MBD2 suppresses SFRP1 expression and promotes colorectal cancer development by blocking MED19 binding to its methylated promoter

Colorectal cancer (CRC) is the third most diagnosed cancer worldwide and the second leading cause of cancer-related deaths. Many researchers have reported that abnormal microRNAs (miRs) were expressed in CRC and participated in the occurrence and progression of CRC. However, there are few reports of miR-887-3p regulating CRC development. In the current study, we investigated the abnormal expression of miR-887-3p and also demonstrated its regulatory role and detailed molecular mechanism in CRC. Initially, miRNA expression data were obtained from TCGA-COAD that consisted of 453 CRC samples and 8 normal tissue samples. These were downloaded and analyzed to compare the expression level of miR-887-3p in CRC tissues to that in normal tissues. Moreover, 32 pairs of surgically resected CRC tumors and para-cancer tissues from our hospital were collected. Quantitative real-time PCR (qRT-PCR) was performed to detect miR-887-3p expression levels in CRC tissues, para-cancer tissues, several CRC cell lines, and an intestinal epithelial cell line. Following miR-887-3p mimic transfection in colon cancer SW480 cell line, the regulatory roles of miR-887-3p on cell proliferation, apoptosis, invasion, migration, and epithelial-mesenchymal transition (EMT) were detected through CCK-8, flow cytometry, transwell assay, and Western blot. After potential targeting protein was predicted by bioinformatic websites, the luciferase reporter assay and Western blot were used to confirm the target of miR-887-3p. The targeting protein expressions were detected by Western blot and qRT-PCR. The relationship between miR-887-3p level and the effect of miR-887-3p on P53 expression was evaluated by Western blot and qRT-PCR. The effects of miR-887-3p on CRC cell growth in vivo by xenograft tumor experiments were investigated, and Ki-67 in tumor tissue was determined by immunohistochemistry. Results. The COAD data demonstrated that the expression levels of miR-887-3p in CRC clinical sample tissues and cell line cultures were remarkably lower than para-cancer normal tissues and NCM460 cells (normal colonic epithelial cell line). Functional experiments demonstrated that overexpression of miR-887-3p in SW480 cells significantly reduced proliferation, migration, invasion, and EMT, and promoted cancer cell apoptosis. Additionally, Western blot, qRT-PCR, and luciferase reporter assays confirmed that DNMT1 was a downstream target of miR-887-3p. Moreover, the blocking of DNMT1 by miR-887-3p mimics also promoted P53 expression. Finally, overexpression of DNMT1 in SW480 cells could partially reverse the regulatory effect of miR-887-3p mimics on CRC cell development. From in vivo experiments, overexpression of miR-887-3p could inhibit tumor growth in CRC xenograft mice and reduce the Ki-67 level. Conclusion. The microRNA miR-887-3p is a potential biomarker of CRC. It inhibited CRC cell proliferation, invasion, and EMT, and promoted cell apoptosis through targeting and downregulating DNMT1 and promoting P53 expression. Therefore, miR-887-3p may be a good biomarker and therapeutic target for CRC treatment.

Objective: To assess whether miR-203 regulates DJ-1 expression, affects colorectal cancer cells through PTEN-PI3K/AKT signaling. Methods: Colorectal cancer (CRC) tissues and adjacent tissues were collected followed by analysis of the level of miR-203, DJ-1 and PTEN. miR-203 and DJ-1 level was measured in HCT116, SW480 and normal colorectal cell NCM460. miR-203 mimic or miR-NC was transfected into HCT116 or SW480 cells followed by measuring the level of miR-203, DJ-1, PTEN, p-AKT as well as cell apoptosis and proliferation. Results: Compared with tumor adjacent tissues, tumor tissues showed significantly lower level of miR-203 and PTEN, and higher level of DJ-1. There is a targeted relationship between miR-203 and DJ-1. Compared with NCM460 cell, HCT116 and SW480 cells displayed significantly lower miR-203 level and higher DJ-1 expression. miR-203 mimic significantly reduced DJ-1 and p-AKT level, increased PTEN expression, cell apoptosis and inhibited cell proliferation. Conclusion: Lower miR-203 and higher DJ-1 level is found in CRC patients. Upregulation of miR-203 inhibits DJ-1 expression, increases PTEN expression, impairs PI3K/AKT signaling, inhibits CRC cell proliferation and promotes apoptosis.

Background: It is well known that beside genetic factors such as mutations, epigenetic mechanisms can also contribute to colorectal cancer formation and progression. Although several genes influenced by DNA methylation and miRNA expression alteration have been identified, our knowledge of the background of gene expression alterations during CRC development remains incomplete. Aims: Our aims were to identify DNA methylation markers and miRNAs playing role in left-sided CRC development on the basis of gene expression alterations along the adenoma-carcinoma formation. Materials & methods: Whole genome expression profiling was performed by using HGU133 Plus 2.0 microarrays (Affymetrix) on healthy colonic (n=49), colorectal adenoma (n=49) and left-sided CRC (n=49) biopsy samples and also on laser microdissected (LCM) epithelial and stromal cells from healthy (n=6) and CRC (n=6) samples. Methylation status of genes with gradually decreasing or increasing expression along the adenoma-carcinoma sequence were analyzed on macrodissected (n=10) and LCM (n=5) healthy colonic, adenomatous biopsy (n=10) and LCM (n=5), macrodissected (n=10) and LCM (n=5) left-sided colorectal cancer samples using bisulfite-sequencing PCR (BS-PCR) followed by pyrosequencing. In silico miRNA prediction for the selected genes with miRWALK algorithm, miRNA expression was analyzed on colorectal cancers (n=3), adenomas (n=3) and normal tissue adjacent to tumor (NAT)(n=3) samples using the Human Panel I + II with the miRCURY Universal RT microRNA PCR protocol (Exiqon). In order to investigate the potential impact of DNA methylation on the protein levels PTGDR and SFRP1 immunohistochemistry experiments were performed. Results: A set of transcripts (18 genes including MAL, SFRP1, SULT1A1, PRIMA1, PTGDR) showed decreasing expression (p≤0,01) in the biopsy samples along the adenoma-carcinoma sequence. COL1A2, SFRP2, SOCS3 showed hypermethylation and THBS2 showed hypomethylation both in the adenomas and tumor samples compared to NAT, while BCL2, PRIMA1 and PTGDR showed hypermethylation only in the CRC group. miR-21 was found to be significantly (p<0,01) upregulated in the adenoma and tumor samples compared to the healthy colonic tissue controls that can potentially influence the expression of genes without remarkable DNA methylation alteration (e.g. BCL2, MAL, PTGS2). Decreasing protein levels of PTGDR and SFRP1 could be observed along the adenoma-carcinoma sequence. Conclusion: Genome-wide gene expression-based screening was found to be a suitable approach for the identification of genes, that can be potentially downregulated by DNA hypermethylation or miRNA upregulation. Hypermethylation of the selected markers (COL1A2, SFRP2, SOCS3) or miR21 upregulation might result in reduced expression and may contribute to the formation of colorectal cancer. Citation Format: Bela Molnar, Balint Peterfia, Alexandra Kalmar, Peter Hollosi, Zsofia Brigitta Nagy, Barnabas Wichmann, Ilona Kovalszky, Zsolt Tulassay. DNA hypermethylation or upregulated miRNA21 expression potentially leads to decreased mRNA expression of COL1A2, SFRP2, SOCS3, BCL2, MAL and PTGS2 in left-sided colorectal adenoma and cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 422. doi:10.1158/1538-7445.AM2014-422

Propofol is a commonly used intravenous anesthetic agent that can also suppress the proliferation of various human cancer types, including colorectal cancer (CRC). The present study aimed to investigate whether propofol could induce the ferroptosis of CRC cells by regulating signal transducer and activator of transcription 3 (STAT3). STAT3 expression in normal and CRC tissues was measured. Human normal colonic epithelial NCM460 cells and human CRC SW480 cells were exposed to different concentrations of propofol and then cell viability was detected. SW480 cells were transfected with a vector overexpressing STAT3 and treated with propofol, and the cell viability, colony formation, cell proliferation, iron level, ROS production and ferroptosis of these cells and control cells were evaluated. Overall, the results showed that STAT3 was highly expressed in CRC tissues. Propofol exerted no marked effect on NCM460 cell viability, but inhibited SW480 cell viability in a concentration-dependent manner. Meanwhile, STAT3 was downregulated by propofol in a concentration-dependent manner. Propofol also inhibited CRC cell proliferation and colony formation, and enhanced cellular iron and ROS levels. Additionally, the expression of proteins involved in ferroptosis was also altered by propofol, including the upregulation of CHAC1 and PTGS2 expression in CRC cells, and the inhibition of GPX4 expression. However, STAT3 overexpression blocked the effect of propofol on CRC cells. In conclusion, propofol may trigger the ferroptosis of CRC cells by downregulating STAT3 expression.

BackgroundSecreted frizzled-related protein 1 (SFRP1) is a member of the SFRPs family that modulates the Wnt signal transduction pathway. Recent studies have showed down-regulation of SFRP1 expression in colorectal cancer (CRC). We aimed to evaluate the effect of SFRP1 on the proliferation, migration, invasion and apoptosis of CRC cells in vitro.Materials and methodsWe used real-time fluorescence quantification (RT-PCR) and Western blotting to detect SFRP1 expression in CRC, pericarcinomatous tissues and CRC cell lines. We assessed the influence of overexpression and knockdown of SFRP1 on CRC cell proliferation, migration, invasion, and apoptosis, Western blotting was used to evaluate protein levels of Wnt, β-catenin, and apoptosis-related proteins.ResultsThe expression of SFRP1 was significantly decreased in CRC tissues. Among the six CRC cell lines (sw-480, sw1116, caco-2, ht-29, colo-205, and hct-116), RT-PCR revealed that sw1116 cells had the lowest expression of SFRP1, while caco-2 cells had the highest SFRP1 expression. SFRP1 overexpression in sw1116 cells significantly suppressed cell proliferation while SFRP1 knockdown in caco-2 cells significantly increase the cell proliferation. In addition, overexpression of SFRP1 in sw1116 cells remarkedly suppressed cell migration and invasion, whereas knockdown of SFRP1 in caco-2 cells resulted in significant enhancement of migration and invasion. Furthermore, SFRP1 overexpression in sw1116 cells promoted cell apoptosis. Western blotting showed that SFRP1 overexpression significantly decreased the protein levels of Wnt, β-catenin and apoptosis-related proteins, including MMP2, MMP9, Twist, CDK1, TGF, and Bcl2.ConclusionOur results demonstrate that SFRP1 suppresses cell proliferation, migration and invasion, and promotes apoptosis in CRC cells.

Secreted Frizzled-Related Protein 3 Regulates Activity-Dependent Adult Hippocampal Neurogenesis

Colorectal cancer (CRC) is one of the most common digestive tract cancers and ~90% of CRC‑related deaths are caused by metastasis. MicroRNA (miR)‑129 has been reported to be involved in the metastasis of various malignant tumors. However, the role of miR‑129 in CRC metastasis remains unclear. The purpose of the present study was to identify the potential functions and mechanisms of action of miR‑129 in CRC progression. The expression of miR‑129 and sex‑determining region Y‑related high‑mobility group‑box 4 (SOX4) was determined in CRC tissues or cell lines by reverse transcription‑quantitative PCR, western blot or immunofluorescence assays. The mechanism underlying the role of miR‑129 in CRC progression was assessed by MTT, wound healing, Transwell, western blot and dual‑luciferase report assays. The results revealed that miR‑129 was significantly decreased, whereas SOX4 was increased, in CRC tissues and cell lines. SW620 and SW480 cells exhibited a higher proliferation, migration and invasion capacity compared with NCM460 cells. miR‑129 overexpression significantly inhibited cell proliferation, migration, invasion and epithelial‑to‑mesenchymal transition (EMT), and it activated the nuclear factor (NF)‑κB signaling pathway in CRC cells, while the inhibition of miR‑129 exerted opposite effects. Additionally, SOX4 was identified as a direct target gene of miR‑129. Taken together, the findings of the present study suggested that miR‑129 may act as a tumor suppressor in CRC by inhibiting CRC cell proliferation, migration, invasion and EMT, in part through targeting the 3'‑untranslated region of SOX4 mRNA, and the mechanism may involve activation of the NF‑κB signaling pathway.

Epithelial-mesenchymal transition (EMT) is related to colorectal cancer invasion and metastasis. Glioma-associated oncogene homolog 1 (Gli1) abnormal expression is associated with EMT, invasion, and metastasis in various cancers. MiR-150 is found downregulated in colorectal cancer pathogenesis. Bioinformatics analysis shows the complementary targeted relationship between miR-150 and the 3'-UTR of Gli1 mRNA. This study explores the role of miR-150 in regulating Gli1 expression, colorectal cancer cell EMT, and invasion. Dual luciferase assay confirmed the targeted relationship between miR-150 and Gli1 predicted by bioinformatics analysis. MiR-150 and Gli1 expressions were compared in NCM460, SW480, and SW620 cells. Cell colony formation and invasion were tested in SW480 and SW620 cells. Anip973 and AGYZ83-a cells were treated by 10 ng/mL TGF-β1 to detect miR-150 and Gli1 expressions. SW620 cells were cultured in vitro and divided into five groups, including miR-NC, miR-150 mimic, si-NC, si-Gli1, and miR-150 mimic + si-Gli1 groups. MiR-150 specifically inhibited Gli1 expression. The level of miR-150 was significantly downregulated, while Gli1 was elevated in SW480 and SW620 cells compared with that in NCM460 cells. SW620 exhibited markedly stronger invasive and colony formation abilities than SW480. The level of miR-150 was apparently reduced, whereas Gli1 was increased in SW620 than that in SW480 cells after the treatment of TGFβ1. MiR-150 mimic and/or si-Gli1 transfection markedly reduced Gli1 and Snail levels, upregulated E-cadherin expression, and attenuated cell colony formation and invasion. Downregulation of miR-150 and elevation of Gli1 promote the development and invasion of colorectal cancer cell EMT. MiR-150 attenuated the progression of colorectal cancer cell EMT via inhibiting Gli1.

Abnormal activation of the Wnt signaling pathway is found in 90% of colorectal cancers (CRCs). Secreted frizzled-related protein 4 (sFRP4) serves as an antagonist of the canonical Wnt signaling pathway. Epigenetic alterations, including changes in DNA methylation and histone methylation, may influence the expression of sFRP4. Polycomb group (PcG) proteins are epigenetic transcriptional repressors that selectively repress gene expression by forming polycomb repressive complexes (PRCs). Enhancer of zeste homolog 2 (EZH2), the core component of PRC2, is a histone-lysine N-methyltransferase that interacts with DNA methyltransferases. In the present study, the promoter DNA methylation status of sFRP4 in CRC cell lines was analyzed and the underlying mechanisms of action governing this modification was investigated. Firstly, the DNA methylation status of the sFRP4 promoter in CRC cell lines was assessed using methylation-specific PCR. Subsequently, the mRNA and protein levels of sFRP4 were measured using real-time qPCR and western blot analysis, respectively, to determine whether the DNA methylation status of the sFRP4 promoter is correlated with its transcriptional levels. To screen for important epigenetic modifiers that may regulate the promoter DNA methylation status of sFRP4, the expression levels of PcG proteins were examined by gene array analysis. ChIP-qPCR was performed to test whether the selected PcG proteins directly bind the promoter region of sFRP4. Finally, the downregulated PcG proteins EZH2, chromobox 7 (CBX7) and jumonji and AT-rich interaction domain containing 2 (JARID2) were identified and their association with sFRP4 expression levels and Wnt/β-catenin signaling pathway activity were investigated. The present study revealed that sFRP4 was hypermethylated in the promoter region and downregulated during the progression of the CRC cell lines from Dukes A to Dukes C. Expression levels of PcG proteins EZH2, CBX7 and JARID2 were upregulated and positively associated with the aberrantly activated Wnt signaling pathway in the CRC cell lines. EZH2, CBX7 and JARID2 were all enriched in the sFRP4 promoter region in CRC cells. EZH2 downregulation did not affect the promoter DNA methylation status of sFRP4 but increased its expression levels and decreased CRC cell proliferation. DNA methylation controls the expression of sFRP4. EZH2 regulates sFRP4 expression without affecting the DNA hypermethylation of the sFRP4 promoter and influences CRC cell proliferation and Wnt/β-catenin signaling pathway activities.

Simple SummaryColorectal cancer is common cancer, and currently used serum markers for detecting colorectal cancer lack excellent diagnostic accuracy. In the present study, we collected matched tumor and adjacent normal tissues and serum from patients and cancer cells to demonstrate the clinical value of DJ-1 in colorectal cancer and the role of DJ-1-induced mitophagy in colorectal cancer progression. Our data indicate that DJ-1 might be clinically valuable as serum and tissue biomarkers for predicting the TNM (tumor-node-metastasis) stage in colorectal cancer patients. Besides, DJ-1 knockdown enhanced intracellular reactive oxygen species generation and damaged mitochondrial accumulation and mitophagy inhibition in metastatic colorectal adenocarcinoma cells. Since DJ-1-induced mitophagy promotes tumor progression, DJ-1 inhibition is a potential therapeutic strategy for colorectal cancer treatment.Colorectal cancer is the second most common cancer and the third cancer-associated death in Taiwan. Currently used serum markers for detecting colorectal cancer lack excellent diagnostic accuracy, which results in colorectal cancer being often recognized too late for successful therapy. Mitophagy is the selective autophagic degradation of damaged or excessive mitochondria. DJ-1 is an antioxidant protein that attenuates oxidative stress and maintains mitochondrial quality through activating mitophagy. Mitophagy activation contributes to anti-cancer drug resistance. However, the role of DJ-1-induced mitophagy in colorectal cancer progression remains unclear. In the present study, we collected matched tumor and adjacent normal tissues and serum from patients and cancer cells to demonstrate the clinical value and physiological function of DJ-1 in colorectal cancer. We found that DJ-1 increased in tumor tissues and serum; it was positively correlated with TNM (tumor-node-metastasis) stages of colorectal cancer patients. Through stable knockdown DJ-1 expression in metastatic colorectal adenocarcinoma cells SW620, DJ-1 knockdown inhibited cancer cell survival, migration, and colony formation. In SW620 cells, DJ-1 knockdown induced an incomplete autophagic response that did not affect ATP production; DJ-1 knockdown enhanced intracellular reactive oxygen species generation and damaged mitochondrial accumulation and mitophagy inhibition. It suggests that DJ-1 knockdown inhibits mitophagy that causes metastatic colorectal adenocarcinoma cells to be unable to remove damaged mitochondria and further enhance cancer cell apoptosis. Our data indicate that DJ-1 might be clinically valuable as serum and tissue biomarkers for predicting the TNM stage in colorectal cancer patients. Since DJ-1-induced mitophagy promotes tumor progression, DJ-1 inhibition is a potential therapeutic strategy for colorectal cancer treatment.

Our previous studies showed that the suppression of colorectal cancer (CRC) cell proliferation by resveratrol (RV) was accompanied by a disproportionate increase of the non-canonical Wnt signaling pathway (N-CAN) components relative to the canonical Wnt signaling pathway (CAN) components. Wnt signaling, which consists of CAN and N-CAN is an intricate ensemble of signaling components involved with various cellular processes. The CAN utilizes β-catenin and stimulates cell proliferation whereas the N-CAN is independent of β-catenin and is instrumental in cell differentiation. These opposing mechanisms act in concert to maintain homeostasis in healthy tissues which is dysregulated in cancer. These observations have led us to propose a novel hypothesis that suppression of CRC proliferation may be mediated by the activation of the non-canonical Wnt pathway by nutritional supplements (NNS) such as RV. As far as we know, no known studies have determined the effects of RV on the non-canonical pathways in human CRC. The aim of this study was to determine whether suppression of CRC cell proliferation by RV involves non-canonical Wnt pathway components. CRC cells derived from surgical specimens (n = 5) were treated with RV (5ug/ml) for 72h. Cell proliferation was determined using MTS assay and gene expression was performed using commercially available quantitative RT-PCR Wnt pathway gene expression arrays. RV significantly increased N-CAN ligand Wnt A and Wnt 11(p<0.01) and CAN ligand Wnt 10A (p<0.05) by 18, 9 and 4-folds, respectively. RV treatment resulted in a significant decrease of CRC cell proliferation (56% inhibition; p<0.01) compared to untreated cells. This inhibition was partially reversed by IWP-2, a Wnt ligand production inhibitor. Conditioned media from CRC cells treated with RV significantly suppressed CRC cell proliferation (p<0.05) compared to cells treated with conditioned media from untreated cells and that from cells treated with conditioned media from RV+IWP treated cells. Proliferation was not different in cells treated with conditioned media from untreated cells, IWP-2 or RV+IWP-2-treated cells. Wnt 9A antibody partially reversed the RV-mediated suppression of CRC cell proliferation relative to cells treated with IgG controls. To the best of our knowledge this is the first known study to show the involvement of a non-canonical Wnt pathway ligand, Wnt9A, in the suppression of CRC proliferation mediated by a NNS, resveratrol. This study suggests the presence of a novel mechanism for the suppression of CRC proliferation which entails activation of the non-canonical pathway. Given the excellent tolerance profile of NNS such as RV, the capacity of such agents to activate the non-canonical pathway to inhibit tumor proliferation, offers the opportunity to investigate the effects of chronic administration of these agents on tumor growth and progression in patients. Citation Format: Irshad Ali, Bani M. Fagla, Donald P. Braun. Suppression of colorectal cancer cell proliferation by resveratrol involves upregulation of the non-canonical ligand Wnt9a. [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 1042. doi:10.1158/1538-7445.AM2015-1042

The secreted frizzled related proteins (SFRPs) are extracellular inhibitors of WNT pathway signaling. Methyl-CpG binding domain protein 2 (MBD2) and enhancer of zeste homolog 2 (EZH2) are core members of the methylated DNA binding domain (MBD) and polycomb group (PcG) protein families for epigenetic regulation, respectively. This study aimed to ascertain the potential role of MBD2 and EZH2 proteins in colorectal cancer (CRC) and its effects on the expression of SFRP. Bioinformatics, real-time quantitative polymerase chain reaction (qPCR) and western blot analysis were used to detect the expression of MBD2, EZH2, and SFRP in CRC cell lines and tissues. The functions of MBD2 and EZH2 in regards to cell proliferation, cell cycle distribution, apoptosis and invasion were examined in CRC cell lines. Methylation-specific PCR (MSP) was used to detect the methylation status of the SFRP promoter. The results revealed that the mRNA expression levels of SFRP were significantly decreased in CRC tissues and cell lines compared to these levels in the adjacent tissues and NCM460, respectively. However, the mRNA levels of EZH2 and MBD2 genes were highly expressed in CRC cell lines. We found that reducing MBD2 and EZH2 expression together remarkably inhibited and decreased the proliferation, migration and invasion abilities of the CRC cell lines compared to reducing one of each. Flow cytometric analysis showed that knockdown of MBD2 and EZH2 together in CRC affected cell apoptosis and the cell cycle progression more effectively than knockdown of one of each. The mRNA expression of SFRP1 was reactivated by silencing of MBD2 but not EZH2 in SW480 and HCT116 cells. SFRP4 and SFRP5 mRNA expression was reactivated by silencing of EZH2 but not MBD2 only in SW480 cells. However, depletion of both MBD2 and EZH2 restored SFRP1, SFRP2, SFRP4, and SFRP5 mRNA expression more effectively in CRC cells. Interestingly, there was no significant change in the methylation status of SFRP1, SFRP2, SFRP4, and SFRP5 gene promoter between before and after interference with MBD2, EZH2, and both. In conclusion, our results suggest that silencing of MBD2 and EZH2 simultaneously was able to rescue the expression of SFRP and inhibit the proliferation of CRC cells more effectively. However, the underlying regulatory mechanism system of MBD2 and EZH2 for SFRP in CRC requires further research.

Objective To explore The role of sFRP2 and Wnt/β-catenin pathway in the development and progression of colorectal cancer. Methods Immunohistochemical staining was used to detect the expression of sFRP2 and β-catenin in colorectal cancer group and normal colorectal mucosa group, to detect the expression level and positive rate. The expression of sFRP2 gene in colorectal cancer cell line HCT116 was up-regulated by plasmid transfection, it was verified by Western blot. Then we conduct CCK-8 method, wound-healing assay and Transwell assay. Then the effects of up regulation of sFRP2 expression on cell proliferation, migration and invasion were analyzed. Results The results of immunohistochemistry showed that the positive rate of sFRP2 expression in normal colorectal mucosa is higher than colorectal cancer group (χ2=35.902, P=0.000); However, The rate of β-catenin membrane expression deficiency and ectopic expression in colorectal cancer group is higher than normal colorectal mucosa (χ2=23.149, P=0.000, χ2=27.002, P=0.000). The positive expression of sFRP2, the loss of expression and the ectopic expression of β-catenin membrane were significantly correlated with the differentiation of tumor tissues (χ2=5.420, P=0.020, χ2=6.472; P=0.011, χ2=7.158, P=0.007), however, it was not associated with sex, age, tumor location, tumor size, tumor stage and lymph node metastasis (P>0.05). The expression of sFRP2 was negatively correlated with β-catenin membrane expression deficiency and ectopic expression; There was a positive correlation between the β-catenin membrane expression deficiency and the ectopic expression. After plasmid transfection, the expression of sFRP2 and β-catenin significantly increased (t=25.430, P=0.001; t=15.000, P=0.001); The proliferation and migration rate of sFRP2 transfection group was significantly slower compared with the control group and the empty plasmid group (t=16.890, P=0.001; t=7.206, P=0.002); Compared with the control group, the number of transmembrane cells in sFRP2 transfected group was significantly fewer than that in the control group (t=25.459, P=0.001), and the cell invasion ability was significantly decreased. Conclusion The interaction between sFRP2 and Wnt/β-catenin pathway plays an important role in the development and progression of colorectal cancer. The up regulation of sFRP2 significantly inhibited the proliferation, migration and invasion of colorectal cancer cell line HCT116. Key words: Colorectal neoplasms; sFRP2; β-catenin; HCT116

The Caulobacter chromosome changes progressively from the fully methylated to the hemimethylated state during DNA replication. These changes in DNA methylation could signal differential binding of regulatory proteins to activate or repress transcription. The gene encoding CtrA, a key cell cycle regulatory protein, is transcribed from two promoters. The P1 promoter fires early in S phase and contains a GAnTC sequence that is recognized by the CcrM DNA methyltransferase. Using analysis of CcrM mutant strains, transcriptional reporters integrated at different sites on the chromosome, and a ctrA P1 mutant, we demonstrate that transcription of the P1 promoter is repressed by DNA methylation. Moreover moving the native ctrA gene to a position near the chromosomal terminus, which delays the conversion of the ctrA promoter from the fully to the hemimethylated state until late in the cell cycle, inhibited ctrA P1 transcription, and altered the time of accumulation of the CtrA protein and the size distribution of swarmer cells. Together, these results show that CcrM-catalyzed methylation adds another layer of control to the regulation of ctrA expression.

Dysregulation of the Wnt pathway in histologically distinct cancers has been studied extensively. The canonical Wnt pathway (CWP) utilizing β-catenin is one of the major drivers for proliferation. In contrast, the capacity of the non-canonical Wnt pathway (NCWP) to regulate proliferation has not been studied extensively in neoplastic tissues. Our earlier studies demonstrated that RV-mediated suppression of colorectal cancer (CRC) cell proliferation correlated with upregulated expression of several NCWP components. These results are consistent with the hypothesis that these upregulated NCWP components suppress CWP activity leading to proliferation inhibition. Of the NCWP components measured, the ligand Wnt9A was most prominently upregulated. Since the CWP utilizes β-catenin for its activity, the purpose of the present study was to determine whether induction of Wnt9A and suppression of CRC proliferation by RV can be correlated directly to modulation of β-catenin message and protein levels. This study was conducted on human CRC cells derived from surgical specimens (n = 5). Cell proliferation and apoptosis were determined by MTS and Caspase 3 assays, respectively. β-catenin protein levels were determined by ELISA and gene expression using quantitative RT-PCR Wnt pathway gene expression arrays. RV increased Wnt9A expression in CRC cells in a concentration-dependent manner. Wnt9A expression was increased by a mean of 8, 36 and 260 fold (p<0.004, ANOVA) relative to media controls with RV concentrations of 5, 10 and 20 μg/ml, respectively. The increase in Wnt9A expression was associated with decreased total β-catenin protein by 21, 51 and 71% (p<0.001, ANOVA), and active β-catenin protein by 17, 22 and 50% (p<0.001, ANOVA). These effects were associated with significant inhibition of proliferation by 35, 54 and 66% relative to media controls with RV concentrations of 5, 10 and 20 μg/ml, respectively (p<0.006, ANOVA) and a significant increase in apoptosis (p<0.01, ANOVA). Significantly, mRNA levels for β-catenin were not affected by any concentration of RV. Finally, the suppression of CRC cell proliferation by RV was reversed by Wnt9A antibody and IWP-2, a Wnt ligand secretion inhibitor (p<0.02, ANOVA). The induction of Wnt9A with subsequent suppression of β-catenin protein levels by resveratrol represents a novel mechanism for the suppression of canonical Wnt pathway-mediated CRC cell proliferation. These results suggest that interventions that stimulate the induction of non-canonical Wnt pathway components, and specifically Wnt9A, may be therapeutically beneficial for controlling proliferation of colorectal cancers. Citation Format: Irshad Ali, Bani Medegan, Donald Braun. Novel mechanism for suppression of human colorectal cancer cell proliferation through induction of Wnt9A and suppression of β-catenin by resveratrol. [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 4606.