with minor allele frequency >0.2 recorded in the HapMap database were genotyped using a polymerase chain reaction-restriction fragment length polymorphism protocol and the TaqMan method. RESULTS: SNP rs17127024 was associated with outcomes of HBV infection in an allele frequency (p=0.014) and genotype distributions (p=0.031), while SNP rs4244165 was associated with outcomes of HBV infection only in genotype distributions (p=0.008). There were no significant differences in allele frequencies and genotype distributions of these SNPs between the response group and the nonresponse group to IFNα therapy. CONCLUSIONS: SNPs rs4244165 and rs17127024 in JAK1 were associated with outcomes of HBV infection, but not with response to IFNα therapy.