Development Of CRC Research Articles

Abstract 1136: Decrease in maturation of stem cells along the neuroendocrine lineage contributes to stem cell overpopulation that drives human CRC development

Abstract Stem cell (SC) overpopulation is thought to drive tumor initiation and progression during colon cancer development, but it is unclear what causes the SC overpopulation. Because most neuroendocrine (NE) cells (NECs) in normal colonic crypts reside within the SC niche at the crypt bottom, we thought that aberrant NECs might be linked to the SC overpopulation. We hypothesized that (i) in normal crypts, SC and NEC populations are anatomically and functionally related, and (ii) during tumorigenesis, changes in these relationships quantitatively correlate with increased SC numbers. We tested this hypothesis - using quantitative immunohistochemical mapping - as a first step towards understanding how interactions between SC and NEC might become altered during colon tumorigenesis. In normal human colonic crypts, most (>80%) cells staining for ALDH1, a colonic SC marker, co-stained for chromogranin-A (CGA) and several other NE markers. Neither ALDH1+ nor CGA+ cells co-stained for MCM2, a marker for proliferating cells. These findings indicate that, in normal colonic crypts, many ALDH1+ cells have NE characteristics. In contrast, the proportion of ALDH1+ cells that co-stained for NE markers progressively decreased (to <20%) during the stepwise progression to cancer (from normal crypts to normal-appearing FAP crypts to adenomatous crypts to colon carcinomas). However, the absolute number of ALDH+ cells progressively increased during this progression, reflecting the extent of the SC overpopulation. These findings suggest that (i) NEC maturation is integral to regulation of SC population size and (ii) a decreased rate of NEC maturation contributes to SC overpopulation during colon tumorigenesis. Citation Format: Bruce M. Boman, Tao Zhang, Shirin Modarai, Lynn Opdenacker, Fields Jeremy. Decrease in maturation of stem cells along the neuroendocrine lineage contributes to stem cell overpopulation that drives human CRC development [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 1136.

Polymorphism in cancer-associated fibroblasts (CAFs) related genes and clinical outcome in metastatic colorectal cancer (mCRC) patients (pts) enrolled in two independent randomized phase III trials: TRIBE and FIRE-3.

645 Background: Tumor microenvironment plays a critical role for CRC development and progression. CAFs are the most dominant cellular components which orchestrate tumor cell proliferation, survival and invasion and have been shown to be associated with prognosis in pts with CRC. We aimed to evaluate whether single-nucleotide polymorphisms (SNPs) in CAFs related genes predict outcome in mCRC pts treated with first-line FOLFIRI/bevacizumab (bev) in TRIBE and FIRE-3 trials. Methods: The OncoArray, a custom array by Illumina including 530K SNP markers, was used to genotype genomic DNA from blood samples and served as our database, from which 15 functional SNPs within 9 genes (OPN, CD44, TCN, CD63, TIMP1, SLIT2, ROBO1, LOXL2, INHBA) related to CAF pathway were analyzed. Results: A total of 451 pts were included. Those treated with FOLFIRI/bev in TRIBE (N = 215, median PFS/OS: 9.7/26.3 months (mts)) and FIRE-3 (N = 107, median PFS/OS: 11.6/31.5 mts) served as discovery and validation cohorts respectively, while FIRE-3 FOLFIRI/cetuximab arm (N = 129, median PFS/OS: 12.8/49.8 mts) was used as the control. In overall pts in discovery set, OPN (osteopontin) rs11728697 any C variants showed longer PFS (median 10.8 vs 9.0 mts; HR = 0.68, 95%CI = 0.49-0.95; p = 0.025) and OS (median 30.6 vs 23.8 mts; HR = 0.69, 95%CI = 0.50-0.95; p = 0.025) than the homozygous wild type genotype (T/T) in multivariable analysis. These data were validated in the FIRE-3 cohort in OS both in univariate (median 49.3 vs 23.7 mts; HR = 0.40, 95%CI = 0.21-0.77; p < 0.001) and in multivariable analyses (HR = 0.31, 95%CI = 0.16-0.60; p < 0.001). However, the favorable impact on outcome was not shown among C allele carriers treated with FOLFIRI/cetuximab in the control arm. Conclusions: Our findings confirm that CAFs play a crucial role in mCRC and suggest that OPN polymorphisms could serve as predictive biomarkers for mCRC pts treated with first-line FOLFIRI/bev.

Exosomal lncRNA 91H is associated with poor development in colorectal cancer by modifying HNRNPK expression

BackgroundExosomes mediated transfer of lncRNA 91H may play a critical role in the development of CRC. However, few studies have proved the mechanism. So we performed this study to deeply explore the biological functions of exosomal 91H in the development and progression of CRC.MethodsThe association between lncRNA 91H and exosomes was detected in vitro and vivo. Then RNA pulldown and RIP were used to detect how lncRNA 91H affect CRC IGF2 express. At last, clinic pathological significance of exosomal 91H was evaluated by Cox proportional hazards model.ResultsWe found that serum lncRNA 91H expression was closely related to cancer exosomes in vitro and vivo which may enhance tumor-cell migration and invasion in tumor development by modifying HNRNPK expression. Then the clinic pathological significance of exosomal 91H was evaluated which demonstrated that CRC patients with high lncRNA 91H expression usually showed a higher risk in tumor recurrence and metastasis than patients with low lncRNA 91H expression (P < 0.05).ConclusionAll these data suggested that exosomal lncRNA 91H enhancing CRC metastasis by modifying HNRNPK expression might be an early plasma-based biomarker for CRC recurrence or metastasis. Further large-scale studies are needed to confirm our findings.

Abstract A35: Somatic mutation profile of a panel of oncogenes and tumor suppressor genes in colorectal cancer in a Brazilian population

Abstract Colorectal cancer (CRC) is the third most frequent cancer worldwide and the fourth leading cause of death due to cancer. The development of this disease usually occurs due to the progression of precursor lesions in the normal intestinal mucosa to tumor. Environment, age, lifestyle, and genetic background are considered risk factors for the development and progression of CRC. The molecular abnormalities involved in the progression of CRC have been well characterized, including mutations, microsatellite instability, and epigenetic changes. Although the molecular profile of colorectal cancer is well known in developed countries, in the Brazilian population information is still very limited. Therefore, in the present study we intended to characterize the mutation profile of a Brazilian population and associate this with their clinicopathologic features. A total of 61 colorectal cancer cases of different stages were included. DNA was extracted from frozen fresh tumors and sequenced using a panel of 298 genes by next-generation sequencing (NGS). The mean age of patients was 63 years old; 57.4% were male. Three patients had CRC in stage 0, 16 in stage I, 29 in stage II, 10 in stage III, and three in stage IV. Considering only nonsynonymous alterations, we found on average 7.24 mutations per patient. The majority were missense (61.8%), followed by nonsense (15.2%) and frameshift mutations (10.9%). The genes most frequently mutated were APC (78.7%), TP53 (72.1%), KRAS (60.7%), PIK3CA (14.8%), and FBXW7 (14.8%). Alterations in tumor suppressor genes such as APC and TP53 were found throughout the entire gene. On the other hand, oncogenes such as KRAS had alterations in hotspots of the gene. In conclusion, the frequency of alterations in genes sequenced in our population was similar to that found in the international literature for other populations. Citation Format: Wellington Santos, Adriane Feijó Evangelista, Ronilson Durães, Marcus Matsushita, Rui Manuel Reis, Denise Peixoto Guimarães. Somatic mutation profile of a panel of oncogenes and tumor suppressor genes in colorectal cancer in a Brazilian population [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A35.

Long non-coding RNA CCAL promotes hepatocellular carcinoma progression by regulating AP-2α and Wnt/β-catenin pathway

ObjectiveLong non-coding RNAs are emerging as key molecules in cancer progression. LncRNA-CCAL has shown to be highly expressed and important in regulating CRC and osteosarcoma development. Nevertheless, the expression and mechanism of CCAL in HCC is still not well understood. MethodsqRT-PCR and ISH were used to evaluate CCAL expression in HCC tissues and cell lines. Histone H3 methylation and acetylation levels across CCAL promoter region were examined by chromatin immunoprecipitation assays. Transfection of Lv-CCAL-shRNAs into HCC cell lines was used to evaluate cellular invasion and proliferation. The influence of CCAL depletion on AP-2α expression and Wnt/β-catenin pathway was analyzed by qRT-PCR, western blot and immunofluorescence. ResultsHigher expression of CCAL was found in HCC tumor tissues compared with normal tissues, and was associated with tumor metastasis and TNM stage. Furthermore, the decreased histone H3 methylation and increased histone H3 acetylation across CCAL promoter region contributed to the upregulation of CCAL in HCC. Moreover, the depletion of CCAL inhibited HCC cellular invasion and proliferation, and promoted cell apoptosis. In addition, CCAL depletion up-regulated AP-2α expression and inhibited Wnt/β-catenin pathway activation. ConclusionsCCAL has an important role in hepatic carcinogenesis and may serve as a new target for HCC diagnosis and treatment.

Feasibility of quantifying SDC2 methylation in stool DNA for early detection of colorectal cancer

BackgroundColorectal cancer (CRC) screening is the most efficient strategy to reduce disease-related mortality. Frequent aberrant DNA methylation is known to occur in selected genes and early during CRC development, which has emerged as a new epigenetic biomarker for early detection of CRC. Previously, we reported that we identified that CpG sites of SDC2 were aberrantly methylated in tumor tissues of most CRC patients through comprehensive methylation analysis and demonstrated a high potential of quantification of SDC2 methylation in blood for early detection of colorectal cancer. In this study, we aim to investigate the feasibility of quantifying SDC2 methylation in stool DNA for the early detection of CRC. The objective of this study was to confirm a high frequency of SDC2 methylation in tumor tissues at various stages of CRC and investigate the feasibility of a quantitative test for SDC2 methylation in fecal DNA by highly sensitive and accurate real-time PCR for early detection of CRC.MethodsBisulfite-pyrosequencing assay was performed to measure the SDC2 methylation status in tissue samples. For methylation analysis in stool DNA, a highly sensitive and accurate method was applied which implements consecutive two rounds of PCR consisting of unidirectional linear target enrichment (LTE) of SDC2 and quantitative methylation-specific real time PCR (qMSP) for SDC2, named as meSDC2 LTE-qMSP assay. Its limit of detection was 0.1% methylation (corresponding to ~ 6 copies in total ~ 6200 genome copies).ResultsPositive SDC2 methylation was observed in 100% of primary tumors, 90.6% of adenomatous polyps, 94.1% of hyperplastic polyps, and 0% of normal tissues. SDC2 methylation level also significantly (P < 0.01) increased according to the severity of lesions. In stool DNA test for SDC2 methylation by LTE-qMSP comparing CRC patients with various stages (I to IV) (n = 50) and precancerous lesions (n = 21) with healthy subjects (n = 22), the overall sensitivity was 90.0% for detecting CRC and 33.3% for detecting small polyps, with a specificity of 90.9%.ConclusionsTaken together, our result indicates that stool DNA-based SDC2 methylation test by LTE-qMSP is a potential noninvasive diagnostic tool for early detection of CRC.

Abstract A17: Molecular and clinicopathological features of native Nigerian colorectal cancer

Abstract Colorectal cancer (CRC) needs no introduction in Africa; once a rare cancer type, it is now the 3rd or 4th most common cancer. However, there are significant differences in the incidence rates, tumor biology, and clinical behavior in comparison to other populations. While the total incidence rates are about one-tenth of those in the developed world, Nigerian CRC occurs at a younger age and rapidly metastasizes. Molecular pathology and genetics of African CRC have not been comprehensively studied. In addition to better understanding of the biology of African CRC, analysis of genetic and molecular biomarkers, including microsatellite instability (MSI), is aimed at improving CRC diagnosis, treatment, and prognosis in Africa. We obtained 83 CRC paraffin-embedded blocks from the University of Ibadan and private pathology laboratories in Ibadan collected in the period from 2007 to 2014. Median age of the cases was 56±14.6 years with equal number of males and females. More than 60% of the patients had stage III and IV CRC. The majority were adenocarcinomas (76%) with a relatively high prevalence of mucinous adenocarcinoma (10%) and signet ring carcinoma (4%). Of all patients, 66% had rectal cancer. Poorly differentiated tumors were observed in 34% of all cases. We found a high prevalence of MSI CRC (43%, 15/35) in the tested Nigerian samples. There were no significant differences in clinicopathological features, including tumor location, between MSI and microsatellite stable (MSS) CRC. Our results are in line with our previous report that found a high prevalence (41%) of MSI CRC in Ghanaian patients. These findings raise important questions about the role of genetic and environmental factors in CRC development in West Africa. Citation Format: David O. Irabor, Marilena Melas, Stephen B. Gruber, Chanjuan Shi, Leon Raskin. Molecular and clinicopathological features of native Nigerian colorectal cancer [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr A17.

Alterations in PGC1\u03b1 expression levels are involved in colorectal cancer risk: a qualitative systematic review

BackgroundColorectal cancer (CRC) is a major global public health problem and the second leading cause of cancer-related death. Mitochondrial dysfunction has long been suspected to be involved in this type of tumorigenesis, as supported by an accumulating body of research evidence. However, little is known about how mitochondrial alterations contribute to tumorigenesis. Mitochondrial biogenesis is a fundamental cellular process required to maintain functional mitochondria and as an adaptive mechanism in response to changing energy requirements. Mitochondrial biogenesis is regulated by peroxisome proliferator-activated receptor gamma coactivator 1-α (PPARGC1A or PGC1α). In this paper, we report a systematic review to summarize current evidence on the role of PGC1α in the initiation and progression of CRC. The aim is to provide a basis for more comprehensive research.MethodsThe literature search, data extraction and quality assessment were performed according to the document Guidance on the Conduct of Narrative Synthesis in Systematic Reviews and the PRISMA declaration.ResultsThe studies included in this review aimed to evaluate whether increased or decreased PGC1α expression affects the development of CRC. Each article proposes a possible molecular mechanism of action and we create two concept maps.ConclusionOur systematic review indicates that altered expression of PGC1α modifies CRC risk. Most studies showed that overexpression of this gene increases CRC risk, while some studies indicated that lower than normal expression levels could increase CRC risk. Thus, various authors propose PGC1α as a good candidate molecular target for cancer therapy. Reducing expression of this gene could help to reduce risk or progression of CRC.

Colorectal neoplasia: Are young and female individuals remain at low risk for it?

ObjectiveIn this study, along with the determination of colorectal adenoma profile, we try to address whether young and female patients remain low-risk groups for colorectal adenoma. MethodIn a retrospective cross-sectional study, records of 15420 patients who had undergone total colonoscopy between 2010 and 2016 for any indication and were referred to Firoozgar Hospital (a referral hospital) in Tehran, Iran, were analyzed. Demographic data as well as characteristics of the lesions were collected from endoscopic and pathologic reports. ResultsRecords of 15420 patients were analyzed, and polypoid lesions were detected in 4542 (29.5%) of the patients. The mean age of the patients was 57 ± 14.4 years of them 61.3% were men. Adenomas were found in 3494 (77.5%) patients, of whom 1912 (54.7%) had the advanced adenoma, 148 (4.2%) had serrated adenomas, and 298 (8.5%) had malignant polyps. Histopathologically, the most common adenoma were the tubular type and the majority of them was located in distal colon. The prevalence of adenoma was comparable in both the genders. It was observed that while gender and number of lesions had significant associations with the type of adenoma, the size of lesions had significant associations with the development of malignancy. ConclusionThe influence of age and gender on CRC development may not have the same power as before. These findings suggest that firstly, the threshold of colonoscopy needs to be increased, and then a screening program should be decided upon for this region regardless of age and gender.

Measuring the Burden of Modifiable Risk Factors for Colorectal Cancer in an Inner City Cohort

Introduction: The rising prevalence of obesity projects it to exceed cigarette smoking as the leading preventable cause of death in the US. Increasing evidence demonstrates a link between obesity and colorectal cancer. With minority populations carrying a disproportionate degree of incidence and mortality secondary to colorectal cancer, this study sought to examine the burden of modifiable risk factors within a predominantly African American (AA) cohort at a major inner city teaching hospital. Methods: All adults who had undergone a screening or diagnostic colonoscopy from 2013 to 2015 were included in this study. A total of 4359 patients were analysed after eliminating the duplicate data.The following variables, BMI categories, cigarette smoking status, ethnicity, Diabetes Mellitus and alcohol intake were analysed to examine if there were differences in risk factors (DM, smoking, BMI and alcohol) between these groups. Results: Our population consisted of 77.3% AA. The prevalence of obesity, BMI ≥ 30, in this cohort, was 44.9%. 44.8% with DM, 19.7% current smokers and 31.1% reporting alcohol consumption. 40 subjects (0.9%) were diagnosed with CRC over the 3 year period. Subjects were more likely to be obese or overweight (77.1%). AA in this population were more likely to be overweight and obese (OR-1.25, 95% CI- 1.06 - 1.47) and to consume alcohol products (OR- 1.45; 95% CI- 1.23 - 1.70) There was no statistical differences in smoking status and DM in AA. In the analysis of predictors of CRC in the study population, gender was independently associated with the development of CRC, with females being less likely to be diagnosed by almost half (OR- 0.45: 95% CI- 0.23- 0.86). When adjusted for all the other predictors including increasing. There was still a statistically significant association between gender, females vs males (OR- 0.46; 95% CI; 0.23- 0.93) and increasing age (OR- 1.06, 95% CI; 1.02- 1.10). Conclusion: Overall incidence of CRC was higher in this population compared to US averages. AA were more likely to have a greater burden of modifiable risk factors (BMI>25 and alcohol intake), but these factors did not significantly predict risk of CRC. It is possible that in low socioeconomic cohorts, other unmeasured confounders like diet and environmental factors may play a greater role as modifiable risk factors than obesity and DM. This study highlights the striking prevalence and additional burden of modifiable risk factors in an underserved, predominantly AA cohort.

Increased expression of interleukin-21 along colorectal adenoma-carcinoma sequence and its predicating significance in patients with sporadic colorectal cancer

The role and significance of interleukin (IL)-21 in the development of sporadic CRC have not been well defined. The aim of this study is therefore to investigate the dynamics of the IL-21 along colorectal adenoma-carcinoma sequence and to evaluate the impact of IL-21 on clinicopathological parameters and CRC prognosis. The real-time PCR results showed that the level of IL-21 in adenomas (n=50) and sporadic CRC (n=50) were significantly higher than that in normal controls (n=18), which were predominately observed in the adenoma/CRC stroma. Analysis revealed that IL-21 level was correlated with the overall survival time in CRC patients. Double immunofluorescence observations confirmed that IL-21 positive cells were mostly natural killer cells and T lymphocytes in the tumor stroma. These results indicate that significant increased IL-21 expression present within the adenoma/CRC microenvironment might have a potential predicating significance for survival time in patients with CRC.

Investigating MicroRNA and transcription factor co-regulatory networks in colorectal cancer

BackgroundColorectal cancer (CRC) is one of the most common malignancies worldwide with poor prognosis. Studies have showed that abnormal microRNA (miRNA) expression can affect CRC pathogenesis and development through targeting critical genes in cellular system. However, it is unclear about which miRNAs play central roles in CRC’s pathogenesis and how they interact with transcription factors (TFs) to regulate the cancer-related genes.ResultsTo address this issue, we systematically explored the major regulation motifs, namely feed-forward loops (FFLs), that consist of miRNAs, TFs and CRC-related genes through the construction of a miRNA-TF regulatory network in CRC. First, we compiled CRC-related miRNAs, CRC-related genes, and human TFs from multiple data sources. Second, we identified 13,123 3-node FFLs including 25 miRNA-FFLs, 13,005 TF-FFLs and 93 composite-FFLs, and merged the 3-node FFLs to construct a CRC-related regulatory network. The network consists of three types of regulatory subnetworks (SNWs): miRNA-SNW, TF-SNW, and composite-SNW. To enhance the accuracy of the network, the results were filtered by using The Cancer Genome Atlas (TCGA) expression data in CRC, whereby we generated a core regulatory network consisting of 58 significant FFLs. We then applied a hub identification strategy to the significant FFLs and found 5 significant components, including two miRNAs (hsa-miR-25 and hsa-miR-31), two genes (ADAMTSL3 and AXIN1) and one TF (BRCA1). The follow up prognosis analysis indicated all of the 5 significant components having good prediction of overall survival of CRC patients.ConclusionsIn summary, we generated a CRC-specific miRNA-TF regulatory network, which is helpful to understand the complex CRC regulatory mechanisms and guide clinical treatment. The discovered 5 regulators might have critical roles in CRC pathogenesis and warrant future investigation.

Gut microbiota and colorectal cancer: insights into pathogenesis for novel therapeutic strategies.

Colorectal cancer (CRC), as a leading cause of cancer-related death, is triggered by the complex interplay of host genetics and environmental factors. Mounting evidence has shed light on the association of the gut microbiota dysbiosis with CRC. In CRC experimental models, certain gut microbial strains have been shown to inhibit or attenuate immune responses, indicating that specific species among intestinal commensal bacteria may play either a pathogenic or a protective role in the development of CRC. Oral intake of probiotics/prebiotics can therefore serve as a therapeutic approach for CRC treatment. Microbiota studies in cancer, however, are still at the early stage, lack of quantitative data for clinic application. Fortunately, sequencing-based technologies are a boon to further investigation on the association of the intestinal bacterial flora and human diseases. This review considers the evidence for the role of the gut microbiota in CRC development and progression, responsiveness to immune system, and the related therapeutic applications of probiotics/prebiotics.

Methylation of TMEM176A is an independent prognostic marker and is involved in human colorectal cancer development

ABSTRACTColorectal cancer (CRC) is the third most common malignancy and the fourth most common cause of cancer related death worldwide. This study was designed to find tumor suppressors involved in CRC development by performing RNA-seq. Eight CRC cell lines and 130 cases of primary CRC samples were used. RNA-seq, methylation-specific PCR (MSP), flow cytometry, transwell assays, and a xenograft mouse model were used. Reduction of TMEM176A expression was confirmed in human CRC cells by RNA-seq. TMEM176A was expressed in LS180 and SW620 cells, loss of TMEM176A expression was observed in LOVO, HCT116, RKO, and DLD1 cells, and reduced TMEM176A expression was found in HT29 and SW480 cells. Unmethylation of the TMEM176A promoter was found in LS180 and SW620 cells, whereas complete methylation was found in LOVO, HCT116, RKO, and DLD1 cells, and partial methylation was found in HT29 and SW480 cells. Promoter region methylation correlated with loss of/reduced expression of TMEM176A. Re-expression of TMEM176A was induced by 5-aza-2′-deoxycytidine. TMEM176A was methylated in 50.77% of primary colorectal cancers. Methylation of TMEM176A was associated with tumor metastasis (P<0.05) and was an independent prognostic factor for 5-year overall survival (OS) according to Cox proportional hazards model analysis (P<0.05). TMEM176A induced apoptosis and inhibited cell migration and invasion in CRC cells. TMEM176A suppressed CRC cell growth both in vitro and in vivo. Our results suggest that expression of TMEM176A is regulated by promoter region methylation. TMEM176A methylation is an independent prognostic marker for 5-year OS in CRC, and may act as a tumor suppressor in CRC.

Abstract 5309: Dietary magnesium is inversely associated with colorectal cancer risk in the Atherosclerosis Risk in Communities study

Abstract Background: Magnesium is a key nutritional mineral required for the regulation of numerous biochemical reactions throughout the body. Main food sources of magnesium include whole grains, nuts, and green vegetables, but more than 50% of Americans do not currently meet their daily recommended magnesium intake. Prior research has shown that those deficient in magnesium have increased susceptibility to oxidative stress and chronic inflammation, leading to endothelial dysfunction and potentially stimulating cancer cell proliferation and invasiveness. Previous epidemiological studies suggested a link between a diet high in magnesium and reduced CRC, especially among women. The American Cancer Society underscores the importance of clarifying the role of dietary magnesium in CRC development overall and by gender. Thus, we prospectively examined the associations between dietary magnesium intake and CRC risk in the ARIC study, overall, and separately in men and women. Methods: The ARIC study followed 15,792 men and women (45-64 years old at baseline) for cancer occurrence from 1987-2006. Participants received medical examinations and completed questionnaires at 4 visits; food frequency data were collected at Visit I (1987-89) and magnesium intake estimated. Cox proportional hazards regression was used to calculate hazard ratios (HR) and 95%CI for CRC associated with dietary magnesium intake in quartiles using two models: Model 1 adjusted for baseline age, race, center, sex, energy intake, BMI, and physical activity. Model 2 included Model 1 covariates as well as intake of alcohol, dietary calcium, dietary fiber, aspirin, CRP levels, and cigarette smoking. Results: The analytic cohort (n=14,160, 54.5% women, 27.3% black and 72.7% white, 45-64 years at baseline) was followed for a median of 15.4 years; 315 CRC cases were identified. Since only the highest quartile of dietary magnesium intake met US recommendations (&amp;gt;300 mg/day), we examined the highest quartile versus the 3 lowest quartiles combined (reference category). In Model 1, the highest quartile of dietary magnesium was associated with lower CRC risk: HR (95%CI) =0.68 (0.47-0.99). The inverse association between magnesium and CRC persisted in Model 2 (HR=0.70; 95% CI 0.48, 1.02). The association followed a similar trend after stratification by colon and rectal cancer cases (HR(95% CI) =0.72 (0.48-1.08) and HR(95% CI)=0.49 (0.23-1.06), respectively) and by men and women (HR(95% CI)=0.75 (0.42-1.21) and HR(95% CI)=0.63 (0.36-1.12) respectively). There was no interaction of dietary magnesium intake with dietary calcium intake, alcohol consumption use or dietary fiber. Conclusion: Our findings corroborate an inverse association between higher dietary magnesium intake and CRC risk, with similar associations observed in men and women. Support: NHLBI, NCI, NPCR Citation Format: Guillaume C. Onyeaghala, Elizabeth Polter, Pamela L. Lutsey, Aaron R. Folsom, Corrine E. Joshu, Elizabeth A. Platz, Anna E. Prizment. Dietary magnesium is inversely associated with colorectal cancer risk in the Atherosclerosis Risk in Communities study [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 5309. doi:10.1158/1538-7445.AM2017-5309

Abstract 2690: MK2 pathway blockade inhibits inflammatory cytokine production and colorectal cancer growth and invasion

Abstract Introduction: Colorectal cancer is the third most common malignancy diagnosed for both men and women in the United States of America. High levels of inflammatory cytokines in colorectal tumors cause increased growth and invasion and carry a higher risk of metastasis. Identifying new targets to control inflammation is important for developing improved treatment approaches. Mitogen-activated protein kinase-activated protein kinase 2 (MK2) is a regulator of pro-inflammatory cytokines that may promote colorectal tumor progression. MK2 signaling is known to induce IL-1, IL-6, and TNF-α production. These pro-inflammatory cytokines are associated with CRC development, invasion, and metastasis. We hypothesized that the MK2 pathway could be an important component of CRC growth, invasion and tumor regrowth. Methods: To investigate this pathway, CT26 CRC cells were examined. Cells were flank-injected into Balb/c mice with and without MK2 inhibitor treatment. At day 19 after tumor injection, tumors were harvested and measured using calipers. Cytokines were quantitated by multiplex bead array in organ culture supernatants and in supernatants from tumor cells plated in fibronectin coated wells. In culture, tumor cell invasion was measured in scratch wound assays containing matrigel. Results: Treating CT26 cells with MK2 inhibitors markedly reduced tumor growth in mice by a mean of 60% compared to vehicle control treated cells. Inflammatory cytokines were also dramatically decreased with MK2 inhibition compared to controls by up to 80%. Cytokines affected included both known MK2 downstream cytokines (IL-1, IL-6, and TNF-α) and also chemokines such as MIP-1α and MCP-1 in both supernatants from cultured tumor cells and in organ culture supernatants. MK2 inhibition also decreased invasion of tumor cells in a cytokine dependent manner. Conclusions: The MK2 pathway regulates production of multiple pro-inflammatory cytokines in colorectal tumors, including several previously unreported chemokines. Inhibition of this pathway markedly decreases tumor growth and invasion. Thus, MK2 may be a promising tumor target to prevent colorectal cancer progression. Citation Format: Anita L. Ray, Amanda S. Peretti, Wade Johnson, Gregory Gan, Ellen J. Beswick. MK2 pathway blockade inhibits inflammatory cytokine production and colorectal cancer growth and invasion [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 2690. doi:10.1158/1538-7445.AM2017-2690

Abstract 1445: Understanding the 8q24 colorectal cancer risk locus via CRISPR/Cas9 scarless genome editing

Abstract Colorectal Cancer (CRC) is one of the leading causes of mortality in the US, being the third most common cancer among both men and women. While an exact cause of CRC has not yet been elucidated, the existence of a clear heritable genetic component has been established. With the recent surge of data from Genome-Wide Association Studies, a handful of CRC-associated loci have been identified, each containing numerous Single Nucleotide Polymorphisms (SNPs). We hypothesize that among the ~25 loci associated with increased risk for CRC, there are driver SNPs functioning to contribute to initiation or progression of CRC development. It is crucial, in order to enhance our understanding CRC tumorigenesis, to extract these functional variants from the pool of associated SNPs and validate their causative significance within the cell. To model these functional SNPs, we have developed a novel “scarless” isolation pipeline for selecting isogenic clones with the desired single-base edit without selection markers following CRISPR-Cas9 mediated HDR. With this pipeline, we have successfully modeled our first selected SNP, rs6983267, from the 8q24 risk locus in HCT-116 cells, producing the heterozygous genotype (G/T) from the parental homozygous risk (G/G) genotype. Rs6983267 is located within a known c-Myc enhancer and has been shown to exert risk-allele specific increase in enhancer activity. Oncogenic c-Myc is known to play a significant role in CRC pathogenesis. In line with previous studies, we observe a slight decrease in c-Myc expression in our heterozygous clones compared to wildtype homozygous risk. Moreover, we observe a preferential binding of the transcription factor in control of c-Myc transcriptional regulation, TCF7L2, to the risk allele over the reference allele while binding of proximal factors such as CTCF show no allelic preference. Interestingly, we also see significant changes in expression of target genes of the WNT signaling pathway. Dysregulation of WNT is a hallmark characteristic of CRC. These changes indicate there is indeed an effect of the risk allele of rs6983267 on overall WNT activity in CRC cells due to increased binding of transcription factor TCF7L2. This novel SNP editing pipeline provides a method to study precise effects of single base changes located anywhere in the genome. Utilizing it to model other risk SNPs will enable the enhancement of our current understanding of GWAS-identified risk loci in CRC pathogenesis at the molecular level. Citation Format: Nicole Coggins, Jacob Stultz, David Segal, Luis Carvajal-Carmona. Understanding the 8q24 colorectal cancer risk locus via CRISPR/Cas9 scarless genome editing [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 1445. doi:10.1158/1538-7445.AM2017-1445

Abstract 240: Dietary patterns and risk of colorectal neoplasia in Puerto Rican Hispanics: A case control study

Abstract Colorectal cancer (CRC) is the leading cause of cancer death in Puerto Rico and second cause of death in the US. It is well accepted that modifiable lifestyle habits such as diet, contribute to CRC development. Diet could reduce up to 70% of CRC cases. Dietary patterns vary according to race/ethnicity. Puerto Rican Hispanics (PRH) have been shown to have a distinct diet compared to US non-Hispanic Whites (NHW). According to the BRFSS, only 17.7% of PRH and 23.4% of NHW consume fruit and vegetables five times per day. The objective of this study was to describe the association of specific food groups consumption with the risk of colorectal neoplasia (CRN: polyps and/or CRC) in PRH. The validated Colon Cancer Family Registry food frequency questionnaire was administered to healthy individuals (controls) and individuals with CRN (cases) recruited through the Puerto Rico Colorectal Cancer Registry (PURIFICAR). Models adjusted by age, gender, educational attainment and family history of CRC were fitted to estimate the odds ratios (OR) with 95% confidence intervals (CI) through a polytomous logistic regression models comparing cases and controls. A total of 577 subjects were included in the study (controls=254; polyps=50; and CRC=273). The frequency of consumption of fruits, low fat dairy products, eggs, pasta/rice, non-fried chicken, and seafood were significantly different between controls and CRN cases (p&amp;lt;0.05). Adjusted models showed that a higher consumption of fruits (OR≥once per day=0.29) and full-fat dairy products (OR≥once per day=0.22) were associated with decreased risk of polyps (p&amp;lt;0.05). A frequency of consumption of 2-4 times per week, when compared to ≤ 1 per week, of full-fat dairy products (OR2-4 times per week=0.51), seafood (OR2-4 times per week=0.49), and red meats (OR2-4 times per week=0.56) was associated with a decreased risk of CRC (p&amp;lt;0.05). Our results showed that a higher consumption of fruits and full-fat dairy products significantly reduce the risk of polyps in PRH. In addition, consumption of seafood, full-fat dairy, and red meats up to 4 times per week may reduce the risk of CRC in PRH. Educational interventions for cancer prevention in PRH should incorporate nutritional information tailor to our community. Citation Format: Julyann Perez-Mayoral, Sachelly Julian, Marievelisse Soto-Salgado, Michael J. Gonzalez, Marcia Cruz-Correa. Dietary patterns and risk of colorectal neoplasia in Puerto Rican Hispanics: A case control study [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 240. doi:10.1158/1538-7445.AM2017-240

Co-targeting of EGFR and autophagy signaling is an emerging treatment strategy in metastatic colorectal cancer

The epidermal growth factor receptor (EGFR) and its associated pathway is a critical key regulator of CRC development and progression. The monoclonal antibodies (MoAbs) cetuximab and panitumumab, directed against EGFR, represent a major step forward in the treatment of metastatic colorectal cancer (mCRC), in terms of progression-free survival and overall survival in several clinical trials. However, the activity of anti-EGFR MoAbs appears to be limited to a subset of patients with mCRC. Studies have highlighted that acquired-resistance to anti-EGFR MoAbs biochemically converge into Ras/Raf/Mek/Erk and PI3K/Akt/mTOR pathways. Recent data also suggest that acquired-resistance to anti-EGFR MoAbs is accompanied by inhibition of EGFR internalization, ubiqutinization, degradation and prolonged downregulation. It is well established that autophagy, a self-cannibalization process, is considered to be associated with resistance to the anti-EGFR MoAbs therapy. Additionally, autophagy induced by anti-EGFR MoAbs acts as a protective response in cancer cells. Thus, inhibition of autophagy after treatment with EGFR MoAbs can result in autophagic cell death. A combination therapy comprising of anti-EGFR MoAbs and autophagy inhibitors would represent a multi-pronged approach that could be evolved into an active therapeutic strategy in mCRC patients.

NR2F2 inhibits Smad7 expression and promotes TGF-β-dependent epithelial-mesenchymal transition of CRC via transactivation of miR-21

Metastasis is one of the most decisive factors influencing CRC patient prognosis and current studies suggest that a molecular mechanism known as EMT broadly regulates cancer metastasis. NR2F2 is a key molecule in the development of CRC, but the roles and underlying mechanisms of NR2F2 in TGF-β induced EMT in CRC remain largely unknown. In the current study, we were interested to examine the role of NR2F2 in the TGF-β-induced EMT in CRC. Here, we found NR2F2 was upregulated in CRC cells and promotes TGF-β-induced EMT in CRC. Using comparative miRNA profiling TGF-β pre-treated CRC cells in which NR2F2 had been knocked down with that of control cells, we identified miR-21 as a commonly downregulated miRNA in HT29 cells treated with TGF-β and NR2F2 siRNA, and its downregulation inhibiting migration and invasion of CRC cells. Moreover, we found NR2F2 could transcriptional activated miR-21 expression by binding to miR-21 promoter in HT29 by ChIP and luciferase assay. In the last, our data demonstrated that Smad7 was the direct target of miR-21 in CRC cells. Thus, NR2F2 could promote TGF-β-induced EMT and inhibit Smad7 expression via transactivation of miR-21, and NR2F2 may be a new common therapeutic target for CRC.