Re: Detection of Hypermethylated Genes in Women With and Without Cervical Neoplasia

Feng et al. (1) examined whether changes in DNA methylation of 20 genes, selected on the basis of their role in cervical cancer, could be used as markers of cervical intraepithelial neoplasia (CIN) and invasive cervical cancer (ICC). The authors found varying frequencies of promoter hypermethylation in these 20 genes in 319 exfoliated cell samples and matched tissue biopsy specimens. For four of these genes (DAPK1, RARB, TWIST1, and CDH13), increasing frequency of hypermethylation was statistically significantly associated with increasing severity of disease. The estimated specificity of the three-gene panel (DAPK, RARB, and TWIST1) was 95%, which is higher than specificities reported for cytology and human papillomavirus (HPV) testing. Although the study was well designed and a molecular genetic test with high specificity such as this one is needed, some of the findings are inconsistent with previously reported results and the conclusions may therefore not be valid

Integrative genomic approaches in cervical cancer: implications for molecular pathogenesis

Cervical cancer (CC) as a single diagnostic entity exhibits differences in clinical behavior and poor outcomes in response to therapy in advanced tumors. Although infection of high-risk human papillomavirus is recognized as an important initiating event in cervical tumorigenesis, stratification of CC into subclasses for progression and response to treatment remains elusive. Existing knowledge of genetic, epigenetic and transcriptional alterations is inadequate in addressing the issues of diagnosis, progression and response to treatment. Recent technological advances in high-throughput genomics and the application of integrative approaches have greatly accelerated gene discovery, facilitating the identification of molecular targets. In this article, we discuss the results obtained by preliminary integrative analysis of DNA copy number increases and gene expression, utilizing the two most common copy number-gained regions of 5p and 20q in identifying gene targets in CC. These analyses provide insights into the roles of genes such as RNASEN, POLS and SKP2 on 5p, KIF3B, RALY and E2F1 at 20q11.2 and CSE1L, ZNF313 and B4GALT5 at 20q13.13. Future integrative applications using additional datasets, such as mutations, DNA methylation and clinical outcomes, will raise the promise of accomplishing the identification of biological pathways and molecular targets for therapies for patients with CC

Mapping common deleted regions on 5p15 in cervical carcinoma and their occurrence in precancerous lesions

BACKGROUND: Previous studies have shown that the short arm of chromosome 5 (5p) exhibit frequent genetic changes in invasive cervical carcinoma (CC), and that these changes arise early during the carcinogenesis, in precancerous lesions. These data therefore suggest that loss of candidate tumor suppressor genes located on 5p is associated with the development of CC. However, the precise location of 5p deletions is not known. RESULTS: We performed a detailed deletion mapping of 5p in 60 cases of invasive CC. We found that 60% of the tumors exhibit a 5p loss of heterozygosity (LOH). The patterns of LOH allowed us to identify two minimal regions of deletions, one at 5p15.3 spanning a 5.5 cM genetic distance and a second site of 7 cM at 5p15.2-15.3. In addition, we also identified 5p deletions in 16% lesions of high-grade cervical intraepithelial neoplasia (CIN). 5p LOH was found in 63% of HPV 16 positive tumors, while only 33% tumors with other HPV-types had 5p LOH. The differences in frequency of 5p LOH between tumors harboring HPV16 in combination with other HPV types and tumors harboring HPV16 DNA alone were significantly higher, suggesting a synergistic effect of high-risk types in causing genomic instability. CONCLUSION: These findings implicate the presence of tumor suppressor gene(s) on 5p relevant to CC tumorigenesis

Comprehensive Molecular Cytogenetic Characterization of Cervical Cancer Cell Lines

We applied a combination of molecular cytogenetic methods, including comparative genomic hybridization (CGH), spectral karyotyping (SKY), and fluorescence in situ hybridization (FISH), to characterize the genetic aberrations in eight widely used cervical cancer (CC) cell lines. CGH identified the most frequent chromosomal losses including 2q, 3p, 4q, 6q, 8p, 9p, 10p, 13q, and 18q; gains including 3q, 5p, 5q, 8q, 9q, 11q, 14q, 16q, 17q, and 20q; and high-level chromosomal amplification at 3q21, 7p11, 8q23– q24, 10q21, 11q13, 16q23– q24, 20q11.2, and 20q13. Several recurrent structural chromosomal rearrangements, including der(5)t(5;8)(p13;q23) and i(5)(p10); deletions affecting chromosome bands 5p11, 5q11, and 11q23; and breakpoint clusters at 2q31, 3p10, 3q25, 5p13, 5q11, 7q11.2, 7q22, 8p11.2, 8q11.2, 10p11.2, 11p11.2, 14q10, 15q10, 18q21, and 22q11.2 were identified by SKY. We detected integration of HPV16 sequences by FISH on the derivative chromosomes involving bands 18p10 and 18p11 in cell line C-4I, 2p16, 5q21, 5q23, 6q, 8q24, 10, 11p11, 15q, and 18p11 in Ca Ski, and normal chromosome 17 at 17p13 in ME-180. FISH analysis was also used further to determine the copy number changes of PIKA3CA and MYC. This comprehensive cytogenetic characterization of eight CC cell lines enhances their utility in experimental studies aimed at gene discovery and functional analysis

Promoter hypermethylation-mediated inactivation of multiple Slit-Robo pathway genes in cervical cancer progression

BACKGROUND: Cervical Cancer (CC) exhibits highly complex genomic alterations. These include hemizygous deletions at 4p15.3, 10q24, 5q35, 3p12.3, and 11q24, the chromosomal sites of Slit-Robo pathway genes. However, no candidate tumor suppressor genes at these regions have been identified so far. Slit family of secreted proteins modulates chemokine-induced cell migration of distinct somatic cell types. Slit genes mediate their effect by binding to its receptor Roundabout (Robo). These genes have shown to be inactivated by promoter hypermethylation in a number of human cancers. RESULTS: To test whether Slit-Robo pathway genes are targets of inactivation at these sites of deletion, we examined promoter hypermethylation of SLIT1, SLIT2, SLIT3, ROBO1, and ROBO3 genes in invasive CC and its precursor lesions. We identified a high frequency of promoter hypermethylation in all the Slit-Robo genes resulting in down regulated gene expression in invasive CC, but the inhibitors of DNA methylation and histone deacetylases (HDACs) in CC cell lines failed to effectively reactivate the down-regulated expression. These results suggest a complex mechanism of inactivation in the Slit-Robo pathway in CC. By analysis of cervical precancerous lesions, we further show that promoter hypermethylation of Slit-Robo pathway occurs early in tumor progression. CONCLUSION: Taken together, these findings suggest that epigenetic alterations of Slit-Robo pathway genes (i) play a role in CC development, (ii) further delineation of molecular basis of promoter methylation-mediated gene regulation provides a potential basis for epigenetic-based therapy in advanced stage CC, and (iii) form epigenetic signatures to identify precancerous lesions at risk to progression

Frequent Promoter Methylation of CDH1, DAPK, RARB, and HIC1 Genes in Carcinoma of Cervix Uteri: Its Relationship to Clinical Outcome

BACKGROUND: Cervical cancer (CC), a leading cause of cancer-related deaths in women worldwide, has been causally linked to genital human papillomavirus (HPV) infection. Although a host of genetic alterations have been identified, molecular basis of CC development is still poorly understood. RESULTS: We examined the role of promoter hypermethylation, an epigenetic alteration that is associated with the silencing tumor suppressor genes in human cancer, by studying 16 gene promoters in 90 CC cases. We found a high frequency of promoter methylation in CDH1, DAPK, RARB, and HIC1 genes. Correlation of promoter methylation with clinical characteristics and other genetic changes revealed the following: a) overall promoter methylation was higher in more advanced stage of the disease, b) promoter methylation of RARB and BRCA1 predicted worse prognosis, and c) the HIC1 promoter methylation was frequently seen in association with microsatellite instability. Promoter methylation was associated with gene silencing in CC cell lines. Treatment with methylation or histone deacetylation-inhibiting agents resulted in profound reactivation of gene expression. CONCLUSIONS: These results may have implications in understanding the underlying epigenetic mechanisms in CC development, provide prognostic indicators, and identify important gene targets for treatment

Identification of Copy Number Gain and Overexpressed Genes on Chromosome Arm 20q by an Integrative Genomic Approach in Cervical Cancer: Potential Role in Progression

Recurrent karyotypic abnormalities are a characteristic feature of cervical cancer (CC) cells, which may result in deregulated expression of important genes that contribute to tumor initiation and progression. To examine the role of gain of the long arm of chromosome 20 (20q), one of the common chromosomal gains in CC, we evaluated CC at various stages of progression using single nucleotide polymorphism (SNP) array, gene expression profiling, and fluorescence in situ hybridization (FISH) anal- yses. This analysis revealed copy number increase (CNI) of 20q in >50% of invasive CC and identified two focal amplicons at 20q11.2 and 20q13.13 in a subset of tumors. We further demonstrate that the acquisition of 20q gain occurs at an early stage in CC development and the high-grade squamous intraepithelial lesions (HSIL) that exhibit 20q CNI are associated (P 5 0.05) with persistence or progression to invasive cancer. We identified a total of 26 overexpressed genes as consequence of 20q gain (N 5 14), as targets of amplicon 1 (N 5 9; two genes also commonly expressed with 20q gain) and amplicon 2 (N 5 6; one gene also commonly expressed with 20q gain). These include a number of functionally important genes in cell cycle regulation (E2F1, TPX2, KIF3B, PIGT, and B4GALT5), nuclear function (CSEL1), viral replication (PSMA7 and LAMA5), methylation and chromatin remodeling (ASXL1, AHCY, and C20orf20), and transcription regulation (TCEA2). Our findings implicate a role for these genes in CC tumorigenesis, represent an important step toward the development of clinically significant biomarkers, and form a framework for testing as molecular therapeutic targets

The aryl hydrocarbon receptor repressor is a putative tumor suppressor gene in multiple human cancers

The aryl hydrocarbon receptor repressor (AHRR) is a bHLH/Per-ARNT-Sim transcription factor located in a region of chromosome 5 (5p15.3) that has been proposed to contain one or more tumor suppressor genes. We report here consistent downregulation of AHRR mRNA in human malignant tissue from different anatomical origins, including colon, breast, lung, stomach, cervix, and ovary, and demonstrate DNA hypermethylation as the regulatory mechanism of AHRR gene silencing. Knockdown of AHRR gene expression in a human lung can- cer cell line using siRNA significantly enhanced in vitro anchorage-dependent and -independent cell growth as well as cell growth after transplantation into immunocompromised mice. In addition, knockdown of AHRR in non-clonable normal human mammary epithelial cells enabled them to grow in an anchorage-independent manner. Further, downregulation of AHRR expression in the human lung cancer cell line conferred resistance to apoptotic signals and enhanced motility and invasion in vitro and angiogenic potential in vivo. Ectopic expression of AHRR in tumor cells resulted in diminished anchorage-dependent and -independent cell growth and reduced angiogenic potential. These results therefore demonstrate that AHRR is a putative new tumor suppressor gene in multiple types of human cancers

Integrative genomics analysis of chromosome 5p gain in cervical cancer reveals target over-expressed genes, including Drosha

Background: Copy number gains and amplifications are characteristic feature of cervical cancer (CC) genomes for which the underlying mechanisms are unclear. These changes may possess oncogenic properties by deregulating tumor-related genes. Gain of short arm of chromosome 5 (5p) is the most frequent karyotypic change in CC. Methods: To examine the role of 5p gain, we performed a combination of single nucleotide polymorphism (SNP) array, fluorescence in situ hybridization (FISH), and gene expression analyses on invasive cancer and in various stages of CC progression. Results: The SNP and FISH analyses revealed copy number increase (CNI) of 5p in 63% of invasive CC, which arises at later stages of precancerous lesions in CC development. We integrated chromosome 5 genomic copy number and gene expression data to identify key target over expressed genes as a consequence of 5p gain. One of the candidates identified was Drosha (RNASEN), a gene that is required in the first step of microRNA (miRNA) processing in the nucleus. Other 5p genes identified as targets of CNI play a role in DNA repair and cell cycle regulation (BASP1, TARS, PAIP1, BRD9, RAD1, SKP2, and POLS), signal transduction (OSMR), and mitochondrial oxidative phosphorylation (NNT, SDHA, and NDUFS6), suggesting that disruption of pathways involving these genes may contribute to CC progression. Conclusion: Taken together, we demonstrate the power of integrating genomics data with expression data in deciphering tumor-related targets of CNI. Identification of 5p gene targets in CC denotes an important step towards biomarker development and forms a framework for testing as molecular therapeutic targets

Role of promoter hypermethylation in Cisplatin treatment response of male germ cell tumors

BACKGROUND: Male germ cell tumor (GCT) is a highly curable malignancy, which exhibits exquisite sensitivity to cisplatin treatment. The genetic pathway(s) that determine the chemotherapy sensitivity in GCT remain largely unknown. RESULTS: We studied epigenetic changes in relation to cisplatin response by examining promoter hypermethylation in a cohort of resistant and sensitive GCTs. Here, we show that promoter hypermethylation of RASSF1A and HIC1 genes is associated with resistance. The promoter hypermethylation and/or the down-regulated expression of MGMT is seen in the majority of tumors. We hypothesize that these epigenetic alterations affecting MGMT play a major role in the exquisite sensitivity to cisplatin, characteristic of GCTs. We also demonstrate that cisplatin treatment induce de novo promoter hypermethylation in vivo. In addition, we show that the acquired cisplatin resistance in vitro alters the expression of specific genes and the highly resistant cells fail to reactivate gene expression after treatment to demethylating and histone deacetylase inhibiting agents. CONCLUSIONS: Our findings suggest that promoter hypermethylation of RASSF1A and HIC1 genes play a role in resistance of GCT, while the transcriptional inactivation of MGMT by epigenetic alterations confer exquisite sensitivity to cisplatin. These results also implicate defects in epigenetic pathways that regulate gene transcription in cisplatin resistant GCT