Wenhao Weng

Circular RNA ciRS-7—A Promising Prognostic Biomarker and a Potential Therapeutic Target in Colorectal Cancer

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.

FOXM1 and FOXQ1 Are Promising Prognostic Biomarkers and Novel Targets of Tumor-Suppressive miR-342 in Human Colorectal Cancer

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.

An update on miRNAs as biological and clinical determinants in colorectal cancer: a bench-to-bedside approach.

Colorectal carcinogenesis represents a sequential progression of normal colonic mucosa from adenoma to carcinoma. It has become apparent that miRNA deregulation contributes to the initiation and progression of colorectal cancer (CRC). These oncogenic or tumor-suppressive miRNAs interact with intracellular signaling networks and lead to alteration of cell proliferation, apoptosis, metastasis and even response to chemotherapeutic treatments. This article aims to review the cutting edge progress in the discovery of the role of novel mechanisms for miRNAs in the development of CRC. We will also discuss the potential use of miRNAs as biomarkers for early diagnosis and prognosis of CRC. Furthermore, with advancements in RNA delivery technology, it is anticipated that manipulation of miRNAs may offer an alternative therapy for CRC treatment.

Novel evidence for a PIWI-interacting RNA (piRNA) as an oncogenic mediator of disease progression, and a potential prognostic biomarker in colorectal cancer.

BackgroundEmerging evidence suggests that PIWI-interacting RNAs (piRNAs) may be important epigenetic regulators of gene expression in human cancers; however, their functional and clinical significance in colorectal cancer (CRC) remains unknown.MethodsWe performed piRNA expression profiling in paired cancer and normal tissues through small RNA-sequencing. The clinical significance of candidate piRNAs was investigated, and independently validated in 771 CRC patients from three independent cohorts. The biological function of piRNAs was characterized in cell lines, followed by identification and validation of downstream target genes in CRC tissues.ResultsWe identified piR-1245 as a novel and frequently overexpressed noncoding RNA in CRC, and its expression significantly correlated with advanced and metastatic disease. Patients with high piR-1245 expression experienced significantly shorter overall survival, and multivariate analysis identified its expression to serve as an independent prognostic biomarker in CRC. Functionally, piR-1245 acts as an oncogene and promotes tumor progression, and gene expression profiling results identified a panel of downstream target-genes involved in regulating cell survival pathway. Based upon piRNA:mRNA sequence complementarity, we identified a panel of tumor suppressor genes (ATF3, BTG1, DUSP1, FAS,NFKBIA, UPP1, SESN2, TP53INP1 and MDX1) as direct targets of piR-1245, and successfully validated an inverse correlation between their expression and piR-1245 in CRC.ConclusionsWe for the first time have identified the role for a PIWI-interacting noncoding RNA, piR-1245, as a novel oncogene and a potential prognostic biomarker in colorectal cancer.

SNORA21 – An Oncogenic Small Nucleolar RNA, with a Prognostic Biomarker Potential in Human Colorectal Cancer

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.

Abstract 768: The ATP6V1C2 (a vacuolar-ATPase gene) is a novel early prognosticator for colorectal cancer

Background: Vacuolar (v) -ATPases are expressed in certain tissues and cell types, and hypothesized to detoxify intracellular environments by expelling protons across plasma membranes, thereby contributing to acidified extracellular environments of solid tumors. Acidic microenvironments suppress anti-cancer cytotoxic T-cells and activate metalloproteinases, promoting tumor cell survival, motility and invasion. Scarce nutrients, hypoxia, and accelerated metabolism that favor pathways that produce excess intracellular protons by promoting glycolysis and lactic acid fermentation, lead to selection of tumor cells that adapted to and countered toxic/acidic cytoplasm. It is unclear whether colorectal cancers (CRCs) upregulate proton-expelling mechanisms, and whether associated genes are prognostic biomarkers. Aim: We hypothesized that adaptations through upregulation of proton transporting genes is prognostic for CRC. This study aims to identify transcriptional adaptations that CRCs undergo in response to acidic microenvironments and evaluate their prognostic value in multiple cohorts of CRC patients. Methods: Comprehensive microarray data from 222 CRCs obtained from The Cancer Genome Atlas were used to screen for genes upregulated in CRCs vs. normal mucosae, and stage III and IV vs. early staged tumors. Filtered genes involved in metabolite transport were tested for their ability to predict survival using Kaplan-Meier survival analysis with a stringent median cutoff. A second cohort was used to corroborate initial findings. A third cohort of matching polyps and adenomas was used to determine if candidate gene upregulation can be detected in pre-cancerous lesions. Functional analysis was performed using siRNA knockdown to determine if the candidate gene is critical for CRC proliferation. Results: Progressive upregulation of ATP6V1C2 from early to late stage CRCs was detected in two separate multinational patient cohorts, where its high expression was correlated with shorter overall survival. Receiver operating characteristic (ROC) curve analysis confirmed that ATP6V1C2 expression successfully discriminated between cancerous and non-cancer tissues. Fisher9s exact test confirmed that high ATP6V1C2 expression correlated with advanced depth of invasion (T stage), and distant and liver metastasis. ATP6V1C2 expression was increased in pre-cancerous polyps. SiRNA knockdown of ATP6V1C2 inhibited cell proliferation in high expressing cell lines (HCT116 and HCT15) but not in low expressing RKO and HT29. Citation Format: Timothy J. Zumwalt, Kunitoshi Shigeyasu, Wenhao Weng, Yoshinaga Okugawa, Jinsei Miyoshi, Ajay Goel. The ATP6V1C2 (a vacuolar-ATPase gene) is a novel early prognosticator for 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 768.

Abstract 4926: Clinical significance of SNORA21, an H/ACA box snoRNA, as a metastasis predicting and prognostic biomarker in colorectal cancer

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.