Mechanisms of myeloid-derived suppressor cell-mediated immunosuppression in colorectal cancer and related therapies

Severe immunosuppression is a hallmark of colorectal cancer(CRC).Myeloid-derived suppressor cells(MDSCs),one of the most abundant components of the tumor stroma,play an important role in the invasion,metastasis,and immune escape of CRC.MDSCs create an immunosuppressive microenvironment by inhibiting the proliferation and activation of immunoreactive cells,including T and natural killer cells,as well as by inducing the proliferation of immunosuppressive cells,such as regulatory T cells and tumor-associated macrophages,which,in turn,promote the growth of cancer cells.Thus,MDSCs are key contributors to the emergence of an immunosup-pressive microenvironment in CRC and play an important role in the breakdown of antitumor immunity.In this narrative review,we explore the mechanisms through which MDSCs contribute to the immunosuppressive microenvironment,the current therapeutic approaches and technologies targeting MDSCs,and the therapeutic potential of modulating MDSCs in CRC treatment.This study provides ideas and methods to enhance survival rates in patients with CRC.

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.

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.

Mobilization of monocytic myeloid-derived suppressor cells is regulated by PTH1R activation in bone marrow stromal cells

Myeloid-derived suppressor cells(MDSCs)are bone marrow(BM)-derived immunosuppressive cells in the tumor microenvironment,but the mechanism of MDSC mobilization from the BM remains unclear.We investigated how BM stromal cell activation by PTH1R contributes to MDSC mobilization.PTH1R activation by parathyroid hormone(PTH)or PTH-related peptide(PTHrP),a tumor-derived counterpart,mobilized monocytic(M-)MDSCs from murine BM without increasing immunosuppressive activity.In vitro cell-binding assays demonstrated thatα4β1 integrin and vascular cell adhesion molecule(VCAM)-1,expressed on M-MDSCs and osteoblasts,respectively,are key to M-MDSC binding to osteoblasts.Upon PTH1R activation,osteoblasts express VEGF-A and IL6,leading to Src family kinase phosphorylation in M-MDSCs.Src inhibitors suppressed PTHrP-induced MDSC mobilization,and Src activation in M-MDSCs upregulated two proteases,ADAM-17 and MMP7,leading to VCAM1 shedding and subsequent disruption of M-MDSC tethering to osteoblasts.Collectively,our data provide the molecular mechanism of M-MDSC mobilization in the bones of tumor hosts.

RASAL2 acts as a tumor suppressor in cervical cancer cells

This study was designed to investigate the roles of RASAL2 in cervical cancer(CC).Methods:Fifty-four CC tissues and 33 adjacent tissues were obtained from CC patients admitted to our hospital between March 2012 and June 2014.Real-time polymerase chain reaction and western blotting were performed to analyze the expression of RASAL2 mRNA and protein in these tissues,CC cell lines,and normal cervical cells.Over-expression and silencing of RASAL2 were induced after transfection,and the migration,invasion,and proliferation of the CC cell lines were examined.Results:RASAL2 mRNA and protein expressions were significantly down-regulated in CC tissues and cell lines than in adjacent tissues and normal cervical cells,respectively.While low RASAL2 expression correlated with advanced stage and metastasis of CC,its over-expression significantly inhibited proliferation and metastasis of CC cells and induced apoptosis.Under in vitro conditions,silencing of RASAL2 expression could significantly increase the proliferation,invasion,and migration of CC cells.Conclusion:RASAL2 functioned as a tumor suppressor in CC,and was down-regulated in CC tissue samples and cell lines.

Comprehensive analysis of cell-extracellular matrix protein Ras suppressor-1 in function and prognosis of gastrointestinal cancers

BACKGROUND Ras suppressor 1(RSU1),a highly conserved protein,plays an important role in actin cytoskeleton remodeling and cell-extracellular matrix adhesion.Aberration of RSU1 activity can cause changes in cell adhesion and migration,thereby enhancing tumor proliferation and metastasis.However,the correlation between RSU1 and gastrointestinal cancers(GICs),as well as its prognostic role related to tumor-infiltrating immune cells(TIICs)remains unclear.AIM To shows RSU1 plays a potential promoting role in facilitating tumor immune escape in GIC.METHODS Differential expression of RSU1 in different tumors and their corresponding normal tissues was evaluated by exploring the Gene Expression Profiling Interactive Analysis(GEPIA)dataset.The correlation between RSU1 expression and prognosis of GIC cancer patients was evaluated by Kaplan-Meier plotter.Then,RSU1-correlated genes were screened and functionally characterized via enrichment analysis.The correlation between RSU1 and TIICs was further characterized using the Tumor Immune Estimation Resource(TIMER).In addition,the correlation between RSU1 and immune cell surface molecules was also analyzed by TIMER.RESULTS High RSU1 expression was associated with poor overall survival of gastric cancer patients,exhibiting a hazard ratio(HR)=1.36,first progression HR=1.53,and post progression survival HR=1.6.Specifically,high RSU1 Levels were associated with prognosis of gastric cancer in females,T4 and N3 stages,and Her-2-negative subtypes.Regarding immune-infiltrating cells,RSU1 expression level was positively correlated with infiltration of CD4+T cells,macrophages,neutrophils,and dendritic cells(DCs)in colorectal adenocarcinoma and stomach adenocarcinoma.RSU1 expression was also predicted to be strongly correlated with immune marker sets in M2 macrophage,DCs and T cell exhaustion in GICs.CONCLUSION In gastrointestinal cancers,RSU1 is increased in tumor tissues,and predicts poor survival of patients.Increased RSU1 may be involved in promoting macrophage polarization,DC infiltration,and T cell exhaustion,inducing tumor immune escape and the development of tumors in GICs.We suggest that RSU1 is a promising prognostic biomarker reflecting immune infiltration level of GICs,as well as a potential therapeutic target for precision treatment through improving the immune response.

Aberrant Methylation in CpG Islands of pl5 and pl6 Tumor Suppressor Genes in Pancreatic Cancer Tissue

Objective： To detect the aberrant methylation patterns in the CpG islands of p16 and p15 tumor suppressor genes, and to analyze its correlation with pancreatic carcinogenesis and with clinicopathological characteristics of patients with pancreatic cancer （PC）. Methods： The methylation-specific polymerase chain reaction （MSP） method was used to monitor methylation patterns in the CpG islands of p15 and p16 genes from 29 cases of PC and 3 cases of chronic pancreatitis （CP） paraffin-embedded tissue, as well as 2 cases of normal liver tissues and 12 cases of normal blood samples. Results： p15 and p16 genes were detected to show unmethylation patterns and no amplification using methylation-specific primers in control group. The aberrant methylation rates of p16 in carcinoma tissue and adjacent noncarcinoma tissue were 37.9% （11 of 29 cases） and 34.5% （10 of 29 cases） respectively. Of the 11 aberrant methylated samples, 5 showed complete methylation and 6 hemimethylation. The methylation rates of p15 gene in carcinoma tissue and adjacent noncarcinoma tissue were 27.5% （8/29） and 24.4% （7/29） respectively. Of the 8 aberrant methylated samples, 3 showed complete methylation and 5 hemimethylation. In 6 PC samples, aberrant methylation in CpG islands of both p15 and p16 genes existed simultaneously. The aberrant methylation patterns in CpG islands of p15 and p16 genes had no close correlation with the clinicopathological characteristics （age, sex, smoking, volume of primary tumor, differentiation, clinical stage and histological classification） of the patients with PC （P〉0.05）. Conclusion： The aberrant methylation in CpG islands of p15 and p16 genes could be regarded as an early molecular event in PC and had no close correlation with the clinicopathological characteristics of the patients with PC.

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.

Overexpression and mutations of tumor suppressor gene p53 in hepatocellular carcinoma

AIMS To examine the prevalance of p53 mutations in hepatocellular carcinoma (HCC) from Chongqing area and the relationship between the p53 mutations and clinicopathological features of HCC,as well as the risk factors. METHODS The overexpression and point mutations of tumor suppressor gene p53 in 38 cases of HCC were detected by a sensitive antigen retrieval fluid (ARF) immunohistochemical method and polymerase chain re- action(PCR)-restriction fragment length polymorphism (RFLP),and single strand conformation polymorphism (SSCP)-silver staining analysis. RESULTS The results showed that 16 of 38 HCCs had positive p53 protein (42.1%),7 HCCs had p53 mutation at 249 (18.4 % ) and 2 HCCS had point muta- tion within exon 7 other than 249. Among 9 cases of HCC with mutations,8 cases demonstrated positive p53 protein,its coincidental rate was 88.9%. The overexpression and mutations of p53 were significantly related to the differentiation and metastasis of HCCs. The frequency of p53 mutations was consistent with high prevalence of HBV and a moderate aflatoxin B1 (AFB1) exposure in our area. CONCLUSIONS The results suggest that AFB1 acts synergistically with HBV in the generation of p53 mutations. Furthermore,dietary exposure to AFB1 may mainly contribute to the tumor specific mutation at codon 249,while HBV may account for other scattered mutations in HCC.

Characterization of six tumorsuppressor genes and microsatellite instability in hepatocellular carcinomain southern African blacks

AIM To analyse cumulative loss of heterozygosity (LOH) of chromosomal regions and tumor suppressor genes in hepatocellular carcinomas (HCCs) from 20 southern African blacks. METHODS p53, RB1, BRCA1, BRCA2, WT1 and E cadherin genes were analysed for LOH, and p53 gene was also analysed for the codon 249 mutation, in tumor and adjacent non tumorous liver tissues using molecular techniques and 10 polymorphic microsatellite markers. RESULTS p53 codon 249 mutation was found in 25% of the subjects, as was expected, because many patients were from Mozambique, a country with high aflatoxin B 1 exposure. LOH was found at the RB1, BRCA2 and WT1 loci in 20%(4/*!20) of the HCCs, supporting a possible role of these genes in HCC. No LOH was evident in any of the remaining genes. Reports of mutations of p53 and RB1 genes in combination, described in other populations, were not confirmed in this study. Change in microsatellite repeat number was noted at 9/*!10 microsatellite loci in different HCCs, and changes at two or more loci were detected in 15%(3/*!20) of subjects. CONCLUSION We propose that microsatellite/genomic instability may play a role in the pathogenesis of a subset of HCCs in black Africans.

Increased frequency and clinical significance of myeloidderived suppressor cells in human colorectal carcinoma

AIM： To investigate the frequency and clinical signifi- cance of the myeloid-derived suppressor cells （MDSC） in human colorectal carcinoma （CRC）. METHODS： Samples of peripheral blood and tumor tis- sue from 49 CRC patients were analyzed. Mononuclear cells were isolated by FicolI-Hypaque density gradient centrifugation and were subjected to a flow cytometry- based immunophenotypic analysis. RESULTS： A considerable increase in the percentage of CD33＋HLA-DR MDSCs was observed in the periph- eral blood （1.89% ：1= 0.75%） and tumor tissues （2.99%±1.29%） of CRC patients as compared with that in theperipheral blood of healthy controls （0.54%±0.35%）. This expanded CD33＋HLA-DR subset exhibited imma- ture myeloid cell markers, but not lineage markers, and showed up-regulation of CD18/CD11b expression as compared with the MDSCs from healthy donors. Fur- ther studies showed that the MDSC proportion in CRC peripheral blood was correlated with nodal metastasis （P = 0.023）, whereas that in tumor tissues was cor- related with nodal/distant metastasis （P = 0.016/P = 0.047） and tumor stage （P = 0.028）, suggesting the involvement of MDSCs in CRC tumor development. CONCLUSION： Characterization of MDSCs in CRC sug- gests the clinical significance of circulating and tumor- infiltrating MDSCs and may provide new insights into the CRC immunotherapy targeting MDSCs.

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.

mi R-382 functions as a tumor suppressor against esophageal squamous cell carcinoma

AIM To explore the effect of miR-382 on esophageal squamous cell carcinoma (ESCC) in vitro and its possible molecular mechanism. METHODS Eca 109 cells derived from human ESCC and Het-1A cells derived from human normal esophageal epithelium were used. Lentivirus-mediated miR-382 was overexpressed in Eca109 cells. The effect of miR-382 on cell proliferation was evaluated by MTT and colony formation assay. For cell cycle analysis, cells were fixed and stained for 30 min with propidium iodide (PI) staining buffer containing 10 mg/mL PI and 100 mg/mL RNase A, and analyzed by BD FACSCalibur (TM) flow cytometer. For cell apoptosis assay, cells were stained with an Annexin V-FITC/PI Apoptosis Detection Kit according to the manufacturer's instructions and analyzed by a dual-laser flow cytometer. Cell invasion and migration abilities were determined through use of transwell chambers, non-coated or pre-coated with matrigel. Levels of proteins related to cell growth and migration were examined by western blotting. RESULTS Endogenous miR-382 was down-regulated in Eca109 cells compared with Het-1A. Introduction of miR-382 not only significantly inhibited proliferation and colony formation, but also arrested cell cycle at the G2/M phase, as well as promoted apoptosis and autophagy in Eca109 cells. Migration, invasion and epithelial-mesenchymal transition of Eca109 cells were suppressed by overexpressing miR-382. Western blotting results showed that miR-382 inhibited the phosphorylation of mTOR and 4E-BP1. CONCLUSION miR-382 functions as a tumor suppressor against ESCC development and metastasis, and could be considered as a potential drug source for the treatment of ESCC patients.

Tumor suppressor and hepatocellular carcinoma

A few signaling pathways are driving the growth of hepatocellular carcinoma.Each of these pathways possesses negative regulators.These enzymes,which normally suppress unchecked cell proliferation,are circumvented in the oncogenic process,either the overactivity of oncogenes is sufficient to annihilate the activity of tumor suppressors or tumor suppressors have been rendered ineffective.The loss of several key tumor suppressors has been described in hepatocellular carcinoma.Here,we systematically review the evidence implicating tumor suppressors in the development of hepatocellular carcinoma.

Tumor suppressor role of mi R-133a in gastric cancer by repressing IGF1R

AIM:To investigate the function and mechanism of mi R-133a in gastric cancer(GC)and its relationship with clinicopathological characteristics of GC.METHODS:A total of 105 GC patients who underwent surgical resection as primary treatment were selected for this study.Real-time quantitative reverse transcriptase polymerase chain(q RT-PCR)was used to examine the expression levels of mi R-133a in human GC and adjacent non-tumor tissues,as well as in GC cell lines(SGC-7901,BGC-823,MGC-803,and AGS)and a human gastric mucosal epithelial cell line(GES-1).The biological role of mi RNA(mi R)-133a was assessed in the GC cell lines using MTT,apoptosis,migration and invasion,and colony formation assays,and xenograft tumorigenesis.q RT-PCR and western blot analyses were used to evaluate the potential target gene expression of mi R-133a.Pearson’s correlation was calculated to evaluate the correlation between mi R-133a and insulinlike growth factor 1 receptor(IGF1R)expression.The regulation of IGF1R by mi R-133a was verified using the luciferase reporter assay.RESULTS:In 80%of the 105 GC patients,the mean expression of mi R-133a was significantly downregulated in tumor tissues compared with adjacent normal tissues(1.215±0.1477 vs 3.093±0.4104,P<0.0001).Downregulation of mi R-133a was significantly correlated with the degree of differentiation(P=0.01),local invasion(P=0.001)and TNM stage(P=0.02)in GC patients.Compared with a control construct,forced expression of mi R-133a in GC cell lines inhibited proliferation(0.4787±0.0219 vs 0.7050±0.0147,P=0.0013 in SGC-7901 cells;and 0.5448±0.0085vs 0.7270±0.0084,P=0.001 in MGC-803 cells);migration(0.6333±0.0233 vs 1.037±0.0584,P=0.003 in SGC-7901 cells;0.6126±0.0311 vs 1.024±0.0456,P=0.0017 in MGC-803 cells);and invasion(0.613±0.0399 vs 1.033±0.0278,P=0.0013 in SGC-7901 cells;0.7433±0.0221 vs 1.017±0.0311,P=0.002 in MGC-803 cells).It also induced apoptosis(18.19%±0.2483%vs 5.887%±0.3837%,P<0.0001 in SGC-7901 cells;22.69%±0.7846%vs9.347%±0.3012%,P<0.0001 in MGC-803 cells).Furthermore,mi R-133a inhibited tumor growth and xenograft tumorigenesis of SGC-7901 cells in vivo.In addition,we identified IGF1R as a regulatory target of mi R-133a in GC.CONCLUSION:This study suggests that mi R-133a is downregulated in GC and functions as a tumor suppressor in vitro and in vivo partly by repressing IGF1R.

PTEN： a default gate-keeping tumor suppressor with a versatile tail

The tumor suppressor PTEN controls a variety of biological processes including cell proliferation, growth, migration, and death. As a master cellular regulator, PTEN itself is also subjected to deliberated regulation to ensure its proper function. Defects in PTEN regulation have a profound impact on carcinogenesis. In this review, we briefly discuss recent advances concerning PTEN regulation and how such knowledge facilitates our understanding and further exploration of PTEN biology. The carboxyl-tail of PTEN, which appears to be associated with multiple types of posttranslational regulation, will be under detailed scrutiny. Further, a comparative analysis of PTEN and p53 suggests while p53 needs to be activated to suppress tumorigenesis （a dormant gatekeeper）, PTEN is probably a constitutive surveillant against cancer development, thus a default gatekeeper.

Circulating myeloid-derived suppressor cells in patients with pancreatic cancer

BACKGROUND： Myeloid-derived suppressor cells （MDSCs） are heterogeneous cell types that suppress T-cell responses in cancer patients and animal models, some MDSC subpopulations are increased in patients with pancreatic cancer. The present study was to investigate a specific subset of MDSCs in patients with pancreatic cancer and the mechanism of MDSCs increase in these patients. METHODS： Myeloid cells from whole blood were collected from 37 patients with pancreatic cancer, 17 with cholangiocarcinoma, and 47 healthy controls. Four pancreatic cancer cell lines were co- cultured with normal peripheral blood mononudear cells （PBMCs） to test the effect of tumor cells on the conversion of PBMCs to MDSCs. Levels of granulocyte-macrophage colony-stimulating factor （GM-CSF） and arginase activity in the plasma of cancer patients were analyzed by enzyme-linked immunosorbent assay. RESULTS： CD14＋/CD11b＋/HLA-DR MDSCs were increased in patients with pancreatic or bile duct cancer compared with those in healthy controls, and this increase was correlated with clinical cancer stage. Pancreatic cancer cell lines induced PBMCs to MDSCs in a dose-dependent manner. GM-CSF and arginase activity levels were significantly increased in the se rum of patients with pancreatic cancer. CONCLUSIONS： MDSCs were tumor related： tumor cells induced PBMCs to MDSCs in a dose-dependent manner and circulating CD14＋/CD11b＋/HLA-DR- MDSCs in pancreatic cancer patients were positively correlated with tumor burden. MDSCs might be useful markers for pancreatic cancer detection and progression.

Science Letters:IGFBP7 plays a potential tumor suppressor role against colorectal carcinogenesis with its expression associated with DNA hypomethylation of exon 1

Insulin-like growth factor binding-protein-7 （IGFBP7） was obtained from our previous colonic adenocarcinoma （CRC） and normal mucosa suppression subtraction hybridization （SSH） cDNA libraries. By RT-PCR and immunohistochemistry, we found that IGFBP7 was overexpressed in CRC tissue compared to normal tissue. However, our in vitro experiments performed in 10 CRC cell lines showed that IGFBP7 expressed only in SW480 and Caco2 cell lines, which implied an underlying reversible regulatory mechanism. Using methylation-specific PCR （MSP） and bisulfite sodium PCR （BSP）, we found that its expression was associated with DNA hypomethylation of exonl. This was further supported by the in vitro study which showed restored IGFBP7 expression after demethylation agent 5-aza-2＇-deoxycytidine treatment. Correlation analysis between IGFBP7 expression and prognosis indicated that overexpression of IGFBP7 in CRC tissue correlated with favourable survival. Investigation of the functional role of IGFBP7 through transfection studies showed that IGFBP7 protein could inhibit growth rate, decrease colony formation activity, and induce apoptosis in RKO and SW620 cells, suggesting it a potential tumor suppressor protein in colorectal carcinogenesis. In conclusion, our study clearly demonstrated that IGFBP7 plays a potential tumor suppressor role against colorectal carcinogenesis and its expression is associated with DNA hypomethylation of exon 1.

Alteration of tumor suppressor gene p16 and Rb in gastric cancinogesis

IM To study the alterations of tumor suppressor gene p16 and Rb in the carcinogenesis of the stomach. METHODS Different mucosal biopsies were endoscopically obtained, all samples were immediately fixed with 10% buffered formalin, embedded with paraffin and sectioned serielly. Alterations of p16 and Rb protein in 12 cases of superficial gastritis, 15 atrophic gastritis, 20 atypical hyperplasia and 40 cancerous tissues were detected by the immunohistochemical method (ABC). RESULTS Different degrees of nuclear immunostaining of p16 and Rb occurred on gastric epithelium in different stages of lesions. With the lesions progressing, the positive immunostaining rate of p16 protein had a decreasing tendency (833%→733%→300%→275%), and on the other hand, that of Rb protein had an increasing tendency (250%→467%→600%→675%). A negative correlationship was found between these two parameters in the gastric cancer. Of 40 cases of gastric cancer, a negative relationship was observed in 20 cases. In comparison with both positive (9 cases) and both negative tissues (11 cases), there was a significant difference (500%,225%,275%) (P<005).CONCLUSION Abnormal expression of p16 and Rb plays an important role in gastric carcinogenesis.

Inactivation of the tumor suppressor Krüppel-like factor 6 (KLF6) by mutation or decreased expression in hepatocellular carcinomas

Background and aim： The Krueppel-like transcription factor KLF6 is a novel tumor-suppressor gene. It was inactivated in human prostate cancer and other tumors tissue, as the result of frequent mutation and loss of heterozygosity （LOH）. However, there is no data reporting the levels of KLF6 both mRNA and protein in hepatocellular carcinomas （HCCs）. We therefore detected mutations and expression of KLF6 in HCC tissues and further observed the effect of it on cell growth in HCC cell lines. Methods： We analyzed the exon-2 ofKLF6 gene by direct DNA sequencing, and detected the expression of KLF6 by RT-PCR and Western blot in 23 HCC tissues and corresponding nontumorous tissues. Loss of growth suppressive effect of the HCC-derived KLF6 mutant was characterized by in vitro growth curves plotted, flow cytometry and Western blotting. Results： KLF6 mutations were found in 2 of 23 HCC tissues and one of mutations was missense. Expression ofKLF6 mRNA or protein was down-regulated in 8 （34.7%） or 9 （39.1%） of 23 HCC tissues. Wild-type KLF6 （wtKLF6） inhibited cellular proliferation and prolonged G1 -S transition by inducing the expression of p21WAF 1 following stable transfection into cultured HepG2 cells, but tumor-derived KLF6 mutant （mKLF6） had no effects. Conclusion： Our findings suggest that KLF6 may be involved in pathogenesis of HCC.