Potential Tumor Suppressor Gene Research Articles

The expression of clock gene CLOCK and its clinical significance in nasopharyngeal carcinoma

Objective: To explore the relationship between expression levels of CLOCK mRNA and protein and the clinical characteristics of patients with nasopharyngeal carcinoma. Methods: The frozen tissue specimens from 33 patients with nasopharyngeal carcinoma in the Affiliated Tumor Hospital of Guizhou Medical University from 2018 to 2019 were collected. Seventeen cases of tissue specimens from patients with nasopharyngeal chronic inflammation in the Affiliated Hospital of Guizhou Medical University in 2019 were collected. From 2008 to 2014, 68 cases of formalin-fixed paraffin-embedding (FFPE) nasopharyngeal carcinoma tissue and 37 cases of FFPE nasopharyngeal chronic inflammation tissue were collected from the Affiliated Tumor Hospital of Guizhou Medical University. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot (WB) were used to detect the mRNA and protein expression levels of CLOCK. The nasopharyngeal carcinoma cells including CNE1, CNE2, 5-8F and the normal nasopharyngeal epithelial cell NP69 were cultured. qRT-PCR was used to detect the expression level of CLOCK mRNA in each cell line at the time points of ZT2, ZT6, ZT10, ZT14, ZT18 and ZT22. The cosine method was used to fit the rhythm of CLOCK gene in nasopharyngeal carcinoma. The protein expression of CLOCK protein was detected by using immunohistochemical method in 68 cases of nasopharyngeal carcinoma and 37 cases of nasopharyngeal chronic inflammation tissue. Survival was analyzed by Kaplan-Meier method and Log rank test, and the influencing factors was analyzed by Cox regression model. Results: The expression levels of CLOCK mRNA in CNE1, CNE2 and 5-8F cells (0.63±0.07, 0.91±0.02 and 0.33±0.04, respectively) were lower than that in NP69 cell (1.00±0.00, P<0.05). The expression levels of CLOCK protein in CNE1, CNE2 and 5-8F cells (0.79±0.06, 0.57±0.05 and 0.74±0.10, respectively) were lower than that of NP69 cells (1.00±0.00, P<0.05). The expressions of CLOCK mRNA in nasopharyngeal carcinoma cells including CEN1, CNE2, 5-8F and normal nasopharyngeal epithelial cell NP69 were different at different time points, with temporal fluctuations. The fluctuation periods of CLOCK mRNA in CNE1, CNE2, 5-8F, and NP69 cells were 16, 14, 22 and 24 hours, respectively. The peak and trough times were ZT10: 40 and ZT18: 40, ZT10 and ZT3, ZT14: 30 and ZT3: 30, ZT12: 39 and ZT0: 39, respectively. CLOCK mRNA and protein expression levels in nasopharyngeal carcinoma tissues (0.37±0.20 and 0.20±0.26, respectively) were lower than those in nasopharyngeal chronic inflammation tissues (1.00±0.00 and 0.51±0.41, respectively, P<0.05). The 1, 3, and 5-year survival rates of patients in the CLOCK protein high expression group (CLOCK protein expression level ≥ 0.178) were 96.2%, 92.1%, and 80.1%, respectively, which were higher than those in the low expression group (CLOCK protein expression level <0.178, 92.9% , 78.6% and 57.1%, respectively, P=0.009). The 1, 3, and 5-year progression-free survival (PFS) rates of patients in the CLOCK protein high expression group were 96.2%, 87.8%, and 87.7%, respectively, which were higher than those in the low expression group (92.7%, 82.2%, and 70.8%, respectively, P=0.105). Compared with the low-expression group (100.0%, 96.9%, and 90.0%, respectively), the 1, 3, and 5-year recurrence-free survival rates of patients in the CLOCK protein high expression group (100.0%, 95.7%, and 95.7%, respectively) were not statistically significant (P=0.514). Compared with the low-expression group (92.7%, 82.2%, and 79.3%), the 1, 3, and 5-year survival rates without metastasis in the CLOCK protein high expression group (96.2%, 92.0%, and 92.0%, respectively) were not statistically significant (P=0.136). CLOCK protein expression and T stage were independent prognostic factors of overall survival (P<0.05). Conclusions: The expression of CLCOK is downregulated in the nasopharyngeal carcinoma cell and nasopharyngeal carcinoma tissues. Clock gene CLOCK is rhythmically expressed in the nasopharyngeal carcinoma cells and normal nasopharyngeal epithelial cells. Compared with normal nasopharyngeal epithelial cells, the fluctuation period of CLOCK in nasopharyngeal carcinoma cells is shortened. The overall survival of patients in the CLOCK protein high expression group is better than that of low expression group. The expression of CLOCK protein is an independent influencing factor for overall survival. CLOCK gene may be a potential tumor suppressor gene in the nasopharyngeal carcinoma.

CRYAB predicts clinical prognosis and is associated with immunocyte infiltration in colorectal cancer.

BackgroundαB-Crystallin (CRYAB) is differentially expressed in various tumors. However, the correlation between CRYAB and immune cell infiltration in colorectal cancer (CRC) remains unclear.Materials & MethodsKaplan–Meier survival curves in The Cancer Genome Atlas (TCGA) were used to evaluate the relationship between CRYAB expression and both overall survival and progression-free survival. The relationships between CRYAB expression and infiltrating immune cells and their corresponding gene marker sets were examined using the TIMER database.ResultsThe expression of CRYAB was lower in CRC tumor tissues than in normal tissues (P < 0.05). High CRYAB gene expression and high levels of CRYAB gene methylation were correlated with high-grade malignant tumors and more advanced tumor, nodes and metastasis (TNM) cancer stages. In addition, in colorectal cancer, there was a positive correlation between CRYAB expression and immune infiltrating cells including neutrophils, macrophages, CD8 + T cells, and CD4 + T cells, as well as immune-related genes including CD2, CD3D, and CD3E. Methylation sites such as cg13084335, cg15545878, cg13210534, and cg15318568 were positively correlated with low expression of CRYAB.ConclusionBecause CRYAB likely plays an important role in immune cell infiltration, it may be a potential tumor-suppressor gene in CRC and a potential novel therapeutic target and predictive biomarker for colorectal cancer (CRC).

Combined oncolytic adenovirus carrying MnSOD and mK5 genes both regulated by survivin promoter has a synergistic inhibitory effect on gastric cancer.

Gastric cancer (GC) is one of the major causes of cancer-related mortality. The use of oncolytic virus for cancer gene-virotherapy is a new approach for the treatment of human cancers. In this study, a novel Survivin promoter-driven recombinant oncolytic adenovirus carrying mK5 or MnSOD gene was constructed, which was modified after deletion of the E1B gene. Human plasminogen Kringle 5 mutant (mK5) and manganese superoxide dismutase (MnSOD) are both potential tumor suppressor genes. By constructing Ad-Surp-mK5 and Ad-Surp-MnSOD oncolytic adenoviruses, we hypothesized that the combination of the two viruses would enhance the therapeutic efficacy of GC as compared to the one virus alone. The results of the in vitro experiments revealed that the combination of adenovirus carrying mK5 and MnSOD gene exhibited stronger cytotoxicity to GC cell lines as compared to the virus alone. Additionally, the virus could selectively kill cancer cells and human somatic cells. Cell staining, flow cytometry, and western blot analysis showed that the combination of two adenoviruses containing therapeutic genes could promote the apoptosis of cancer cells. In vivo experiments further verified that Ad-Surp-mK5 in combination with Ad-Surp-MnSOD exhibited a significant inhibitory effect on the growth of GC tumor xenograft as compared to the virus alone, and no significant difference was observed in the bodyweight of treatment and the normal mice. In conclusion, the combination of our two newly constructed recombinant oncolytic adenoviruses containing mK5 or MnSOD therapeutic genes could significantly inhibit gastric cancer growth by inducing apoptosis, suggestive of its potential for GC therapy.

PCBP1 regulates the transcription and alternative splicing of metastasis\u2011related genes and pathways in hepatocellular carcinoma

PCBP1 is a multifunctional RNA-binding protein (RBP) expressed in most human cells and is involved in posttranscriptional gene regulation. PCBP1 regulates the alternative splicing, translation and RNA stability of many cancer-related genes and has been identified as a potential tumour suppressor gene. PCBP1 inhibits the invasion of hepatocellular carcinoma (HCC) cells, but there are few studies on the specific regulatory target and mechanism of RBPs in HCC, and it is unclear whether PCBP1 plays a role in tumour metastasis as a splicing factor. We analysed the regulation of gene expression by PCBP1 at the transcriptional level. We obtained and analysed PCBP1-knockdown RNA-seq data and eCLIP-seq data of PCBP1 in HepG2 cells and found that PCBP1 widely regulates the alternative splicing and expression of genes enriched in cancer-related pathways, including extracellular matrix, cell adhesion, small molecule metabolic process and apoptosis. We validated five regulated alternative splicing events affected by PCBP1 using RT-qPCR and found that there was a significant difference in the expression of APOC1 and SPHK1 between tumour and normal tissues. In this study, we provided convincing evidence that human PCBP1 profoundly regulates the splicing of genes associated with tumour metastasis. These findings provide new insight into potential markers or therapeutic targets for HCC treatment.

TMOD-13. IDENTIFYING DRIVERS IN THE CONVERGING SYNTENIC REGIONS OF SPONTANEOUS CANINE AND PEDIATRIC HIGH-GRADE GLIOMA USING IMAGING BASED CRISPR-CAS9 ARRAY SCREEN

Abstract Gliomas occur in companion dogs at rates comparable to humans, with short-snouted breeds such as boxers being more susceptible than others. The natural progression of cancer in the immuno-competent host allows companion dogs diagnosed with sporadic glioma as an optimal model for preclinical testing of therapeutic approaches with human relevance, including immunotherapies. We have recently performed comprehensive genomic and epigenetic characterization of glioma in dogs to their human counterparts and found strong convergent evolution – shared somatic mutations and aneuploidies - among syntenic regions, including those of known pediatric glioma drivers, e.g., PDGFRA, MYC, PIK3CA. Here, using arrayed CRISPR-Cas9 imaging based phenotypic screen, we will probe potential oncogenic drivers and tumor suppressor genes within syntenic aneuploidies and thus outline functional versus non-functional heterogeneity of cancer aneuploidy. Specifically, we are conducting arrayed knockout screen (one gene per well) of 400+ genes within syntenic aneuploidies across canine (n=2) and pediatric (n=2) high-grade glioma cell lines. We will first capture images by high-speed confocal imaging system at three time points post-transduction of single guide RNAs (2 per gene) targeting each of 400+ genes in their separate wells. Then, using high-throughput image analysis and semi-supervised machine learning methods, we will measure well-based phenotypic features (viability, growth, and morphology) from these images. Genes will be ranked per cross-validated predicted probability in yielding either proliferating or slow-growing cell type based on learned phenotypic features using image data of knockout cells from and across wells. The top ranked genes will then be linked to oncogenes and tumor suppressors based on pathway and ontology analysis. We expect that we will see convergence of the most impactful molecular abnormalities (based on their knockout phenotypes) on mechanisms or candidate signaling pathways for the development of new drugs and repurposing of existing drugs for kids and dogs with high-grade glioma.

CCR5 is a prognostic biomarker and an immune regulator for triple negative breast cancer.

This study aims to explore the clinical implications and potential mechanistic functions of CCR5 in triple negative breast cancer. Briefly, we demonstrated that CCR5 is overexpressed in TNBC and is associated with better prognosis of TNBC. CCR5 expression is positively correlated with tumor immune cell infiltration and tumor immune response related pathways. Multi-omics data analyses identified CCR5 associated genomic and proteomic changes. CCR5 overexpression was associated with better overall survival in TNBC patients with TP53 mutation. We also summarized the latest findings on ICB efficacy related genes and explored the association between CCR5 and those genes. These results indicated that CCR5 is a potential tumor suppressor gene and individualized therapeutic strategy could be established based on multi-omics background and expression pattern of ICB related genes. In conclusion, CCR5 is associated with better survival of TNBC patients with TP53 mutation, which may exert its roles through tumor immune environment.

The Loss of Heterozygosity of FHIT Gene in Sporadic Breast Cancer

The loss of heterozygosity (LOH) is a genetic event that can change gene function. FHIT is a potential tumor suppressor gene. Although the precise FHIT molecular mechanism of action is not well understood, evidences suggest that Fhit protein reduced levels are involved in mammary carcinogenesis. The aim of this study was to investigate if FHIT LOH could influence on sporadic breast cancer (BC) biological behavior, through its association with prognostic factors for sporadic BC.Tumor tissue and peripheral blood samples were analyzed using the microsatellite marker D3S1300. The findings were associated with clinicopathological parameters including overall survival. LOH was detected in 31.1%(52/167) of the informative BC’ cases. Considering clinical and pathological characteristics we have found no significant association with FHIT LOH status. The mean follow-up time was 80 months. After the Cox regression analysis two parameters remained associated with BC’s risk of death: TNM stage III and IV - HR = 3.74(95% CI, 1.16-12.1) P=0.027 and disease relapse HR = 3.14(CI 95% 1.26-7.80) P =0.014. This study shows that FHIT LOH by itself is not a prognostic factor for sporadic BC. Further researches are required to elucidate the functional role of FHIT LOH concerning to BC.

Association of PARK-2 Non-synonyms Polymorphisms and Their In Silico Validation Among North Indian Colorectal Cancer Patients.

PARK2 is a potential tumour suppressor gene and its genetic alterations (regionic loss) are common across many human cancers. The association of PARK2 germline variations (SNPs) with Parkinson's has been shown, but their association in development and progression of cancer remains elusive. The aim of this study was to identify association of PARK2 polymorphisms (rs1801474, rs1801334) with colorectal cancer in a case control study design. This case control study included a total of 650 genetically unrelated subjects comprising 300 colorectal cancer cases and 350 healthy controls belonging to North Indian. Both SNPs were analyzed using the PCR-RFLP assay. Statistical analysis for describing risk and association was performed using SPSS-17 software. Structural deviations due to non- synonymous substitutions (S167N and D394N) were analyzed using MD simulations. The genotype distributions of both the SNPs were in Hardy-Weinberg equilibrium. For both the polymorphisms, the allelic model showed statistically significant risk with OR ~ 1.3. Many of the associations remained significant even after Bonferroni correction (P < 0.00125). The result suggested that both S167N and D394N were deviated from wild type and structures and were stable after 5ns. The average value of RMSD for backbone atoms was calculated from 5 to 10ns molecular dynamics simulation data. In conclusion, our study revealed a significant association of PARK2 SNPs with colorectal cancer as well as their relations with other clinical parameters highlighting their contribution towards colorectal cancer susceptibility in North Indian population.

PI3K-AKT, JAK2-STAT3 pathways and cell\u2013cell contact regulate maspin subcellular localization

BackgroundMaspin (SERPINB5) is a potential tumor suppressor gene with pleiotropic biological activities, including regulation of cell proliferation, death, adhesion, migration and gene expression. Several studies indicate that nuclear localization is essential for maspin tumor suppression activity. We have previously shown that the EGFR activation leads to maspin nuclear localization in MCF-10A cells. The present study investigated which EGFR downstream signaling molecules are involved in maspin nuclear localization and explored a possible role of cell–cell contact in this process.MethodsMCF-10A cells were treated with pharmacological inhibitors against EGFR downstream pathways followed by EGF treatment. Maspin subcellular localization was determined by immunofluorescence. Proteomic and interactome analyses were conducted to identify maspin-binding proteins in EGF-treated cells only. To investigate the role of cell–cell contact these cells were either treated with chelating agents or plated on different cell densities. Maspin and E-cadherin subcellular localization was determined by immunofluorescence.ResultsWe found that PI3K-Akt and JAK2-STAT3, but not MAP kinase pathway, regulate EGF-induced maspin nuclear accumulation in MCF-10A cells. We observed that maspin is predominantly nuclear in sparse cell culture, but it is redistributed to the cytoplasm in confluent cells even in the presence of EGF. Proteomic and interactome results suggest a role of maspin on post-transcriptional and translation regulation, protein folding and cell–cell adhesion.ConclusionsMaspin nuclear accumulation is determined by an interplay between EGFR (via PI3K-Akt and JAK2-STAT3 pathways) and cell–cell contact.DeaMyif71VSQHA76EZpJtKVideo

Identification of the molecular function of tripartite motif containing 58 in human lung cancer.

Lung cancer is a major public health problem worldwide, with a high associated incidence and mortality. In the present study, novel epigenetic signatures were identified through genome-wide DNA methylation microarrays. The results revealed that tripartite motif containing 58 (TRIM58), a potential tumor suppressor gene exhibited high methylation and low expression in lung cancer tissue samples compared with normal tissues. Receiver operating characteristic curve analysis demonstrated that TRIM58 may be a promising early diagnostic indicator of lung cancer. In addition, the present study analyzed the role of TRIM58 in tumorigenesis and development in lung cancer A549 cells. Wound healing assay and transwell migration assay were used to investigate cell migration, and flow cytometry analysis was used to detect apoptosis. Silencing TRIM58 accelerated the proliferation and migration of lung cancer cells. In contrast, the overexpression of TRIM58 significantly inhibited the proliferation and migration of lung cancer cells and promoted apoptosis. Gene set enrichment analysis revealed that TRIM58 expression was negatively correlated with MYC targets, G2M checkpoints and the mTORC1 signaling pathway. These results of the present study suggested that TRIM58, a potential tumor suppressor gene may serve as a novel diagnostic biomarker and therapeutic target in human lung cancer.

Abstract 2091: DNA methylation enhances apoptosis resistance of renal cancer cells

Abstract Epigenetic modifications are important not only for carcinogenesis but also for metastasis of clear cell renal cell carcinoma (ccRCC). We previously reported that inflammation-related signaling is constitutively activated in advanced ccRCC through the formation of super enhancer. In addition to histone modification, methylation of the primary structure of DNA is implicated in the progression of ccRCC. Recent studies revealed that DNA hypermethylation was frequently observed in ccRCC and correlated with poor prognosis. However, unlike in other cancers, methylated genes responsible for cancer progression is still unclear in ccRCC. In this study, we tried to identify a target(s) for DNA methyltransferases (DNMTs) in ccRCC cells based on the genome-wide analysis. Its function for the regulation of cellular survival was examined. Our preclinical model of ccRCC using the serial orthotopic inoculation model exhibited the up-regulation of DNMT3B in advanced ccRCC. Pre-treatment of advanced ccRCC with 5-aza-dC attenuated a formation of a primary tumor through the induction apoptosis. DNA methylated sites were analyzed using methylation array in reference to RNA-sequencing data. As a result, ubiquinol cytochrome c reductase hinge protein (UQCRH), one of the components of mitochondrial complex III, was extracted as a methylation target in advanced ccRCC. Immunohistochemical analysis revealed that the expression of UQCRH in human ccRCC tissue was lower than normal adjacent tissue. Silencing of UQCRH attenuated the cytochrome c release in response to apoptotic stimuli and resulted in enhancement of primary tumor formation in vivo. Our results imply that DNA methylation induces the decreased expression of potential tumor suppressor gene UQCRH, which is essential for the completion of apoptotic process of ccRCC cells. These findings may contribute to the understanding of molecular mechanisms involved in the drug resistance of ccRCC cells and provide an important insight for the improvement of the efficacy of existing medicines. Citation Format: Kosuke Miyakuni, Jun Nishida, Shogo Ehata, Kohei Miyazono. DNA methylation enhances apoptosis resistance of renal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2091.

JMJD3 Is a Potential Tumor Suppressor Gene That Cooperates with TP53 in Chromosome 17p13

Introduction:Hemizygous deletion of 17p13 (17p13(del)) has been identified in up to 30% of multiple myeloma (MM) patients and is a powerful prognostic marker. We have previously identified that TP53 demonstrates haploinsufficiency, which could render an aggressive nature in the 17p13(del) MM cells (Teoh et al., Leukemia, 2014). Importantly, there are various other genes that are also located within the minimal deleted region of 17p13. Therefore, we postulated that beyond TP53 deletion alone, the adverse effects conferred by 17p13(del) may be due to the concomitant loss of other potential tumor suppressor genes. Herein, we sought to identify a novel gene that can co-operate with TP53 in mediating growth and proliferation of MM cells and found KDM6B/JMJD3, which is a histone demethylase, to be of particular interest. The role of JMJD3 in cancer has remained controversial, not to mention, unknown in MM.Methods:We utilised publicly available MM patient datasets and various human myeloma cell lines (HMCLs) of differential p53 status as our study models. We created a dynamic system to elucidate the potential role of JMJD3 by performing a single knockdown of either p53 or JMJD3 as well as double knockdown of both p53 and JMJD3 in an isogenic HMCL by using the lentivirus transduction mechanism. The former approach was taken so as to enable us to study the independent tumor suppressive role of JMJD3. These cell lines were subjected to selection agents (puromycin) and stable cell lines were established within three to four weeks. Functional assays (CTG, colony formation and cell cycle) were done on these stable HMCLs to investigate the phenotypes upon loss of expression of JMJD3 or/and p53.Results:Comprehensive gene mapping on patient datasets has unveiled 33 genes to be residing within the minimally deleted 17p13 (0.7Mbp). Besides TP53, we identified JMJD3 to be our gene of interest. We interrogated the impact of 17p13(del) on JMJD3 expression and found that loss of the chromosomal copy number led to a substantial reduction in JMJD3 expression in both MM patients and HMCLs, denoting a possible biological significance of JMJD3 in this cytogenetic aberration. Consistently, our functional analyses then revealed JMJD3 to possess potential tumor suppressor properties. Even in the presence of WT-p53, single knockdown of JMJD3 promoted a more proliferative cellular state, suggesting that independently of TP53, loss of JMJD3 alone was already able to increase MM tumorigenicity. Nevertheless, concurrent knockdown of both p53 and JMJD3 could actually lead to a significantly higher cellular growth and proliferation. These results could also be replicated when JMJD3 was depleted in p53-deficient cells, thus, proving that concomitant loss of both genes had synergistic oncogenic effects to the MM cells. Interestingly, we observed that JMJD3 expression was not associated with the global histone methylation status. Protein localization assay demonstrated that majority of JMJD3 is located within the cytoplasm, hence, its tumor suppressive properties in these HMCLs are deemed to be demethylase-independent. Additionally, we identified positive p53-JMJD3 protein interactions, hinting the possibility of both proteins being a cofactor for each other.Conclusions:Beyond the classical belief, our preliminary data indicates that 17p13(del) is actually so much more than just TP53 itself. Although JMJD3 could exert tumor suppressor properties independently of p53, our results underline the importance of combined effects of dose reduction of two tumor suppressor genes within 17p13. Our study is important in shedding some lights to the doom and gloom of the disease biology of the high-risk patients with 17p13(del). DisclosuresFonseca:Novartis: Consultancy; Bayer: Consultancy; Celgene Corporation: Consultancy, Research Funding; Pharmacyclics: Consultancy; AMGEN: Consultancy; Takeda: Consultancy; Sanofi: Consultancy; Jansen: Consultancy; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Mayo Clinic & Dr Fonseca: Patents & Royalties: Prognostication of myeloma via FISH, ~$2000/year; Merck: Consultancy; Bristol-Myers Squibb: Consultancy. Chng:Janssen China R&D: Research Funding.

ZFP36L1, a Novel Potential Tumor Supressor Gene in Myelofibrosis, Epigenetically Deregulated By Methylation of the Enhancer Region

IntroductionMyelofibrosis (MF) is known for frequent alterations in genes involved in epigenetic machinery, particularly in the regulation of DNA methylation. Enhancer regions experience changes in DNA methylation throughout myelopoiesis and may also experience aberrant DNA methylation in hematologic neoplasms, leading to altered expression of relevant genes. We aim to describe the DNA methylome of MF focusing on dynamic changes of DNA methylation in enhancer-regions, in order to identify new potential gene candidates with epigenetic deregulation who might have a role in the pathogenesis of MF and become therapeutic targets for this disease.MethodsDNA methylation profiles of 39 MF samples compared to 8 healthy controls were analysed using the Infinum Human-methylation 450K BeadChip platform. Differentially methylated CpG dinucleotides (DMCs) were validated with bisulfite sequencing. Enhancer regions were defined using publicly available data from the ChromHMM algorithm (H3K27Ac and H3K4Me3; Wijetunga, Nat Comm, 2014;5:5195). DNA methylation of DMCs mapped to enhancer regions was correlated to public gene expression datasets (GSE26049). The subgroup of genes with hypermethylated enhancers and downregulated expression was further studied, selecting candidate genes for epigenetic regulation through enhancer DNA methylation. Luciferase reporter assay using a CpG-free vector (pCPGL) was performed to confirm the functional effect of hypermethylated enhancers. SET2 cell line (JAK2V617F mutated) was used as in vitro model for pharmacologic treatment and lentiviral induction of selected gene candidates.ResultsWe found a total of 35216 differentially methylated CpGs (DMCs) between MF and controls (FDR <0.05). DMCs were significantly enriched to enhancer regions accounting for 8764 probes (24.8%; p<0.001 vs global DMC). 4182 enhancer-specific DMCs were hypermethylated (27.6%), comprising 2131 genes, whereas the remaining 10936 enhancer DMC were hypomethylated. Among genes showing reduced expression and enhancer hypermethylation in MF samples, we identified the ZFP36L1 gene as potential candidate. Moreover, gene targets of ZFP36L1 showed a significant enrichment among the genes overexpressed in MF indicating that ZFP36L1 downregulation could play an important role in MF. A novel algorithm to search for genes with putative 5´UTR AU rich elements was used (DREME motif discovery algorithm; E-value=4.5*10-15).We detectedabsence of DNA methylation in the promoter region of ZFP36L1 in SET2 cell line, MF samples and healthy controls, whereas the enhancer region showed intermediate DNA methylation in healthy controls and hypermethylation in SET2 and MF samples. DNA hypermethylation of the enhancer correlated with reduced ZFP36L1 gene expression (p<0.05). 5-azacytidine partially rescued ZFP36L1 expression in SET2 cell line, which was associated with a reduction of enhancer DNA methylation. Luciferase reporter assays with the pCPGL-vector showed that DNA methylation of the ZFP36L1 enhancer region abrogated the luciferase activity, demonstrating for the first time that this enhancer region regulates the expression of ZFP36L1 in MF.Finally, we demonstrated that lentiviral induction of ZFP36L1 in SET2 cell line, significantly reduced cell proliferation and increased apoptosis (Annexin V). We observed similar results by treatment of SET2 cells with PMA, a known inductor of ZFP36L1 . Additionally, we found that rescue of ZFP36L1 expression was associated with a decrease in CDK6, suggesting a potential role of ZFP36L1-CDK6 axis in the pathogenesis of MF.ConclusionWe detected a significant inappropriate DNA methylation of enhancer regions in MF, showing a novel pathogenetic mechanism for this disease that might be extrapolated to other haematologic malignancies. Aberrant DNA methylation of enhancer regions affects genes with potential functional implications. ZFP36L1 is an example of this phenomenon, harbouring DNA hypermethylation of the enhancer region and constituting a potential tumour suppressor gene with inappropriate epigenetic silencing. ZFP36L1 might be relevant for the control of cell cycle progression in MF through modulation of the expression of CDK6 . DisclosuresNo relevant conflicts of interest to declare.

ZNF677 downregulation by promoter hypermethylation as a driver event through gastric tumorigenesis

Gastric cancer (GC) is one of the leading causes of cancer-related death worldwide, due to poor prognosis and treatment failure; demanding new diagnostic and therapeutic targets. Therefore, in the present study, the methylation and expression status of ZNF677, as a promising tumor suppressor, were investigated in GC. Gene Expression Omnibus (GEO) datasets were used to initially evaluate ZNF677 expression and methylation in GC samples. Confirmation was performed on fifty internal samples, including gastric tumors and adjacent normal specimens, using q-MSP and q-PCR methods. Further validations were done using The Cancer Genome Atlas (TCGA) data on human cancers. The obtained results in silico and experimentally illustrated that ZNF677 is significantly hypermethylated and downregulated through gastric tumorigenesis. ZNF677 methylation levels were also correlated with perineural invasion (p = 0.0382) in internal samples. Furthermore, Spearman's correlation analysis showed that ZNF677 methylation is negatively (r = −0.4614, p < 0.0001) correlated with its mRNA expression levels. ROC curve analysis also illustrated the high diagnostic value of ZNF677 methylation for early detection of GC (AUC = 0.8592). Gene set enrichment analysis further revealed that ZNF677 participates in the regulation of cellular processes such as cell proliferation in GC. Moreover, in addition to hypermethylation in other malignancies, including breast, lung, and colorectal cancers, ZNF677 was hypermethylated in precancerous gastric tissues with intestinal metaplasia, indicating its methylation as a driver event through tumorigenesis. Taken together, our results suggest ZNF677 as a potential tumor suppressor gene, which could be considered as a diagnostic and therapeutic target for GC.

TSHZ3 functions as a tumor suppressor by DNA methylation in colorectal cancer

ObjectiveTeashirt zinc finger homeobox 3 (TSHZ3) is currently reported to be aberrantly expressed in several tumors, but the detailed functions and epigenetic mechanisms of TSHZ3 in colorectal cancer (CRC) remain unclear. Materials and methodsIn this study, the TSHZ3 expression in 118 CRC and normal adjacent tissues (NATs) was evaluated, and the methylation status of the TSZH3 promoter region in CRC tissues and cell lines was also analyzed. ResultsThe results of PCR analysis showed that TSHZ3 was significantly down-regulated in CRC tissues, and patients with low TSHZ3 levels had a poorer 5-year overall survival (OS) rate. Analyzing the promoter sequence (−1000∼0) by MethPrimer, TSHZ3 promoter was found to harbor abundant of CpG islands. The methylation specific PCR (MSP) analysis presented a relatively hypermethylated status of THSZ3 promoter in CRC samples. The data of MSP and bisulfite sequencing PCR (BSP) also confirmed that CpG sites of TSHZ3 promoter were methylated in CRC cells, and the DNA methyltransferase (DNMT) inhibitor 5-aza-2′-deoxycytidine (5-Aza) could effectively restored the TSHZ3 expression in vitro. Functionally, the proliferation, apoptosis and metastasis of CRC cells were regulated by TSZH3 over-expression, and the suppressing effects of TSHZ3 in CRC were also confirmed in a xenograft mouse model. ConlusionsOur results indicated that promoter methylation was one of the mechanisms contributing to the down-regulation of TSHZ3 in CRC, and TSZH3 might served as a potential tumor suppressor gene in the development and progression of CRC.

Identification of significant genes as prognostic markers and potential tumor suppressors in lung adenocarcinoma via bioinformatical analysis

BackgroundLung adenocarcinoma (LAC) is the predominant histologic subtype of lung cancer and has a complicated pathogenesis with high mortality. The purpose of this study was to identify differentially expressed genes (DEGs) with prognostic value and determine their underlying mechanisms.MethodsGene expression data of GSE27262 and GSE118370 were acquired from the Gene Expression Omnibus database, enrolling 31 LAC and 31 normal tissues. Common DEGs between LAC and normal tissues were identified using the GEO2R tool and Venn diagram software. Next, the Database for Annotation, Visualization, and Integrated Discovery (DAVID) was used to analyze the Gene Ontology and Kyoto Encyclopedia of Gene and Genome (KEGG) pathways. Then, protein-protein interaction (PPI) network of DEGs was visualized by Cytoscape with Search Tool for the Retrieval of Interacting Genes and central genes were identified via Molecular Complex Detection. Furthermore, the expression and prognostic information of central genes were validated via Gene Expression Profiling Interactive Analysis (GEPIA) and Kaplan-Meier analysis, respectively. Finally, DAVID, real-time PCR and immunohistochemistry were applied to re-analyze the identified genes, which were also further validated in two additional datasets from ArrayExpress database.ResultsFirst, 189 common DEGs were identified among the two datasets, including 162 downregulated and 27 upregulated genes. Next, Gene Ontology and KEGG pathway analysis of the DEGs were conducted through DAVID. Then, PPI network of DEGs was constructed and 17 downregulated central genes were identified. Furthermore, the 17 downregulated central genes were validated via GEPIA and datasets from ArrayExpress, and 12 of them showed a significantly better prognosis. Finally, six genes were identified significantly enriched in neuroactive ligand-receptor interactions (EDNRB, RXFP1, P2RY1, CALCRL) and Rap1 signaling pathway (TEK, P2RY1, ANGPT1) via DAVID, which were further validated to be weakly expressed in LAC tissues via RNA quantification and immunohistochemistry analysis.ConclusionsThe low expression pattern and relation to prognosis indicated that the six genes were potential tumor suppressor genes in LAC. In conclusion, we identified six significantly downregulated DEGs as prognostic markers and potential tumor suppressor genes in LAC based on integrated bioinformatics methods, which could act as potential molecular markers and therapeutic targets for LAC patients.

SPRY4 suppresses proliferation and induces apoptosis of colorectal cancer cells by repressing oncogene EZH2.

Colorectal cancer (CRC), a common malignant tumor in the digestive tract, is a leading cause of cancer-related death. SPRY4 has been reported to act as a tumor suppressor gene in various tumors. This study aims to assess the role of SPRY4 in colorectal cancer (CRC) and uncover its underlying mechanisms. Firstly, the expression levels of SPRY4 were measured in CRC cell lines. SPRY4-overexpressing or silencing plasmids were transfected into CRC cells to regulate its expression level. CCK-8, colony formation, EdU assay, wound-healing and Transwell assays were performed to determine cell proliferation, invasion and migration abilities. Then, apoptosis was measured by flow cytometry analysis, and the expression of apoptosis-related protein was analyzed by western-blotting. Next, the in vivo tumorigenesis assay was performed in nude mice. According to the results, there was a lower expression of SPRY4 in CRC cell lines compared with normal cell line, and the overexpression of SPRY4 significantly suppressed cell proliferation, migration and invasion, and promoted apoptosis in SW480 cells. Moreover, the enhanced proliferation, invasion and migration upon SPRY4 silencing was reversed by EZH2 inhibition. In addition, we found that the overexpression of SPRY4 inhibited tumorigenesis in vivo by diminishing the size and weight of the tumors. Our study indicates that SPRY4 might be a potential tumor suppressor gene and prognostic factor for patients with CRC.

Genome-Wide Methylation Mapping Using Nanopore Sequencing Technology Identifies Novel Tumor Suppressor Genes in Hepatocellular Carcinoma.

Downregulation of multiple tumor suppressor genes (TSGs) plays an important role in cancer formation. Recent evidence has accumulated that cancer progression involves genome-wide alteration of epigenetic modifications, which may cause downregulation of the tumor suppressor gene. Using hepatocellular carcinoma (HCC) as a system, we mapped 5-methylcytosine signal at a genome-wide scale using nanopore sequencing technology to identify novel TSGs. Integration of methylation data with gene transcription profile of regenerated liver and primary HCCs allowed us to identify 10 potential tumor suppressor gene candidates. Subsequent validation led us to focus on functionally characterizing one candidate—glucokinase (GCK). We show here that overexpression of GCK inhibits the proliferation of HCC cells via induction of intracellular lactate accumulation and subsequently causes energy crisis due to NAD+ depletion. This suggests GCK functions as a tumor suppressor gene and may be involved in HCC development. In conclusion, these data provide valuable clues for further investigations of the process of tumorigenesis in human cancer.

Bioinformatics analysis reveals that ANXA1 and SPINK5 are novel tumor suppressor genes in patients with oral squamous cell carcinoma.

BackgroundOral squamous cell carcinoma (OSCC) is a solid tumor of squamous epithelial origin. Currently, surgery is still the main treatment for OSCC, with radiotherapy and chemotherapy as important adjuvant treatments. However, the problem of poor prognosis of OSCC patients still exists in clinical practice. To explore further potential biomarkers or treatment targets in OSCC patients, this study used a high-throughput gene expression database to study the potential molecular mechanisms of OSCC carcinogenesis.MethodsThe GEO database related to OSCC was searched and analyzed using GEO2R. Oncomine and the Human Protein Atlas were used to evaluate the expression level of differentially-expressed genes (DEGs). The cBioPortal dataset was used to analyze the mutations of the potential DEGs and patient survival.ResultsThree GEO datasets, GSE146483, GSE138206, and GSE148944, were downloaded and 7 DEGs were found in common in OSCC tissues. Using Oncomine and the Human Protein Atlas, ANXA1, IL1RN, and SPINK5 were decreased in cancer tissues, while protein levels of APOE and IFI35 were increased accordingly. Interestingly, low levels of ANXA1 and SPINKS were associated with the TNM stage of OSCC patients. No mutations in DEGs were found in OSCC patients, based on the cBioPortal dataset. Survival analysis indicated OSCC patients with high MSR1 had poor overall survival (OS), while low expression of CXCR4, ANXA1, IL1RN, and SPINK5 also predicted poor OS in OSCC patients.ConclusionsOur findings uncovered 7 potential biomarkers of OSCC patients, with ANXA1 and SPINK5 serving as potential tumor suppressor genes in OSCC.

Aryl hydrocarbon receptor interacting protein (AIP) significantly influences prognosis of pancreatic carcinoma

IntroductionAryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor. Aryl hydrocarbon receptor interacting protein (AIP) in one of AHR ligands. The aim of this study is to analyze the prognostic influence of AIP in pancreatic carcinoma. Material and methodsRetrospective case series with immunohistochemical analysis of AIP. We have estimated a multivariate Cox's model for the outcome (progression free and overall survival). Results204 patients were included in the study. As expected prognosis was poor and 67.8% died of disease. As for AIP 9.8% of the cases showed nuclear staining of the epithelial tumor cells and 59.4% a cytoplasmic one. Stroma was stained in 53.1% of the cases.Univariate survival analysis revealed a significantly worse prognosis of patients with cytoplasmic AIP expression (stroma and epithelium), but nuclear expression was associated to a better prognosis. In the multivariate analysis stromal AIP expression was an independent prognosticator of progression free survival, together with pT stage, histological grade and history of diabetes. DiscussionAIP Is a conserved cochaperone protein binding to many proteins. AIP has been proposed as a potential tumor suppressor gene. To date, no study has analyzed the immunohistochemical expression of AIP in pancreatic carcinoma. Our results indicate that both epithelial and stromal cytoplasmic expression of AIP is associated to bad prognosis, while nuclear translocation seems to improve prognosis. ConclusionAlthough we must deepen into the complex signaling pathways underlying this potential association, our results open a way to inhibiting AHR as a potential target against pancreatic carcinoma.