Adenomatous Polyposis Coli Promoter Research Articles

Wnt/β-catenin Signaling in Endometrioid Endometrial Cancer and Precursor Lesions

Endometrioid endometrial carcinoma (EEC) is most common invasive malignancy of female genital tract. In spite of advances in diagnosis and treatment, incidence of EEC has been rising because of increasing aging population and prevalence of obesity. Therefore, there is need of research exploring the underlying molecular mechanism of EEC and its precursors. Wnt/ β-catenin signaling are involved in the progression and invasion of EEC. However, the potential role of Wnt/ β-catenin signaling and its effectors in endometrial cancer and precursor lesions remains to be investigated. In the present study a total of 95 samples of endometrial biopsies were assessed histopathologically along with β-catenin expression by immunohistochemistry and promoter methylation of adenomatous polyposis coli (APC) gene by bisulfite method. Women age ranged from 35 to 70 years whose endometrial biopsy and resected samples were obtained. Expression of β-catenin was observed in 28/39; 72% of EEC, 23/37; 62% of precancer cases and 4/19; 21% of normal proliferative endometrium. APC promoter was completely unmethylated in all controls (14/14) and precancer (10/10) groups, however, only one case (1/13, 8%) of cancer was methylated. The present study showed that expression of β-catenin was upregulated in EEC and precursor lesions suggesting involvement of Wnt/ β-catenin signaling from an early stage of development of cancer from precancer. The information might offer a potential therapeutic target for EEC and could also be used as a screening tool in endometrial hyperplasia (EH) cases for detecting potentially precancerous lesions.

Clinical significance of SPOP and APC gene alterations in colorectal cancer in Indian population.

Speckle-Type Poz Protein (SPOP) involved in the regulation of proteasome-mediated degradation of several oncoproteins, resulting in cancer initiation and progression. Mutations in Adenomatous Polyposis Coli (APC) gene is reported in most sporadic and hereditary colorectal cancer (CRC). Identifying the cellular changes involved in carcinogenesis when APC is mutated is an important issue that needs attention. The tumor suppressive function of SPOP and APC has long been a major focus in the research field of colorectal cancer. However, the clinical significance of SPOP and APC gene alteration in CRC has not been established to date. Mutational analysis was performed by single-strand conformational polymorphism followed by Sanger sequencing, methylation status by methylation-specific PCR, and protein expression by immunohistochemistry on 142 tumor tissues along with their adjacent non-cancerous specimens. The overall survival (OS) and recurrence free survival (RFS) were estimated by Kaplan-Meier Curve. Mutation rates of APC and SPOP gene were 2.8% and 11.9% while that of promoter hypermethylation were 37% and 47%, respectively. The grade of differentiation and Lymph node metastasis were significantly correlated with APC methylation pattern (p ≤ 0.05). The down regulation of APC was more often seen in colonic cancer compared to rectal cancer (p = 0.07) and more commonly in T3-4 depth of invasion (p = 0.07) and in patients without lymphovascular and perineural invasion (p = 0.007, p = 0.08 respectively). The median overall survival and recurrence free survival (RFS) was 67 & 36months while 3-yr and 5-yr OS and RFS were 61.1% & 56.4% and 49.2% & 44.8%, respectively. APC promoter methylation had a better overall survival (p = 0.035) while loss of SPOP expression had a worse survival (p = 0.09). Our findings reveal high percentage of SPOP gene mutations in CRC. A significant link is found between promoter hyper methylationand protein expression in all mutant cases of APC and SPOP, suggesting that both genes may be associated in the development of colorectal cancer in people of Indian decent. Hypermethylation of APC gene and loss of SPOP expression have shown an association with disease prognosis and could be further studied looking at its potential role in planning adjuvant treatment in CRC patients.

A pan-cancer analysis on the carcinogenic effect of human adenomatous polyposis coli.

Adenomatous polyposis coli (APC) is the most commonly mutated gene in colon cancer and can cause familial adenomatous polyposis (FAP). Hypermethylation of the APC promoter can also promote the development of breast cancer, indicating that APC is not limited to association with colorectal neoplasms. However, no pan-cancer analysis has been conducted. We studied the location and structure of APC and the expression and potential role of APC in a variety of tumors by using The Cancer Genome Atlas and Gene Expression Omnibus databases and online bioinformatics analysis tools. The APC is located at 5q22.2, and its protein structure is conserved among H. sapiens, M. musculus with C. elaphus hippelaphus. The APC identity similarity between homo sapiens and mus musculus reaches 90.1%. Moreover, APC is highly specifically expressed in brain tissues and bipolar cells but has low expression in most cancers. APC is mainly expressed on the cell membrane and is not detected in plasma by mass spectrometry. APC is low expressed in most tumor tissues, and there is a significant correlation between the expressed level of APC and the main pathological stages as well as the survival and prognosis of tumor patients. In most tumors, APC gene has mutation and methylation and an enhanced phosphorylation level of some phosphorylation sites, such as T1438 and S2260. The expressed level of APC is also involved in the level of CD8+ T-cell infiltration, Tregs infiltration, and cancer-associated fibroblast infiltration. We conducted a gene correlation study, but the findings seemed to contradict the previous analysis results of the low expression of the APC gene in most cancers. Our research provides a comparative wholesale understanding of the carcinogenic effects of APC in various cancers, which will help anti-cancer research.

APC Promoter Methylation in Gastrointestinal Cancer.

The adenomatous polyposis coli (APC) gene, known as tumor suppressor gene, has the two promoters 1A and 1B. Researches on APC have usually focused on its loss-of-function variants causing familial adenomatous polyposis. Hypermethylation, however, which is one of the key epigenetic alterations of the APC CpG sequence, is also associated with carcinogenesis in various cancers. Accumulating studies have successively explored the role of APC hypermethylation in gastrointestinal (GI) tumors, such as in esophageal, colorectal, gastric, pancreatic, and hepatic cancer. In sporadic colorectal cancer, the hypermethylation of CpG island in APC is even considered as one of the primary causative factors. In this review, we systematically summarized the distribution of APC gene methylation in various GI tumors, and attempted to provide an improved general understanding of DNA methylation in GI tumors. In addition, we included a robust overview of demethylating agents available for both basic and clinical researches. Finally, we elaborated our findings and perspectives on the overall situation of APC gene methylation in GI tumors, aiming to explore the potential research directions and clinical values.

APC Promoter Hypermethylation as a Prognostic Marker in Breast Cancer Patients.

Background:Adenomatous polyposis coli (APC) promoter hypermethylation implicated in breast cancer development through Wnt signaling pathway, hypermethylation may result in inactivation of APC expression. This study aimed to investigated whether hypermethylation of APC promoter, the aggressive behavior of breast cancer cells, and correlated them with clinicopathological parameters and survival. Methods:Sixty-one fresh tissues of breast tumor were evaluated for APC promoter hypermethylation with methylation-specific PCR techniques (MS-PCR) and APC mRNA expression level analysis by quantitative real-time reverse transcription-PCR. Results:Our results show aberrant APC hypermethylation status was founded in 27 of 61 cases (44%), and significantly associated with chemotherapy treatment (OR= 6.9, 95%CI=1.5-31.01, P = 0.01), distant metastasis (OR = 5.52, 95%CI = 1.27-24.08, P = 0.04) as well as APC methylated status also associated with shorter overall survival than those without (8.4 and 11.0 years respectively, P = 0.02). Conclusion:The findings indicated hypermethylation of APC promoter may be used as a useful prognostic biomarker in breast cancer patients.

Role of a Rare Variant in APC Gene Promoter 1B Region in Classic Familial Adenomatous Polyposis

Background: Familial adenomatous polyposis (FAP) is most commonly caused by germline variants in the adenomatous polyposis coli (APC) gene. Although definite pathogenic variants could be detected in the majority of individuals with FAP, there are still numerous variant-negative FAP patients. Method: We utilized a 139-gene next-generation sequencing (NGS) panel and multiplex ligation-dependent probe amplification (MLPA) to detect pathogenic variants in FAP patients and found a variant-negative pedigree. Through whole-exome sequencing (WES), we identified a point variant in the noncoding region in the APC gene. Finally, we used Sanger sequencing to analyze its pedigree cosegregation and a dual-luciferase reporter (DLR) assay to assess its function. Results: With the exception of 2 variants of undetermined significance (VUS), WES showed no pathogenic or likely pathogenic variants. After performing MLPA, the pedigree was still variant-negative. Interestingly, through WES, a point variant c.-190G>A located in the promoter 1B region of the APC gene was identified in 3 affected individuals. Moreover, a variant carrier was found during screening of the family with Sanger sequencing. Through the DLR assay, we further confirmed that the variant c.-190G>A caused significant suppression of downstream transcription of APC. Conclusions: The variant (c.-190G>A) in the APC promoter 1B region is able to cause FAP with a classic phenotype, but this kind of variant in the noncoding region could be missed by conventional genetic testing. Thus, utilizing sequencing technologies covering a larger area can help us to further explore the pathogenesis in variant-negative FAP cases.

Association of APC gene promoter methylation and the risk of gastric cancer: A meta-analysis and bioinformatics study.

The methylation status of the adenomatous polyposis coli (APC) promoter has been shown to be associated with the occurrence of gastric cancer, but this finding remains controversial. The aim of this study was to investigate the relationship between methylation of the APC gene promoter and gastric cancer. We searched the Web of Science, EMBASE, Medline, and Cochrane Central Register of Controlled Trials (CENTRAL) databases from the date of creation until August 1, 2019. According to the inclusion criteria, the relationship between the methylation status of the APC gene promoter and gastric cancer was investigated. The incidence of APC promoter methylation in the tissues or blood of patients with and without gastric cancer was compared. The results are expressed as the odds ratio (OR) and 95% confidence interval (CI). The pooled OR of each study was estimated using a fixed effects model or a random effects model to generate forest plots. We further validated the results using the MethHC database. Eight studies (985 samples) were included. Our meta-analysis showed that the incidence of APC promoter methylation in patients with gastric cancer was higher than that of patients without gastric cancer (OR = 3.86, 95% CI 1.71-8.74, P = .001). Methylation of the APC promoter is associated with the incidence of gastric cancer, and it increases the risk of gastric cancer. This study provides a new strategic direction for research on gastric cancer. Methylation of the APC promoter may be a potential biomarker for the diagnosis of gastric cancer, but the results of this work require further confirmation.

Promoter epigenetics of APC gene and its implication in sporadic breast cancer patients from South Indian population

BackgroundThe adenomatous polyposis coli (APC) gene is a tumor suppressor gene associated with both familial and sporadic cancer, whose protein plays an important role in the WNT signal transduction pathway. WNT signaling is a key developmental pathway involved in embryonic development, cell differentiation, cell proliferation and tissue maintenance in adults. APC protein acts as an antagonist of the WNT signaling pathway by binding and regulating the β-catenin protein. Impaired APC gene function usually leads to a lack of degradation of β-catenin, which could cause uncontrolled cell growth and lead to tumorigenic transformation. APC gene may be inactivated by aberrant DNA methylation of CpG islands in their promoter regions. ObjectiveHence the present study was designed to determine the role of promoter methylation of APC genes in sporadic breast cancer patients from South Indian population. MethodsDNA methylation analyses of APC gene was performed by methylation-specific polymerase chain reaction (MSP).·Fifty biopsy samples of breast tumor and their corresponding non-malignant portions as controls were studied comparatively. APC mRNA expression analysis was also done using real time PCR. ResultsThirty eight percent (38%) cancerous tissue samples (19/50) showed promoter hypermethylation in APC gene; however, none of normal tissues showed promoter hypermethylation. The difference in methylation frequency between cancerous and normal tissue was statistically significant (p = 0.0001). APC promoter methylation was positively associated with lymph node involvement (p = 0.048), APC Promote hyper methylation and decreased mRNA expression was significantly associated (p = 0.0034). Lower APC-mRNA expression was associated with patient's age above 50 years (p = 0.044). ConclusionIn conclusion, our data showed that promoter hypermethylation of APC gene was a prognostic factor in breast cancer patients from South Indian population. The loss of APC gene function usually leads to an accumulation of β-catenin, which may promote tumorigenesis.

Wnt/β‑catenin pathway activation and silencing of the APC gene in HPV‑positive human cervical cancer‑derived cells.

Although persistent infections with high-risk human papilloma virus (HPV) constitute the most significant cofactor for the development of cervical cancer, they are insufficient on their own. Mutations or epigenetic inactivation of the tumor suppressor adenomatous polyposis coli (APC), the two acting as prominent oncogenic mechanisms in a number of types of cancer, are frequently associated with aberrant activation of the Wnt/β-catenin pathway. According to these observations, it was hypothesized that APC alteration may lead to β-catenin deregulation and the abnormal expression of direct targets of the Wnt pathway in HPV-infected cervical cancer cells. The present study confirmed that the stabilization of β-catenin correlates with enhanced transcriptional activity of the β-catenin/T-cell factor complex in cervical cancer cell lines. Sequence analysis of the ‘hot-spot’ in the mutation cluster region did not exhibit genetic alterations that may be associated with APC gene inactivation. In addition, it was identified that there was a good correlation with the hypermethylation status of the APC promoter 1A and the abnormal accumulation of endogenous β-catenin in cell lines and biopsies infected with HPV16, although not HPV18. Removal of the epigenetic markers led to an increase in APC levels and a reduction of β-catenin expression in two transcriptional targets of the Wnt pathway: Matrix metalloproteinase-7 and vascular endothelial growth factor. The present study suggested that the increase in Wnt activity in certain cervical cancer-derived cells may be associated with an alteration in the methylation status of the APC gene promoter 1A.

The loss-of-function of DNA methyltransferase 1 by siRNA impairs the growth of non-small cell lung cancer with alleviated side effects via reactivation of RASSF1A and APC in vitro and vivo.

Hypermethylation of tumor suppressor genes (TSGs) promoters by DNA methyltransferase (DNMT) can be observed in almost all cancers which represent a hallmark of carcinogenesis, including lung cancer. DNMT inhibitors (e.g.5-Aza-CR/CdR) reactivate TSGs to exert anti-cancer activity and have been applied into the clinical. However, it is cytotoxic even at low concentrations, which might be not directly related to DNA methylation. We here investigated an alternative strategy in the lung cancer therapy and aimed to estimate and compare its efficiency and side effects of knockdown of DNMT1 in vitro and in vivo. Lung cancer tissues (n=20) showed enhanced expression of DNMT1 than corresponding non-neoplastic tissues. Similar results were found in lung cancer cell lines A549 and H538. The treatment of 5-Aza-CR or knockdown of DNMT1 in vitro could inhibit the expressions of DNMT1 but restore the TSGs expressions including the Ras association domain family 1A (RASSF1A) and the adenomatous polyposis coli (APC) via the demethylation of its promoter region, which results in the decreased proliferation, increased apoptosis and impaired ability of migration. Importantly, knockdown of DNMT1 by siRNA in vivo also effectively demethylated the RASSF1A and APC promoter, elevated their expressions and limited tumor growth, which functioned like 5-Aza-CR but with alleviated side effects, suggesting that knockdown of DNMT1 might be potential strategy for the treatment of lung cancer with better tolerability.

The effect of EBV on WIF1, NLK, and APC gene methylation and expression in gastric carcinoma and nasopharyngeal cancer.

Epstein-Barr virus (EBV) is an important DNA tumor virus that is associated with approximately 10% of gastric carcinomas and 99% of nasopharyngeal cancers (NPC). DNA methylation and microRNAs (miRNAs) are the most studied epigenetic mechanisms that can prompt disease susceptibility. This study aimed to detect the effect of EBV on Wnt inhibitory factor 1 (WIF1), Nemo-like kinase (NLK), and adenomatous polyposis coli (APC) gene methylation, and expression in gastric carcinoma and NPC. The WIF1, NLK, and APC gene mRNA expression levels were measured by real-time quantitative RT-PCR in four EBV-positive cell lines and four EBV-negative cell lines. Bisulfite genomic sequencing or methylation-specific PCR was used to detect the methylation status of the WIF1, NLK, and APC promoters. All cell lines were treated with 5-azacytidine (5-aza-dC), miR-BART19-3p mimics or an inhibitor, and analyzed by flow cytometry and MTT cell proliferation assays. The WIF1, NLK, and APC promoters were hypermethylated in all eight cell lines. 5-Aza-dC displayed a growth inhibitory effect on cells . After transfection with miR-BART19-3p mimics, the expression of WIF1, and APC decreased, and the cellular proliferation rate increased. After transfection with the miR-BART19-3p inhibitor, the expression levels were higher, and the cell growth was inhibited. In the NPC and GC cell lines, the promoters of WIF1, NLK, and APC are highly methylated, and the expression of these three genes is regulated by miR-BART19-3p. The activity of the Wnt pathway in EBV-associated tumors may be enhanced by miR-BART19-3p.

Does adenomatous polyposis coli gene promoter 1A methylation increase non-small cell lung cancer risk? A meta-analysis.

BackgroundThe promoter region of the adenomatous polyposis coli (APC) gene is hypermethylated in several types of cancers, including non‐small cell lung cancer (NSCLC). The prevalence of methylation in the promoter region of this gene in tumor tissues and autologous controls has not been consistent in previous studies. We evaluated the frequency of APC gene promoter 1A methylation between tumor tissues and autologous controls in NSCLC patients by meta‐analysis.MethodsOpen published studies of APC gene promoter 1A methylation between tumor tissues and autologous samples in NSCLC patients were identified using a systematic search. Odds ratios (OR) and 95% confidence intervals (CI) of APC gene promoter 1A methylation in lung cancer tissues versus autologous controls were calculated. Fourteen studies, involving a total of 1345 patients and 2182 samples, were finally included.ResultsThe pooled proportion of APC promoter 1A methylation was 0.62 (95% CI 0.52–072) and 0.34 (95% CI 0.21–0.50) in cancer tissues and autologous controls, respectively. The APC gene promoter 1A methylation rate in cancer tissues was much higher than in autologous controls, with a pooled OR of 3.66 (95% CI 2.12–6.33). A strong and significant correlation of APC gene promoter 1A methylation between tumor tissues and autologous controls was detected (correlation coefficient rpearson = 0.77; P = 0.0013).ConclusionThe proportion of APC promoter 1A methylation in lung cancer tissues was higher than in autologous controls, indicating that promoter 1A methylation of the APC gene may play an important role in NSCLC carcinogenesis.

APC hypermethylation for early diagnosis of colorectal cancer: a meta-analysis and literature review.

Adenomatous polyposis coli (APC) promoter hypermethylation has been frequently observed in colorectal cancer (CRC). The association between APC promoter methylation and clinicopathological significance in CRC is under investigation. We performed a meta-analysis to quantitatively evaluate the significance of APC methylation in CRC. The study included a total of 24 articles and 2025 CRC patients. The frequency of APC promoter hypermethylation was significantly higher in colorectal adenoma than in normal colorectal tissue, OR was 5.76, 95% CI, 2.45-13.56; p<0.0001, I2=0%. APC promoter more frequently hypermethylated in CRC stage I compared to normal colorectal tissue, OR was 13.42, 95% CI, 3.66-49.20; p<0.0001, I2=31%. The risk of incidence of CRC was significantly correlated to APC promoter hypermethylation, pooled OR was 9.80, 95%CI, 6.07-15.81; p<0.00001, I2=43%. APC methylation was not associated with grade, stage of CRC as well as tumor location, patients’ gender, and smoking behavior. The results indicate that APC promoter hypermethylation is an early event in carcinogenesis of CRC, could be a valuable diagnostic marker for early-stage CRC. APC methylation is not significantly associated with overall survival in patients with CRC. APC is a potential drug target for development of personalized treatment.

Association between APC promoter methylation and clinicopathological features of patients with hepatocellular carcinoma: a meta-analysis with PRISMA guideline

Background: The adenomatous polyposis coli ( APC ) has been reported as a key tumor suppressor gene in hepatocellular carcinoma (HCC). However, the clinical significance of APC promoter methylation in HCC remains unclear. This meta-analysis was conducted to assess the relationship between APC promoter methylation and clinicopathological characteristics of patients with HCC. Methods: Eligible publications were identified by online electronic databases (PubMed, Embase, EBSCO and the Cochrane Library). The combined odds ratios (ORs) with their corresponding 95% confidence intervals (95% CIs) were calculated and summarized under the random-effects model. Results: Final 21 available studies were included in the current study. APC promoter methylation was significantly higher in HCC than in normal live tissues, liver cirrhosis, and healthy blood samples (OR=11.46, P 0.1). APC promoter methylation was shown to be associated with hepatitis B virus (HBV), and hepatitis C virus (HCV) infection status of patients with HCC (OR=2.86, P=0.005; OR=3.50, P=0.001; respectively), but not correlated with clinical stage, gender, and vascular invasion status (P>0.1). Conclusions: APC promoter methylation may be correlated with HCC, especially for patients with HBV and HCV infection status. Promoter methylation of the APC may be a potential noninvasive biomarker using blood samples in the detection of HCC. More studies with large samples sizes are needed to validate our findings in the future.

Activation of the Wnt/β-catenin pathway is common in wilms tumor, but rarely through β-catenin mutation and APC promoter methylation.

The Wnt/β-catenin pathway is known to be crucial for the regulation of embryogenesis and cell differentiation, and its constitutive activation is associated with a wide range of malignancies. There are two major principles for an activated Wnt/β-catenin pathway. The first is caused by the failure of the destruction complex, mainly due to the decreased expression of the tumor suppressor gene adenomatous polyposis coli (APC); the second is the mutation of the β-catenin (CTNNB1) protein itself. Wilms tumors (WTs) are also thought to be malignancies with a high rate of Wnt/β-catenin pathway activation. The aim of this study was to analyze a large cohort of WT for activated Wnt/β-catenin pathway. The transcription of axis inhibition protein 2 (AXIN2) and APC was analyzed by real-time PCR. Expression was compared with those in healthy renal tissues as a control. Methylation status of the APC promoter was measured by pyrosequencing and correlated with APC expression. Finally, the mutations of CTNNB1 itself were detected by Sanger sequencing. The analysis was done in a cohort of 103 WTs, treated in our institution. There was a significant overexpression of AXIN2 in WTs (P<0.0001), with 33 (32%) tumors showing higher expression (median+3× SD) than normal kidney tissue. In contrast, the expression of APC as well as its promoter methylation did not differ from control (P=0.78; P=0.82). Finally, there were only seven (6.8%) mutations detectable in CTNNB1, and five out of seven were seen in WTs with AXIN2 overexpression. The finding that AXIN2, one of the major Wnt target genes, is overexpressed in our cohort of WTs, is indicative for the activation of the Wnt/β-catenin pathway. However, neither the alteration of APC nor frequent CTNNB1 mutations were seen in our analyses. Therefore, other mechanisms might be responsible for the common activation of the Wnt/β-catenin pathway.

Hypermethylated APC in serous carcinoma based on a meta-analysis of ovarian cancer.

BackgroundThe reduced expression of the Adenomatous polyposis coli (APC) gene, a tumor suppressor gene, through promoter hypermethylation has been reported to play a key role in the carcinogenesis. However, the correlation between APC promoter hypermethylation and ovarian cancer (OC) remains to be clarified.MethodsA comprehensive literature search was carried out in related research databases. The overall odds ratio (OR) and corresponding 95 % confidence interval (CI) were used to evaluate the effects of APC promoter hypermethylation on OC and clinicopathological characteristics.ResultsUltimately, 12 eligible studies were used in our study, including 806 OC samples, 429 normal controls, 109 benign lesions and 75 LMP samples. The pooled OR showed that APC promoter hypermethylation was significantly higher in OC than in normal and benign controls (OR = 6.18 and OR = 3.26, respectively). No significant correlation was observed between OC and low malignant potential (LMP) tumors (P = 0.436). In the comparison of OC and normal controls, subgroup analysis based on race showed that the overall OR of APC promoter hypermethylation was significant and similar in Asians and Caucasians (OR = 8.34 and OR = 5.39, respectively). A subgroup analysis based on sample type found that the pooled OR was significantly higher in blood than in tissue (OR = 18.71 and OR = 5.74, respectively). A significant association was not observed between APC promoter hypermethylation and tumor grade or tumor stage. The pooled OR indicated that APC promoter hypermethylation was significantly lower in serous carcinoma than in non-serous carcinoma (OR = 0.56, P = 0.02). No obvious publication bias was detected by Egger’s test (all P > 0.05).ConclusionsAPC promoter hypermethylation may be linked to the increased risk of OC. It was associated with histological type, but not with tumor grade or tumor stage. Moreover, hypermethylated APC may be a noninvasive biomarker using blood samples. Future studies are required to validate these results.

APC promoter is frequently methylated in pancreatic juice of patients with pancreatic carcinomas or periampullary tumors.

Early detection of pancreatic and periampullary neoplasms is critical to improve their clinical outcome. The present authors previously demonstrated that DNA hypermethylation of adenomatous polyposis coli (APC), histamine receptor H2 (HRH2), cadherin 13 (CDH13), secreted protein acidic and cysteine rich (SPARC) and engrailed-1 (EN-1) promoters is frequently detected in pancreatic tumor cells. The aim of the present study was to assess their prevalence in pancreatic juice of carcinomas of the pancreas and periampullary area. A total of 135 pancreatic juices obtained from 85 pancreatic cancer (PC), 26 ampullary carcinoma (AC), 10 intraductal papillary mucinous neoplasm (IPMN) and 14 chronic pancreatitis (CP) patients were analyzed. The methylation status of the APC, HRH2, CDH13, SPARC and EN-1 promoters was analyzed using methylation specific-melting curve analysis (MS-MCA). Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations were also tested with allele-specific quantitative polymerase chain reaction amplification. Out of the 5 promoters analyzed, APC (71%) and HRH2 (65%) were the most frequently methylated in PC juice. APC methylation was also detected at a high frequency in AC (76%) and IPMN (80%), but only occasionally observed in CP (7%). APC methylation had a high sensitivity (71-80%) for all types of cancer analyzed. The panel (where a sample scored as positive when ≥2 markers were methylated) did not outperform APC as a single marker. Finally, KRAS detection in pancreatic juice offered a lower sensitivity (50%) and specificity (71%) for detection of any cancer. APC hypermethylation in pancreatic juice, as assessed by MS-MCA, is a frequent event of potential clinical usefulness in the diagnosis of pancreatic and periampullary neoplasms.

Methylation status of the APC and RASSF1A promoter in cell-free circulating DNA and its prognostic role in patients with colorectal cancer.

DNA methylation is the most frequent epigenetic alteration. Using methylation-specific polymerase chain reaction (MSP), the methylation status of the adenomatous polyposis coli (APC) and Ras association domain family 1 isoform A (RASSF1A) genes was examined in cell-free circulating DNA from 155 plasma samples obtained from patients with early and advanced colorectal cancer (CRC). APC and RASSF1A hypermethylation was frequently observed in both early and advanced disease, and was significantly associated with a poorer disease outcome. The methylation status of the APC and RASSF1A promoters was investigated in cell-free DNA of patients with CRC. Using MSP, the promoter methylation status of APC and RASSF1A was examined in 155 blood samples obtained from patients with CRC, 88 of whom had operable CRC (oCRC) and 67 had metastatic CRC (mCRC). The frequency of APC methylation in patients with oCRC was 33%. Methylated APC promoter was significantly associated with older age (P=0.012), higher stage (P=0.014) and methylated RASSF1A status (P=0.050). The frequency of APC methylation in patients with mCRC was 53.7%. In these patients, APC methylation was significantly associated with methylated RASSF1A status (P=0.016). The frequency of RASSF1A methylation in patients with oCRC was 25%. Methylated RASSF1A in oCRC was significantly associated with higher stage (P=0.021). The frequency of RASSF1A methylation in mCRC was 44.8%. Methylated RASSF1A in mCRC was associated with moderate differentiation (P=0.012), high levels of carcinoembryonic antigen (P=0.023) and methylated APC status (P=0.016). Patients with an unmethylated APC gene had better survival in both early (81±5 vs. 27±4 months, P<0.001) and advanced disease (37±7 vs. 15±3 months, P<0.001), compared with patients with methylated APC. Patients with an unmethylated RASSF1A gene had better survival in both early (71±6 vs. 46±8 months, P<0.001) and advanced disease (28±4 vs. 16±3 months, P<0.001) than patients with methylated RASSF1A. The observed significant correlations between APC and RASSF1A promoter methylation status and survival may be indicative of a prognostic role for these genes in CRC, which requires additional testing in larger studies.

Quantitative assessment of the association between APC promoter methylation and breast cancer.

Adenomatous polyposis coli (APC) is an important tumor suppressor gene in breast cancer. However, there were inconsistent conclusions in the association between APC promoter methylation and breast cancer. Hence, we conducted a meta-analysis to quantitatively assess the clinicopathological significance and diagnosis role of APC methylation in breast cancer. In total, 3172 samples from 29 studies were performed in this study. The odds ratio (OR) of APC methylation was 5.92 (95% CI = 3.16–11.07) in breast cancer cases compared to controls,. The APC promoter methylation was associated with cancer stage (OR = 0.47, 95% CI = 0.28–0.80, P = 0.006), lymph node metastases (OR = 0.55, 95% CI = 0.36–0.84, P = 0.005) and ER status (OR = 1.34, 95% CI = 1.03–1.73, P = 0.003) in breast cancer. Furthermore, the sensitivity and specificity for all included studies were 0.444 (95% CI: 0.321–0.575, P < 0.0001) and 0.976 (95% CI: 0.916–0.993, P < 0.0001), respectively. These results suggested that APC promoter methylation was associated with breast cancer risk, and it could be a valuable biomarker for diagnosis, treatment and prognosis of breast cancer.

Epigenetic regulation of APC in the molecular pathogenesis of gallbladder cancer.

Background & objectives:Loss of function of adenomatous polyposis coli (APC) has been reported in cancer. The two promoters of APC, 1A and 1B also have roles in cancer. But, the epigenetic role of APC promoters is not yet clear in gallbladder cancer (GBC) and gallstone diseases (GSD). We undertook this study to determine the epigenetic role of APC in GBC and GSD.Methods:Methylation-specific (MS)-PCR was used to analyze the methylation of APC gene. The expression of APC gene was studied by semi-quantitative PCR, real-time PCR and immunohistochemistry (IHC) in GBC, GSD and adjacent normal tissues.Results:Of the two promoters, APC 1A promoter was found methylated in 96 per cent GBC (P=0.0155) and 80 per cent GSD (P=0.015). Exon 1 was downregulated in grade II (P=0.002) and grade III (P=0.0001) of GBC, while exon 2 was normally expressed. Scoring analysis of IHC revealed 0 or negativity in 34.48 per cent (P=0.057) and 1+ in 24.14 per cent (P=0.005) GBC cases suggesting loss of APC expression.Interpretation & conclusions:The present findings indicate epigenetic silencing of APC in advanced GBC. The methylation pattern, followed by expression analysis of APC may be suggested for diagnostic, prognostic and therapeutic purposes in GBC in future.