Human Sporadic Colorectal Cancer Research Articles

Abstract 4201: miR-124 suppresses colorectal tumor progression in a mouse xenograft model via targeting KITENIN.

Abstract MicroRNAs (miRNAs) are increasingly implicated in modulating the progression of various cancers. Although there is emerging evidences that some miRNAs can function as oncogenes or tumour suppressors, the regulation of colorectal cancer (CRC) progression by miRNA is not fully understood. KITENIN (KAI1 C-terminal interacting tetraspanin, Vangl1) is not only a major component of PCP pathway but also a functional protein promoting CRC cell motility and invasiveness. Previously, we observed that KITENIN acts as a metastasis-enhancing protein in a mouse model of colon cancer and that KITENIN is highly expressed in sporadic human CRC tissues. But the underlying regulation mechanisms of aberrant expression of KITENIN in CRC are not clearly understood. Here we tried to identify the miRNAs, which modulate the expression of KITENIN, using luciferase assay and with computational prediction of miRNA targeting KITENIN. We identified several miRNAs and one of them is miR-124. miR-124 negatively regulated KITENIN expression through binding to the 3′-untranslated region of KITENIN. We further examined whether increased expression of miR-124 affects colorectal tumor progression via targeting KITENIN. Ectopic expression of miR-124 was found to suppress the migration and invasiveness of various CRC cells. miR-124 also inhibited tumor growth in a mouse xenograft model. These findings provide the experimental evidence to a possible therapeutic effect of miR-124 on suppressing colorectal tumor progression and identification of miR-124-mediated regulation of KITENIN might provide a promising therapeutic target in treating CRC. Citation Format: So-Yeon Park, Jeong A Bae, Somy Yoon, Hangun Kim, Kyung Keun Kim. miR-124 suppresses colorectal tumor progression in a mouse xenograft model via targeting KITENIN. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4201. doi:10.1158/1538-7445.AM2013-4201

Oxidative stress in the pathogenesis of colorectal cancer: cause or consequence?

There is a growing support for the concept that reactive oxygen species, which are known to be implicated in a range of diseases, may be important progenitors in carcinogenesis, including colorectal cancer (CRC). CRC is one of the most common cancers worldwide, with the highest incidence rates in western countries. Sporadic human CRC may be attributable to various environmental and lifestyle factors, such as dietary habits, obesity, and physical inactivity. In the last decades, association between oxidative stress and CRC has been intensively studied. Recently, numerous genetic and lifestyle factors that can affect an individual's ability to respond to oxidative stress have been identified. The aim of this paper is to review evidence linking oxidative stress to CRC and to provide essential background information for accurate interpretation of future research on oxidative stress and CRC risk. Brief introduction of different endogenous and exogenous factors that may influence oxidative status and modulate the ability of gut epithelial cells to cope with damaging metabolic challenges is also provided.

Silibinin, a natural flavonoid, modulates the early expression of chemoprevention biomarkers in a preclinical model of colon carcinogenesis

The flavonolignan silibinin, the major biologically active compound of the milk thistle (Silybum marianum), has been shown to possess anticancer properties in a variety of epithelial cancers. The present study investigated the potential of silibinin as a chemopreventive agent in colon carcinogenesis. The rat azoxymethane (AOM)-induced colon carcinogenesis model was used because of its molecular and clinical similarities to sporadic human colorectal cancer. One week after AOM injection (post-initiation), Wistar rats received daily intragastric feeding of 300 mg silibinin/kg body weight per day until their sacrifice after 7 weeks of treatment. Silibinin-treated rats exhibited a 2-fold reduction in the number of AOM-induced hyperproliferative crypts and aberrant crypt foci in the colon compared to AOM-injected control rats receiving the vehicle. Silibinin-induced apoptosis in the colon mucosal cells was demonstrated by flow cytometry after propodium iodide staining and by colorimetric measurement of caspase-3 activity. Mechanisms involved in silibinin-induced apoptosis included the downregulation of the anti-apoptotic protein Bcl-2 and upregulation of the pro-apoptotic protein Bax, inverting the Bcl-2/Bax ratio to <1. This modulation already takes place at the mRNA expression level as shown by real-time RT-PCR. Furthermore, silibinin treatment significantly (P<0.01) decreased the genetic expression of biomarkers of the inflammatory response such as IL1β, TNFα and their downstream target MMP7, all of them shown to be upregulated during colon carcinogenesis. The downregulation of MMP7 protein was confirmed by western blot analysis. The present findings show the ability of silibinin to shift the disturbed balance between cell renewal and cell death in colon carcinogenesis in rats previously injected with the carcinogen AOM. Silibinin administered via intragastric feeding exhibited potent pro-apoptotic, anti-inflammatory and multi-targeted effects at the molecular level. The effective reduction of preneoplastic lesions by silibinin supports its use as a natural agent for colon cancer chemoprevention.

Proximal Fluid Proteome Profiling of Mouse Colon Tumors Reveals Biomarkers for Early Diagnosis of Human Colorectal Cancer

Early detection of colorectal cancer (CRC) and its precursor lesions is an effective approach to reduce CRC mortality rates. This study aimed to identify novel protein biomarkers for the early diagnosis of CRC. Proximal fluids are a rich source of candidate biomarkers as they contain high concentrations of tissue-derived proteins. The FabplCre;Apc(15lox/+) mouse model represents early-stage development of human sporadic CRC. Proximal fluids were collected from normal colon and colon tumors and subjected to in-depth proteome profiling by tandem mass spectrometry. Carcinoembryonic antigen (CEA) and CHI3L1 human serum protein levels were determined by ELISA. Of the 2,172 proteins identified, quantitative comparison revealed 192 proteins that were significantly (P < 0.05) and abundantly (>5-fold) more excreted by tumors than by controls. Further selection for biomarkers with highest specificity and sensitivity yielded 52 candidates, including S100A9, MCM4, and four other proteins that have been proposed as candidate biomarkers for human CRC screening or surveillance, supporting the validity of our approach. For CHI3L1, we verified that protein levels were significantly increased in sera from patients with adenomas and advanced adenomas compared with control individuals, in contrast to the CRC biomarker CEA. These data show that proximal fluid proteome profiling with a mouse tumor model is a powerful approach to identify candidate biomarkers for early diagnosis of human cancer, exemplified by increased CHI3L1 protein levels in sera from patients with CRC precursor lesions.

Tissue expression of Toll-like receptors 2 and 4 in sporadic human colorectal cancer

BackgroundToll-like receptors (TLRs) play an important role in innate immunity by sensing a variety of pathogens and inducing acquired immunity. To test our hypothesis that dysregulation of innate immune responses acts to trigger carcinogenesis, we studied the expression of TLR2 and 4 in sporadic human colorectal cancer tissue.MethodsIn specimens of cancerous and noncancerous colorectal tissue obtained at surgery, mRNA expression levels of TLR2 and 4 were quantified by TaqMan real-time polymerase chain reaction and compared between the two types of tissue. To confirm TLR2 and TLR4 protein expression levels, immunohistochemical analysis was performed using the same samples.ResultsTLR2 mRNA expression was significantly higher in cancerous tissue than in noncancerous tissue, while TLR4 mRNA expression did not differ significantly. Immunohistochemical analysis revealed stronger staining for TLR2 in cancerous mucosal epithelial cells than in noncancerous tissue. Staining for TLR4 in the lamina propria of the mucosa was equally weakly positive in noncancerous tissue and cancerous tissue. This TLR-specific difference in expression suggested that such expression does not only reflect a local inflammatory response to cancer infiltration, i.e., if this was the case, both TLR2 and 4 expression would probably be up-regulated. Our results suggest that TLR2 expression might be involved in sporadic colorectal carcinogenesis, whereas TLR4 is not.

Morphological and Molecular Alterations in 1,2 Dimethylhydrazine and Azoxymethane Induced Colon Carcinogenesis in Rats

The dimethyhydrazine (DMH) or azoxymethane (AOM) model is a well-established, well-appreciated, and widely used model of experimental colon carcinogenesis. It has many morphological as well as molecular similarities to human sporadic colorectal cancer (CC), which are summarized and discussed in this paper. In addition, the paper combines present knowledge of morphological and molecular features in the multistep development of CC recognized in the DMH/AOM rat model. This understanding is necessary in order to accurately identify and interpret alterations that occur in the colonic mucosa when evaluating natural or pharmacological compounds in DMH/AOM rat colon carcinogenesis. The DMH/AOM model provides a wide range of options for investigating various initiating and environmental factors, the role of specific dietary and genetic factors, and therapeutic options in CC. The limitations of this model and suggested areas in which more research is required are also discussed.

Differential expression of hMLH1 in sporadic human colorectal cancer tumors and distant metastases

Somatic defects in the mismatch repair system constitute an important pathway in colorectal carcinogenesis. We have examined the expression of mismatch repair proteins in sporadic stage IV colorectal tumors and their derived metastases. Sporadic tumors were further examined for differences in expression between the tumor transition zone and the invasive front. Expression of hMSH2, hMLH1, and hPMS2 was screened immunohistochemically in 92 stage IV tumors and derived liver metastases. In cases with loss of mismatch repair protein expression, lymph node metastases were also examined. Clinicopathological parameters and Ki-67 staining indexes were evaluated and compared. Four tumors displayed a complete loss of hMLH1/hPMS2 expression at the transition zone; however, three of these expressed both proteins at the invasive front and in liver and lymph node metastases. A further four were predominantly hMLH1/hPMS2 negative at the transition zone, but with distinct subclones of hMLH1/hPMS2-expressing cells at the transition zone. All of these tumors expressed hMLH1/hPMS2 at the invasive front and in liver metastases, with three also expressing hMLH/hPMS2 in lymph node metastases. No significant difference in the proliferative index was observed for the hMLH1/hPMS2-compromised group. In stage IV tumors re-expression of hMLH1/hPMS2 occurred, leading to different patterns of expression within the primary tumor and between tumor and metastases. This may have functional importance for the chemosensitivity of metastases compared to the primary tumor.

Correlative Study of Microvessel Density and 5-Lipoxygenase Expression in Human Sporadic Colorectal Cancer

5-Lipoxygenase (5-LO) is an arachidonic acid- metabolizing enzyme, which has been demonstrated to exert a role in colorectal cancer tumorigenesis. Its activity in promoting neoangiogenesis in colorectal malignancies has been also recently theorized on the basis of in vitro studies. To investigate whether any correlation existed between 5-LO immunoexpression amount and the quantity of neoangiogenesis, as reflected by microvessel density (MVD) in human sporadic surgically resected colorectal adenocarcinomas. A total of 45 formalin-fixed, paraffin-embedded colorectal adenocarcinomas were submitted to the immunohistochemical procedures for 5-LO and CD105, which represent specific markers for neoangiogenesis and which were used in the assessment of MVD. CD105-positive, intratumoral, newly formed vessels were present in 45 of 45 cases with variable MVD values. A 5-LO-positive immunohistochemical reaction was also found in 45 of 45 cases. A significantly higher MVD was evident in cases displaying a high 5-LO amount in comparison with those characterized by a low 5-LO expression (28.33 vs 19.44 vessels per mm(2); P = .02). In addition, a positive significant correlation emerged between 5-LO immunoexpression amount and the MVD counts (r = 0.2986, P = .04). Our study demonstrates the existence of a relationship between 5-LO expression and the neoangiogenesis process as reflected by intratumoral MVD in human sporadic colorectal adenocarcinomas, thus suggesting that 5-LO may modulate the formation of blood vessels in these neoplasias.

Correlative Study of Microvessel Density and 5-Lipoxygenase Expression in Human Sporadic Colorectal Cancer

Abstract Context.—5-Lipoxygenase (5-LO) is an arachidonic acid– metabolizing enzyme, which has been demonstrated to exert a role in colorectal cancer tumorigenesis. Its activity in promoting neoangiogenesis in colorectal malignancies has been also recently theorized on the basis of in vitro studies. Objective.—To investigate whether any correlation existed between 5-LO immunoexpression amount and the quantity of neoangiogenesis, as reflected by microvessel density (MVD) in human sporadic surgically resected colorectal adenocarcinomas. Design.—A total of 45 formalin-fixed, paraffin-embedded colorectal adenocarcinomas were submitted to the immunohistochemical procedures for 5-LO and CD105, which represent specific markers for neoangiogenesis and which were used in the assessment of MVD. Results.—CD105-positive, intratumoral, newly formed vessels were present in 45 of 45 cases with variable MVD values. A 5-LO–positive immunohistochemical reaction was also found in 45 of 45 cases. A significantly higher MVD w...

A Therapeutic Cancer Vaccine Targeting Carcinoembryonic Antigen in Intestinal Carcinomas

A genetic vaccine platform based on DNA electroporation (DNA-EP) and adenovirus (Ad) was used to generate immune response against human carcinoembryonic antigen (CEA) and antitumor effects in murine models with spontaneous tumors arising in an orthotopic location. CEA transgenic (CEA.Tg) mice treated with the carcinogen 1,2-dimethylhydrazine developed CEA-overexpressing tumors that resembled human sporadic colorectal cancer. APC1638N/CEA hybrid mice, generated by crossing mice carrying the adenomatous polyposis coli (Apc1638N) gene mutation with CEA.Tg mice, are representative of human familial polyposis and develop polyps that overexpress the antigen. In both models, the DNA-EP/Ad vaccine succeeded in breaking immune tolerance and achieved significant antitumor effects in therapeutic settings. Our data suggest that genetic vaccines targeting CEA may be feasible strategies against gut tumors that overexpress the antigen. In addition, these models are powerful systems for evaluating antigen-specific tumor immunity and assessing therapeutic vaccine strategies for human colorectal cancer.

The pro-apoptotic K-Ras 4A proto-oncoprotein does not affect tumorigenesis in the Apc Min/+mouse small intestine

BackgroundAlterations in gene splicing occur in human sporadic colorectal cancer (CRC) and may contribute to tumour progression. The K-ras proto-oncogene encodes two splice variants, K-ras 4A and 4B, and K-ras activating mutations which jointly affect both isoforms are prevalent in CRC. Past studies have established that splicing of both the K-ras oncogene and proto-oncogene is altered in CRC in favour of K-ras 4B. The present study addressed whether the K-Ras 4A proto-oncoprotein can suppress tumour development in the absence of its oncogenic allele, utilising the ApcMin/+ (Min) mouse that spontaneously develops intestinal tumours that do not harbour K-ras activating mutations, and the K-rastmΔ4A/tmΔ4A mouse that can express the K-ras 4B splice variant only. By this means tumorigenesis in the small intestine was compared between ApcMin/+, K-ras+/+ and ApcMin/+, K-rastmΔ4A/tmΔ4A mice that can, and cannot, express the K-ras 4A proto-oncoprotein respectively.MethodsThe relative levels of expression of the K-ras splice variants in normal small intestine and small intestinal tumours were quantified by real-time RT-qPCR analysis. Inbred (C57BL/6) ApcMin/+, K-ras+/+ and ApcMin/+, K-rastmΔ4A/tmΔ4A mice were generated and the genotypes confirmed by PCR analysis. Survival of stocks was compared by the Mantel-Haenszel test, and tumour number and area compared by Student's t-test in outwardly healthy mice at approximately 106 and 152 days of age. DNA sequencing of codons 12, 13 and 61 was performed to confirm the intestinal tumours did not harbour a K-ras activating mutation.ResultsThe K-ras 4A transcript accounted for about 50% of K-ras expressed in the small intestine of both wild-type and Min mice. Tumours in the small intestine of Min mice showed increased levels of K-ras 4B transcript expression, but no appreciable change in K-ras 4A transcript levels. No K-ras activating mutations were detected in 27 intestinal tumours derived from Min and compound mutant Min mice. K-Ras 4A deficiency did not affect mouse survival, or tumour number, size or histopathology.ConclusionThe K-Ras 4A proto-oncoprotein does not exhibit tumour suppressor activity in the small intestine, even though the K-ras 4A/4B ratio is reduced in adenomas lacking K-ras activating mutations.

Genomic profiles of colorectal cancers differ based on patient smoking status

Human sporadic colorectal cancer is the result of a lengthy somatic evolutionary process facilitated by various forms of genomic instability. Such instability arises endogenously from mutations in genes whose role is to preserve genomic integrity, and exogenously from environmental agents that generate genomic damage. We have found that cigarette smoking shifts the genomic profiles and genomic instability patterns of colorectal carcinomas. The genomic profiles of 57 consecutive cancers were examined; 31 cases were current or former smokers and 26 were nonsmokers. Genome-wide allelotypes of 348 markers were examined, along with comparative genomic hybridization (CGH) on ordered BAC microarrays, microsatellite instability, and inter-(simple sequence repeat) polymerase chain reaction instability. Tumors from nonsmokers exhibited losses of heterozygosity, particularly on chromosomes 14 and 18, whereas tumors from smokers exhibited a more diffuse pattern of allelic losses. Tumors from smokers exhibited higher overall rates of loss of heterozygosity, but showed lower rates of background microsatellite instability (MSI-L). On BAC array CGH, higher levels of generalized amplifications and deletions were observed in tumors from smokers, differentially affecting male smokers. In the transforming growth factor-β signaling pathway, MADH4 mutations were more common in tumors from smokers, whereas transforming growth factor-β RII mutations were more common among nonsmokers.

Frequent rearrangements and amplification of the CDX2 homeobox gene in human sporadic colorectal cancers with chromosomal instability

The expression of the CDX2 gene, a crucial regulator of gut homeostasis, is altered in human colorectal cancers in parallel with de-differentiation. Here, we have investigated the chromosomal status of CDX2 in human sporadic colorectal cancers with the phenotype of chromosomal instability. Allelic imbalance determination showed frequent rearrangements at the CDX2 locus. The rearrangements correlated with CDX2 gene amplification, as assessed by quantitative PCR analysis. However, they were not predictive of the Cdx2 protein pattern. These data suggest that mechanisms other than structural alterations at the CDX2 locus account for the change of expression in colorectal cancers.

Prognostic value of p16INK4a and p14ARF gene hypermethylation in human colon cancer

The INK4a/ARF locus (9p21) encodes two unique and unrelated cell cycle regulators, p16 INK4a and p14 ARF. This study was performed to evaluate the methylation status of p16 INK4a and p14 ARF genes, as well as its association with p16 and p53 expression, microsatellite instability (MI) status, and various clinicopathologic parameters in sporadic colorectal cancer. Sixty-five cases of primary colorectal adenocarcinoma with a series of clinicopathological parameters were obtained. We performed methylation-specific PCR of p16 INK4a and p14 ARF genes in colorectal cancer paraffin blocks with its paired normal samples, as well as immunohistochemical stainings for p16 and p53, and MI analysis. Aberrant methylations of p16 INK4a and p14 ARF gene were present in 21 (32.3%) and 33 (50.8%) out of 65 cases, respectively. p16 INK4a aberrant methylation was correlated with p16 negativity ( P = 0.021 ) and p53 overexpression ( P = 0.007 ). p16 INK4a aberrant methylation was more frequently present in poorly differentiated adenocarcinomas ( P = 0.002 ). Aberrant methylation of p14 ARF gene occurred more frequently in patients under 50 years of age and in left-sided colon cancers, and was not statistically significant. Compared with the group with simultaneous absence of methylation in both promoters, the group showing concomitant alterations in both p16 INK4a and p14 ARF genes ( n = 10 ) more frequently presented lymph node metastasis ( P = 0.020 ) and higher tumor grade ( P = 0.014 ). There was no correlation between p16 INK4a and p14 ARF gene hypermethylation or MI status. This study suggests that simultaneous hypermethylation of both p16 INK4a and p14 ARF genes is greater prognostic value in sporadic human colorectal cancer.

KRAS, p53 and BRAF Gene Mutations and Aneuploidy in Sporadic Colorectal Cancer Progression

Background: The origin and mechanisms of chromosomal instability are still widely unknown. We previously investigated a limited number of human sporadic colorectal cancers (CRCs) and observed a statistically different occurrence of KRAS and p53 mutations among predetermined subgroups of tumors with different degrees of DNA aneuploidy. The aim of the present study was to further verify these observations by including BRAF gene analysis and by investigating a larger series of cases subdivided into Dukes' stages A to D to reconstruct some form of chronological modulation for events during CRC progression. Methods: KRAS, p53, BRAF mutations and flow cytometric DNA Index were evaluated by established techniques in a series of 135 human sporadic CRCs. Results: p53, KRAS and BRAF mutations were found in 39%, 34%, and 4% of tumors, respectively. The frequency of p53 mutations increased from 15% for stage A to 48% for stage D and was highest in near-diploid (DI 1.6) tumors. A similar correlation between gene mutations and DI values was observed for KRAS. The simultaneous presence of KRAS and p53 mutations was observed in only 11% of cases. Moreover, the co-occurrence of p53 and KRAS mutations was only observed in near-diploid and high-aneuploid tumors. Conclusion: Our findings suggest that KRAS and p53 gene mutations, which are rarely simultaneous and are associated with specific DI aneuploid values, do not represent a synergistic evolutionary pathway but may influence mechanisms of chromosomal instability.

Poly(ADP-Ribose) Polymerase-1 Is a Component of the Oncogenic T-Cell Factor-4/β-Catenin Complex

T-cell factor (TCF)-4 regulates a certain set of genes related to growth and differentiation of intestinal epithelial cells. Aberrant transactivation of these TCF-4-regulated genes by beta-catenin protein plays a crucial role in early intestinal carcinogenesis, and the transcriptional machinery of the TCF-4/beta-catenin complex is likely to contain targets for molecular therapy. We explored the molecular composition of the TCF-4/beta-catenin transcriptional complex by means of proteomics. A protein of approximately 112 kilodaltons was consistently coimmunoprecipitated with FLAG-tagged TCF-4 transiently expressed in HEK293 cells, and the protein was identified by mass spectrometry as poly(ADP-ribose) polymerase-1 (PARP-1). PARP-1 physically interacted with TCF-4 and augmented the transcriptional activity of the beta-catenin/TCF-4 complex. Knockdown of PARP-1 by RNA interference significantly suppressed both transcriptional activity and proliferation by colorectal cancer cells. Auto-polyADP-ribosylation of the PARP-1 protein induced by DNA damage inhibited the functional interaction of PARP-1 with TCF-4. PARP-1 was overexpressed in the intestinal adenomas of patients with familial adenomatous polyposis and multiple intestinal polyposis mice. The expression of PARP-1 was closely associated with the accumulation of beta-catenin and with the undifferentiated status of intestinal epithelial cells. In this study, we identified PARP-1 as a novel coactivator of the beta-catenin/TCF-4 complex. Although PARP-1 has been believed to play a protective role against carcinogenesis, these expression patterns and functional properties of PARP-1 were highly suggestive of its participation in early colorectal carcinogenesis.

Vascular endothelial growth factor and enhanced angiogenesis do not promote metastatic conversion of a newly established azoxymethane‐induced colon cancer cell line

The organo-specific carcinogen, azoxymethane (AOM), produces colon tumors in mice that share many pathological features with sporadic human colorectal cancer (CRC). An important distinction between AOM-induced CRC and human CRC is lack of mucosal invasion in the murine model. To assess the role of the microenvironment in preventing the invasive phenotype, multiple benign in situ adenocarcinomas were harvested from AOM-treated mice and cultured in vitro. However, tumor cell growth was extremely limiting under standard culturing conditions. Thus, we injected tumor cells directly into nude mice and performed two serial transplants, and successfully explanted a rapidly growing epithelial tumor cell line (AJ02nm(0)). When injected subcutaneously (sc) into nude mice, AJ02nm(0) cells formed well-differentiated adenocarcinomas with minimal tumor invasive capacity. To define whether metastatic and invasive potential were related to lack of angiogenic stimuli, the AJ02nm(0) cells were transfected to overexpress murine vascular endothelial growth factor-164 (VEGF(164)). AJ02nm-VEGF cells produced rapidly growing tumors in nude mice that exhibited extensive pseudo-epithelial ductal architecture and supporting vasculature, but without increased invasive potential compared to controls. The established murine colon epithelial cell line provides a useful experimental model to further elaborate genetic and epigenetic factors that may promote or inhibit colon tumorigenesis and metastasis.

Up-regulation of hnRNP A1 gene in sporadic human colorectal cancers

We have previously reported that the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), a major hnRNP, binds to G-rich repetitive sequences and quadruplex (G4') structures in DNA, including the 5'-TTAGGG-3' telomere repeat and 5'-GGCAG-3' short-tandem-repeat. DNA synthesis arrest at the (GGG) sites within these repeats in vitro was retrieved by the addition of the hnRNP A1 protein or its N-terminal proteolytic product, UP1, in a dose-dependent manner. Therefore, functional perturbation of hnRNP A1 may abrogate the genomic stability of telomere repeats and other G-rich sequences, independent of its major role in transcriptional and translational regulation. In the present study, we conducted genetic and expression analysis of the hnRNP A1 gene in sporadic human colorectal cancers to clarify its possible involvement in human carcinogenesis. Of 30 lesions, one harbored a mutation at the -11 position from the translation initiation site, but none in the coding region. A single nucleotide polymorphism, an A or G-allele, was found in the 5' upstream promoter region of the gene. Quantitative gene expression analysis revealed that 60% (18/30) of cases showed over-expression of hnRNP A1 in cancer tissues by 2-fold or greater, compared to their normal colon tissues, with values of 78, 64 and 40% for clinicopathological stages II, III and IV, respectively. Although the biological consequences of hnRNP A1 overexpression in colorectal cancers remain to be clarified, it could contribute to maintenance of telomere repeats in cancer cells with enhanced cell proliferation. Alternatively, since the variations in the stoichiometry of hnRNP family proteins are considered to affect cell-specific gene expression, quantitative alteration of hnRNP A1 could result in facilitation of transformation of colon epithelial cells as a consequence of transcriptional and translational perturbation.

Genetic alterations and expression of inhibitor of growth 1 in human sporadic colorectal cancer

To explore the effect and significance of inhibitor of growth 1 (ING1) gene in carcinogenesis and progression of human sporadic colorectal cancer. mRNA expression, mutation, and loss of heterozygosity (LOH) of ING1 gene in 35 specimens of sporadic colorectal cancer tissues and the matched normal mucous membrane tissues were detected by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), PCR-single strain conformation polymorphism (PCR-SSCP) and PCR-simple sequence length polymorphism (PCR-SSLP) using microsatellite markers, respectively. The average ratios of light intensities of p33(ING1b) and p47(ING1a) mRNA expression in the cancerous tissues were significantly lower than those in normal tissues. The difference between the two mRNA splices was not significant in the matched tissues. In addition, the ratios of light intensities of p33(ING1b) and p47(ING1a) mRNA expression in the cancerous tissues of Dukes' stages C and D were significantly lower than those in cancerous tissues of Dukes' stages A and B. However, no mutation of ING1 gene was detected in all 35 cases; only 4 cases of LOH (11.4%) were found. p33(ING1b) and p47(ING1a) mRNA expressions are closely related with the carcinogenesis and progression of human sporadic colorectal cancer. No mutation of ING1 gene is found, and there are only few LOH in sporadic colorectal cancers. These might not be the main reasons for the down regulation of ING1 expression. Its low expression may happen in transcription or post-transcription.

Alpha2HS-glycoprotein, an antagonist of transforming growth factor beta in vivo, inhibits intestinal tumor progression.

Transforming growth factor (TGF)-beta1 is associated with tumor progression and resistance to chemotherapy in established cancers, as well as host immune suppression. Here, we show that the serum glycoprotein alpha2-HS-glycoprotein (AHSG) blocks TGF-beta1 binding to cell surface receptors, suppresses TGF-beta signal transduction, and inhibits TGF-beta-induced epithelial-mesenchymal transition, suggesting that AHSG may play a role in tumor progression. In 66 consecutive sporadic human colorectal cancer specimens, we observed a 3-fold depletion of ASHG in tumor compared with normal tissue, whereas levels of other abundant plasma proteins, albumin and transferrin, were equivalent. Using the Multiple intestinal neoplasia/+ (Min/+) mouse model of intestinal tumorigenesis, we found twice as many intestinal polyps overall, twice as many large polyps (>3 mm diameter), and more progression to invasive adenocarcinoma in Min/+ Ahsg-/- mice than in littermates expressing Ahsg. Phosphorylated Smad2 was more abundant in the intestinal mucosa and tumors of Min/+ mice lacking Ahsg, demonstrating increased TGF-beta signaling in vivo. Furthermore, TGF-beta-mediated suppression of immune cell function was exaggerated in Ahsg-/- animals, as shown by inhibition of macrophage activation and reduction in 12-O-tetradecanoylphorbol 13-acetate-induced cutaneous inflammation. Reconstitution of Ahsg-/- mice with bovine Ahsg suppressed endogenous TGF-beta-dependent signaling to wild-type levels, suggesting that therapeutic enhancement of AHSG levels may benefit patients whose tumors are driven by TGF-beta.