Normal Colonic Mucosa Samples Research Articles

Panomics reveals patient individuality as the major driver of colorectal cancer progression

BackgroundColorectal cancer (CRC) is one of the most prevalent cancers, with over one million new cases per year. Overall, prognosis of CRC largely depends on the disease stage and metastatic status. As precision oncology for patients with CRC continues to improve, this study aimed to integrate genomic, transcriptomic, and proteomic analyses to identify significant differences in expression during CRC progression using a unique set of paired patient samples while considering tumour heterogeneity.MethodsWe analysed fresh-frozen tissue samples prepared under strict cryogenic conditions of matched healthy colon mucosa, colorectal carcinoma, and liver metastasis from the same patients. Somatic mutations of known cancer-related genes were analysed using Illumina's TruSeq Amplicon Cancer Panel; the transcriptome was assessed comprehensively using Clariom D microarrays. The global proteome was evaluated by liquid chromatography-coupled mass spectrometry (LC‒MS/MS) and validated by two-dimensional difference in-gel electrophoresis. Subsequent unsupervised principal component clustering, statistical comparisons, and gene set enrichment analyses were calculated based on differential expression results.ResultsAlthough panomics revealed low RNA and protein expression of CA1, CLCA1, MATN2, AHCYL2, and FCGBP in malignant tissues compared to healthy colon mucosa, no differentially expressed RNA or protein targets were detected between tumour and metastatic tissues. Subsequent intra-patient comparisons revealed highly specific expression differences (e.g., SRSF3, OLFM4, and CEACAM5) associated with patient-specific transcriptomes and proteomes.ConclusionOur research results highlight the importance of inter- and intra-tumour heterogeneity as well as individual, patient-paired evaluations for clinical studies. In addition to changes among groups reflecting CRC progression, we identified significant expression differences between normal colon mucosa, primary tumour, and liver metastasis samples from individuals, which might accelerate implementation of precision oncology in the future.

Abstract 477: Elevated EVL methylation level in the normal colon mucosa is a potential risk biomarker for developing metachronous polyps

Abstract The purpose of this study is to evaluate the use of molecular markers for more accurately predicting the individual risk of metachronous polyps or cancer during colonoscopic surveillance. Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related death in the United States. The risk of CRC varies among people; individuals who develop adenomas are at heightened risk of forming metachronous adenomas and interval cancer, compared to those without a personal history of adenoma or CRC. Yet, even in these high risk individuals, only 25-30% will develop subsequent adenomas. To date, few studies have evaluated the use of molecular markers for predicting the risk of metachronous lesions. We have demonstrated that aberrantly methylated mir342/EVL is present at a higher frequency in the normal colon of people with CRC compared to people with no colon cancer or polyps, suggesting it may indicate a field cancerization process. Further, we developed an ultra-sensitive quantitative Methylation-specific ddPCR (MS-ddPCR) assay to accurately determine the level of rare EVL methylation in the normal colon mucosa. To investigate the correlation of EVL methylation in normal mucosa and the risk of developing polyps during the follow-up colonoscopy, we conducted a retrospective cohort study (N=136 subjects) and EVL methylation status was determined in normal colon mucosa samples from these subjects. Among them, 71 subjects had polyps detected within 12 years of the index colonoscopy, and 11 subjects were polyp-free at all subsequent colonoscopic exams done within 8-16 years of the index colonoscopy (54 subjects did not meet the criteria for follow up, so were excluded from the following analysis). Applying the standard Inverse Probability Weighted Logistic Regression (IPWLR) model, we found that higher level of EVL methylation in the normal colon mucosa at the time of the index colonoscopy (which was when the tissue sample was collected) indicated an increased risk of having polyps detected within 12 years, compared to subjects with no polyps detected by colonoscopy done within 8-16 years (Odds Ratio (95% CI) = 4.35 (1.14, 16.60); p-value = 0.0317). Our results suggest that EVL methylation level in the normal colon mucosa can be used as a risk biomarker for metachronous adenomas. Citation Format: Ming Yu, Ting Wang, Kelly T. Carter, Kelsey Baker, Mary W. Redman, Kathy Vickers, Lynda Ann Dzubinski, Robert E. Schoen, William M. Grady. Elevated EVL methylation level in the normal colon mucosa is a potential risk biomarker for developing metachronous polyps [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 477.

DNA Methylation Signatures and the Contribution of Age-Associated Methylomic Drift to Carcinogenesis in Early-Onset Colorectal Cancer.

Simple SummaryThe role of DNA methylation in the carcinogenesis of colorectal cancer (CRC) diagnosed <50 years of age (early-onset CRC or EOCRC) is currently unknown. In this study, we investigated the genome-wide DNA methylation of 97 tumour and 54 normal colonic mucosa samples from people with EOCRC with the aim of identifying unique DNA methylation signatures and determining the role of ageing-related DNA methylation drift and age-acceleration in EOCRC aetiology. We found extensive DNA methylation alterations associated with EOCRC carcinogenesis, including a unique signature comprising 234 loci compared with CRCs from people >50 years of age. CpGs that undergo ageing-related methylation drift were significantly altered in EOCRC, and accelerated ageing was also evident in normal mucosa from people with EOCRC. Our study is the first study to identify unique DNA methylation changes in EOCRC, contributing novel information that may aid future efforts towards EOCRC prevention.We investigated aberrant DNA methylation (DNAm) changes and the contribution of ageing-associated methylomic drift and age acceleration to early-onset colorectal cancer (EOCRC) carcinogenesis. Genome-wide DNAm profiling using the Infinium HM450K on 97 EOCRC tumour and 54 normal colonic mucosa samples was compared with: (1) intermediate-onset CRC (IOCRC; diagnosed between 50–70 years; 343 tumour and 35 normal); and (2) late-onset CRC (LOCRC; >70 years; 318 tumour and 40 normal). CpGs associated with age-related methylation drift were identified using a public dataset of 231 normal mucosa samples from people without CRC. DNAm-age was estimated using epiTOC2. Common to all three age-of-onset groups, 88,385 (20% of all CpGs) CpGs were differentially methylated between tumour and normal mucosa. We identified 234 differentially methylated genes that were unique to the EOCRC group; 13 of these DMRs/genes were replicated in EOCRC compared with LOCRCs from TCGA. In normal mucosa from people without CRC, we identified 28,154 CpGs that undergo ageing-related DNAm drift, and of those, 65% were aberrantly methylated in EOCRC tumours. Based on the mitotic-based DNAm clock epiTOC2, we identified age acceleration in normal mucosa of people with EOCRC compared with normal mucosa from the IOCRC, LOCRC groups (p = 3.7 × 10−16) and young people without CRC (p = 5.8 × 10−6). EOCRC acquires unique DNAm alterations at 234 loci. CpGs associated with ageing-associated drift were widely affected in EOCRC without needing the decades-long accrual of DNAm drift as commonly seen in intermediate- and late-onset CRCs. Accelerated ageing in normal mucosa from people with EOCRC potentially underlies the earlier age of diagnosis in CRC carcinogenesis.

Nkx2.5 Functions as a Conditional Tumor Suppressor Gene in Colorectal Cancer Cells via Acting as a Transcriptional Coactivator in p53-Mediated p21 Expression.

NK2 homeobox 5 (Nkx2.5), a homeobox-containing transcription factor, is associated with a spectrum of congenital heart diseases. Recently, Nkx2.5 was also found to be differentially expressed in several kinds of tumors. In colorectal cancer (CRC) tissue and cells, hypermethylation of Nkx2.5 was observed. However, the roles of Nkx2.5 in CRC cells have not been fully elucidated. In the present study, we assessed the relationship between Nkx2.5 and CRC by analyzing the expression pattern of Nkx2.5 in CRC samples and the adjacent normal colonic mucosa (NCM) samples, as well as in CRC cell lines. We found higher expression of Nkx2.5 in CRC compared with NCM samples. CRC cell lines with poorer differentiation also had higher expression of Nkx2.5. Although this expression pattern makes Nkx2.5 seem like an oncogene, in vitro and in vivo tumor suppressive effects of Nkx2.5 were detected in HCT116 cells by establishing Nkx2.5-overexpressed CRC cells. However, Nkx2.5 overexpression was incapacitated in SW480 cells. To further assess the mechanism, different expression levels and mutational status of p53 were observed in HCT116 and SW480 cells. The expression of p21WAF1/CIP1, a downstream antitumor effector of p53, in CRC cells depends on both expression level and mutational status of p53. Overexpressed Nkx2.5 could elevate the expression of p21WAF1/CIP1 only in CRC cells with wild-type p53 (HCT116), rather than in CRC cells with mutated p53 (SW480). Mechanistically, Nkx2.5 could interact with p53 and increase the transcription of p21WAF1/CIP1 without affecting the expression of p53. In conclusion, our findings demonstrate that Nkx2.5 could act as a conditional tumor suppressor gene in CRC cells with respect to the mutational status of p53. The tumor suppressive effect of Nkx2.5 could be mediated by its role as a transcriptional coactivator in wild-type p53-mediated p21WAF1/CIP1 expression.

Evaluation of EZH2 and ERRα in colorectal carcinoma: an immunohistochemical study.

The combined immunohistochemical evaluation of EZH2 (enhancer of zeste homolog 2) and ERRα (estrogen-related receptor α), in relation to clinicopathological prognostic factors and patients' outcome, has not been performed yet in colorectal carcinoma (CRC). In order to achieve this aim, 120 samples were extracted; 60 cases of CRC; and 60 samples from normal colonic tissue. Our study showed that 63.3% and 38.3% of CRC cases reveal high EZH2 and high ERRα nuclear expression, respectively. 6.6% and 8.3% of normal colonic mucosa samples express low EZH2 and low ERRα nuclear expression, respectively. High EZH2 and high ERRα expression correlate with late tumor stages (p = 0.001 each), high grade (p = 0.001, p = 0.009 respectively), positive lymph node involvement (p = 0.001, p = 0.002 respectively) and larger tumor size (p = 0.001 each). There is a moderate highly statistically significant agreement (κ = 0.467, p = 0.001) between EZH2 and ERRα immunohistochemical expression. By Kaplan Meier analysis, high EZH2 and high ERRα show statistically significant shorter overall survival, and progression free survival than cases with low EZH2 and low ERRα immunohistochemical expression, respectively. Thus, EZH2 and ERRα might serve as potential promising prognostic markers in CRC.

Nuclear Expression Loss of SSBP2 Is Associated with Poor Prognostic Factors in Colorectal Adenocarcinoma.

Single-stranded DNA binding protein 2 (SSBP2) is involved in DNA damage response and may induce growth arrest in cancer cells, having a potent tumor suppressor role. SSBP2 is ubiquitously expressed and the loss of its expression has been reported in various tumor types. However, the correlation between SSBP2 expression and colorectal cancer (CRC) prognosis remains unclear. SSBP2 nuclear expression was evaluated immunohistochemically in 48 normal colonic mucosae, 47 adenomas, 391 primary adenocarcinomas, and 131 metastatic carcinoma tissue samples. The clinicopathological factors, overall survival (OS), and recurrence-free survival were evaluated, and associations with the clinicopathological parameters were analyzed in 391 colorectal adenocarcinoma patients. A diffuse nuclear SSBP2 expression was detected in all normal colonic mucosa and adenoma samples. SSBP2 expression loss was observed in 131 (34.3%) primary adenocarcinoma and 100 (76.3%) metastatic carcinoma samples. SSBP2 expression was significantly associated with poor prognostic factors, such as vascular invasion (p = 0.005), high pT category (p = 0.045), and shorter OS (p = 0.038), using univariate survival analysis. Nuclear SSBP2 expression loss was significantly observed in colorectal carcinoma and metastatic carcinoma tissues, being associated with poor prognostic factors. SSBP2 acts as a tumor suppressor and may be used as a CRC prognostic biomarker.

Retraction Note: MALAT2-activated long noncoding RNA indicates a biomarker of poor prognosis in gastric cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Dysfunctional epigenetic aging of the normal colon and colorectal cancer risk

BackgroundChronological age is a prominent risk factor for many types of cancers including colorectal cancer (CRC). Yet, the risk of CRC varies substantially between individuals, even within the same age group, which may reflect heterogeneity in biological tissue aging between people. Epigenetic clocks based on DNA methylation are a useful measure of the biological aging process with the potential to serve as a biomarker of an individual’s susceptibility to age-related diseases such as CRC.MethodsWe conducted a genome-wide DNA methylation study on samples of normal colon mucosa (N = 334). Subjects were assigned to three cancer risk groups (low, medium, and high) based on their personal adenoma or cancer history. Using previously established epigenetic clocks (Hannum, Horvath, PhenoAge, and EpiTOC), we estimated the biological age of each sample and assessed for epigenetic age acceleration in the samples by regressing the estimated biological age on the individual’s chronological age. We compared the epigenetic age acceleration between different risk groups using a multivariate linear regression model with the adjustment for gender and cell-type fractions for each epigenetic clock. An epigenome-wide association study (EWAS) was performed to identify differential methylation changes associated with CRC risk.ResultsEach epigenetic clock was significantly correlated with the chronological age of the subjects, and the Horvath clock exhibited the strongest correlation in all risk groups (r > 0.8, p < 1 × 10−30). The PhenoAge clock (p = 0.0012) revealed epigenetic age deceleration in the high-risk group compared to the low-risk group.ConclusionsAmong the four DNA methylation-based measures of biological age, the Horvath clock is the most accurate for estimating the chronological age of individuals. Individuals with a high risk for CRC have epigenetic age deceleration in their normal colons measured by the PhenoAge clock, which may reflect a dysfunctional epigenetic aging process.

Proteome analysis of formalin-fixed paraffin-embedded colorectal adenomas reveals the heterogeneous nature of traditional serrated adenomas compared to other colorectal adenomas.

Traditional serrated adenoma (TSA) remains the least understood of all the colorectal adenomas, although these lesions have been associated with a significant cancer risk, twice that of the conventional adenoma (CAD) and of the sessile serrated adenoma (SSA/P). This study was performed to investigate the proteomic profiles of the different colorectal adenomas to better understand the pathogenesis of TSA. We performed a global quantitative proteome analysis using the label-free quantification (LFQ) method on 44 colorectal adenoma (12 TSAs, 15 CADs, and 17 SSA/Ps) and 17 normal colonic mucosa samples, archived as formalin-fixed paraffin-embedded blocks. Unsupervised consensus hierarchical clustering applied to the whole proteomic profile of the 44 colorectal adenomas identified four subtypes: C1 and C2 were well-individualized clusters composed of all the CADs (15/15) and most of the SSA/Ps (13/17), respectively. This is consistent with the fact that CADs and SSA/Ps are homogeneous and distinct colorectal adenoma entities. In contrast, TSAs were subdivided into C3 and C4 clusters, consistent with the more heterogeneous entity of TSA at the morphologic and molecular levels. Comparison of the proteome expression profile between the adenoma subtypes and normal colonic mucosa further confirmed the heterogeneous nature of TSAs, which overlapped either on CADs or SSA/Ps, whereas CADs and SSAs formed homogeneous and distinct entities. Furthermore, we identified LEFTY1 a new potential marker for TSAs that may be relevant for the pathogenesis of TSA. LEFTY1 is an inhibitor of the Nodal/TGFβ pathway, which we found to be one of the most overexpressed proteins specifically in TSAs. This finding was confirmed by immunohistochemistry. Our study confirms that CADs and SSA/Ps form homogeneous and distinct colorectal adenoma entities, whereas TSAs are a heterogeneous entity and may arise from either SSA/Ps or from normal mucosa evolving through a process related to the CAD pathway. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Diabetes-mediated promotion of colon mucosa carcinogenesis is associated with mitochondrial dysfunction.

Type 2 diabetes mellitus (T2DM) has been associated with an increased risk of cancer, including colon cancer (CC). However, we recently reported no influence of T2DM on CC prognosis, suggesting that any effect might be at the early stages of tumor development. We hypothesized that T2DM may create an environment in the healthy tissue, which acts as a carcinogenesis driver in agreement with the field of cancerization concept. Here, we focused on early carcinogenesis by analyzing paired tumor and normal colonic mucosa samples from the same patients. The proteome of CC and paired mucosa was quantitatively analyzed in 28 individuals (12 diabetics and 16 nondiabetics) by mass spectrometry with isobaric labeling. Out of 3076 identified proteins, 425 were differentially expressed at the tumor in diabetics compared with nondiabetics. In the adjacent mucosa, 143 proteins were differentially expressed in diabetics and nondiabetics. An enrichment analysis of this signature pointed to mitochondria, ribosome, and translation. Only six proteins were upregulated by diabetes both in tumor and mucosa, of which five were mitochondrial proteins. Differential expression in diabetic versus nondiabetic mucosa was confirmed for MRPL53, MRPL18, and TIMM8B. Higher levels of MRPL18, TIMM8B, and EIF1A were also found in normal colon epithelial cells exposed to high‐glucose conditions. We conclude that T2DM is associated with specific molecular changes in the normal mucosa of CC patients, consistent with field of cancerization in a diabetic environment. The mitochondrial protein signature identifies a potential therapeutic target that could underlie the higher risk of CC in diabetics.

The expression of brown fat-associated proteins in colorectal cancer and the relationship of uncoupling protein 1 with prognosis.

Colorectal carcinoma is one of the most common types of malignancy and a leading cause of cancer related death. The aberrant expression of a brown fat-like phenotype in cancer cells has been previously implicated in tumour growth. Therefore, the expression of brown fat-associated proteins in colorectal cancer could be associated with tumour prognosis. Monoclonal antibodies to brown fat-associated proteins CIDEA, ELOVL3, ELOVL5, and UCP1 were developed. The antibodies were used to profile the expression of protein targets by immunohistochemistry in a discovery cohort comprising 50 normal colonic mucosa samples and 274 primary colorectal cancers and a validation cohort comprising 549 colorectal cancers. Immunostaining for UCP1 was observed in the majority of colorectal tumours while no immunostaining was observed in normal colonic mucosa (p < 0.001). The expression of UCP1 was significantly associated with better overall survival in both the discovery cohort (HR = 0.615, 95%CI = 0.416-0.909, χ2 = 6.119, p = 0.013) and the validation cohort (HR = 0.629, 95%CI = 0.480-0.825, χ2 = 11.558, p = 0.001). Furthermore, UCP1 was independently prognostic in multivariate analysis (p = 0.004). This study has identified the brown fat-like phenotype as a novel pathway associated with survival in colorectal cancer. The expression of UCP1 was identified as a significant prognostic biomarker for colorectal cancer.

Molecular evidence of field cancerization initiated by diabetes in colon cancer patients.

The potential involvement of type 2 diabetes mellitus (T2DM) as a risk factor for colon cancer (CC) has been previously reported. While several clinical studies show a higher incidence of CC and a lower survival rate in diabetics, others report no association. Our own experience indicates that diabetes does not seem to worsen the prognosis once the tumor is present. Despite this controversy, there are no wide‐spectrum molecular studies that delve into the impact of T2DM‐related mechanisms in colon carcinogenesis. Here, we present a transcriptomic and proteomic profiling of paired tumor and normal colon mucosa samples in a cohort of 42 CC patients, 23 of which have T2DM. We used gene set enrichment and network approaches to extract relevant pathways in diabetics, referenced them to current knowledge, and tested them using in vitro techniques. Through our transcriptomics approach, we identified an unexpected overlap of pathways overrepresented in diabetics compared to nondiabetics, in both tumor and normal mucosa, including diabetes‐related metabolic and signaling processes. Proteomic approaches highlighted several cancer‐related signaling routes in diabetics found only in normal mucosa, not in tumors. An integration of the transcriptome and proteome analyses suggested the deregulation of key pathways related to colon carcinogenesis which converged on tumor initiation axis TEAD/YAP‐TAZ as a potential initiator of the process. In vitro studies confirmed upregulation of this pathway in nontumor colon cells under high‐glucose conditions. In conclusion, T2DM associates with deregulation of cancer‐related processes in normal colon mucosa adjacent to tissue which has undergone a malignant transformation. These data support that in diabetic patients, the local microenvironment in normal colon mucosa may be a factor driving field cancerization promoting carcinogenesis. Our results set a new framework to study links between diabetes and colon cancer, including a new role of the TEAD/YAP‐TAZ complex as a potential driver.

PO-127 Role of the NEDD4 family of E3 ubiquitin ligases in colorectal cancer pathogenesis and their potential as biomarkers

IntroductionColorectal cancer (CRC) is a global health challenge. There is limited response to standard oncological treatment and few stratified treatment options based on prognostic and/or predictive factors. The four biologically distinct and gene expression-based consensus molecular subtypes (CMS) provide prognostic stratification independent of cancer stage, and represent a new potential paradigm for stratified treatment. In this project, we are analysing the biomarker and drug target potential of members of the HECT (homologous to E6AP C-terminus) family of E3 ubiquitin ligases in CRC.Material and methodsA total of 412 primary CRCs and 51 normal colonic mucosa samples were subjected to genome wide expression analysis at exon level resolution using either the Affymetrix Human Exon Array or the Affymetrix Human Transcriptome Array. The CRISPR/Cas9 system was applied to generate NEDD4 knock-out Caco2 cell lines.Results and discussionsWe identified NEDD4 (neural precursor cell-expressed developmentally down-regulated 4) as significantly overexpressed in CRC, while the NEDD4 homolog NEDD4L was significantly down regulated. Furthermore, we found that NEDD4 is differentially expressed between the different CMS classes, with high expression particularly in the epithelial and canonical CMS2 group. CRISPR/Cas9-mediated knock-out of NEDD4 in the CRC cell line Caco2 was found to result in altered growth characteristics.ConclusionThe data indicate that NEDD4 and NEDD4L are differently expressed in CRC samples compared to normal colonic mucosa, and that knock-out of NEDD4 in Caco2 cells affects their growth characteristics.

Expression of Zinc Finger and BTB Domain-containing 7A in Colorectal Carcinoma.

Previous studies have revealed that zinc finger and BTB domain-containing 7A (ZBTB7A), an important proto-oncogene, plays multiple roles in carcinogenesis and is up-regulated in several human malignancies. However, the expression of ZBTB7A in colorectal carcinoma (CRC) has seldom been documented. In this study, we investigated the differential expression of ZBTB7A in CRC cell lines and tissues. Expression levels of ZBTB7A mRNA and protein were examined in CRC cell lines. ZBTB7A protein expression was also evaluated in tissue samples of normal colonic mucosa, high-grade dysplasia, and CRC using immunohistochemical staining. All CRC cell lines exhibited significantly higher ZBTB7A mRNA expression levels than did normal colonic epithelial cells. The ZBTB7A protein expression levels were clearly higher in the CRC cell lines than in the normal colonic epithelial cells. Consistent with the cell line data, immunostaining revealed that there were significant differences in ZBTB7A protein expression between tissue samples of CRC and normal colonic mucosa (p=0.048) and high-grade dysplasia (p=0.015). In addition, metastatic CRC exhibited significantly higher ZBTB7A protein expression levels than primary CRC (p=0.027). We demonstrated that ZBTB7A expression is up-regulated in CRC cell lines and tissues. Our data suggest that ZBTB7A is involved in the development and progression of CRC.

Downregulation of phosphorylated MKK4 is associated with a poor prognosis in colorectal cancer patients.

Mitogen-activated protein kinase kinase 4 (MKK4) is a key mediator of Jun N-terminal kinase signaling and influences malignant metastasis. Here, we used immunohistochemistry to assess phosphorylated MMK4 (pMKK4) levels and examine their association with the clinicopathological features of a pilot set of patient samples consisting of normal colonic mucosa (NCM), colorectal adenoma (CA), and colorectal cancer (CRC) tissues. pMKK4 levels were also assessed in a validation set of CRC cases with accompanying follow-up data to confirm their clinicopathological and prognostic significance. pMKK4 levels, which were high in 79.17% of NCM samples, were downregulated in 33.33% of CA and 63.54% of CRC samples. pMKK4 downregulation was associated with metastasis, especially to the liver. In the validation set, pMKK4 downregulation was associated with increases in invasive depth, lymph node metastasis, distant metastasis, and TNM stage. Univariate analysis indicated that pMKK4 score, tumor differentiation, and TNM stage were correlated with disease-free survival and overall survival. Multivariate analysis indicated that decreased pMKK4 expression was an independent risk factor for disease-free survival in CRC patients. These results suggest that CRC patients with low pMKK4 immunochemistry scores should be monitored carefully for early detection of possible recurrences, especially liver metastasis.

Targeted Proteomics for Multiplexed Verification of Markers of Colorectal Tumorigenesis

Targeted proteomic methods can accelerate the verification of multiple tumor marker candidates in large series of patient samples. We utilized the targeted approach known as selected/multiple reaction monitoring (S/MRM) to verify potential protein markers of colorectal adenoma identified by our group in previous transcriptomic and quantitative shotgun proteomic studies of a large cohort of precancerous colorectal lesions. We developed SRM assays to reproducibly detect and quantify 25 (62.5%) of the 40 selected proteins in an independent series of precancerous and cancerous tissue samples (19 adenoma/normal mucosa pairs; 17 adenocarcinoma/normal mucosa pairs). Twenty-three proteins were significantly up-regulated (n = 17) or downregulated (n = 6) in adenomas and/or adenocarcinomas, as compared with normal mucosa (linear fold changes ≥ ±1.3, adjusted p value <0.05). Most changes were observed in both tumor types (up-regulation of ANP32A, ANXA3, SORD, LDHA, LCN2, NCL, S100A11, SERPINB5, CDV3, OLFM4, and REG4; downregulation of ARF6 and PGM5), and a five-protein biomarker signature distinguished neoplastic tissue from normal mucosa with a maximum area under the receiver operating curve greater than 0.83. Other changes were specific for adenomas (PPA1 and PPA2 up-regulation; KCTD12 downregulation) or adenocarcinoma (ANP32B, G6PD, RCN1, and SET up-regulation; downregulated AKR1B1, APEX1, and PPA1). Some changes significantly correlated with a few patient- or tumor-related phenotypes. Twenty-two (96%) of the 23 proteins have a potential to be released from the tumors into the bloodstream, and their detectability in plasma has been previously reported. The proteins identified in this study expand the pool of biomarker candidates that can be used to develop a standardized precolonoscopy blood test for the early detection of colorectal tumors.

Human Colorectal Cancer Stage-dependent Global DNA Hypomethylation of Cancer-associated Fibroblasts.

Cancer-associated fibroblasts (CAFs) play an important role in tumor development and progression. The prevailing consensus favors the view that a specific epigenetic signature underpins the stable CAF phenotype. The aim of the present study was to assess global DNA methylation in CAFs during the adenoma-carcinoma sequence in non-familial sporadic human colorectal cancer (CRC). Immunohistochemical staining of nuclear 5-methylcytosine (5'-meCyt) was performed in matched samples of colonic tumor tissue and normal colonic mucosa excised from six patients with adenomas and four with adenocarcinomas. The staining intensity was expressed semi-quantitatively as the immunohistochemical staining score (ISS). ISS values of human colonic CAFs and adenomatous samples were 14.00±2.2 and 14.08±1.8, respectively, showing no statistically significant difference. In contrast, a marked trend was found towards global DNA hypomethylation in CAFs from adenocarcinomatous specimens compared to matched normal mucosa: ISS: 9.25±2.44 (range=6-11) vs. 16.17±0.75, respectively, p<0.03. Final stages of cancer development in CRC are associated with global DNA hypomethylation in stromal CAFs.

Prognostic value of 5-microRNA based signature in T2-T3N0 colon cancer.

The role of adjuvant chemotherapy in stage T2-T3N0 colon cancer (CC) is controversial and there are currently no reliable factors allowing for individual selection of patients with high risk of relapse for such therapy. We searched for microRNA-based signature with prognostic significance in this group. We assessed by qRT-PCR expression of 754 microRNAs (miRNAs) in tumour samples from 85 stage pT2-3N0 CC patients treated with surgery alone. MiRNA expression was compared between two groups of patients: 40 and 45 patients who did and did not develop distant metastases after resection, respectively. Additionally, miRNA expression was compared between CC and normal colon mucosa samples and between the mismatch repair (MMR) competent and deficient tumours. Low expression of miR-1300 and miR-939 was significantly correlated with shorter distant metastasis-free survival (DMFS) in Cox univariate analysis (p.adjusted = 0.049). The expression signature of five miRNAs (miR-1296, miR-135b, miR-539, miR-572 and miR-185) was found to be prognostic [p = 1.28E−07, HR 8.4 (95 % CI: 3.81–18.52)] for DMFS and cross-validated in a leave-one-out analysis, with the sensitivity and specificity of 74 and 78 %, respectively. The expression of miR-592 was significantly associated with the MMR status (p.adjusted <0.01). The expression of several novel miRNAs were found to be tumour specific, e.g. miR-888, miR-523, miR-18b, miR-302a, miR-423-5p, miR-582-3p (p < 0.05). We developed a miRNA expression signature that may be predictive for the risk of distant relapse in early stage CC and confirmed previously reported association between miR-592 expression and MMR status.Electronic supplementary materialThe online version of this article (doi:10.1007/s10585-016-9810-1) contains supplementary material, which is available to authorized users.

Low expression of PKCα and high expression of KRAS predict poor prognosis in patients with colorectal cancer.

The current study aimed to determine the association between protein kinase Cα (PKCα) and Kirsten rat sarcoma viral oncogene homolog (KRAS) expression and the response to folinic acid, 5-fluorouracil and oxaliplatin (FOLFOX regimen) in patients with colorectal cancer (CRC). The protein levels of PKCα and KRAS were analyzed by immunohistochemistry in tissue samples from patients with CRC and in non-cancerous tissues, including 152 cases of colorectal adenocarcinoma, 30 cases of colorectal adenoma and 20 normal colonic mucosa samples. The association between PKCα and KRAS expression and clinicopathological features was analyzed. The rates of positive PKCα protein expression in patients with poorly, moderately and well-differentiated adenocarcinoma were 16.7% (6/36), 40.0% (24/60), and 57.1% (32/56), respectively (P<0.013). The rate of positive KRAS expression in CRC patients was significantly higher than in patients with colon adenoma and normal colon mucosa (P<0.001). Expression levels of KRAS were associated with the degree of differentiation of CRC (P<0.001). Expression of PKCα was negatively correlated with KRAS expression in CRC tissues. The mean progression-free survival (PFS) times in patients with high and low expression of PKCα were 43.9 and 38.8 months, respectively (P<0.001). The mean PFS times were 38.5 and 45.5 months in patients with high and low expression of KRAS, respectively (P=0.001). In conclusion, low PKCα and high KRAS expression predicted relatively poor prognosis in patients with CRC.

SNHG16 is regulated by the Wnt pathway in colorectal cancer and affects genes involved in lipid metabolism

It is well established that lncRNAs are aberrantly expressed in cancer where they have been shown to act as oncogenes or tumor suppressors. RNA profiling of 314 colorectal adenomas/adenocarcinomas and 292 adjacent normal colon mucosa samples using RNA-sequencing demonstrated that the snoRNA host gene 16 (SNHG16) is significantly up-regulated in adenomas and all stages of CRC. SNHG16 expression was positively correlated to the expression of Wnt-regulated transcription factors, including ASCL2, ETS2, and c-Myc. In vitro abrogation of Wnt signaling in CRC cells reduced the expression of SNHG16 indicating that SNHG16 is regulated by the Wnt pathway. Silencing of SNHG16 resulted in reduced viability, increased apoptotic cell death and impaired cell migration. The SNHG16 silencing particularly affected expression of genes involved in lipid metabolism. A connection between SNHG16 and genes involved in lipid metabolism was also observed in clinical tumors. Argonaute CrossLinking and ImmunoPrecipitation (AGO-CLIP) demonstrated that SNHG16 heavily binds AGO and has 27 AGO/miRNA target sites along its length, indicating that SNHG16 may act as a competing endogenous RNA (ceRNA) “sponging” miRNAs off their cognate targets. Most interestingly, half of the miRNA families with high confidence targets on SNHG16 also target the 3′UTR of Stearoyl-CoA Desaturase (SCD). SCD is involved in lipid metabolism and is down-regulated upon SNHG16 silencing. In conclusion, up-regulation of SNHG16 is a frequent event in CRC, likely caused by deregulated Wnt signaling. In vitro analyses demonstrate that SNHG16 may play an oncogenic role in CRC and that it affects genes involved in lipid metabolism, possible through ceRNA related mechanisms.