Colorectal Adenoma Cells Research Articles

The early predictive effect of low expression of the ITGA4 in colorectal cancer.

The early diagnosis of colorectal cancer (CRC) is very important for the prognosis of patients. It has been suggested that the cytosine-phosphate-guanine (CpG) island of itga4 is highly methylated in colorectal adenoma cell lines AA/C1, Vaco 235 and so on. So the purpose of our study is to explore the diagnostic accuracy and related mechanism of integrin alpha 4 (ITGA4) in early CRC. The Cancer Genome Atlas (TCGA) database was used to analyze the relationship between the expression of ITGA4 and the clinicopathological features and the overall survival rate of the disease. Then, the interaction protein and function enrichment region of ITGA4 were analyzed. Finally, the infiltration of related immune cells was analyzed. Compared with normal tissues, the expression of ITGA4 in colon adenocarcinoma and rectum adenocarcinoma (COAD-READ) tumor tissues was lower (P<0.05). The overall survival rate of COAD-READ patients with low ITGA4 level was lower than that of patients with high ITGA4 expression (P<0.05), and expression of ITGA4 had a more significant predictive effect in the early stage of tumor development. The results of protein network and enrichment analysis suggested that ITGA4 was closely related to ITGB2 and might be involved in the inflammatory reaction and inflammatory tumor transformation process in the carcinogenesis of inflammatory bowel disease (IBD), which was verified by another independent sequence. In terms of immune infiltration, the expression level of ITGA4 was positively correlated with the infiltration level of intestinal macrophages (Th17), immature dendritic cells (IDC), dendritic cells (DC), mast cells, and eosinophils in COAD-READ, and significantly negatively correlated with CD56bright natural killer (NK) cells. The low expression of ITGA4 was related to the poor prognosis of COAD-READ. Findings showed that ITGA4 might participate in the inflammatory reaction and inflammatory tumor transformation process in the carcinogenesis of IBD, and that ITGA4 was related to the infiltration of immune cells, macrophages, syndactyls, and CD56bright NK cells. The expression of ITGA4 could be used as an early predictor of CRC. However, the mechanism of ITGA4 promoting tumor progression in CRC still needs further research.

Role of Vitamin D in Preventing Colorectal Carcinogenesis

Introduction: Colorectal carcinoma is one of the cancers with a high disease burden globally. Previous observational studies have found a connection between colorectal cancer incidence with sunlight exposure and vitamin D levels. Subsequent studies investigated this relationship further and found various anti-tumoral pathways regulated by vitamin D in colorectal tissue. This paper aims to elucidate the actions of those pathways in preventing the malignant transformation of the colorectal cell by reviewing relevant literature. Methods: A search was conducted on several medical literature electronic databases for original research studying the effects of vitamin D treatment on colorectal adenoma and colorectal cancer and its underlying anti-tumoral mechanism. A total of 122 studies were included for evaluation. Results: Twenty-seven studies passed for analysis. These in vitro and in vivo study reveals that vitamin D treatment can suppress cell proliferation, induce apoptosis, maintain cellular differentiation, reduce the pro-inflammatory response, inhibit angiogenesis, and hinder metastatic progression in colorectal cancer and colorectal adenoma cells by regulating associated gene transcription or directly prevents activation of selected signalling pathways. Five studies have also shown that adding calcium to vitamin D treatment increases the anti-tumoral activity of vitamin D through cross-talk between both of their pathways. Conclusion: Vitamin D could potentially impede colorectal cancer transformation and growth through interaction with various signalling pathways and regulating gene transcription. Further clinical studies are needed to confirm whether vitamin D can be used as the basis of targeted colorectal cancer therapy using its inherent anti-tumoral properties.

Accumulation of Paneth Cells in Early Colorectal Adenomas Is Associated with Beta-Catenin Signaling and Poor Patient Prognosis.

Background: Previous studies in mice indicated that Paneth cells and c-Kit-positive goblet cells represent the stem cell niche of the small intestine and colon, respectively, partly by supporting Wnt and Notch activation. Whether these cell populations play a similar role in human intestinal cancer remains unexplored. Methods: We performed histopathological evaluation and immunohistochemical analysis of early colorectal adenomas and carcinoma adenoma from patients at the Hospital del Mar in Barcelona. We then determined the possible correlation between the different parameters analyzed and with patient outcomes. Results: Paneth cells accumulate in a subset of human colorectal adenomas directly associated with Notch and Wnt/β-catenin activation. Adenoma areas containing Paneth cells display increased vessel density in the lamina propria and higher levels of the stem cell marker EphB2. In an in-house cohort of 200 colorectal adenoma samples, we also observed a significant correlation between the presence of Paneth cells and Wnt activation. Kaplan–Meier analysis indicated that early adenoma patients carrying Paneth cell-positive tumors display reduced disease-free survival compared with patients with Paneth cell-free lesions. Conclusions: Our results indicate that Paneth cells contribute to the initial steps of cancer progression by providing the stem cell niche to adenoma cells, which could be therapeutically exploited.

CEUS-Based Radiomics Can Show Changes in Protein Levels in Liver Metastases After Incomplete Thermal Ablation.

ObjectiveTo investigate the ability of contrast-enhanced ultrasound (CEUS)-based radiomics combined with machine learning to detect early protein changes after incomplete thermal ablation.MethodsHCT-26 colorectal adenoma cells were engrafted into the livers of 80 mice, which were randomly divided into 4 groups for palliative laser ablation. Changes in heat shock protein (HSP) and apoptosis-related protein expression in the tumors were assessed. SCID mice subjected to CEUS and ultrasonography were divided into training (n=56) and test (n=24) datasets. Then, 102 features from seven feature groups were extracted. We use the least absolute shrinkage and selection operator (LASSO) feature selection method to fit the machine learning classifiers. The feature selection methods and four classifiers were combined to determine the best prediction model.ResultsThe areas under the receiver-operating characteristic curves (AUCs) of the classifiers in the test dataset ranged from 0.450 to 0.932 (median: 0.721). The best score was obtained from the model in which the omics data of CEUS was analyzed in the arterial phase by random forest (RF) classification.ConclusionsA machine learning model, in which radiomics characteristics are extracted by multimodal ultrasonography, can accurately, rapidly and noninvasively identify protein changes after ablation.

A Combined Proteomics and Mendelian Randomization Approach to Investigate the Effects of Aspirin-Targeted Proteins on Colorectal Cancer.

Evidence for aspirin's chemopreventative properties on colorectal cancer (CRC) is substantial, but its mechanism of action is not well-understood. We combined a proteomic approach with Mendelian randomization (MR) to identify possible new aspirin targets that decrease CRC risk. Human colorectal adenoma cells (RG/C2) were treated with aspirin (24 hours) and a stable isotope labeling with amino acids in cell culture (SILAC) based proteomics approach identified altered protein expression. Protein quantitative trait loci (pQTLs) from INTERVAL (N = 3,301) and expression QTLs (eQTLs) from the eQTLGen Consortium (N = 31,684) were used as genetic proxies for protein and mRNA expression levels. Two-sample MR of mRNA/protein expression on CRC risk was performed using eQTL/pQTL data combined with CRC genetic summary data from the Colon Cancer Family Registry (CCFR), Colorectal Transdisciplinary (CORECT), Genetics and Epidemiology of Colorectal Cancer (GECCO) consortia and UK Biobank (55,168 cases and 65,160 controls). Altered expression was detected for 125/5886 proteins. Of these, aspirin decreased MCM6, RRM2, and ARFIP2 expression, and MR analysis showed that a standard deviation increase in mRNA/protein expression was associated with increased CRC risk (OR: 1.08, 95% CI, 1.03-1.13; OR: 3.33, 95% CI, 2.46-4.50; and OR: 1.15, 95% CI, 1.02-1.29, respectively). MCM6 and RRM2 are involved in DNA repair whereby reduced expression may lead to increased DNA aberrations and ultimately cancer cell death, whereas ARFIP2 is involved in actin cytoskeletal regulation, indicating a possible role in aspirin's reduction of metastasis. Our approach has shown how laboratory experiments and population-based approaches can combine to identify aspirin-targeted proteins possibly affecting CRC risk.

Endoscopically Unresectable Paneth Cell (PC)–Containing Adenomas and the Risk of Developing Colorectal Neoplasia

Abstract Objectives Whether the presence of Paneth cells (PCs) in colorectal adenomas indicates an increased risk of colorectal neoplasia or not is controversial. We examined the clinicopathologic features of PC-containing adenomas (PCAs) that were surgically removed, focusing on the risk of developing subsequent colorectal neoplasia on follow-up. Methods A retrospective cohort of 154 patients with endoscopically unresectable colorectal adenomas who underwent surgical removal was retrieved. Archived pathology slides were evaluated for the presence of PC, villous features, and high-grade dysplasia. Demographic and clinical data were obtained by reviewing electronic medical records. A minimum 12 months of follow-up was considered valid follow-up data. Fisher’s exact test and Student t test were performed when indicated (P &lt; .05 was considered statistically significant). Results PCAs were identified in 84 out of 154 cases (54.5%), commonly in the proximal as compared to distal colorectum (60.7% vs 38.1%, P = .018). There was no significant difference in patient age (mean, 66.0 vs 63.8), gender (M:F, 42:42 vs 35:35), adenoma size (mean, 3.18 cm vs 2.78 cm), villous features (69.0% vs 68.6%), and high-grade dysplasia (44.0% vs 35.7%) between PCA and non-PCA groups. After the mean follow-up duration of 65.8 months (range 12-169), 11 out of 30 patients (36.7%) had recurrent colorectal neoplasia, including 2 adenocarcinomas in the PCA group, as compared to 8 out of 27 (29.6%) in non-PCA group with no adenocarcinoma (P &gt; .05). No statistical difference in the risk of developing colorectal neoplasia was noted between proximal PCA (24%), distal PCA (33.3%), and non-PCA (29.6%) groups. Conclusion PCAs are more common in the proximal colon, consistent with normal anatomic distribution of PC in the colon. No association was observed between PCA and high-grade dysplasia or colorectal neoplasia risk in endoscopically unresectable adenomas. PCA location was not a significant marker for future adverse outcomes.

Abstract 2965: Functionally informed genome-wide interaction analysis of nonsteroidal anti-inflammatory drugs on colorectal cancer risk

Abstract Background Regular use of aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) is associated with lower risk of colorectal cancer (CRC). Genome-wide interaction analysis (GxE) has identified a few variants that may modify the effects of NSAIDs on CRC risk. However, limited statistical power remains a concern. Restricting analyses by using functional genomic information to aggregate variants into biologically relevant sets can reduce the number of tests, thereby increasing statistical power. We tested the interactions between variant models of gene expression and NSAIDs use on CRC risk. Methods Functional weights of each variant were estimated using PrediXcan based on jointly measured transcriptomes and genomes data of transverse colon tissues from the Genotype-Tissue Expression (GTEx) Project for all genes with sufficient heritability (≥1%). A mixed-effects model was used to assess the GxE effects in a gene among 9,917 incident CRC cases and 10,533 controls from 17 (nested) case-control studies from the Colon Cancer Family Registry (CCFR) and Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO). GxE analysis was done by modeling the interaction between predicted gene expression and NSAID use (fixed effects), and residual variant-specific GxE effects that are not accounted for (random effects). Regular user of aspirin and/or non-aspirin NSAIDs was compared to non-regular users. False discovery rate (FDR) was used to account for multiple comparison and FDR≤0.2 was considered genome-wide significant. Secondary analysis was performed on significant genes and duration of use among users. Results Regular use of aspirin was higher among controls (30.3%) compared to cases (24.3%). Among the 4,842 genes tested, SORD significantly modified the effect of regular use of aspirin on CRC risk (p-interaction = 1.45×10-5; FDR=0.07). The SORD gene encodes sorbitol dehydrogenase (SORD), which oxidizes sorbitol to fructose in the polyol pathway. Decreased SORD concentrations were previously observed to dramatically increase in colorectal adenoma cells when compared to normal mucosa cells, suggesting the involvement of dysregulated polyol metabolisms in colorectal tumorigenesis. However, the duration of aspirin use was not statistically significantly associated with SORD gene expression on CRC risk (p=0.149) among 5,560 aspirin users. No significant interactions were observed between genetically determined colon gene expression levels and any NSAID use or non-aspirin NSAID use at FDR&amp;lt;0.2. Conclusions Incorporating functional information, we discovered a novel gene that may interact with aspirin use to confer CRC risk. These findings provide preliminary support for new biological insights that could help understand the chemopreventive mechanisms of aspirin on CRC. We aim to replicate these findings in additional studies. Citation Format: Xiaoliang Wang, Yu-Ru Su, Andrew T. Chan, Stephanie Bien, Sonja I. Bernt, Hermann Brenner, Graham Casey, Jenny Chang-Claude, Steven J. Gallinger, Robert W. Haile, Tabitha A. Harrison, Michael Hoffmeister, Mark A. Jenkins, Amit Joshi, Yi Lin, Noralane M. Lindor, Loic Le Marchand, Hongmei Nan, Polly A. Newcomb, John D. Potter, Martha L. Slattery, Steve N. Thibodeau, Emily White, Li Hsu, Ulrike Peters. Functionally informed genome-wide interaction analysis of nonsteroidal anti-inflammatory drugs on colorectal cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2965.

Hotspot KRAS exon 2 mutations in CD166 positive colorectal cancer and colorectal adenoma cells.

Colorectal cancer (CRC) is the third most common cancer and the fourth leading cause of cancer deaths worldwide. Recent studies have shown that cancer stem cells (CSCs) are an important cause of tumor recurrence and metastasis. We hypothesized that CSCs marker CD166-positive CRC and colorectal adenoma (CAD) cells consist of more hotspot mutations than CD166-negative CRC and colorectal adenoma cells. To verify this, formalin fixed paraffin embedded tissue specimens from 42 patients each with CRC and CAD were recruited and CD166 immunohistochemical (IHC) staining followed by macrodissection was performed. DNA extracted was used for quantitative polymerase chain reaction detection on a somatic mutation array. Results showed that the immunoreactivity of CD166 protein had significant difference among CRC, CAD, and normal colorectal epithelial tissues (NCET) (P < 0.0001, Kruskal-Wallis test). Moreover, nucleotide changes were found in APC, KRAS, P53, PIK3CA, FBXW7 and SRC genes. Among those genes, KRAS exon 2 mutations were validated in another cohort of 70 CRC and 72 CAD specimens. Results showed that the difference in percentage of KRAS exon 2 mutations between CD166 positive and CD166 negative CRC specimens was significant (P < 0.05, chi-square test). Long term follow-up of the CRC patients showed that CD166-positive KRAS exon 2 mutations was useful in discriminating CRC patients with worse outcome. This study has provided evidence that KRAS exon 2 mutations are concentrated in CD166-positive cancer cells, with prognostic significance in CRC, and those mutations are also detected in CAD.

Beneficial Phytochemicals with Anti-Tumor Potential Revealed through Metabolic Profiling of New Red Pigmented Lettuces (Lactuca sativa L.).

The present study aimed to compare polyphenols among red lettuce cultivars and identify suitable cultivars for the development and utilization of healthy vegetables. Polyphenols, mineral elements, and antioxidant activity were analyzed in the leaves of six red pigmented lettuce (Lactuca sativa L.) cultivars; thereafter, we assessed the anti-tumor effects of cultivar B-2, which displayed the highest antioxidant activity. Quadrupole–Orbitrap mass spectrometry analysis revealed four classes of polyphenols in these cultivars. The composition and contents of these metabolites varied significantly among cultivars and primarily depended on leaf color. The B-2 cultivar had the highest antioxidant potential than others because it contained the highest levels of polyphenols, especially anthocyanin, flavone, and phenolic acid; furthermore, this cultivar displayed anti-tumor effects against the human lung adenocarcinoma cell line A549, human hepatoma cell line Bel7402, human cancer colorectal adenoma cell line HCT-8, and HT-29 human colon cancer cell line. Hence, the new red-leaf lettuce cultivar B-2 has a distinct metabolite profile, with high potential for development and utilization of natural phytochemical and mineral resources in lettuces and can be used as a nutrient-dense food product.

LGR5 expression is regulated by EGF in early colorectal adenomas and governs EGFR inhibitor sensitivity

Background:LGR5 serves as a co-receptor for Wnt/β-catenin signalling and marks normal intestinal stem cells; however, its role in colorectal cancer (CRC) remains controversial. LGR5+ cells are known to exist outside the stem cell niche during CRC progression, and the requirement for epidermal growth factor (EGF) signalling within early adenomas remains to be fully elucidated.Methods:Epidermal growth factor and gefitinib treatments were performed in EGF-responsive LGR5+ early adenoma RG/C2 cells. 2D growth assays were measured using an IncuCyte. LGR5 or MEK1/2 silencing studies were executed using siRNA and LGR5 expression was assessed by qRT–PCR and immunoblotting. Ki67 level and cell cycle status were analysed by flow cytometry.Results:Epidermal growth factor suppresses expression of LGR5 at both the transcript and protein level in colorectal adenoma and carcinoma cells. Suppression of LGR5 reduces the survival of EGF-treated adenoma cells by increasing detached cell yield but also inducing a proliferative state, as evidenced by elevated Ki67 level and enhanced cell cycle progression. Repression of LGR5 further increases the sensitivity of adenoma cells to EGFR inhibition.Conclusions:LGR5 has an important role in the EGF-mediated survival and proliferation of early adenoma cells and could have clinical utility in predicting response of CRC patients to EGFR therapy.

Paneth Cell in Adenomas of the Distal Colorectum Is Inversely Associated with Synchronous Advanced Adenoma and Carcinoma.

Recent studies have linked appearance of Paneth cells in colorectal adenomas to adenoma burden and male gender. However, the clinical importance of Paneth cells’ associations with synchronous advanced adenoma (AA) and colorectal carcinoma (CRC) is currently unclear. We performed a comprehensive case-control study using 1,900 colorectal adenomas including 785 from females, and 1,115 from males. We prospectively reviewed and recorded Paneth cell status in the colorectal adenomas consecutively collected between February 2014 and June 2015. Multivariable logistic regression analyses revealed that, in contrast to the adenomas without Paneth cells, the Paneth cell-containing adenomas at distal colorectum were inversely associated with presence of a synchronous AA or CRC (odds ratio [OR] 0.39, P = 0.046), whereas no statistical significance was reached for Paneth cell-containing proximal colorectal adenomas (P = 0.33). Synchronous AA and CRC were significantly associated with older age (60 + versus <60 years, OR 1.60, P = 0.002), male gender (OR 1.42, P = 0.021), and a history of AA or CRC (OR 2.31, P < 0.001). However, synchronous CRC was not associated with Paneth cell status, or a history of AA or CRC. Paneth cell presence in the adenomas of distal colorectum may be a negative indicator for synchronous AA and CRC, and seems to warrant further studies.

EphB6 overexpression and Apc mutation together promote colorectal cancer.

The erythropoietin-producing hepatocyte (Eph) family tyrosine kinases play important roles in tumorigenesis and cancer aggression. In this study, we investigated the role of EphB6 in oncogenic transformation of colorectal epithelial cells in vitro and in vivo. EphB6 is upregulated in human colorectal cancer (CRC) tissues as compared to normal tissues, and its overexpression promotes proliferation, migration and invasion by IMCE colorectal adenoma cells, in which one Apc allele is mutated. EphB6 overexpression together with Apc mutation leads to the development of colorectal tumors in vivo. Expression microarrays using mRNAs and lncRNAs isolated from EphB6-overexpresssing IMCE and control cells revealed a large number of dysregulated genes involved in cancer-related functions and pathways. The present study is the first to demonstrate that EphB6 overexpression together with Apc gene mutations may enhance proliferation, invasion and metastasis by colorectal epithelial cells. Microarray data and pathway analysis of differentially expressed genes provided insight into possible EphB6-regulated mechanisms promoting tumorigenesis and cancer progression. EphB6 overexpression may represent a novel, effective biomarker predictive of cell proliferation, invasion and metastasis patterns in CRC tumors.

PTH-278 Aspirin enhances the response to radiation in colorectal cancer cell lines

Introduction Advances in surgical resection, neo-adjuvant therapies and efforts to ensure early detection have led to a reduction in colorectal cancer mortality rates since the 1970s. Despite this, around 40% of patients die from recurrence or metastatic disease, making it the second most common cause of cancer related death in the UK. Poor response to adjuvant treatment for colorectal cancer continues to pose a significant problem. Colorectal Cancer Stem Cells (CSC), are thought to be responsible for fuelling resistance and driving recurrent disease.1The identification of a reliable CSC marker, LGR5, 2has enabled further investigation and it has been demonstrated that both the function and survival of colorectal CSCs is driven by COX-2/PGE2and BCL-3/NFκB signalling pathways.3 Therefore, we aimed to determine whether Aspirin, through inhibiting PGE2production and BCL-3 expression, can enhance the response to radiotherapy, in colorectal cancer cells. Method Colorectal carcinoma (HCA7/P, LS174T, LoVo) and adenoma (AN/C1, RG/C2) cell lines were treated with Aspirin, (1–2 mM, 24–96 h). Cell survival (cell yield and apoptosis) and protein expression (analysed by Western blotting), were determined. Colorectal cancer cells were seeded at low densities and treated with combinations of Aspirin and/or γ-radiation (Cs-137 source). Cell survival was determined by crystal violet cell viability assay. Results 2 mM Aspirin decreased cell yield (e.g. LoVo- Control 26.7 × 106vs. 2 mM Aspirin 9.4 × 106, p = 0.002) and increased apoptosis (e.g. Lovo- Control 7.8 × 106vs. 2 mM Aspriin 19.5 × 106, p = 0.014) in carcinoma and adenoma cell lines at 72 and 96 h. Western analysis showed Aspirin treatment reduced the expression of CSC related markers in both colorectal carcinoma and adenoma cell lines. 2mM Aspirin increased the sensitivity to 2.5Gy radiation in colorectal carcinoma cells, leading to an approximate two-fold decrease in cell survival, (e.g. HCA7/P- 2.5Gy radiation 81.9% survival vs. 43.4% for 2.5Gy and 2 mM Aspirin combined). Suppression of BCL-3 expression, in addition to Aspirin, further enhanced response to radiation. Conclusion Aspirin decreases cell survival and increases apoptosis in colorectal carcinoma and adenoma cell lines. Furthermore, it enhances the response to radiation, reducing colorectal cancer cell survival. BCL-3 suppression may modulate this response. Clinical translation, to further explore Aspirin’s role as a novel adjunct to radiation, is underway in our multi-centre prospective cohort study, (ASPIRE). Disclosure of interest None Declared. References O’Brien CA. Nature 2007;445(7123):106–110 Barker N. Nature 2007;449(7165):1003–7 Al-Kharusi MRA. Carcinogenesis 2013;34(5):1150–1157

BCL-3 expression promotes colorectal tumorigenesis through activation of AKT signalling

ObjectiveColorectal cancer remains the fourth most common cause of cancer-related mortality worldwide. Here we investigate the role of nuclear factor-κB (NF-κB) co-factor B-cell CLL/lymphoma 3 (BCL-3) in promoting colorectal tumour...

Inhibition of β-catenin signalling promotes DNA damage elicited by benzo[a]pyrene in a model of human colon cancer cells via CYP1 deregulation.

Deregulation of Wnt/β-catenin signalling plays an important role in the pathogenesis of colorectal cancer. Interestingly, this pathway has been recently implicated in transcriptional control of cytochrome P450 (CYP) family 1 enzymes, which are responsible for bioactivation of a number of dietary carcinogens. In the present study, we investigated the impact of inhibition of Wnt/β-catenin pathway on metabolism and genotoxicity of benzo[a]pyrene (BaP), a highly mutagenic polycyclic aromatic hydrocarbon and an efficient ligand of the aryl hydrocarbon receptor, which is known as a primary regulator of CYP1 expression, in cellular models derived from colorectal tumours. We observed that a synthetic inhibitor of β-catenin, JW74, significantly increased formation of BaP-induced DNA adducts in both colorectal adenoma and carcinoma-derived cell lines. Using the short interfering RNA (siRNA) targeting β-catenin, we then found that β-catenin knockdown in HCT116 colon carcinoma cells significantly enhanced formation of covalent DNA adducts by BaP and histone H2AX phosphorylation, as detected by (32)P-postlabelling technique and immunocytochemistry, respectively, and it also induced expression of DNA damage response genes, such as CDKN1A or DDB2. The increased formation of DNA adducts formed by BaP upon β-catenin knockdown corresponded with enhanced production of major BaP metabolites, as well as with an increased expression/activity of CYP1 enzymes. Finally, using siRNA-mediated knockdown of CYP1A1, we confirmed that this enzyme plays a major role in formation of BaP-induced DNA adducts in HCT116 cells. Taken together, the present results indicated that the siRNA-mediated inhibition of β-catenin signalling, which is aberrantly activated in a majority of colorectal cancers, modulated genotoxicity of dietary carcinogen BaP in colon cell model in vitro, via a mechanism involving up-regulation of CYP1 expression and activity.

Characterization of LGR5 stem cells in colorectal adenomas and carcinomas.

LGR5 is known to be a stem cell marker in the murine small intestine and colon, however the localization of LGR5 in human adenoma samples has not been examined in detail, and previous studies have been limited by the lack of specific antibodies. Here we used in situ hybridization to specifically examine LGR5 mRNA expression in a panel of human adenoma and carcinoma samples (n = 66). We found that a small number of cells express LGR5 at the base of normal colonic crypts. We then showed that conventional adenomas widely express high levels of LGR5, and there is no evidence of stereotypic cellular hierarchy. In contrast, serrated lesions display basal localization of LGR5, and the cellular hierarchy resembles that of a normal crypt. Moreover, ectopic crypts found in traditional serrated adenomas show basal LGR5 mRNA, indicating that they replicate the stem cell organization of normal crypts with the development of a cellular hierarchy. These data imply differences in the stem cell dynamics between the serrated and conventional pathways of colorectal carcinogenesis. Furthermore we noted high LGR5 expression in invading cells, with later development of a stem cell niche in adenocarcinomas of all stages.

PTEN expression in colorectal adenomas: Relationship to morphology and cell heterogeneity

IntroductionColorectal adenomas are the most frequent benign colorectal tumors. These tumors are characterized by dysplasia, low and high grade, the latter having the potential to evolve to adenocarcinoma.The aim of this study was to study the expression patterns of PTEN protein in a series of colorectal adenomas and the relationships to cell proliferation and CD133, marker of stem phenotype. MethodsColorectal adenomas were studied for the immunohistochemical expression of PTEN on tissue microarrays. PTEN expression was analysed with regard to morphological features and with regard to the Ki67 and CD133-positive cell compartments by using the Kendall rank-correlation test. ResultsPTEN was expressed in 92% adenomas, either in a cytoplasmic or nuclear pattern. Cytoplasmic PTEN was correlated to cytoplasmic CD133 (p=0.02, tau 0.191) while nuclear PTEN to decreased adenoma size and to tubular architecture (p=0.01, τ−0.184 and p=0.01, τ−0.183). Nuclear PTEN was also correlated to low grade intraepithelial neoplasia, while global PTEN (nuclear or cytoplasmic) was correlated to the presence of a decreased Ki67-positive component but with marginal significance (p=0.06, τ−0.144 and p=0.07, τ−0.213). ConclusionThe results of this study suggest a role for PTEN in colorectal adenoma morphogenesis and cell protein heterogeneity, being correlated to decreased size, tubular architecture and a high CD133-positive component.

Comprehensive suppression of all apoptosis-induced proliferation pathways as a proposed approach to colorectal cancer prevention and therapy.

Mutations in the WNT/beta-catenin pathway are present in the majority of all sporadic colorectal cancers (CRCs), and histone deacetylase inhibitors induce apoptosis in CRC cells with such mutations. This apoptosis is counteracted by (1) the signaling heterogeneity of CRC cell populations, and (2) the survival pathways induced by mitogens secreted from apoptotic cells. The phenomena of signaling heterogeneity and apoptosis-induced survival constitute the immediate mechanisms of resistance to histone deacetylase inhibitors, and probably other chemotherapeutic agents. We explored the strategy of augmenting CRC cell death by inhibiting all survival pathways induced by the pro-apoptotic agent LBH589, a histone deacetylase inhibitor: AKT, JAK/STAT, and ERK signaling. The apoptosis-enhancing ability of a cocktail of synthetic inhibitors of proliferation was compared to the effects of the natural product propolis. We utilized colorectal adenoma, drug-sensitive and drug-resistant colorectal carcinoma cells to evaluate the apoptotic potential of the combination treatments. The results suggest that an effective approach to CRC combination therapy is to combine apoptosis-inducing drugs (e.g., histone deacetylase inhibitors, such as LBH589) with agents that suppress all compensatory survival pathways induced during apoptosis (such as the cocktail of inhibitors of apoptosis-associated proliferation). The same paradigm can be applied to a CRC prevention approach, as the apoptotic effect of butyrate, a diet-derived histone deacetylase inhibitor, is augmented by other dietary agents that modulate survival pathways (e.g., propolis and coffee extract). Thus, dietary supplements composed by fermentable fiber, propolis, and coffee extract may effectively counteract neoplastic growth in the colon.

Abstract 37: TP53 coding and noncoding variants in cancer susceptibility: From Li-Fraumeni syndrome to Lynch syndrome

Abstract The present study is to generate a mouse model of a p53 noncoding germline polymorphism that predisposes human carriers to multiple types of cancer. p53, encoded by TP53, is recognized as the guardian of the human genome. Three distinct sets of alterations occur at the TP53 locus, which spans ∼20kb in the human genome: tumor-associated somatic mutations, germline mutations causing Li-Fraumeni syndrome, and germline polymorphisms. The first two types of TP53 mutations occur in the coding sequence (CDS) and produce mutant p53 proteins that lack most or all of the normal tumor suppressive functions and often confer oncogenic properties. By contrast, only a few of the &amp;gt;200 naturally occurring germline variants of TP53 in human populations cause measurable perturbation of p53 function. More than 90% of TP53 germline variants occur in 10 introns alleged to have no cancer-related biological consequences. None of the 20 variants in the TP53 CDS have been reproducibly linked to cancer predisposition. Genome-wide association studies (GWAS) reported no significant association between any germline TP53 polymorphism and any cancer with a P value ≤ 10-7 (a threshold for genome-wide significance) until a single nucleotide polymorphism (SNP) rs78378222 was found to be associated strongly with skin basal cell carcinoma. rs78378222 is located in the fifth nucleotide of the TP53 polyadenylation signal (PAS) that is required for polyadenylation machinery recognition and subsequent cleavage and polyadenylation of mRNA. The risk-associated minor allele is a “C”, resulting in an alternative PAS (AATACA) instead of the canonical PAS (AATAAA). This variant is also associated with an increased risk of colorectal adenoma, prostate cancer, glioma, and esophageal squamous cell carcinoma. It is notable that cancer susceptibility of this p53 noncoding variant does not mirror that of p53 coding germline mutations (patients with Li-Fraumeni syndrome develop predominantly breast cancer, brain tumors, acute leukemia, sarcoma, and adrenal cortical carcinoma). Specifically, carriers with the p53 PAS variant are not predisposed to breast cancer. The p53 PAS variant is uniquely positioned among cancer-susceptibility alleles: (i) low frequency (∼2% in the general populations), (ii) intermediate cancer risk (odds ratio 1.39-3.54), and (iii) predisposing carriers to multiple types of cancer. To mimic this naturally occurring human variant, we have generated a mouse line with a p53 gene carrying the alternative PAS (AATACA) using zinc-finger nuclease technology. The resultant mutant p53 allele is termed p531755C as the 1755th nucleotide of mouse p53 reference mRNA is the ortholog of human rs78378222. We demonstrate that heterozygote p53+/1755C and homozygote p531755C/1755C mice treated with azoxymethane (AOM) and dextran sodium sulfate (DSS) developed more colon tumors than the p53+/+ controls and homozygous mice developed more advanced tumors than heterozygous mice. With data from murine tumorigenesis and human epidemiology, we propose that this p53 noncoding germline variant is a cancer susceptibility allele associated with Lynch syndrome rather than Li-Fraumeni syndrome. Citation Format: Qipan Deng, Yong Li. TP53 coding and noncoding variants in cancer susceptibility: From Li-Fraumeni syndrome to Lynch syndrome. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr 37. doi:10.1158/1538-7445.CANSUSC14-37

576: Modulating the Wnt-pathway in colorectal adenoma cells by 1,25-dihydroxyvitamin D3

576: Modulating the Wnt-pathway in colorectal adenoma cells by 1,25-dihydroxyvitamin D3