Adjacent Normal Colonic Mucosa Research Articles

Novel method of image analysis that combines Radon and Fourier image transformations for the purpose of differentiating between malignant and benign colonic biopsies.

Colorectal cancer is the third most common type of cancer. It develops slowly as a polyp that can turn into a cancerous tumor. This study aimed to develop a decision-making algorithm of microscopic images using texture analysis that is orientation free, to be used for automated classification of normal and neoplastic (malignant or premalignant) colonic biopsies. Forty-nine colonic adenocarcinomas, 41 adenomas and a control group of adjacent normal colonic mucosa were included in the texture analysis. Radon transform followed by the Fast Fourier transform were applied to the images. Subsequently, the gray level co-occurrence matrix (GLCM) transform was applied allowing the extraction of four textural variables (homogeneity, contrast, correlation, and entropy). For classification and prediction of the diagnosis, a statistical multivariate regression model and a neural network (NNET) model were used and compared. The statistical model provided a sensitivity of 71.3% and a specificity of 50% (Area under the ROC curve: 0.67) for classifying the neoplastic and the normal images, respectively. The NNET model was superior to the statistical model and produced a sensitivity of 97.9% and specificity of 88% (Area under the ROC curve: 0.92). To our knowledge, this is the first study that used a combination of Radon, FFT, and GLCM transformations in order to overcome the tissue orientation problem in texture analysis of microscopic images of colonic biopsies. The NNET classifier trained by the extracted textural features proved to be superior to the statistical classifier, thus predicting colonic neoplasia with high accuracy. RESEARCH HIGHLIGHTS: We propose a novel decision-making algorithm of orientation invariant image texture analysis, fast and easily implemented for automated differentiation between benign and neoplastic epithelial tumors of the colon. This method can reduce the turnaround time allowing to prioritize the biopsies during their examination and diagnosis by the pathologist.

Fueling the flames of colon cancer - does CRP play a direct pro-inflammatory role?

Systemic inflammation, diagnostically ascribed by measuring serum levels of the acute phase reactant C-reactive protein (CRP), has consistently been correlated with poor outcomes across cancer types. CRP exists in two structurally and functionally distinct isoforms, circulating pentameric CRP (pCRP) and the highly pro-inflammatory monomeric isoform (mCRP). The aim of this pilot study was to map the pattern of mCRP distribution in a previously immunologically well-defined colon cancer (CC) cohort and explore possible functional roles of mCRP within the tumor microenvironment (TME). Formalin-fixed, paraffin-embedded (FFPE) tissue samples from 43 stage II and III CC patients, including 20 patients with serum CRP 0-1 mg/L and 23 patients with serum CRP >30 mg/L were immunohistochemically (IHC) stained with a conformation-specific mCRP antibody and selected immune and stromal markers. A digital analysis algorithm was developed for evaluating mCRP distribution within the primary tumors and adjacent normal colon mucosa. mCRP was abundantly present within tumors from patients with high serum CRP (>30 mg/L) diagnostically interpreted as being systemically inflamed, whereas patients with CRP 0-1 mg/L exhibited only modest mCRP positivity (median mCRP per area 5.07‰ (95%CI:1.32-6.85) vs. 0.02‰ (95%CI:0.01-0.04), p<0.001). Similarly, tissue-expressed mCRP correlated strongly with circulating pCRP (Spearman correlation 0.81, p<0.001). Importantly, mCRP was detected exclusively within tumors, whereas adjacent normal colon mucosa showed no mCRP expression. Double IHC staining revealed colocalization of mCRP with endothelial cells and neutrophils. Intriguingly, some tumor cells also colocalized with mCRP, suggesting a direct interaction or mCRP expression by the tumor itself. Our data show that the pro-inflammatory mCRP isoform is expressed in the TME of CC, primarily in patients with high systemic pCRP values. This strengthens the hypothesis that CRP might not only be an inflammatory marker but also an active mediator within tumors.

Abstract LB126: Functional limitations in respiratory complex I underlie mitochondrial bioenergetics in human colorectal adenocarcinoma

Abstract Typified by oxidative phosphorylation (OXPHOS), mitochondria catalyze a wide variety of cellular processes now understood to be critical for malignant growth. As such, there is considerable interest in targeting mitochondrial OXPHOS in cancer. The present project was based on the premise that the development of efficacious mitochondrial-targeted anti-cancer compounds requires answering two fundamental questions: 1) is mitochondrial bioenergetics in fact different between cancer and non-cancer cells? and 2) If so, what are the underlying mechanisms? To address these questions, we developed a methodological workflow designed to integrate in vitro functional readouts of mitochondrial flux with mass-spectrometry-based proteomics. This allowed us to directly quantitate both total respiratory capacity and OXPHOS kinetics on a per mitochondrion basis. Experiments were performed in isolated mitochondria prepared from freshly resected human colorectal adenocarcinomas (COAD). Relative to adjacent normal colonic mucosa, tumor mitochondria consistently presented with partial disruptions in respiratory complex I (CI). Surprisingly, CI functional limitations did not impair absolute oxidative flux. Instead, the tumor mitochondrial network displayed clear evidence of intrinsic remodeling. Such remodeling maintained respiratory competence at the expense of decreased oxidative ATP synthesis (i.e., OXPHOS inefficiency or increased ‘uncoupling’). Given recent findings demonstrating that mitochondrial OXPHOS is dispensable for tumor growth, our data raise the intriguing possibility that the requirement for mitochondria in COAD may have little to do with oxidative ATP production, but instead reflect a requirement for accelerated mitochondrial flux to support other aspects of anabolic growth (e.g., extracellular nutrient uptake, NAD+ regeneration, nucleotide synthesis). In this respect, rather than functioning as energy producers, COAD mitochondria may primarily function as ‘energy sinks’. Citation Format: Ilya Boykov, McLane M. Montgomery, James T. Hagen, Hannah S. Coalson, Kelsey H. Fisher-Wellman. Functional limitations in respiratory complex I underlie mitochondrial bioenergetics in human colorectal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB126.

Investigation of miRNA dysregulation and association with immune cell profile during malignant transformation of colorectal cells

BackgroundColorectal cancer (CRC) is one of the most prevalent and life-threatening cancer among the world. Accumulated somatic mutations during malignant transformation process endow cancer cells with increased growth, invasiveness and immunogenicity. These highly immunogenic cancer cells develop multiple strategies to evade immune attack. Through post-transcriptional regulation, microRNAs (miRNAs) not only participate in cancer development and progression but also manipulate anti-cancer immune response. This study aims to identify miRNAs associated with the colorectal cell malignant transformation process and their association with immune cell population using synchronous adjacent normal, polyp and CRC specimens. MethodsWe conducted a Low Density Array to compare the miRNA expression profile of synchronous colorectal adenoma, adenocarcinoma and adjacent normal colon mucosa collected from 8 patients, in order to identify candidate miRNAs involved in CRC progression. These findings were further validated in 14 additional patients and GEO dataset GSE41655. The relative abundance of dendritic cells, natural killer cells, neutrophil and macrophage was determined and correlated with dysregulated miRNA levels. ResultsMicroRNA microarray identified 39 miRNAs aberrantly expressed during the colorectal cell transformation process. Seven novel miRNAs were shortlisted, and dysregulation of miR-149-3p, miR-192-3p, miR-335-5p and miR-425 were further validated by the qPCR validation experiment and data retrieved from the GEO dataset. Furthermore, these miRNAs demonstrated certain associations with level of dendritic cells, natural killer cells, neutrophil and macrophage within the polyp or CRC specimens. ConclusionThis study revealed miRNA dysregulated during stepwise malignant transformation of colorectal mucosal cells and their association with immune cell population.

Abstract 2089: Epigenetic regulation of sprouty2 in colorectal cancer

Abstract Background: The specific role of Sprouty-2 in colorectal cancer (CRC) as either a tumor suppressor or an oncogene is unclear. This is further obscured by the fact that no known driver mutations of Sprouty-2 have been reported. Therefore, for the first time, alterations of DNA 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) were investigated to better understand the regulation of Sprouty-2 in CRC. Methods: Sprouty-2 transcript expression was evaluated in CRC patient samples and Sprouty-2 protein expression was evaluated in CRC cells. The DNA methylation status spanning Sprouty-2 gene was evaluated using combined bisulfite restriction analysis (COBRA). The 5hmC status in the Sprouty-2 gene body and promoter was analyzed using previously performed nano-hmC-seal data from colon cancers and adjacent normal colonic mucosa. Publicly available RNA-seq and methylation data from The Cancer Genome Atlas (TCGA) were extracted from tumors from CRC patients. Results: Variable Sprouty-2 mRNA expression was observed among CRC patient tumors compared to adjacent normal appearing colonocytes. For the first time, differential methylation between adjacent normal colon and CRC samples was identified in 2 CTCF binding sites and in the promoter near the Transcriptional Start Site (TSS) of Sprouty-2, where the 5mC status in 1 of the CTCF binding sites correlated with transcript abundance. In CRC, this is the first study to reveal increases of 5hmC deposition in the gene body and promoter region of Sprouty-2 compared to normal colon. In metastatic CRC, an inverse correlation of increased Sprouty-2 promoter 5mC and decreased mRNA expression was found in patients registered in TCGA. Conclusions: In terms of tumor growth, epigenetic aberrations including Sprouty-2 promoter hypomethylation and increased 5hmC may play a prominent role in positive regulation of Sprouty-2 in CRC. In metastatic stages of CRC, increases in Sprouty-2 promoter 5mC may downregulate Sprouty-2 expression, which may ultimately label Sprouty-2 as a tumor suppressor in CRC. Citation Format: Alexei Stuckel, Sharad Khare. Epigenetic regulation of sprouty2 in colorectal cancer [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 2089.

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.

Comparative Study of Organoids from Patient-Derived Normal and Tumor Colon and Rectal Tissue.

Colon and rectal tumors, often referred to as colorectal cancer, show different gene expression patterns in studies that analyze whole tissue biopsies containing a mix of tumor and non-tumor cells. To better characterize colon and rectal tumors, we investigated the gene expression profile of organoids generated from endoscopic biopsies of rectal tumors and adjacent normal colon and rectum mucosa from therapy-naive rectal cancer patients. We also studied the effect of vitamin D on these organoid types. Gene profiling was performed by RNA-sequencing. Organoids from a normal colon and rectum had a shared gene expression profile that profoundly differed from that of rectal tumor organoids. We identified a group of genes of the biosynthetic machinery as rectal tumor organoid-specific, including those encoding the RNA polymerase II subunits POLR2H and POLR2J. The active vitamin D metabolite 1α,25-dihydroxyvitamin D3/calcitriol upregulated stemness-related genes (LGR5, LRIG1, SMOC2, and MSI1) in normal rectum organoids, while it downregulated differentiation marker genes (TFF2 and MUC2). Normal colon and rectum organoids share similar gene expression patterns and respond similarly to calcitriol. Rectal tumor organoids display distinct and heterogeneous gene expression profiles, with differences with respect to those of colon tumor organoids, and respond differently to calcitriol than normal rectum organoids.

Enhanced CXCR4 Expression Associates with Increased Gene Body 5-Hydroxymethylcytosine Modification but not Decreased Promoter Methylation in Colorectal Cancer

In colorectal cancer (CRC), upregulation of the C-X-C motif chemokine receptor 4 (CXCR4) is correlated with metastasis and poor prognosis, highlighting the need to further elucidate CXCR4’s regulation in CRC. For the first time, DNA methylation and 5-hydroxymethylcytosine aberrations were investigated to better understand the epigenetic regulation of CXCR4 in CRC. CXCR4 expression levels were measured using qPCR and immunoblotting in normal colon tissues, primary colon cancer tissues and CRC cell lines. Publicly available RNA-seq and methylation data from The Cancer Genome Atlas (TCGA) were extracted from tumors from CRC patients. The DNA methylation status spanning CXCR4 gene was evaluated using combined bisulfite restriction analysis (COBRA). The methylation status in the CXCR4 gene body was analyzed using previously performed nano-hmC-seal data from colon cancers and adjacent normal colonic mucosa. CXCR4 expression levels were significantly increased in tumor stromal cells and in tumor colonocytes, compared to matched cell types from adjacent normal-appearing mucosa. CXCR4 promoter methylation was detected in a minority of colorectal tumors in the TCGA. The CpG island of the CXCR4 promoter showed increased methylation in three of four CRC cell lines. CXCR4 protein expression differences were also notable between microsatellite stable (MSS) and microsatellite instable (MSI) tumor cell lines. While differential methylation was not detected in CXCR4, enrichment of 5-hydroxymethylcytosine (5hmC) in CXCR4 gene bodies in CRC was observed compared to adjacent mucosa.

175 Clinical Usefulness of SMOC1 as a Diagnostic Marker for Colorectal Serrated Lesions and Colorectal Precancerous Lesions

INTRODUCTION: Colorectal serrated lesions (SLs) include hyperplastic polyp (HP), traditional serrated adenoma (TSA) and sessile serrated adenoma/polyp (SSA/P). SSA/Ps are well known precursors of colorectal cancer (CRC) characterized by BRAF mutation and microsatellite instability (MSI). However, clinical and molecular characteristics of TSAs are not fully understood. We have reported that epigenetic silencing of SMOC1 is associated with development of traditional serrated adenomas (TSAs) and that SMOC1 may play a role in neoplastic pathways in TSAs and conventional adenomas. 1 We aimed to clarify the clinical usefulness of SMOC1 in colorectal tumors. METHODS: Expression of SMOC1 was analyzed in a series of 127 colorectal tumors and adjacent normal colonic mucosa by immunohistochemistry (IHC). The staining intensity of SMOC1 was assessed as strong (3), moderate (2), weak (1) or negative (0). The proportions of positively stained tumor cells were assessed as 0 to 10 for each staining intensity. Because neoplasm heterogeneity caused varying degrees of immunoreactivity in respective specimens, we used the sum of each intensity × proportion as an IHC score (e.g., intensity × proportion = (3) × 5 + (2) × 3 + (1) × 1 + (0) × 1 = IHC score 22, scale of 0 to 30). RESULTS: Our IHC analysis revealed that SMOC1 is abundantly expressed in normal colon, HPs and SSA/Ps, while it was significantly downregulated in TSAs, high-grade adenomas and CRCs. Among serrated lesions, SMOC1 is expressed in HPs, SSA/Ps and SSA/Ps with CRC, while it was significantly downregulated in TSAs and TSAs with CRC. Notably, in TSAs which consisted of flat and protruding subcomponents, SMOC1 expression levels were lower in the protruding portions than those in the flat portions (P &lt; 0.001). CONCLUSION: These results suggest that downregulated expression of SMOC1 could be a hallmark of TSAs and conventional adenomas at high risk of developing CRC.

Downregulated IGFBP7 facilitates liver metastasis by modulating epithelial‑mesenchymal transition in colon cancer.

Distant metastasis is a major cause of cancer-associated mortality in patients with colon cancer. Insulin-like growth factor binding protein 7 (IGFBP7) has been identified as a crucial inhibitor of human cancer. However, the role of IGFBP7 in the pathogenesis of metastatic colon cancer has not been investigated. In the present study, the expression of IGFBP7 in 81 pairs of colon cancer tissues and adjacent normal tissues were investigated using immunohistochemistry. Furthermore, 24 pairs of primary colon cancer and matched liver metastasis tissues were analyzed. LοVο cells with IGFBP7-knockdown and HT-29 cells with IGFBP7-overexpression were employed. The expression levels of E-cadherin, N-cadherin and Vimentin were quantified and compared. Significant alterations in the expression of IGFBP7 between late stage (III + IV) colon cancer and adjacent normal colonic mucosa were observed. (P=0.031). The association between IGFBP7 and epithelial-mesenchymal transition (EMT) markers were validated in primary colon cancer and matched liver metastasis tissues. The invasive front of liver metastatic colon tissues revealed reduced IGFBP7 expression. Additionally, knockdown of IGFBP7 in LοVο cells resulted in decreased E-cadherin, and increased N-cadherin and Vimentin expression compared with the control group. Overexpression of IGFBP7 in HT-29 cells induced an upregulation of E-cadherin; however, the N-cadherin and Vimentin levels were decreased. In conclusion, the results of the present study suggested that IGFBP7 may prevent colon cancer metastasis by inhibiting EMT, and serves as a potential diagnostic marker and therapeutic target for patients with colon cancer.

Over-Expression of Cyclooxygenase-2 in Colorectal Cancer Patients.

Background:Colorectal carcinoma (CRC) is the most common neoplasm of the gastrointestinal tract. COX-2 plays an important role in CRC development and is a key target for the regression of colorectal tumorigenesis by non-steroidal anti-inflammatory drugs. The present study was conducted to examine the relationship of the levels of COX-2 in CRC patients with the clinico-pathological parameters and also to assess its usefulness as a potential biomarker for diagnosis of CRC. Methods:Prior to surgery, 30 CRC patients were enrolled and the samples from colon tumors and surrounding tissues were taken after they underwent surgical intervention at PGIMER, Chandigarh. mRNA expression levels of COX-2 were examined in 30 CRC and adjacent normal colonic mucosa by quantitative polymerase chain reaction (qPCR). The expression of COX-2 was assessed by immunohistochemical method using rabbit polyclonal antibodies against human COX-2 protein. Results:The quantitative relative expression of COX-2 mRNA was observed to be significantly higher (p<0.05) in colorectal cancer tissues as compared to adjacent normal colon tissues. Also, female CRC patients showed significantly higher (p<0.009) expression of COX-2 mRNA vis-a-vis male colorectal cancer patients. This is the first study which has reported a direct relationship between COX-2 mRNA expressions in male colorectal cancer patients versus females. Further, immunohistochemistry of COX-2 confirmed the quantitative real time-PCR findings. Conclusion:Our study shows that COX-2 over expression in colorectal carcinoma patients is closely associated with clinico-pathological parameters and is more pronounced in males versus females. Further, COX-2 mRNA expression can serve as a potential biomarker for the diagnosis of CRC.

The Influence of Tumor Microenvironment on ATG4D Gene Expression in Colorectal Cancer Patients

Despite great progress in research on the subject, the involvement of autophagy in colorectal cancer (CRC) pathogenesis (initiation, progression, metastasis) remains obscure and controversial. Autophagy is a catabolic process, fundamental to cell viability and connected with degradation/recycling of proteins and organelles. In this study, we aimed at investigating the relative expression level of mRNA via Real-Time PCR of 16 chosen genes belonging to Atg8 mammalian orthologs and their conjugation system, comprising GABARAP, GABARAPL1, GABARAPL2, MAP1LC3A, MAP1LC3B, MAP1LC3C, ATG3, ATG7, ATG10, ATG4A, ATG4B, ATG4C, ATG4D, and three genes encoding proteins building the multimeric ATG16L1 complex, namely ATG5, ATG12, and ATG16L1, in 73 colorectal tumors and paired adjacent normal colon mucosa. Our study demonstrated the relative downregulation of all examined genes in CRC tissues in comparison to adjacent noncancerous mucosa, with the highest rate of expression in both tumor and non-tumor tissues observed for GAPARBPL2 and the lowest for MAP1LC3C. Moreover, in patients with advanced-stage tumors and high values of regional lymph nodes, statistically significant downregulation of ATG4D expression in adjacent normal cells was observed. Our study confirms the role of autophagy genes as cancer suppressors in colorectal carcinogenesis. Furthermore, in regard to the ATG4D gene, we observed the influence of tumor microenvironments on gene expression in adjacent colon mucosa.

Abstract 5178: MUC13 is a novel molecular signature, for early detection and metastatic colorectal cancer

Abstract Objective: Colorectal cancer (CRC) is a leading cause of cancer mortality affecting over a million people every year. Biological markers for early detection and distant metastatic disease in patients with CRC are not well defined. We have identified transmembrane mucin MUC13, which is expressed in normal colon mucosa to be highly expressed in colorectal cancer, but the underlying pathways and the signaling mechanisms involved in CRC pathogenesis are not known. Our studies suggest MUC13 correlation with demographic and clinicopathologic characteristics. Materials and Methods: Retrospective institutional tumor registry was reviewed to identify patients with resected colon adenocarcinoma. Archived FFPE tissue blocks were reviewed by an experienced pathologist. Selected representative tissue blocks were serially sectioned at 4 µm. IHC staining was performed using an in-house MAb for MUC13. Slides were digitally scanned and analyzed qualitatively as well as quantitatively using a modified H-score based on the intensity of expression and percentage of stained cells. MUC13 was correlated with disease stage, aggressiveness and inflammatory markers, indicating poor prognosis in CRC patients. MUC13 splice variants were probed using novel designed probes. MUC13 SNPs were identified using unbiased approach by analyzing dpGAP database. Results: 196 tissues, of which 56.1% were female, 52% were white, and the median age at resection was 70. 38 (19.4%) were stage I, 64 (32.7%) stage II, 84 (42.9%) stage III, and 10 (5.1%) stage IV. 100% of colon adenocarcinoma tissues stained positively for MUC13, including definitive tumor epithelial staining, little-to-no background stromal staining, and mild staining of adjacent normal colon mucosa. Typical colon adenocarcinoma cells exhibited strong apical membranous staining with varying degrees of cytoplasmic staining. Advanced stage tumors were noted to more frequently exhibit basolateral and/or circumferential membranous staining compared to early stage tumors which more frequently displayed apical membrane staining alone. Five MUC13 transcript variants were identified by database analysis. Two of the protein coding MUC13 variants (long and short) showed differential expression in aggressive cell lines and patient tissues. Cyclic turnover between the short and long isoforms were observed during Anoikis resistance, cortisol, alcohol and BAP treatments. SNPs have been associated with increased risk of cancer incidence or fatality. Relevant MUC13 SNPs have been identified through systematic analysis of patient databases (dpGAP database). Verification and correlation of identified SNPs with patient disease stage and prognosis will also be presented. Conclusion: This is the first study to correlate MUC13 expression with disease outcome. Alternative transcripts and single nucleotide polymorphisms gave genetic insight of the role of MUC13 in CRC pathogenesis. Citation Format: Manish K. Tripathi, Chidi Zacheaus, Kyle Doxtater, Zachary Stiles, Fatemeh Keramatnia, Nadeem Zafar, Mahul Amin, Meena Jaggi, Subhash Chauhan. MUC13 is a novel molecular signature, for early detection and metastatic colorectal cancer [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 5178.

Differential Regulation of LET-7 by LIN28B Isoform-Specific Functions.

The RNA-binding protein LIN28B plays an important role in development, stem cell biology, and tumorigenesis. LIN28B has two isoforms: the LIN28B-long and -short isoforms. Although studies have revealed the functions of the LIN28B-long isoform in tumorigenesis, the role of the LIN28B-short isoform remains unclear and represents a major gap in the field. The LIN28B-long and -short isoforms are expressed in a subset of human colorectal cancers and adjacent normal colonic mucosa, respectively. To elucidate the functional and mechanistic aspects of these isoforms, colorectal cancer cells (Caco-2 and LoVo) were generated to either express no LIN28B or the -short or -long isoform. Interestingly, the long isoform suppressed LET-7 expression and activated canonical RAS/ERK signaling, whereas the short isoform did not. The LIN28B-long isoform-expressing cells demonstrated increased drug resistance to 5-fluorouracil and cisplatin through the upregulation of ERCC1, a DNA repair gene, in a LET-7-dependent manner. The LIN28B-short isoform preserved its ability to bind pre-let-7, without inhibiting the maturation of LET-7, and competed with the LIN28B-long isoform for binding to pre-let-7 Coexpression of the short isoform in the LIN28B-long isoform-expressing cells rescued the phenotypes induced by the LIN28B-long isoform.Implications: This study demonstrates the differential antagonistic functions of the LIN28B-short isoform against the LIN28B-long isoform through an inability to degrade LET-7, which leads to the novel premise that the short isoform may serve to counterbalance the long isoform during normal colonic epithelial homeostasis, but its downregulation during colonic carcinogenesis may reveal the protumorigenic effects of the long isoform. Mol Cancer Res; 16(3); 403-16. ©2018 AACR.

Successful identification of a predictive biomarker for lymph node metastasis in colorectal cancer using a proteomic approach.

Colorectal cancer (CRC)-associated mortality is primarily caused by lymph node (LN) and distant metastasis, highlighting the need for biomarkers that predict LN metastasis and facilitate better therapeutic strategies. We used an Isobaric Tags for Relative and Absolute Quantification (iTRAQ)-based comparative proteomics approach to identify novel biomarkers for predicting LN metastasis in CRC patients. We analyzed five paired samples of CRC with or without LN metastasis, adjacent normal mucosa, and normal colon mucosa, and differentially expressed proteins were identified and subsequently validated at the protein and/or mRNA levels by immunohistochemistry and qRT-PCR, respectively. We identified 55 proteins specifically associated with LN metastasis, from which we selected ezrin for further analysis and functional assessment. Expression of ezrin at both the protein and mRNA levels was significantly higher in CRC tissues than in adjacent normal colonic mucosa. In univariate analysis, high ezrin expression was significantly associated with tumor progression and poor prognosis, which was consistent with our in vitro findings that ezrin promotes the metastatic capacity of CRC cells by enabling cell invasion and migration. In multivariate analysis, high levels of ezrin protein and mRNA in CRC samples were independent predictors of LN metastasis. Our data thus identify ezrin as a novel protein and mRNA biomarker for predicting LN metastasis in CRC patients.

Abstract 1971: ITGBL1 is a novel epithelial mesenchymal transition-associated prognostic biomarker in colorectal cancer

Abstract Purpose: Colorectal cancer (CRC) ranks as the third leading cancer worldwide, and its incidence continues to rise gradually, highlighting the need to stratify the risk of recurrence after curative surgery. Recently, several genes have been identified which appear to associate with metastasis, as they mediate epithelial-to-mesenchymal transition (EMT) in cancer. This study aimed to identify novel EMT and cancer recurrence-associated biomarkers through systematic and comprehensive discovery and validated strategy in multiple, independent CRC cohorts. Experimental Design: Two independent gene expression microarray datasets (n =173 and n =307 respectively) were used to identify novel metastasis-recurrence biomarkers for CRC. Following carefully selection and prioritization of biomarkers, we selected a candidate gene and validated its performance as a recurrence marker in a large testing cohort (n=566), and two independent clinical validation cohorts (n=201, n=475, respectively). To confirm the protein expression of ITGBL1 in cancer, immunohistochemistry (IHC) was performed in paired 33 primary CRCs and adjacent normal mucosa, as well as a subset of liver and lung metastases tissues. In addition, we used Gene Set Enrichment Analysis (GSEA) to determine the functional role of ITGBL1 in CRC. Results: During the discovery step, gene expression profiles from differentially expressed genes between recurrence positive and negative primary CRCs, as well as evaluation of the metastatic sites compared with primary CRC, identified ITGBL1 as a most promising candidate biomarker. High expression of ITGBL1 associated with poor overall survival (OS) in stage I-IV patients and worse disease-free survival (DFS) in stage I-III patients. Subgroup validation of these results in two large and independent patient cohorts confirmed these findings and demonstrated that high ITGBL1 expression correlated with shorter DFS in stage II and III CRC patients. In addition, high ITGBL1 expression emerged as an independent prognostic factor for DFS in stage II and III patients. IHC analysis revealed that both early stage CRCs and adjacent normal colonic mucosa displayed low ITGBL1 expression, while ITGBL1 expression gradually increased from tumor surface to the invasive front in late stage cancer, indicating that ITGBL1 may facilitate EMT process and promote a more aggressive phenotype in CRC. Conclusions: High expression of ITGBL1 in primary tumors was associated with tumor recurrence in CRC patients after curative surgery. Collectively, we have identified ITGBL1 as a novel EMT-associated biomarker which could be used for risk stratification for metastatic potential in CRC. Citation Format: Takatoshi Matsuyama, Toshiaki Ishikawa, Naoki Takahashi, Yasuhide Yamada, Masamichi Yasuno, Tatsuyuki Kawano, Hiroyuki Uetake, Ajay Goel. ITGBL1 is a novel epithelial mesenchymal transition-associated prognostic biomarker in colorectal cancer [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 1971. doi:10.1158/1538-7445.AM2017-1971

MCM2 expression in serrated polyps demonstrates aberrant cellular proliferation

In normal colonic epithelium, the proliferative zone is limited to the lower half of the colonic crypt. Evaluating the changes in the colonic epithelial proliferation can be useful in understanding pathophysiology of various diseases. Our aim was to investigate the proliferative compartment of serrated polyps (SPs) using MCM2, a protein involved in DNA replication, and assess for changes along the SP spectrum. Immunohistochemistry was performed on serrated polyps (16 microvesicular-type hyperplastic polyps (HP), 58 sessile serrated adenomas (SSA), 7 SSAs with dysplasia) and 6 sections of normal colon using anti-MCM2 antibody. Multiple sections of normal colon showed the following pattern for MCM2 and Ki-67 staining: positive nuclear staining of the lower half of the colonic crypts and/or slightly expanded to the lower two-thirds of the crypt. By MCM2, SPs show expansion of the proliferative compartments; 81.3% of HPs and 100% of SSAs showed some degree of full crypt MCM2 staining. SSAs with dysplasia showed consistent diffuse polyp staining. Aberrant staining in adjacent normal mucosa was also seen in SSAs with dysplasia and in a subset of non-dysplastic SSAs. By using MCM2, we show that serrated polyps exhibit changes in proliferation during progression along the pathway. HPs and SSAs show a similar highly proliferative profile. Aberrant proliferative cell staining patterns in adjacent normal colonic mucosa as seen in SSAs with dysplasia and a subset of SSAs suggest a field effect phenomenon. This indicates that changes in the colonic micro-environment may promote adenoma morphogenesis and predisposition to malignancy.

Proteomics analysis of differential protein expression identifies heat shock protein 47 as a predictive marker for lymph node metastasis in patients with colorectal cancer.

The discovery of biomarkers to predict the potential for lymph node (LN) metastasis in patients with colorectal cancer (CRC) is essential for developing improved strategies for treating CRC. In the present study, they used isobaric tags for relative and absolute quantitation to conduct a proteomic analysis designed to identify novel biomarkers for predicting LN metastasis in patients with CRC. They identified 60 differentially expressed proteins specifically associated with LN metastasis in CRC patients and classified the molecular and functional characteristics of these proteins by bioinformatic approaches. A literature search led them to select heat shock protein 47 (HSP47) as the most suitable candidate biomarker for predicting LN metastasis. Validation analysis by immunohistochemistry showed that HSP47 expression in patients with CRC and the number of HSP47-positive spindle cells in the tumor stroma were significantly higher compared with those in adjacent normal colonic mucosa, and the number of the latter cells increased with tumor progression. Further, the number of HSP47-positive spindle cells in stroma was a more informative marker for identifying LN metastasis than HSP47expression. Multivariate analysis identified spindle cells that expressed elevated levels of HSP47 as an independent predictive biomarker for CRC with LN metastasis. Moreover, these cells served as an independent marker of disease-free and overall survival of patients with CRC. Their data indicate that the number of HSP47-positive spindle cells in the stroma of CRC may serve as a novel predictive biomarker of LN metastasis, early recurrence and poor prognosis.

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.

A novel miR-203-DNMT3b-ABCG2 regulatory pathway predisposing colorectal cancer development.

Colorectal cancer (CRC) is a major cause of mortality and morbidity worldwide. The majority of studies to date focused on genetic mutations and epigenetic changes that drive the CRC carcinogenesis process. Xenobiotic transporters play an important role in safeguarding our body from external toxic substances. These transporters lining the gastrointestinal tract protect us from dietary carcinogens. This study aimed to investigate the downregulation of an efflux transporter ABCG2 in CRC versus normal colon mucosa, so as to shed light on its relevance to CRC initiation and progression. We found that ABCG2 expression is at least 50-fold lower in adenomatous polyps and colon carcinoma specimens obtained from CRC patients than in their matched pair of adjacent normal colon mucosa. The underlying mechanism(s) for ABCG2 under-expression in CRC is currently not known. To this end, aberrant promoter methylation of ABCG2 has been reported to cause its repression in a few cancer types including renal carcinoma and multiple myeloma. In this study, miR-203 was found to be downregulated in all polyps and CRC specimens, relative to adjacent normal colon mucosa. We demonstrated that the de novo DNA methyltransferase DNMT3b is a direct target of miR-203. Importantly, by relieving the repression on DNMT3b, the lower expression of miR-203 in CRC caused ABCG2 promoter methylation and remarkable lower ABCG2 expression in colon cancer cell lines and the patient CRC specimens. The restoration of ABCG2 function via modulating this new microRNA-methylation mechanism in precancerous cells may represent an attractive strategy to delay the carcinogenesis process. © 2016 Wiley Periodicals, Inc.