Search

Clear cell renal cell carcinoma (ccRCC) is the most prevalent type of malignancy in adults. However, the clinical significance of tumor suppressor genes (TSG) is largely elusive. Herein, the expression profile TSGs and its clinical response in ccRCC were investigated. A total of 603 ccRCC samples from two cohorts (TCGA and ICGC) were retrieved in this study. Three molecular subtypes (C1, C2, and C3) were identified based on the TSGs expression profile in the TCGA dataset. Through Weighted Gene Correlation Network Analysis (WGCNA), six modules associated with three subtypes were identified. Pathway enrichment for the modules revealed that crucial pathways including p53 signaling and immune-related pathways were significantly enriched. We further focused on the relationship between immune infiltration level and subtypes, and found that subtype C1 was associated with higher immune infiltration level, subtype C2 was corresponding with medium immune infiltration level, whereas subtype C3 was correlated with lower immune infiltration level. Interestingly, C2 have a better survival outcome, while C1 and C3 showed a poor prognosis. Considering their survival difference, we then performed a differentially expression analysis between C2 and C1&3, and a total of 99 differentially expressed tumor suppressor genes (DETSGs) were identified. According to these DETSGs, 59 potential compounds with 28 mechanisms of action (MOA) were predicted using the Connectivity Map (CMap) database. Among these compounds, leflunomide, naftopidil, and ribavirin were the most prospective compounds for the treatment of ccRCC. In addition, we found that subtype C2 is more sensitive to sorafenib and sunitinib drugs, and C2 have more likelihood to be responded to immunotherapy. In summary, the three subtypes hinged on the tumor suppressor gene expression for ccRCC might contribute to understanding the underlying molecular mechanisms of ccRCC. Also, its potential compounds might offer guidelines for developing a novel treatment strategy of ccRCC.

Rationale: Breast cancer (BrCa) is the most common cancer worldwide, and the 5-year relative survival rate has declined in patients diagnosed at stage IV. Advanced BrCa is considered as incurable, which still lack effective treatment strategies. Identifying and characterizing new tumor suppression genes is important to establish effective prognostic biomarkers or therapeutic targets for late-stage BrCa. Methods: RNA-seq was applied in BrCa tissues and normal breast tissues. Through analyzing differentially expressed genes, DRD2 was selected for further analysis. And expression and promoter methylation status of DRD2 were also determined. DRD2 functions were analyzed by various cell biology assays in vitro. Subcutaneous tumor model was used to explore DRD2 effects in vivo. A co-cultivated system was constructed to investigate interactions of DRD2 and macrophages in vitro. WB, IHC, IF, TUNEL, qRT-PCR, Co-IP, Antibody Array, and Mass Spectrum analysis were further applied to determine the detailed mechanism. Results: In BrCa, DRD2 was found to be downregulated due to promoter methylation. Higher expression of DRD2 positively correlated with longer survival times especially in HER2-positive patients. DRD2 also promoted BrCa cells sensitivity to Paclitaxel. Ectopic expression of DRD2 significantly inhibited BrCa tumorigenesis. DRD2 also induced apoptosis as well as necroptosis in vitro and in vivo. DRD2 restricted NF-κB signaling pathway activation through interacting with β-arrestin2, DDX5 and eEF1A2. Interestingly, DRD2 also regulated microenvironment as it facilitated M1 polarization of macrophages, and triggered GSDME-executed pyroptosis. Conclusion: Collectively, this study novelly manifests the role of DRD2 in suppressing BrCa tumorigenesis, predicting prognosis and treatment response. And this study further reveals the critical role of DRD2 in educating M1 macrophages, restricting NF-κB signaling pathway and triggering different processes of programmed cell death in BrCa. Taking together, those findings represent a predictive and therapeutic target for BrCa.

The paternally imprinted neuronatin (NNAT) gene has been identified as a target of aberrant epigenetic silencing in diverse cancers, but no association with pediatric bone cancers has been reported to date. In screening childhood cancers, we identified aberrant CpG island hypermethylation in a majority of osteosarcoma (OS) samples and in 5 of 6 human OS cell lines studied but not in normal bone-derived tissue samples. CpG island hypermethylation was associated with transcriptional silencing in human OS cells, and silencing was reversible upon treatment with 5-aza-2’-deoxycytidine. Expression of NNAT was detectable in osteoblasts and chondrocytes of human bone, supporting a potential role in bone homeostasis. Enforced expression of NNAT in human OS cells lacking endogenous expression resulted in significant reduction in colony formation and in vitro migration compared to nonexpressor control cells. We next analyzed the effect of NNAT expression on intracellular calcium homeostasis and found that was associated with an attenuated decay of calcium levels to baseline following ATP-induced release of calcium from endoplasmic reticulum (ER) stores. Furthermore, NNAT expression was associated with increased cytotoxicity in OS cells from thapsigargin, an inhibitor of calcium reuptake into ER and an inducer of the ER stress response. These results suggest a possible tumor suppressor role for NNAT in human osteosarcoma. Additional study is needed ascertain sensitization to ER stress-associated apoptosis as a mechanism of NNAT-dependent cytotoxicity. In that case, epigenetic modification therapy to effect NNAT transcriptional derepression may represent a therapeutic strategy potentially of benefit to a majority of osteosarcoma patients.

Disruption of the circadian rhythm is a risk factor for cancer, while glioma is a leading contributor to mortality worldwide. Substantial efforts are being undertaken to decrypt underlying molecular pathways. Our understanding of the mechanisms through which disrupted circadian rhythm induces glioma development and progression is incomplete. We, therefore, examined changes in the expression of glioma-related genes in the mouse brain after chronic jetlag (CJL) exposure. A total of 22 candidate tumor suppressor (n= 14) and oncogenes (n= 8) were identified and analyzed for their interaction with clock genes. Both the control and CJL groups were investigated for the expression of candidate genes in the nucleus accumbens, hippocampus, prefrontal cortex, hypothalamus, and striatum of wild type, Bmal1-/- and Cry1/2 double knockout male mice. We found significant variations in the expression of candidate tumor suppressor and oncogenes in the brain tissues after CJL treatment in the wild type, Bmal1-/- and Cry1/2 double knockout mice. In response to CJL treatment, some of the genes were regulated in the wild type, Bmal1-/- and Cry1/2 similarly. However, the expression of some of the genes indicated their association with the functional clock. Overall, our result suggests a link between CJL and gliomas risk at least partially dependent on the circadian clock. However, further studies are needed to investigate the molecular mechanism associated with CJL and gliomas.

// Benjamin Garcia-Bloj 1, 2, 4 , Colette Moses 1, 2 , Agustin Sgro 1 , Janice Plani-Lam 1 , Mahira Arooj 1, 5 , Ciara Duffy 1, 2 , Shreyas Thiruvengadam 1 , Anabel Sorolla 1 , Rabab Rashwan 1 , Ricardo L. Mancera 5 , Andrea Leisewitz 4 , Theresa Swift-Scanlan 6 , Alejandro H. Corvalan 3, 4 , Pilar Blancafort 1, 2 1 Cancer Epigenetics group, The Harry Perkins Institute of Medical Research, Perth, WA, 6009, Australia 2 School of Anatomy, Physiology and Human Biology, The University of Western Australia, Perth, WA, 6009, Australia 3 Advanced Center for Chronic Diseases (ACCDiS), Pontificia Universidad Catolica de Chile, Santiago, RM, 8330034, Chile 4 Faculty of Medicine, School of Medicine, Pontificia Universidad Catolica de Chile, Santiago, RM, 8330034, Chile 5 School of Biomedical Sciences, Curtin Health Innovation Research Institute, Curtin University, Perth WA 6845, Australia 6 School of Nursing, Virginia Commonwealth University, Richmond, Virginia, 23298, USA Correspondence to: Pilar Blancafort, email: pilar.blancafort@uwa.edu.au Keywords: CRISPR/dCas9, ZF, TALE, tumor suppressor genes, gene reactivation Received: June 01, 2016 Accepted: July 19, 2016 Published: August 09, 2016 ABSTRACT The aberrant epigenetic silencing of tumor suppressor genes (TSGs) plays a major role during carcinogenesis and regaining these dormant functions by engineering of sequence-specific epigenome editing tools offers a unique opportunity for targeted therapies. However, effectively normalizing the expression and regaining tumor suppressive functions of silenced TSGs by artificial transcription factors (ATFs) still remains a major challenge. Herein we describe novel combinatorial strategies for the potent reactivation of two class II TSGs, MASPIN and REPRIMO , in cell lines with varying epigenetic states, using the CRISPR/dCas9 associated system linked to a panel of effector domains (VP64, p300, VPR and SAM complex), as well as with protein-based ATFs, Zinc Fingers and TALEs. We found that co-delivery of multiple effector domains using a combination of CRISPR/dCas9 and TALEs or SAM complex maximized activation in highly methylated promoters. In particular, CRISPR/dCas9 VPR with SAM upregulated MASPIN mRNA (22,145-fold change) in H157 lung cancer cells, with accompanying re-expression of MASPIN protein, which led to a concomitant inhibition of cell proliferation and induction of apoptotic cell death. Consistently, CRISPR/dCas9 VP64 with SAM upregulated REPRIMO (680-fold change), which led to phenotypic reprogramming in AGS gastric cancer cells. Altogether, our results outlined novel sequence-specific, combinatorial epigenome editing approaches to reactivate highly methylated TSGs as a promising therapy for cancer and other diseases.

Lysosomal-associated protein multispanning transmembrane 5 (LAPTM5) is a membrane protein that localizes to intracellular vesicles. It has been previously demonstrated that LAPTM5 expression level is decreased in neuroblastoma (NB) cells, and excessive accumulation of LAPTM5 was shown to induce lysosomal cell death in these cells. However, the pathological expression and role of LAPTM5 in other types of human cancers are largely unknown. Here, we found that LAPTM5 mRNA level is frequently decreased in various cancer cell lines, and its low expression in patients with esophageal squamous cell carcinoma (ESCC) and non-small cell lung cancer (NSCLC) was significantly correlated with poor prognosis. Furthermore, we showed that overexpression of LAPTM5 in several cancer cells induces lysosomal cell death due to lysosomal destabilization, indicated by leakage of lysosomal cathepsin D into the cytosol as well as impairment of autophagy. These findings suggest that the inactivation of LAPTM5 may contribute to tumorigenesis in a subset of human cancers.

// Abdulkhaleg Ibrahim 1, 2, * , Mahmoud Alhosin 3, 4, * , Christophe Papin 1 , Khalid Ouararhni 1 , Ziad Omran 5 , Mazin A. Zamzami 3, 4 , Abdulrahman Labeed Al-Malki 3 , Hani Choudhry 3, 4 , Yves Mely 6 , Ali Hamiche 1 , Marc Mousli 6 and Christian Bronner 1 1 Institut De Genetique Et De Biologie Moleculaire Et Cellulaire (IGBMC), INSERM U1258 CNRS UMR 7104, Universite de Strasbourg, Illkirch, France 2 BioTechnology Research Center (BTRC), Tripoli, Lybia 3 Department of Biochemistry, Cancer Metabolism and Epigenetic Unit, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia 4 Cancer and Mutagenesis Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia 5 College of Pharmacy, Umm Al-Qura University, Makkah, Saudi Arabia 6 CNRS UMR 7021 Laboratoire de Bioimagerie et Pathologies, Universite de Strasbourg, Faculte de Pharmacie, Illkirch, France * These authors contributed equally to this work Correspondence to: Mahmoud Alhosin, email: malhaseen@kau.edu.sa Christian Bronner, email: bronnerc@igbmc.fr Keywords: apoptosis; thymoquinone; tumor suppressor genes; ubiquitination; UHRF1 Received: November 30, 2017 Accepted: May 19, 2018 Published: June 19, 2018 ABSTRACT Down-regulation of UHRF1 (Ubiquitin-like containing PHD and Ring Finger 1) in Jurkat cells, induced by natural anticancer compounds such as thymoquinone, allows re-expression of tumor suppressor genes such as p73 and p16 INK4A . In order to decipher the mechanisms of UHRF1 down-regulation, we investigated the kinetic of expression of HAUSP (herpes virus-associated ubiquitin-specific protease), UHRF1, cleaved caspase-3 and p73 in Jurkat cells treated with thymoquinone. We found that thymoquinone induced degradation of UHRF1, correlated with a sharp decrease in HAUSP and an increase in cleaved caspase-3 and p73. UHRF1 concomitantly underwent a rapid ubiquitination in response to thymoquinone and this effect was not observed in the cells expressing mutant UHRF1 RING domain, suggesting that UHRF1 commits an auto-ubiquitination through its RING domain in response to thymoquinone treatment. Exposure of cells to Z-DEVD, an inhibitor of caspase-3 markedly reduced the thymoquinone-induced down-regulation of UHRF1, while proteosomal inhibitor MG132 had no such effect. The present findings indicate that thymoquinone induces in cancer cells a fast UHRF1 auto-ubiquitination through its RING domain associated with HAUSP down-regulation. They further suggest that thymoquinone-induced UHRF1 auto-ubiquitination followed by its degradation is a key event in inducing apoptosis through a proteasome-independent mechanism.

Tumor suppressor genes (TSGs) are commonly inactivated by somatic mutation and/or promoter methylation; yet, recent high-throughput genomic studies have not identified key TSGs inactivated by both mechanisms. We pursued an integrated molecular analysis based on methylation binding domain sequencing (MBD-seq), 450K Methylation arrays, whole exome sequencing, and whole genome gene expression arrays in primary head and neck squamous cell carcinoma (HNSCC) tumors and matched uvulopalatopharyngoplasty tissue samples (UPPPs). We uncovered 186 down-regulated genes harboring cancer specific promoter methylation including PAX1 and PAX5 and we identified 10 key tumor suppressor genes (GABRB3, HOXC12, PARP15, SLCO4C1, CDKN2A, PAX1, PIK3AP1, HOXC6, PLCB1, and ZIC4) inactivated by both promoter methylation and/or somatic mutation. Among the novel tumor suppressor genes discovered with dual mechanisms of inactivation, we found a high frequency of genomic and epigenomic alterations in the PAX gene family of transcription factors, which selectively impact canonical NOTCH and TP53 pathways to determine cell fate, cell survival, and genome maintenance. Our results highlight the importance of assessing TSGs at the genomic and epigenomic level to identify key pathways in HNSCC, deregulated by simultaneous promoter methylation and somatic mutations.

CDC42 is an oncogenic Rho GTPase overexpressed in colorectal cancer (CRC). Although CDC42 has been shown to regulate gene transcription, the specific molecular mechanisms regulating the oncogenic ability of CDC42 remain unknown. Here, we have characterized the transcriptional networks governed by CDC42 in the CRC SW620 cell line using gene expression analysis. Our results establish that several cancer-related signaling pathways, including cell migration and cell proliferation, are regulated by CDC42. This transcriptional signature was validated in two large cohorts of CRC patients and its clinical relevance was also studied. We demonstrate that three CDC42-regulated genes offered a better prognostic value when combined with CDC42 compared to CDC42 alone. In particular, the concordant overexpression of CDC42 and silencing of the putative tumor suppressor gene CACNA2D2 dramatically improved the prognostic value. The CACNA2D2/CDC42 prognostic classifier was further validated in a third CRC cohort as well as in vitro and in vivo CRC models. Altogether, we show that CDC42 has an active oncogenic role in CRC via the transcriptional regulation of multiple cancer-related pathways and that CDC42-mediated silencing of CACNA2D2 is clinically relevant. Our results further support the use of CDC42 specific inhibitors for the treatment of the most aggressive types of CRC, This work has been supported by grants to JCL from Ministerio de Ciencia e Innovación (SAF2008- 03750, RD06-0020-0016 and RD12/0036/0019) and to DGO Cancer Institute New South Wales (2017/CDF625). FVM is a National Breast Cancer Foundation/Cure Cancer Australia Foundation Postdoctoral Training Fellow.

Aberrant hypermethylation of CpG islands in tumor suppressor genes (TSGs) contributes to colorectal tumorigenesis. To identify new colorectal cancer (CRC) screening marker, we investigated DNA methylation alterations in novel TSGs. Using HumanMethylation450 BeadChip arrays, CpG regions in BEND5 were the most highly methylated among all genomic regions in 26 colorectal tumors compared to paired non-neoplastic tissues from a Taiwan cohort. Therefore, BEND5 was selected for further analysis. Quantitative methylation-specific real-time PCR revealed that 86.7% (117/135) of CRC patients exhibited hypermethylated BEND5. Real-time reverse transcription PCR identified that BEND5 mRNA expression was downregulated in 68% (32/47) of the analyzed samples. BEND5 hypermethylation was associated with poor overall survival (OS) in Taiwan patients with early-stage CRC (P = 0.037). In a CRC tissue set from South Korea, OS was higher in patients with high BEND5 protein expression than in those with low BEND5 protein expression (P = 0.037) by using immunohistochemistry assays. Consistently, BEND5 hypermethylation was associated with poor OS in patients with early-stage CRC in The Cancer Genome Atlas (TCGA) data set (P = 0.003). Multivariate Cox proportional hazards regression analysis further supported that hypermethylation of BEND5 genes was significantly associated with OS in Taiwan and TCGA CRC patients (P = 0.023 and 0.033, respectively). Finally, the cell model assay with transient transfection of BEND5 or si-BEND5 knockdown indicated that BEND5 inhibited cancer cell proliferation. In conclusion, epigenetic alteration in the candidate TSG BEND5 contributes to colorectal cancer development and is a prognostic marker of CRC.

Loss of 3p11-p14 is a frequent event in epithelial cancer and a candidate prognostic biomarker in cervical cancer. In addition to loss, promoter methylation can participate in gene silencing and promote tumor aggressiveness. We have performed a complete mapping of promoter methylation at 3p11-p14 in two independent cohorts of cervical cancer patients (n = 149, n = 121), using Illumina 450K methylation arrays. The aim was to investigate whether hyperm-ethylation was frequent and could contribute to gene silencing and disease aggressiveness either alone or combined with loss. By comparing the methylation level of individual CpG sites with corresponding data of normal cervical tissue, 26 out of 41 genes were found to be hypermethylated in both cohorts. The frequency of patients with hypermethylation of these genes was found to be higher at tumor stages of 3 and 4 than in stage 1 tumors. Seventeen of the 26 genes were transcriptionally downregulated in cancer compared to normal tissue, whereof 6 genes showed a significant correlation between methylation and expression. Integrated analysis of methylation, gene dosage, and expression of the 26 hypermethylated genes identified 3 regulation patterns encompassing 8 hypermethylated genes; a methylation driven pattern (C3orf14, GPR27, ZNF717), a gene dosage driven pattern (THOC7, PSMD6), and a combined methylation and gene dosage driven pattern (FHIT, ADAMTS9, LRIG1). In survival analysis, patients with both hypermethylation and loss of LRIG1 had a worse outcome compared to those harboring only hypermethylation or none of the events. C3orf14 emerged as a novel methylation regulated suppressor gene, for which knockdown was found to promote invasive growth in human papilloma virus (HPV)-transformed keratinocytes. In conclusion, hypermethylation at 3p11-p14 is common in cervical cancer and may exert a selection pressure during carcinogenesis alone or combined with loss. Information on both events could lead to improved prognostic markers.

C13ORF18 is frequently hypermethylated in cervical cancer but not in normal cervix and might serve as a biomarker for the early detection of cervical cancer in scrapings. As hypermethylation is often observed for silenced tumor suppressor genes (TSGs), hypermethylated biomarker genes might exhibit tumor suppressive activities upon re-expression. Epigenetic drugs are successfully exploited to reverse TSG silencing, but act genome-wide. Artificial Transcription Factors (ATFs) provide a gene-specific approach for re-expression of silenced genes. Here, we investigated the potential tumor suppressive role of C13ORF18 in cervical cancer by ATF-induced re-expression.Five zinc finger proteins were engineered to bind the C13ORF18 promoter and fused to a strong transcriptional activator. C13ORF18 expression could be induced in cervical cell lines: ranging from >40-fold in positive (C13ORF18-unmethylated) cells to >110-fold in negative (C13ORF18-methylated) cells. Re-activation of C13ORF18 resulted in significant cell growth inhibition and/or induction of apoptosis. Co-treatment of cell lines with ATFs and epigenetic drugs further enhanced the ATF-induced effects. Interestingly, reactivation of C13ORF18 led to partial demethylation of the C13ORF18 promoter and decreased repressive histone methylation. These data demonstrate the potency of ATFs to re-express and potentially demethylate hypermethylated silenced genes. Concluding, we show that C13ORF18 has a TSG function in cervical cancer and may serve as a therapeutic anti-cancer target. As the amount of epimutations in cancer exceeds the number of gene mutations, ATFs provide promising tools to validate hypermethylated marker genes as therapeutic targets. (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Made available in DSpace on 2013-09-27T14:54:30Z (GMT). No. of bitstreams: 1 WOS000299828200002.pdf: 4061341 bytes, checksum: 619a18898a4a748ec384f1463d41e000 (MD5) Previous issue date: 2011-12-01 Made available in DSpace on 2013-09-30T18:38:00Z (GMT). No. of bitstreams: 1 WOS000299828200002.pdf: 4061341 bytes, checksum: 619a18898a4a748ec384f1463d41e000 (MD5) Previous issue date: 2011-12-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:50:32Z No. of bitstreams: 1 WOS000299828200002.pdf: 4061341 bytes, checksum: 619a18898a4a748ec384f1463d41e000 (MD5) Made available in DSpace on 2014-05-20T13:50:32Z (GMT). No. of bitstreams: 1 WOS000299828200002.pdf: 4061341 bytes, checksum: 619a18898a4a748ec384f1463d41e000 (MD5) Previous issue date: 2011-12-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) NIH/NCI Epigenetic mechanisms are frequently deregulated in cancer cells and can lead to the silencing of genes with tumor suppressor activities. The isoform A of the Ras-association domain family member 1 (RASSF1A) gene is one of the most frequently silenced transcripts in human tumors; however, few studies have simultaneously investigated epigenetic abnormalities associated with the 3p21.3 tumor suppressor gene cluster flanking RASSF1 (i.e., SEMA3B, HYAL3, HYAL2, HYAL1, TUSC2, RASSF1, ZMYND10, NPRL2, TMEM115 and CACNA2D2). This study aimed to investigate the role of epigenetic changes to these genes in 17 breast cancer cell lines and in three non-tumorigenic epithelial breast cell lines (184A1, 184B5 and MCF 10A) and to evaluate the effect on gene expression of treatment with the demethylating agent 5-Aza -2'-deoxycytidine and/or Trichostatin A (TSA), a histone deacetylase inhibitor. We report that, although the RASSF1A isoform was determined to be epigenetically silenced in 15 of the 17 breast cancer cell lines, all the cell lines expressed the RASSF1C isoform. Five breast cancer cell lines overexpressed RASSF1C when compared with the normal epithelial cell line 184A1. Furthermore, the genes HYAL1 and CACNA2D2 were significantly overexpressed after the treatments. After the combined treatment, RASSF1A re-expression was accompanied by an increase in expression levels of the flanking genes. The Spearman's correlation coefficient indicated a positive co-regulation of the following gene pairs: RASSF1 and TUSC2 (r = 0.64, p = 0.002), RASSF1 and ZMYND10 (r = 0.58, p = 0.07), RASSF1 and NPRL2 (r = 0.48, p = 0.03), ZMYND10 and NPRL2 (r = 0.71; p = 0.0004) and NPRL2 and TMEM115 (r = 0.66, p = 0.001). Interestingly, the genes TUSC2, NPRL2 and TMEM115 were found to be unmethylated in each of the untreated cell lines. Chromatin immunoprecipitation using antibodies against the acetylated and trimethylated lysine 9 of histone H3 demonstrated low levels of histone methylation in these genes, which are located closest to RASSF1. These results provide evidence that epigenetic repression is involved in the downregulation of multiple genes at 3p21.3 in breast cancer cells. São Paulo State Univ UNESP, Lab Epigenet, Dept Genet, Biosci Inst, São Paulo, Brazil Georgetown Univ, Med Ctr, Dept Oncol, Washington, DC 20007 USA São Paulo State Univ UNESP, Lab Epigenet, Dept Genet, Biosci Inst, São Paulo, Brazil FAPESP: 07/59110-9 NIH/NCI: P30-CA51008

Identification of mutations of the genes that give cancer a selective advantage is an important step towards research and clinical objectives. As such, there has been a growing interest in developing methods for identification of driver genes and their temporal order within a single patient (intra-tumor) as well as across a cohort of patients (inter-tumor). In this paper, we develop a probabilistic model for tumor progression, in which the driver genes are clustered into several ordered driver pathways. We develop an efficient inference algorithm that exhibits favorable scalability to the number of genes and samples compared to a previously introduced ILP-based method. Adopting a probabilistic approach also allows principled approaches to model selection and uncertainty quantification. Using a large set of experiments on synthetic datasets, we demonstrate our superior performance compared to the ILP-based method. We also analyze two biological datasets of colorectal and glioblastoma cancers. We emphasize that while the ILP-based method puts many seemingly passenger genes in the driver pathways, our algorithm keeps focused on truly driver genes and outputs more accurate models for cancer progression., Author summary Cancer is a disease caused by the accumulation of somatic mutations in the genome. This process is mainly driven by mutations in certain genes that give the harboring cells some selective advantage. The rather few driver genes are usually masked amongst an abundance of so-called passenger mutations. Identification of the driver genes and the temporal order in which the mutations occur is of great importance towards research and clinical objectives. In this paper, we introduce a probabilistic model for cancer progression and devise an efficient inference algorithm to train the model. We show that our method scales favorably to large datasets and provides superior performance compared to an ILP-based counterpart on a wide set of synthetic data simulations. Our Bayesian approach also allows for systematic model selection and confidence quantification procedures in contrast to the previous non-probabilistic progression models. We also study two large datasets on colorectal and glioblastoma cancers and validate our inferred model in comparison to the ILP-based method.

Grazia Lazzari,1 Giuseppe Buono,2 Benedetto Zannino,2 Giovanni Silvano1 1Radiation Oncology Unit, San Giuseppe Moscati Hospital, Taranto, 74100, Italy; 2Medical Oncology Unit, San Rocco Hospital, Sessa Aurunca, Caserta, 81037, ItalyCorrespondence: Grazia LazzariRadiation Oncology Unit, San Giuseppe Moscati Hospital, Taranto, 74100, ItalyEmail lazzarigrazia@gmail.comAbstract: BRCA1, BRCA2, TP53 and ATM gene mutations are the most studied tumour suppressor genes (TSGs) influencing the loco-regional approach to breast cancer (BC). Due to altered radio sensitivity of mutated cancer cells, mastectomy has always been advised in most patients with BC linked to TSGs mutations in order to avoid or minimize the use of adjuvant radiotherapy (ART). Whether ART is safe or not in these carriers is still debated. As a result, this issue has been widely discussed in the recent ASTRO and ASCO papers, yielding important and useful recommendations on the use of ART according to the mutational status. In this review, we have highlighted the impact of these mutations on local control, toxicities, second tumors, and contralateral breast cancers (CBCs) after ART to solve remaining doubts and encourage the safe use of ART when indicated.Keywords: radiation, radiosensitivity, tumor suppressor genes

BRCA1, BRCA2, TP53 and ATM gene mutations are the most studied tumour suppressor genes (TSGs) influencing the loco-regional approach to breast cancer (BC). Due to altered radio sensitivity of mutated cancer cells, mastectomy has always been advised in most patients with BC linked to TSGs mutations in order to avoid or minimize the use of adjuvant radiotherapy (ART). Whether ART is safe or not in these carriers is still debated. As a result, this issue has been widely discussed in the recent ASTRO and ASCO papers, yielding important and useful recommendations on the use of ART according to the mutational status. In this review, we have highlighted the impact of these mutations on local control, toxicities, second tumors, and contralateral breast cancers (CBCs) after ART to solve remaining doubts and encourage the safe use of ART when indicated.

BackgroundPancreatic cancer continues to be one of the most aggressive malignant tumors. Work in recent years in cancer molecular biology has revealed that metabolic reprogramming is an additional hallmark of cancer that is involved in the pathogenesis of cancers, and is intricately linked to gene mutations.Main textHowever, though oncogenes such asKRASandc-Mycplay important roles in the process, and have been extensively studied, no substantial improvements in the prognosis of pancreatic cancer have seen. Therefore, some scientists have tried to explain the mechanisms of abnormal cancer metabolism from the perspective of tumor suppressor genes. In this paper, we reviewed researches about how metabolic reprogramming was regulated by tumor suppressor genes in pancreatic cancer and their clinical implications.ConclusionAbnormal metabolism and genetic mutations are mutually causal and complementary in tumor initiation and development. A clear understanding of how metabolic reprogramming is regulated by the mutated genes would provide important insights into the pathogenesis and ultimately treatment of pancreatic cancer.

Colorectal cancer (CRC) is one of the most prevalent types of cancer in the world and ranks second in cancer deaths in the US. Despite the recent improvements in screening and treatment, the number of deaths associated with CRC is still very significant. The complexities involved in CRC therapy stem from multiple oncogenic mutations and crosstalk between abnormal pathways. This calls for using advanced molecular genetics to understand the underlying pathway interactions responsible for this cancer. In this paper, we construct the CRC pathway from the literature and using an existing public dataset on healthy vs tumor colon cells, we identify the genes and pathways that are mutated and are possibly responsible for the disease progression. We then introduce drugs in the CRC pathway, and using a boolean modeling technique, we deduce the drug combinations that produce maximum cell death. Our theoretical simulations demonstrate the effectiveness of Cryptotanshinone, a traditional Chinese herb derivative, achieved by targeting critical oncogenic mutations and enhancing cell death. Finally, we validate our theoretical results using wet lab experiments on HT29 and HCT116 human colorectal carcinoma cell lines.

ObjectClinically, the effective treatment options available to thyroid cancer (THCA) patients are very limited. Elucidating the features of tumor suppressor genes (TSGs) and the corresponding signal transduction cascade may provide clues for the development of new strategies for targeted therapy of THCA. Therefore, this paper aims to explore the mechanism of ZNF24 underlying promoting THCA cell senescence at molecular level.MethodsWe performed RT-PCR and Western Blotting for evaluating associated RNA and protein expression. CCK8, colony forming, wound healing and Transwell chamber assays were conducted to examine THCA cell proliferation, invasion and migration. β-galactosidase staining assay was performed to detect THCA cells senescence. The size and volume of xenotransplanted tumors in nude mice are calculated to asses ZNF24 effect in vivo.ResultsEctopic expression of ZNF24 significantly inhibited the cell viability, colony forming, migration and invasion abilities of THCA cell lines (K1/GLAG-66i and BCPAPi) (P < 0.05). ZNF24 induced BCPAPi cells senescence through regulating Wnt signaling pathway. ZNF24 inhibited Wnt signaling pathway activition by competitively binding β-catenin from LEF1/TCF1-β-catenin complex. In nude mice, both Ectopic expression of ZNF24 and 2,4-Da (the strong β-catenin/Tcf-4 inhibitor) treatment significantly decreased both the size and weight of xenotransplanted tumors when compared with control mice (P < 0.05).ConclusionResults obtained in vivo and in vitro reveal the role of ZNF24 in significantly suppressing THCA tumorigenesis and invasion by regulating Wnt signaling pathway.

EBV-associated gastric cancer (EBVaGC) is characterized by high frequency of DNA methylation. In this study, we investigated how epigenetic alteration of host genome contributes to pathogenesis of EBVaGC through the analysis of transcriptomic and epigenomic datasets from NIH TCGA (The Cancer Genome Atlas) consortium. We identified that immune related genes (IRGs) is a group of host genes preferentially silenced in EBV-positive gastric cancers through DNA hypermethylation. Further functional characterizations of selected IRGs reveal their novel antiviral activity against not only EBV but also KSHV. In particular, we showed that metallothionein-1 (MT1) and homeobox A (HOXA) gene clusters are down-regulated via EBV-driven DNA hypermethylation. Several MT1 isoforms suppress EBV lytic replication and release of progeny virions as well as KSHV lytic reactivation, suggesting functional redundancy of these genes. In addition, single HOXA10 isoform exerts antiviral activity against both EBV and KSHV. We also confirmed the antiviral effect of other dysregulated IRGs, such as IRAK2 and MAL, in scenario of EBV and KSHV lytic reactivation. Collectively, our results demonstrated that epigenetic silencing of IRGs is a viral strategy to escape immune surveillance and promote viral propagation, which is overall beneficial to viral oncogenesis of human gamma-herpesviruses (EBV and KSHV), considering that these IRGs possess antiviral activities against these oncoviruses., Author summary Epstein-Barr virus (EBV), one of the human gamma-herpesviruses, is a well-defined viral agent that strongly associates with malignancies of lymphoid and epithelial origin. EBV-associated gastric carcinoma (EBVaGC) is the most common malignancy caused by EBV infection. In this paper, we identified that beyond tumor suppressor genes, immune related genes (IRGs) are another group of host genes that are extensively DNA hypermethylated and epigenetically silenced in EBVaGC comparing to non-EBV GC. For certain IRGs, such as metallothionein-1 (MT1) and homeobox A (HOXA) genes, the whole gene clusters undergo DNA hypermethylation in EBVaGC, while it also occurs for isolated individual IRGs, such as IRAK2 and MAL. Considering the critical role of innate immunity in antiviral control, we expected that silencing of IRGs would benefit EBV viral replication and propagation. Indeed, our further investigation of several IRGs (MT1G, HOXA10, IRAK2, and MAL) confirmed their antiviral activities against EBV lytic replication. Some of them also suppress lytic replication of another human gamma-herpesvirus, Kaposi’s sarcoma-associated herpesvirus (KSHV), indicating their broad antiviral spectrum. Collectively, our results demonstrated that epigenetic silencing of antiviral IRGs is an efficient viral strategy utilized by oncoviruses, such as EBV, to escape immune surveillance and promote viral propagation, which is overall beneficial to viral oncogenesis.

Copy number variation (CNV) comprises a recently discovered kind of variation involving deletion and duplication of DNA segments of variable size, ranging from a few hundred basepairs to several million. By altering gene dosage levels or disrupting proximal or distant regulatory elements CNVs create human diversity. They represent also an important factor in human evolution and play a role in many disorders including cancer. Array-based comparative genomic hybridization as well as expression arrays are powerful and suitable methods for determination of copy number variations or gene expression changes in the human genome. In paper I we established a 32K clone-based genomic array, covering 99% of the current assembly of the human genome with high resolution and applied it in the profiling of 71 healthy individuals from three ethnic groups. Novel and previously reported CNVs, involving ~3.5% of the genome, were identified. Interestingly, 87% of the detected CNV regions overlapped with known genes indicating that they probably have phenotypic consequences. In papers II through IV we applied this platform to different tumor types, namely two collections of brain tumors, glioblastoma (paper II) and medulloblastoma (paper III), and a set of bladder carcinoma (paper IV) to identify chromosomal alterations at the level of DNA copy number that could be related to tumor initiation/progression. Tumors of the central nervous system represent a heterogeneous group of both benign and malignant neoplasms that affect both children and adults. Glioblastoma and medulloblastoma are two malignant forms. Glioblastoma often affects adults while the embryonal tumor medulloblastoma is the most common malignant brain tumor among children. The detailed profiling of 78 glioblastomas, allowed us to identify a complex pattern of aberrations including frequent and high copy number amplicons (detected in 79% of samples) as well as a number of homozygously deleted loci. These regions encompassed not only previously reported oncogenes and tumor suppressor genes but also numerous novel genes. In paper III, a subset of 26 medulloblastomas was analyzed using the same genomic array. We observed that alterations involving chromosome 17, especially isochromosome 17q, were the most common genomic aberrations in this tumor type, but copy number alterations involving other chromosomes: 1, 7 and 8 were also frequent. Focal amplifications, on chromosome 1 and 3, not previously described, were also detected. These loci may encompass novel genes involved in medulloblastoma development. In paper IV we examined for the presence of DNA copy number alterations and their effect on gene expression in a subset of 21 well-characterized Ta bladder carcinomas, selected for the presence or absence of recurrences. We identified a number of novel genes as well as a significant association between amplifications and high-grade and recurrent tumors which might be clinically useful. The results derived from these studies increase our understanding of the genetic alterations leading to the development of these tumor forms and point out candidate genes that may be used in future as targets for new diagnostic and therapeutic strategies.

DNA Methylation is an epigenetic phenomenon in which methyl groups are added to the cytosines, thereby altering the physio-chemical properties of the DNA region and influencing gene expression. Aberrant DNA methylation in a set of genes or across the genome results in many epigenetic diseases including cancer. In this paper, we use entropy to analyze the extent and distribution of DNA methylation in Tumor Suppressor Genes (TSG's) and Oncogenes related to a specific type of cancer (viz.) KIRC (Kidney-renal-clear-cell-carcinoma). We apply various mathematical transformations to enhance the different regions in DNA methylation distribution and compare the resultant entropies for healthy and tumor samples. We also obtain the sensitivity and specificity of classification for the different mathematical transformations. Our findings show that it is not just the measure of methylation, but the distribution of the methylation levels in the genes that are significant in cancer.

Comparative genomic hybridization (CGH) is a modern genetic method which enables a genome‐wide survey of chromosomal imbalances. For each chromosome region, one obtains the information whether there is a loss or gain of genetic material, or whether there is no change at that region. Usually it is not possible to evaluate all 46 chromosomes of a metaphase, therefore several (up to 20 or more) metaphases are analyzed per individual, and expressed as average. Mostly one does not study one individual alone but groups of 20–30 individuals. Therefore, large amounts of data quickly accumulate which must be put into a logical order. In this paper we present the application of a self‐organizing map (Genecluster) as a tool for cluster analysis of data from pT2N0 prostate cancer cases studied by CGH. Self‐organizing maps are artificial neural networks with the capability to form clusters on the basis of an unsupervised learning rule, i.e., in our examples it gets the CGH data as only information (no clinical data). We studied a group of 40 recent cases without follow‐up, an older group of 20 cases with follow‐up, and the data set obtained by pooling both groups. In all groups good clusterings were found in the sense that clinically similar cases were placed into the same clusters on the basis of the genetic information only. The data indicate that losses on chromosome arms 6q, 8p and 13q are all frequent in pT2N0 prostatic cancer, but the loss on 8p has probably the largest prognostic importance.