APC mRNA Research Articles

PRMT6-mediated transcriptional activation of ythdf2 promotes glioblastoma migration, invasion, and emt via the wnt–β-catenin pathway

BackgroundProtein arginine methyltransferase 6 (PRMT6) plays a crucial role in various pathophysiological processes and diseases. Glioblastoma (GBM; WHO Grade 4 glioma) is the most common and lethal primary brain tumor in adults, with a prognosis that is extremely poor, despite being less common than other systemic malignancies. Our current research finds PRMT6 upregulated in GBM, enhancing tumor malignancy. Yet, the specifics of PRMT6’s regulatory processes and potential molecular mechanisms in GBM remain largely unexplored.MethodsPRMT6’s expression and prognostic significance in GBM were assessed using glioma public databases, immunohistochemistry (IHC), and immunoblotting. Scratch and Transwell assays examined GBM cell migration and invasion. Immunoblotting evaluated the expression of epithelial-mesenchymal transition (EMT) and Wnt-β-catenin pathway-related proteins. Dual-luciferase reporter assays and ChIP-qPCR assessed the regulatory relationship between PRMT6 and YTHDF2. An in situ tumor model in nude mice evaluated in vivo conditions.ResultsBioinformatics analysis indicates high expression of PRMT6 and YTHDF2 in GBM, correlating with poor prognosis. Functional experiments show PRMT6 and YTHDF2 promote GBM migration, invasion, and EMT. Mechanistic experiments reveal PRMT6 and CDK9 co-regulate YTHDF2 expression. YTHDF2 binds and promotes the degradation of negative regulators APC and GSK3β mRNA of the Wnt-β-catenin pathway, activating it and consequently enhancing GBM malignancy.ConclusionsOur results demonstrate the PRMT6-YTHDF2-Wnt-β-Catenin axis promotes GBM migration, invasion, and EMT in vitro and in vivo, potentially serving as a therapeutic target for GBM.

5-Methylcoumarin-4β-glucoside mitigated colon tumor progression in mice with AOM/DSS-induced colon carcinogenesis

ContextThe in vitro cytotoxicity profile of 5-Methylcoumarin-4β-glucoside (5-MC4βG) has been demonstrated to inhibit the proliferation of HT-29 adenocarcinoma cells. Aim: The need for newer and affordable chemotherapeutic agents is critical. Main methodsWe investigated the effect of 5-MC4βG on an azoxymethane /dextran sodium sulfate-induced colon carcinogenesis model in six groups of laboratory BALB/c mice for six weeks. While the first and second groups of mice served as vehicle and disease controls respectively, the third, fourth, and fifth groups were administered oral graded doses (25, 50, and 100 mg/kg) of 5-MC4βG. The sixth group was treated with 5-Flourouracil (15 mg/kg). Key findingsThe 100 mg/kg dose of 5-MC4βG upregulated APC mRNA expression by two-fold and downregulated β-catenin mRNA expression by two-fold compared to their respective disease controls. Furthermore, tumorigenic gene transcripts (MDM2, BCL2, CDK1, and cyclin D1) were downregulated by one-fold (except cyclin D1 which was downregulated by two-fold), whereas pro-apoptotic genes (p53, Bax, and Casp3) were upregulated by two-fold following treatment with 100 mg/kg dose of 5-MC4βG. At the metabolic level, the bioactive compound lowered the expression of classical tumor markers; tissue polypeptide antigen, tumor-associated glycoprotein 72, and carcinoembryonic antigen by at least half. Histologically, 5-MC4βG intervention revealed a reduction in neoplastic cells associated with cellular necrosis. Significance5-MC4βG reduced colon carcinogenesis in mice. Thus, this compound may be a promising candidate for colorectal cancer chemotherapy. Further development of 5-MC4βG will hopefully lead to the development of a potential anti-colon cancer drug candidate.

Azithromycin induces read-through of the nonsense Apc allele and prevents intestinal tumorigenesis in C3B6F1 ApcMin/+ mice

Therapeutic strategies that promote read-through of a mutant gene have proved effective for certain non-neoplastic diseases. However, the efficacy of this approach is unproven regarding neoplastic diseases with germline nonsense mutations, including familial adenomatous polyposis. Here we examined the cancer-preventive efficacy of the macrolide antibiotic azithromycin, with a reported read-through effect, on intestinal tumorigenesis in C3B6F1 ApcMin/+ mice harboring a nonsense Apc mutation resulting in a truncated Apc protein. Mice were given drinking water lacking azithromycin or containing 0.0125–0.2 mg/mL azithromycin from 3 weeks of age. The small intestine and cecum were analyzed for pathological changes and alterations of intestinal flora. Azithromycin suppressed the number of tumors and the proportion of adenocarcinomas, with the most effective drinking-water concentration being 0.0125 mg/mL. Furthermore, azithromycin recovered the cellular level of full-length Apc, resulting in downregulation of β-catenin and cyclin D1. Conversely, the effect of azithromycin on the diversity of the intestinal microbiota depended on the drinking-water concentration. These results suggest that the balance between azithromycin-mediate read-through of mutant Apc mRNA and antibacterial effects influences intestinal tumorigenesis. Thus, azithromycin is a potential anticancer agent for familial adenomatous polyposis patients harboring nonsense mutations.

A Single Variant in Pri-miRNA-155 Associated with Susceptibility to Hereditary Breast Cancer Promotes Aggressiveness in Breast Cancer Cells.

Variants in genes encoding for microRNAs have been associated with their deregulation in breast cancer (BC). Sequencing of microRNAs deregulated in BC was performed using DNA from Chilean patients with a strong family history and negative for mutations in BRCA1/BRCA2. Seventeen variants were identified, three of which were selected for a case-control association study: rs376491654 (miR-335), rs755634302 (miR-497), and rs190708267 (miR-155). For rs190708267 C>T, the heterozygous T allele was detected in four BC cases and absent in controls, while homozygous TT cases were not detected. Variants were modelled in silico, cloned in a plasmid, expressed in BC cell lines, and functional in vitro assays were performed. Overexpression of the miR-155-T allele increased mature miR-155-5p levels in both BC cell lines, suggesting that its presence alters pre-miR-155 processing. Moreover, BC cells overexpressing the miR-155-T allele showed increased proliferation, migration, and resistance to cisplatin-induced death compared to miR-155-C overexpressing cells. Of note, the 3′UTR of APC, GSK3β, and PPP1CA genes, all into the canonical Wnt signaling pathway, were identified as direct targets. APC and GSK3β mRNA levels decreased while PP1 levels increased. These results suggest a pathogenic role of the variant rs190708267 (miR-155) in BRCA 1/2 negative BC, conferring susceptibility and promoting traits of aggressiveness.

Constitutional chromothripsis of the APC locus as a cause of genetic predisposition to colon cancer

PurposeApproximately 20% of patients with clinical familial adenomatous polyposis (FAP) remain unsolved after molecular genetic analysis of the APC and other polyposis genes, suggesting additional pathomechanisms.MethodsWe applied multidimensional genomic analysis...

Investigation of the expression levels of CPEB4, APC, TRIP13, EIF2S3, EIF4A1, IFNg, PIK3CA and CTNNB1 genes in different stage colorectal tumors

Background/aimThe aim of the study is to assess expression levels of CPEB4, APC, TRIP13, EIF2S3, EIF4A1, IFNg, PIK3CA and CTNNB1 genes in tumors and peripheral bloods of colorectal cancer patients in stages I–IV.Materials and methodsThe mRNA levels of the genes were determined in tumor tissues and peripheral blood samples of 45 colorectal cancer patients and colon tissues and peripheral blood samples of 5 healthy individuals. Real-time polymerase chain reaction method was used for the analysis.ResultsThe mRNA level of the CPEB4 gene was significantly downregulated in colorectal tumor tissues and was upregulated in the peripheral blood of colorectal cancer patients relative to the controls (P < 0.05). APC mRNA level was significantly downregulated in tissues and upregulated in the peripheral blood (P < 0.05). TRIP13 mRNA level was upregulated in peripheral blood and also significantly upregulated in colorectal tumor tissues (P < 0.05). EIF2S3 mRNA level was upregulated in tissues and also significantly upregulated in peripheral blood (P < 0.05). PIK3CA mRNA level was downregulated in tissues and upregulated in peripheral blood. EIF4A1 mRNA level was downregulated in tissues and significantly upregulated in peripheral blood (P < 0.05). CTNNB1 mRNA level was downregulated in tissues and upregulated in peripheral blood. IFNg mRNA level was upregulated in both colorectal cancer tumor tissues and peripheral blood.Conclusion:TRIP13 and CPEB4 mRNA up regulation in the peripheral blood of patients with colorectal cancer may be a potential target for early stage diagnosis. In addition to this evaluation, although there is not much study on EIF2S3 and EIF4A1 mRNA changes in cases with colorectal cancer, upregulation in peripheral blood draws attention in our study. These data will shed light on the new comprehensive studies.

Characterizing the cytotoxic effects and several antimicrobial phytocompounds of Argemone mexicana.

Commonly called the Mexican prickly poppy, Argemone mexicana is a stress-resistant member of the Papaveraceae family of plants that has been used in traditional medicine for centuries by indigenous communities in Mexico and Western parts of the United States. This plant has been exploited to treat a wide variety of ailments, with reported antimicrobial and antioxidant properties, as well as cytotoxic effects against some human cancer cell lines. Due to its various therapeutic uses and its abundance of secondary metabolites, A. mexicana has great potential as a drug discovery candidate. Herein, the germination conditions of A. mexicana are described and the cytotoxic activities of different parts (seeds, leaves, inner vs. outer roots) of the plant from methanol or hexane extracts are preliminarily characterized against cells of seven unique organisms. When comparing 1 mg of each sample normalized to background solvent alone, A. mexicana methanol outer root and leaf extracts possessed the strongest antimicrobial activity, with greatest effects against the Gram-positive bacteria tested, and less activity against the Gram-negative bacteria and fungi tested. Additionally, using the MTT colorimetric assay, the outer root methanol and seed hexane extracts displayed pronounced inhibitory effects against human colon cancer cells. Quantification of c-MYC (oncogene) and APC (tumor suppressor) mRNA levels help elucidate how the A. mexicana root methanol extract may be affecting colon cancer cells. After ultra-performance liquid chromatography coupled with mass spectrometry and subsequent nuclear magnetic resonance analysis of the root and leaf methanol fractions, two main antibacterial compounds, chelerythrine and berberine, have been identified. The roots were found to possess both phytocompounds, while the leaf lacked chelerythrine. These data highlight the importance of plants as an invaluable pharmaceutical resource at a time when antimicrobial and anticancer drug discovery has plateaued.

S3125 Risk Assessment in Hereditary Colorectal Cancer Family Using APC and MSH2 mRNA Gene Expression and Bayesian Analysis

S3125 Risk Assessment in Hereditary Colorectal Cancer Family Using APC and MSH2 mRNA Gene Expression and Bayesian Analysis

Effects of latanoprost on the expression of TGF-β1 and Wnt / β-catenin signaling pathway in the choroid of form-deprivation myopia rats

In this study, we investigated the effect of latanoprost on the expression of TGF- β1 and Wnt / β - Catenin signal pathway in the choroid of form-deprivation myopia model rats. Forty rats were randomly divided into two groups: the control group and the FDM model group. Each group had 20 rats. The FDM model group was established by feeding latanoprost daily for 28 days. 15 rats in each group were used to measure the length of the ocular axis and the level of TGF-β1 in choroidal tissue; the remaining 5 rats in each group were used for choroidal fibroblast culture. After modeling, the rats were killed, the length of the ocular axis was measured with a vernier caliper, and the level of TGF - β1 protein and mRNA in the choroidal tissue of each group were measured with RT-PCR method. Results showed that compared with the control group, there was a significant difference in the axial length of the FDM model group (P&lt; 0.05). There was a significant difference in the expression of TGF- β1 protein and mRNA between the two groups (P&lt;0.05). The cultured cells were identified as choroidal fibroblasts by immunocytochemistry. There was no significant difference (P&gt;0.05) in the comparison of GSK3 β protein in choroidal fibroblasts of rats in each group. TGF-β 1 and APC protein in FDM group were significantly lower than those in the control group (P&lt;0.05), while dcl3, p21-gsk3 β and β - Catenin proteins were significantly higher (P&lt;0.05), and there was no significant difference (P&gt;0.05) in the ratio of various indexes protein in FDM + ddk1 group and the comparison of TGF - β1 and APC protein in FDM + ddk1 group and FDM group The expression of dcl3, p21-gsk3 β and β - Catenin decreased significantly (P&lt;0.05). There was no significant difference in the expression of GSK3 β mRNA in the choroidal fibroblasts of each group (P&gt;0.05). The expression of TGF - β 1 and APC mRNA in FDM group was significantly lower than that in the control group (P&lt;0.05), while the expression of dcl3, p21-gsk3 β and β-catenin mRNA in FDM + ddk1 group was significantly higher than that in the control group (P&lt;0.05) &gt;In FDM + ddk1 group, TGF-β 1 and APC mRNA were significantly lower than those in FDM group (P&lt;0.05), while dcl3, p21-gsk3 β and β-Catenin mRNA were significantly higher (P&lt;0.05).

MiR-590-3p regulates osteogenic differentiation of human mesenchymal stem cells by regulating APC gene

The present study aimed to investigate miR-590-3p's role in osteogenic differentiation of human mesenchymal stem cells (hMSC). HMSC were cultured and transfected with empty vector, miR-590-3p vector to construct control hMSC and miR-590-3p overexpression hMSC. MiR-590-3p suppressed hMSC was created with oligonucleotide transient transfection. All 3 groups of cells were analyzed for their osteogenic level as well as Wnt/β-catenin signaling pathway activities. The results of alizarin red staining indicated that overexpression of miR-590-3p significantly enhanced mineralized deposition. Osteogenic markers including ALP, OC and SOST were also up-regulated. The result of dual-luciferase and western blot analysis indicated that miR-590-3p bound to 3′UTR of APC mRNA selectively. By suppressing mRNA level of APC, over-expressed miR-590-3p stabilized β-catenin and increased the transcription activities of downstream target genes. These result suggested that miR-590-3p can promote osteogenic differentiation via suppressing APC expression and stabilizing β-catenin.

Abstract 4491: Pseudoexons may underlie unbalanced expression of APC alleles in familial adenomatous polyposis

Abstract Familial Adenomatous Polyposis (FAP) is caused by germline mutations in the APC gene. Clinically, the disease can be subdivided into classical FAP with hundreds or thousands of polyps throughout the colon and rectum and Attenuated Familial Adenomatous Polyposis (AFAP) with a milder polyposis. Conventional screening protocols fail to detect any APC mutation in a considerable fraction of adenomatous polyposis families, especially those representing AFAP. We investigated 56 FAP and AFAP families from Finland that had remained APC mutation-negative after protein truncation test, exon-specific Sanger sequencing, and multiplex ligation-dependent probe amplification. Of particular interest were four families with constitutionally unbalanced mRNA expression of APC alleles by Single Nucleotide Primer Extension. These families were interrogated by next generation sequencing of the whole genomes (WGS) and RNA (RNA-seq); additionally, APC promoter methylation was addressed by methylation-specific multiplex ligation-dependent probe amplification. RNA-seq raised the suspicion of a pseudoexon (erroneous inclusion of intronic sequence in the mature mRNA) in three families. A pseudoexon between exons 5 and 6 of the APC gene was present in one family and a pseudoexon between exons 10 and 11 in two families. By WGS, the exon 5-6 pseudoexon consisted of c.646-1806 T&amp;gt;G, which generated a cryptic splice site leading to a 127-bp intronic insertion in the APC mRNA. This pseudoexon was associated with classical FAP. Two alternative genetic changes underlay the exon 10-11 pseudoexon: c.1408+729 A&amp;gt;G was present in one family (AFAP) and c.1408+731 C&amp;gt;T in another family (FAP); both created a new splice donor site leading to an identical 83-bp insertion in APC mRNA. The exon 10-11 pseudoexon resulting from c.1408+731 C&amp;gt;T has previously been described in two unrelated German patients with FAP; the remaining two pseudoexon-associated mutations are novel. All three pseudoexons were predicted to cause premature stop codons leading to APC protein truncation. Sanger sequencing was used to confirm the mutations and to test their presence in the entire series of 56 families; no additional mutation-positive cases were detected. Taken together, deep intronic mutations of the APC gene may explain a proportion of FAP and AFAP families that screen mutation-negative by traditional methods. Specifically, such mutations accounted for three out of four families with the unbalanced expression phenotype of APC in our study. We further conclude that RNA-seq is an effective method to reveal deep intronic mutations and is worth considering in apparent mutation-negative families. Citation Format: Taina Tuulikki Nieminen, Walter Pavicic, Noora Porkka, Heikki J. Jarvinen, Anna Lepisto, Paivi Peltomaki. Pseudoexons may underlie unbalanced expression of APC alleles in familial adenomatous polyposis. [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 4491.

MiR-142 regulates the tumorigenicity of human breast cancer stem cells through the canonical WNT signaling pathway.

MicroRNAs (miRNAs) are important regulators of stem and progenitor cell functions. We previously reported that miR-142 and miR-150 are upregulated in human breast cancer stem cells (BCSCs) as compared to the non-tumorigenic breast cancer cells. In this study, we report that miR-142 efficiently recruits the APC mRNA to an RNA-induced silencing complex, activates the canonical WNT signaling pathway in an APC-suppression dependent manner, and activates the expression of miR-150. Enforced expression of miR-142 or miR-150 in normal mouse mammary stem cells resulted in the regeneration of hyperproliferative mammary glands in vivo. Knockdown of endogenous miR-142 effectively suppressed organoid formation by BCSCs and slowed tumor growth initiated by human BCSCs in vivo. These results suggest that in some tumors, miR-142 regulates the properties of BCSCs at least in part by activating the WNT signaling pathway and miR-150 expression.

APC alterations are frequently involved in the pathogenesis of acinar cell carcinoma of the pancreas, mainly through gene loss and promoter hypermethylation

Genetic and epigenetic alterations involved in the pathogenesis of pancreatic acinar cell carcinomas (ACCs) are poorly characterized, including the frequency and role of gene-specific hypermethylation, chromosome aberrations, and copy number alterations (CNAs). A subset of ACCs is known to show alterations in the APC/β-catenin pathway which includes mutations of APC gene. However, it is not known whether, in addition to mutation, loss of APC gene function can occur through alternative genetic and epigenetic mechanisms such as gene loss or promoter methylation. We investigated the global methylation profile of 34 tumor suppressor genes, CNAs of 52 chromosomal regions, and APC gene alterations (mutation, methylation, and loss) together with APC mRNA level in 45 ACCs and related peritumoral pancreatic tissues using methylation-specific multiplex ligation probe amplification (MS-MLPA), fluorescence in situ hybridization (FISH), mutation analysis, and reverse transcription-droplet digital PCR. ACCs did not show an extensive global gene hypermethylation profile. RASSF1 and APC were the only two genes frequently methylated. APC mutations were found in only 7 % of cases, while APC loss and methylation were more frequently observed (48 and 56 % of ACCs, respectively). APC mRNA low levels were found in 58 % of cases and correlated with CNAs. In conclusion, ACCs do not show extensive global gene hypermethylation. APC alterations are frequently involved in the pathogenesis of ACCs mainly through gene loss and promoter hypermethylation, along with reduction of APC mRNA levels.

Sa1686 Characteristics of and Associations Among the HER2 Signal-Related Factors Thought to Be Predictive Factors for the Effectiveness of Trastuzumab Therapy in Gastric Cancer

Background: Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine that is highly up-regulated in inflammatory bowel disease and reduces the expression of the intestinal specific homeodomain transcription factor, CDX2. CDX2 is a master regulator of the intestinal homeostasis and is considered to be a tumor suppressor. WNT/ β-catenin signaling is critical for intestinal cell proliferation, but decreased CDX2 expression contributes to an abnormal increased activation of WNT signaling and progression of cancer. Furthermore, low CDX2 expression is associated with enhanced epithelial-to-mesenchymal transition (EMT) in colorectal cancer (CRC). Inflammation is often observed at the invasive front of the colon cancer, and it has been suggested that the inflammatory microenvironment around a tumor also induces EMT. Hence, it is likely that down-regulation of CDX2 expression in the invasive front in CRC contributes to enhanced tumorigenicity by regulating genes associated with the WNT/β-catenin pathway. Aim: To determine the role of TNFα in the regulation of CDX2 expression in CRC and its influence onWNT/β-catenin signaling. Methods: Carcinoma specimens were obtained from patients undergoing surgical resection of the rectum. Ten cases with tumor cell buddings were selected, and the expression patterns of TNFα and CDX2 at the invasive front were evaluated on immunostained slides. The mechanisms behind TNF-α-mediated down-regulation of CDX2 were investigated in vitro by selective inhibitors, and the impact of TNF-α on APC, AXIN2 and GSK3β expression were analyzed by quantitative real-time PCR (qPCR) in Caco-2 cells. Subsequently, in vivo CDX2-DNA interactions to APC, AXIN2 and GSK3β gene regulatory elements were investigated by chromatin immunoprecipitation in relation to the TNF-α status (presence vs. absence). Results: Immunohistochemical staining showed reduced CDX2 positive-cells in tumor buddings in areas with TNF-α expression in the surrounding inflammatory cells. TNF-α treatment showed a dosedependent decrease of CDX2 mRNA and protein expression dependent on p38 and NFκB, but not on JNK and ERK signaling pathways. Down-regulation of CDX2 leads to significantly decreased levels of APC (p,0.05), AXIN2 (p,0.01), and GSK3β (p,0.05) mRNA. In line with these results, TNF-α treatment impaired the ability of CDX2 to interact and activate the expression of APC (p,0.05), AXIN2 (p,0.01), and GSK3β (p,0.05). Conclusions: This study suggests that TNF-α has a tumor-promoting effect on CRC by influencing the activity of WNT signaling through down-regulation of CDX2 expression. These findings provide a novel insight into the molecular regulation of genes in the β-catenin degradation complex in response to the pro-inflammatory cytokine TNFα in cancer cells.

Sa1683 Five Different Subclasses of Colorectal Neoplasias Identified by Integrated Genetic and Epigenetic Analysis: Association With Polypoid and Nonpolypoid Carcinogenesis

Background: Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine that is highly up-regulated in inflammatory bowel disease and reduces the expression of the intestinal specific homeodomain transcription factor, CDX2. CDX2 is a master regulator of the intestinal homeostasis and is considered to be a tumor suppressor. WNT/ β-catenin signaling is critical for intestinal cell proliferation, but decreased CDX2 expression contributes to an abnormal increased activation of WNT signaling and progression of cancer. Furthermore, low CDX2 expression is associated with enhanced epithelial-to-mesenchymal transition (EMT) in colorectal cancer (CRC). Inflammation is often observed at the invasive front of the colon cancer, and it has been suggested that the inflammatory microenvironment around a tumor also induces EMT. Hence, it is likely that down-regulation of CDX2 expression in the invasive front in CRC contributes to enhanced tumorigenicity by regulating genes associated with the WNT/β-catenin pathway. Aim: To determine the role of TNFα in the regulation of CDX2 expression in CRC and its influence onWNT/β-catenin signaling. Methods: Carcinoma specimens were obtained from patients undergoing surgical resection of the rectum. Ten cases with tumor cell buddings were selected, and the expression patterns of TNFα and CDX2 at the invasive front were evaluated on immunostained slides. The mechanisms behind TNF-α-mediated down-regulation of CDX2 were investigated in vitro by selective inhibitors, and the impact of TNF-α on APC, AXIN2 and GSK3β expression were analyzed by quantitative real-time PCR (qPCR) in Caco-2 cells. Subsequently, in vivo CDX2-DNA interactions to APC, AXIN2 and GSK3β gene regulatory elements were investigated by chromatin immunoprecipitation in relation to the TNF-α status (presence vs. absence). Results: Immunohistochemical staining showed reduced CDX2 positive-cells in tumor buddings in areas with TNF-α expression in the surrounding inflammatory cells. TNF-α treatment showed a dosedependent decrease of CDX2 mRNA and protein expression dependent on p38 and NFκB, but not on JNK and ERK signaling pathways. Down-regulation of CDX2 leads to significantly decreased levels of APC (p,0.05), AXIN2 (p,0.01), and GSK3β (p,0.05) mRNA. In line with these results, TNF-α treatment impaired the ability of CDX2 to interact and activate the expression of APC (p,0.05), AXIN2 (p,0.01), and GSK3β (p,0.05). Conclusions: This study suggests that TNF-α has a tumor-promoting effect on CRC by influencing the activity of WNT signaling through down-regulation of CDX2 expression. These findings provide a novel insight into the molecular regulation of genes in the β-catenin degradation complex in response to the pro-inflammatory cytokine TNFα in cancer cells.

Sa1682 TNF-α-Mediated Down-Regulation of CDx2 Decreases the Expression of β-Catenin Degradation Complex Genes in Colorectal Cancer

Background: Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine that is highly up-regulated in inflammatory bowel disease and reduces the expression of the intestinal specific homeodomain transcription factor, CDX2. CDX2 is a master regulator of the intestinal homeostasis and is considered to be a tumor suppressor. WNT/ β-catenin signaling is critical for intestinal cell proliferation, but decreased CDX2 expression contributes to an abnormal increased activation of WNT signaling and progression of cancer. Furthermore, low CDX2 expression is associated with enhanced epithelial-to-mesenchymal transition (EMT) in colorectal cancer (CRC). Inflammation is often observed at the invasive front of the colon cancer, and it has been suggested that the inflammatory microenvironment around a tumor also induces EMT. Hence, it is likely that down-regulation of CDX2 expression in the invasive front in CRC contributes to enhanced tumorigenicity by regulating genes associated with the WNT/β-catenin pathway. Aim: To determine the role of TNFα in the regulation of CDX2 expression in CRC and its influence onWNT/β-catenin signaling. Methods: Carcinoma specimens were obtained from patients undergoing surgical resection of the rectum. Ten cases with tumor cell buddings were selected, and the expression patterns of TNFα and CDX2 at the invasive front were evaluated on immunostained slides. The mechanisms behind TNF-α-mediated down-regulation of CDX2 were investigated in vitro by selective inhibitors, and the impact of TNF-α on APC, AXIN2 and GSK3β expression were analyzed by quantitative real-time PCR (qPCR) in Caco-2 cells. Subsequently, in vivo CDX2-DNA interactions to APC, AXIN2 and GSK3β gene regulatory elements were investigated by chromatin immunoprecipitation in relation to the TNF-α status (presence vs. absence). Results: Immunohistochemical staining showed reduced CDX2 positive-cells in tumor buddings in areas with TNF-α expression in the surrounding inflammatory cells. TNF-α treatment showed a dosedependent decrease of CDX2 mRNA and protein expression dependent on p38 and NFκB, but not on JNK and ERK signaling pathways. Down-regulation of CDX2 leads to significantly decreased levels of APC (p,0.05), AXIN2 (p,0.01), and GSK3β (p,0.05) mRNA. In line with these results, TNF-α treatment impaired the ability of CDX2 to interact and activate the expression of APC (p,0.05), AXIN2 (p,0.01), and GSK3β (p,0.05). Conclusions: This study suggests that TNF-α has a tumor-promoting effect on CRC by influencing the activity of WNT signaling through down-regulation of CDX2 expression. These findings provide a novel insight into the molecular regulation of genes in the β-catenin degradation complex in response to the pro-inflammatory cytokine TNFα in cancer cells.

Abstract 1110: Musashi 1 stabilizes and blocks translation of Adenomatous Polyposis Coli mRNA in intestinal cells.

Abstract Most colorectal cancers are thought to be initiated by mutation of the tumor suppressor Adenomatous polyposis coli gene (APC). APC mutations that result in loss of function lead to deregulation of Wnt signaling and inappropriate proliferation within intestinal crypts. Understanding APC regulation within normal intestinal cells is important for developing methods to restore function in a pathological state. Using cultured human colonocytes, we have previously shown that translation of APC can be blocked by the RNA binding protein, Musashi 1 (MSI1) and that MSI1 is a Wnt target. We propose that a double negative feedback loop between APC and MSI1 functions in maintenance of intestinal cell homeostasis. To explore the MSI1/APC interaction in vivo, we analyzed intestinal tissue from MSI1 knock-out mice (Msi1-/-). We found higher levels of APC and less Wnt signal activation in intestines from the Msi1-/- mice. There were also more enterocytes within the intestinal crypts of Msi1-/-mice, indicating an increase in differentiation. These three observations provide confirmation of the proposed APC/MSI1 double negative feedback loop in vivo. Evidence from cultured human cells and mouse models supports a role for an APC/MSI1 double negative feedback loop in the response of intestinal cells to Wnt signaling. However, the underlying mechanisms remain to be determined. Proteins that bind to a specific motif in the 3’UTR of target mRNA can induce translation inhibition or promotion as well as stabilization or destabilization of the mRNA. We already established that MSI1 blocks APC translation; however, the effect of MSI1 on APC mRNA stability was unknown. Here, we show that MSI1 binding to the 3’ UTR of APC mRNA stabilizes the message. In contrast, Numb, another established MSI1 target, does not appear to be stabilized by MSI1 binding. If MSI1 bound to the 3’UTR of APC mRNA stabilizes the message, but inhibits translation, then something likely leads to MSI1 release when APC translation resumes. One possibility is that MSI1 protein is inherently unstable. In this case, MSI1 binds to APC mRNA and blocks translation but is rapidly degraded and therefore releases APC mRNA for translation unless additional MSI1 is made. Because MSI1 is a Wnt target, a cell stimulated by Wnt ligand should continually replace the MSI1, therefore APC mRNA translation would remain blocked until the Wnt signal is removed. Our data showing that MSI1 has a relatively long half-life does not support this model. Current work is aimed to explore whether MSI1 release from APC mRNA is regulated by post-translational modifications or other protein-protein interactions. In conclusion, by acting as a Wnt antagonist, APC blocks MSI1 transcription. On the other hand, MSI1 binds to and stabilizes APC mRNA while also blocking APC translation. The MSI/APC double negative feedback loop appears important for maintaining homeostasis of intestinal epithelial cells. Citation Format: Andy R. Wolfe, Erick Spears, Amanda Ernlund, Kristi L. Neufeld. Musashi 1 stabilizes and blocks translation of Adenomatous Polyposis Coli mRNA in intestinal cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1110. doi:10.1158/1538-7445.AM2013-1110

Expression analysis of MET, EGFR, and HER2, and K-ras mutation status in patients with stage II/III gastric cancer enrolled in the ACTS-GC study.

40 Background: Exploratory biomarker analysis was conducted to identify factors related to outcomes of patients enrolled in the ACTS-GC study, a randomized controlled trial comparing surgery followed by adjuvant S-1 therapy with surgery alone in 1,059 patients with stage II/III gastric cancer. Methods: Formalin-fixed paraffin-embedded specimens obtained from 105 patients, an institutional subset of this biomarker study of 829 patients, were extensively examined for EGFR and HER2 by immunohistochemistry (IHC), MET amplification by FISH, and K-ras mutation status (codon 12, 13) by PNA-enriched direct sequencing. The expressions of 63 genes involved in pyrimidine metabolic pathway, growth factor signaling pathway, apoptosis, DNA repair, etc., were measured by quantitative real-time RT-PCR. The relations of the expressions of these genes to clinicopathological features such as histological type, T grade, N grade, tumor stage, gender, and age were also investigated. Results: The rates of K-ras mutation, EGFR overexpression, HER2 overexpression, and MET amplification were 6.7% (7/105), 14.3% (15/105), 11.4% (12/105), and 21.0% (22/105), respectively. These factors were almost mutually exclusive. Co-expressions were seen in 2 (EGFR &amp; HER2), 4 (HER2 &amp; MET), 5 (MET and EGFR) and 1 (EGFR &amp; HER2 &amp; MET) of the 105 patients. MET amplification and EGFR overexpression were associated with worse outcomes. In contrast, KRAS and HER2 status were not associated with overall survival or relapse-free survival. Expression levels of PCAM1, ESR1, MUC2, CAV1, ITGB3, and APC mRNA were significantly higher in diffuse-type cancer than in intestinal-type cancer, whereas expression levels of HER2, E2F1, LDHA and TOP2A mRNA were significantly higher in intestinal-type than in diffuse-type cancer (FDR P&lt;0.05). Associations of other clinically significant genes with clinicopathological features other than histologic type were not screened. Conclusions: Many clinical, pathological, molecular, and genetic factors have been investigated with a growing understanding of the molecular pathogenesis of gastric cancer. We review and discuss such prognostic markers in the ACTS-GC study.

Inhibition of DNA methyltransferase activity and expression by treatment with the pan-deacetylase inhibitor panobinostat in hepatocellular carcinoma cell lines

BackgroundHepatocellular carcinoma (HCC) still represents an unmet medical need. Epigenetic inactivation of tumor suppressor genes like RASSF1A or APC by overexpression of DNA methyltransferases (DNMTs) has been shown to be common in HCC and to be linked to the overall prognosis of patients. Inhibitors of protein and histone deacetylases (DACi) have been demonstrated to possess strong anti-tumor effects in HCC models.MethodsWe therefore investigated whether DACi also has any influence on the expression and activity of DNMTs and methylated target genes in HepG2 and Hep3B cell culture systems and in a xenograft model by immunohistochemistry, westernblotting, RT-qPCR and methylation-specific PCR.ResultsOur findings demonstrate a rapid inhibition of DNMT activity 6 h after treatment with 0.1 μM of the pan-DACi panobinostat. A downregulation of DNMT mRNAs and protein were also observed at later points in time. This loss of DNMT activity and expression was paralleled by a diminished methylation of the target genes RASSF1A and APC and a concomitant re-expression of APC mRNA and protein. Analysis of HepG2 xenograft specimens confirmed these results in vivo.ConclusionWe suggest a dual mode of action of DACi on DNA methylation status: a rapid inhibition of enzyme activity due to interference with posttranslational acetylation and a delayed effect on transcriptional control of DNMT genes by HDAC or miRNA mechanisms.

Prelinical evaluation of selective inhibitors of nuclear export (SINE) CRM1 (XPO1) inhibitors in prostate cancer (PrCa).

e15200 Background: CRM1 (XPO1) is the sole exportin mediating transport of multiple tumor suppressor proteins (TSP) including p53, pRB, FOXO, APC and NPM1 out of the nucleus, abrograting their function. Many tumor types show overexpression of CRM1, thus extinguishing TSP function. CRM1 inhibition forces nuclear accumulation of TSPs inducing apoptosis in cancer cells. Methods: MSKCC PrCa gene database was used for mRNA analyses. Inhibition of CRM1 mediated NE was determined by IHC. MTT assays were used to determine cytotoxicity across divergent PrCa lines: LAPC-4 (p53wt, AR+, low Akt/mTOR); LnCaP (p53wt, AR+, high Akt/mTOR); LnCaP-C81 and LnCaP-C4-2B (p53wt, AR+, androgen independent, high Akt/mTOR); 22rv1 (p53wt, AR+, androgen independent, low Akt/mTOR); PC3 (p53-del, AR–, high Akt/mTOR), DU145 (p53mut, AR–, low Akt/mTOR). BPH1 and prostatic epithelial line EPN were used as non-neoplastic controls. 22rv1 xenografts were initiated in the flanks of SCID mice and treatment begun when tumors reached ~150mm3. Results: Analyses of nuclear pore complex (NPC)-related mRNA levels shows coordinate regulation of transcripts and allow PrCa to be segregated into high and low NPC expression. Low NPC expressing tumors tended to have reduced AR expression; APC, NPM1, and RB1 mRNA levels were also low (p&lt;10e-12) and p16INK4A levels were elevated. As NE is dysregulated in PrCa, we assessed the effects of SINE, irreversible CRM1 inhibitors, across PrCa cell lines. SINE block CRM1 mediated nuclear export of TSPs with IC50 values &lt;100 nM in all tested cell lines. SINE are selectively cytotoxic for neoplastic (EC50 10-1000nM) versus non-neoplastic lines (EC 50 &gt;5-20µM). SINE cytotoxic effects are independent all tested genetic alterations. In the aggressive, androgen independent 22rv1 xenograft, oral SINE KPT-251 showed dose-dependent inhibition of tumor growth and synergy with cisplatin. Conclusions: These data show that PrCa shows coordinate NPC regulation, and that selective blockade of CRM1 dependent NE represents a completely novel, neoplasia-selective and well-tolerated target for use as single agent or in combination with chemotherapy for PrCa.