Therapeutic Target For Colon Cancer Research Articles

Low expression of bestrophin-2 is associated with poor prognosis in colon cancer.

The purpose of this research was to confirm the prognostic value of bestrophin-2 (BEST2), one of the hub genes in colon cancer, via bioinformatics analysis and validation in public databases and immunohistochemistry detection. The GEO2R online tool and Venn diagram software were utilized to identify differentially expressed genes (DEGs) from expression profiles, including GSE20916, GSE44861 and GSE74602, from the Gene Expression Omnibus (GEO). The overall survival (OS) and disease-free survival (DFS) of colon cancer patients from The Cancer Genome Atlas (TCGA) were analyzed through Kaplan-Meier survival curves. Verification of the significance of BEST2 in colon cancer was based on TCGA, Genotype Tissue Expression (GTEx) and 10 datasets from GEO. BEST2 expression was detected with immunohistochemistry (IHC) in 330 colon tissue samples on microarrays including 165 colon cancerand 165 adjacent normal tissues. For further validation, comprehensive analysis from tissue microarrays and multiple datasets was performed by the summarizing of receiver operating characteristic (SROC) curves and the standard mean differences (SMDs). BEST2 expression in various kinds of colon cancer tissues and cell lines in the context of pancancer was obtained from the Expression Atlas database. The CBioPortal database was queried to identify BEST2 gene alterations and mutation status in colon cancer. Correlated genes (CEGs) with BEST2 and DEGs from public database data were assembled for functional and pathway enrichment analysis. We identified 85 DEGs from the three datasets and screened out BEST2 as a prognostic predictor via the TCGA database. Colon cancer patients with high expression of BEST2 had better survival than patients with low BEST2 (HR=0.5, P=0.006) as shown in Kaplan-Meier survival curves in GEPIA. In all, 1463 colon cancer tissues and 1023 colon normal tissues were gathered via public databases as well as in-house tissue microarrays. The comprehensiveexpression analysis suggested low-expression of BEST2 in colon cancer (SMD=-2.48, 95% CI [-3.15- -1.80]) and the notable efficacy of BEST2 expression in differentiating colon cancer from noncancer samples (AUC=0.97). Gene alteration status of BEST2 occurred in 5% of colon cancer cases, mostly missense mutations and deep deletions. Genes positively correlated with BEST2 and DEGs primarily aggregated in pathways such as anion absorption, digestive juice secretion, cAMP signaling and so on (P<0.05). Ampleevidencesupportsthe role of BEST2 in distinguishing colon cancer from normal tissues in this research. Low expression of BEST2 is correlated with a shorter OS, which implies that BEST2 can be employed as a potential biomarker and therapeutictarget in colon cancer.

Silencing ESRP1 expression promotes caspase-independent cell death via nuclear translocation of AIF in colon cancer cells

Epithelial splicing regulatory protein 1 (ESRP1) is overexpressed in the majority of cancer types, while downregulated in a few cancers, thus it has emerged as a tumorigenic or a tumor suppressor depending on disease context and cell type. Moreover, the underlying molecular mechanism of ESRP1 is poorly understood in cancer progression. Here, we initially analyzed Clinical Proteomic Tumor Analysis Consortium (CPTAC), colon tissue microarray, and colon cancer cells to evaluate the ESRP1 expression levels in colorectal cancer subtypes. The association between the expression of ESRP1 and cell death signaling pathways was evaluated in colon cancer cells. Furthermore, silencing ESRP1 was performed to detect the relation between ESRP1 and apoptosis-inducing factor (AIF). Subsequently, translocation of AIF and apoptosis were analyzed by immunofluorescence assay and FACS, respectively. ESRP1 is found to be expressed at high levels in the early stage, and gradually decreases with the increasing colorectal cancer stage, wherein epithelial cell to mesenchymal cell transition (EMT) occurs during cancer progression. Moreover, ESRP1 silencing in HCT116 colorectal cancer cells reveals the translocation of the caspase-independent cell death marker AIF to the nucleus, thereby enhancing the DNA damage response, which inevitably induces cancer cell death. Our results demonstrate that silencing ESRP1 in colorectal cancer cells promotes HCT116 cell death by inducing caspase-independent cell death via regulation of CD44 alternative splicing. Collectively, our findings provide an insight into ESRP1 as a therapeutic target in colon cancer.

A Novel Defined Necroptosis-Related miRNAs Signature for Predicting the Prognosis of Colon Cancer.

ObjectiveThis study aims at exploring the relationship between necroptosis-related miRNAs and colon cancer prognosis.MethodsWe downloaded the miRNA sequencing data from the TCGA, and eight differentially expressed necroptosis-related miRNAs were screened. Then, we used Cox regression analysis to establish a prediction model of necroptosis-related miRNA. Finally, the prognosis related miRNAs were used to predict the target genes, and functional analysis was used to explore the potential mechanism of these target genes.ResultsThe miRNA-seq data of 444 COAD cases were downloaded from TCGA. We identified 8 differentially expressed miRNAs (has-miR-16-5p, has-miR-141-3p, has-miR-148a-3p, has-miR-425-5p, has-miR-7-5p, has-miR-223-3p, has-miR-200a-5p, and has-miR-500a-3p), then Cox analysis was performed for determining eight-miRNA signature prognostic biomarkers with obviously different OS. The area under the curve (AUC) of receiver operating characteristic (ROC) curve for predicting 1-, 3-, and 5-year survival were 0.663, 0.653 and 0.639, respectively. The multivariate analysis also implied that the risk score was an independent prognostic factor considering other confounding factors (HR = 1.847, 95% CI = 1.197–2.848, P = 0.006). According to the Kaplan–Meier analysis, the expression of hsa-miR-500a-3p (P = 0.003), hsa-miR-16-5p (P = 0.004) and hsa-miR-148a-3p (P = 0.035) significantly affected OS outcomes. We predicted the target genes of these three miRNAs and then screened 10 hub genes (CCND1, SMAD3, SMAD2, CDK1, TGFB2, CDC25A, CHEK1, VEGFA, CCNE1, WEE1). In addition, CHEK1 was associated with the survival prognosis.ConclusionOur study demonstrated that necroptosis is closely associated with colon cancer, and the model of eight necroptosis-related miRNAs are potentially useful prognostic biomarkers and therapeutic targets for colon cancer.

M2 Macrophage Derived Extracellular Vesicle-Mediated Transfer of MiR-186-5p Promotes Colon Cancer Progression by Targeting DLC1.

Colon cancer (CC) is one of the most common malignances in digestive tract. M2-polarized macrophages within the tumor microenvironment could facilitate CC cell growth by transferring molecules via extracellular vesicles, but the mechanisms are not fully elucidated. The current study aims to identify the possible effectors in M2 macrophage-derived extracellular vesicles (M2-EVs) and reveal related molecular mechanisms. In our study, we validated the promotion effects of M2-EVs on the proliferation and motility of CC cells, which was found to be dependent on the EVs enclosed molecules by a mild EVs digestion assay. Then we found that miR-186-5p was enriched in M2-EVs and was responsible for the tumor promoting functions of M2-EVs. Furthermore, mechanism investigation revealed M2-EVs transferring miR-186-5p inhibited DLC1 expression by targeting its 3'UTR, and restored DLC1 successfully neutralized the tumor-promoting effects of M2-EVs transferring miR-186-5p via inhibiting the β-catenin pathway. Our study revealed that M2-EVs facilitates the growth and motility of CC cells by delivering the enclosed miR-186-5p, which directly targets DLC1 mRNAs and facilitates their degradation, which could provide a potential biomarker and therapeutic target for CC.

Role of Long Noncoding RNA Regulator of Reprogramming in Colon Cancer Progression via Epidermal Growth Factor Receptor Signaling.

Background: Long intergenic noncoding RNA regulator of reprogramming (linc-ROR) is a novel long noncoding RNA that exhibits significant effects on cancer progression. This research presented that linc-ROR had a crucial part in promoting biological characteristics associated with worse prognosis in colon cancer. Method: Bioinformatics analysis was performed to predict signaling pathways related to linc-ROR. In addition, western blot, quantitative reverse transcription-polymerase chain reaction, RNA-pulldown, cell proliferation assays, colony formation assays, wound healing assays, and transwell assays were applied to detect the role and regulation of particular molecules. Results: Our results showed that the knockdown of linc-ROR reduced cell invasion, proliferative ability, and migration in colon cancer. Further evaluation verified that downregulating linc-ROR inhibited the activation of epidermal growth factor receptor (EGFR) signaling. In addition, cbl-b, a kind of E3 ubiquitin ligase that increases the degradation of EGFR, was found to be a potential linc-ROR target. Conclusions: Based on our findings, it was presented that linc-ROR served a role as a tumor-promoting factor via repressing the ubiquitination and degradation of EGFR signaling, which indicated that it could be a possible prognostic marker and therapeutic target for colon cancer.

MiR-142-3p Regulates Tumor Cell Autophagy and Promotes Colon Cancer Progression by Targeting TP53INP2

Objectives: Colon cancer (CC) is the third largest cancer worldwide. Investigation of the molecular mechanism of CC progression helps to explore novel therapeutic targets. We attempted to understand the modulatory mechanism of miR-142-3p in CC cell autophagy and CC progression, which will lay a theoretical groundwork for seeking potential diagnostic and therapeutic targets for CC. Methods: Through bioinformatics methods, miRNA expression data were subjected to differential analysis for identification of target miRNA. Downstream target mRNAs were predicted, and gene set enrichment analysis was completed. qRT-PCR assessed gene expression in cells. Cell counting kit-8, cell doubling time calculation, colony formation, and flow cytometry were used to assess cellular biological functions. Dual-luciferase assay was used for targeting relationship validation of the target miRNA and mRNA. Western blot was performed to evaluate expression of proteins related to HEDGEHOG signaling pathway and autophagy. Results: miR-142-3p was markedly highly expressed in CC, and high miR-142-3p expression in CC patients was implicated with relatively poor prognosis. Overexpressing miR-142-3p facilitated proliferation and inhibited apoptosis of CC cells, whereas silencing it produced an opposite result. miR-142-3p targeted and decreased TP53INP2 level. TP53INP2 overexpression suppressed the HEDGEHOG signaling pathway and induced the activation of CC cell autophagy. Rescue experiments revealed that influence of the miR-142-3p inhibitor on CC cell proliferation and apoptosis could be reversed by silencing TP53INP2. Conclusion: miR-142-3p hampered tumor cell autophagy and promoted CC progression via targeting TP53INP2, which will offer a fresh research orientation for the diagnosis of CC.

RNF2 promotes the progression of colon cancer by regulating ubiquitination and degradation of IRF4

Ring finger protein 2 (RNF2), as a well-known E3 ligase, has an oncogenic role in various cancers. The role of RNF2 in colon cancer is still unknown. The aim of this work is to determine the biological role of RNF2 in colon cancer. We first examined the expression of RNF2 and interferon regulatory factor 4 (IRF4) in colon cancer patients and colon cancer cell lines (SW480 and HCT116). Compared with normal tumor-adjacent tissues, RNF2 was up-regulated whereas IRF4 was down-regulated in the colon cancer tissues. RNF2 was also up-regulated in colon cancer cells with respect to human fetal colon epithelial cells. RNF2 overexpression enhanced the ability of proliferation, migration and invasion of SW480 cells, whereas RNF2 knockdown caused an opposite result in HCT116 cells. Furthermore, a tumor xenograft model was constructed to verify the impact of RNF2 overexpressed-SW480 cells on tumor growth. RNF2 up-regulation elevated Ki-67 proliferation index, accelerated the growth of tumor tissues, and led to severe colon tissue damage in the tumor xenograft mice. In addition, RNF2 interacted with IRF4, and repressed IRF4 protein expression. IRF4 was a substrate of RNF2, and RNF2 promoted the ubiquitination and degradation of IRF4. RNF2 overexpression increased the ability of proliferation, migration and invasion in SW480 cells by promoting the ubiquitination and degradation of IRF4. In conclusion, this work demonstrated that RNF2 promoted tumor growth in colon cancer by regulating ubiquitination and degradation of IRF4. Thus, RNF2 may be served as a potential therapeutic target for colon cancer.

MiR-1236-3p Inhibits the Proliferation, Invasion, and Migration of Colon Cancer Cells and Hinders Epithelial-Mesenchymal Transition by Targeting DCLK3.

BackgroundDysregulated microRNAs (miRNAs) are common in human cancer and are involved in the proliferation, promotion, and metastasis of tumor cells. Therefore, this study aimed to evaluate the expression and biological function of miR-1236-3p in colon cancer.MethodsThis study screened the miRNA in normal and colon cancer tissues through array analysis. In addition, quantitative Reverse Transcription–Polymerase Chain Reaction (qRT-PCR) analysis was performed to validate the expression of miR-1236-3p in normal and tumor tissues from colon cancer patients and cancer cell lines. Online predicting algorithms and luciferase reporter assays were also employed to confirm Doublecortin Like Kinase 3 (DCLK3) was the target for miR-1236-3p. Moreover, the impact of miR-1236-3p on the progression of colon cancer was evaluated in vitro and in vivo. Western blotting and qRT-PCR were also performed to investigate the interactions between miR-1236-3p and DCLK3.ResultsMiR-1236-3p was significantly downregulated in colon cancer tissues and its expression was associated with the TNM stage and metastasis of colon. In addition, the in vitro and in vivo experiments showed that miR-1236-3p significantly promoted cancer cell apoptosis and inhibited the proliferation, invasion, and migration of cancer cells. The results also showed that miR-1236-3p hindered Epithelial–mesenchymal Transition (EMT) by targeting DCLK3. Moreover, the expression of DCLK3 mediated the effects of miR-1236-3p on the progression of cancer.ConclusionsMiR-1236-3p functions as a tumor suppressor in colon cancer by targeting DCLK3 and is therefore a promising therapeutic target for colon cancer.

Integrated Bioinformatics Analysis to Identify Abnormal Methylated Differentially Expressed Genes for Predicting Prognosis of Human Colon Cancer.

ObjectiveTo identify the value of key differentially expressed genes (DEGs) regulated by differentially methylated regions (DMRs) in predicting the prognosis of human colon cancer.Materials and MethodsRNA sequencing data and DNA methylation data of 455 colon adenocarcinoma (COAD) cases and 41 normal controls were downloaded from The Cancer Genome Atlas (TCGA). Gene Ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed by the DAVID database. To identify the hub genes regulated by methylation, univariate Cox and multivariate Cox regression analyses were carried out. A nomogram based on the risk score was built to identify the power of the hub genes to predict prognosis in patients with colon cancer.ResultsA total of 133 DEGs regulated by DMRs were identified through analyzing RNA sequencing data and DNA methylation data from TCGA. GO functional enrichment and KEGG pathway enrichment analysis showed the genes involved in the initiation and progression of colon cancer. Univariate Cox regression analysis and multivariate Cox regression analysis focused on the seven hub genes (CDH4, CR2, KRT85, LGI4, NPAS4, RUVBL1 and SP140) associated with overall survival, the expression of which negatively correlated with their methylation level. The risk score and nomogram model showed that the hub genes served as potential biomarkers for the prognosis prediction of patients with colon cancer.ConclusionOur findings suggest that the DEGs regulated by DMRs are involved in the carcinogenesis and development of colon cancer, and the aberrantly methylated DEGs associated with overall survival of patients may be potential diagnostic and therapeutic targets for colon cancer.

Colorectal cancer cells utilize autophagy to maintain mitochondrial metabolism for cell proliferation under nutrient stress.

Cancer cells reprogram cellular metabolism to maintain adequate nutrient pools to sustain proliferation. Moreover, autophagy is a regulated mechanism to break down dysfunctional cellular components and recycle cellular nutrients. However, the requirement for autophagy and the integration in cancer cell metabolism is not clear in colon cancer. Here, we show a cell-autonomous dependency of autophagy for cell growth in colorectal cancer. Loss of epithelial autophagy inhibits tumor growth in both sporadic and colitis-associated cancer models. Genetic and pharmacological inhibition of autophagy inhibits cell growth in colon cancer–derived cell lines and patient-derived enteroid models. Importantly, normal colon epithelium and patient-derived normal enteroid growth were not decreased following autophagy inhibition. To couple the role of autophagy to cellular metabolism, a cell culture screen in conjunction with metabolomic analysis was performed. We identified a critical role of autophagy to maintain mitochondrial metabolites for growth. Loss of mitochondrial recycling through inhibition of mitophagy hinders colon cancer cell growth. These findings have revealed a cell-autonomous role of autophagy that plays a critical role in regulating nutrient pools in vivo and in cell models, and it provides therapeutic targets for colon cancer.

Study on effects of miR-141-3p in proliferation, migration, invasion and apoptosis of colon cancer cells by inhibiting Bcl2.

This study aimed to investigate the relationship between miR-141-3p and B lymphocyte-2 gene (Bcl2) gene and its biological behavior on colon cancer cell line SW480. qRT-PCR was used to detect the expression level of miR-141-3p in colon cancer tissues and adjacent tissues, as well as in colon cancer cell line and normal human colonic epithelial cell line FHC. MTT assay, wound assay, and Transwell demonstrated the effects of miR-141-3p on colon cancer proliferation, migration and invasion. Targetscan7.1 predictive software and dual luciferase reporter assays were used to detect the targeted regulation of miR-141-3p on the apoptosis-related gene Bcl2. MTT assay, wound assay, Transwell and flow cytometry were used to detect the effect of Bcl2 on miR-141-3p on colon cancer proliferation, migration, invasion and apoptosis. Compared with adjacent tissues, the expression of miR-141-3p in colon cancer tissues was significantly down-regulated. Colon cancer patients with low expression of miR-141-3p had poorer prognosis. Compared with normal colonic epithelial cells, miR-141-3p expression was significantly down-regulated in colon cancer cell lines, and overexpression of miR-141-3p significantly attenuated the proliferation, migration and invasion of colon cancer cells. Knockdown of miR-141-3p significantly promoted the proliferation, migration and invasion of colon cancer cells. miR-141-3p targets the negative regulation of Bcl2. Knockdown of Bcl2 significantly attenuated the promotion of miR-141-3p inhibitor on proliferation, migration and invasion of colon cancer cells and inhibition of apoptosis. Knockdown of Bcl2 significantly enhanced the inhibition effect of miR-141-3p inhibitor on proliferation, migration and invasion of colon cancer cells. In conclusion, miR-141-3p can inhibit the cancer by regulating Bcl2, and miR-141-3p has the potential to become a potential therapeutic target for colon cancer.

Overexpression of the transcribed ultraconserved region Uc.138 accelerates colon cancer progression

Ultraconserved regions (UCRs) are 481 genomic sequences with 100% identity across humans, rats, and mice. Increasing evidence suggests that non-coding RNAs transcribed from UCRs are involved in various diseases, especially cancers. The human transformer 2β gene (TRA2B) encodes a UCR (uc.138) that spans exon 2 and its neighboring introns. TRA2B4 RNA is the only transcript that contains the whole exon 2 among five spliced TRA2B RNA variants (TRA2B1-5). TRA2B4 is upregulated in colon cancer cell lines, although it is not translated to Tra2β protein because of its nuclear retention. Nevertheless, the clinical significance and biological functions of uc.138 in colon cancer cells remain unclear. In this study, RNA in situ hybridization showed that TRA2B4 was predominantly overexpressed in the nucleus of colon adenocarcinoma and adenoma. Overexpression of TRA2B4 in colon cancer HCT116 cells promoted cell proliferation by changing the expression of G2/M-related cell cycle regulators. Moreover, TRA2B4 increased migration and cell viability in a uc.138 sequence-dependent manner. TRA2B4 significantly enhanced tumorigenesis in vivo. Taken together, uc.138 encoded in TRA2B4 plays an oncogenic role in tumor progression and may become a potential biomarker and therapeutic target in colon cancer.

Quantitative Proteomics Analysis of Berberine-Treated Colon Cancer Cells Reveals Potential Therapy Targets.

Simple SummaryColon cancer is one of the most common malignant tumors and beberine has been found to exert potential anti-colon cancer activity in vitro and in vivo. In this study, by using proteomics and bioinformatics approaches, we report that berberine may inhibit the proliferation of colon cancer cells by regulating mitochondrial translation and ribosome biogenesis, as well as by promoting calcium mobilization and metabolism of fat-soluble vitamins. Moreover, GTPase ERAL1 and mitochondrial ribosomal proteins MRPL11, 15, 30, 37, 40, and 52 have great potential to serve as potential therapeutic targets for colon cancer treatment.Colon cancer is one of the most lethal malignancies worldwide. Berberine has been found to exert potential anti-colon cancer activity in vitro and in vivo, although the detailed regulatory mechanism is still unclear. This study aims to identify the underlying crucial proteins and regulatory networks associated with berberine treatment of colon cancer by using proteomics as well as publicly available transcriptomics and tissue array data. Proteome profiling of berberine-treated colon cancer cells demonstrated that among 5130 identified proteins, the expression of 865 and 675 proteins were changed in berberine-treated HCT116 and DLD1 cells, respectively. Moreover, 54 differently expressed proteins that overlapped in both cell lines were mainly involved in mitochondrial protein synthesis, calcium mobilization, and metabolism of fat-soluble vitamins. Finally, GTPase ERAL1 and mitochondrial ribosomal proteins including MRPL11, 15, 30, 37, 40, and 52 were identified as hub proteins of berberine-treated colon cancer cells. These proteins have higher transcriptional and translational levels in colon tumor samples than that of colon normal samples, and were significantly down-regulated in berberine-treated colon cancer cells. Genetic dependency analysis showed that silencing the gene expression of seven hub proteins could inhibit the proliferation of colon cancer cells. This study sheds a light for elucidating the berberine-related regulatory signaling pathways in colon cancer, and suggests that ERAL1 and several mitochondrial ribosomal proteins might be promising therapeutic targets for colon cancer.

Fibrotic Phenotype of Peritumour Mesenteric Adipose Tissue in Human Colon Cancer: A Potential Hallmark of Metastatic Properties.

The impact of tumour associated stroma on cancer metastasis is an emerging field. However, cancer associated genes in peritumoral adipose tissue (pAT) in human colon cancer have not been explored. The aim of this study was to identify differentially expressed genes (DEGs) associated with cancer pathways in mesenteric pAT compared with adjacent adipose tissue. In total, nine patients with colon cancer pathological stage T2/T4 were employed in this study. DEGs were identified in 6 patients employing Nanostring PanCancer Pathway Panel and pathway enrichment analyses were performed. Differential expression of the 5 most up-regulated and 2 down regulated genes was validated with qRT-PCR. Results showed collagen type I alpha 1 chain (COL1A1) p = 0.007; secreted frizzled related protein (SFRP2) p = 0.057; fibroblast growth factor 7 (FGF7) not significant (ns); phospholipase A2, group IIA (PLA2G2A) ns; nerve growth factor receptor (NGFR) ns; lymphoid enhancer binding factor 1 (LEF1) p = 0.03; cadherin 1, Type 1, E-cadherin (epithelial) (CDH1) 0.09. Results have highlighted down-regulation of the Wingless/Integrated (Wnt) pathway in mesenteric pAT compared to distal adipose tissue. Highly upregulated genes in mesenteric pAT were involved in extracellular matrix (ECM)-receptor interactions and focal adhesion. Highly down regulated genes were involved in the cell cycle. Immunohistochemistry revealed differential distribution of COL1A1 showing maximum levels in tumour tissue and gradually decreasing in distant adipose tissue. COL1A1 and down regulation of Wnt pathway may have a role in local invasion and distant metastasis. COL1A1 may represent a stromal prognostic biomarker and therapeutic target in colon cancer.

CircPPP1R12A promotes the progression of colon cancer through regulating CTNNB1 via sponging miR-375.

Circular RNAs (circRNAs) have been identified as potential biomarkers for many cancer, including colon cancer (CC). However, the function and mechanism of circPPP1R12A in CC have not been fully elucidated. Quantitative real-time PCR was employed to assess the expression of circPPP1R12A, microRNA (miR)-375 and catenin beta-1 (CTNNB1). The proliferation, apoptosis, migration and invasion of cells were determined using colony formation assay, flow cytometry, wound healing assay and transwell assay. The protein levels of cell cyclin-related markers and CTNNB1 were detected by western blot analysis. The interaction between miR-375 and circPPP1R12A or CTNNB1 was verified by dual-luciferase reporter assay. Xenograft models were built to evaluate the effect of circPPP1R12A silencing and CTNNB1 overexpression on CC tumor growth in vivo. Our results showed that circPPP1R12A was a highly expressed circRNA in CC tissues and cells. Silenced circPPP1R12A suppressed the proliferation, promoted the apoptosis, and inhibited the migration and invasion of CC cells. MiR-375 could be sponged by circPPP1R12A, and its inhibitor could reverse the inhibition of circPPP1R12A silencing on CC progression. Furthermore, CTNNB1 was a target of miR-375, and its overexpression also abolished the suppression of miR-375 on CC progression. Moreover, circPPP1R12A indirectly regulated CTNNB1 expression by sponging miR-375. Importantly, circPPP1R12A knockdown reduced the tumor growth of CC in vivo, and this effect also could be reversed by overexpressing CTNNB1. Our study proposed that circPPP1R12A might play an oncogenic role in CC, which could act as a potential therapeutic target for CC.

EHD2 Overexpression Suppresses the Proliferation, Migration, and Invasion in Human Colon Cancer

Background: To investigate how EHD2 influences the development of colon cancer. Methods: Immunohistochemistry of 90 colon cancer tissue specimens were determined the expression of EHD2. The lentivirus-EHD2-transfected colon cancer cells were conducted to evaluate the biological behaviors. Results: EHD2 was closely associated with clinic pathological parameters (p < 0.001). EHD2 upregulation was relative with a longer overall survival. The results of the univariate and multivariate analyses indicated that EHD2 could be an independent prognosis marker. EHD2 overexpression suppressed cell invasion and proliferation, but enhanced cell apoptosis and cell cycle arrest. Conclusions: EHD2 might represent a therapeutic target of colon cancer. IMPACT STATEMENT What is already known on this subject? Membrane trafficking is crucial for cell proliferation, differentiation and apoptosis, especially tumorigenesis and development. EHD2 proteins play an important role in the regulation of membrane trafficking in endocytosis. EHD2 has been suggested to participate in the occurrence of some malignancies. What are the new findings? EHD2 could be an independent prognosis marker in colon cancer. EHD2 overexpression suppressed cell invasion and proliferation, but enhanced cell apoptosis and cell cycle arrest in vitro. EHD2 overexpression markedly increased the expression of EMT marker E-cadherin in colon cancer. How might it impact on clinical practice in the foreseeable future? EHD2 overexpression may inhibit tumorigenesis in colon cancer through the modulation of E-cadherin, the critical marker of EMT which is closely related to invasion and distant metastasis of tumor cells.

Schisandrin B Attenuates Colitis-Associated Colorectal Cancer through SIRT1 Linked SMURF2 Signaling.

Colon cancer, a common type of malignant tumor, seriously endangers human health. However, due to the relatively slow progress in diagnosis and treatment, the clinical therapeutic technology of colon cancer has not been substantially improved in the past three decades. The present study was designed to investigate the effects and involved mechanisms of schisandrin B in cell growth and metastasis of colon cancer. C57BL/6 mice received AOM and dextran sulfate sodium. Mice in treatment groups were gavaged with 3.75-30 mg/kg/day of schisandrin B. Transwell chamber migration, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, immunoprecipitation (IP) and immunofluorescence were conducted, and HCT116 cell line was employed in this study. Data showed that schisandrin B inhibited tumor number and tumor size in the AOD+DSS-induced colon cancer mouse model. Schisandrin B also inhibited cell proliferation and metastasis of colon cancer cells. We observed that schisandrin B induced SMURF2 protein expression and affected SIRT1 in vitro and in vivo. SMURF2 interacted with SIRT1 protein, and there was a negative correlation between SIRT1 and SMURF2 expressions in human colorectal cancer. The regulation of SMURF2 was involved in the anticancer effects of schisandrin B in both in vitro and in vivo models. In conclusion, the present study revealed that schisandrin B suppressed SIRT1 protein expression, and SIRT1 is negatively correlated with the induction of SMURF2, which inhibited cell growth and metastasis of colon cancer. Schisandrin B could be a leading compound, which will contribute to finding novel potential agents and therapeutic targets for colon cancer.

Overexpression of ZNF460 predicts worse survival and promotes metastasis through JAK2/STAT3 signaling pathway in patient with colon cancer.

Zinc finger proteins (ZNFs) are a class of protein containing zinc finger domains, and they play an important role in tumor progression. However, as a member of the ZNFs family, the effect of ZNF460 in colon cancer remains unclear. In this study, we found that the expression of ZNF460 protein were markedly increased in clinical colon cancer tissues compared with para-cancer non-cancerous tissues by tissue immunohistochemistry (IHC) and western blot (WB). We also confirmed this result at the mRNA and protein levels of ZNF460 through bioinformatics analysis. In addition, high expression of ZNF460 was correlated with increased depth of invasion (P<0.05), increased lymph node metastasis (P<0.05), distant metastasis (P<0.05) and high blood serum CA19-9 level (P<0.05). High expression of ZNF460 predicted poor overall survival (OS) and recurrence free survival (RFS) in patients with colon cancer. Moreover, multivariate analyses revealed that ZNF460 was an independent prognostic factor in both OS (hazard ratio [HR]: 1.636; 95% confidence interval [CI], 1.028-2.603; P = 0.038) and RFS (HR: 2.215; 95% CI: 1.227-3.997; P = 0.008). The knockdown of ZNF460 suppressed the invasion and metastasis of colon cancer cells in vitro. Mechanistically, we revealed that ZNF460 promotes the activation of the JAK2/STAT3 signaling pathway in colon cancer cells. Taken together, overexpression of ZNF460 predicted worse survival and promoted metastasis through JAK2/STAT3 signaling pathway in patient with colon cancer, and could be a novel therapeutic target in colon cancer.

MiR-219a-1 inhibits colon cancer cells proliferation and invasion by targeting MEMO1

ABSTRACT Colon cancer is the third most common cancer worldwide. Many miRNAs have been reported to be involved in colon cancer progression. However, there are only a few studies on the role of miR-219a-1 in colon cancer, and the molecular mechanisms involved remain unclear. The aim of this study was to investigate the miR-219a-1 level in patients with colon cancer and to explore both the effects and regulatory mechanisms of miR-219a-1 in the malignancy of colon cancer cells. Real-time PCR and western blot analysis were used to analyze the expression levels of miR-219a-1 and mediator of ErbB2-driven cell motility 1. Cell Counting Kit-8, transwell and wound-healing assays were performed to investigate the malignant ability of colon cancer cells. A luciferase assay was performed to explore whether miR-219a-1 could directly bind to 3ʹ-UTR region of MEMO1. miR-219a-1 was found to be downregulated in colon cancer cell lines and in patients with colon cancer. Additionally, miR-219a-1 could inhibit colon cancer cell proliferation, invasion and migration. We identified MEMO1 as a novel potential target gene of miR-219a-1. Luciferase assays showed that miR-219a-1 could directly bind to 3′-UTR of MEMO1. Overexpression of miR-219a-1 in colon cancer cells could inhibit the expression of MEMO1. Furthermore, MEMO1 was upregulated in patients with colon cancer, which was inversely correlated with miR-219a-1 levels. In conclusion, our study revealed that miR-219a-1 exerts anti-tumor effects and regulates colon cancer cell proliferation, invasion and migration by targeting MEMO1, suggesting that miR-219a-1 could act as a therapeutic target in colon cancer.

Abstract A23: Dissecting the SMAD4 metastasis suppressor complex to identify novel prognostic biomarkers and therapeutic targets for colon cancer

Abstract Colon cancer is the second most lethal cancer in men and women in the United States and metastasis is the primary cause of mortality. Hence, there is an urgent need to elucidate the molecular mechanisms underlying metastasis to identify new prognostic biomarkers and therapeutic targets. Previous studies reported that SMAD4 is either mutated frequently (10-20%) or exhibits low expression, which is associated with poor prognosis, presence of metastases, and resistance to 5-fluorouracil (5-FU), the common chemotherapeutic agent for colon cancer. Our analyses confirmed that the probability of overall survival (OS) and relapse-free survival (RFS) is worse in patients with low levels of SMAD4 expression and found that it is associated with resistance to both 5-FU and irinotecan, two of the most widely used chemotherapeutic agents for treating colon cancer. Since our previous studies found that SMAD4 interacts with and inhibits HIF1α, leading to reduced expression of HIF1α target genes such as VEGF (angiogenesis) and GLUT1 (glucose uptake), we hypothesized that SMAD4 forms a metastasis suppressor complex to inhibit colon cancer progression. Here, we report the identification of the components in the SMAD4 metastasis suppressor complex using co-immunoprecipitation and mass spectrometry, with potential roles in regulating metastasis and for conferring resistance to chemotherapy. We will discuss the characterization of candidate genes, which include PI3K/mTOR pathway genes that might serve as biomarkers in predicting disease progression/prognosis and as therapeutic targets to treat metastatic colon cancer. Citation Format: Chen Khuan Wong, Sam Thiagalingam. Dissecting the SMAD4 metastasis suppressor complex to identify novel prognostic biomarkers and therapeutic targets for colon cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr A23.