The role of tazarotene-induced gene 1 in carcinogenesis:is it a tumor suppressor gene or an oncogene?

Tazarotene-induced gene 1(TIG1)is induced by a derivative of vitamin A and is known to regulate many important biological processes and control the development of cancer.TIG1 is widely expressed in various tissues;yet in many cancer tissues,it is not expressed because of the methylation of its promoter.Additionally,the expression of TIG1 in cancer cells inhibits their growth and invasion,suggesting that TIG1 acts as a tumor suppressor gene.However,in some cancers,poor prognosis is associated with TIG1 expression,indicating its protumor growth characteristics,especially in promoting the invasion of inflammatory breast cancer cells.This review comprehensively summarizes the roles of the TIG1 gene in cancer development and details the mechanisms through which TIG1 regulates cancer development,with the aim of understanding its various roles in cancer development.

Cloning and Expression Analysis of a Human Putative Tumor Suppressor Gene Homologue from Rice

A gene homologous to the human Putative tumor suppressor gone QM, designated OSQM1, was isolated from rice (Oryza sativa L.) genomic DNA library using through homology screening. It contained 4 exons and 3 introns, encoding a protein of 219 amino acids with 46 basic amino acids, leading to a high isoelectric point of 11.02. Homology search showed that this gene existed in eukaryotes and highly conserved throughout eukaryotes, suggesting an essential role of this gene. Northern Not analysis showed that it was expressed in various rice organs, but at lower level in developing flower and callus tissue than in other vegetative organs. Its expression levels in roots and leaves were influenced by different environmental factors.

Inactivation of RASSF1A, the tumor suppressor gene at 3p21.3 in extrahepatic cholangiocarcinoma

AIM: To evaluate the genetic and epigenetic inactivation mechanism of the RASSF1A tumor suppressor gene at 3p21.3 in extrahepatic cholangiocarcinoma. METHODS: RT-PCR was used to investigate the transcriptional expressing and re-expression of RASSFIA. RASSFIA mutation was analyzed with SSCP and selective sequencing. PCR was performed to detect the loss of heterozygosity (LOH) at the region of chromosome 3p21.3. Genomic DNA were modificated bisulfite and the frequency of methylation of CpG islands in RASSFIA promoter were evaluated by methylation specific PCR (MS-PCR). RESULTS: In all 48 samples and one cell lines of extrahepatic cholangiocarcinoma, the RASSFIA mutation is rare (6.12%, 3/49), 33 samples (68.75%) and QBC-939 cell lines (X2= 14.270, P= 0.001<0.01) showed RASSFIA express inactivation with LOH at microsatellite loci D3S4604. Among these 33 samples and QBC-939, 28 of 33 (84.85%) tumor samples and 1 cell lines were methylated for majority of 16 CpGs, the average frequency is 73.42%. CONCLUSION: The data we present suggest that RASSFIA which we have been searching for at 3p21.3 may be one of the key tumor suppressor gene and play an important role in the pathogenesis of extrahepatic cholangiocarcinoma, and the promoter methylation and allelic loss are the major mechanism for inactivation of RASSFIA.

Aberrant methylation of the 3q25 tumor suppressor gene PTX3 in human esophageal squamous cell carcinoma

AIM:To identify the novel methylation-silenced gene pentraxin 3(PTX3) in esophageal squamous cell carcinoma(ESCC).METHODS:PTX3 mRNA expression was examined in six human ESCC cell lines,one human immortalized normal esophageal epithelial cell line,primary ESCC tumor tissue,and paired adjacent nontumor tissue using reverse transcription polymerase chain reaction(RTPCR).Semi-quantitative immunohistochemistry was used to examine cellular localisation and protein levels.Methylation specific PCR and bisulphite genomic sequencing were employed to investigate the methylation of the candidate gene.RESULTS:In the majority of ESCC cell lines,we found that PTX3 expression was down-regulated due to gene promoter hypermethylation,which was further confirmed by bisulphite genomic sequencing.Demethylation treatment with 5-aza-2'-deoxycytidine restored PTX3 mRNA expression in ESCC cell lines.Methylation was more common in tumor tissues(85%) than in adjacent nontumor tissues(25%)(P < 0.01).CONCLUSION:PTX3 is down-regulated through promoter hypermethylation in ESCC,and could potentially serve as a biomarker of ESCC.

CHROMOSOME 3 MAY HARBOR MULTIPLE TUMOR SUPPRESSOR GENES ASSOCIATED WITH PRIMARY GLIOBLASTOMA MULTIFORME

Objective: To investigate whether deletion of chromosome 3 is involved in the carcinogenesis of primary glioblastoma multiforme (GBM) and to localize the possible common deletion region in the aforementioned chromosome. Methods: PCR based microsatellite polymorphism analyses were performed to detect loss of heterozygosity (LOH). Twenty-three loci on chromosome 3 were examined in 20 cases of GBM. Fluorescence-labeled primers and Perkin Elmer 377 DNA Sequencer were applied. Results: 50% informative cases of GBM displayed LOH on chromosome 3. 50% of informative cases displayed LOH on 3q and 35% on 3p. 25.6% of informative loci showed LOH in our series, in which frequent LOH were observed in the chromosomal region from loci D3S1614 (42.9%) to D3S1565 (35.3%) on 3q24–27 and at loci D3S1569 (35.3%) on 3q22–23 and D3S1289 (33.3%) on 3p14.1–14.3. Conclusion: Loss of genetic material on chromosome 3 may play an important part in the tumorigenesis of GBM. The chromosomal regions from loci D3S1614 to D3S1565 on 3q24–27 and at loci D3S1569 on 3q22–23 and D3S1289 on 3p14.1–14.3 are potential sites for novel tumor suppressor genes associated with GBM.

CHROMOSOME 17P MAY HARBOR MULTIPLE TUMOR SUPPRESSOR GENES ASSOCIATED WITH PRIMARY GLIOBLASTOMA MULTIFORME

Objective: To investigate whether deletion of chromosome 17 is involved in the carcinogenesis of primary glioblastoma multiforme and to localize the possible common deletion region in the aforementioned chromosome. Methods: Polymerase chain reaction-based microsatellite analysis was used to assess loss of heterozygosity (LOH) on chromosome 17 in 20 primary glioblastoma multiforme (GBM). Fifteen fluorescent dye-labeled polymorphic markers were used. Results: Thirteen of twenty (65%) GBM displayed LOH on at least one marker of chromosome 17p. Two tumors showed either LOH or non-informativeness on all markers tested. The most frequent LOH was observed at loci including D17s799 (53.3%), D17s1852 (53.8%), D17s938 (63.20/o), D17s831 (55.6%). The loci D17s831 (on 17p13) and D17s799–D17s1852 (17p11.2–p12) are distal and proximal to p53 respectively. The frequencies of LOH at all loci examined on chromosome 17q were relatively low (<30%). None of informative loci exhibited microsatellite instability in this study. Conclusion: Loss of genetic material on chromosome 17p may play an important role in the pathogenesis of GBM. Besides the well-known TSG p53 on 17p, other unknown TSCs associated with GBM may be present on the chromosomal regions 17p13 and 17p11.2–p12, which are distal and proximal to p53 respectively.

Are there tumor suppressor genes on chromosome 4p in sporadic colorectal carcinoma?

AIM: To study the candidate tumor suppressor genes (TSG) on chromosome 4p by detecting the high frequency of loss of heterozygosity (LOH) in sporadic colorectal carcinoma in Chinese patients.METHODS: Seven fluorescent labeled polymorphic microsatellite markers were analyzed in 83 cases of colorectal carcinoma and matched normal tissue DNA by PCR. PCR products were eletrophoresed on an ABI 377 DNA sequencer. Genescan 3.7 and Genotype 3.7 software were used for LOH scanning and analysis. The same procedure was performed by the other six microsatellite markers spanning D4S3013 locus to make further detailed deletion mapping. Comparison between LOH frequency and clinicopathological factors was performed by χ2 test.RESULTS: Data were collected from all informative loci. The average LOH frequency on 4p was 24.25%, and 42.3% and 35.62% on D4S405 and D4S3013 locus, respectively. Adjacent markers of D4S3013 displayed a low LOH frequency (< 30%) by detailed deletion mapping. Significant opposite difference was observed between LOH frequency and tumor diameter on D4S412 and D4S1546 locus (0% vs 16.67%, P = 0.041; 54.55% vs 11.11%, P = 0.034, respectively). On D4S403 locus, LOH was significantly associated with tumor gross pattern (11.11%, 0, 33.33%, P = 0.030). No relationship was detected on other loci compared with clinicopathologial features.CONCLUSION: By deletion mapping, two obvious high frequency LOH regions spanning D4S3013 (4p15.2) and D4S405 (4p14) locus are detected. Candidate TSG, which is involved in carcinogenesis and progression of sporadic colorectal carcinoma on chromosome 4p, may be located between D4S3017 and D4S2933 (about 1.7 cm).

Promoter methylation of tumor suppressor genes in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma(ESCC) is a prevalent and fatal cancer in China and other Asian countries.Epigenetic silencing of key tumor suppressor genes(TSGs) is critical to ESCC initiation and progression.Recently,many novel TSGs silenced by promoter methylation have been identified in ESCC,and these genes further serve as potential tumor markers for high-risk group stratification,early detection,and prognosis prediction.This review summarizes recent discoveries on aberrant promoter methylation of TSGs in ESCC,providing better understanding of the role of disrupted epigenetic regulation in tumorigenesis and insight into diagnostic and prognostic biomarkers for this malignancy.

Tumor suppressor genes on frequently deleted chromosome 3p in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is among the most common malignancies in southern China.Deletion of genomic DNA,which occurs during the complex pathogenesis process for NPC,represents a pivotal mechanism in the inactivation of tumor suppressor genes (TSGs).In many circumstances,loss of TSGs can be detected as diagnostic and prognostic markers in cancer.The short arm of chromosome 3 (3p) is a frequently deleted chromosomal region in NPC,with 3p21.1-21.2 and 3p25.2-26.1 being the most frequently deleted minimal regions.In recent years,our research group and others have focused on the identification and characterization of novel target TSGs at 3p,such as RASSF1A,BLU,RBMS3,and CHL1,in the development and progression of NPC.In this review,we summarize recent findings of TSGs at 3p and discuss some of these genes in detail.A better understanding of TSGs at 3p will significantly improve our understanding of NPC pathogenesis,diagnosis,and treatment.

Recent progress in the study of methylated tumor suppressor genes in gastric cancer

Gastric cancer is one of the most common malignancies and a leading cause of cancer mortality worldwide.The pathogenesis mechanisms of gastric cancer are still not fully clear.Inactivation of tumor suppressor genes and activation of oncogenes caused by genetic and epigenetic alterations are known to play significant roles in carcinogenesis.Accumulating evidence has shown that epigenetic silencing of the tumor suppressor genes,particularly caused by hypermethylation of CpG islands in promoters,is critical to carcinogenesis and metastasis.Here,we review the recent progress in the study of methylations of tumor suppressor genes involved in the pathogenesis of gastric cancer.We also briefly describe the mechanisms that induce tumor suppressor gene methylation and the status of translating these molecular mechanisms into clinical applications.

Hepatocellular carcinoma mouse models:Hepatitis B virusassociatedhepatocarcinogenesis and haploinsufficienttumor suppressor genes

The multifactorial and multistage pathogenesis of hepatocellular carcinoma(HCC)has fascinated a wide spectrum of scientists for decades.While a number of major risk factors have been identified,their mechanistic roles in hepatocarcinogenesis still need to be elucidated.Many tumor suppressor genes(TSGs)have been identified as being involved in HCC.These TSGs can be classified into two groups depending on the situation with respect to allelic mutation/loss in the tumors:the recessive TSGs with two required mutated alleles and the haploinsufficient TSGs with one required mutated allele.Hepatitis B virus(HBV)is one of the most important risk factors associated with HCC.Although mice cannot be infected with HBV due to the narrow host range of HBV and the lack of a proper receptor,one advantage of mouse models for HBV/HCC research is the numerous and powerfulgenetic tools that help investigate the phenotypic effects of viral proteins and allow the dissection of the dose-dependent action of TSGs.Here,we mainly focus on the application of mouse models in relation to HBV-associated HCC and on TSGs that act either in a recessive or in a haploinsufficient manner.Discoveries obtained using mouse models will have a great impact on HCC translational medicine.

Relationship between hepatitis B virus associated primary hepatocellular carcinoma and alteration of tumor suppressor gene p53

Objective: To explore the changes and significance of tumor suppressor gene p53 in primary hepatocellu-lar carcinoma (PHC ) with hepatitis B virus (HBV ) infection. Methods: Tumor tissues and surrounding nontumortissues of sixteen PHC cases were studied by Southern hybridization to detect the state of HBV-DNA in tissues, byimmunohistochemical staining to determine HBsAg, HBxAg and p53 protein, and by PCR directed sequencing toanalyse the point mutation of p53 gene exons 5 to 8. Results: Among the 16 cases. 13 cases were HBV-DNA posi-tive, 10 tumor cases and 13 nontumor tissues cases HBxAg positive, and 9 cases posltive for p53 protein. The se-quencing of p53 gene point mutation was found in 5 cases, only one of which was sited at codon 249 G to T. Con-clusion: The mutation of p53 gene codon 249 is infrequent in HBV related PHC,indicating the accumulation of p53protein in cells may be associated with expression of HBxAg. HBxAg binding to p53 protein and inactivation of p53function play important roles in the development of PHC.

Effect of 5-Aza-2'-deoxycytidine on the P16 tumor suppressor gene in hepatocellular carcinoma cell line HepG2

INTRODUCTIONHepatocellular carcinoma (HCC) is one of the mostcommon human malignancies worldwide[1,2], and isclosely associated with infection of HBV and HCVand contamination of aflatoxin B1[3-6]. Althoughthe molecular mechanisms of hepatocarcinogenesisremain poorly understood, an increasing number ofgenetic abnormalities have been recognized[7-10],for example, the p16 gene[11,12] the p53gene[13-18], the E-cadherin gene[19], and the c-mycgene[20].

Expression and significance of tumor suppressor gene p16 in human ovarian neoplasm

To observe the relationship between tumor suppressor gene p16 expression and ovarian cancer occurrence and development. Metbods: Using ABC immunohistochemistry method, we investigated the expression of p16 in 72 cases of ovarian neoplasm. Results: The positive rates of p16 in malignant, benign, borderline tumors and normal ovarian tissue were 7. 89%, 60.00%, 66. 67% and 83. 33%, respectively (P<0.01). In the cases whose tumors were more malignant and poorly differentiated, and who relapsed and died, the positive stainings were not discovered. Conclusiou: p16 is well related with the occurrence and development of malignant ovarian tumor.

Prokaryotic expression, purification of a novel candidate tumor suppressor gene FUS1 and characterization of its polyclonal antibodies

FUS1 is a novel candidate tumor suppressor gene identified in human chromosome 3p21.3. Its expression showed significantly reduction or even loss in lung cancer and other types of cancers. In order to further investigate the biological function of FUS1 protein, FUS1 cDNA from MRC-5 cells was amplified by RT-PCR and cloned into prokaryotic expression vector pQE-30. The recombinant expression plasmids were transformed into M15 strain and grown at 20℃ or 37℃. SDS–PAGE analysis revealed that the accumulation of the recombinant protein FUS1 (rFUS1) in inclusion body forms reached maxium amount when induced with 0.5 mM IPTG for 5 h at 37℃. The inclusion bodies were solubilized in 2M urea and purified by a 6 &#215;His tagged affinity column under denaturing condition. The purified rFUS1 was identified by electrospray ionization-mass spectrometry (ESI-MS) and tested for purity by HPLC chromatography. The purified rFUS1 proteins were then used to immunize rabbits to obtain anti-human FUS1 polyclonal antibodies, which were suitable to detect both the recombinant exogenous FUS1 and the endogenous FUS1 from tissues and cells by western blot and immunohistochemistry, Available purified rFUS1 proteins and self-prepared polyclonal antibodies against FUS1 may provide effective tools for further studies on biological function and application of FUS1.

Construction of cloning vector of tumor suppressor gene Fhit

Objective: To clone tumor suppressor gene Fhit from normal human appendix’s tissue and construct its cloning vector. Methods: Extract human total RNA with TRIzol reagent and amplification the Fhit gene with reverse transcription-PCR (RT-PCR), then clone Fhit into a pGEM-T easy cloning vector, lastly identify the pGEM-Fhit vector with polymerase chain reaction (PCR), endonuclease digestion and DNA sequencing. Results: Fhit complementary DNA (cDNA) was cloned into pGEM-T vector, and DNA sequencing was the same as reported. Conclusions: The cloning vector pGEM-Fhit was constructed successfully and builds up a good foundation going deep into the study of Fhit.

Modeling human brain rhabdoid tumor by inactivating tumor suppressor genes in induced pluripotent stem cells

Atypical teratoid/rhabdoid tumor(ATRT)is a rare childhood malignancy that originates in the central nervous system.Over ninety-five percent of ATRT patients have biallelic inactivation of the tumor suppressor gene SMARCB1.ATRT has no standard treatment,and a major limiting factor in therapeutic development is the lack of reliable ATRT models.We employed CRISPR/Cas9 gene-editing technology to knock out SMARCB1 and TP53 genes in human episomal induced pluripotent stem cells(Epi-iPSCs),followed by brief neural induction,to generate an ATRT-like model.The dual knockout Epi-iPSCs retained their stemness with the capacity to differentiate into three germ layers.High expression of OCT4 and NANOG in neurally induced knockout spheroids was comparable to that in two ATRT cell lines.Beta-catenin protein expression was higher in SMARCB1-deficient cells and spheroids than in normal Epi-iPSC-derived spheroids.Nucleophosmin,Osteopontin,and Ki-67 proteins were also expressed by the SMARCB1-deficient spheroids.In summary,the tumor model resembled embryonal features of ATRT and expressed ATRT biomarkers at mRNA and protein levels.Ribociclib,PTC-209,and the combination of clofilium tosylate and pazopanib decreased the viability of the ATRT-like cells.This disease modeling scheme may enable the establishment of individualized tumor models with patient-specific mutations and facilitate high-throughput drug testing.

Lecithin-cholesterol acyltransferase is a potential tumor suppressor and predictive marker for hepatocellular carcinoma metastasis

BACKGROUND Hepatocellular carcinoma(HCC)is a major cause of cancer mortality worldwide,and metastasis is the main cause of early recurrence and poor prognosis.However,the mechanism of metastasis remains poorly understood.AIM To determine the possible mechanism affecting HCC metastasis and provide a possible theoretical basis for HCC treatment.METHODS The candidate molecule lecithin-cholesterol acyltransferase(LCAT)was screened by gene microarray and bioinformatics analysis.The expression levels of LCAT in clinical cohort samples was detected by quantitative realtime polymerase chain reaction and western blotting.The proliferation,migration,invasion and tumor-forming ability were measured by Cell Counting Kit-8,Transwell cell migration,invasion,and clonal formation assays,respectively.Tumor formation was detected in nude mice after LCAT gene knockdown or overexpression.The immunohistochemistry for Ki67,E-cadherin,N-cadherin,matrix metalloproteinase 9 and vascular endothelial growth factor were performed in liver tissues to assess the effect of LCAT on HCC.Gene set enrichment analysis(GSEA)on various gene signatures were analyzed with GSEA version 3.0.Three machine-learning algorithms(random forest,support vector machine,and logistic regression)were applied to predict HCC metastasis in The Cancer Genome Atlas and GEO databases.RESULTS LCAT was identified as a novel gene relating to HCC metastasis by using gene microarray in HCC tissues.LCAT was significantly downregulated in HCC tissues,which is correlated with recurrence,metastasis and poor outcome of HCC patients.Functional analysis indicated that LCAT inhibited HCC cell proliferation,migration and invasion both in vitro and in vivo.Clinicopathological data showed that LCAT was negatively associated with HCC size and metastasis(HCC size≤3 cm vs 3-9 cm,P<0.001;3-9 cm vs>9 cm,P<0.01;metastatic-free HCC vs extrahepatic metastatic HCC,P<0.05).LCAT suppressed the growth,migration and invasion of HCC cell lines via PI3K/AKT/mTOR signaling.Our results indicated that the logistic regression model based on LCAT,TNM stage and the serum level of α-fetoprotein in HCC patients could effectively predict high metastatic risk HCC patients.CONCLUSION LCAT is downregulated at translational and protein levels in HCC and might inhibit tumor metastasis via attenuating PI3K/AKT/mTOR signaling.LCAT is a prognostic marker and potential therapeutic target for HCC.

Screening of tumor suppressor genes on 1q31.1-32.1 in Chinese patients with sporadic colorectal cancer

Background As a model for both multistep and multipathway carcinogenesis, colorectal neoplastic progression provides paradigms for researching both oncogenes and tumor suppressor genes （TSGs）. However, the mechanism of colorectal cancer （CRC） is not completely understood, and many genes may be involved in the colorectal carcinogenesis. The purpose of this study was to screen for the potential TSGs on chromosome 1q31.1-32.1 in Chinese patients with sporadic colorectal cancer, to explore whether colorectal cancer in the Chinese population has unique genetic alterations and determine whether other putative TSGs exist and contribute to colon carcinogenesis. Methods Six polymorphic microsatellite markers, at a density of approximately one marker in every 1.6 cM, were chosen for refined loss of heterozygosity （LOH） mapping of 1q31.1-32.1. Eighty-three colorectal cancer patients＇ tumor and normal DNA were analyzed via polymerase chain reaction （PCR） for these microsatellite markers. PCR products were eletrophoresed on an ABI 377 DNA sequencer. Genescan 3.1 and Genotype 2.1 software were used for LOH scanning and analysis. On the basis of refined LOH mapping results, we undertook a microarray-based expression screening to identify tumor association genes in 19 of the CRC cases. Results The average LOH frequency of 1q31.1-32.1 was 24.41%, with the highest frequency of 36.73% （18/49） at D1S2622, and the lowest of 16.42% （11/67） at D1S412. A minimal region of frequent deletion was located within a 2 cM genomic segment at D1S413-D1S2622. There was no significant association between LOH of any marker in the studied regions and the clinicopathological data （patient sex, age, tumor size, growth pattern, or Dukes stage）. On the basis of refined mapping results, we chose 25 genes located in the D1S413-D1S2622 （1q31.3-32.1） region and presented a microarray-based high throughput screening approach in 19 sporadic CRC cases to identify candidate CRC related tumor suppressor genes. This study found 4 significantly down-expressed genes, including CSRP1, LMOD1, PPP1R12B and CFHL3. There was no significant association between expression levels of CFHL3, CSRP1, LMOD1, PPP1R12B and the clinicopathological data. By database searching, CSRP1 was hypothesized to be a colorectal cancer related tumor suppressor gene. Conclusions Through detailed deletion mapping, we found that the 1q31.3-32.1 region might harbor one or more colorectal cancer related tumor suppressor gene（s）. And by microarray-based high-throughput screening of candidate genes located in this region and by subsequent database searching, we present the first evidence that CSRP1 might be involved in the progression of CRC.

Expression level of novel tumor suppressor gene FATS is associated with the outcome of node positive breast cancer

Background Recently, we reported the identification of a previously uncharacterized and evolutionarily conserved gene, fragile-site associated tumor suppressor （FATS）, at a frequently deleted region in irradiation （IR）-induced tumors. However, the role of FATS in breast cancer development and its clinical significance has not been defined. The aim of this study was to determine the role of FATS in breast cancer development and to evaluate its clinical significance in breast cancer. Methods The expression level of FATS mRNA was determined in 106 breast carcinomas and 23 paired normal breast tissues using quantitative real time reverse transcription-polymerase chain reaction （RT-PCR）. The relationship between FATS expression and clinicopathological parameters were also analyzed. Results The mRNA level of FATS was down-regulated in breast cancer compared with paired normal tissues. Low expression of FATS was correlated with high nuclear grade. There was a tendency to a favorable outcome for patients with high expression of FATS （P=0.346）. However, low expression of FATS was associated with poor outcome of breast cancer patients with node positive （P=0.011）. Furthermore, the mRNA level of FATS showed an independent value in predicting the outcome of breast cancer patients with positive lymph nodes. Conclusion FATS is involved in the carcinogenesis and development of breast cancer and could be a potential biomarker and prognostic factor for breast cancer therapy.