Profiling alternatively spliced mRNA isoforms for prostate cancer classification

BACKGROUND: Prostate cancer is one of the leading causes of cancer illness and death among men in the United States and world wide. There is an urgent need to discover good biomarkers for early clinical diagnosis and treatment. Previously, we developed an exon-junction microarray-based assay and profiled 1532 mRNA splice isoforms from 364 potential prostate cancer related genes in 38 prostate tissues. Here, we investigate the advantage of using splice isoforms, which couple transcriptional and splicing regulation, for cancer classification. RESULTS: As many as 464 splice isoforms from more than 200 genes are differentially regulated in tumors at a false discovery rate (FDR) of 0.05. Remarkably, about 30% of genes have isoforms that are called significant but do not exhibit differential expression at the overall mRNA level. A support vector machine (SVM) classifier trained on 128 signature isoforms can correctly predict 92% of the cases, which outperforms the classifier using overall mRNA abundance by about 5%. It is also observed that the classification performance can be improved using multivariate variable selection methods, which take correlation among variables into account. CONCLUSION: These results demonstrate that profiling of splice isoforms is able to provide unique and important information which cannot be detected by conventional microarrays

A Global View of Cancer-Specific Transcript Variants by Subtractive Transcriptome-Wide Analysis

BACKGROUND: Alternative pre-mRNA splicing (AS) plays a central role in generating complex proteomes and influences development and disease. However, the regulation and etiology of AS in human tumorigenesis is not well understood. METHODOLOGY/PRINCIPAL FINDINGS: A Basic Local Alignment Search Tool database was constructed for the expressed sequence tags (ESTs) from all available databases of human cancer and normal tissues. An insertion or deletion in the alignment of EST/EST was used to identify alternatively spliced transcripts. Alignment of the ESTs with the genomic sequence was further used to confirm AS. Alternatively spliced transcripts in each tissue were then subtractively cross-screened to obtain tissue-specific variants. We systematically identified and characterized cancer/tissue-specific and alternatively spliced variants in the human genome based on a global view. We identified 15,093 cancer-specific variants of 9,989 genes from 27 types of human cancers and 14,376 normal tissue-specific variants of 7,240 genes from 35 normal tissues, which cover the main types of human tumors and normal tissues. Approximately 70% of these transcripts are novel. These data were integrated into a database HCSAS (http://202.114.72.39/database/human.html, pass:68756253). Moreover, we observed that the cancer-specific AS of both oncogenes and tumor suppressor genes are associated with specific cancer types. Cancer shows a preference in the selection of alternative splice-sites and utilization of alternative splicing types. CONCLUSIONS/SIGNIFICANCE: These features of human cancer, together with the discovery of huge numbers of novel splice forms for cancer-associated genes, suggest an important and global role of cancer-specific AS during human tumorigenesis. We advise the use of cancer-specific alternative splicing as a potential source of new diagnostic, prognostic, predictive, and therapeutic tools for human cancer. The global view of cancer-specific AS is not only useful for exploring the complexity of the cancer transcriptome but also widens the eyeshot of clinical research

A Machine Learning Model for Discovery of Protein Isoforms as Biomarkers

Prostate cancer is the most common cancer in men. One in eight Canadian men will be diagnosed with prostate cancer in their lifetime. The accurate detection of the disease’s subtypes is critical for providing adequate therapy; hence, it is critical for increasing both survival rates and quality of life. Next generation sequencing can be beneficial when studying cancer. This technology generates a large amount of data that can be used to extract information about biomarkers. This thesis proposes a model that discovers protein isoforms for different stages of prostate cancer progression. A tool has been developed that utilizes RNA-Seq data to infer open reading frames (ORFs) corresponding to transcripts. These ORFs are used as features for classificatio. A quantification measurement, Adaptive Fragments Per Kilobase of transcript per Million mapped reads (AFPKM), is proposed to compute the expression level for ORFs. The new measurement considers the actual length of the ORF and the length of the transcript. Using these ORFs and the new expression measure, several classifiers were built using different machine learning techniques. That enabled the identification of some protein isoforms related to prostate cancer progression. The biomarkers have had a great impact on the discrimination of prostate cancer stages and are worth further investigation

Transcriptome-Wide Analysis Of Hypoxic Cancer Cells Identify Alternative Splicing As A Mechanism To Inhibit Translation

Cellular adaptation to hypoxia involves downregulation of energy-consuming processes such as macromolecular synthesis, and leads to tumor aggressiveness and resistance to therapies for many solid cancers. To delineate mechanisms underlying this process, I carried out a transcriptome-wide study to measure hypoxia-mediated changes in gene expression and alternative splicing in in vivo and in vitro models of hypoxic head and neck carcinoma (HNC) cells. These data represent the first nucleotide-resolution study of the hypoxic transcriptome of HNC cells in vivo and in vitro. This investigation uncovered a global downregulation of genes known to regulate RNA processing, including a significant number of genes involved in splicing catalysis. Exon-level analyses classified \u3e1,000 mRNAs to be affected by alternative splicing, and led to the discovery of a unique retained intron in the master regulator of translation initiation, EIF2B5. In this dissertation, I will describe a previously uncharacterized mechanism by which a hypoxia-mediated retained intron in EIF2B5 leads to a truncated isoform that opposes full-length eIF2Bε to inhibit translation. A functional investigation of this hypoxia-induced isoform confirmed that expression of the truncated 65kDa isoform of eIF2Bε confers a survival advantage to HNC cells under conditions of hypoxia. Moreover, expression of this isoform was observed in solid tumors of patients with HNC in a stage-dependent manner. Additional work to uncover -cis and -trans regulators of EIF2B5 splicing identified several factors that influence intron retention in EIF2B5: a weak splice site with an alternate splice site at the retained intron, hypoxia-induced expression of the splicing factor SRSF3, and increased binding of total and phospho-Ser2 RNA polymerase II (RNAPII) specifically at the intron retained under hypoxia. Altogether, these data reveal differential splicing as a previously uncharacterized mode of translational control under hypoxia and are supported by a model in which hypoxia-induced changes to co-transcriptional processing lead to selective retention of an intron containing a premature-termination codon in EIF2B5

Androgen receptor signaling regulates the transcriptome of prostate cancer cells by modulating global alternative splicing

Androgen receptor (AR), is a transcription factor and a member of a hormone receptor superfamily. AR plays a vital role in the progression of prostate cancer and is a crucial target for therapeutic interventions. While the majority of advanced-stage prostate cancer patients will initially respond to the androgen deprivation, the disease often progresses to castrate-resistant prostate cancer (CRPC). Interestingly, CRPC tumors continue to depend on hyperactive AR signaling and will respond to potent second-line antiandrogen therapies, including bicalutamide (CASODEX®) and enzalutamide (XTANDI®). However, the progression-free survival rate for the CRPC patients on antiandrogen therapies is only 8–19 months. Hence, there is a need to understand the mechanisms underlying CRPC progression and eventual treatment resistance. Here, we have leveraged next-generation sequencing and newly developed analytical methodologies to evaluate the role of AR signaling in regulating the transcriptome of prostate cancer cells. The genomic and pharmacologic stimulation and inhibition of AR activity demonstrates that AR regulates alternative splicing within cancer-relevant genes. Furthermore, by integrating transcriptomic data from in vitro experiments and in prostate cancer patients, we found that a significant number of AR-regulated splicing events are associated with tumor progression. For example, we found evidence for an inadvertent AR-antagonist-mediated switch in IDH1 and PL2G2A isoform expression, which is associated with a decrease in overall survival of patients. Mechanistically, we discovered that the epithelial-specific splicing regulators (ESRP1 and ESRP2), flank many AR-regulated alternatively spliced exons. And, using 2D invasion assays, we show that the inhibition of ESRPs can suppress AR-antagonist-driven tumor invasion. Our work provides evidence for a new mechanism by which AR alters the transcriptome of prostate cancer cells by modulating alternative splicing. As such, our work has important implications for CRPC progression and development of resistance to treatment with bicalutamide and enzalutamide

Loss of Endocan tumorigenic properties after alternative splicing of exon 2

<p>Abstract</p> <p>Background</p> <p>Endocan was originally described as a dermatan sulfate proteoglycan found freely circulating in the blood. Endocan expression confers tumorigenic properties to epithelial cell lines or accelerate the growth of already tumorigenic cells. This molecule is the product of a single gene composed of 3 exons. Previous data showed that endocan mRNA is subject to alternative splicing with possible generation of two protein products. In the present study we identified, and functionally characterized, the alternative spliced product of the endocan gene: the exon 2-deleted endocan, called endocanΔ2.</p> <p>Methods</p> <p>Stable, endocanΔ2-overexpressing cell lines were generated to investigate the biological activities of this new alternatively spliced product of endocan gene. Tumorigenesis was studied by inoculating endocan and endocanΔ2 expressing cell lines subcutaneously in SCID mice. Biochemical properties of endocan and endocanΔ2 were studied after production of recombinant proteins in various cell lines of human and murine origin.</p> <p>Results</p> <p>Our results showed that the exon 2 deletion impairs synthesis of the glycan chain, known to be involved in the pro-tumoral effect of endocan. EndocanΔ2 did not promote tumor formation by 293 cells implanted in the skin of severe combined immunodeficient (SCID) mice.</p> <p>Conclusion</p> <p>Our results emphasize the key role of the polypeptide sequence encoded by the exon 2 of endocan gene in tumorigenesis, and suggest that this sequence could be a target for future therapies against cancer.</p

Alternative splicing and its role in pathologies of the endocrine system

Różnicowe składanie pierwotnego transkryptu (splicing alternatywny pre-mRNA) jest procesem polegającym na usuwaniu rejonów, które nie kodują dojrzałej cząsteczki mRNA (intronów) i łączeniu rejonów kodujących (eksonów) w różnych konfiguracjach, dzięki czemu powstają cząsteczki mRNA o różnej sekwencji nukleotydów. Różnicowemu składaniu podlegają transkrypty prawie wszystkich kodujących białka genów człowieka, przyczyniając się do powstawania wielkiej różnorodności izoform białek kodowanych przez stosunkowo niewielką liczbę genów. Proces splicingu alternatywnego w istotny sposób przyczynia się do prawidłowego funkcjonowania komórek, również tych biorących udział w sygnalizacji hormonalnej. Zaburzenia różnicowego składania pre-mRNA prowadzą do nieprawidłowości w procesach komórkowych i w konsekwencji mogą prowadzić do zaburzeń endokrynnych. Zaburzenia różnicowego składania premRNA wynikają z mutacji w tak zwanych miejscach splicingowych bądź z nieprawidłowego działania czynników splicingowych, czyli białek zaangażowanych w regulację tego procesu. Niniejsza praca ma na celu przedstawienie roli różnicowego składania pre-mRNA w patologiach układu endokrynnego, takich jak nowotwory endokrynne (guzy przysadki, rak tarczycy, prostaty, jajnika, piersi, insulinoma), izolowany niedobór hormonu wzrostu, dysgenezja gonad. Omówione zostały także przykłady patologii niezwiązanych bezpośrednio z układem endokrynnym, w których wykryto zaburzenia alternatywnego splicingu transkryptów genów biorących udział w sygnalizacji hormonalnej. Artykuł kończy przegląd możliwości diagnostycznych i terapeutycznych w chorobach endokrynnych, wykorzystujących proces różnicowego składania pre-mRNA. (Endokrynol Pol 2011; 62 (2): 160&#8211;170)Alternative splicing of pre-mRNA is a process in which noncoding regions of primary transcript are removed and coding regions are joined in different manners to produce mRNA molecules of different sequences. Alternative splicing affects nearly all human genes and is a key source of diversity of proteins coded by a relatively small number of genes. Since alternative splicing is of crucial importance for the proper functioning of cells, including those involved in hormonal signalling, aberrations of alternative splicing can lead to disruption of cellular mechanisms and in consequence result in serious endocrine pathologies. Disturbances of alternative splicing include mutations of consensus splice regulatory sites and improprieties in the action of splicing factors, the proteins involved in regulating the process. In consequence of disturbed alternative splicing, improperly spliced mRNA and protein isoforms can be produced which can lead to disruption of function of their wild type counterparts. This review aims to discuss the role of alternative splicing in pathologies of the endocrine system and gives examples that highlight the importance of this process in the proper functioning of hormones, hormone receptors and other factors involved in hormonal regulation. The examples given include endocrine-related tumours (pituitary tumours, cancers of the thyroid, prostate, ovary and breast, and insulinoma), isolated growth hormone deficiency, and Frasier syndrome. Non-endocrine pathologies in which aberrant alternative splicing of transcripts of genes involved in hormonal signalling have been detected are also described. Finally, we discuss future perspectives on the possible usage of alternative splicing in diagnostics and therapy. (Pol J Endocrinol 2011; 62 (2): 160&#8211;170

Alternative splicing discriminates molecular subtypes and has prognostic impact in diffuse large B-cell lymphoma

Effect of alternative splicing (AS) on diffuse large B-cell lymphoma (DLBCL) pathogenesis and survival has not been systematically addressed. Here, we compared differentially expressed genes and exons in association with survival after chemoimmunotherapy, and between germinal center B-cell like (GCB) and activated B-cell like (ABC) DLBCLs. Genome-wide exon array-based screen was performed from samples of 38 clinically high-risk patients who were treated in a Nordic phase II study with dose-dense chemoimmunotherapy and central nervous system prophylaxis. The exon expression profile separated the patients according to molecular subgroups and survival better than the gene expression profile. Pathway analyses revealed enrichment of AS genes in inflammation and adhesion-related processes, and in signal transduction, such as phosphatidylinositol signaling system and adenosine triphosphate binding cassette transporters. Altogether, 49% of AS-related exons were protein coding, and domain prediction showed 28% of such exons to include a functional domain, such as transmembrane helix domain or phosphorylation sites. Validation in an independent cohort of 92 DLBCL samples subjected to RNA-sequencing confirmed differential exon usage of selected genes and association of AS with molecular subtypes and survival. The results indicate that AS events are able to discriminate GCB and ABC DLBCLs and have prognostic impact in DLBCL.Peer reviewe