Identification of a prognostic alternative splicing signature in oral squamous cell carcinoma

Alternative splicing (AS) is critically associated with tumorigenesis and patient's prognosis. Here, we systematically analyzed survival-associated AS signatures in oral squamous cell carcinoma (OSCC) and evaluated their prognostic predictive values. Survival-related AS events were identified by univariate and multivariate Cox regression analyses using OSCC data from the TCGA head neck squamous cell carcinoma data set. The Percent Spliced In calculated by SpliceSeq from 0 to 1 was used to quantify seven types of AS events. A predictive model based on AS events was constructed by least absolute shrinkage and selection operator Cox regression assay and further validated using a training-testing cohort design. Patient survival was estimated using the Kaplan–Meier method and compared with Log-rank test. The receiver operating characteristics curve area under the curves was used to evaluate the predictive abilities of these predictive models. Furthermore, gene–gene interaction networks and the splicing factors (SFs)-AS regulatory network was generated by Cytoscape. A total of 825 survival-related AS events within 719 genes were identified in OSCC samples. The integrative predictive model was better at predicting outcomes of patients as compared to those models built with the individual AS event. The predictive model based on three AS-related genes also effectively predicted patients’ survival. Moreover, seven survival-related SFs were detected in OSCC including RBM4, HNRNPD, and HNRNPC, which have been linked to tumorigenesis. The SF-AS network revealed a significant correlation between survival-related AS genes and these SFs. Our findings revealed a systemic portrait of survival-associated AS events and the splicing network in OSCC, suggesting that AS events might serve as novel prognostic biomarkers and therapeutic targets for OSCC.     

Epstein-Barr virus mRNAs produced by alternative splicing.

The structure of Epstein-Barr virus mRNAs transcribed in B95-8 cells has been studied by cDNA cloning and sequencing. We present here the analysis of four cDNAs. The corresponding mRNAs are probably transcribed from a single promoter located in the US region. They are produced by alternative splicing of exons transcribed from the US, IR and UL regions. The exons are spread over 100 kbp. The exons from the IR region constitute a unit which is repeated several times. The cDNAs share the exons from the US and IR regions. Some of the cDNAs also share some of the exons from the UL region. Each cDNA contains a long open reading frame or the 5' end of a long open reading frame which ends several hundred nucleotides downstream on the viral genome. The 5' untranslated regions are unusually long. Three mRNA species differing in their 5' untranslated regions may encode for the nuclear antigen EBNA-1. The other mRNAs encode for polypeptides which may not have any common region.     

Expression pattern of squamous cell carcinoma antigen in oesophageal dysplasia and squamous cell carcinoma

The aim of the present study was to evaluate the tissue expression of squamous cell carcinoma antigen (SCCA) in oesophageal dysplasia and squamous cell carcinoma (SCC) with reference to its clinico-pathologic and prognostic significance. Immunohistochemistry using SCCA polyclonal antibody was performed on SCCs from 61 surgical oesophagectomies. Fifteen cases of low-grade dysplasia (LGD) and 37 non-coexistent high-grade dysplasia (HGD) were also sampled from these materials, together with sixteen chronic cases of oesophagitis. SCCA immunoreactivity was present in the maturative compartments of all normal epithelia and oesophagitis. LGDs showed no SCCA immunoreactivity in the dysplastic proliferative component but only in the superficial normal layers. In 94.6% of HGDs, no SCCA immunoreactivity was detected throughout the thickness of the epithelium. In SCCs, SCCA expression higher than 25% was found in 54% of cases. SCCA positivity showed an inverse correlation with histological grade, whereas no statistically significant correlation was found with TNM classifications, stage, or survival. SCCA is not expressed in early oesophageal carcinogenesis but, in SCC, it represents an indicator of histologic differentiation. In differentiated SCC, SCCA may represent a negative factor for cancer invasiveness, through inhibition of proteases.     

SOX2 modulates alternative splicing in transitional cell carcinoma

Aberrant alternative splicing of key cellular regulators may play a pivotal role in cancer development. To investigate the potential influence of altered alternative splicing on the development of transitional cell carcinoma (TCC), splicing activity in the TCC cell lines TSGH8301 and BFTC905 was examined using the SV40-immortalized uroepithelial cell line SV-HUC-1 as a reference. Our results indicate a significant alteration in splice site selection in the TCC cell lines. By gene expression profiling and subsequent validation, we discovered that sex-determining region Y-box protein 2 (SOX2) is specifically upregulated in BFTC905. Furthermore, ectopic expression of SOX2 modulates alternative splicing of the splicing reporter in vivo. More significantly, using an in vitro pull-down assay, it was found that SOX2 exhibits RNA-binding capability. Our observations suggest that SOX2 modulates alternative splicing by functioning as a splicing factor.     

Characterization of new human c-myc mRNA species produced by alternative splicing

Characterization of two human c-myc cDNAs corresponding to the mRNAs 2.5 and 3.1 kb in length transcribed from PO previously demonstrated the existence of alternative acceptor sites at the end of intron 1 and intron 2, respectively [Bentley, D.L. and Groudine, M. (1986) Mol. Cell. Biol. 6, 3481–3489]. We investigated the use of these alternative acceptor sites in each c-myc mRNA species. We characterized cDNAs corresponding to c-myc mRNAs transcribed in the SW613-S human carcinoma cell line. The use of the alternative acceptor site at the end of intron I was demonstrated in two out of 10 cDNAs corresponding to the 3.1-kb mRNA transcribed from PO and in three out of 10 cDNAs corresponding to the mRNAs transcribed from P1 or P2. The use of this acceptor site is therefore not restricted to the 2.5-kb mRNA transcribed from PO. The mRNAs resulting from the use of this acceptor site would encode for a variant form of the p67 polypeptide lacking one amino-acid residue. Conversely, the use of the alternative acceptor site at the end of intron 2 was not found in any of the cDNAs corresponding to the mRNAs transcribed from PO (0/10), from P1 or P2 (0/10) and from P3 (0/10). In the course of this study, we isolated a cDNA corresponding to another new c-myc mRNA species. This mRNA is produced by alternative splicing within intron 1 and encodes only for p64.     

Systematic analysis of survival-associated alternative splicing signatures in clear cell renal cell carcinoma

Alternative splicing (AS) constitutes a major reason for messenger RNA (mRNA) and protein diversity. Increasing studies have shown a link to splicing dysfunction associated with malignant neoplasia. Systematic analysis of AS events in kidney cancer remains poorly reported. Therefore, we generated AS profiles in 533 kidney renal clear cell carcinoma (KIRC) patients in The Cancer Genome Atlas (TCGA) database using RNA-seq data. Then, prognostic models were developed in a primary cohort (N = 351) and validated in a validation cohort (N = 182). In addition, splicing networks were built by integrating bioinformatics analyses. A total of 11 268 and 8083 AS variants were significantly associated with patient overall survival time in the primary and validation KIRC cohorts, respectively, including STAT1, DAZAP1, IDS, NUDT7, and KLHDC4. The AS events in the primary KIRC cohorts served as candidate AS events to screen the independent risk factors associated with survival in the primary cohort and to develop prognostic models. The area under the curve of the receiver-operator characteristic curve for prognostic prediction in the primary and validation KIRC cohorts was 0.84 and 0.82 at 2500 days of overall survival, respectively. In addition, splicing correlation networks revealed key splicing factors (SFs) in KIRC, such as HNRNPH1, HNRNPU, KHDBS1, KHDBS3, SRSF9, RBMX, SFQ, SRP54, HNRNPA0, and SRSF6. In this study, we analyzed the AS landscape in the TCGA KIRC cohort and detected predictors (prognostic) based on AS variants with high performance for risk stratification of the KIRC cohort and revealed key SFs in splicing networks, which could act as underlying mechanisms.     

Truncated P-cadherin is produced in oral squamous cell carcinoma

Cadherins belong to a family of homophilic cell-cell adhesion proteins that are responsible for the establishment of a precise cell architecture and tissue integrity. Moreover, experimental data suggest that loss of intercellular adhesion is inversely correlated with cellular differentiation. Furthermore, dedifferentiation is closely linked to tumor progression. Recently, we have shown that a secreted 50 kDa N-terminal fragment of P-cadherin plays a role in the progression of malignant melanoma. In this study, we have detected both the full-length and the truncated versions of P-cadherin in cell lysates of differentiated head and neck oral squamous cell carcinoma cell lines, whereas in cell lysates of dedifferentiated cell lines, we detected only the truncated 50 kDa version of P-cadherin. Treatment of the cell lines with a recombinantly expressed biotinylated, soluble 50 kDa form of the N-terminal part of P-cadherin revealed a major effect on cell aggregation and migration of oral squamous cell carcinoma cells. However, the 50 kDa N-terminal fragment of P-cadherin did not show any influence on cell proliferation in 2D and 3D cell culture. These results suggest that generation of truncated P-cadherin during the progression of oral squamous carcinoma attenuates tissue integrity, facilitates cellular separation, and leads to the acquisition of a more migratory phenotype.