Event In Colorectal Carcinogenesis Research Articles

Abstract B025: Role of clock and clock-controlled gene methylation in colorectal adenoma risk and regulation of global DNA methylation: Pilot study

Abstract Dysregulation of the clock and clock-controlled genes, which have epigenetic and tumor suppressor properties, may drive colorectal adenoma and tumor formation. Global DNA hypomethylation (measured by LINE-1) is an early event in colorectal carcinogenesis. The potential role of clock genes in regulating LINE-1 methylation has not been described. The goals of this exploratory pilot were to: identify DNA methylation sites (CpGs) in candidate clock and clock-controlled gene promotors to target in an adenoma case-control study among average risk screening colonoscopy patients; and evaluate possible relationships between the candidate gene methylation and global DNA methylation (LINE-1). Initially, in silico analyses were performed on candidate clock (PER1, PER2, PER3, RORA) and clock-controlled (IL-6, TNF-alpha, MTNR1A, MLH1) gene sequences. Methylation data in the Cancer Genome Atlas (TCGA) were queried with the MEXPRESS and Wanderer search engines to identify CpGs with differential methylation between colon adenocarcinoma and control tissue (p<0.05). These CpGs were further evaluated for differential methylation in leukocyte DNA from 22 histologically confirmed adenoma cases relative to 22 controls (no polyp or normal biopsy), as well as 12 adenoma tissue DNA samples relative to 12 adjacent normal gastrointestinal (GI) tissue biopsies. All participants provided informed consent. Next generation sequencing (NGS) quantified percent methylation at target CpGs (Ion S5™ sequencer with Ion Torrent S5 server for sequence alignment). LINE-1 was quantified via pyrosequencing. Statistical comparisons of methylation at each CpG in leukocyte DNA (cases versus controls) were performed via Wilcoxon Rank-Sum tests. CpG methylation in adenomas versus normal GI tissue was assessed via Wilcoxon signed rank tests. A total of 826 CpGs were interrogated, and 155 differentially methylated sites were identified; RORA was the most prevalent gene represented (34% of CpGs identified), followed by MLH1 (19%) and TNF-alpha (12%). An additional 442 CpGs in proximity to the original 155 sites, but without in silico TCGA data, were also sequenced, and another 170 CpGs were identified. Frequently represented genes included RORA (35%), MTNR1A (18%), TNF-alpha (11%) and PER2 (11%). LINE-1 hypomethylation was observed in colorectal adenomas (66±5%) relative to normal GI tissue (71±2%, n=23, p<0.001); no differences were noted in leukocyte DNA. Serial linear regression with a gene-level false discovery rate was used to identify target CpGs that predict LINE-1 methylation. Seven target CpGs in adenoma (IL-6, PER2), and 2 CpGs in leukocyte DNA (RORA), were associated with LINE-1. This pilot study yielded a set of evidence-based target CpGs (N=325) in candidate clock and clock-controlled genes with differential methylation in colorectal adenoma or cancer patients, and 9 of those were related to LINE-1 methylation. Understanding the role of clock and clock-controlled genes in colorectal adenoma formation may lead to novel strategies for colorectal cancer prevention or treatment. Citation Format: James B. Burch, Jonathan Wells, Joshua Mercadel, Morgan Krupa, Venkat Kothandaraman, Thomas Hurley, Angela Murphy, Stephen Lloyd, Scott Strayer, Gauri Sathe, James Hébert. Role of clock and clock-controlled gene methylation in colorectal adenoma risk and regulation of global DNA methylation: Pilot study [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B025.

Promoter hypermethylation of GALR1 acts as an early epigenetic susceptibility event in colorectal carcinogenesis.

Epigenetics play an essential role in colorectal neoplasia process. There is a need to determine the appropriateness of epigenetic biomarkers for early detection as well as expand our understanding of the carcinogenic process. Therefore, the aim of the study was to assess how DNA methylation pattern of GALR1 gene evolves in a sample set representing colorectal neoplastic progression. The study was designed into three phases. Firstly, Methylation status of GALR1 was assessed with genome-wide DNA methylation beadchip and pyrosequencing assays in colorectal lesions and paired normal tissues. Then, linear mixed-effects modeling analyses were applied to describe the trend of DNA methylation during the progression of colorectal neoplasia. In the third phase, quantitative RT-PCR was used to examine GALR1 expression in patients with precursor lesion and colorectal cancer. We found that significant hypermethylation of GALR1 promoter was a widely existent modification in CRCs (P < 0.001). When further examined methylation pattern of GALR1 during neoplastic progression of CRC, we found that DNA methylation level of GALR1 showed a significant stepwise increase from normal to hyperplastic polyps, to adenomas and to carcinoma samples (P < 0.001). Besides, loss of mRNA expression is a common accompaniment to adenomas and carcinomas. Public omics data analyses showed an inverse correlation between gene expression and DNA methylation (P < 0.001). Our findings indicate that epigenetic alteration of GALR1 promoter is gradually accumulated during the colorectal neoplastic progression. It can potentially be a promising biomarker used for screening and surveillance of colorectal cancer.

A Chemical Link between Meat Consumption and Colorectal Cancer Development?

O6-carboxymethylguanine (O6-CMG) is a mutagenic DNA adduct that forms at increased levels when people eat meat. It has been studied as a potential initiating event in colorectal carcinogenesis. It can arise from alkylation of guanine in DNA by electrophilic degradation products of N-nitroso compounds. There is significant data regarding biochemical and cellular process, including DNA repair and translesion DNA synthesis that control O6-CMG accumulation, persistence, and mutagenicity. Mutation spectra arising from the adduct closely resemble common mutations in colorectal cancer; however, gaps remain in understanding the biochemical processes that regulate how and where the damage persists in the genome. Addressing such questions relies on advances in chemistry such as synthesis approaches and bioanalytical methods. Results of research in this area help advance our understanding of the toxicological relevance of O6-CMG-modified DNA. Further attention should focus on understanding how a combination of genetic and environmental factors control its biological persistence and how this information can be used as a basis of biomoniotoring and prevention efforts to help mitigate colon cancer risk.

IMPACT OF KRAS MUTATIONS IN CLINICAL FEATURES IN COLORECTAL CANCER.

ABSTRACT Background: KRAS mutations are important events in colorectal carcinogenesis, as well as negative predictors of response to EGFR inhibitors treatment. Aim: To investigate the association of clinical-pathological features with KRAS mutations in colorectal cancer patients treated. Methods: Data from 69 patients with colorectal cancer either metastatic at diagnosis or later, were retrospectively analyzed. The direct sequencing and pyrosequencing techniques were related to KRAS exon 2. The mutation diagnosis and its type were determined. Results: KRAS mutation was identified in 43.4% of patients. The most common was c.35G>T (p.G12V), c.35G>A (p.G12D) and c.38G>A (p.G13D). No correlation was found between KRAS mutation and age (p=0.646) or gender (p=0.815). However, mutated group had higher CEA levels at admission (p=0.048) and codon 13 mutation was associated with involvement of more than one metastatic site in disease progression (p=0.029). Although there was no association between primary tumor site and mutation diagnosis (p=0.568), primary colon was associated with worse overall survival (p=0.009). Conclusion: The KRAS mutation was identified in almost half of patients. Mutated KRAS group had higher levels of CEA at admission and the mutation at codon 13 was associated with involvement of more than one metastatic site in the course of the disease. Colon disease was associated with the worst overall survival.

PI3K/ Akt/ mTOR Pathway as a Therapeutic Target for Colorectal Cancer: A Review of Preclinical and Clinical Evidence

Phosphoinositide 3-kinase (PI3Ks) is a member of intracellular lipid kinases and involved in the regulation of cellular proliferation, differentiation and survival. Overexpression of the PI3K/Akt/mTOR signalling has been reported in various forms of cancers, especially in colorectal cancers (CRC). Due to their significant roles in the initiation and progression events of colorectal cancer, they are recognized as a striking therapeutic target. The present review is aimed to provide a detailed outline on the role of PI3K/Akt/mTOR pathway in the initiation and progression events of colorectal cancers as well as its function in drug resistance. Further, the role of PI3K/Akt/mTOR inhibitors alone and in combination with other chemotherapeutic drugs, in alleviating colorectal cancer is also discussed. The review contains preclinical and clinical evidence as well as patent literature of the pathway inhibitors which are natural and synthetic in origin. The data were obtained from PubMed/Medline databases, Scopus and Google patent literature. PI3K/Akt/mTOR signalling is an important event in colorectal carcinogenesis. In addition, it plays significant roles in acquiring drug resistance as well as metastatic initiation events of CRCs. Several small molecules of natural and synthetic origin have been found to be potent inhibitors of CRCs by effectively downregulating the pathway. Data from various clinical studies also support these pathway inhibitors and several among them are patented. Inhibitors of the PI3K/mTOR pathway have been successful for the treatment of primary and metastatic colorectal cancers, rendering the pathway as a promising clinical cancer therapeutic target.

The Interplay between Colon Cancer Cells and Tumour-Associated Stromal Cells Impacts the Biological Clock and Enhances Malignant Phenotypes.

Cancer cells interrelate with the bordering host microenvironment that encompasses the extracellular matrix and a nontumour cellular component comprising fibroblasts and immune-competent cells. The tumour microenvironment modulates cancer onset and progression, but the molecular factors managing this interaction are not fully understood. Malignant transformation of a benign tumour is among the first crucial events in colorectal carcinogenesis. The role of tumour stroma fibroblasts is well-described in cancer, but less well-characterized in benign tumours. In the current work we utilized fibroblasts isolated from tubulovillous adenoma, which has high risk for malignant transformation, to study the interaction between benign tumour stroma and the circadian clock machinery. We explored the role of the biological clock in this interplay taking advantage of an experimental model, represented by the co-culture of colon cancer cells with normal fibroblasts or tumour-associated fibroblasts, isolated from human colorectal tumour specimens. When co-cultured with tumour-associated fibroblasts, colon cancer cells showed alterations in their circadian and metabolic parameters, with decreased apoptosis, increased colon cancer cell viability, and increased resistance to chemotherapeutic agents. In conclusion, the interactions among colon cancer cells and tumour-associated fibroblasts affect the molecular clockwork and seem to aggravate malignant cell phenotypes, suggesting a detrimental effect of this interplay on cancer dynamics.

Prognostic Relevance of SFRP1 Gene Promoter Methylation in Colorectal Carcinoma

Background:The development of colorectal carcinoma (CRC) involves many genetic and epigenetic alterations and methylation being an important epigenetic event has been described as a diagnostic and prognostic biomarker. Secreted Frizzled- Related Protein 1 (SFRP1) gene regulates diverse physiological processes via the Wnt signaling. Promoter hypermethylation of SFRP1 gene is an epigenetic regulation mechanism that downregulates SFRP1 protein level in the tumor, and happens to be one of the significant events in colorectal carcinogenesis. We studied the clinicopathological relationship of CRC including survival outcomes with SFRP1 gene promoter methylation.Methods:We evaluated promoter methylation status of SFRP1 gene by methylation-specific PCR (MS-PCR) in the tumor tissue in 54 cases of stage II-III CRC patients in north India. The MS-PCR result was further validated by bisulfite sequencing.Results:SFRP1 gene was methylated in 72.2% cases and un-methylated in 27.8%. We found, that SFRP1 gene methylation in tumor was associated with lymph node invasion (p=0.05). The mean overall survival was 22.318 months and 45.173 months respectively for patients with methylated and unmethylated SFRP1 gene (p= 0.010, log rank test), (HR = 17.313, 95% CI: 2.021-148.290 P=0.009).Conclusion:Study indicates that promoter methylation of SFRP1 gene is associated with lymph-node metastasis and poor mean overall survival and it can be a prognostic marker in CRC.

Analysis of Differentially Expressed lncRNAs and mRNAs for the Identification of Hypoxia-Regulated Angiogenic Genes in Colorectal Cancer by RNA-Seq.

BackgroundHypoxia is an important feature of solid tumors and related to a perturbed blood supply in pathophysiologies. The aim of our research was to analyze the hypoxia response and elaborate its potential functions in colorectal cancer.Material/MethodsThe lncRNAs and mRNAs expression profile were analyzed in colorectal cancer cell line SW480 by RNA sequencing, and the functions and pathways of differentially expressed genes were screened by Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analysis.ResultsIn this study, 77 lncRNAs and 1327 mRNAs were identified as differentially expressed. We discovered several novel lncRNAs, such as RP11-126K1.2, RP3-438O4.4, LINC01119, CTB-22K21.2, RP11-798M19.6, and RP11-2B6.3, which had not been previously reported in regulation by hypoxia. KEGG and GO analyses identified that the differentially expressed changes in mRNAs were mainly related to regulation of basic metabolic processes and gene transcription processes and were involved in several classical pathways which were linked to cancer.ConclusionsTaken together, the present findings elucidate a set of differentially expressed lncRNAs and mRNAs involved in the hypoxia response process of colorectal cancer, which may serve as a candidate diagnostic biomarker and help to explain the mechanism of initial event in colorectal carcinogenesis in colorectal cancer.

MiR-486-5p Downregulation Marks an Early Event in Colorectal Carcinogenesis.

MicroRNAs are dysregulated in colorectal cancer and subsets correlated with advanced tumor stage and metastasis. Data are lacking on microRNA dysregulation from early to late-stage disease. The purpose of this study was to identify a microRNA signature associated with the primary tumor and metastatic site in stage IV disease and to examine whether the signature is evident in earlier stages. A microRNA profile was generated and then explored in normal colon tissue (n = 5), early stage (stage I and II; n = 10), and late-stage (stage III and IV; n = 14) colorectal primary tumors via polymerase chain reaction to delineate molecular events that may promote colorectal carcinogenesis. Genome-wide microRNA expression profiling was performed. A total of 14 patient-matched stage IV primary colorectal cancer tumors and corresponding liver metastases were included. MicroRNA array technology was used to identify microRNA expression-predictive metastatic potential in the primary tumor. A distinct 9-member signature group of microRNAs was concurrent in stage IV primary colorectal cancer and their corresponding liver metastases, when compared with surrounding unaffected colon and liver tissue (microRNA-18b, microRNA-93, microRNA-182, microRNA-183, microRNA21, microRNA-486-5p, microRNA-500a, microRNA-552, and microRNA-941). Of the microRNA panel, only microRNA486-5p was differentially expressed in early stage colorectal cancer samples compared with normal tissue (p = 0.001) and additionally differentially expressed between late-stage colorectal cancer samples and normal tissue (p < 0.01). Our microRNA profile was generated in a small subset of patients and will require validation in more samples. We identified a distinct microRNA signature in primary colon and matched metastatic disease. On additional investigation, 1 microRNA was differentially expressed in both early and late-stage cancer patient samples, and it may herald an early event in colorectal carcinogenesis. This study warrants additional investigation with a larger patient cohort to better understand the effect of microRNAs in carcinogenesis. See Video Abstract at http://links.lww.com/DCR/A723.

Inhibitory effect of selenomethionine on carcinogenesis in the model of human colorectal cancer in vitro and its link to the Wnt/β-catenin pathway.

Selenium compounds have been implicated as anticancer agents; however, the mechanism of their inhibitory action against cancer development has not been extensively investigated. A constitutive activation of the Wnt/β-catenin pathway is a central event in colorectal carcinogenesis. In this pathway, excessive cell proliferation is initiated by generation of β-catenin followed by overexpression of proto-oncogenes, such as c-Myc. It is believed that under physiological conditions the level of c-Myc is efficiently controlled by accessibility of the β-catenin protein through the process of phosphorylation by glycogen synthase kinase 3β (GSK-3β). Here, we determined whether selenomethionine (SeMet) can inhibit cell growth and affect the Wnt/β-catenin pathway in the HT-29 human colorectal cancer cells in vitro. The effective cytotoxic doses of SeMet have been selected after 48 h of incubation of this compound with colorectal cancer HT-29 cell line. MTT assay was used to assess cell viability and the protein and mRNA levels of β-catenin and c-Myc were determined by Western blotting and qPCR, respectively. SeMet potently inhibited growth of HT-29 cells, significantly decreased level of the β-catenin protein and mRNA concentration, down-regulated the c-Myc gene expression and up-regulated the pro-apoptotic Bax protein level. Moreover, SeMet increased the level of GSK-3β phosphorylated at serine 9 (S9) and significantly increased the level of β-catenin phosphorylated at S33 and S37. We conclude that SeMet suppresses growth of HT-29 colorectal cancer cells by a mechanism linked to the Wnt/β-catenin pathway, however, degradation of β-catenin may occur independently of GSK-3β catalytic activity and its phosphorylation status.

PO-334 Distribution of copy number alterations defines clonal populations involved in the evolutionary transition from adenoma-to-carcinoma in colorectal cancer

Introduction Colorectal cancer progresses in a multi-step manner with adenoma being the most well-known precursor lesion. Malignant polyps, which are adenomas containing a focus of adenocarcinoma, are suitable models to study the evolution of colorectal cancer progression. Although the mutations that accrue during the adenoma-to-carcinoma transition have been previously described, the evolutionary dynamics of copy number alterations (CNAs) involved in this malignant transformation have not been fully understood. Material and methods Here, we performed low-pass whole genome sequencing and targeted deep sequencing of the benign and malignant portion of 23 cases. To validate subclonal copy number alterations we performed single-cell FISH analysis using six probes on chromosomes 5, 7, 8, 13, 18, and 20. We applied FISHtrees to reconstruct the phylogenetic tree of 200 single cells from the adenoma and 200 single cells from the adenocarcinoma portion. Results and discussions By inferring a consensus phylogenetic tree from the same lesion, we deciphered a pattern of branched evolution in all samples evaluated. Analysis of FISH data showed that the levels of genomic heterogeneity were highest in malignant polyps compared to non-progressed adenomas, with the adenoma component having a lower degree of chromosomal instability than the adenocarcinoma. Nevertheless, we observed different patterns of clonal evolution including neutral dynamics and punctuated evolution. We estimated which genomic SCNA showed the greatest allelic expansion comparing the carcinoma to the adenoma and defined this expansion as the fitness gain for the alteration. Genomic gains affecting chromosomes 7, 13, and 20 showed the highest fitness values. Interestingly, whole genome duplication, confirmed by image cytometry, appeared to be a decisive step in the transition from adenoma to carcinoma in 35% of the cases. Lastly, in half of the malignant polyps studied the main clone in the carcinoma component was already present in the adenoma, although at a low frequency and only detectable by single cell analysis. Conclusion In conclusion our data suggest that CNAs are early events in colorectal carcinogenesis that can determine the evolutionary transition from adenoma to adenocarcinoma.

Mutant KRAS promotes liver metastasis of colorectal cancer, in part, by upregulating the MEK-Sp1-DNMT1-miR-137-YB-1-IGF-IR signaling pathway

Although the role of insulin-like growth factor-I receptor (IGF-IR) in promoting colorectal liver metastasis is known, the mechanism by which IGF-IR is upregulated in colorectal cancer (CRC) is not defined. In this study, we obtained evidence that mutant KRAS transcriptionally activates IGF-IR gene expression through Y-box-binding protein (YB)-1 upregulation via a novel MEK-Sp1-DNMT1-miR-137 pathway in CRC cells. The mechanistic link between the tumor suppressive miR-137 and the translational regulation of YB-1 is intriguing because epigenetic silencing of miR-137 represents an early event in colorectal carcinogenesis due to promoter hypermethylation. This proposed signaling axis was further verified by the immunohistochemical evaluations of liver metastases from a cohort of 46 KRAS mutant CRC patients, which showed a significant correlation in the expression levels among Sp1, miR-137, YB-1, and IGF-1R. Moreover, suppression of the expression of YB-1 and IGF-IR via genetic knockdown or the pharmacological inhibition of MEK hampers KRAS-driven colorectal liver metastasis in our animal model studies. From a translational perspective, the identification of this KRAS-driven pathway might provide a mechanistic rationale for the use of a MEK inhibitor as an adjuvant, in combination with standard of care, to prevent the recurrence of colorectal liver metastasis in KRAS mutant CRC patients after receiving liver resection, which warrants further investigation.

Trefoil factor 1 gene alternations and expression in colorectal carcinomas.

Aberrant expression of the trefoil factor family (TFF) has been recognized to be involved in the development and/or progression of various solid tumors. Increased trefoil factor 1 (TFF1) expression is found associated with tumor progression in some tumors, and TFF1 missense mutations have been detected in gastric cancer. The aim of the study was to analyze TFF1 alternations and expression in colorectal carcinoma and their correlation with cancer progression and pathological aspects. TFF1 mutations were detected in colorectal carcinomas by DNA sequencing. TFF1 mRNA and protein levels in subsets of the primary tumors were determined using quantitative reverse transcription polymerase chain reaction and immunohistochemistry analyses. The serum level of TFF1 was also detected by enzyme-linked immunosorbent assay for patients with colorectal carcinoma. Five variants were detected in the 5'-untranslation region and intron 1 of TFF1. TFF1 expression was increased in colorectal carcinoma compared to paired distal colonic mucosa. Immunohistochemistry in primary colorectal carcinoma showed no significant differences in tumor TFF1 levels with respect to clinicopathological parameters such as the patient's sex, cancer differentiation, stage and lymph node metastasis. However, serum TFF1 levels were significantly elevated in patients with colorectal carcinoma compared to healthy individuals. The results indicate that TFF1 missense mutations seem to be a rare event in colorectal carcinogenesis. Serum TFF1 may be a potential useful marker for patients with colorectal carcinoma.

Nutrition and lipidomic profile in colorectal cancers.

Background: Adherence to a healthy diet has been reported to be essential for the primary prevention of colorectal cancer, through a reduction of tissue inflammation, a low concentration of circulating lipoproteins and lower levels of serum cholesterol. Since an altered expression of the fatty acids pattern has been demonstrated to be a crucial event in colorectal carcinogenesis, lipidomic analysis is considered able to identify early diagnostic and prognostic biomarkers of complex diseases such as colorectal cancer. Methods: cell membrane fatty acid profile and serum lipoproteins pattern were evaluated by gas chromatography and electrophoresis method respectively. Results: There is a close association between diet and lipidomic profile in colorectal cancer, both in pre-clinical and clinical studies. A modified serum lipoproteins pattern has been demonstrated to be predominant in intestinal tumors. Conclusions: The study of fatty acids profile in cell membrane and the evaluation of serum lipoproteins subfractions could be useful to have an integrate vision on the interactions between lipids and the pathogenesis of colorectal cancer and to understand the mechanisms of action and the consequences of these interactions on human health status. (www.actabiomedica.it)

Extract from Butea monosperma inhibits β-catenin/Tcf signaling in SW480 human colon cancer cells

The colorectal cancer (CRC) is the fourth leading cause of cancer death in worldwide. It has been found that >90% of CRC is caused by aberrant activation of canonical Wnt/β-catenin signaling pathway. Continuous activation of this pathway believes to be an initiating event in colorectal carcinogenesis. There are growing evidences suggest that traditional herbal plant medicines are being raised as a complementary alternative treatment for cancer. In this series, Butea (B) monosperma has been illustrated as a valuable traditional medicinal plant with >45 medicinal traits. Therefore, by targets this pathway using n-butanol fraction of B. monosperma flower extract (NBF-BMFE) could be a better therapeutic strategy for treating CRC. In this present study, we evaluate the inhibitory effect of NBF-BMFE against over activated Wnt signaling mediated colon cancer cells (SW480). Interestingly, the in vitro finding described that the NBF-BMFE had good antiproliferative effect against SW480 human colon cancer cells. Moreover, it showed significant level of down regulated expression in Wnt signaling proteins such as β-catenin, adenomatous polyposis coli (APC), glycogen synthase kinase 3β (GSK-3β), cyclin D1 and c-myc in time-dependent manner. Further, the in silico results of NBF-BMFE derived compounds have shown good binding interaction with target sites of β-catenin, APC and GSK-3β protein. In conclusion, NBF-BMFE may be used as an effective inhibitor for Wnt signaling targeted combined chemotherapeutic agents against CRC.

Abstract 2925: Distribution of copy number alterations defines clonal populations involved in colorectal cancer evolution

Abstract Colorectal cancer progresses in a multi-step manner with adenoma being the most well-known precursor lesion. Malignant polyp, an adenoma that contains a focus of adenocarcinoma, is a suitable model to study the colorectal tumor evolution. Although the mutations that lead adenoma to evolve into carcinoma have been previously described, the copy number changes involved in this malignant transformation have not been fully explored. To understand how these genomic alterations contribute to carcinogenesis, we used sequential fluorescence in situ hybridization using probes for the oncogenes EGFR, MYC, CDX2, and ZNF217 and the tumor suppressor genes SMAD7 and APC in order to analyze the copy number changes in 23 cases of malignant polyps and 10 cases of low grade dysplasia (LGD) adenomas based on single cell analyses. We found the levels of genomic heterogeneity increased from LGD adenomas to malignant polyps, with the adenoma component having a lower degree of chromosomal instability than the adenocarcinoma component. Despite the intercellular heterogeneity, we observed different patterns of evolution. The gain of ZNF217 was the earliest event as it was found to be in some cases of the LGD adenoma, and was also important in the neoplastic transformation of malignant polyps. Other notable genomic imbalances we observed during the malignant transformation were the gain of CDX2 and the loss of SMAD7. Interestingly, in a significant proportion of cases we observed the gain of all loci analyzed to be the decisive step in the transition from adenoma to carcinoma, suggesting a whole genome duplication event. Lastly, in half of the malignant polyps studied the main clone in the carcinoma component was already present in the adjacent adenoma component, although in some cases at a low frequency only detected by single cell analysis. Our data suggest that copy number changes are early events in colorectal carcinogenesis that can determine the evolution from adenoma to carcinoma. Citation Format: Isabel Quintanilla, Elena Asensio, Maria Pellisé, Antoni Castells, Miriam Cuatrecasas, Jordi Camps. Distribution of copy number alterations defines clonal populations involved in colorectal cancer evolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2925. doi:10.1158/1538-7445.AM2017-2925

P-239 - Prognostic role of TRAP1 protein network in human metastatic colorectal carcinoma

Introduction: Background. Colorectal cancer (CRC) is a molecularly heterogeneous disease with high mortality rate. Since drug resistance is the limiting factor for successful treatment of patients in metastatic setting, the identification of prognostic and predictive factors for therapy selection is a major clinical need. We previously characterized the biological relevance of TRAP1, a molecular chaperone belonging to HSP90 family, upregulated in 50-60% of human colorectal carcinomas (CRCs). In particular, TRAP1 is involved in control of several tumor cellular functions as intracellular signaling, cell cycle, apoptosis, stemness and bioenergetics through co-translational regulation of a network of client proteins (i.e., F1ATPase, TBP7, IF2α, EF1G, IF4A, IF4E, EF1A, BRAF, AKT, 18kDa Sorcin, CDK1/2, MAD2, βCatenin). Nevertheless, the prognostic significance of TRAP1 in CRC is still unclear and needs further investigation. Thus, the clinical and biological significance of TRAP1 and its protein network was analyzed in human CRCs. Methods: Materials and Methods. The expression levels of TRAP1 and/or its client proteins were quantified by immunoblot analysis in 60 surgical specimens of CRCs at different stages and in adjacent noncancerous mucosas and, by immunohistochemistry, in 9 colorectal adenomatous polyps, 11 in situ carcinomas and 55 metastatic CRCs (mCRC). The association between TRAP1 protein network and clinical and pathological characteristics were analyzed. Results: Results. TRAP1 upregulation represents an early event in colorectal carcinogenesis, being increased at the transition between low- and high-grade adenomas, in in situ carcinomas and in about 60% CRCs. Indeed, a significant association was observed between TRAP1 protein level and its gene copy number in a subgroup of colorectal tumors (data from the TCGA database). Furthermore, TRAP1 was co-upregulated with its network of client proteins (i.e., TBP7, p-IF2α, IF2α, eF1G, iF4A, IF4E, eF1A, BRAF, CDK1/2, MAD2 and βCatenin) in 60 human CRCs. Indeed, Spearman Rank correlation test showed a significant co-expression between TRAP1 and several client proteins, independently from tumor grade and stage. Noteworthy, the hierarchical cluster analysis of mCRCs identified a cohort of tumors with upregulation of a 7-protein TRAP1 signature (i.e., TRAP1, IF2α, eF1A, TBP7, MAD2, CDK1 and βCatenin) and a significantly shorter overall survival (HR 5.4; 95% C.I. 1.1-26.6; p = 0.037). Consistently, TRAP1 prognostic value was confirmed in a validation cohort of 55 metastatic colorectal tumors. Finally, TRAP1 positive expression and its prognostic value were more evident in left colon cancers. Conclusion: This study provides the first comprehensive proteomic evaluation of TRAP1 protein network in colorectal tumors, showing the clinical relevance of its activation as a mechanism to coordinate a number of signaling pathway responsible for tumor progression and clinical aggressiveness.

Determinants of metastatic competency in colorectal cancer.

Colorectal cancer (CRC) is one of the most common cancer types and represents a major therapeutic challenge. Although initial events in colorectal carcinogenesis are relatively well characterized and treatment for early‐stage disease has significantly improved over the last decades, the mechanisms underlying metastasis – the main cause of death – remain poorly understood. Correspondingly, no effective therapy is currently available for advanced or metastatic disease. There is increasing evidence that colorectal cancer is hierarchically organized and sustained by cancer stem cells, in concert with various stromal cell types. Here, we review the interplay between cancer stem cells and their microenvironment in promoting metastasis and discuss recent insights relating to both patient prognosis and novel targeted treatment strategies. A better understanding of these topics may aid the prevention or reduction of metastatic burden.

D18 - “TRAP1 7-protein signature” predicts clinical outcome in human metastatic colorectal carcinoma

Background: TRAP1 is a member of the HSP90 molecular chaperone family, upregulated in 50-60% of human colorectal carcinomas (CRCs) and a key regulator of several tumor cellular functions through co-traslational regulation of its client proteins (i.e., F1ATPase, TBP7, IF2&agr;, EF1G, IF4A, IF4E, EF1A, BRAF, AKT, 18kDa Sorcin, CDK1/2, MAD2, &bgr;-catenin). Indeed, TRAP1 is involved in control of intracellular signaling, cell cycle, apoptosis, stemness and bioenergetics. Thus, the clinical and biological significance of TRAP1 and its protein network was analyzed in human CRCs. Materials and methods: TRAP1 and its client proteins were quantified by immunoblot and densitometric analysis in 60 surgical specimens of CRCs at different stages, in adjacent noncancerous mucosas and, by immunohistochemistry, in 10 colorectal adenomatous polyps. The association between TRAP1 protein network and clinical and pathological characteristics were analyzed. Results: TRAP1 upregulation represents an early event in colorectal carcinogenesis, being increased in transformed adenomas. Indeed, a direct correlation was observed between TRAP1 mRNA and gene copy number by analyzing the TCGA database. Interestingly, Spearman Rank correlation test showed a significant co-expression between TRAP1 and several client proteins (i.e., TBP7, p-IF2&agr;, IF2&agr;, eF1G, iF4A, IF4E, eF1A, BRAF, CDK1/2, MAD2 and &bgr;-catenin) in our series, independently from tumor grade and stage. In particular, hierarchical cluster analysis of metastatic CRCs identified a 7-protein signature (i.e., TRAP1, IF2&agr;, eF1A, TBP7, MAD2, CDK1/2 and &bgr;-catenin) which is associated with an unfavorable overall survival, independently from major clinical covariates (Kaplan Meier survival analysis, HR 4.5; p= 0.04). Conclusions: We identified a distinct and prognostically relevant “7-protein TRAP1 signature” in human metastatic CRC.

Downregulation of the Deiminase PADI2 Is an Early Event in Colorectal Carcinogenesis and Indicates Poor Prognosis.

Peptidyl arginine deiminases (PADI) are a family of enzymes that catalyze the poorly understood posttranslational modification converting arginine residues into citrullines. In this study, the role of PADIs in the pathogenesis of colorectal cancer was investigated. Specifically, RNA expression was analyzed and its association with survival in a cohort of 98 colorectal cancer patient specimens with matched adjacent mucosa and 50 controls from donors without cancer. Key results were validated in an independent collection of tumors with matched adjacent mucosa and by mining of a publicly available expression data set. Protein expression was analyzed by immunoblotting for cell lines or IHC for patient specimens that further included 24 cases of adenocarcinoma with adjacent dysplasia and 11 cases of active ulcerative colitis. The data indicate that PADI2 is the dominantly expressed PADI enzyme in colon mucosa and is upregulated during differentiation. PADI2 expression is low or absent in colorectal cancer. Frequently, this occurs already at the stage of low-grade dysplasia. Mucosal PADI2 expression is also low in ulcerative colitis. The expression level of PADI2 in tumor and adjacent mucosa correlates with differential survival: low levels associate with poor prognosis. Downregulation of PADI2 is an early event in the pathogenesis of colorectal cancer associated with poor prognosis and points toward a possible role of citrullination in modulating tumor cells and their microenvironment. Mol Cancer Res; 14(9); 841-8. ©2016 AACR.