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Accumulating evidence suggests that dysregulation of transcriptional enhancers plays a significant role in cancer pathogenesis. Herein, we performed a genome-wide discovery of enhancer elements in colorectal cancer (CRC). We identified PVT1 locus as a previously unrecognized transcriptional regulator in CRC with a significantly high enhancer activity, which ultimately was responsible for regulating the expression of MYC oncogene. High expression of the PVT1 long-non-coding RNA (lncRNA) transcribed from the PVT1 locus was associated with poor survival among patients with stage II and III CRCs (p PVT1 locus inversely correlated with the reduced expression of the corresponding the PVT1 lncRNA, as well as MYC gene expression. Bioinformatic analyses of CRC-transcriptomes revealed that the PVT1 locus may also broadly impact the expression and function of other key genes within two key CRC-associated signaling pathways – the TGFβ/SMAD and Wnt/β-Catenin pathways. We conclude that the PVT1 is a novel oncogenic enhancer of MYC and its activity is controlled through epigenetic regulation mediated through aberrant methylation in CRC. Our findings also suggest that the PVT1 lncRNA expression is a promising prognostic biomarker and a potential therapeutic target in CRC.

Accumulating evidence suggests that aspirin has anti-tumorigenic properties in colorectal cancer (CRC). Herein, we undertook a comprehensive and systematic series of in vivo animal experiments followed by 3D-mathematical modeling to determine the kinetics of aspirin’s anti-cancer effects on CRC growth. In this study, CRC xenografts were generated using four CRC cell lines with and without PIK3CA mutations and microsatellite instability, and the animals were administered with various aspirin doses (0, 15, 50, and 100 mg/kg) for 2 weeks. Cell proliferation, apoptosis and protein expression were evaluated, followed by 3D-mathematical modeling analysis to estimate cellular division and death rates and their impact on aspirin-mediated changes on tumor growth. We observed that aspirin resulted in a dose-dependent decrease in the cell division rate, and a concomitant increase in the cell death rates in xenografts from all cell lines. Aspirin significantly inhibited cell proliferation as measured by Ki67 staining (P < 0.05–0.01). The negative effect of aspirin on the rate of tumor cell proliferation was more significant in xenograft tumors derived from PIK3CA mutant versus wild-type cells. A computational model of 3D-tumor growth suggests that the growth inhibitory effect of aspirin on the tumor growth kinetics is due to a reduction of tumor colony formation, and that this effect is sufficiently strong to be an important contributor to the reduction of CRC incidence in aspirin-treated patients. In conclusion, we provide a detailed kinetics of aspirin-mediated inhibition of tumor cell proliferation, which support the epidemiological data for the observed protective effect of aspirin in CRC patients.

Accumulating evidence suggests that cancer cells with stem cell-like features have higher resistance to chemotherapeutic agents. Herein, we identified T-lymphoma invasion and metastasis-inducing protein-1 (TIAM1) as one of the Wnt-signaling associated genes which drives self-renewal and its expression is upregulated by cancer associated fibroblasts (CAFs). TIAM1 expression was assessed in resected colorectal cancer (CRC) tissues from 300 patients who did or did not respond to chemotherapy. siRNA and CRISPR/Cas9 was used to examine whether the inhibition of TIAM1 affects chemosensitivity of CRC. We demonstrate that stemness through Wnt signaling regulates chemosensitivity and this phenomenon occurs exclusively in cancer stem cells. Subsequently, we established patient-derived CAFs and tested whether the drug sensitivity of CRC cell lines is altered with CAF-derived conditioned medium. High-TIAM1 expression correlated significantly with poor prognosis of CRC patients, and was overexpressed in patients who did not respond to chemotherapy. We demonstrated that the inhibition of TIAM1 enhanced sensitivity to chemotherapeutic drugs and reduced tumor invasiveness in a series of experiments in vitro. Moreover, CAF-derived conditioned media increased stemness and chemoresistance in CRC cell lines through TIAM1 overexpression. In addition, we validated TIAM1 associated drug sensitivity using a xenograft model. We have demonstrated that TIAM1 is overexpressed in CRC tumors from patients who did not respond to chemotherapeutic drugs and levels of TIAM1 expression served as an independent prognostic factor. Mechanistically, CAFs enhanced CRC chemoresistance through TIAM1 overexpression. Collectively, these results suggest that TIAM1 regulates chemosensitivity in tumors and stroma and thus may be an attractive therapeutic target.

Background Emerging evidence indicates that small nucleolar RNAs (snoRNAs) play a central role in oncogenesis. Herein, we systematically evaluated expression profiles of snoRNAs in colorectal cancer (CRC) and investigated their clinical and functional role in this malignancy. Methods We compared expression levels of snoRNAs between cancer and normal tissues using publicly available datasets and identified the most differentially expressed and commonly upregulated snoRNAs in CRC. These results were examined in 489 colorectal tissues to assess their clinical significance, followed by a series of in vitro and in vivo experiments to evaluate the functional role of candidate snoRNAs. Results Using multiple RNA profiling datasets, we identified consistent overexpression of SNORA21 in CRC. In the clinical validation cohorts, the expression level of SNORA21 was upregulated in colorectal adenomas and cancers. Furthermore, elevated SNORA21 emerged as an independent factor for predicting poor survival. Both in vitro and in vivo experiments revealed that CRISPR/Cas9-mediated inhibition of SNORA21 expression resulted in decreased cell proliferation and invasion through modulation of multiple cancer related pathways. Conclusions We systematically identified SNORA21 as a key oncogenic snoRNA in CRC, which plays an important role in cancer progression, and might serve as an important prognostic biomarker in CRC., Highlights • Systematic and comprehensive analysis revealed that SNORA21 was significantly upregulated in CRC. • Elevated SNONA21 expression was related to metastasis and predicted poor prognosis in CRC patients. • SNORA21 is a promising cancer prognostic biomarker and a potential therapeutic target in CRC. Emerging evidence indicates that small nucleolar RNAs (snoRNAs) might play a central role in oncogenesis; however, the clinical significance and functional roles of snoRNAs in colorectal cancer (CRC) remain unclear. Herein, using multiple publicly available datasets, we systematically assessed expression profiles of snoRNAs in CRC and elucidated that the high expression of SNORA21 was associated with distant metastasis and poor overall survival. A series of in vitro and in vivo experiments revealed oncogenic role for SNORA21. Our findings indicate that SNORA21 is a promising cancer prognostic biomarker and a potential therapeutic target in CRC.

Purpose: Colorectal cancer is one of the most common malignancies worldwide. Recently, a novel circular RNA, ciRS-7, was proposed to be a potential miR-7 sponge. As miR-7, a putative tumor-suppressor, regulates the expression of several important drivers of colorectal cancer, we analyzed the clinical significance of ciRS-7 in colorectal cancer patients. Experimental Design: Initially, we evaluated the expression levels of ciRS-7 in a training cohort comprising of 153 primary colorectal cancer tissues and 44 matched normal mucosae. We subsequently confirmed its clinical relevance in an independent validation cohort (n = 165), and evaluated the effect of ciRS-7 on miR-7, and its target genes EGFR and RAF1. Functional analyses were performed in cell lines and an animal model to support clinical findings. Results: Our data revealed that ciRS-7 was significantly upregulated in colorectal cancer tissues compared with matched normal mucosae (P = 0.0018), and its overexpression was associated with poor patient survival (P = 0.0224 and 0.0061 in the training and validation cohorts, respectively). Multivariate survival analysis revealed that ciRS-7 emerged as an independent risk factor for overall survival (P = 0.0656 and 0.0324 in the training and validation cohorts, respectively). Overexpression of ciRS-7 in HCT116 and HT29 cells led to the blocking of miR-7 and resulted in a more aggressive oncogenic phenotype, and ciRS-7 overexpression permitted the inhibition of miR-7 and subsequent activation of EGFR and RAF1 oncogenes. Conclusions: CiRS-7 is a promising prognostic biomarker in colorectal cancer patients and may serve as a therapeutic target for reducing EGFR-RAF1 activity in colorectal cancer patients. Clin Cancer Res; 23(14); 3918–28. ©2017 AACR.

Chemoresistance in cancer is linked to a subset of cancer cells termed “cancer stem cells” (CSCs), and in particular, those expressing the CD44 variant appear to represent a more aggressive disease phenotype. Herein, we demonstrate that CD44v6 represents a CSC population with increased resistance to chemotherapeutic agents, and its high expression is frequently associated with poor overall survival (OS) and disease-free survival (DFS) in patients with colorectal cancer (CRC). CD44v6(+) cells showed elevated resistance to chemotherapeutic drugs and significantly high tumor initiation capacity. Inhibition of CD44v6 resulted in the attenuation of self-renewal capacity and resensitization to chemotherapeutic agents. Of note, miRNA profiling of CD44v6(+) spheroid-derived CSCs identified a unique panel of miRNAs indicative of high self-renewal capacity. In particular, miR-1246 was overexpressed in CD44v6(+) cells, and associated with poor OS and DFS in CRC patients. We demonstrate that CD44v6(+) CSCs induced chemoresistance and enhance tumorigenicity in CRC cells, and this was in part orchestrated by a distinct panel of miRNAs with dysregulated profiles. These findings suggest that specific miRNAs could serve as therapeutic targets as well as promising prognostic biomarkers in patients with colorectal neoplasia.

BACKGROUND Circulating microRNAs (miRNAs) are attracting major interest as potential non-invasive biomarkers for colorectal cancer (CRC). This study aimed to identify a novel serum miRNA biomarker for the early detection and/or evaluating prognosis of CRC patients. PATIENTS AND METHODS Comprehensive miRNA array analysis was carried out using serum samples from patients with colorectal neoplasia and healthy controls. Next, to verify whether the candidate miRNA possessed a secretory potential, we screened miRNA expression levels in culture medium from 2 CRC cell lines, followed by serum analysis from 12 stage IV CRC, 12 adenoma, and 12 control subjects. Thereafter, we validated expression of candidate miRNAs in 179 primary CRC tissues, as well as serum samples from an independent cohort of 211 CRCs, 56 adenomas, and 57 control subjects. RESULTS Through microarray analysis, we identified significantly higher levels of miRNA-1290 (miR-1290) in serum from patients with colorectal adenomas and cancers. We verified miR-1290 overexpression in serum of CRC patients in a training cohort. In the validation cohort, serum miR-1290 levels were significantly up-regulated in patients with colorectal adenomas (P < 0.0001) and cancers (P < 0.0001). Serum miR-1290 levels could robustly distinguish adenoma [area under the curve (AUC) = 0.718] and CRC patients (AUC = 0.830) from normal subjects. High miR-1290 expression in serum and tissue was significantly associated with tumor aggressiveness and poor prognosis. Moreover, serum miR-1290 levels were an independent prognostic factor [hazard ratio (HR) = 4.51; 95% confidence interval (CI) = 1.23-23.69; P = 0.0096] and an independent predictor for tumor recurrence (hazard ratio = 3.92; 95% confidence interval = 1.11-25.14; P = 0.032) in CRC. CONCLUSIONS Serum miR-1290 is a novel biomarker for early detection, recurrence, and prognosis in CRC.

Diagnostic strategies, particularly non-invasive blood-based screening approaches, are gaining increased attention for the early detection and attenuation of mortality associated with colorectal cancer (CRC). However, the majority of current screening approaches are inadequate at replacing the conventional CRC diagnostic procedures. Yet, due to technological advances and a better understanding of molecular events underlying human cancer, a new category of biomarkers are on the horizon. Recent evidence indicates that cells release a distinct class of small vesicles called ‘exosomes’, which contain nucleic acids and proteins that reflect and typify host-cell molecular architecture. Intriguingly, exosomes released from cancer cells have a distinct genetic and epigenetic makeup, which allows them to undertake their tumorigenic function. From a clinical standpoint, these unique cancer-specific fingerprints present in exosomes appear to be detectable in a small amount of blood, making them very attractive substrates for developing cancer biomarkers, particularly noninvasive diagnostic approaches.

Resistance to cytotoxic chemotherapy is a major cause of mortality in colorectal cancer (CRC) patients. A small subset of cancer cells, termed "cancer stem cells" (CSCs), are believed to be key contributors of chemoresistance and tumor recurrence. Recently, epigallocatechin-3-gallate (EGCG), an active catechin present in green tea, has been shown to suppress CSC growth in various cancers, but whether it can specifically target CSCs and subsequently sensitize chemoresistant CRC cells to standard of care chemotherapeutic treatments remains unknown. Herein, we investigated the chemosensitizing effects of EGCG in 5-fluorouracil (5FU)-resistant (5FUR) CRC cells and spheroid-derived CSCs (SDCSCs), and interrogated the underlying molecular mechanisms responsible for its chemopreventive activity. EGCG enhanced 5FU-induced cytotoxicity and inhibited proliferation in 5FUR cell lines through enhancement of apoptosis and cell cycle arrest. The 5FUR cells showed higher spheroid forming capacity compared to parental cells, indicating higher CSC population. EGCG treatment in these cells resulted in suppression of SDCSC formation and enhanced 5FU sensitivity to SDCSCs. Furthermore, EGCG suppressed Notch1, Bmi1, Suz12, and Ezh2, and upregulated self-renewal suppressive-miRNAs, miR-34a, miR-145, and miR-200c, which are some of the key pathways targeted in 5FUR CRC cells. These findings were validated in vivo, wherein EGCG treatment resulted in inhibited tumor growth in a SDCSC xenograft model. Collectively our data provide novel and previously unrecognized evidence for EGCG-induced sensitization to 5FU through targeting of CSCs in CRC. Our data highlight that in addition to its chemopreventive ability, EGCG may serve as an adjunctive treatment to conventional chemotherapeutic drugs in CRC patients.

Objectives: MicroRNA (miR)-503 is downregulated in several cancers and plays a tumor-suppressive role in carcinogenesis. However, the miR-503 expression pattern, its clinical significance and its molecular mechanism in colorectal cancer (CRC) have not been investigated. Methods: We analyzed miR-503 expression in normal mucosa (n = 20), adenoma (n = 27) and CRC (n = 20). We quantified miR-503 expression in an independent cohort (n = 191) and investigated the clinical significance of miR-503 in CRC. CRC cell lines were transfected with anti-miR-503 to assess its function and target gene. Results: miR-503 expression increased according to the adenoma-carcinoma sequence. High miR-503 expression was significantly associated with large tumor size, serosal invasion, lymphatic and venous invasion as well as lymph node metastasis. CRC patients with high miR-503 expression had significantly earlier relapse and poorer prognosis than those with low expression. miR-503 was an independent recurrence marker in stage I/II CRC. In vitro, attenuated miR-503 expression resulted in inhibition of proliferation, invasion and migration and acquisition of anoikis of CRC cells. The putative target gene (calcium-sensing receptor) was significantly upregulated after miR-503 attenuation. Conclusions: miR-503 acts as an ‘onco-miR' in CRC. High miR-503 expression is associated with early recurrence and poor prognosis in CRC.

Combining anti-cancer agents in cancer therapies is becoming increasingly popular due to improved efficacy, reduced toxicity and decreased emergence of resistance. Here, we test the hypothesis that dietary agents such as oligomeric proanthocyanidins (OPCs) and curcumin cooperatively modulate cancer-associated cellular mechanisms to inhibit carcinogenesis. By a series of in vitro assays in colorectal cancer cell lines, we showed that the anti-tumorigenic properties of the OPCs-curcumin combination were superior to the effects of individual compounds. By RNA-sequencing based gene-expression profiling in six colorectal cancer cell lines, we identified the cooperative modulation of key cancer-associated pathways such as DNA replication and cell cycle pathways. Moreover, several pathways, including protein export, glutathione metabolism and porphyrin metabolism were more effectively modulated by the combination of OPCs and curcumin. We validated genes belonging to these pathways, such as HSPA5, SEC61B, G6PD, HMOX1 and PDE3B to be cooperatively modulated by the OPCs-curcumin combination. We further confirmed that the OPCs-curcumin combination more potently suppresses colorectal carcinogenesis and modulated expression of genes identified by RNA-sequencing in mice xenografts and in colorectal cancer patient-derived organoids. Overall, by delineating the cooperative mechanisms of action of OPCs and curcumin, we make a case for the clinical co-administration of curcumin and OPCs as a treatment therapy for patients with colorectal cancer.

Adenosine-to-inosine (A-to-I) RNA editing, a process mediated by adenosine deaminases that act on the RNA (ADAR) gene family, is a recently discovered epigenetic modification dysregulated in human cancers. However, the clinical significance and the functional role of RNA editing in colorectal cancer (CRC) remain unclear. We have systematically and comprehensively investigated the significance of the expression status of ADAR1 and of the RNA editing levels of antizyme inhibitor 1 (AZIN1), one of the most frequently edited genes in cancers, in 392 colorectal tissues from multiple independent CRC patient cohorts. Both ADAR1 expression and AZIN1 RNA editing levels were significantly elevated in CRC tissues when compared with corresponding normal mucosa. High levels of AZIN1 RNA editing emerged as a prognostic factor for overall survival and disease-free survival and were an independent risk factor for lymph node and distant metastasis. Furthermore, elevated AZIN1 editing identified high-risk stage II CRC patients. Mechanistically, edited AZIN1 enhances stemness and appears to drive the metastatic processes. We have demonstrated that edited AZIN1 functions as an oncogene and a potential therapeutic target in CRC. Moreover, AZIN1 RNA editing status could be used as a clinically relevant prognostic indicator in CRC patients.

Purpose Despite recent advances in colorectal cancer (CRC) treatment, the prognosis of patients suffering from this malignancy still remains substandard, and metastatic recurrence following curative surgery is the leading cause of mortality. Therefore, it is imperative to identify prognostic markers to predict the clinical outcome of CRC patients. Recent evidence revealed the new role of small nucleolar RNAs (snoRNAs) in oncogenesis. Herein, we systematically evaluated dysregulation of snoRNAs in CRC and clarified their biomarker potential and biological significance in CRC. Experimental design We analysed expression levels of 4 snoRNAs in 274 colorectal tissues from 3 independent cohorts and 6 colon cancer cell lines. The functional characterisation for the role of SNORA42 in CRC was investigated through a series of in vitro and in vivo experiments. Results In the screening phase, expression levels of all four snoRNAs were significantly elevated in CRC tissues than in corresponding normal mucosa. In the clinical validation cohort, increased SNORA42 expression was an independent prognostic factor for overall survival and disease-free survival, and was a risk factor for distant metastasis. SNORA42 expression negatively correlated with overall survival in an additional independent cohort and identified the patients with high risk for recurrence and poor prognosis in stage II CRC. Furthermore, in vitro and in vivo analyses showed that SNORA42 overexpression resulted in enhanced cell proliferation, migration, invasion, anoikis resistance and tumorigenicity. Conclusions SNORA42 appears to be a novel oncogene and could serve as a promising predictive biomarker for recurrence and prognosis in patients with CRC.

Although the anticancer properties of oligomeric proanthocyanidins (OPCs) from grape seeds have been well recognized, the molecular mechanisms by which they exert anticancer effects are poorly understood. In this study, through comprehensive RNA-sequencing-based gene expression profiling in multiple colorectal cancer cell lines, we for the first time illuminate the genome-wide effects of OPCs from grape seeds in colorectal cancer. Our data revealed that OPCs affect several key cancer-associated genes. In particular, genes involved in cell cycle and DNA replication were most significantly and consistently altered by OPCs across multiple cell lines. Intriguingly, our in vivo experiments showed that OPCs were significantly more potent at decreasing xenograft tumor growth compared with the unfractionated grape seed extract (GSE) that includes the larger polymers of proanthocyanidins. These findings were further confirmed in colorectal cancer patient-derived organoids, wherein OPCs more potently inhibited the formation of organoids compared with GSE. Furthermore, we validated alteration of cell cycle and DNA replication-associated genes in cancer cell lines, mice xenografts as well as patient-derived organoids. Overall, this study provides an unbiased and comprehensive look at the mechanisms by which OPCs exert anticancer properties in colorectal cancer.

Approximately 30–50% of colorectal cancer (CRC) patients who undergo curative resection subsequently experience tumor recurrence or metastasis. Although microRNAs (miRNAs) are a class of small noncoding RNAs frequently deregulated in various human malignancies, it remains unknown if these can help predict recurrence and metastasis in CRC patients. MiRNAs were initially screened using miRNA-microarray and miRNA-seq datasets with or without recurrence. Candidate miRNAs were then tested in two independent cohorts of 111 stage II/III and 139 stage I-III CRC patients, as well as serum samples and matched primary and metastatic liver tissues. An animal model of peritoneal dissemination was used to assess the oncogenic role of the target miRNA. Four candidate miRNAs were identified during the initial screening, and we subsequently validated upregulation of miR-139-5p in two independent clinical cohorts, wherein it associated with poor recurrence-free survival. Moreover, miR-139-5p were also upregulated in the serum of recurrence-positive CRC patients and yielded significantly shorter recurrence-free survival. Intriguingly, miR-139-5p was upregulated in metastatic liver tissues and negatively correlated with genes associated with epithelial-mesenchymal transition. Lastly, we showed that miR-139-5p overexpression enhanced peritoneal dissemination in a mouse model. In conclusion, we identified miR-139-5p as a novel biomarker for tumor recurrence and metastasis in CRC.

Population studies suggest that greater dietary fiber intake may lower colorectal cancer (CRC) risk, possibly through the colonic bacterial fermentative production of butyrate. Butyrylated starch delivers butyrate to the colon of humans with potential to reduce CRC risk but high doses may exacerbate risk through promoting epithelial proliferation. Here we report the effects of increasing dietary butyrylated high amylose maize starch (HAMSB) on azoxymethane (AOM) induced distal colonic DNA damage, cell proliferation, mucus layer thickness and apoptosis in rats. Five groups of 15 rats were fed AIN-93G based diets containing 0–40% HAMSB for 4 weeks then injected with (AOM) and killed 6 hours later. Large bowel total SCFA, acetate and butyrate pools and hepatic portal venous plasma total SCFA, acetate and butyrate concentrations were higher with greater HAMSB intake. Distal colonic epithelial apoptotic index and colonic mucus thickness increased, while DNA single strand breaks decreased dose-dependently with greater HAMSB intake. Colonocyte proliferation rates were unaffected by diet. These data suggest that increasing large bowel butyrate may reduce the risk of CRC in a dose dependent manner by enhancing apoptotic surveillance in the colonic epithelium for damaged cells without promoting the risk of tumorigenesis through increased cell proliferation.

This study was designed to determine how aspirin influences the growth kinetics and characteristics of cultured colorectal cancer cells that harbor a variety of different mutational backgrounds, including PIK3CA- and KRAS-activating mutations, and the presence or absence of microsatellite instability. Colorectal cancer cell lines (HCT116, HCT116 + Chr3/5, RKO, SW480, HCT15, CACO2, HT29, and SW48) were treated with pharmacologically relevant doses of aspirin (0.5–10 mmol/L) and evaluated for proliferation and cell-cycle distribution. These parameters were fitted to a mathematical model to quantify the effects and understand the mechanism(s) by which aspirin modifies growth in colorectal cancer cells. We also evaluated the effects of aspirin on key G0–G1 cell-cycle genes that are regulated by the PI3K–Akt pathway. Aspirin decelerated growth rates and disrupted cell-cycle dynamics more profoundly in faster growing colorectal cancer cell lines, which tended to be PIK3CA mutants. Additionally, microarray analysis of 151 colorectal cancer cell lines identified important cell-cycle regulatory genes that are downstream targets of PIK3 and were also dysregulated by aspirin treatment (PCNA and RB1). Our study demonstrated what clinical trials have only speculated, that PIK3CA-mutant colorectal cancers are more sensitive to aspirin. Aspirin inhibited cell growth in all colorectal cancer cell lines regardless of mutational background, but the effects were exacerbated in cells with PIK3CA mutations. Mathematical modeling combined with bench science revealed that cells with PIK3CA-mutations experience significant G0–G1 arrest and explains why patients with PIK3CA mutant colorectal cancers may benefit from aspirin use after diagnosis. Cancer Prev Res; 10(3); 208–18. ©2017 AACR.

Purpose: Dysregulated expression of miRNAs has emerged as a hallmark feature in human cancers. Exportin-5 (XPO5), a karyopherin family member, is a key protein responsible for transporting precursor miRNAs from the nucleus to the cytoplasm. Although XPO5 is one of the key regulators of miRNA biogenesis, its functional role and potential clinical significance in colorectal cancer remains unclear. Experimental Design: The expression levels of XPO5 were initially assessed in three genomic datasets, followed by determination and validation of the relationship between XPO5 expression and clinicopathologic features in two independent colorectal cancer patient cohorts. A functional characterization of XPO5 in colorectal cancer was examined by targeted gene silencing in colorectal cancer cell lines and a xenograft animal model. Results: XPO5 is upregulated, both at mRNA and protein levels, in colorectal cancers compared with normal tissues. High XPO5 expression is associated with worse clinicopathologic features and poor survival in colorectal cancer patient cohorts. The siRNA knockdown of XPO5 resulted in reduced cellular proliferation, attenuated invasion, induction of G1–S cell-cycle arrest, and downregulation of key oncogenic miRNAs in colorectal cancer cells. These findings were confirmed in a xenograft animal model, wherein silencing of XPO5 resulted in the attenuation of tumor growth. Conclusions: XPO5 acts like an oncogene in colorectal cancer by regulating the expression of miRNAs and may be a potential therapeutic target in colorectal cancer. Clin Cancer Res; 23(5); 1312–22. ©2016 AACR.

Purpose: Colorectal cancer ranks as the third most frequent cancer type, and its incidence continues to rise gradually worldwide, highlighting the need to identify previously unrecognized molecular events that propel development of this malignancy. Recent evidence suggests that dysregulated expression of FOX family of transcription factors may be critical in various genetic disorders as well as cancer; however, the functional and clinical significance of this pathway in colorectal cancer remains unclear. Experimental Design and Results: Herein, we performed a systematic and comprehensive discovery step by evaluating the expression of FOX family members, and identified that FOXM1 and FOXQ1 are frequently overexpressed in colorectal cancer. We subsequently confirmed these findings in two large testing cohorts (n = 550) and an independent clinical validation cohort (n = 134), in which high expression of FOXM1 and FOXQ1 emerged as an independent prognostic factor in colorectal cancer patients. We corroborated these findings by performing functional assays in which knockdown of FOXM1 and FOXQ1 resulted in inhibited cell proliferation and suppressed migration and invasion in colorectal cancer cells. Furthermore, using bioinformatic approaches, we identified miR-342 as a novel regulator of both FOXM1 and FOXQ1. Overexpression or inhibition of miR-342 modulated the expression of both genes and contributed to phenotypic alterations in colorectal cancer cells, which was subsequently validated in a xenograft animal model. Conclusions: Collectively, we have firstly identified FOXM1 and FOXQ1 as promising prognostic biomarkers in colorectal cancer patients, and provided novel evidence that therapeutic targeting of these genes or miR-342 may be a potential treatment approach in colorectal cancer patients. Clin Cancer Res; 22(19); 4947–57. ©2016 AACR.

Introduction: Colorectal cancer (CRC) is the third most frequent malignancy in males and second most common disease in females worldwide. CRC pathogenesis is intimately associated to lifestyles, such as diet, obesity, and smoking. Multiple evidences have revealed that these risk factors can trigger specific epigenetic alterations and subsequently promote carcinogenesis. Recently, adenosine (A)-to-inosine (I) RNA editing has been shown to be a potential epigenetic event in human cancers. One of the most important RNA editing gene targets is the antizyme inhibitor 1 (AZIN1), and edited AZIN1 promotes accumulation of ornithine decarboxylase and polyamines, leading to promotion and development of carcinogenesis. However, the oncogenic role and the clinical significance of RNA editing in CRC has not been investigated, which led us to undertake this present study. Materials and methods: We systematically and comprehensively investigated RNA editing in the AZIN1 gene using the RNA editing site specific PCR (RESSqPCR) in CRC patients. We further validates these results using multiple independent cohorts of CRC patients comprising of 329 colorectal cancer and adenoma patients. In addition, we performed a series of functional assays to elucidate the functional role of AZIN1 RNA editing in CRC pathogenesis. Results: Using RESSqPCR, AZIN1 RNA editing levels were analyzed in two CRC cohorts. AZIN1 editing levels were significantly higher in cancer tissues at all stages (I thru IV) compared with normal mucosa. Additionally, AZIN1 was highly edited in colorectal adenoma tissues compared to adjacent normal mucosa, suggesting this epigenetic event to be critical in normal-adenoma-carcinoma cascade. Additionally, the expression of RNA editing enzyme (ADAR1) was also upregulated in cancerous tissues compared to normal mucosa, and positively correlated with AZIN1 editing levels. To interrogate whether AZIN1 editing has an oncogenic role, overexpression of edited-AZIN1 in CRC cell lines resulted in increased cell proliferation, invasion, migration, and stemness. More importantly, AZIN1 editing was significantly enhanced in cancer stem cells, suggesting its importance for the development and maintenance of stemness features. Finally, establishment of xenograft tumors in an animal model resulted in significantly larger tumors in edited vs. wild type-AZIN1 groups. Taken together, these results highlight the oncogenic role of AZIN1 RNA editing in CRC. Conclusion: Our systematic and comprehensive study, which is first of its kind, reveals that AZIN1 RNA editing is novel epigenetic alteration that promotes an oncogenic behavior in colorectal cancer. In addition to its functional role, AZIN1 editing levels may be one of the important facilitators of adenoma-carcinoma sequence in CRC and serve as an important clinical biomarker in this disease. Citation Format: Kunitoshi Shigeyasu, Shusuke Toden, Yoshinaga Okugawa, Jinsei Miyoshi, Takeshi Nagasaka, Toshiyoshi Fujiwara, Leilei Chen, Ajay Goel. Novel evidence for AZIN1 RNA editing-mediated oncogenic role 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 3356. doi:10.1158/1538-7445.AM2017-3356

Background and Aims: Accumulating evidence suggests that a subset of cancer cells also known as the “cancer stem cells” (CSCs) influence various clinical outcomes in cancer, including tumor recurrence, metastasis and resistance to chemotherapy. Recently stemness has been recognized as a dynamic state governed by epigenetic modifiers including miRNAs. Despite identification of several self-renewal associated miRNAs, miRNA profile of CSCs remains unclear. Herein, we characterized miRNA expression of CSCs with high vs. low CD44v6 expression through RNA-Seq. Subsequently, we investigated the clinical significance of a novel miRNA identified from this systematic discovery approach. Methods: Colorectal CSCs from HCT116 and HT29 cells were grown as spheroid-derived cancer stem cells (SDCSCs). CD44v6+ and CD44v6- CSCs were subdivided by FACS and characterized by small RNA-Seq. Differentially expressed miRNAs were subsequently confirmed in CD44v6+ CSCs and chemoresistant cells. The expression of one such candidate, miR-1246, was assessed in a clinical cohort (n = 144) by qRT-PCR. Results: MiRNA profiling identified a unique overall pattern of CD44v6+ SDCSCs indicative of high self-renewal capacity. We noted that a panel of established self-renewal suppressive-miRNAs were downregulated (including miR-34a, 101 and 200 family) in CD44v6+ CSCs, and discovered upregulation of previously unreported miRNAs (miR-1246, 3605, 3182 and 4284). KEGG pathway analysis indicated that these miRNAs regulate Akt-MAPK and Wnt signaling pathways. Subsequently, we selected miR-1246 and validated its expression in CD44v6+ SDCSCs and chemoresistant cells. Clinically, the expression of miR-1246 was significantly elevated in CRC tissues compared to corresponding normal mucosa, and this occurred in a stage-dependent manner in primary CRCs. Furthermore, the expression of CD44v6 positively correlated with miR-1246 in CRC tissues. High miR-1246 expression resulted in poor disease free and overall survival. Conclusion: Using a systematic and comprehensive approach, we have identified a unique network of dysregulated miRNAs in CD44v6 CSCs indicative of high degree of stemness features in cancer. In particular, we identified miR-1246 to be frequently overexpressed in CSCs as well as chemoresistant cells and its expression was associated with poor prognosis in CRC patients. Collectively, we have identified a unique group of previously unreported miRNAs which appear to have important mechanistic roles in CSCs and could serve as a promising predictive biomarkers for recurrence and prognosis in patients with CRC. Citation Format: Shusuke Toden, Takatoshi Matsuyama, Elizabeth Hutchins, Kendall Jensen, Ajay Goel. Identification of a novel network of miRNAs that regulate stemness 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 3355. doi:10.1158/1538-7445.AM2017-3355

Background: Colorectal cancer (CRC) remains a prevalent malignancy worldwide with a high mortality rate. Over the last decade, the roles of several types of noncoding RNAs in oncogenesis have been clarified. Emerging evidence indicates that not only microRNAs, but small nucleolar RNAs (snoRNAs) appear to be dysregulated in various malignancies and they play a critical role in modulating cell transformation, tumor progression and metastasis. Functionally, cancer associated snoRNAs are subdivided into four major categories based on their oncogenic functions: 1) an independent function from imprinting gene in cancer initiation and progression 2) Activation of human telomerase 3) Inhibition of ribosomal maturation 4) Act directly as oncogene or tumor suppressor. Despite these exciting evidences, the role of snoRNAs in CRC remains limited and their clinical significance remains largely unexplored in this malignancy. Aims: We aimed to clarify the clinical relevance of snoRNAs in tumors and evaluate their prognostic potential in CRC. Furthermore, we investigated the functional role of snoRNAs in CRC tumorigenesis. Methods: Initially, we screened for the most commonly differentially expressed snoRNAs from publicly available microarray and RNA-sequence databases for other malignancies. Three clinical cohorts (a total 345 CRC tissues) were then used to select/validate the candidate snoRNAs. The prognostic potential of the candidate snoRNAs were evaluated and the association with clinicopathological features were assessed. CRISPR/Cas9 system was used to knockdown the target snoRNA in multiple CRC cell lines to determine the functional role of the target snoRNA in a series of in vitro assays. Results: In the discovery phase, we identified four differentially overexpressed snoRNAs in human cancers. We then confirmed overexpression of three snoRNAs (SNORA21, SNORA34 and SNORD66) in CRC compared to adjacent normal tissues. Of these three snoRNAs, high SNORA21 expression in tumors resulted in poor prognosis (p = 0.0086, Log-rank test). In an independent validation cohort, we confirmed high SNORA21 expression results in poor overall survival (p = 0.0060), especially in stage IV CRC (p = 0.0272). Clinicopathological analysis showed positive association between SNORA21 and vascular invasion and metastasis (p = 0.030 and 0.011, by Kruskal-Wallis test and Mann-Whitney U test, respectively) indicating that SNORA21 could be involved in metastasis. Moreover, multivariate analysis revealed that increased SNORA21 expression was an independent prognostic factor for overall survival. Using CRISPR/Cas9 stable SNORA21 knocked-down cell lines, we demonstrated that SNORA21 suppression resulted in reduced cell proliferation and colony forming capacity. Conclusion: We firstly identified SNORA21 as a novel oncogene which could also be a potential prognostic marker in patients with CRC. Citation Format: Kazuhiro Yoshida, Shusuke Toden, Wenhao Weng, Takeshi Nagasaka, Toshiyoshi Fujiwara, Ajay Goel. Clinical significance of SNORA21, an H/ACA box snoRNA, as a metastasis predicting and prognostic biomarker in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4926.

Introduction: An enhancer is a short stretch of DNA that can be targeted by specific proteins to activate gene transcription, and recent evidence suggests that enhancers can act as key regulators of oncogenes in cancer. Recently, the FANTOM5 project comprehensively characterized enhancer activities in various cell types. Interestingly, several enhancers coding for lncRNAs were commonly up-regulated in multiple cancer types, suggesting that these enhancers may play an important oncogenic role through enhancer-promoter-lncRNA interaction, and may help mediate chemoresistance. However, specific lncRNAs with oncogenic enhancer function and their clinical significance in colorectal cancer (CRC) remains unexplored. Aims: We analyzed genome-wide cancer-specific enhancer activation in multiple cancers to identify potential oncogenic lncRNAs, and assessed their clinical significance in two independent patient cohorts with CRC. We thereafter evaluated the functional role of candidate enhancer lncRNAs in CRC. Methods: Using the FANTOM5 enhancer database, cancer-specific enhancer activity was analyzed in cancers of the colon, stomach, lung, prostate and melanoma. LncRNA candidates that encode these cancer-specific enhancers were identified. We then screened these lncRNAs in two independent CRC cohorts, as well as the APC(Min/+) mouse model. The functional role of target lncRNAs was assessed in parental and chemoresistant CRC cell lines. The oncogenic enhancer activity was confirmed using the chromatin conformation capture (3C) assay. Results: We identified ten lncRNA candidates with consistent enhancer activities in all five cancer types. In particular, PVT1 appeared to have one of the highest enhancer activities. Using two independent CRC cohorts, we confirmed upregulation of PVT1 in cancer tissues. Patients with high-PVT1 expression had shorter overall survival in Stage II and III CRCs, suggesting that the PVT1 expression could discriminate high-risk patients, and identify those who may benefit from adjuvant chemotherapy. Intriguingly, we discovered that PVT1 was also upregulated in human colorectal adenomas as well as adenomas isolated from APC(Min/+) mice, indicating that PVT1 may be involved in the early stages of the adenoma-carcinoma sequence. The expression level of PVT1 significantly correlated with c-MYC expression in CRC specimens and the 3C assay showed that the enhancer region in PVT1 can target c-MYC. Finally, we demonstrated that PVT1 and c-MYC were co-overexpressed in several chemoresistant CRC cell lines, and that suppression of PVT1 led to a significant reduction in proliferation of chemoresistant cells, further suggesting that PVT1 may play a key role in chemoresistance. Conclusion: PVT1 acts as an oncogenic enhancer of c-MYC in CRC. High expression of PVT1 served as a predictive marker for identifying high-risk Stage II and III CRC patients. PVT1 could serve as a potential therapeutic target in chemoresistant CRCs. Citation Format: Kunitoshi Shigeyasu, Shusuke Toden, Takeshi Nagasaka, Toshiyoshi Fujiwara, C. Richard Boland, Ajay Goel. Genome-wide enhancer analysis identifies PVT1 as an oncogenic enhancer of MYC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 952.

OBJECTIVES: The development of colorectal cancer (CRC) is a highly complex process which needs coordinated regulation in the gene regulatory network. Forkhead box (FOX) proteins constitute a large family of transcriptional regulators. Accumulating evidence indicates that several FOX family members are critical regulators of tumor progression. However, the role of FOX proteins in CRC remains vastly unknown. miRNAs are single stranded small non-coding RNAs which can regulate a variety of biological processes in various human diseases including cancer. AIMS: Herein, we aimed to identify the expression patterns of FOX family members in CRC, followed by extensive characterization for their functional and mechanistic role in this malignancy. In addition, we sought to identify specific miRNAs that are responsible for dysregulated expression of FOX proteins in CRC. METHODS: The web-based database ONCOMINE was used to identify most highly expressed FOX family members in CRC. A cohort of 178 frozen tissues including 134 primary CRC tissues, and 44 matched normal mucosa tissues were collected to analyze the expression levels of FOX genes. For functional analysis, cell proliferation, migration and invasion assays were performed following siRNA knockdown of specific FOX proteins in CRC cells. The potential FOX-targeting miRNAs were initially identified using in-silico approaches, followed by luciferase reporter assays to validate their binding to downstream target genes. RESULTS: We systematically analyzed the expression profiles of FOX family members across 24 studies in cancer and normal tissues, and identified FOXM1, FOXQ1 and FOXA2 as the most highly expressed FOX genes in CRC. The over-expression of FOXM1 and FOXQ1 in CRC was confirmed in a cohort of CRC and matched adjacent normal tissues. Furthermore, the analysis of 134 CRC samples revealed that high FOXM1 and FOXQ1 expression resulted in worse overall survival. We next identified miR-342 as a potential regulator of both FOXM1 and FOXQ1, and subsequently validated its direct binding to the 3’UTR regions of both genes. Furthermore, the expression of miR-342 in CRC tissues inversely correlated with both FOXM1 and FOXQ1 expression. In-vitro functional analysis revealed that suppression of FOXM1 or overexpression of miR-342 inhibited CRC cell proliferation, while FOXM1 and FOXQ1 knockdown or miR-342 overexpression suppressed migration and invasion capacity of CRC cell lines. The stable overexpression of miR-342 reduced FOXM1 and FOXQ1 expression and significantly inhibited tumor growth in CRC xenografts in nude mice. CONCLUSIONS: FOXM1 and FOXQ1 promote cancer progression and act as potential prognostic factors in colon cancer. The downregulation of miR-342 in tumor tissues may contribute to the CRC development through inhibition of FOXM1 and FOXQ1 expression, suggesting that miR-342 could be used as a therapeutic target in colorectal cancer. Citation Format: Wenhao Weng, Yoshinaga Okugawa, Shusuke Toden, Ajay Goel. FOXM1 and FOXQ1 are novel oncogenes in colorectal cancer and can be therapeutically targeted through miRNA-based therapeutics. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1125.

Commentary to:Enhanced acute apoptotic response to azoxymethane-induced DNA damage in the rodent colonic epithelium by Tyrian purple precursors: A potential colorectal cancer chemopreventativeChantel B. Westley, Cassandra M. McIver, Catherine A. Abbott, Richard K. Le Leu and Kirsten Benkendorff

Non-starch polysaccharides (NSP; major components of dietary fibre) have been rather disappointing in the prevention and management of large bowel inflammatory diseases (IBD) or colorectal cancer (CRC). Resistant starch (RS) is that starch which escapes small intestinal digestion and enters the large bowel. RS contributes to total dietary fibre and could be as important as NSP in promoting large bowel health and preventing IBD and CRC. Indeed, it appears that some societies historically at low risk for these conditions eat relatively little NSP but have diets high in RS through their culinary practices. RS acts largely through its large bowel bacterial fermentation products which are, in adults, short chain fatty acids (SCFA). Collectively, SCFA have several non-specific positive actions on large bowel physiology including lowering of luminal pH. Of the major acids, butyrate has attracted the most attention. It is a major metabolic fuel for and promoter of a normal phenotype in colonocytes. Recent data from our laboratory support the latter suggestion. We have shown that, in rats, higher dietary protein (as casein, red meat or soy) increases colonocyte genetic damage and thinning of the colonic mucus barrier. However, feeding of RS as a high amylose maize starch opposed both of these changes in proportion to increased colonic butyrate. These data accord with prospective population data showing lower CRC risk with consumption of total dietary fibre. RS intakes appear to be low in most affluent industrialized countries, so increasing its consumption by modifying consumer foods is one strategy to improve public health. CSIRO and its partners are developing new high amylose cereal cultivars for this purpose. Colonic delivery of specific SCFA could also be useful clinically and we have shown that acetylated, propionylated and butyrylated starches resist small intestinal amylolysis. The bound SCFA are released by the large bowel microflora, raising their digesta levels, with the greatest increase being in the esterified acid. Feeding studies with butyrylated starch in rats have confirmed the opposition of diet-induced genetic damage, supporting a role for this SCFA in lowering risk of CRC and IBD. Further human and animal interventions are planned to determine the potential of these new types of RS in enhancing colonic health.

Markov analytical method. Automated p53 immunohistochemistry staining using clone D07 was used to correlate detected TP53 mutations with tissue expression. Results: The amount of DNA extracted for analysis varied from 2-50ng, including some highly degraded DNA material. TP53 was mutated in 40.5% (15/37) of the cases screened; 28.6% of NDBE (2/ 7), 71.4% of LGD (5/7), 44.4% of HGD (4/9), 33.3% of IMC (2/6) and 25% of IEA (2/8). Interestingly, only 40% (6/15) of cases showed both TP53 mutations and p53 aberrations (overor absence of expression), 60% (9/15) had mutations but no protein aberration, and 45% (5/11) had p53 aberration but no mutational abnormality. The data quality was excellent with all cases having a depth of coverage of at least x800 (mean coverage x1200 (figure 1). The level of p53 staining however did not correlate with the NGS mutation data (figure 2). Strong, normal and absent p53 staining was seen in tissues examined. Conclusion: In the UK BE guidelines, p53 IHC is used when the tissue is suspicious but not diagnostic for dysplasia. These results have shown discordance between detection of TP53 using mutation analysis and current IHC methods for p53 detection. Furthermore, we identified patients with TP53 mutations in NDBE, which may pre-empt progression to EA. Identifying early genomic aberrations driving the development of EA from BE holds the key to accurately risk stratifying BE patients into more aggressive treatment strategies or frequent surveillance regimes. Reference: (1) Weaver et al. Nat Genet. 2014 Aug;46(8):837-43.