A Germline Variant on Chromosome 4q31.1 Associates with Susceptibility to Developing Colon Cancer Metastasis

We tested for germline variants showing association to colon cancer metastasis using a genome-wide association study that compared Ashkenazi Jewish individuals with stage IV metastatic colon cancers versus those with stage I or II non-metastatic colon cancers. In a two-stage study design, we demonstrated significant association to developing metastatic disease for rs60745952, that in Ashkenazi discovery and validation cohorts, respectively, showed an odds ratio (OR) = 2.3 (P = 2.73E-06) and OR = 1.89 (P = 8.05E-04) (exceeding validation threshold of 0.0044). Significant association to metastatic colon cancer was further confirmed by a meta-analysis of rs60745952 in these datasets plus an additional Ashkenazi validation cohort (OR = 1.92; 95% CI: 1.28–2.87), and by a permutation test that demonstrated a significantly longer haplotype surrounding rs60745952 in the stage IV samples. rs60745952, located in an intergenic region on chromosome 4q31.1, and not previously associated with cancer, is, thus, a germline genetic marker for susceptibility to developing colon cancer metastases among Ashkenazi Jews.     

Anosmin-1 involved in neuronal cell migration is hypoxia inducible and cancer regulated

Functional expression of KAL1 gene is critical in the migration of GnRH neurons from the olfactory placode to the hypothalamus in embryogenesis. This gene thus far has not been shown to play a functional role in any other physiological or pathological process either in the developed brain or in peripheral tissues. We show here that KAL1 gene expression is decreased in early stage and increased in later stages of cancers. Screening of colon, lung and ovarian cancer cDNA panels indicated significant decrease in KAL1 expression in comparison to corresponding uninvolved tissues. However, KAL1 expression increased with the progression of cancer from early (I and II) stages to later (III and IV) stages of the cancer. There was a direct correlation between the TGF-β and KAL1 expression in colon cancer cDNA. Using colon cancer cell lines, we showed that TGF-β induces KAL1 gene expression and secretion of anosmin-1 protein (KAL1 coded protein). We further report that hypoxia induces anosmin-1 expression; anosmin-1 protects cancer cells from apoptosis activated by hypoxia and increases cancer cell mobility. Using siRNA technique we found that KAL1 expression following hypoxia is hypoxia-inducible factor (HIF-1) α dependent. Our results suggest that KAL1 gene expression plays an important role in cancer metastasis and protection from apoptosis.     

中国人结肠癌nm23H1基因遗传不稳定性的研究

Techniques such as DNA extraction from paraffin-embedded tissues, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), ordinary silver stain, Envision immunohistochemistry and Leica-Qwin computer imaging techniques were used to study microsatellite instability (MSI) and loss of heterozygosity (LOH) of locus D17S396 at the 17th chromosome of Chinese patients and their influence on the expression of gene nm23H1, and to clarify the relationship between the genetic instability of gene nm23H1 and the development of colon cancer, which may provide experimental basis for clinical treatment. In our experiments, the frequency of MSI, LOH and nm23H1 protein reacted positive of 30 cases of colon cancer were 26.67%, 20.00% and 53.33% respectively. In tumor node metastasis (TNM) staging, the positive frequency of MSI (43.75%) and nm23H1 protein (81.25%) in stage I + II were more than those (MSI 7.14%, p < 0.05 and nm23H1 21.43%, p < 0.01) in stage III + IV, while the frequency of LOH (35.71%), which had a rising trend along with the Duke's staging increasing, was higher than that of LOH (6.25%, p < 0.05) in stage I + II. The positive frequency of nm23H1 protein in the group of tubular adenocarcinoma (60.00%) was distinctively higher than that in the group of mucoid adenocarcinoma (20.00%, p < 0.01), showing a rising trend along with the increase of the differentiation degree of tubular adenocarcinoma. Furthermore, the positive frequency of nm23H1 protein in MSI positive group was also higher than MSI negative group (p < 0.05). And there was no difference in nm23H1 protein expression analyzed by computer imaging techniques. The results of experiments indicated that both MSI and LOH controlled the development of sporadic colon cancer independently in different paths. LOH occurred mostly in the late period of sporadic colon cancer and endowed with it a high aggressive and poor prognosis. In contrast, MSI was an early period molecule marker of sporadic colon cancer. Increasing the amount of nm23H1 protein expression could effectively restrain colon cancer metastasis and improved prognosis of sporadic colon cancer patients.     

Molecular nature of chromosome 5q loss in colorectal tumors and desmoids from patients with familial adenomatous polyposis

Summary Familial adenomatous polyposis (FAP), which includes familial polyposis coli (FPC) and the Gardner syndrome (GS), is a genetically determined premalignant disease of the colon inherited by a locus (APC) mapping within 5q15–q22. To elucidate the role of 5q loss in FAP tumorigenesis, we analysed 51 colorectal tumors and seven desmoids from 19 cases of FPC and five GS patients, as well as 15 sporadic colon cancers. RFLP analysis revealed a high incidence of allelic deletion in hereditary colon cancers as well as in sporadic colon cancers with a peak at the APC locus. APC loss resulted primarily from interstitial deletion or mitotic recombination. Combined tumor and pedigree analysis in a GS family revealed loss of normal 5q alleles in three tumors, including a desmoid tumor, which suggests the involvement of hemizygosity or homozygosity of the defective APC gene in colon carcinogenesis and, possibly, in extracolonic neoplasms associated with FAP.     

S100A4基因在结肠癌中的表达及意义

目的：通过检测S100A4基因在结肠癌细胞系及结肠癌组织中的表达,探讨其与结肠癌的关系。方法：运用RT-PCR法检测不同结肠癌细胞系中S100A4基因的表达情况;通过原位杂交和免疫组化方法检测61例结肠癌标本中S100A4基因的表达。结果：结肠癌细胞系Lovo及HT29均有S100A4基因表达。S100A4蛋白和RNA在结肠癌中表达率分别为36．1％和34.4％,而在正常结肠组织中不表达（p〈0．05）。临床分期晚比临床分期早的患者S100A4表达明显增高（p〈0.05）;有淋巴结转移的患者比无淋巴结转移的患者S100A4表达明显增高（p〈0．05）。此外,S100A4表达还与肿瘤大小,病理学分级,肉眼分型等相关。结论：结肠癌中S100A4基因表达增高,而且与肿瘤的侵袭及转移密切相关,是判断结肠癌生物学行为及预后的有价值的指标。     

Mutations in the APC tumour suppressor gene cause chromosomal instability

Two forms of genetic instability have been described in colorectal cancer: microsatellite instability and chromosomal instability. Microsatellite instability results from mutations in mismatch repair genes; chromosomal instability is the hallmark of many colorectal cancers, although it is not completely understood at the molecular level. As truncations of the Adenomatous Polyposis Coli (APC) gene are found in most colorectal tumours, we thought that mutations in APC might be responsible for chromosomal instability. To test this hypothesis, we examined mouse embryonic stem (ES) cells homozygous for Min (multiple intestinal neoplasia) or Apc1638T alleles. Here we show that Apc mutant ES cells display extensive chromosome and spindle aberrations, providing genetic evidence for a role of APC in chromosome segregation. Consistent with this, APC accumulates at the kinetochore during mitosis. Apc mutant cells form mitotic spindles with an abundance of microtubules that inefficiently connect with kinetochores. This phenotype is recapitulated by the induced expression of a 253-amino-acid carboxy-terminal fragment of APC in microsatellite unstable colorectal cancer cells. We conclude that loss of APC sequences that lie C-terminal to the beta-catenin regulatory domain contributes to chromosomal instability in colorectal cancer.     

The clinical impact of miRNA34a and P53 gene expression in colon cancer

ObjectiveTo study the potential role of miRNA34a gene expression and its relationship with P53 gene expression, fate, stage, metastasis and overall survival of colorectal cancer.Patients and methodsThis study was carried out 30 patients with colon adenocarcinoma, 30 patients with benign colon polyp and 30 apparently healthy persons served as controls. All participants were subjected to full history taking, general clinical examination. Complete blood count, liver and kidney function, determination of serum tumor markers were done. Estimation of microRNA 34a and P53 Gene expression by real-time PCR were done.ResultsThere was a significant negative relationship between serum tumor markers and micro RNA 34a gene expression in cancer patients. Also, there was a statistically significant positive relationship between miRNA34a gene expression and P53 gene expression in both patients groups. The diagnostic accuracy of miRNA34a gene expression was both sensitive and specific for colon cancer. MiRNA34a and P53 gene expression had statistically significant relation with tumor stage and presence of metastases.ConclusionIt can be concluded that the level of miRNA34a can be used to differentiate between colon cancers and begin adenomas. MiRNA34a can be used as a prognostic marker in colon cancer.     

Role of heterozygous APC mutation in niche succession and initiation of colorectal cancer--a computational study

Mutations in the adenomatous polyposis coli (APC) gene are found in most colorectal cancers. They cause constitutive activation of proliferative pathways when both alleles of the gene are mutated. However studies on individuals with familial adenomatous polyposis (FAP) have shown that a single mutated APC allele can also create changes in the precancerous colon crypt, like increased number of stem cells, increased crypt fission, greater variability of DNA methylation patterns, and higher somatic mutation rates. In this paper, using a computational model of colon crypt dynamics, we evolve and investigate a hypothesis on the effect of heterozygous APC mutation that explains these different observations. Based on previous reports and the results from the computational model we propose the hypothesis that heterozygous APC mutation has the effect of increasing the chances for a stem cell to divide symmetrically, producing two stem cell daughters. We incorporate this hypothesis into the model and perform simulation experiments to investigate the consequences of the hypothesis. Simulations show that this hypothesis links together the changes in FAP crypts observed in previous studies. The simulations also show that an APC(+/-) stem cell gets selective advantages for dominating the crypt and progressing to cancer. This explains why most colon cancers are initiated by APC mutation. The results could have implications for preventing or retarding the onset of colon cancer in people with inherited or acquired mutation of one APC allele. Experimental validation of the hypothesis as well as investigation into the molecular mechanisms of this effect may therefore be worth undertaking.     

CARMA3 is overexpressed in colon cancer and regulates NF-κB activity and cyclin D1 expression

CARMA3 was recently reported to be overexpressed in cancers and associated with the malignant behavior of cancer cells. However, the expression of CARMA3 and its biological roles in colon cancer have not been reported. In the present study, we analyzed the expression pattern of CARMA3 in colon cancer tissues and found that CARMA3 was overexpressed in 30.8% of colon cancer specimens. There was a significant association between CARMA3 overexpression and TNM stage (p=0.0383), lymph node metastasis (p=0.0091) and Ki67 proliferation index (p=0.0035). Furthermore, knockdown of CARMA3 expression in HT29 and HCT116 cells with high endogenous expression decreased cell proliferation and cell cycle progression while overexpression of CARMA3 in LoVo cell line promoted cell proliferation and facilitated cell cycle transition. Further analysis showed that CARMA3 knockdown downregulated and its overexpression upregulated cyclin D1 expression and phospho-Rb levels. In addition, we found that CARMA3 depletion inhibited p-IκB levels and NF-κB activity and its overexpression increased p-IκB expression and NF-κB activity. NF-κB inhibitor BAY 11-7082 reversed the role of CARMA3 on cyclin D1 upregulation. In conclusion, our study found that CARMA3 is overexpressed in colon cancers and contributes to malignant cell growth by facilitating cell cycle progression through NF-κB mediated upregulation of cyclin D1.     

癌组织中的遗传不稳定性的RAPD分析（简报）

In five kinds of tumors, total 128 specimens were analyzed by RAPD (random amplified polymorphic DNA) PCR with nine 10-base arbitrary primers for detecting instabilities of DNA and chromosome and screening new molecular markers coupled to putative or unknown oncogenes and/or tumor suppressor genes. Bands representing instabilities have been recovered and purified from agarose and cloned into pCAPs vector, and further labeled by DIG as probes for analysis of Southern blot, Northern blot and Sequencing. Results revealed that sample 5 and 3 of the gastric cancers showed the highest genomic changes and the average detectability in five sorts of cancers was up to at least 40% (42.2%-49.4%), and that there were significant differences in the ability of each primer to detect genomic instability, which ranged from 27% to 68%. Despite the highest detectability of genetic instability (68%) in tumor tissues, primer 2 could produce stable profiles of DNA bands in normal tissue genome with good reproducibility. On the contrary, primer 8 was of the lowest one (27%). Band B of single copy found to be allelic losses in gastric and colon cancers according to RFLP analysis was of a novel sequence and registered by Gen-Bank (Accession Number AF151005). Therefore the genetic instabilities often concentrated on some special locuses of chromosome e.g. repetitive sequences etc. and coupled to carcinogenesis. It was impossible or difficult to get great achievements for cancer treatments with the procedure of gene therapy only to one oncogene or one tumor suppressor gene because the extensive DNA variations occurred during the progression of tumor. RAPD assay connected with other techniques was a good tool for the detection of genomic instabilities and direct screening of some new molecular markers related to tumor suppressor genes or oncogenes.     

胃癌中APC基因I1307K突变及蛋白质表达

为探讨肿瘤抑制基因APC结构及表达异常与胃癌发生、发展的关系,采用ARMS PCR检测胃癌中APC基因I1307K突变存在与否,免疫组织化学方法分析胃癌中APC蛋白表达水平。结果表明,在 62例胃癌高发区易感人群血液标本及45例胃癌中未检测到I1307K突变;胃癌（早期、进展期）中APC蛋白表达阳性率显著低于正常黏膜,进展期胃癌中APC蛋白表达阳性率显著低于早期胃癌,淋巴结转移阳性的胃癌中APC蛋白表达阳性率显著低于淋巴结转移阴性者。因此认为I1307K突变可能与国人胃癌发生无明显相关;APC蛋白低表达与胃癌发生、进展及淋巴结转移密切相关。 Abstract:In order to explore the correlation of the abnormalities of tumor suppressor gene APC with the carcinogenesis and progression of gastric cancer.The I1307K mutation of APC gene in gastric cancer was analysed using Amplification Refractory Mutation System PCR(ARMS ,PCR),also the expression of APC protein in gastric cancer of different stages was detected by immunohistochemical method.We found that there wasn't I1307K mutation of APC gene in 62 cases of blood samples of susceptible population in high incidence areas of gastric cancer and 45 cases of gastric cancer tissues.The positive rates of APC protein in gastric cancer (both early and progressive gastric cancer) were significantly lower than that in normal mucosa,the positive rates of APC protein in progressive gastric cancer were significantly lower than that in early gastric cancer,the positive rates of APC protein in gastric cancer with lymph node metastasis were significantly lower than that in gastric cancer without lymph node metastasis.So it was thought that there might be no correlation between the I1307K mutation of APC gene and carcinogenesis of gastric cancer in China,but the decreased expression of APC protein was closely related to the carcinogenesis,progression and lymph node metastasisof gastric cancer.     

Genomic Instability in Precancerous Lesions before Inactivation of Tumor Suppressors p53 and APC in Patients

The etiology and significance of genomic instability (GIN), a hallmark of human cancers, remains controversial. The paradigm that inactivation of tumor suppressors (e.g. p53 or adenomatous polyposis coli (APC) genes) leads to GIN is largely based on experiments in vitro and in animal models. It remains unclear whether GIN is a cause or a result of cancer, particularly in patients. Precancerous Barrett’s esophagus (BE) provides a clinical model to investigate GIN in cancer progression. We analyzed specimens from endoscopic biopsies or esophagectomies in patients with BE (10 cases), BE-associated esophageal adenocarcinoma (10 cases), or with normal gastro-esophageal junction (5 cases). Chromosomal enumeration probe Cep 7, 11, 12, 17 and 18 were detected by fluorescence in situ hybridization (FISH). Expression of p53 and APC were determined by immunohistochemistry. Increased p53 expression, a measurement of p53 mutations, was observed in BE with high grade dysplasia (HGD) and in BE-associated esophageal cancer (EC). The expression of wild type APC was decreased in BE with HGD and in advanced EC. Chromosomal abnormalities were found in all EC samples. Numeric changes of chromosome 7, 11 and 12 were observed in BE in 14%, 64% and 43% of cases, respectively. Aneusomy of chromosome 11 and 12 were found in ME and in BE without dysplasia, in the presence of normal expression pattern of p53 and APC. Our results suggest that GIN is an early event that occurs at precancerous stages prior to changes in tumor suppressor genes (p53 and APC) in BE-associated tumorigenesis in patients, suggesting that GIN may serve as a causative link between chronic inflammation and cancer.     

p16基因甲基化状态与散发性大肠癌的相关性研究

为探讨p1 6基因甲基化状态与散发性大肠癌发生发展的关系 ,用甲基化特异性的聚合酶链反应 (methylati omspecificPCR ,MSP)结合测序检测散发性大肠癌及相应癌旁组织p1 6基因甲基化状态。研究发现p1 6基因在散发性大肠癌中甲基化率为 2 8 9% (1 3 4 5 ) ,有 8例癌及癌旁组织都发生了甲基化 ;有淋巴结及远处转移的甲基化率为5 0 % (8 1 6 ) ,高于无转移的甲基化率 2 0 8(5 2 4 ) (P <0 0 5 )。p1 6基因高甲基化是散发性大肠癌中常见的分子改变之一 ,大肠癌中p1 6基因高甲基化可能发生在癌变早期并与大肠癌的恶性进展有相关性     

Analysis of the candidate tumor suppressor Ris-1 in primary human breast carcinomas

Frequent chromosome 3 losses have been described in several tumors types, which strongly suggest the presence of one or several tumor suppressor genes. Recently, a novel candidate tumor suppressor gene termed Ris-1 (for Ras-induced senescence 1) has been identified at chromosomal position 3p21.3. Ris-1 has been proposed to participate in anti-tumor responses that resemble cellular senescence and that are elicited by oncogenes such as Ras. To analyze the role of Ris-1 as a putative tumor suppressor gene in human breast cancer, we have performed a real-time quantitative analysis of its mRNA expression in 60 patients. Moreover, we carried out a first approach to evaluate the most common inactivation mechanism that can affect expression levels of tumor suppressor genes (mutation, promoter hypermethylation and allelic losses). Furthermore, a correlation study between expression as well as inactivating mechanisms of Ris-1 and several clinico-pathological parameters of the tumors was designed, with the objective of appraising the prognostic value of Ris-1 status. Decreased expression of Ris-1 was observed in 23% of the cases and overexpressed Ris-1 was detected in 15% of the primary breast tumors. Our data showed high frequency of LOH (30%) at one of the markers used. Nevertheless, a polymorphism related with the expression levels was described. Statistically significant correlations were found between decreased Ris-1 expression and negative progesterone receptors, as well as between overexpressing Ris-1 tumors and high histological grade. Despite all these data, we conclude that the suggested role of Ris-1 as tumor suppressor gene is not evident, at least in breast cancer. Future and larger series studies in different tumor types are necessary to clarify Ris-1 function in human cancer.     

Adenomatous polyposis coli (APC) differentially regulates beta-catenin phosphorylation and ubiquitination in colon cancer cells

Most colorectal cancers have mutations of the adenomatous polyposis coli (APC) gene or the beta-catenin gene that stabilize beta-catenin and activate beta-catenin target genes, leading ultimately to cancer. The molecular mechanisms of APC function in beta-catenin degradation are not completely known. APC binds beta-catenin and is involved in the Axin complex, suggesting that APC regulates beta-catenin phosphorylation. Some evidence also suggests that APC regulates beta-catenin nuclear export. Here, we examine the effects of APC mutations on beta-catenin phosphorylation, ubiquitination, and degradation in the colon cancer cell lines SW480, DLD-1, and HT29, each of which contains a different APC truncation. Although the current models suggest that beta-catenin phosphorylation should be inhibited by APC mutations, we detected significant beta-catenin phosphorylation in these cells. However, beta-catenin ubiquitination and degradation were inhibited in SW480 but not in DLD-1 and HT29 cells. The ubiquitination ofbeta-catenin in SW480 cells can be rescued by exogenous expression of APC. The APC domains required for beta-catenin ubiquitination were analyzed. Our results suggest that APC regulates beta-catenin phosphorylation and ubiquitination by distinct domains and by separate molecular mechanisms.