Oncogenic Role In Colorectal Cancer Research Articles

DEAD-box helicase 27 promotes colorectal cancer growth and metastasis and predicts poor survival in CRC patients

Copy number alterations (CNAs) are crucial for colorectal cancer (CRC) development. In this study, DEAD box polypeptide 27 (DDX27) was identified to be highly amplified in both TCGA CRC (474/615) and primary CRC (47/103), which was positively correlated with its mRNA overexpression. High DDX27 mRNA (N = 199) and protein expression (N = 260) predicted poor survival in CRC patients. Ectopic expression of DDX27 increased CRC cells proliferation, migration and invasion, but suppressed apoptosis. Conversely, silencing of DDX27 exerted opposite effects in vitro and significantly inhibited murine xenograft tumor growth and lung metastasis in vivo. Up-regulation of DDX27 enhanced and prolonged TNF-α-mediated NF-κB signaling. Nucleophosmin (NPM1) was identified as a binding partner of DDX27. DDX27 increased nuclear NPM1 and NF-κB-p65 interaction to enhance DNA binding activity of NF-κB. Silencing NPM1 abrogated DDX27-activating NF-κB signaling and its tumor-promoting function. Together, DDX27 is overexpressed and plays a pivotal oncogenic role in CRC.

Abstract 3356: Novel evidence for AZIN1 RNA editing-mediated oncogenic role in colorectal cancer

Abstract 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

Long non-coding RNA FBXL19-AS1 plays oncogenic role in colorectal cancer by sponging miR-203

Long non-coding RNAs (lncRNAs) have emerged as critical regulators of the progression of human cancers, including colorectal cancer (CRC). The study of genome-wide lncRNA expression patterns in metastatic CRC could provide novel mechanism underlying CRC carcinogenesis. In here, we determined the lncRNA expression profiles correlating to CRC with or without lymph node metastasis (LNM) based on microarray analysis. We found that 2439 lncRNAs and 1654 mRNAs were differentially expressed in metastatic CRC relative to primary CRC. Among these dysregulated lncRNAs, FBXL19-AS1 was the most significantly upregulated lncRNA in metastatic tumors. Functionally, knockdown of FBXL19-AS1 played tumor-suppressive effects by inhibiting cell proliferation, migration and invasion in vitro and tumor growth and metastasis in vivo. Overexpression of FBXL19-AS1 was markedly correlated with TNM stage and LNM in CRC. Bioinformatics analysis predicted that miR-203 was potentially targeted by FBXL19-AS1, which was confirmed by dual-luciferase reporter assay. Pearson's correlation analysis showed that miR-203 expression was negatively related to FBXL19-AS1 in tumor tissues. Finally, miR-203 inhibition abrogated the effect of FBXL19-AS1 knockdown on the proliferation and invasion of LoVo cells. Our results reveal the cancer-promoting effect of FBXL19-AS1, acting as a molecular sponge in negatively modulating miR-203, which might provide a new insight for understanding of CRC development.

711 - Ring Finger Protein 6 Exerts an Oncogenic Role in Colorectal Cancer by Activating WNT/β-Catenin Pathway Through TLE3 Ubiquitin Degradation

711 - Ring Finger Protein 6 Exerts an Oncogenic Role in Colorectal Cancer by Activating WNT/β-Catenin Pathway Through TLE3 Ubiquitin Degradation

Clinical relevance of the transcriptional signature regulated by CDC42 in colorectal cancer

CDC42 is an oncogenic Rho GTPase overexpressed in colorectal cancer (CRC). Although CDC42 has been shown to regulate gene transcription, the specific molecular mechanisms regulating the oncogenic ability of CDC42 remain unknown. Here, we have characterized the transcriptional networks governed by CDC42 in the CRC SW620 cell line using gene expression analysis. Our results establish that several cancer-related signaling pathways, including cell migration and cell proliferation, are regulated by CDC42. This transcriptional signature was validated in two large cohorts of CRC patients and its clinical relevance was also studied. We demonstrate that three CDC42-regulated genes offered a better prognostic value when combined with CDC42 compared to CDC42 alone. In particular, the concordant overexpression of CDC42 and silencing of the putative tumor suppressor gene CACNA2D2 dramatically improved the prognostic value. The CACNA2D2/CDC42 prognostic classifier was further validated in a third CRC cohort as well as in vitro and in vivo CRC models. Altogether, we show that CDC42 has an active oncogenic role in CRC via the transcriptional regulation of multiple cancer-related pathways and that CDC42-mediated silencing of CACNA2D2 is clinically relevant. Our results further support the use of CDC42 specific inhibitors for the treatment of the most aggressive types of CRC.

Dual effect of WISP-1 in diverse pathological processes.

Wnt-1 inducible signaling pathway-1 (WISP-1), also known as CCN-4, belongs to the connective tissue growth factor (CTGF) family. WISP-1 is primarily expressed in embryonic stem cells and is involved in adult organ development. WISP-1 participates in many cellular processes, including proliferation, differentiation, apoptosis and adhesion. In addition, WISP-1 plays an important role in diverse pathophysiological processes, such as embryonic development, inflammation, injury repairs and cancers. Recent studies showed that WISP-1 was highly correlated with tumor progression and malignant transformation, whereas it played an oncogenic role in colorectal cancer, cholangiocarcinoma, hepatocellular carcinoma and breast cancer. However, interestingly, WISP-1 exerts a tumor-suppressing role in lung and prostate cancers. WISP-1 promotes cell proliferation, adhesion, motility, invasion, metastasis and epithelial-to-mesenchymal transition via particular signaling pathways. In this review, we discussed the structure, expression profile, functions, clinical significance and potential mechanisms of WISP-1 in cancer and non-neoplastic diseases.

Downregulation of SMC1A inhibits growth and increases apoptosis and chemosensitivity of colorectal cancer cells

ObjectiveThe structural maintenance of chromosomes (SMC) 1A protein is a component of the cohesin multiprotein complex that is essential for sister chromatid cohesion. SMC1A gene mutations have been reported in colorectal cancer. This study aimed to investigate the role of SMC1A gene expression in colorectal cancer in vitro.MethodsSMC1A gene expression was silenced by lentivirus-mediated infection with small interfering RNA (siRNA) in the human colorectal cancer cell line HT-29. Cell proliferation rates, SMC1A mRNA and protein levels, apoptosis and chemosensitivity to oxaliplatin were evaluated using routine in vitro assays, real-time polymerase chain reaction, Western blotting and flow cytometry.ResultsKnockdown of SMC1A protein and mRNA levels resulted in the inhibition of cell proliferation, an increased rate of apoptosis and enhanced chemosensitivity to oxaliplatin in HT-29 cells.ConclusionsThe findings of this study suggest that SMC1A plays an oncogenic role in colorectal cancer and that it might be a promising target for colorectal cancer therapy.

Increased TEAD4 expression and nuclear localization in colorectal cancer promote epithelial-mesenchymal transition and metastasis in a YAP-independent manner.

Dysregulation of the Hippo pathway occurs in a variety of cancers and often correlates with a poor prognosis. To further explore the potential role of Hippo pathway dysregulation in tumor development and progression, we investigated its downstream transcription factor TEAD4 in colorectal cancer (CRC). Increased expression and nuclear localization of TEAD4 were found in a significant portion of CRC tissues, in association with metastasis and a poor prognosis. In CRC cells, TEAD4 knockdown induced the mesenchymal-epithelial transition and decreased cell mobility in vitro and metastasis in vivo. Microarray analysis revealed that TEAD4 promoted cell adhesion and upregulated the epithelial-mesenchymal transition-related transcriptome in CRC cells. Vimentin was identified as a new direct target gene mediating TEAD4 function in CRC cells, whereby forced vimentin expression markedly reversed TEAD4-knockdown-induced cell morphological changes and decreased mobility. Interestingly, rescued expression of both WT TEAD4 and a Y429H mutant can reverse the mesenchymal-epithelial transition and increase vimentin expression, cell mobility and metastatic potential in TEAD4-knockdown CRC cells. The discrepant expression of YAP and TEAD4 in CRC tissues, the rescue ability of TEAD4 mutant defect in YAP binding and no effect on vimentin expression by YAP knockdown in CRC cells, all implicated a YAP-independent manner of TEAD4 function in CRC. Furthermore, vimentin positively correlated and CDH1 reversely correlated with the level of TEAD4 in CRC tissues and xenograft tumors. Our results suggest that TEAD4 nuclear expression can serve as a biomarker for CRC progression and poor prognosis. The transcription factor TEAD4 regulates a pro-metastasis transcription program in a YAP-independent manner in CRC, thus providing a novel mechanism of TEAD4 transcriptional regulation and its oncogenic role in CRC, independently of the Hippo pathway.

Identification of the DEAD box RNA helicase DDX3 as a therapeutic target in colorectal cancer.

Identifying druggable targets in the Wnt-signaling pathway can optimize colorectal cancer treatment. Recent studies have identified a member of the RNA helicase family DDX3 (DDX3X) as a multilevel activator of Wnt signaling in cells without activating mutations in the Wnt-signaling pathway. In this study, we evaluated whether DDX3 plays a role in the constitutively active Wnt pathway that drives colorectal cancer. We determined DDX3 expression levels in 303 colorectal cancers by immunohistochemistry. 39% of tumors overexpressed DDX3. High cytoplasmic DDX3 expression correlated with nuclear β-catenin expression, a marker of activated Wnt signaling. Functionally, we validated this finding in vitro and found that inhibition of DDX3 with siRNA resulted in reduced TCF4-reporter activity and lowered the mRNA expression levels of downstream TCF4-regulated genes. In addition, DDX3 knockdown in colorectal cancer cell lines reduced proliferation and caused a G1 arrest, supporting a potential oncogenic role of DDX3 in colorectal cancer. RK-33 is a small molecule inhibitor designed to bind to the ATP-binding site of DDX3. Treatment of colorectal cancer cell lines and patient-derived 3D cultures with RK-33 inhibited growth and promoted cell death with IC50 values ranging from 2.5 to 8 μM. The highest RK-33 sensitivity was observed in tumors with wild-type APC-status and a mutation in CTNNB1. Based on these results, we conclude that DDX3 has an oncogenic role in colorectal cancer. Inhibition of DDX3 with the small molecule inhibitor RK-33 causes inhibition of Wnt signaling and may therefore be a promising future treatment strategy for a subset of colorectal cancers.

Abstract 3570: Identification of the DEAD box RNA helicase DDX3 as a therapeutic target in colorectal cancer

Abstract Over 85% of colorectal cancers is driven by aberrations in the Wnt-signaling pathway. Thus, identifying druggable targets in this pathway can be beneficial for optimizing colorectal cancer treatment. Within this context, a member of the RNA helicase gene family, DDX3, has been identified to exhibit oncogenic properties in breast and lung carcinomas as well as medulloblastomas. Notably, recent studies have identified DDX3 as a multilevel activator of Wnt-signaling in both normal and transformed cells without activating mutations in the Wnt signaling pathway. In this study, we evaluated whether DDX3 also plays a role in the constitutionally activated Wnt-signaling that drives colorectal cancer and therefore could be a potential therapeutic target in this cancer type. To determine if DDX3 is expressed in colorectal cancers, we immunohistochemically stained a cohort of 303 Dutch and German colorectal cancer patients. We found 40.4% of these tumors to overexpress DDX3 in comparison to the surrounding normal tissue. DDX3 expression was found predominantly in the cytoplasm and occasionally in the nucleus. High cytoplasmic DDX3 expression correlated with nuclear Beta-catenin expression, a marker of activated Wnt-signaling. The presence of nuclear DDX3 expression correlated with shorter overall survival (HR = 2.38, 95% CI 1.45-3.93, p < 0.001). Functionally, we validated these findings in vitro and found that inhibition of DDX3 with siRNA resulted in reduced proliferation and a G1-arrest in the HCT116 and HT29 colorectal cancer cell lines. This finding further supports the potential oncogenic role of DDX3 in colorectal cancer. With respect to targeting DDX3, we developed a small molecule inhibitor of DDX3, referred to as RK-33. RK-33 is designed to bind to the ATP-binding site of DDX3 and abrogate its functional activity. As proof of principle, we demonstrated that RK-33 binds preferentially to DDX3 and not to DDX5 and DDX17, other members of the RNA helicase family. Moreover, RK-33 inhibited the helicase activity in an in vitro assay. Furthermore, treatment of colorectal cancer cell lines and patient derived 3D- tumor cell cultures indicated that RK-33 inhibits growth and promotes cell death with IC-50 values ranging from 2.5 to 8 uM. To further elucidate the mechanism of RK-33, we studied if inhibition of DDX3 with RK-33 could cause inhibition of Wnt-signaling in colorectal cancer cell lines. Treatment with RK-33 indeed resulted in reduced TCF-reporter activity and lowered the mRNA expression levels of the Wnt-signaling downstream target genes AXIN-2, C-MYC, CCND1 and BIRC5A. Overall, we conclude that DDX3 has an oncogenic role in colorectal cancer. Inhibition of DDX3 with the small molecule inhibitor RK-33 causes potent inhibition of Wnt-signaling and is a promising future treatment strategy in colorectal cancer. Citation Format: Marise R. Heerma van Voss, Farhad Vesuna, Kari Trumpi, Justin Brilliant, Liudmila L. Kodach, Folkert H.M. Morsink, G. Johan A. Offerhaus, Horst Buerger, Elsken van der Wall, Paul J. van Diest, Venu Raman. Identification of the DEAD box RNA helicase DDX3 as a therapeutic target in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3570. doi:10.1158/1538-7445.AM2015-3570

Increased Sushi repeat-containing protein X-linked 2 is associated with progression of colorectal cancer.

Sushi repeat-containing protein X-linked 2 (SRPX2) is a novel chondroitin sulfate proteoglycan overexpressed in gastrointestinal cancer. Its role in tumor biology remains unknown. The aim of this study was to investigate the expression of SRPX2 in colorectal cancer and its potential association with cancer progression. The expression of SRPX2 and its clinicopathological significance was evaluated using immunohistochemistry in a tissue microarray including 88 colon cancer and pairing normal tissues. The impact of SRPX2 on behavior of colorectal cancer cells and possible mechanism was explored using gene transfection and silencing. Strong staining of SRPX2 was noted in 71 (80.7 %) of 88 colon cancer specimen and 30 (34.1 %) of 88 adjacent normal tissues (P < 0.001). The expression of SRPX2 was significantly correlated with histological differentiation grade (P = 0.003), infiltration depth (P = 0.003), and clinical stage (P = 0.006). The expression of SRPX2 was significantly higher in HCT116 than in HT29 and SW480 cells. Suppression of endogenous SRPX2 expression by small interfering ribonucleic acid (siRNA) in HCT116 cells resulted in significant reduction in the ability of cell proliferation, adhesion, migration, and invasion. Up-regulation of endogenous SRPX2 in SW480 cells significantly promoted the migration and invasion of SW480 cells. In addition, inhibition of SRPX2 by siRNA led to notable down-regulation of β-catenin, matrix metalloproteinase (MMP)-2, and MMP-9. These findings indicate that overexpressed SRPX2 exerts an oncogenic role in colorectal cancer. SRPX2 may promote the invasion of colorectal cancer through MMP-2 and MMP-9 modulated by Wnt/β-catenin pathway.

MiR-203 Suppresses ZNF217 Upregulation in Colorectal Cancer and Its Oncogenicity

Zinc finger protein 217 (ZNF217) is essential for cell proliferation and has been implicated in tumorigenesis. However, its expression and exact roles in colorectal cancer (CRC) remain unclear. In this study, we demonstrated that ZNF217 expression was aberrantly upregulated in CRC tissues and associated with poor overall survival of CRC patients. In addition, we found that ZNF217 was a putative target of microRNA (miR)-203 using bioinformatics analysis and confirmed that using luciferase reporter assay. Moreover, in vitro knockdown of ZNF217 or enforced expression of miR-203 attenuated CRC cell proliferation, invasion and migration. Furthermore, combined treatment of ZNF217 siRNA and miR-203 exhibited synergistic inhibitory effects. Taken together, our results provide new evidences that ZNF217 has an oncogenic role in CRC and is regulated by miR-203, and open up the possibility of ZNF217- and miR-203-targeted therapy for CRC.

NIRF is frequently upregulated in colorectal cancer and its oncogenicity can be suppressed by let-7a microRNA

Np95 ICBP90 RING finger (NIRF) is essential for the regulation of cell proliferation and has been implicated in tumorigenesis. However, the role of NIRF in colorectal cancer (CRC) remains unclear. In this study, we demonstrated that NIRF expression was aberrantly increased in CRC tissues and associated with poor overall survival. Bioinformatics analysis indicated that NIRF was a putative target of the microRNA let-7a, which was confirmed by luciferase reporter assay. We then demonstrated in vitro that enforced expression of let-7a, or knockdown of NIRF, led to reduced CRC cell proliferation due to cell cycle arrest at the G0/G1 phase and reduced cell migration. Finally, an in vivo tumorigenicity assay in nude mice showed that synthetic let-7a suppressed NIRF expression and reduced tumor growth. Taken together, our results provide new evidence that NIRF has an oncogenic role in CRC. This opens up the possibility of targeting NIRF and let-7a for CRC therapy.