Aberrant RNA splicing in cancer; expression changes and driver mutations of splicing factor genes.

Alternative splicing is a widespread process contributing to structural transcript variation and proteome diversity. In cancer, the splicing process is commonly disrupted, resulting in both functional and non-functional end-products. Cancer-specific splicing events are known to contribute to disease progression; however, the dysregulated splicing patterns found on a genome-wide scale have until recently been less well-studied. In this review, we provide an overview of aberrant RNA splicing and its regulation in cancer. We then focus on the executors of the splicing process. Based on a comprehensive catalog of splicing factor encoding genes and analyses of available gene expression and somatic mutation data, we identify cancer-associated patterns of dysregulation. Splicing factor genes are shown to be significantly differentially expressed between cancer and corresponding normal samples, and to have reduced inter-individual expression variation in cancer. Furthermore, we identify enrichment of predicted cancer-critical genes among the splicing factors. In addition to previously described oncogenic splicing factor genes, we propose 24 novel cancer-critical splicing factors predicted from somatic mutations.

Profiling of the small RNA populations in human testicular germ cell tumors shows global loss of piRNAs.

BACKGROUND: Small non-coding RNAs play essential roles in gene regulation, however, the interplay between RNA groups, their expression levels and deregulations in tumorigenesis requires additional exploration. In particular, a comprehensive analysis of microRNA (miRNA), PIWI-interacting RNAs (piRNAs), and tRNA-derived small RNAs in human testis and testicular germ cell tumor (TGCT) is lacking. RESULTS: We performed small RNA sequencing on 22 human TGCT samples from 5 histological subtypes, 3 carcinoma in situ, and 12 normal testis samples. miRNA was the most common group among the sequences 18-24 nt in length and showed histology-specific expression. In normal samples, most sequences 25-31 nucleotides in length displayed piRNA characteristics, whereas a large proportion of the sequences 32-36 nt length was derived from tRNAs. Expression analyses of the piRNA population demonstrated global loss in all TGCT subtypes compared to normal testis. In addition, three 5' small tRNA fragments and 23 miRNAs showed significant (p < 10(-6)) differential expression in cancer vs normal samples. CONCLUSIONS: We have documented significant changes in the small RNA populations in normal adult testicular tissue and TGCT samples. Although components of the same pathways might be involved in miRNA, piRNA and tRNA-derived small RNA biogenesis, our results showed that the response to the carcinogenic process differs between these pathways, suggesting independent regulation of their biogenesis. Overall, the small RNA deregulation in TGCT provides new insight into the small RNA interplay.

CLC and IFNAR1 are differentially expressed and a global immunity score is distinct between early- and late-onset colorectal cancer.

Colorectal cancer (CRC) incidence increases with age, and early onset of the disease is an indication of genetic predisposition, estimated to cause up to 30% of all cases. To identify genes associated with early-onset CRC, we investigated gene expression levels within a series of young patients with CRCs who are not known to carry any hereditary syndromes (n=24; mean 43 years at diagnosis), and compared this with a series of CRCs from patients diagnosed at an older age (n=17; mean 79 years). Two individual genes were found to be differentially expressed between the two groups, with statistical significance; CLC was higher and IFNAR1 was less expressed in early-onset CRCs. Furthermore, genes located at chromosome band 19q13 were found to be enriched significantly among the genes with higher expression in the early-onset samples, including CLC. An elevated immune content within the early-onset group was observed from the differentially expressed genes. By application of outlier statistics, H3F3A was identified as a top candidate gene for a subset of the early-onset CRCs. In conclusion, CLC and IFNAR1 were identified to be overall differentially expressed between early- and late-onset CRC, and are important in the development of early-onset CRC.

The testicular germ cell tumour transcriptome.

Testicular germ cell tumours (TGCTs) are characterized by young age of onset and a complex pattern of histological subtypes. Transcriptomic studies have tried to uncover the gene expression patterns underlying this. Here, we present a systematic review of transcriptome studies of TGCTs of adolescents and young adults and identify genes common across the various studies, both for TGCTs in general as well as the histological subtypes, hence elucidating both transcriptional changes associated with malignant transformation and differentiation patterns. A meta-analysis of this type adds power and significance to the genes thus found, where most studies have included only a limited number of samples. Both known (KRAS, MYCN and TPD52) and novel (CCT6A, IGFBP3 and SALL2) cancer genes are implicated in TGC tumorigenesis. Gene expression patterns characteristic to embryonic stem cells are also found deregulated in TGC tumorigenesis. This is reflected in how pluripotent embryonal carcinoma cells commonly differentiate into a variety of embryonic and extra-embryonic histological types, each with unique transcriptomes. The embryonal carcinomas in particular are found to overexpress pluripotency genes, while gene signatures for seminomas, teratomas and yolk sac tumours were also identified. This underlines the distinctive transcriptomic programme across histological subtypes, especially striking given that the TGCT genome is largely similar across the same subtypes.

The pluripotency transcription factor Krüppel-like factor 4 is strongly expressed in intratubular germ cell neoplasia unclassified and seminoma.

Germ cell tumors of the testis are the most frequent tumors in men between 20 and 40 years. Their most common subtype is the seminoma, which arises like the embryonal carcinoma from an intratubular germ cell neoplasia unclassified (IGCNU), i.e. fetal germ cells that escaped from the control of the developing testicular stem cell niche, eventually leading to a fully developed seminoma (or embryonal carcinoma). The molecular causes for the development of an IGCNU are still unknown. However, IGCNU cells share the expression of several factors with primordial germ cells and gonocytes and, interestingly, also with pluripotent embryonic stem (ES) cells and induced pluripotent stem (iPS) cells. One factor playing important roles in both iPS and ES cells is the transcription factor Krüppel-like factor 4 (KLF4). This study examined KLF4 expression data from 179 human testicular samples including normal controls and seminoma, deposited in Gene Expression Omnibus repository for microarray data at the National Centre for Biotechnology Information. Immunohistochemistry was used to detect KLF4 protein expression in IGCNU (n = 6), seminoma (n = 14) and fetal human testes (n = 14). Microarray data from three independent sources suggest higher mRNA expression in seminoma than in normal testis. Normal spermatogonia, which are the stem cells of spermatogenesis, controlled by their stem cell niche, do not express KLF4. In contrast, IGCNU and seminoma cells strongly express KLF4. In conclusion, this finding suggests that KLF4 may be an important factor for the maintenance of the developmental and the tumorigenic potential of IGCNU as well as for the malignancy of seminoma.

Novel epigenetically deregulated genes in testicular cancer include homeobox genes and SCGB3A1 (HIN-1).

Testicular germ cell tumours (TGCTs) are classified into two main histological subgroups: seminomas and non-seminomas. The latter comprise several subtypes: embryonal carcinomas, yolk sac tumours, choriocarcinomas, and teratomas. These embryonal and extra-embryonal-like differentiation lineages represent a caricature of early normal development, and inactivation of gene expression through promoter hypermethylation may therefore be of particular importance in germ cell tumourigenesis. The promoter methylation status of ten candidate genes-CDH13, DLX6, EMX2, HOXA9, HOXB5, MSX1, MSX2, RASSF1A, RUNX3, and SCGB3A1 (alias HIN-1)-was assessed by methylation-specific PCR in seven intratubular germ cell neoplasias and 55 primary TGCTs. Furthermore, by a discovery-based global approach, comparing cDNA microarray expression profiles of two germ cell tumour cell lines before and after treatment with the demethylating agent 5-aza-2'-deoxycytidine, a gene list of potentially epigenetic targets was identified, from which CGGBP1, CGRRF1, SMARCC2, SORBS1, and XPA were analysed further. Overall, the non-seminomas were significantly more often methylated than were seminomas (p < 0.001). The three most frequently methylated genes among this subtype were SCGB3A1 (54%), RASSF1A (29%), and HOXA9 (26%). CDH13 and HOXB5 were methylated at low frequencies (10-15%), and EMX2, MSX1, RUNX3, SORBS1, and XPA only rarely (<10%). In conclusion, this study has identified several novel epigenetically deregulated target genes in TGCT development, including homeobox genes and SCGB3A1, suggesting that epigenetic inactivation of key genes in normal development also has an important role in TGCTs.

Frequent promoter hypermethylation of the O6-Methylguanine-DNA Methyltransferase (MGMT) gene in testicular cancer.

Testicular germ cell tumours are classified into two major histological subgroups, seminomas and nonseminomas. All tumours display several recurrent chromosomal aberrations, but few target genes have been identified. Previous studies have shown that genome-wide hypermethylation of CpG islands is significantly more prevalent in nonseminomas than in seminomas. We have studied two potential target genes in testicular cancer. A series of 70 tumours were analysed for methylation of CpG sites in the O(6)-methylguanine-DNA methyltransferase (MGMT) gene promoter, and in exon 1alpha of the cyclin-dependent kinase inhibitor 2A gene (CDKN2A). In addition, eight microsatellite markers within and flanking these genes at chromosome arms 10q and 9p, respectively, were analysed for allelic imbalances. Allele alterations were frequently seen at 9p loci (47 out of 70, 67%), but none of the tumours (none out of 55) showed methylation of CDKN2A. On the other hand, a high frequency of MGMT promoter methylation (32 out of 69, 46%) was found, as well as allelic imbalances at 10q markers (50 out of 70, 71%). A significantly higher methylation frequency was found in nonseminomas (24 out of 35, 69%) compared to seminomas (eight out of 33, 24%) (P=0.0003, Fisher's exact test). Immunohistochemical analysis of the MGMT protein in a subgroup (n=20) of the testicular tumours supported the hypothesis of gene silencing being the functional consequence of the promoter methylation. In summary, our data suggest that inactivation of MGMT contributes to development of nonseminomatous testicular cancer.

Familial/bilateral and sporadic testicular germ cell tumors show frequent genetic changes at loci with suggestive linkage evidence.

Testicular germ cell tumor (TGCT) is the most common tumor type among adolescent and young adult males. Familial clustering and bilateral disease are suggestive of a genetic predisposition among a subgroup of these patients, but susceptibility genes for testicular cancer have not yet been identified. However, suggestive linkage between disease and genetic markers has been reported at loci on chromosome arms 3q, 5q, 12q, 18q, and Xq. We have analyzed primary familial/bilateral (n=20) and sporadic (n=27) TGCTs, including 28 seminomas and 19 nonseminomas, for allelic imbalance (AI) within the autosomal regions. DNA from all tumors were analyzed by fluorescent polymerase chain reaction of 22 polymorphic loci at 3q27-ter, 5q13-35.1, 12q21-ter, and 18q12--ter. All tumor genotypes were evaluated against their corresponding constitutional genotypes. The percentages of TGCTs with genetic changes at 3q, 5q, 12q, and 18q, were 79%, 36%, 53% and 43%, respectively. The frequencies at 3q and 12q in nonseminomas were significantly higher than in seminomas (P=.003 and P=.004). In order to evaluate changes at hemizygous Xq loci, five loci were analyzed by co-amplification with an autosomal reference marker known to reveal retained heterozygosity in the tumor DNA. Gain of Xq sequences was seen in more than 50% of the tumors. The degree of amplification varied among the loci in each of five tumors, and based on these breakpoints, a common region of overlapping gains was found at Xq28. No significant differences were found between the frequencies of genetic changes in familial/bilateral versus sporadic tumors, an observation speaking in disfavor of the existence of a single susceptibility gene for TGCT in any of the analyzed regions. Our data suggest that gain of genetic material at distal Xq and losses at 5q and 18q contribute to establishment of seminomas, whereas imbalances at 3q as well as gain at distal part of 12q are associated with further progression into nonseminomas.

Evaluation of loss of heterozygosity/allelic imbalance scoring in tumor DNA.

Loss of heterozygosity and allelic imbalance in tumors are usually detected by either radioactive labeling of PCR products with subsequent scoring of autoradiographs or by a semi-quantitative fluorescence-based protocol. Polymorphic microsatellite loci are the most common marker type used in these studies. Even though no consensus exists as to how to evaluate such data, results are often compared directly between studies applying the two different protocols. In the present study, we analyzed twice by each protocol three loci in 60 blood/tumor pairs, finding good correlation between the results obtained by the two methods. However, a higher sensitivity and the possibility to correct for stutter peaks were among several advantages inherent in the fluorescence labeling approach. In addition, we determined the cut-off level for allelic imbalance scoring by the fluorescent primer protocol, by repeated analysis of 485 constitutional heterozygous genotypes at 20 different dinucleotide repeat loci. Based on the standard deviation, we found that allelic imbalance should be scored whenever the peak height of one allele in tumor DNA is reduced to less than 0.84 of its value in constitutional DNA, relative to the other allele. Applying this cut-off value, more imbalances are detected than by the visual scoring of autoradiographs. Our data therefore suggest that a lower threshold value (0.75) must be used when results from both fluorescent and radioactive assays are compared.