Tumor-specific Splicing Research Articles

Perturbation of cytoskeleton dynamics by the opposing effects of Rac1 and Rac1b

Rac1, a ubiquitously expressed member of the Rho GTPase family, plays a pivotal role in the regulation of multiple cellular processes including cytoskeleton reorganization, cell growth, differentiation and motility. Here we show that the tumor-specific splice variant of Rac1, Rac1b, negatively regulates Rac1 activity. The expression of Rac1b in HeLa cells interferes with Rac1 activation by PDGF, leads to a reduction in membrane-bound Rac1 and promotes an increase in Rho activity. The antagonistic relationship between Rac1 and Rac1b perturbs the regulatory circuitry that controls actin cytoskeleton dynamics thereby leading to tumor-linked alterations in cell morphology and motility.

Identification of Alternative Splicing Events Regulated by the BCR-ABL Oncoprotein

Post-transcriptional modifications, including alternative splicing, are critical in determining the protein inventory of normal human cells. A number of malignancy-related and apoptosis-related genes have de-regulated alternative splicing due to mutation or deletion of genomic splice sites or through other, less well understood mechanisms. Chronic myelogenous leukemia (CML) is characterized by a translocation that produces the dominant acting BCR-ABL oncoprotein, which is responsible for genomic instability, resistance to apoptosis, cellular proliferation, differentiation abnormalities, and many other acquired cellular changes. It is likely that additional undiscovered properties of the BCR-ABL oncogene contribute to CML pathogenesis. Imatinib, an inhibitor of the BCR-ABL tyrosine kinase, induces a high rate of complete cytogenetic remission in CML. However, the ability of imatinib to eradicate the leukemia stem cell is unknown. Moreover, additional treatment targets will need to be identified to optimize therapies for imatinib-refractory patients. We used gene array analysis to investigate whether alternative splicing may be important in the biology and pathogenesis of CML. Two analyses were conducted, examining the splicing patterns of a total of 587 genes. 570 of the genes have been implicated in apoptosis, and 17 of the genes play a role in hematologic malignancies. In the first experiment, splicing profiles of all 587 genes (5832 independent splice events) were evaluated using a customized ExonHit array. Splicing profiles of three CML cell lines were compared to a well characterized primary thymus control tissue. Dye swaps confirmed experimental consistency. Bioconductor software was used to select events with greater than two-fold differential expression. We have identified 124 candidate splice forms in 91 genes that are differentially regulated in at least two of the three CML cell lines compared with control hematopoietic tissue. 88 of the 91 are implicated in apoptosis, while the remaining three are among the genes implicated in hematologic malignancies. The candidate genes identified by these means include WT1, RUNX1, PFDN5, and BRCA1. In a second experiment, the same 587 genes were examined in two CML cell lines treated with imatinib and compared to untreated controls. Prior pilot experiments identified a dose of imatinib that inhibited BCR-ABL but did not cause apoptosis during the course of the experiment. Using the same experimental procedures and analysis, we identified 41 candidate splice forms in 41 genes that were differentially expressed in imatinib-treated CML cell lines. Among these candidate genes, 40 are from the group of apoptosis-related genes, and one is from the group of genes related to hematologic malignancies. Candidate genes include MAPK3, FES, DCC, and TGFB1. Combining the two experiments, a group of 122 genes and 163 splice events were identified as potential BCR-ABL directed, imatinib-sensitive candidates. We performed validation experiments for one of these, WT1. Imatinib treatment consistently altered the ratios of specific splice forms present in CML cells. These findings are consistent with BCR-ABL dependent splicing of WT1 in CML. Because WT1 isoforms have differential effects on the survival, proliferation, and differentiation of leukemia cells, regulation of WT1 splicing by BCRABL may play an important role in the pathogenesis of CML. Overall, our results suggest that the BCR-ABL oncoprotein may cause significant dysregulation of mRNA splicing as part of the process of neoplastic transformation. We are currently validating selected genes among the 122 candidates in primary CML samples, performing bioinformatics analysis of the specific tumor-specific splice site anatomy to gain insights into potential upstream regulators, and investigating apoptosis regulation by selected candidate splice regulated targets.

Alternative Splicing in Colon, Bladder, and Prostate Cancer Identified by Exon Array Analysis

Alternative splicing enhances proteome diversity and modulates cancer-associated proteins. To identify tissue- and tumor-specific alternative splicing, we used the GeneChip Human Exon 1.0 ST Array to measure whole-genome exon expression in 102 normal and cancer tissue samples of different stages from colon, urinary bladder, and prostate. We identified 2069 candidate alternative splicing events between normal tissue samples from colon, bladder, and prostate and selected 15 splicing events for RT-PCR validation, 10 of which were successfully validated by RT-PCR and sequencing. Furthermore 23, 19, and 18 candidate tumor-specific splicing alterations in colon, bladder, and prostate, respectively, were selected for RT-PCR validation on an independent set of 81 normal and tumor tissue samples. In total, seven genes with tumor-specific splice variants were identified (ACTN1, CALD1, COL6A3, LRRFIP2, PIK4CB, TPM1, and VCL). The validated tumor-specific splicing alterations were highly consistent, enabling clear separation of normal and cancer samples and in some cases even of different tumor stages. A subset of the tumor-specific splicing alterations (ACTN1, CALD1, and VCL) was found in all three organs and may represent general cancer-related splicing events. In silico protein predictions suggest that the identified cancer-specific splice variants encode proteins with potentially altered functions, indicating that they may be involved in pathogenesis and hence represent novel therapeutic targets. In conclusion, we identified and validated alternative splicing between normal tissue samples from colon, bladder, and prostate in addition to cancer-specific splicing events in colon, bladder, and prostate cancer that may have diagnostic and prognostic implications.

RAP80/UIMC1 as cancer-associated antigen: Alternative splice variants and their immunogenicity

We have identified RAP80/UIMC1, the protein highly expressed in testis, as a new cancer-associated antigen. Sera from 5% to 10% of patients with different types of cancer contain specific antibodies to RAP80/UIMC1. In order to investigate the possible reasons for RAP80/UIMC1 immunogenicity, we characterized its numerous splice isoforms and mapped immunogenic regions of the protein. The majority of RAP80/UIMC1 transcripts was detected both in normal tissues and in colon tumors. There are several RAP80/UIMC1 isoforms that are predominantly expressed in testis, however we did not observe elevated expression of these transcripts in tumors from seropositive patients. We mapped the major immunogenic region of RAP80/UIMC1 to the central part of the protein encoded by exon 9 which is present in a number of ubiquitous splice forms. Thus, based on our data, autoreactivity to RAP80/UIMC1 is related to reasons other than overexpression or tumor-specific splicing.

Activation of Tumor-Specific Splice Variant Rac1b by Dishevelled Promotes Canonical Wnt Signaling and Decreased Adhesion of Colorectal Cancer Cells

Rac1b is a tumor-specific splice variant of the Rac1 GTPase that displays limited functional similarities to Rac1. We have shown previously a novel cross-talk between Rac1 and beta-catenin, which induces canonical Wnt pathway activation in colorectal cancer cells. This prompted us to investigate if Rac1b, frequently overexpressed in colon tumors, contributes to Wnt pathway dysregulation. We show that Rac1b overexpression stimulates Tcf-mediated gene transcription, whereas depletion of Rac1b results in decreased expression of the Wnt target gene cyclin D1. Reconstitution experiments revealed an important difference between Rac1 and Rac1b such that Rac1b was capable of functionally interacting with Dishevelled-3 (Dvl-3) but not beta-catenin to mediate synergistic induction of Wnt target genes. In agreement, Dvl-3 but not beta-catenin caused increased activation of Rac1b levels, which may explain the functional cooperativity displayed in transcription assays. Furthermore, we show that Rac1b negatively regulates E-cadherin expression and results in decreased adhesion of colorectal cancer cells. RNA interference-mediated suppression of Rac1b resulted in reduced expression of Slug, a specific transcriptional repressor of E-cadherin, and a concomitant increase in E-cadherin transcript levels was observed. Intriguingly, mutation of the polybasic region of Rac1b resulted in complete loss of Rac1b stimulatory effects on transcription and suppressive effects on adhesion, indicating the importance of nuclear and membrane localization of Rac1b. Our results suggest that Rac1b overexpression may facilitate tumor progression by enhancing Dvl-3-mediated Wnt pathway signaling and induction of Wnt target genes specifically involved in decreasing the adhesive properties of colorectal cancer cells.

197 POSTER High throughput experimental verification of predicted tissue and tumor specific splice isoforms

197 POSTER High throughput experimental verification of predicted tissue and tumor specific splice isoforms

High-throughput experimental verification of predicted tissue-specific and tumor-specific splice isoforms

Alternative splicing of transcripts may lead to different mRNA species and therefore to potentially different proteins. Any failure or error in the splicing control mechanism can be involved in a number of pathological processes, such as cancer. Splice isoforms that are disease specific could therefore serve as excellent diagnostic markers, which are easily identifiable by PCR. Computational prediction of alternative splice variants has been highly facilitating the identification of novel splice isoforms. Our prediction strategy is based on the genomic mapping (SpliceNest) of EST consensus sequences and library annotation provided in the GeneNest database. This revealed 427 genes with at least one tissue-specific transcript as well as 1120 genes showing tumor-specific isoforms. Out of these genes, a subset of predicted isoforms was experimentally verified by an RT-PCR screening approach. We have set up an experimental strategy that allows us to screen expression of genes in up to 112 different human tissues of multiple developmental stages and cell lines. Within this project, the electrophoretic separation of RT-PCR products turned out to be the bottleneck impeding the switch from a medium-throughput to a high-throughput strategy. To circumvent the limitations of DNA slab gel analysis, a laboratory prototype of an automated on-chip electrophoresis system that allows high-throughput analysis of DNA fragments was implemented in the workflow. In our experimental set-up, we analyzed RT-PCR samples on 4 × 96-well plates within a defined sequence of consecutive one-on-one measurements. The high-throughput experimental verification of computationally predicted tissue specific isoforms revealed a high success rate in confirming their expression in the respective tissue. However, low expression levels of the respective transcript and the limited sensitivity of the experimental method can explain failed detection of the restricted expression pattern. The combination of computational prediction of alternative splicing events with high-throughput experimental verification facilitates the efficient detection of tissue-specific and tumor-specific transcripts.

Strengths and weaknesses of EST-based prediction of tissue-specific alternative splicing.

BackgroundAlternative splicing contributes significantly to the complexity of the human transcriptome and proteome. Computational prediction of alternative splice isoforms are usually based on EST sequences that also allow to approximate the expression pattern of the related transcripts. However, the limited number of tissues represented in the EST data as well as the different cDNA construction protocols may influence the predictive capacity of ESTs to unravel tissue-specifically expressed transcripts.MethodsWe predict tissue and tumor specific splice isoforms based on the genomic mapping (SpliceNest) of the EST consensus sequences and library annotation provided in the GeneNest database. We further ascertain the potentially rare tissue specific transcripts as the ones represented only by ESTs derived from normalized libraries. A subset of the predicted tissue and tumor specific isoforms are then validated via RT-PCR experiments over a spectrum of 40 tissue types.ResultsOur strategy revealed 427 genes with at least one tissue specific transcript as well as 1120 genes showing tumor specific isoforms. While our experimental evaluation of computationally predicted tissue-specific isoforms revealed a high success rate in confirming the expression of these isoforms in the respective tissue, the strategy frequently failed to detect the expected restricted expression pattern. The analysis of putative lowly expressed transcripts using normalized cDNA libraries suggests that our ability to detect tissue-specific isoforms strongly depends on the expression level of the respective transcript as well as on the sensitivity of the experimental methods. Especially splice isoforms predicted to be disease-specific tend to represent transcripts that are expressed in a set of healthy tissues rather than novel isoforms.ConclusionsWe propose to combine the computational prediction of alternative splice isoforms with experimental validation for efficient delineation of an accurate set of tissue-specific transcripts.

CTAGE: A Cutaneous T Cell Lymphoma Associated Antigen Family with Tumor-Specific Splicing

cTAGE-1 is a cutaneous-T-cell-lymphoma-specific tumor antigen recently identified by serologic identification of antigens by recombinant expression cloning. This study was aimed at identifying and characterizing related genes. Rapid amplification of cDNA ends and DNA screening led to five new members of the cTAGE gene family belonging to four different genes, two of which were differentially spliced (cTAGE-1/2 and cTAGE-5). Expression analysis using reverse transcription polymerase chain reaction revealed that cTAGE-1, cTAGE-1B, and cTAGE-5A expression was restricted to testis and tumor tissues, whereas the other cTAGE members were found in two to eight other normal tissues (of 27 tissues tested). Tumor-specific protein expression of cTAGE-5 was confirmed by Western blotting. Sero-reactivity against cTAGE-1, cTAGE-4, cTAGE-5A, and cTAGE-5B was found only in tumor patients (cutaneous T cell lymphoma and melanoma). The immunogenic epitope of cTAGE-1 was determined by using epitope mapping and sera of two cutaneous T cell lymphoma patients. Moreover, cTAGE-1, cTAGE-4, cTAGE-5A, and cTAGE-5B could be detected in most types of tumor tissues and cell lines at variable frequencies, including those of cutaneous T cell lymphoma, melanoma, head and neck squamous cell carcinoma, breast carcinoma, and colon carcinoma. We conclude that cTAGE-1 and cTAGE-5 are new cancer germline antigens and that tumor-specific splicing of cTAGE genes may lead to further candidate proteins for specific immunotherapy of cutaneous T cell lymphoma and other malignancies.

Differential expression of estrogen receptor-beta (ER beta) in human pituitary tumors: functional interactions with ER alpha and a tumor-specific splice variant.

The mitogenic and regulatory effects of estrogen (E2) in adenohypophysial cells are known to be mediated through the nuclear estrogen receptor (ER alpha). Expression of ER alpha and several of its messenger ribonucleic acid (RNA) alternate splice variants has been shown to be restricted to prolactinomas and gonadotroph tumors. However, little is known about gene expression patterns of the novel nuclear hormone receptor ER beta in the neoplastic pituitary. ER beta has high homology to ER alpha in the DNA- and ligand-binding domains, but encodes a distinct transcriptional activating function-1 (AF-1) domain. Using RT-PCR analysis of total RNA from 38 human pituitary adenomas, we found that ER beta messenger RNA was coexpressed with ER alpha and its splice variants in 60% of prolactinomas, 100% of mixed GH/PRL tumors, and 29% of gonadotroph tumors. ER beta gene expression was not limited to ER alpha-positive tumor subtypes, however, and was also found in 100% of null cell tumors, 80% of somatotroph tumors, and 60% of corticotroph tumors. Because ER beta is coexpressed with ER alpha and its splice variants in prolactinomas and gonadotroph tumors, we functionally characterized the potential interactions between ER beta and ER alpha. We also examined the potential cooperative effects on ER beta-mediated gene expression of a tumor-specific truncated delta 5ER alpha splice variant that has been shown to be coexpressed in the majority of ER alpha-positive tumors. This exon 5 splice variant encodes the AF-1 domain as well as regions critical for DNA binding and nuclear localization, but lacks the ligand-binding and AF-2 domains. Mammalian expression vectors encoding ER alpha, delta 5ER alpha, and/or ER beta complementary DNAs were transiently transfected along with an E2 response element promoter-luciferase (ERELuc) reporter into human ER alpha/ER beta-negative osteosarcoma U2-OS cells. ER beta was less potent than ER alpha in activating E2-stimulated ERELuc activity (4-vs. 14-fold relative to basal control levels). However, when delta 5ER alpha was coexpressed with ER beta or ER alpha, E2-stimulated ERELuc activity was markedly increased to 8- and 57-fold, respectively, relative to basal control levels when each full-length isoform was expressed alone. Finally, coexpression of ER beta with ER alpha did not significantly alter the E2-stimulated ERELuc activity induced by ER alpha alone. Cotreatment with tamoxifen markedly inhibited all E2-stimulated ERELuc responses to baseline levels. Together, these data suggest that ER beta has a minor role in mediating E2 responses in ER alpha-positive tumors, but may be the main mediator of E2-stimulated gene expression when expressed alone in somatotroph, corticotroph, and null cell tumors. This low, but significant, level of ER beta trans-activation potential may be enhanced by coexpression of delta 5ER alpha in neoplastic pituitary. Therefore, E2-mediated gene expression in normal and neoplastic pituitary appears to be highly dependent on the expression of ER alpha and ER beta isoforms, which have varying transcriptional activities.

Differential Expression of Estrogen Receptor- (ER ) in Human Pituitary Tumors: Functional Interactions with ER and a Tumor-Specific Splice Variant

Differential Expression of Estrogen Receptor- (ER ) in Human Pituitary Tumors: Functional Interactions with ER and a Tumor-Specific Splice Variant

Tumor-specific expression and alternate splicing of messenger ribonucleic acid encoding activin/transforming growth factor-beta receptors in human pituitary adenomas.

Activin, a member of the transforming growth factor-beta (TGF beta) cytokine family, acts as a pituitary cell mitogen via a novel family of receptor-linked serine/threonine (Ser/Thr) kinases. Pituitary tumors synthesize activin subunits, and the autocrine action of these growth factors may modulate tumor proliferation. We, therefore, investigated the expression of activin/TGF beta type I receptor messenger ribonucleic acids (mRNAs), designated ALK1 through ALK5 (ALK = activin receptor-like kinase), and type II receptor mRNAs using RT-PCR in 34 human pituitary adenomas of all phenotypes and normal pituitary tissue. ALK2 and ALK5, specific mediators of activin and TGF beta signals, respectively, were found to be expressed only in tumor and not in normal pituitary cells, and ALK2 expression was found only in tumors of a mammosomatotroph cell lineage. ALK1, ALK3, and ALK4 mRNAs were found in both normal and neoplastic pituitary cells. The alternatively spliced cytoplasmic domain of ALK4 consists of 11 kinase subdomains, that are critical for modulating receptor function and intracellular signaling. Truncated forms of the ALK4 cytoplasmic domain lacking these subdomains may attenuate activin signal transduction and affect both tumor phenotype and proliferation via the formation of inactive type I/type II complexes. Three truncated ALK4 receptor mRNAs generated by alternate splicing of the cytoplasmic Ser/Thr kinase domain were found to be tumor specific. One of these truncated receptor mRNAs, ALK4-5, is a novel splice variant that has not been previously described. Expression of the ActRII and T beta RII type II receptor mRNAs, which specifically bind activin and TGF beta, respectively, was highly prevalent among all tumor subtypes and normal pituitary tissue. However, ActRIIB, an activin-specific type II receptor that displays a 3- to 4-fold higher affinity for ligand than ActRII, was expressed in 94% of tumors, but was not prevalent in normal tissue. These data are the first to demonstrate tumor-specific expression of Ser/Thr kinase receptors mRNAs and their splice variants in human pituitary adenomas.