Bystander elimination of antigen loss variants in established tumors

Cancers express antigens that are targets for specific cytotoxic T lymphocytes (CTLs). However, cancer cells are genetically unstable. Consequently, sub-populations of cancer cells that no longer express the target antigen may escape destruction by CTLs and grow progressively. We show that cytotoxic T cells indirectly eliminate these antigen loss variants (ALVs) in a model system when the parental cancer cells express sufficient antigen to be effectively cross-presented by the tumor stroma. When the parental tumor expressed lower levels of antigen, cytotoxic T cells eradicated the antigen-positive parental cancer cells, but the ALVs escaped, grew and killed the host. By contrast, when the parental tumor expressed higher levels of antigen, cytotoxic T cells eradicated not only the parental cancer cells but also the ALVs. This 'bystander' elimination of ALVs required stromal cells expressing major histocompatibility complex (MHC) molecules capable of presenting the antigen, and occurred in tumors showing evidence of stromal destruction. ALVs were apparently eliminated indirectly when tumor-specific CTLs killed stromal cells that were cross-presenting antigen produced by and released from antigen-positive cancer cells. These results highlight the general importance of targeting the tumor stroma to prevent the escape of variant cancer cells.     

Differential expression of miRNAs in colorectal cancer: comparison of paired tumor tissue and adjacent normal mucosa using high-throughput sequencing

We present the results of a global study of dysregulated miRNAs in paired samples of normal mucosa and tumor from eight patients with colorectal cancer. Although there is existing data of miRNA contribution to colorectal tumorigenesis, these studies are typically small to medium scale studies of cell lines or non-paired tumor samples. The present study is to our knowledge unique in two respects. Firstly, the normal and adjacent tumor tissue samples are paired, thus taking into account the baseline differences between individuals when testing for differential expression. Secondly, we use high-throughput sequencing, thus enabling a comprehensive survey of all miRNAs expressed in the tissues. We use Illumina sequencing technology to perform sequencing and two different tools to statistically test for differences in read counts per gene between samples: edgeR when using the pair information and DESeq when ignoring this information, i.e., treating tumor and normal samples as independent groups. We identify 37 miRNAs that are significantly dysregulated in both statistical approaches, 19 down-regulated and 18 up-regulated. Some of these miRNAs are previously published as potential regulators in colorectal adenocarcinomas such as miR-1, miR-96 and miR-145. Our comprehensive survey of differentially expressed miRNAs thus confirms some existing findings. We have also discovered 16 dysregulated miRNAs, which to our knowledge have not previously been associated with colorectal carcinogenesis: the following significantly down-regulated miR-490-3p, -628-3p/-5p, -1297, -3151, -3163, -3622a-5p, -3656 and the up-regulated miR-105, -549, -1269, -1827, -3144-3p, -3177, -3180-3p, -4326. Although the study is preliminary with only eight patients included, we believe the results add to the present knowledge on miRNA dysregulation in colorectal carcinogenesis. As such the results would serve as a robust training set for validation of potential biomarkers in a larger cohort study. Finally, we also present data supporting the hypothesis that there are differences in miRNA expression between adenocarcinomas and neuroendocrine tumors of the colon.     

Association of caucasian-identified variants with colorectal cancer risk in singapore chinese

Background

Genome-wide association studies (GWAS) in Caucasians have identified fourteen index single nucleotide polymorphisms (iSNPs) that influence colorectal cancer (CRC) risk.

Methods

We investigated the role of eleven iSNPs or surrogate SNPs (sSNPs), in high linkage disequilibrium (LD, r²≥0.8) and within 100 kb vicinity of iSNPs, in 2,000 age- and gender-matched Singapore Chinese (SCH) cases and controls.

Results

Only iSNP rs6983267 at 8q24.21 and sSNPs rs6695584, rs11986063, rs3087967, rs2059254, and rs7226855 at 1q41, 8q23.3, 11q23.1, 16q22.1 and 18q21.1 respectively showed evidence of association with CRC risk, with odds ratios (OR) ranging from 1.13 to 1.40. sSNP rs827401 at 10p14 was associated with rectal cancer risk (OR = 0.74, 95% CI 0.63–0.88) but not disease prognosis (OR = 0.91, 95% CI 0.69–1.20). Interestingly, sSNP rs3087967 at 11q23.1 was associated with CRC risk in men (OR = 1.34, 95% CI 1.14–1.58) but not women (OR = 1.07, 95% CI: 0.88–1.29), suggesting a gender-specific role. Half of the Caucasian-identified variants, including the recently fine-mapped BMP pathway loci, BMP4, GREM1, BMP2 and LAMA 5, did not show any evidence for association with CRC in SCH (OR ∼1; p-value >0.1). Comparing the results of this study with that of the Northern and Hong Kong Chinese, only variants at chromosomes 8q24.21, 10p14, 11q23.1 and 18q21.1 were replicated in at least two out of the three Chinese studies.

Conclusions

The contrasting results between Caucasians and Chinese could be due to different LD patterns and allelic frequencies or genetic heterogeneity. The results suggest that additional common variants contributing to CRC predisposition remained to be identified.     

Age-related increase in colorectal cancer stem cells in macroscopically normal mucosa of patients with adenomas: A risk factor for colon cancer

It is becoming increasingly evident that cancer stem cells play a vital role in development and progression of cancers and relapse following chemotherapy. The present study examines the presence of cancer stem-like cells (CSC) in adenomatous polyps and in normal appearing colonic mucosa in humans during aging. The number of polyps was found to increase linearly with advancing age (r² = 0.92, p < 0.02). Immunohistochemical analysis revealed co-localization of CSC markers CD44 and CD166 in colonic polyps. Real-time RT-PCR analysis of normal appearing mucosa from subjects with adenomatous polyps showed an age-related rise in CSC as evidenced by the increased expression of CD44, CD166 and ESA. A similar phenomenon was also observed for EGFR. In addition, the expression each CSC marker was found to be about 2-fold higher in subjects with 3–4 polyps than those with 1–2 polyps. In conclusion, our results show that colon cancer stem-like cells are present in the premalignant adenomatous polyps as well in normal appearing colonic mucosa. Moreover, our observation of the age-related rise in CSC in macroscopically normal colonic mucosa suggests a predisposition of the organ to developing colorectal cancer.     

Comparative proteome analysis of breast cancer and adjacent normal breast tissues in human

Two-dimensional polyacrylamide gel assisted laser desorption/ionization electrophoresis （2D-PAGE） and matrixtandem time-of-flight mass spectrometry （MALDI-TOF/TOF-MS）, incorporated with online database searching, were performed to investigate differential proteins of breast cancer and adjacent normal breast tissues. Considering that serum albumin is abundantly presented in normal control samples, 15 differential spots detected in 11 out of 12 （91.7%） breast cancer samples were identified by online SIENA-2DPAGE database searching and MALDI-TOF/TOF-MS analysis. The results indicate that pathological changes of breast cancer are concerned with augmentation of substance metabolism, promotion of proteolytic activity, decline of activity of some inhibitors of enzymes, and so on. Some important proteins involved in the pathological process of breast cancer with changed expression may be useful biomarkers, such as alpha-l-antitrypsin, EF- 1-beta, cathepsin D, TCTP, SMT3A, RPS12, and PSMA1, among which SMT3A, RPSl2, and PSMA1 were first reported for breast cancer in this study.     

Proteomic analysis of the aging-related proteins in human normal colon epithelial tissue

In order to screen the aging related proteins in human normal colon epithelia, the comparative proteomics analysis was applied to get the two-dimensional electrophoresis (2-DE) profiles with high resolution and reproducibility from normal colon epithelial tissues of young and aged people. Differential proteins between the colon epithelia of two age groups were found with PDQuest software. The thirty five differential protein-spots were identified by peptide mass fingerprint (PMF) based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and database searching. Among them there are sixteen proteins which are significantly up-regulated in the colonic mucosal epithelia of young people group, which include ATP synthase beta chain, electron transfer flavoprotein alpha-subunit, catalase, glutathione peroxidase 1, annexin A2 and heat shock cognate 71 kDa protein, etc.; There are nineteen proteins which are significantly up-regulated in the colonic mucosal epithelia of aged people group, which include far upstream element-binding protein 1, nucleoside diphosphate kinase B, protein disulfide-isomerase precursor and VDAC-2, etc.. The identified differential proteins appear to be involved in metabolism, energy generation, chaperone, antioxidation, signal transduction, protein folding and apoptosis. The data will help to understand the molecular mechanisms of human colon epithelial aging.     

Single-cell genomic profile-based analysis of tissue differentiation in colorectal cancer

Science China Life Sciences - Colorectal cancer (CRC) progression is associated with cancer cell dedifferentiation and sternness acquisition. Several methods have been developed to identify...     

Effects of fish oil supplementation on prostaglandins in normal and tumor colon tissue: modulation by the lipogenic phenotype of colon tumors

Dietary fish oils have potential for prevention of colon cancer, and yet the mechanisms of action in normal and tumor colon tissues are not well defined. Here we evaluated the impact of the colonic fatty acid milieu on the formation of prostaglandins and other eicosanoids. Distal tumors in rats were chemically induced to model inflammatory colonic carcinogenesis. After 21 weeks of feeding with either a fish oil diet containing an eicosapentaenoic acid/ω-6 fatty acid ratio of 0.4 or a Western fat diet, the relationships between colon fatty acids and prostaglandin E₂ (PGE₂) concentrations were evaluated. PGE₂ is a key proinflammatory mediator in the colon tightly linked with the initiation and progression of colon cancer. The fish oil vs. the Western fat diet resulted in reduced total fatty acid concentrations in serum but not in colon. In the colon, the effects of the fish oil on fatty acids differed in normal and tumor tissue. There were distinct lipodomic patterns consistent with a lipogenic phenotype in tumors. In tumor tissue, the eicosapentaenoic acid/arachidonic acid ratio, cyclooxygenase-2 expression and the mole percent of saturated fatty acids were significant predictors of inter-animal variability in colon PGE₂ after accounting for diet. In normal tissues from either control rats or carcinogen-treated rats, only diet was a significant predictor of colon PGE₂. These results show that the fatty acid milieu can modulate the efficacy of dietary fish oils for colon cancer prevention, and this could extend to other preventive agents that function by reducing inflammatory stress.     

Glutathione S-transferases in normal and malignant human colon tissue

This study focuses on the GST composition of a tissue intrinsically resistant to chemotherapy, the human colon. GSTs were purified from matched pairs of colon tissue (normal and tumor) using glutathione affinity chromatography. The mean GST activity of colon tumors was 1.5-fold higher than that of normal tissue, with tumors of the sigmoid colon showing the greatest increase (2.3-fold). Two-dimensional gel electrophoresis and Western blot analysis of purified enzymes demonstrated the presence of all three GST classes (alpha, mu and pi) in colon, with GST pi being both the predominant isozyme in normal and malignant tissues. The level of alpha class subunits was the same in normal and tumor tissues, while the mu class subunits were decreased in tumors. A protein copurifying with GSTs from both normal and tumor tissue did not crossreact with GST antibodies, but instead reacted with a polyclonal antibody to glyoxylase I. This enzyme existed as a dimer in its native state. Upon boiling, monomeric subunits were produced with a molecular mass of 22.6 kDa and an isoelectric point more acidic than GST pi. Increased amounts of glyoxylase I were also found in tumor vs. normal colon. The apparent elevated levels of these glutathione-associated detoxifying enzymes in colon tumors may contribute to their intrinsic drug resistance.     

Expression of protein kinase C isoforms in cancerous breast tissue and adjacent normal breast tissue

The role of protein kinase C (PKC) in the carcinogenesis of human breast tissue has been studied at the molecular level for more than two decades. In this study, we employed Western blotting to determine the presence of PKC isoforms in cancerous and normal breast tissues. The results indicate significant expression of a conventional PKC (PKCα) and two atypical PKCs (PKC ζ and λ/ι) in both breast tumors and adjacent normal breast tissue. For the α,ζ and λ/ι isoforms, the expression of individual isoforms was higher in the breast tumors than in the adjacent normal breast tissue. Although the correlation coefficient was low, significant linear correlation was found among the activities of the isoforms. The data suggest a potential new direction in cancer chemotherapy, namely the blockage of the signal transduction pathway of specific PKC isoforms.     

Gene promoter methylation in colorectal cancer and healthy adjacent mucosa specimens

We evaluated the promoter methylation levels of the APC, MGMT, hMLH1, RASSF1A and CDKN2A genes in 107 colorectal cancer (CRC) samples and 80 healthy adjacent tissues. We searched for correlation with both physical and pathological features, polymorphisms of folate metabolism pathway genes (MTHFR, MTRR, MTR, RFC1, TYMS, and DNMT3B), and data on circulating folate, vitamin B12 and homocysteine, which were available in a subgroup of the CRC patients. An increased number of methylated samples were found in CRC respect to adjacent healthy tissues, with the exception of APC, which was also frequently methylated in healthy colonic mucosa. Statistically significant associations were found between RASSF1A promoter methylation and tumor stage, and between hMLH1 promoter methylation and tumor location. Increasing age positively correlated with both hMLH1 and MGMT methylation levels in CRC tissues, and with APC methylation levels in the adjacent healthy mucosa. Concerning gender, females showed higher hMLH1 promoter methylation levels with respect to males. In CRC samples, the MTR 2756AG genotype correlated with higher methylation levels of RASSF1A, and the TYMS 1494 6bp ins/del polymorphism correlated with the methylation levels of both APC and hMLH1. In adjacent healthy tissues, MTR 2756AG and TYMS 1494 6bp del/del genotypes correlated with APC and MGMT promoter methylation, respectively. Low folate levels were associated with hMLH1 hypermethylation. Present results support the hypothesis that DNA methylation in CRC depends from both physiological and environmental factors, with one-carbon metabolism largely involved in this process.     

Lifestyle factors and Ki-ras mutations in colon cancer tumors

Heterogeneity in colon tumors implies that environmental, lifestyle, or genetic factors influence the type of mutations seen in tumors. In this study we evaluate the association between previously identified risk factors for colon cancer and Kirsten-ras (Ki-ras) mutations in tumors. The presence of Ki-ras mutations in codons 12 and 13 were determined in a population-based case-control study of colon cancer. Participants were between 30 and 79 years of age at time of diagnosis and include both men and women. Questionnaire data were used to obtain information on lifestyle factors. Valid study data and Ki-ras mutational status were available from 1428 cases of colon cancer, data from 2410 controls were available for comparative purposes. Participants with Ki-ras mutations were more likely to have proximal rather than distal tumors. Cigarette smoking, use of aspirin and/or NSAIDs, use of vitamin/mineral supplements, and consumption of caffeine were associated with both Ki-ras+ and Ki-ras- tumors; the associations were not confounded by dietary intake or other lifestyle factors. Among men, but not among women, those with low levels of physical activity were more likely to have a tumor with a Ki-ras mutation than one without a Ki-ras mutation. However, among women, those with a larger BMI were more likely to have a Ki-ras mutation in their tumor. Given the limited information available on what causes Ki-ras mutations, the information generated from this study indicates that these factors previously associated with colon cancer work through other disease pathways.     

Rare germline copy number variants in colorectal cancer predisposition characterized by exome sequencing analysis

正Colorectal cancer(CRC)is one of the most common neoplasms and an important cause of mortality worldwide(http://globocan.iarc.fr/).Approximately 35%of the variation in CRC susceptibility is likely due to heritable factors(Lichtenstein et al.,2000).Genetic variations in the human genome include single nucleotide variants(SNVs),short insertions and deletions,and larger structural vari-     

Germline EPHB2 receptor variants in familial colorectal cancer

Familial clustering of colorectal cancer occurs in 15-20% of cases, however recognized cancer syndromes explain only a small fraction of this disease. Thus, the genetic basis for the majority of hereditary colorectal cancer remains unknown. EPHB2 has recently been implicated as a candidate tumor suppressor gene in colorectal cancer. The aim of this study was to evaluate the contribution of EPHB2 to hereditary colorectal cancer. We screened for germline EPHB2 sequence variants in 116 population-based familial colorectal cancer cases by DNA sequencing. We then estimated the population frequencies and characterized the biological activities of the EPHB2 variants identified. Three novel nonsynonymous missense alterations were detected. Two of these variants (A438T and G787R) result in significant residue changes, while the third leads to a conservative substitution in the carboxy-terminal SAM domain (V945I). The former two variants were found once in the 116 cases, while the V945I variant was present in 2 cases. Genotyping of additional patients with colorectal cancer and control subjects revealed that A438T and G787R represent rare EPHB2 alleles. In vitro functional studies show that the G787R substitution, located in the kinase domain, causes impaired receptor kinase activity and is therefore pathogenic, whereas the A438T variant retains its receptor function and likely represents a neutral polymorphism. Tumor tissue from the G787R variant case manifested loss of heterozygosity, with loss of the wild-type allele, supporting a tumor suppressor role for EPHB2 in rare colorectal cancer cases. Rare germline EPHB2 variants may contribute to a small fraction of hereditary colorectal cancer.