HSPA6 is an ulcerative colitis susceptibility factor that is induced by cigarette smoke and protects intestinal epithelial cells by stabilizing anti-apoptotic Bcl-XL.

BACKGROUND: Cigarette smoking ameliorates ulcerative colitis (UC) and aggravates Crohn's disease (CD). Cigarette smoke suppresses inflammation-induced apoptosis in intestinal epithelial cells (DLD-1), which may explain its protective effect in UC. Here, we performed transcriptome profiling of cigarette smoke extract (CSE)-exposed DLD-1 and Jurkat cells (T-lymphocytes) and related this to UC susceptibility genes with protective functions in the intestinal epithelium. METHODS: CSE-regulated genes in DLD-1 and Jurkat cells were identified by Illumina microarrays and compared to genes in UC susceptibility loci. Colon biopsies were analyzed by immunohistochemistry for cell-specific expression of HSPA6. CSE-induced gene expression was analyzed by Q-PCR, Western blotting and immunofluorescence microscopy. Protein (HSPA6/Bcl-XL) interactions were analyzed by immunoprecipitation. RESULTS: CSE changed the expression of 536 and 2560 genes in DLD-1 and Jurkat cells, respectively. The "response to unfolded protein" was one of the most significantly affected gene sets with prominent induction (20.3-fold) of heat shock protein A6 (HSPA6). Six CSE-induced genes in DLD-1 cells were located in UC-susceptibility loci, including HSPA6 (rs1801274). HSPA6 is highly expressed in the human colonic epithelium. CSE caused a dose-dependent strong (>100-fold at 30% CSE for 6h), but transient induction of HSPA6 mRNA and protein in DLD-1 cells. HSPA6 co-immune precipitated with anti-apoptotic Bcl-XL, protein levels of which were increased while mRNA levels were unchanged. CONCLUSIONS: HSPA6 is a cigarette smoke-induced UC-susceptibility gene. The HSPA6 risk locus is associated with decreased HSPA6 expression. HSPA6 provides epithelial protection by stabilizing anti-apoptotic Bcl-XL, thereby contributing to the beneficial effect of cigarette smoking in UC.

Genome-wide methylation profiling identifies hypermethylated biomarkers in high-grade cervical intraepithelial neoplasia.

Epigenetic modifications, such as aberrant DNA promoter methylation, are frequently observed in cervical cancer. Identification of hypermethylated regions allowing discrimination between normal cervical epithelium and high-grade cervical intraepithelial neoplasia (CIN2/3), or worse, may improve current cervical cancer population-based screening programs. In this study, the DNA methylome of high-grade CIN lesions was studied using genome-wide DNA methylation screening to identify potential biomarkers for early diagnosis of cervical neoplasia. Methylated DNA Immunoprecipitation (MeDIP) combined with DNA microarray was used to compare DNA methylation profiles of epithelial cells derived from high-grade CIN lesions with normal cervical epithelium. Hypermethylated differentially methylated regions (DMRs) were identified. Validation of nine selected DMRs using BSP and MSP in cervical tissue revealed methylation in 63.2-94.7% high-grade CIN and in 59.3-100% cervical carcinomas. QMSP for the two most significant high-grade CIN-specific methylation markers was conducted exploring test performance in a large series of cervical scrapings. Frequency and relative level of methylation were significantly different between normal and cancer samples. Clinical validation of both markers in cervical scrapings from patients with an abnormal cervical smear confirmed that frequency and relative level of methylation were related with increasing severity of the underlying CIN lesion and that ROC analysis was discriminative. These markers represent the COL25A1 and KATNAL2 and their observed increased methylation upon progression could intimate the regulatory role in carcinogenesis. In conclusion, our newly identified hypermethylated DMRs represent specific DNA methylation patterns in high-grade CIN lesions and are candidate biomarkers for early detection.

Involvement of the TGF-beta and beta-catenin pathways in pelvic lymph node metastasis in early-stage cervical cancer.

PURPOSE: Presence of pelvic lymph node metastases is the main prognostic factor in early-stage cervical cancer patients, primarily treated with surgery. Aim of this study was to identify cellular tumor pathways associated with pelvic lymph node metastasis in early-stage cervical cancer. EXPERIMENTAL DESIGN: Gene expression profiles (Affymetrix U133 plus 2.0) of 20 patients with negative (N(0)) and 19 with positive lymph nodes (N(+)), were compared with gene sets that represent all 285 presently available pathway signatures. Validation immunostaining of tumors of 274 consecutive early-stage cervical cancer patients was performed for representatives of the identified pathways. RESULTS: Analysis of 285 pathways resulted in identification of five pathways (TGF-ß, NFAT, ALK, BAD, and PAR1) that were dysregulated in the N(0), and two pathways (ß-catenin and Glycosphingolipid Biosynthesis Neo Lactoseries) in the N(+) group. Class comparison analysis revealed that five of 149 genes that were most significantly differentially expressed between N(0) and N(+) tumors (P < 0.001) were involved in ß-catenin signaling (TCF4, CTNNAL1, CTNND1/p120, DKK3, and WNT5a). Immunohistochemical validation of two well-known cellular tumor pathways (TGF-ß and ß-catenin) confirmed that the TGF-ß pathway (positivity of Smad4) was related to N(0) (OR: 0.20, 95% CI: 0.06-0.66) and the ß-catenin pathway (p120 positivity) to N(+) (OR: 1.79, 95%CI: 1.05-3.05). CONCLUSIONS: Our study provides new, validated insights in the molecular mechanism of lymph node metastasis in cervical cancer. Pathway analysis of the microarray expression profile suggested that the TGF-ß and p120-associated noncanonical ß-catenin pathways are important in pelvic lymph node metastasis in early-stage cervical cancer.

Gene promoter methylation patterns throughout the process of cervical carcinogenesis.

OBJECTIVES: To determine methylation status of nine genes, previously described to be frequently methylated in cervical cancer, in squamous intraepithelial lesions (SIL). METHODS: QMSP was performed in normal cervix, low-grade (L)SIL, high-grade (H)SIL, adenocarcinomas and squamous cell cervical cancers, and in corresponding cervical scrapings. RESULTS: Only CCNA1 was never methylated in normal cervices and rarely in LSILs. All other genes showed methylation in normal cervices, with CALCA, SPARC and RAR-beta(2) at high levels. Methylation frequency of 6 genes (DAPK, APC, TFPI2, SPARC, CCNA1 and CADM1) increased with severity of the underlying cervical lesion. DAPK showed the highest increase in methylation frequency between LSIL and HSIL (10% vs. 40%, p<0.05), while CCNA1 and TFPI2 were most prominently methylated in cervical cancers compared to HSILs (25% vs. 52%, p<0.05, 30% vs. 58%, p<0.05). CADM1 methylation in cervical cancers was related to depth of invasion (p<0.05) and lymph vascular space involvement (p<0.01), suggesting a role in invasive potential of cervical cancers. Methylation ratios in scrapings reflected methylation status of the underlying lesions (p<0.05). CONCLUSION: Methylation of previously reported cervical cancer specific genes frequently occurs in normal epithelium. However, frequency of methylation increases during cervical carcinogenesis, with CCNA1 and DAPK as the best markers to distinguish normal/LSIL from HSIL/cancer lesions.

Methylation markers for CCNA1 and C13ORF18 are strongly associated with high-grade cervical intraepithelial neoplasia and cervical cancer in cervical scrapings.

PURPOSE: Recently, we reported 13 possible cervical cancer-specific methylated biomarkers identified by pharmacologic unmasking microarray in combination with large-genome computational screening. The aim of the present study was to perform an in-depth analysis of the methylation patterns of these 13 candidate genes in cervical neoplasia and to determine their diagnostic relevance. EXPERIMENTAL DESIGN AND RESULTS: Five of the 13 gene promoters (C13ORF18, CCNA1, TFPI2, C1ORF166, and NPTX1) were found to be more frequently methylated in frozen cervical cancer compared with normal cervix specimens. Quantitative methylation analysis for these five markers revealed that both CCNA1 and C13ORF18 were methylated in 68 of 97 cervical scrapings from cervical cancer patients and in only 5 and 3 scrapings, respectively, from 103 healthy controls (P < 0.0005). In cervical scrapings from patients referred with an abnormal Pap smear, CCNA1 and C13ORF18 were methylated in 2 of 43 and 0 of 43 CIN 0 (no cervical intraepithelial neoplasia) and in 1 of 41 and 0 of 41 CIN I, respectively. Furthermore, 8 of 43 CIN II, 22 of 43 CIN III, and 3 of 3 microinvasive cancer patients were positive for both markers. Although sensitivity for CIN II or higher (for both markers 37%) was low, specificity (96% and 100%, respectively) and positive predictive value (92% and 100%, respectively) were high. CONCLUSION: Methylation of CCNA1 and C13ORF18 in cervical scrapings is strongly associated with CIN II or higher-grade lesions. Therefore, these markers might be used for direct referral to gynecologists for patients with a methylation-positive scraping.

Discovery of DNA methylation markers in cervical cancer using relaxation ranking.

BACKGROUND: To discover cancer specific DNA methylation markers, large-scale screening methods are widely used. The pharmacological unmasking expression microarray approach is an elegant method to enrich for genes that are silenced and re-expressed during functional reversal of DNA methylation upon treatment with demethylation agents. However, such experiments are performed in in vitro (cancer) cell lines, mostly with poor relevance when extrapolating to primary cancers. To overcome this problem, we incorporated data from primary cancer samples in the experimental design. A strategy to combine and rank data from these different data sources is essential to minimize the experimental work in the validation steps. AIM: To apply a new relaxation ranking algorithm to enrich DNA methylation markers in cervical cancer. RESULTS: The application of a new sorting methodology allowed us to sort high-throughput microarray data from both cervical cancer cell lines and primary cervical cancer samples. The performance of the sorting was analyzed in silico. Pathway and gene ontology analysis was performed on the top-selection and gives a strong indication that the ranking methodology is able to enrich towards genes that might be methylated. Terms like regulation of progression through cell cycle, positive regulation of programmed cell death as well as organ development and embryonic development are overrepresented. Combined with the highly enriched number of imprinted and X-chromosome located genes, and increased prevalence of known methylation markers selected from cervical (the highest-ranking known gene is CCNA1) as well as from other cancer types, the use of the ranking algorithm seems to be powerful in enriching towards methylated genes.Verification of the DNA methylation state of the 10 highest-ranking genes revealed that 7/9 (78%) gene promoters showed DNA methylation in cervical carcinomas. Of these 7 genes, 3 (SST, HTRA3 and NPTX1) are not methylated in normal cervix tissue. CONCLUSION: The application of this new relaxation ranking methodology allowed us to significantly enrich towards methylation genes in cancer. This enrichment is both shown in silico and by experimental validation, and revealed novel methylation markers as proof-of-concept that might be useful in early cancer detection in cervical scrapings.

Assessment of gene promoter hypermethylation for detection of cervical neoplasia.

Current cervical cancer screening is based on morphological assessment of Pap smears and associated with significant false negative and false positive results. Previously, we have shown that detection of hypermethylated genes in cervical scrapings using quantitative methylation-specific PCR (QMSP) is a promising tool for identification of squamous cell cervical cancer. Aim of the present pilot-study was to evaluate presence of hypermethylated genes in cervical carcinogenesis, both in squamous cell as well as adenocarcinomas. Cervical scrapings were obtained from 30 patients diagnosed with cervical cancer (20 squamous cell carcinomas and 10 adenocarcinomas) and 19 women with histologically normal cervices. The scraped cells were used for determination of promoter hypermethylation by QMSP for 12 genes and for morphological assessment. Overall, CALCA, DAPK, ESR1, TIMP3, APC and RAR-beta2 promoters were significantly more often hypermethylated in cancers than in controls, while adenocarcinomas were more often hypermethylated above the highest control ratio for APC, TIMP3 and RASSF1A promoters. Combining 4 genes (CALCA, DAPK, ESR1 and APC) yielded a sensitivity of 89% (with all adenocarcinomas identified), equal to cytomorphology (89%) and high-risk human papilloma virus (Hr-HPV; 90%). The 4-gene QMSP proved theoretically superior to cytomorphology as well as Hr-HPV in specificity (100% vs. 83 and 68%, respectively), because cytology identified 3 controls as moderate or severe dyskaryosis and 6 controls were positive for Hr-HPV. In conclusions, QMSP of 4 gene promoters combined appears to have comparable sensitivity and potentially better specificity in comparison to "classic" cytomorphological assessment and Hr-HPV detection. QMSP holds promise as a new diagnostic tool for both squamous cell carcinoma and adenocarcinoma of the cervix.