Changes in SLIT2 expression are associated with the migration of human ovarian clear cell carcinoma cells.

Ovarian clear cell carcinoma (OCCC) is characterized by a poor survival of patients, which is mainly due to metastasis and treatment failure. Slit guidance ligand 2 (SLIT2), a secreted protein, has been reported to modulate the migration of neural cells and human cancer cells. However, the effect of changes in SLIT2 expression on the regulation of cell migration in OCCC remains unknown. The present study examined alterations in SLIT2 expression using OCCC cell models, including low- and high-mobility SKOV3 cells, as well as OCCC tissues. DNA methylation analysis suggested that promoter hypermethylation was responsible for the low expression levels of SLIT2 in OCCC cells. The demethylating agent 5-Aza-deoxycytosine was able to restore SLIT2 expression at both the mRNA and protein levels in high-mobility SKOV3 cells that harbored the relevant methylated promoter. Overexpression of SLIT2 inhibited the migration of high-mobility OCCC cells, as well as decreased the protein expression levels of ß-catenin, phosphorylated (p)AKT and snail family transcriptional repressor 1 (SNAI1). On the other hand, knockdown of SLIT2 increased the migration of low-mobility OCCC cells, and enhanced the protein expression levels of ß-catenin, pAKT and SNAI1. Overall, the results of the present study provided evidence that low expression levels of SLIT2 were associated with increased OCCC cell migration, and that SLIT2 may act as a suppressor gene of cancer cell migration.

Growth-arrest-specific 7C protein inhibits tumor metastasis via the N-WASP/FAK/F-actin and hnRNP U/ß-TrCP/ß-catenin pathways in lung cancer.

Growth-arrest-specific 7 (GAS7) belongs to a group of adaptor proteins that coordinate the actin cytoskeleton. Among human GAS7 isoforms, only GAS7C possesses a Src homology 3 domain. We report here that GAS7C acts as a migration suppressor and can serve as a prognostic biomarker in lung cancer. GAS7C overexpression reduces lung cancer migration, whereas GAS7C knockdown enhances cancer cell migration. Importantly, ectopically overexpressed GAS7C binds tightly with N-WASP thus inactivates the fibronectin/integrin/FAK pathway, which in turn leads to the suppression of F-actin dynamics. In addition, overexpression of GAS7C sequesters hnRNP U and thus decreases the level of ß-catenin protein via the ß-TrCP ubiquitin-degradation pathway. The anti-metastatic effect of GAS7C overexpression was also confirmed using lung cancer xenografts. Our clinical data indicated that 23.6% (25/106) of lung cancer patients showed low expression of GAS7C mRNA which correlated with a poorer overall survival. In addition, low GAS7C mRNA expression was detected in 60.0% of metastatic lung cancer patients, indicating an association between low GAS7C expression and cancer progression. A significant inverse correlation between mRNA expression and promoter hypermethylation was also found, which suggests that the low level of GAS7C expression was partly due to promoter hypermethylation. Our results provide novel evidence that low GAS7C correlates with poor prognosis and promotes metastasis in lung cancer. Low GAS7C increases cancer cell motility by promoting N-WASP/FAK/F-actin cytoskeleton dynamics. It also enhances ß-catenin stability via hnRNP U/ß-TrCP complex formation. Therefore, GAS7C acts as a metastasis suppressor in lung cancer.

Global methylation silencing of clustered proto-cadherin genes in cervical cancer: serving as diagnostic markers comparable to HPV.

Epigenetic remodeling of cell adhesion genes is a common phenomenon in cancer invasion. This study aims to investigate global methylation of cell adhesion genes in cervical carcinogenesis and to apply them in early detection of cancer from cervical scraping. Genome-wide methylation array was performed on an investigation cohort, including 16 cervical intraepithelial neoplasia 3 (CIN3) and 20 cervical cancers (CA) versus 12 each of normal, inflammation and CIN1 as controls. Twelve members of clustered proto-cadherin (PCDH) genes were collectively methylated and silenced, which were validated in cancer cells of the cervix, endometrium, liver, head and neck, breast, and lung. In an independent cohort including 107 controls, 66 CIN1, 85 CIN2/3, and 38 CA, methylated PCDHA4 and PCDHA13 were detected in 2.8%, 24.2%, 52.9%, and 84.2% (P < 10(-25) ), and 2.8%, 24.2%, 50.6%, and 94.7% (P < 10(-29) ), respectively. In diagnosis of CIN2 or more severe lesion of the cervix, a combination test of methylated PCDHA4 or PCDHA13 from cervical scraping had a sensitivity, specificity, positive predictive value, and negative predictive value of 74.8%, 80.3%, 73%, and 81.8%, respectively. Testing of this combination from cervical scraping is equally sensitive but more specific than human papillomavirus (HPV) test in diagnosis of CIN2 or more severe lesions. The study disclosed a collective methylation of PCDH genes in cancer of cervix and other sites. At least two of them can be promising diagnostic markers for cervical cancer noninferior to HPV.

Testing for methylated PCDH10 or WT1 is superior to the HPV test in detecting severe neoplasms (CIN3 or greater) in the triage of ASC-US smear results.

OBJECTIVE: Management of equivocal Papanicolaou smear result remains to be challenging even with the aid of human papillomavirus test. Recently, 3 novel methylation-silenced genes, PAX1, WT1, and PCDH10, have been found to be specifically associated with cervical cancer. We compared the performances of methylation test of these genes with human papillomavirus tests in triage of equivocal Papanicolaou smear result. STUDY DESIGN: Two hundred twenty-two women with Papanicolaou smear results of atypical cells of undetermined significance nested to a multicenter, nation-wide cohort (the T1899 cohort) were studied. Status of cervical neoplasm was diagnosed with colposcopic biopsy. Status of gene methylation was determined by methylation-specific polymerase chain reaction. High-risk human papillomavirus DNA was detected by polymerase chain reaction-reverse line blot hybridization and Hybrid Capture 2. RESULTS: Cervical intraepithelial neoplasm 1, cervical intraepithelial neoplasm 2, cervical intraepithelial neoplasm 3, carcinoma in situ, carcinoma, and normal cervix were diagnosed in 58, 17, 14, 10, 1, and 120 women, respectively. Methylation of PCDH10, WT1, and PAX1 was highly associated with the severity of cervical neoplasm (P < 10⁻9, < 10⁻7, and < 10⁻5, respectively). In comparison with a negative test result, the odds ratio (95% confidence intervals) for cervical intraepithelial neoplasm 3 or more severe neoplasms for women tested positive for methylation of these 3 genes were 26.4 (9.0-77.3), 18.1 (6.9-47.2), and 10.3 (4.1-25.9), respectively; whereas those positive for human papillomavirus polymerase chain reaction and Hybrid Capture 2 were 10.5 (3.5-31.9) and 5.6 (2.3-21.4). In triage for atypical cells of undetermined significance, each methylation test had less colposcopy referral and false-positive rates, but higher false-negative rate than the human papillomavirus tests. With a combination test of PCDH10 or WT1 methylation, a comparable false-negative rate (P = .62) but much less false-positive rate (P = .002) and colposcopy referral rate (P < 10⁻6) were achieved. CONCLUSION: In triage of atypical cells of undetermined significance Papanicolaou smear results, methylation test of WT1 and PCDH10 is superior to human papillomavirus test in this multicenter cohort. Comparing to current human papillomavirus triage, the new test has only one third of false positivity and half of colposcopy referral, with no compromise of the sensitivity in diagnosis of cervical intraepithelial neoplasm 3 or more severe neoplasms.

Quantitative analysis of methylation status of the PAX1 gene for detection of cervical cancer.

OBJECTIVE: Although aided by high-risk human papillomavirus (HPV) DNA test, early detection of cervical cancer is still a challenge. Hypermethylation of the paired boxed gene 1 (PAX1) was recently reported as a characteristic of cervical cancer. This study designed a quantitative measure of PAX1 methylation and compared its efficacy to the currently available Hybrid Capture 2 (HC2) HPV test in detection of cervical cancer. METHODS: Using real-time quantitative methylation-specific polymerase chain reaction, we measured the percentage of PAX1 methylation in cervical scrapings obtained from a hospital-based cohort of women with cervical neoplasia of different severities and compared the efficacy of diagnosis of cervical cancer to that of the HC2 HPV test. RESULTS: From 73 cervical scrapings, with diagnoses of normal (n = 17), cervical intraepithelial neoplasm 1 (CIN1; n = 10), CIN2 (n = 18), CIN3 (n = 14), and invasive cancer (n = 14), the percentage of PAX1 methylation was determined. The percent of methylated reference of invasive cancer (mean [SE], 56.7 [7.1]) was significantly higher than CIN3 (6.5 [2.3]) and the other milder lesions (1.0 [0.3]; P < 0.0001). At a cutoff percent of methylated reference value of 4.5, PAX1 methylation was found in 100% of invasive cancer tissue as compared with 0% of normal tissue, 10% of CIN1, 11% of CIN2, and 43% of CIN3 (P < 0.0001). As a comparison, the HC2 HPV test result was positive in 5.9% of normal tissue, 70% of CIN1, 55.6% of CIN2, 71.4% of CIN3, and 100% of invasive cancer. In addition to cancer tissue, methylation of PAX1 was also found in normal tissue adjacent to the cancer lesion (9/11, 82%) but much less in the remote normal tissues (2/5, 40%), indicating a field methylation. CONCLUSIONS: In this hospital-based study, quantitative measurement of PAX1 hypermethylation in cervical scrapings is highly sensitive and is more specific than HC2 in detection of cervical cancer.

Prognostic significance of X-ray cross-complementing group 1 T-77C polymorphism in resected non-small cell lung cancer.

OBJECTIVE: A novel T-77C polymorphism in the promoter region of the DNA repair gene X-ray cross-complementing group 1 (XRCC1) may modulate its transcription to increase the risk of lung cancer. Here, we attempt to clarify: (i) whether the XRCC1 T-77C polymorphism was associated with lung cancer risk in Taiwanese and (ii) whether this polymorphism could act as a prognostic indicator to predict the clinical outcome of non-small-cell lung cancer (NSCLC) patients. METHODS: A total of 294 primary lung cancer patients and 288 potential controls were recruited into our study. Clinical data were collected. The genotypes of XRCC1 T-77C were identified by polymerase chain reaction. RESULTS: Our case-control study showed that the XRCC1 T-77C polymorphism was not associated with the risk of lung cancer in Taiwanese patients. To verify the impact of the XRCC1 T-77C polymorphism on the clinical outcome of NSCLC, survival analysis showed that patients with TT had a lower survival rate than those with the TC + CC genotype (33.1% versus 48.8%, P = 0.031). The Cox regression analysis further indicated that patients with the TT genotype had a 1.84-fold risk compared with those with the TC + CC genotype (95% CI, 1.16-2.86, P = 0.008). CONCLUSION: Our results suggest that XRCC1 T-77C variants (TC + CC) may act as a favorable prognostic indicator of resected NSCLC.