Despite their extensive clinical and pathologic heterogeneity, all malignant germ cell tumors (GCT) are thought to originate from primordial germ cells. However, no common biological abnormalities have been identified to date. We profiled 615 microRNAs (miRNA) in pediatric malignant GCTs, controls, and GCT cell lines (48 samples in total) and re-analyzed available miRNA expression data in adult gonadal malignant GCTs. We applied the bioinformatic algorithm Sylamer to identify miRNAs that are of biological importance by inducing global shifts in mRNA levels. The most significant differentially expressed miRNAs in malignant GCTs were all from the miR-371-373 and miR-302 clusters (adjusted P < 0.00005), which were overexpressed regardless of histologic subtype [yolk sac tumor (YST)/seminoma/embryonal carcinoma (EC)], site (gonadal/extragonadal), or patient age (pediatric/adult). Sylamer revealed that the hexamer GCACTT, complementary to the 2-to 7-nucleotide miRNA seed AAGUGC shared by six members of the miR-371-373 and miR-302 clusters, was the only sequence significantly enriched in the 3′-untranslated region of mRNAs downregulated in pediatric malignant GCTs (as a group), YSTs and ECs, and in adult YSTs (all versus nonmalignant tissue controls; P < 0.05). For the pediatric samples, downregulated genes containing the 3′-untranslated region GCACTT showed significant overrepresentation of Gene Ontology terms related to cancer-associated processes, whereas for downregulated genes lacking GCACTT, Gene Ontology terms generally represented metabolic processes only, with few genes per term (adjusted P < 0.05). We conclude that the miR-371-373 and miR-302 clusters are universally overexpressed in malignant GCTs and coordinately downregulate mRNAs involved in biologically significant pathways. Cancer Res; 70(7); 2911-23. ©2010 AACR.

Integration of high-risk human papillomavirus (HRHPV) into the host genome is a key event in cervical neoplastic progression. Integration is associated with deregulated expression of the viral oncogenes E6 and E7 and acquisition of a selective growth advantage for cells containing integrants. Overexpression of the viral transcriptional regulator E2 from heterologous promoters has an inhibitory effect on transcription from integrated HRHPV. Therefore, we hypothesized that loss of E2-expressing episomes from cells in which integration had previously occurred would be required for such cells to gain a growth advantage. Using the unique W12 model of cervical squamous carcinogenesis, we show that cells containing integrated HPV16 reproducibly emerged during long-term culture when there had been a rapid fall in episome numbers. During the period of emergence, it is possible to isolate single-cell clones containing an intracellular mixture of the integrant being selected and episomes at reduced load. The lower level of E2 expression seen in such cells is associated with partial inhibition of transcription from the HPV16 integrant. Full deregulation is not observed until complete loss of E2-expressing episomes occurs. Microarray analysis showed that episome loss was closely associated with endogenous activation of antiviral response genes that are also inducible by the type I IFN pathway. Taken together, our results indicate that episome loss, associated with induction of antiviral response genes, is a key event in the spontaneous selection of cervical keratinocytes containing integrated HPV16. We conclude that cervical carcinogenesis requires not only HRHPV integration, but also loss of inhibitory episomes.human papillomavirus ͉ cervix ͉ integration ͉ interferon ͉ progression I ntegration of high-risk human papillomavirus (HRHPV) into the host genome is an important step in cervical neoplastic progression (1, 2). Integrated viral genomes from which HRHPV early genes are transcribed have been detected in Ϸ87.5% of cervical malignancies (3). Integration usually causes deletion or disruption of the viral regulatory E2 gene, while retaining a variable segment including the E6 and E7 oncogenes and the upstream regulatory region (4, 5). Overexpression of E2 from heterologous promoters in cells harboring integrated HRHPV can repress the early promoter of the integrated virus, causing a sharp reduction in E6 and E7 expression (6). Thus, HRHPV integration and disruption͞deletion of E2 leads to increased expression of the viral oncogenes (7,8). Cells containing integrated HRHPV acquire a growth advantage over cells harboring episomal HRHPV (the natural viral state in productive infections) and show increased genomic instability (9-11).Cervical keratinocyte cell lines established from precursor low-grade squamous intraepithelial lesions have indicated that episomal HRHPV genomes are maintained at Ϸ100 copies per cell in the basal region of an infected epithelium (12, 13). Viral integration is therefore most likely to occur in cel...

Gain of chromosome 5p is seen in over 50% of advanced cervical squamous cell carcinomas (SCCs), although the genes responsible for the selective advantage provided by this abnormality are poorly understood. In the W12 cervical carcinogenesis model, we observed that 5p gain was rapidly selected over approximately 15 population doublings and was associated with the acquisition of a growth advantage and invasiveness. The most significantly upregulated transcript following 5p gain was the microRNA (miRNA) processor Drosha. In clinically progressed cervical SCC, Drosha copy-number gain was seen in 21/36 clinical samples and 8/10 cell lines and there was a significant association between Drosha transcript levels and copy-number gain. Other genes in the miRNA processing pathway, DGCR8, XPO5 and Dicer, showed infrequent copy-number gain and over-expression. Drosha copy-number and expression were not elevated in pre-malignant cervical squamous intraepithelial lesions. Importantly, global miRNA profiling showed that Drosha over-expression in cervical SCC appears to be of functional significance. Unsupervised principal component analysis of a mixed panel of cervical SCC cell lines and clinical specimens showed clear separation according to Drosha over-expression. miRNAs most significantly associated with Drosha over-expression are implicated in carcinogenesis in other tissues, suggesting that they regulate fundamental processes in neoplastic progression. Our evidence suggests that copy-number driven over-expression of Drosha and consequent changes in miRNAs are likely to be important contributors to the selective advantage provided by 5p gain in cervical neoplastic progression.

BackgroundWe hypothesised that differences in microRNA expression profiles contribute to the contrasting natural history and clinical outcome of the two most common types of malignant germ cell tumour (GCT), yolk sac tumours (YSTs) and germinomas.ResultsBy direct comparison, using microarray data for paediatric GCT samples and published qRT-PCR data for adult samples, we identified microRNAs significantly up-regulated in YSTs (n = 29 paediatric, 26 adult, 11 overlapping) or germinomas (n = 37 paediatric). By Taqman qRT-PCR we confirmed differential expression of 15 of 16 selected microRNAs and further validated six of these (miR-302b, miR-375, miR-200b, miR-200c, miR-122, miR-205) in an independent sample set. Interestingly, the miR-302 cluster, which is over-expressed in all malignant GCTs, showed further over-expression in YSTs versus germinomas, representing six of the top eight microRNAs over-expressed in paediatric YSTs and seven of the top 11 in adult YSTs. To explain this observation, we used mRNA expression profiles of paediatric and adult malignant GCTs to identify 10 transcription factors (TFs) consistently over-expressed in YSTs versus germinomas, followed by linear regression to confirm associations between TF and miR-302 cluster expression levels. Using the sequence motif analysis environment iMotifs, we identified predicted binding sites for four of the 10 TFs (GATA6, GATA3, TCF7L2 and MAF) in the miR-302 cluster promoter region. Finally, we showed that miR-302 family over-expression in YST is likely to be functionally significant, as mRNAs down-regulated in YSTs were enriched for 3' untranslated region sequences complementary to the common seed of miR-302a~miR-302d. Such mRNAs included mediators of key cancer-associated processes, including tumour suppressor genes, apoptosis regulators and TFs.ConclusionsDifferential microRNA expression is likely to contribute to the relatively aggressive behaviour of YSTs and may enable future improvements in clinical diagnosis and/or treatment.

Malignant germ cell tumors (GCT) of childhood are rare and heterogeneous neoplasms thought to arise from primordial germ cells. They vary substantially in their natural history and show important clinical differences from their adult counterparts. To address the biological basis for these observations, we have undertaken a comprehensive analysis of global gene expression patterns in pediatric malignant GCTs and compared these findings with published data on adult testicular GCTs (TGCT). Our study included 27 primary tumors and assessed the principal malignant histologic types of pediatric GCT, yolk sac tumor (YST; n = 18), and seminoma (n = 9). Analysis of Affymetrix U133A GeneChip data was performed using the statistical software environment R, including gene set enrichment analysis, with cross-validation at the RNA and protein level. Unsupervised analysis showed complete separation of YSTs and seminomas by global gene expression profiles and identified a robust set of 657 discriminatory transcripts. There was no segregation of tumors of the same histology arising at different sites or at different ages within the pediatric range. In contrast, there was segregation of pediatric malignant GCTs and adult malignant TGCTs, most notably for the YSTs. The pediatric seminomas were significantly enriched for genes associated with the self-renewing pluripotent phenotype, whereas the pediatric YSTs were significantly enriched for genes associated with a differentiation and proliferation phenotype. We conclude that histologic type is the key discriminator in pediatric malignant GCTs and that the observed clinical differences between malignant GCTs of children and adults are mirrored by significant differences in global gene expression.

Although gain of chromosome 5p is one of the most frequent DNA copy-number imbalances in cervical squamous cell carcinoma (SCC), the genes that drive its selection remain poorly understood. In a previous cross-sectional clinical study, we showed that the microRNA processor Drosha (located on chromosome 5p) demonstrates frequent copy-number gain and overexpression in cervical SCC, associated with altered microRNA profiles. Here, we have conducted gene depletion/overexpression experiments to demonstrate the functional significance of up-regulated Drosha in cervical SCC cells. Drosha depletion by RNA interference (RNAi) produced significant, specific reductions in cell motility/invasiveness in vitro, with a silent RNAi-resistant Drosha mutation providing phenotype rescue. Unsupervised hierarchical clustering following global profiling of 319 microRNAs in 18 cervical SCC cell line specimens generated two groups according to Drosha expression levels. Altering Drosha levels in individual SCC lines changed the group into which the cells clustered, with gene depletion effects being rescued by the RNAi-resistant mutation. Forty-five microRNAs showed significant differential expression between the groups, including four of 14 that were differentially expressed in association with Drosha levels in clinical samples. miR-31 up-regulation in Drosha-overexpressing samples/cell lines was the highest-ranked change (by adjusted p value) in both analyses, an observation validated by northern blotting. These functional data support the role of Drosha as an oncogene in cervical SCC, by affecting expression of cancer-associated microRNAs that have the potential to regulate numerous protein-coding genes.

Malignant germ cell tumours (MGCTs) of childhood are a rare group of neoplasms that comprise many histological subtypes and arise at numerous different sites. Genomic imbalances have been described in these tumours but, largely because of the paucity of cases reported in the literature, it is unclear how they relate to abnormalities in adult MGCTs and impact on potential systems for classifying GCTs. We have used metaphase-based comparative genomic hybridisation to analyse the largest series of paediatric MGCTs reported to date, representing 34 primary tumours (22 yolk sac tumours (YSTs), 11 germinomatous tumours and one metastatic embryonal carcinoma) occurring in children from birth to age 16, including 17 ovarian MGCTs. The large dataset enabled us to undertake statistical analysis, with the aim of identifying associations worthy of further investigation between patterns of genomic imbalance and clinicopathological parameters. The YSTs showed an increased frequency of 1p-(P ¼ 0.003), 3p þ (P ¼ 0.02), 4qÀ (P ¼ 0.07) and 6qÀ (P ¼ 0.004) compared to germinomatous tumours. Gain of 12p, which is invariably seen in adult MGCTs, was present in 53% of primary MGCTs of children aged 5 -16 and was also observed in four of 14 YSTs affecting children less than 5. Two of these cases (14% of MGCTs in children less than 5) showed gain of the 12p11 locus considered to be particularly relevant in adult MGCTs. Gain of 12p showed a significant association with gain of 12q. Conversely, MGCTs without 12p gain displayed a significantly increased frequency of loss on 16p (P ¼ 0.04), suggesting that this imbalance may contribute to tumour development in such cases. This data provides new insight into the biology of this under-investigated tumour group and will direct future studies on the significance of specific genetic abnormalities.

Methylation of APC and LOH 5q21-22 in YSTs and teratomas provide evidence for involvement of APC in the accumulation of β-catenin and activation of the WNT pathway. Our additional analyses suggest that APC is unlikely to be solely responsible for the formation and progression of childhood GCTs.