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Alternative Splicing in Lung Cancer

  • Ruben Pio
    Correspondence
    Division of Oncology, CIMA-University of Navarra, Pio XII 55, Pamplona 31008, Spain
    Affiliations
    Division of Oncology, Center for Applied Medical Research and School of Medicine, University of Navarra, 31080 Pamplona, Spain

    Department of Biochemistry, Center for Applied Medical Research and School of Medicine, University of Navarra, 31080 Pamplona, Spain
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  • Luis M. Montuenga
    Affiliations
    Division of Oncology, Center for Applied Medical Research and School of Medicine, University of Navarra, 31080 Pamplona, Spain

    Department of Histology and Pathology, Center for Applied Medical Research and School of Medicine, University of Navarra, 31080 Pamplona, Spain
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      Abstract

      Alterations in alternative splicing affect essential biologic processes and are the basis for a number of pathologic conditions, including cancer. In this review we will summarize the evidence supporting the relevance of alternative splicing in lung cancer. An example that illustrates this relevance is the altered balance between Bcl-xL and Bcl-xS, two splice variants of the apoptosis regulator Bcl-x. Splice modifications in cancer-related genes can be associated with modifications either in cis-acting splicing regulatory sequences or in trans-acting splicing factors. In fact, lung tumors show abnormal expression of splicing regulators such as ASF/SF2 or some members of the heterogeneous nuclear ribonucleoprotein family. The potential significance of alternative splicing as a target for lung cancer diagnosis or treatment will also be discussed.

      Keywords

      Alternative splicing is a major contributor to transcriptome and proteome diversity. Multiple transcripts are generated from a single mRNA precursor by means of alternative splicing. The alternative use of exons in each transcript may have a profound effect on the biologic characteristics of the final protein by adding or deleting functional domains, changing its stability, controlling its location, or modifying its protein-protein interactions. Alternative splicing may also influence mRNA translation, location, and stability. Alternative splicing is not a rare event in normal cells, but a central regulatory mechanism. Recent data from genome-wide studies suggest that more than 90% of human genes undergo alternative splicing.
      • Wang ET
      • Sandberg R
      • Luo S
      • et al.
      Alternative isoform regulation in human tissue transcriptomes.
      Alterations in alternative splicing have been shown to affect essential biologic processes and many disease conditions, including cancer. Mutations in splicing regulatory elements within the nucleotide sequences and alterations in the expression of splicing regulatory factors have been extensively reported in cancer. Moreover, the expression of several alternatively spliced gene products has also been linked to the development of neoplastic diseases.
      • Kalnina Z
      • Zayakin P
      • Silina K
      • Linē A
      Alterations of pre-mRNA splicing in cancer.
      • Venables JP
      Unbalanced alternative splicing and its significance in cancer.
      In the recent years, powerful techniques for genome-wide identification and analysis of alternative splicing isoforms have been developed.
      • Johnson JM
      • Castle J
      • Garrett-Engele P
      • et al.
      Genome-wide survey of human alternative pre-mRNA splicing with exon junction microarrays.
      • Clark TA
      • Sugnet CW
      • Ares MJ
      Genomewide analysis of mRNA processing in yeast using splicing-specific microarrays.
      • Anton MA
      • Gorostiaga D
      • Guruceaga E
      • et al.
      SPACE: an algorithm to predict and quantify alternatively spliced isoforms using microarrays.
      These technologies will allow for the identification of novel cancer-related markers and targets for therapy. It is, therefore, not surprising that alternative splicing in cancer is now emerging as a growing and promising field in basic and translational oncology.
      • Pajares MJ
      • Ezponda T
      • Catena R
      • Calvo A
      • Pio R
      • Montuenga LM
      Alternative splicing: an emerging topic in molecular and clinical oncology.

      Relevance of Alternative Splicing in Lung Cancer

      Several reviews have addressed the relevance of alternative splicing in cancer-related genes.
      • Kalnina Z
      • Zayakin P
      • Silina K
      • Linē A
      Alterations of pre-mRNA splicing in cancer.
      • Brinkman BM
      Splice variants as cancer biomarkers.
      • Venables JP
      Aberrant and alternative splicing in cancer.
      • Skotheim RI
      • Nees M
      Alternative splicing in cancer: noise, functional, or systematic?.
      Table 1 lists genes that have alternative splice variants associated with lung cancer. Splicing variants from cancer-related genes may have a critical impact on lung cancer cell biology.
      • Schwerk C
      • Schulze-Osthoff K
      Regulation of apoptosis by alternative pre-mRNA splicing.
      This is well illustrated by the significance of alternative splicing in the function of the apoptosis regulator Bcl-x. The first coding exon of this gene contains an alternative 5′ splice site (Figure 1). If the Bcl-x pre-mRNA is spliced to include the whole exon, the translated product is Bcl-xL, a protein that inhibits apoptosis. If, conversely, the pre-mRNA is alternatively spliced eliminating a 3′-end portion of the exon, Bcl-xS is produced, a shorter protein with proapoptotic activity. A higher proportion of Bcl-xL is frequently found in both non-small cell lung cancer (NSCLC) and small cell lung cancer, deregulating the balance between proapoptotic and antiapoptotic signals and potentially contributing to tumor progression.
      • Reeve JG
      • Xiong J
      • Morgan J
      • Bleehen NM
      Expression of apoptosis-regulatory genes in lung tumour cell lines: relationship to p53 expression and relevance to acquired drug resistance.
      • Groeger AM
      • Esposito V
      • De Luca A
      • et al.
      Prognostic value of immunohistochemical expression of p53, bax, Bcl-2 and Bcl-xL in resected non-small-cell lung cancers.
      • Karczmarek-Borowska B
      • Filip A
      • Wojcierowski J
      • et al.
      Estimation of prognostic value of Bcl-xL gene expression in non-small cell lung cancer.
      The alternative splicing of CD44 is also biologically relevant in lung cancer. CD44 is a multifunctional surface glycoprotein with an important role in cell adhesion and migration. The human CD44 gene contains 10 variable exons (v1 to v10) that can be alternatively spliced to generate many different CD44 protein isoforms. The most common CD44 isoform is the standard CD44 (CD44s), in which all the variable exons are skipped. CD44s function as a cell-cell and cell-matrix adherence molecule, and its down-regulation has been associated with metastasis in many malignancies.
      • Naor D
      • Nedvetzki S
      • Golan I
      • Melnik L
      • Faitelson Y
      CD44 in cancer.
      In fact, Pirinen et al.
      • Pirinen R
      • Hirvikoski P
      • Bohm J
      • et al.
      Reduced expression of CD44v3 variant isoform is associated with unfavorable outcome in non-small cell lung carcinoma.
      showed that up-regulation of total CD44 and of CD44v3 were predictors of favorable outcome in NSCLC patients. Enhanced expression of other splice variants can be associated with tumor progression and metastasis. Conflicting results have been reported on the significance of CD44v6 in the outcome of NSCLC patients, with studies suggesting that its expression is related to reduced survival,
      • Hirata T
      • Fukuse T
      • Naiki H
      • Hitomi S
      • Wada H
      Expression of CD44 variant exon 6 in stage I non-small cell lung carcinoma as a prognostic factor.
      • Nguyen VN
      • Mirejovsky T
      • Melinova L
      • Mandys V
      CD44 and its v6 spliced variant in lung carcinomas: relation to NCAM, CEA, EMA and UP1 and prognostic significance.
      and studies showing no relationship with prognosis.
      • Givehchian M
      • Woerner SM
      • Lacroix J
      • et al.
      Expression of CD44 splice variants in normal respiratory epithelium and bronchial carcinomas: no evidence for altered CD44 splicing in metastasis.
      • Carbognani P
      • Spaggiari L
      • Romani A
      • et al.
      Expression of human CD44v6 in non-small-cell lung cancer.
      Inconsistent results on the prognostic significance of CD44 or its splice forms have been also published in other human cancers. This example underlines the importance of standardization and interlaboratory validation of reagents and protocols used to analyze cancer-specific splicing isoforms.
      • Pajares MJ
      • Ezponda T
      • Catena R
      • Calvo A
      • Pio R
      • Montuenga LM
      Alternative splicing: an emerging topic in molecular and clinical oncology.
      TABLE 1Examples of Genes With Splice Variants Associated With Lung Cancer
      GeneLung Cancer HistologyFunctionReferences
      Actinin-4SCLCCytoskeleton binding56
      Bcl-xNSCLC/SCLCApoptosis12–14
      CD44NSCLC/SCLC
      • Multifunctional
      • receptor
      17,18
      CEACAM-1NSCLCAdhesion52
      CAIXNSCLCpH regulation57
      Cyclin D1NSCLCCell cycle24,58
      FHITNSCLCNucleotide metabolism59
      FibronectinNSCLC/SCLC
      • Adhesion
      • Angiogenesis
      44,60,61
      KLF6AdenocarcinomaTranscription factor43
      MDM2NSCLCProliferation62,63
      MesothelinMesotheliomaAdhesion64
      NF2/merlinMesotheliomaMembrane stabilizing protein65
      NRSFSCLCTranscription factor66
      PPARγSquamous cell carcinomaNuclear hormone receptor67
      TSG101SCLCReceptor trafficking68
      VEGFNSCLCAngiogenesis69
      XAGE-1AdenocarcinomaUnknown53,54
      SCLC, small cell lung cancer; NSCLC, non-small cell lung cancer.
      Figure thumbnail gr1
      FIGURE 1Bcl-x is an apoptosis regulator with two main isoforms (Bcl-xL and Bcl-xS) generated by alternative 5′ splice site selection. Interestingly, the two splice variants have opposing functions: Bcl-xL is proapoptotic whereas Bcl-xS is antiapoptotic. Several transacting splicing factors regulate the balance between the two splice variants. In lung cancer, abnormal splicing of the Bcl-x mRNA gives rise to a predominant production of Bcl-xL. Various strategies for diagnosis or treatment of lung cancer can be proposed based on the predominant expression of the cancer-related splice isoform (either from Bcl-x or from any other gene with splice isoforms involved in lung cancer).
      Splicing changes associated with genetic polymorphisms are also relevant for lung cancer risk. Several case-control studies have found an association between the cyclin D1 (CCND1) G870A polymorphism and an increased risk of lung cancer.
      • Gautschi O
      • Ratschiller D
      • Gugger M
      • Betticher DC
      • Heighway J
      Cyclin D1 in non-small cell lung cancer: a key driver of malignant transformation.
      CCND1 is a protein that controls G1-S phase progression through its interaction with cyclin-dependent kinases (CDKs). In addition, CCND1 can act independently of the CDKs, modulating migration, invasion, differentiation, inflammation, and angiogenesis.
      • Gautschi O
      • Ratschiller D
      • Gugger M
      • Betticher DC
      • Heighway J
      Cyclin D1 in non-small cell lung cancer: a key driver of malignant transformation.
      The G870A polymorphism does not change the amino acid sequence of CCND1, but modulates its splice pattern. The presence of the A870-allele favors splicing into transcript b, which encodes for the cyclin D1b splice variant. Cyclin D1b induces malignant transformation,
      • Lu F
      • Gladden AB
      • Diehl JA
      An alternatively spliced cyclin D1 isoform, cyclin D1b, is a nuclear oncogene.
      • Solomon DA
      • Wang Y
      • Fox SR
      • et al.
      Cyclin D1 splice variants. Differential effects on localization, RB phosphorylation, and cellular transformation.
      and its expression has been associated with poor outcome in lung cancer patients.
      • Li R
      • An SJ
      • Chen ZH
      • et al.
      Expression of cyclin D1 splice variants is differentially associated with outcome in non-small cell lung cancer patients.

      Splicing Regulatory Proteins in Lung Cancer

      The mechanisms leading to aberrant alternative splicing in lung cancer are poorly understood. Splice modifications in cancer-related genes can be associated with modifications in intronic and exonic cis-acting splicing regulatory elements or in trans-acting splicing factors. There are several examples of the importance of cis-acting splicing regulatory motifs in lung cancer, such as the polymorphism in CCND1 described above, or the somatic intronic mutations of Met kinase that lead to an alternatively spliced transcript.
      • Kong-Beltran M
      • Seshagiri S
      • Zha J
      • et al.
      Somatic mutations lead to an oncogenic deletion of met in lung cancer.
      Nevertheless, it is reasonable to assume that the majority of splicing alterations in lung cancer are due to modifications in the concentration, localization, composition, or activity of RNA binding proteins acting as splicing regulatory factors. The heterogeneous nuclear ribonucleoprotein (hnRNP) family is a group of RNA binding proteins involved in splicing and in other aspects of RNA metabolism: stabilization, nucleocytoplasmic transport, translation, and transcriptional regulation. Overexpression of hnRNP proteins has been reported in several malignancies. Abnormal expression of hnRNP proteins in NSCLC clinical samples and animal models suggests that tumors develop hnRNP profiles that may contribute to lung carcinogenesis.
      • Pino I
      • Pio R
      • Toledo G
      • et al.
      Altered patterns of expression of members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family in lung cancer.
      • Zerbe LK
      • Pino I
      • Pio R
      • et al.
      Relative amounts of antagonistic splicing factors, hnRNP A1 and ASF/SF2, change during neoplastic lung growth: implications for pre-mRNA processing.
      • Peebles KA
      • Dwyer-Nield LD
      • Malkinson AM
      Altered expression of splicing factor, heterogeneous nuclear ribonucleoprotein A2/B1, in mouse lung neoplasia.
      In fact, overexpression of hnRNP A2/B1 was proposed in the past as a potential lung cancer early detection biomarker.
      • Tockman MS
      • Mulshine JL
      • Piantadosi S
      • et al.
      Prospective detection of preclinical lung cancer: results from two studies of heterogeneous nuclear ribonucleoprotein A2/B1 overexpression.
      • Sueoka E
      • Sueoka N
      • Iwanaga K
      • et al.
      Detection of plasma hnRNP B1 mRNA, a new cancer biomarker, in lung cancer patients by quantitative real-time polymerase chain reaction.
      It could be argued that the overexpression of these and other RNA binding proteins mostly responds to the increased metabolic rate associated with tumor proliferation, however, some studies have demonstrated that these proteins are also involved in the malignant transformation of the cells. ASF/SF2, an important splicing regulator, is up-regulated in many tumors and can act as an oncogene.
      • Karni R
      • de Stanchina E
      • Lowe SW
      • Sinha R
      • Mu D
      • Krainer AR
      The gene encoding the splicing factor SF2/ASF is a proto-oncogene.
      On the other hand, our group has shown that hnRNP E4 (PCBP4 or ±CP-4) is down-regulated in lung cancer and may function as a tumor suppressor gene.
      • Pio R
      • Zudaire I
      • Pino I
      • et al.
      Alpha CP-4, encoded by a putative tumor suppressor gene at 3p21, but not its alternative splice variant alpha CP-4a, is underexpressed in lung cancer.
      • Castano Z
      • Vergara-Irigaray N
      • Pajares MJ
      • Montuenga LM
      • Pio R
      Expression of alpha CP-4 inhibits cell cycle progression and suppresses tumorigenicity of lung cancer cells.

      Impact of Alternative Splicing in Lung Cancer Therapy and Detection

      Drugs that modulate alternative splicing have been proposed as potential therapeutic tools for lung cancer. Recently, the splice variant K-Ras 4A has been identified as the main mediator of the oncogenic activity of mutant K-Ras in lung carcinogenesis. This provides the rationale for designing targeted therapies specific for this cancer-associated splice variant.
      • To MD
      • Wong CE
      • Karnezis AN
      • Del Rosario R
      • Di Lauro R
      • Balmain A
      Kras regulatory elements and exon 4A determine mutation specificity in lung cancer.
      Therapies for this and other cancer-related genes can be aimed to modify the activity of splicing factors or be directed at reversing specific abnormal splicing events. The latter can be achieved by the use of antisense oligonucleotides that shift alternatively spliced isoforms toward the therapeutically favorable one.
      • Gleave ME
      • Monia BP
      Antisense therapy for cancer.
      Synthetically modified oligonucleotides are favored over regular nucleotide backbones for their stability and low toxicity in vivo. Synthetically modified oligonucleotides which modify the splicing pattern of apoptosis-related genes may induce apoptosis or sensitize cells to treatment with chemo or radiotherapy. The ability to alter the Bcl-x splicing is particularly interesting, given that the two splice variants have opposing functions. In fact, antisense oligonucleotides targeting the Bcl-xL 5′ splice site to specifically inhibit Bcl-xL have been used against lung cancer cells.
      • Leech SH
      • Olie RA
      • Gautschi O
      • et al.
      Induction of apoptosis in lung-cancer cells following bcl-xL anti-sense treatment.
      • Simoes-Wust AP
      • Hopkins-Donaldson S
      • Sigrist B
      • Belyanskaya L
      • Stahel RA
      • Zangemeister-Wittke U
      A functionally improved locked nucleic acid antisense oligonucleotide inhibits Bcl-2 and Bcl-xL expression and facilitates tumor cell apoptosis.
      Other antisense oligonucleotides which induce the expression of Bcl-xS sensitize cells to apoptosis in response to treatment.
      • Taylor JK
      • Zhang QQ
      • Wyatt JR
      • Dean NM
      Induction of endogenous Bcl-xS through the control of Bcl-x pre-mRNA splicing by antisense oligonucleotides.
      Boon-Unge et al. used a different strategy to switch the splicing pattern of Bcl-x. They screened 1040 drugs searching for compounds able to regulate Bcl-x splicing in the lung adenocarcinoma cell line A549 and found that a compound named Emetine down-regulated the mRNA levels of Bcl-xL with a concomitant increase in the mRNA levels of Bcl-xS.
      • Boon-Unge K
      • Yu Q
      • Zou T
      • Zhou A
      • Govitrapong P
      • Zhou J
      Emetine regulates the alternative splicing of Bcl-x through a protein phosphatase 1-dependent mechanism.
      Previously, Chalfant et al.
      • Chalfant CE
      • Rathman K
      • Pinkerman RL
      • et al.
      De novo ceramide regulates the alternative splicing of caspase 9 and Bcl-x in A549 lung adenocarcinoma cells. Dependence on protein phosphatase-1.
      showed that the chemotherapeutic agent gemcitabine increases the generation of ceramide in A549 cells, which induces the dephosphorylation of splicing factors, elevating the levels of the proapoptotic splice variants Bcl-xS and caspase-9, with a parallel reduction in the expression of the antiapoptotic forms Bcl-xL and caspase-9S. Other anticancer drugs can also induce apoptosis in association with a Bcl-x splicing switch in lung cancer cells.
      • Sen S
      • Sharma H
      • Singh N
      Curcumin enhances Vinorelbine mediated apoptosis in NSCLC cells by the mitochondrial pathway.
      • Chan HC
      • Kuo SC
      • Liu SC
      • Liu CH
      • Hsu SL
      4-Fluoro-N-butylphenylacetamide: a synthetic phenylacetate derivative that upregulates Bcl-X(S), activates caspase cascade and induces apoptosis in human squamous lung cancer CH27 cells.
      Inhibition of KLF6-SV1, a splicing variant of the tumor suppressor gene KLF6, may also have therapeutic potential. DiFeo et al.
      • DiFeo A
      • Feld L
      • Rodriguez E
      • et al.
      A functional role for KLF6-SV1 in lung adenocarcinoma prognosis and chemotherapy response.
      have recently reported that KLF6-SV1 is overexpressed in lung adenocarcinomas and is associated with poor clinical outcome. KLF6-SV1 overexpression abrogates the proapoptotic effect of cisplatin, whereas targeted reduction of KLF6-SV1, either alone or in combination with cisplatin, results in a marked increase in apoptosis.
      • DiFeo A
      • Feld L
      • Rodriguez E
      • et al.
      A functional role for KLF6-SV1 in lung adenocarcinoma prognosis and chemotherapy response.
      Alternative splice variants can also be used as targets for imaging and selective delivery of bioactive molecules to tumors. A fibronectin splice variant (ED-B), containing an additional domain inserted by alternative splicing, accumulates around new-forming blood vessels, but is absent in normal vasculature in adult tissues. Antibodies specific for ED-B have been designed to selectively deliver therapeutic molecules to tumor neovasculature both in animal models and patients.
      • Santimaria M
      • Moscatelli G
      • Viale GL
      • et al.
      Immunoscintigraphic detection of the ED-B domain of fibronectin, a marker of angiogenesis, in patients with cancer.
      • Lo KM
      • Lan Y
      • Lauder S
      • et al.
      huBC1-IL12, an immunocytokine which targets EDB-containing oncofetal fibronectin in tumors and tumor vasculature, shows potent anti-tumor activity in human tumor models.
      Cancer specific alternative splicing forms can potentially be used as diagnostic biomarkers.
      • Brinkman BM
      Splice variants as cancer biomarkers.
      Any splice variant found exclusively in cancer cells may be a candidate. At the mRNA level, variants would be easily detected by polymerase chain reaction using splice variant-specific primers. Antibodies that specifically detect particular splice variants could be generated and applied in immunoassays. However, to our knowledge, none of these strategies has been tested to date for diagnosis of lung cancer, which may be attributed to the absence of appropriate candidates. The use of high-throughput technologies to identify splicing patterns of many genes simultaneously, will undoubtedly push forward the discovery of lung cancer biomarkers with potential use for diagnosis.
      • Calarco JA
      • Saltzman AL
      • Ip JY
      • Blencowe BJ
      Technologies for the global discovery and analysis of alternative splicing.
      In this sense, whole genome exon arrays have already been used to detect splice variants differentially expressed in several tumor types.
      • Gardina PJ
      • Clark TA
      • Shimada B
      • et al.
      Alternative splicing and differential gene expression in colon cancer detected by a whole genome exon array.
      • French PJ
      • Peeters J
      • Horsman S
      • et al.
      Identification of differentially regulated splice variants and novel exons in glial brain tumors using exon expression arrays.
      • Thorsen K
      • Sorensen KD
      • Brems-Eskildsen AS
      • et al.
      Alternative splicing in colon, bladder, and prostate cancer identified by exon array analysis.
      • Cheung HC
      • Baggerly KA
      • Tsavachidis S
      • et al.
      Global analysis of aberrant pre-mRNA splicing in glioblastoma using exon expression arrays.
      Xi et al.
      • Xi L
      • Feber A
      • Gupta V
      • et al.
      Whole genome exon arrays identify differential expression of alternatively spliced, cancer-related genes in lung cancer.
      have recently applied this technology to analyze alternative splicing events in lung adenocarcinoma. The authors confirmed the already reported overexpression of a CEACAM1 variant,
      • Wang L
      • Lin SH
      • Wu WG
      • et al.
      C-CAM1, a candidate tumor suppressor gene, is abnormally expressed in primary lung cancers.
      and, for the first time, found overexpression of specific variants for ERG, CDKN2A, and CDH3.
      • Xi L
      • Feber A
      • Gupta V
      • et al.
      Whole genome exon arrays identify differential expression of alternatively spliced, cancer-related genes in lung cancer.
      Exon arrays are not specifically designed to identify alternative splicing and some events may have been missed. For this reason, more powerful tools, such as splicing specific microarrays or deep sequencing technologies, should be applied in the future to characterize new splice variants suitable to be used as lung cancer biomarkers.
      Finally, abnormal alternative splicing can generate protein antigens recognizable by the immune system. Autoantibody responses have been described to XAGE-1 protein in patients with lung adenocarcinoma.
      • Nakagawa K
      • Noguchi Y
      • Uenaka A
      • et al.
      XAGE-1 expression in non-small cell lung cancer and antibody response in patients.
      • Ali Eldib AM
      • Ono T
      • Shimono M
      • et al.
      Immunoscreening of a cDNA library from a lung cancer cell line using autologous patient serum: identification of XAGE-1b as a dominant antigen and its immunogenicity in lung adenocarcinoma.
      XAGE-1 belongs to the family of cancer-testis antigens. Four transcript variants XAGE-1a, XAGE-1b, XAGE-1c, and XAGE-1d have been identified. XAGE-1b is the dominant antigen recognized by sera from lung adenocarcinoma patients. Immunogenicity of XAGE-1 alternatively spliced forms suggests that they might be used as vaccine targets for lung cancer.
      • Wang T
      • Fan L
      • Watanabe Y
      • et al.
      L552S, an alternatively spliced isoform of XAGE-1, is over-expressed in lung adenocarcinoma.
      Detection of these autoantibodies may be also used for early detection or diagnosis in lung cancer patients.

      CONCLUSIONS

      There is abundant evidence on the importance of alternative splicing in lung cancer. The impact in diagnosis and treatment is still minimal. A better understanding of the mechanisms regulating alternative splicing in lung cancer and the identification of new cancer-associated splice variants will undoubtedly lead to the development of new strategies for lung cancer detection and treatment (Figure 1). Splicing-specific microarrays, designed to explore global splicing changes, are very powerful tools to discover splice variants differentially expressed in tumors, and may be used to identify new candidates for lung cancer diagnosis, prognosis, and therapy. Finally, considering that the splicing machinery is altered in lung cancer, it is important to keep in mind that many splice variants found in lung cancer tissues would likely be “passengers” and not “drivers” of malignant transformation. Functional studies will be required to confirm the biologic relevance of the splice variants found in lung cancer.

      ACKNOWLEDGMENT

      Supported by “UTE project CIMA” and the Spanish “Red Temática de Investigación Cooperativa en Cáncer” (RTICC RD06/0020/0066).

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