Splice variants of zinc finger protein 695 mRNA associated to ovarian cancer
- Sergio Juárez-Méndez1,
- Alejandro Zentella-Dehesa3, 4,
- Vanessa Villegas-Ruíz5,
- Oscar Alberto Pérez-González1,
- Mauricio Salcedo5,
- Ricardo López-Romero5,
- Edgar Román-Basaure7,
- Minerva Lazos-Ochoa6,
- Víctor Edén Montes de Oca-Fuentes4,
- Guelaguetza Vázquez-Ortiz†5, 8Email author and
- José Moreno†2Email author
© Juárez-Méndez et al.; licensee BioMed Central Ltd. 2013
Received: 28 May 2013
Accepted: 24 August 2013
Published: 5 September 2013
Studies of alternative mRNA splicing (AS) in health and disease have yet to yield the complete picture of protein diversity and its role in physiology and pathology. Some forms of cancer appear to be associated to certain alternative mRNA splice variants, but their role in the cancer development and outcome is unclear.
We examined AS profiles by means of whole genome exon expression microarrays (Affymetrix GeneChip 1.0) in ovarian tumors and ovarian cancer-derived cell lines, compared to healthy ovarian tissue. Alternatively spliced genes expressed predominantly in ovarian tumors and cell lines were confirmed by RT-PCR.
Among several significantly overexpressed AS genes in malignant ovarian tumors and ovarian cancer cell lines, the most significant one was that of the zinc finger protein ZNF695, with two previously unknown mRNA splice variants identified in ovarian tumors and cell lines. The identity of ZNF695 AS variants was confirmed by cloning and sequencing of the amplicons obtained from ovarian cancer tissue and cell lines.
Alternative ZNF695 mRNA splicing could be a marker of ovarian cancer with possible implications on its pathogenesis.
KeywordsOvarian cancer Alternative mRNA splicing ZNF695
Ovarian Cancer (OC) is the sixth most prevalent form of cancer worldwide, which has a high mortality rate because at the time of diagnosis nearly 70% of cases are at an advanced stage, leading to a 5 year survival below 30% . The classification of OC depends on its cellular origin, with approximately two-thirds belonging to the epithelial serous type . Similar to other types of cancer, OC is characterized by changes in gene expression profiles [3–6], including under and overexpression, or even de novo gene expression [7, 8].
Alternative splicing (AS) provides a critical and flexible layer of regulation, intervening in many biological processes, such as the diversity of proteins.
AS has major impact on the cell phenotype as a single pre-mRNA spliced in different ways can give rise to different mature mRNA transcripts (variants) that are translated onto distinct proteins varying in functions [9–11]. Apparently, over 90% of human genes have two or more splice variants [9, 10], greatly increasing the complexity of both the transcriptome and the proteome [12, 13]. Therefore, AS could play an important role in gene regulation both in health and disease. In cancer, AS could affect the cellular processes related to tumor progression, including inhibition of apoptosis, tumor invasiveness, metastasis and angiogenesis .
Among the genes with well established AS patterns whose derived alternative proteins affect tumor cell behavior is the SRPK1 kinase that in breast, colonic and pancreatic carcinomas phosphorylates the splicing factor SF2/ASF, allowing import to the nucleus, where it modulates AS of multiple target mRNAs, such as BIN1, S6K1, MNK2, contributing to tumor progression [15, 16]. Additional cancer types with apparent alterations of alternative splicing, include: gastric, colon and bladder carcinomas , hepatocarcinoma , prostatic cancer [19, 20], multiple myeloma , breast cancer, and OC , where most cancer-associated transcript variants belong to genes related to processes such as cellular transformation [23, 24], adhesion, proliferation, migration and invasion [25–28]. In OC, a new, previously unknown, variant of p53 mRNA transcript variant (p53δ) was identified  whereas variants of the NR4A1, a nuclear receptor involved in steroidogenesis, and MRRF, a mitochondrial protein, were identified in prostatic cancer . Although the extent and pathophysiological meaning of this has yet to be established, there is little doubt that the study of alternative splicing can lead to a better understanding of the mechanisms of cancer development, and to the identification of new biomarkers for the diagnosis, epidemiological studies of prevalence, prognosis, and therapeutic responses.
The aim of the present study was to identify the whole genome profile of alternatively spliced mRNA in ovarian cancer and cell lines by high-density microarrays. Among the spectrum of several ovarian cancer-associated alternatively spliced genes, one mRNA, coding for ZNF695, a zinc finger protein, had the most significantly overexpression in OC with two prominent splice variants that were not present in normal ovarian tissue. These variants were cloned and sequenced. Here we describe some of the characteristics of ZNF695 mRNA splicing variants associated to ovarian cancer.
Data set and specimens
All investigations were performed in accordance with the Declaration of Helsinki with approval by the Central Research Committee of the Mexican Institute of Social Security and The Ethics Committee of Centro Médico Siglo XXI, Mexican Institute of Social Security. After informed consent was obtained, normal ovarian tissue (HOT), borderline ovarian tumor (BOT), malignant epithelial ovarian tumors stages III and IV (MOT) tissues were collected by the clinical partners at the Oncology Hospital, National Medical Center Siglo XXI, IMSS, and at the General Hospital of Mexico SSA (Secretaría de Salud) from patients with diagnosed ovarian cancer, or healthy ovarian tissue from patients who underwent abdominal surgery for hysterectomy due to uterine myomatosis with no evidence of ovarian pathology. Routinely, during this type of procedure, in patients over 45 years old both ovaries are removed, and only one in patients under 45.
Cancer and corresponding normal tissue specimens were cut into three fragments and snapped frozen in liquid nitrogen, one of which was stored in RNA LatterR (Qiagen, Valencia, CA, USA) at −70°C for a maximum of two months until RNA was purified, and the other two remaining fragments were formalin-fixed, paraffin-embedded, sliced, mounted on slides, and stained with HE. Only tissue samples with >80% tumor cells or normal epithelial cells (MOT or HOT, respectively), according to the histopathological examination were included for analysis.
Moreover, we also included for study ovarian cancer cell lines (OCL) NIH: OVCAR-3 , SK-OV-3 , TOV-112D  and TOV-21G , kindly provided by Dr. Laura Díaz-Cueto, Research Unit on Reproductive Medicine, Instituto Mexicano del Seguro Social (IMSS).
Samples were disrupted using a TissueLyser™ system (Qiagen, Valencia, CA, USA) for 60s at 30 Hz. Total RNA was obtained with RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and total RNA concentration was quantified using a NanoDrop ND-1000 spectrophotometer and RNA quality was visualized and measured on an Agilent RNA 6000 Nano Assays in an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
Microarray GeneChip 1.0 assay
The microarray used for these studies was Affymetrix GeneChip 1.0, which contains over 750,000 probe sets representing all exons of ~28,800 annotated genes. Sample amplification and preparation for microarray hybridization was performed according to Affymetrix specifications (http://media.affymetrix.com/support/downloads/manuals/wt_expressionkit_manual.pdf). In brief, 100 ng total RNA was reverse transcribed to cDNA, amplified by in vitro transcription and reverse transcribed to cDNA again. Fragments between 40 and 70 bp were generated enzymatically, labelled and hybridized onto the microarray chips in an Affymetrix hybridization oven at 60 rpms, 45°C for 17 hours. Chips were washed according to the established protocols (Affymetrix, Santa Clara, CA, USA) with GeneChip fluidics station 450, and finally they were scanned with an Affymetrix 7G GeneChip scanner. The raw data (CEL files) will be deposited in Gene Expression Omnibus (GEO).
Microarray analysis was achieved by means of CEL files of the Partek Genomics Suite 6.5 v software (Partek Incorporated, Saint Louis, MO). Probe sets were summarized by means of Median Polish and normalized by quantiles with no probe sets excluded from analysis. Background noise correction was achieved by means Robust Multi-chip Average (RMA) and data were log2 transformed. Data grouping and categorization was achieved by principal component analysis (PCA). Differentially expressed exons were detected by means of Alternative Splicing ANOVA with the healthy control samples as the baseline. Moreover, BOT, MOT and OCL were also examined against HOT by the Geometric least squares means model. Hierarchical clustering was based on the dissimilarity of samples (Euclidian method) by means of average linkage.
Reverse transcription PCR
For linear cDNA synthesis, 1 μg total RNA was predigested with 1 U DNAse, 1 × DNAse buffer, 5 mM EDTA, after which it was incubated at 37°C for 30 min and at 65°C for an additional 10 min. Thereafter, samples were placed in master mix containing: 40 U Ribolock RNAse inhibitor, 0.2 μg random hexamer primers, 20 mM dNTP’s mix, 40 U M-Mulv reverse transcriptase (RT), and 1 × M-Mulv RT Buffer (Thermo Scientific).
Conditions for endpoint PCR amplification were: 5′ CGAATGAGAGCTGGCAAAGGCAAA 3′ Fwd., 5′ ACGCCAAGTGCCGTACAATTCATC 3′ Rev. primers (housekeeping gene RPL4) 7.5 mM, 1 × Taq buffer, 2 mM MgCl2, 0.4 mM dNTP’s, 1.25 U Taq Pol, 2 ul cDNA; whereas ZNF695 was amplified with primers: 5′ GCCTTTGTCTCCTTGCGGC 3′ Fwd. 5′ GGCTGTCTTCTCTGTGTTCACGTT 3′ Rev. 12.5 mM, 1 × Taq buffer, 3 mM MgCl2, 0.4 mM dNTP’s, 1.25 U Taq Pol, 2 ul cDNA. In both cases mix reactions were initially incubated at 95°C for 5 min, and then were run for 40 cycles at 94°C 45 s, 59°C 45 s, 72°C 60 s; and finally at 72°C for 5 min.
PCR product purification and cloning
PCR products were separated by electrophoresis (2.5% agarose gels) and extracted by means of Gel extraction kitTM (Qiagen, Valencia, CA, USA). The extracted products were ligated into pGem-T Easy Vector™ (Promega, Madinson, WI) by incubating overnight in 1.5 mL Eppendorf tubes with 2 × Rapid Ligation Buffer (T4 ligase), pGEM-T Easy Vector, PCR product and T4 DNA ligase at 4°C.
Recombinant plasmid DNA was purified with Wizard Plus Miniprep DNA Purification System™ (Promega) and selected clones were sequenced with M13 oligonucleotide and BigDye Terminator 3.1 cycle sequencing kit (Applied Biosystems), and sequenced in an Applied Biosystems Abi Prism 3130 genetic analyzer automated sequencer. Subsequently, the PCR amplicon sequences were assembled and checked against the transcript sequences annotated in the NCBI nucleotide database.
Expression microarray assays
A total of 14 samples with an RNA integrity number (RIN) ≥ 8 were hybridized in GeneChip 1.0 microarrays according to the MIAME guidelines. Histopathological classification of tissues was as follows: healthy ovarian tissue (HOT) n = 4, benign ovarian tumors (BOT) n = 2, (malignant) serous epithelial ovarian tumors in stages III and IV (MOT) n = 4, and ovarian cell lines (OCL) n = 4. As a prerequisite, healthy tissue had to be free of any visible alteration, whereas all tumor tissues, benign or malignant, selected for study contained at least 90% tumor cells.
Gene expression differences among groups
BOT vs. HOT
MOT vs. HOT
OCL vs. HOT
MOT vs. BOT
OCL vs. BOT
OCL vs. MOT
BOT + MOT + OCL vs. HOT
MOT + OCL vs. HOT
MOT + OCL vs. BOT
HOT + MOT + OCL vs. BOT
MOT + OCL vs. BOT + HOT
Exon analysis identifies two major ovarian cancer-associated, differentially spliced transcripts of gene ZNF695
Once we had examined the relative OC-associated gene expression profiles, it was important to examine whether some of the overexpressed genes reflected only quantitative differences or if there were also qualitative differences among them. To achieve this, we performed exon analysis of genes overexpressed in both MOT and OCL. As differential exon usage cannot be easily examined in suppressed genes, we exclusively examined overexpressed genes.
Main genes whit potential alternative splicing
# of markers
Fold-Change(OCL and MOT vs. BOT and HOT)
ZNF695 splice variants in OC
ZNF695 encode a zinc finger protein with as yet unknown functions and its gene contains six exons located in chromosome 1q cytogenetic positions 247,148,625-247,171,358. This gene has six possible transcripts of which two (ZNF695-003, and ZNF695-006) encode complete ORFs yielding a 515 and a 172 amino acid length proteins, respectively; whereas the other four transcripts encode products thought to undergo nonsense-mediated decay, a process that detects nonsense mutations and prevents the expression of truncated transcripts (http://www.ensembl.org/Human/Search/Results?q=ZNF695;site=ensembl;facet_species=Human).
Understanding the origin of malignancy is one of the greater challenges of modern science. Among malignant tumors, OC represents a major problem because little is known about its pathogenesis, which is also difficult to identify in early stages as it goes asymptomatic over long periods of time to be detectable only in late stages, almost always beyond any possibility of remission [33, 34]. Alternative exon splicing is a biological process of major importance, because gene changes leading to altered splicing can affect normal cell and tissue function [19, 20, 27, 35], including malignant transformation . The current studies were carried out to examine whether OC could be associated to particular exon-splicing state and if so, to identify differentially spliced transcripts present in OC but absent in healthy ovarian tissue. With the exon array data set presented here, we identified nine overexpressed genes with differential exon profiles associated to OC, one such gene, ZNF695, coding for a largely uncharacterized zinc finger protein, is the most representative, with three transcripts differentially expressed by MOT and OCL, one corresponding to the whole protein, a second ORF corresponding to a shorter peptide and a third, with lower but significant expression that corresponds to a long non-coding mRNA. These results likely provide a useful biomarker of malignant transformation in women suspected to have OC and open the study of the role of these transcripts in cell proliferation and malignant transformation.
Alternative splicing is a major source of protein diversity, bioinformatics-based methods indicate that >90% human genes could be subject to AS [10, 12, 13] with an estimate of several million different proteins, and some individual proteins having over 1000 variants due only to AS . This process can differ during distinct cellular functional or developmental stages [38, 39]. It is, therefore, not surprising that AS has also been found altered during malignant transformation , which could be either a general marker of cancer or limited to certain cancer types. Moreover, cancer-associated AS could be the clue to understand the basis of malignant transformation, tumor behavior [23, 41], and even for the identification of potential therapeutic targets .
We found that OC tissue has indeed a signature of alternatively spliced genes. Although we do not know yet whether these changes are indeed related only to OC or they are general markers of cancer. Of the >270 differentially spliced genes found in OC, nine were highly significant, but we decided to focus on the most significantly expressed gene with differential AS, the zinc finger protein ZNF695.
The zinc finger protein family (ZNF) spans over 700 members with many functional roles within the cell, including regulation of gene expression, which is achieved by different means. For instance, ZNF transcription factors bind to DNA by means of C2H2 zinc finger domains, constituting a subfamily of ZNF . Although some ZNF members act only as repressors, others solely as act as activators, most of them can apparently be either repressors or activators depending on the particular status of the cell. Moreover, some ZNF play roles in signal transduction and many other cellular functions. ZNF695, which we found here to be differentially expressed and spliced in OC belongs to the C2H2 subfamily of ZNF and also contains Krüpple-associated box (KRAB) domains, which characteristically identify gene repressors [44–46]. Because these genes, including ZNF695 contain two or more functional domains, changes affecting only one domain can have dramatic consequences [45, 47]. On one hand, repressors could lose their regulatory function or even turn in the opposite direction and become activators . KRAB domains in ZNF proteins serve to bind co-repressors, which in turn mediate transcription repression . Of the ZNF695 splice variants we found here to be preferentially associated to OC, isoforms 2 and 3 have incomplete KRAB domains that are essential for interactions with co-repressors which suggests that such AS pattern could be related to carcinogenesis. The third variant lacked the initial translation codon; hence, it is unlikely to yield a translational product.
Unfortunately, as yet, almost nothing is known about ZNF695 in humans or in other species, with the closest homologues having up to 64% identity. Therefore, at present it is not possible to predict how ZNF695 could play a role in OC development, if at all. One could envision that because the alternative forms found in OC have incomplete KRAB domains, this potential repressor could function as an activator and turn on cell proliferation and, hence, malignant transformation. The other possibility would be to function as dominant negative variants, but this seems unlikely because normal ovarian tissue does not express ZNF695 in any of its isoforms ad OC cells express only the alternative splice variants. Therefore, we consider ZNF695 splice variants 1/2, 4 and 5 as potential oncogenes playing a role in the pathogenesis of OC.
Health ovarian tissue
Borderline ovarian tumor
Malignant ovarian tumor
Ovarian cell lines
Principal component analysis
False discovery ratio
This work was supported in part by a Basic Science grant 61742 form SEP-CONACyT México. Sergio Juárez-Méndez was financially supported by a scholarship provided by the National Council of Science and Technology (CONACyT) and IMSS. This work constitutes a partial fulfillment of the graduate program in PhD Biological Science of National Autonomous University of México (UNAM) of Sergio Juárez-Méndez. We finally thank Dr. Carlos Pérez-Plasencia, Faculty of Superior Studies, Ixtacala, UNAM for critical review of the manuscript and Dr. Laura Díaz-Cueto, Research Unit on Reproductive Medicine, Instituto Mexicano del Seguro Social (IMSS) for kindly providing cell lines.
- Bast RC Jr, Hennessy B, Mills GB: The biology of ovarian cancer: new opportunities for translation. Nat Rev Cancer 2009, 9: 415–428. 10.1038/nrc2644PubMed CentralPubMedView ArticleGoogle Scholar
- Scully RE: Classification of human ovarian tumors. Env Health Perspect 1987, 73: 15–25.View ArticleGoogle Scholar
- Lancaster JM, Dressman HK, Clarke JP, Sayer RA, Martino MA, Cragun JM, Henriott AH, Gray J, Sutphen R, Elahi A, et al.: Identification of genes associated with ovarian cancer metastasis using microarray expression analysis. Int J Gynecol Cancer 2006, 16: 1733–1745. 10.1111/j.1525-1438.2006.00660.xPubMedView ArticleGoogle Scholar
- Maxwell GL, Chandramouli GV, Dainty L, Litzi TJ, Berchuck A, Barrett JC, Risinger JI: Microarray analysis of endometrial carcinomas and mixed mullerian tumors reveals distinct gene expression profiles associated with different histologic types of uterine cancer. Clin Cancer Res 2005, 11: 4056–4066. 10.1158/1078-0432.CCR-04-2001PubMedView ArticleGoogle Scholar
- Tinker AV, Boussioutas A, Bowtell DD: The challenges of gene expression microarrays for the study of human cancer. Cancer Cell 2006, 9: 333–339. 10.1016/j.ccr.2006.05.001PubMedView ArticleGoogle Scholar
- Zorn KK, Bonome T, Gangi L, Chandramouli GV, Awtrey CS, Gardner GJ, Barrett JC, Boyd J, Birrer MJ: Gene expression profiles of serous, endometrioid, and clear cell subtypes of ovarian and endometrial cancer. Clin Cancer Res 2005, 11: 6422–6430. 10.1158/1078-0432.CCR-05-0508PubMedView ArticleGoogle Scholar
- Keita M, Bachvarova M, Morin C, Plante M, Gregoire J, Renaud MC, Sebastianelli A, Trinh XB, Bachvarov D: The RUNX1 transcription factor is expressed in serous epithelial ovarian carcinoma and contributes to cell proliferation, migration and invasion. Cell Cycle 2013, 12: 972–986. 10.4161/cc.23963PubMed CentralPubMedView ArticleGoogle Scholar
- Treeck O, Schuler S, Haring J, Skrzypczak M, Lattrich C, Ortmann O: icb-1 Gene counteracts growth of ovarian cancer cell lines. Cancer Lett 2013, 335: 441–446. 10.1016/j.canlet.2013.02.049PubMedView ArticleGoogle Scholar
- Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K, Dewar K, Doyle M, FitzHugh W, et al.: Initial sequencing and analysis of the human genome. Nature 2001, 409: 860–921. 10.1038/35057062PubMedView ArticleGoogle Scholar
- Modrek B, Resch A, Grasso C, Lee C: Genome-wide detection of alternative splicing in expressed sequences of human genes. Nucleic Acids Res 2001, 29: 2850–2859. 10.1093/nar/29.13.2850PubMed CentralPubMedView ArticleGoogle Scholar
- Graveley BR: Alternative splicing: increasing diversity in the proteomic world. Trends Genet 2001, 17: 100–107. 10.1016/S0168-9525(00)02176-4PubMedView ArticleGoogle Scholar
- Carninci P: Constructing the landscape of the mammalian transcriptome. J Exp Biol 2007, 210: 1497–1506. 10.1242/jeb.000406PubMedView ArticleGoogle Scholar
- Schmucker D, Clemens JC, Shu H, Worby CA, Xiao J, Muda M, Dixon JE, Zipursky SL: Drosophila Dscam is an axon guidance receptor exhibiting extraordinary molecular diversity. Cell 2000, 101: 671–684. 10.1016/S0092-8674(00)80878-8PubMedView ArticleGoogle Scholar
- Venables JP: Unbalanced alternative splicing and its significance in cancer. Bioessays 2006, 28: 378–386. 10.1002/bies.20390PubMedView ArticleGoogle Scholar
- Hayes GM, Carrigan PE, Miller LJ: Serine-arginine protein kinase 1 overexpression is associated with tumorigenic imbalance in mitogen-activated protein kinase pathways in breast, colonic, and pancreatic carcinomas. Cancer Res 2007, 67: 2072–2080. 10.1158/0008-5472.CAN-06-2969PubMedView ArticleGoogle Scholar
- Akgul C, Moulding DA, Edwards SW: Alternative splicing of Bcl-2-related genes: functional consequences and potential therapeutic applications. Cell Mol Life Sci 2004, 61: 2189–2199.PubMedView ArticleGoogle Scholar
- Chen LL, Sabripour M, Wu EF, Prieto VG, Fuller GN, Frazier ML: A mutation-created novel intra-exonic pre-mRNA splice site causes constitutive activation of KIT in human gastrointestinal stromal tumors. Oncogene 2005, 24: 4271–4280. 10.1038/sj.onc.1208587PubMedView ArticleGoogle Scholar
- Wang XQ, Luk JM, Leung PP, Wong BW, Stanbridge EJ, Fan ST: Alternative mRNA splicing of liver intestine-cadherin in hepatocellular carcinoma. Clin Cancer Res 2005, 11: 483–489.PubMedGoogle Scholar
- Narla G, Difeo A, Reeves HL, Schaid DJ, Hirshfeld J, Hod E, Katz A, Isaacs WB, Hebbring S, Komiya A, et al.: A germline DNA polymorphism enhances alternative splicing of the KLF6 tumor suppressor gene and is associated with increased prostate cancer risk. Cancer Res 2005, 65: 1213–1222. 10.1158/0008-5472.CAN-04-4249PubMedView ArticleGoogle Scholar
- Thorsen K, Sorensen KD, Brems-Eskildsen AS, Modin C, Gaustadnes M, Hein AM, Kruhoffer M, Laurberg S, Borre M, Wang K, et al.: Alternative splicing in colon, bladder, and prostate cancer identified by exon array analysis. Mol Cell Proteomics 2008, 7: 1214–1224. 10.1074/mcp.M700590-MCP200PubMedView ArticleGoogle Scholar
- Adamia S, Reiman T, Crainie M, Mant MJ, Belch AR, Pilarski LM: Intronic splicing of hyaluronan synthase 1 (HAS1): a biologically relevant indicator of poor outcome in multiple myeloma. Blood 2005, 105: 4836–4844. 10.1182/blood-2004-10-3825PubMed CentralPubMedView ArticleGoogle Scholar
- Mazoyer S, Puget N, Perrin-Vidoz L, Lynch HT, Serova-Sinilnikova OM, Lenoir GM: A BRCA1 nonsense mutation causes exon skipping. Am J Hum Genet 1998, 62: 713–715. 10.1086/301768PubMed CentralPubMedView ArticleGoogle Scholar
- Singh A, Karnoub AE, Palmby TR, Lengyel E, Sondek J, Der CJ: Rac1b, a tumor associated, constitutively active Rac1 splice variant, promotes cellular transformation. Oncogene 2004, 23: 9369–9380. 10.1038/sj.onc.1208182PubMedView ArticleGoogle Scholar
- Zhou YQ, He C, Chen YQ, Wang D, Wang MH: Altered expression of the RON receptor tyrosine kinase in primary human colorectal adenocarcinomas: generation of different splicing RON variants and their oncogenic potential. Oncogene 2003, 22: 186–197. 10.1038/sj.onc.1206075PubMedView ArticleGoogle Scholar
- Bauer TW, Fan F, Liu W, Johnson M, Parikh NU, Parry GC, Callahan J, Mazar AP, Gallick GE, Ellis LM: Insulinlike growth factor-I-mediated migration and invasion of human colon carcinoma cells requires activation of c-Met and urokinase plasminogen activator receptor. Ann Surg 2005, 241: 748–756. discussion 756–748 10.1097/01.sla.0000160699.59061.92PubMed CentralPubMedView ArticleGoogle Scholar
- Brembeck FH, Rosario M, Birchmeier W: Balancing cell adhesion and Wnt signaling, the key role of beta-catenin. Curr Opin Genet Dev 2006, 16: 51–59. 10.1016/j.gde.2005.12.007PubMedView ArticleGoogle Scholar
- Cheng C, Sharp PA: Regulation of CD44 alternative splicing by SRm160 and its potential role in tumor cell invasion. Mol Cell Biol 2006, 26: 362–370. 10.1128/MCB.26.1.362-370.2006PubMed CentralPubMedView ArticleGoogle Scholar
- Wong MP, Cheung N, Yuen ST, Leung SY, Chung LP: Vascular endothelial growth factor is up-regulated in the early pre-malignant stage of colorectal tumour progression. Int J Cancer 1999, 81: 845–850. 10.1002/(SICI)1097-0215(19990611)81:6<845::AID-IJC1>3.0.CO;2-5PubMedView ArticleGoogle Scholar
- Hofstetter G, Berger A, Fiegl H, Slade N, Zoric A, Holzer B, Schuster E, Mobus VJ, Reimer D, Daxenbichler G, et al.: Alternative splicing of p53 and p73: the novel p53 splice variant p53delta is an independent prognostic marker in ovarian cancer. Oncogene 2010, 29: 1997–2004. 10.1038/onc.2009.482PubMedView ArticleGoogle Scholar
- Hamilton TC, Young RC, McKoy WM, Grotzinger KR, Green JA, Chu EW, Whang-Peng J, Rogan AM, Green WR, Ozols RF: Characterization of a human ovarian carcinoma cell line (NIH: OVCAR-3) with androgen and estrogen receptors. Cancer Res 1983, 43: 5379–5389.PubMedGoogle Scholar
- Fogh J, Wright WC, Loveless JD: Absence of HeLa cell contamination in 169 cell lines derived from human tumors. J Natl Cancer Inst 1977, 58: 209–214.PubMedGoogle Scholar
- Provencher DM, Lounis H, Champoux L, Tetrault M, Manderson EN, Wang JC, Eydoux P, Savoie R, Tonin PN, Mes-Masson AM: Characterization of four novel epithelial ovarian cancer cell lines. In vitro Cell Dev Biol Anim 2000, 36: 357–361. 10.1290/1071-2690(2000)036<0357:COFNEO>2.0.CO;2PubMedView ArticleGoogle Scholar
- Hoskins WJ: Prospective on ovarian cancer: why prevent? J Cell Biochem Suppl 1995, 23: 189–199.PubMedView ArticleGoogle Scholar
- Nguyen HN, Averette HE, Hoskins W, Sevin BU, Penalver M, Steren A: National survey of ovarian carcinoma. VI. Critical assessment of current International Federation of Gynecology and Obstetrics staging system. Cancer 1993, 72: 3007–3011. 10.1002/1097-0142(19931115)72:10<3007::AID-CNCR2820721024>3.0.CO;2-NPubMedView ArticleGoogle Scholar
- Venables JP, Klinck R, Koh C, Gervais-Bird J, Bramard A, Inkel L, Durand M, Couture S, Froehlich U, Lapointe E, et al.: Cancer-associated regulation of alternative splicing. Nat Struct Mol Biol 2009, 16: 670–676. 10.1038/nsmb.1608PubMedView ArticleGoogle Scholar
- Zardi L, Carnemolla B, Siri A, Petersen TE, Paolella G, Sebastio G, Baralle FE: Transformed human cells produce a new fibronectin isoform by preferential alternative splicing of a previously unobserved exon. EMBO J 1987, 6: 2337–2342.PubMed CentralPubMedGoogle Scholar
- Missler M, Sudhof TC: Neurexins: three genes and 1001 products. Trends Genet 1998, 14: 20–26. 10.1016/S0168-9525(97)01324-3PubMedView ArticleGoogle Scholar
- Joseph R, Dou D, Tsang W: Neuronatin mRNA: alternatively spliced forms of a novel brain-specific mammalian developmental gene. Brain Res 1995, 690: 92–98. 10.1016/0006-8993(95)00621-VPubMedView ArticleGoogle Scholar
- Chen CD, Kobayashi R, Helfman DM: Binding of hnRNP H to an exonic splicing silencer is involved in the regulation of alternative splicing of the rat beta-tropomyosin gene. Genes Dev 1999, 13: 593–606. 10.1101/gad.13.5.593PubMed CentralPubMedView ArticleGoogle Scholar
- Merdzhanova G, Gout S, Keramidas M, Edmond V, Coll JL, Brambilla C, Brambilla E, Gazzeri S, Eymin B: The transcription factor E2F1 and the SR protein SC35 control the ratio of pro-angiogenic versus antiangiogenic isoforms of vascular endothelial growth factor-A to inhibit neovascularization in vivo . Oncogene 2010, 29: 5392–5403. 10.1038/onc.2010.281PubMedView ArticleGoogle Scholar
- Guo M, Liu W, Serra S, Asa SL, Ezzat S: FGFR2 isoforms support epithelial-stromal interactions in thyroid cancer progression. Cancer Res 2012, 72: 2017–2027. 10.1158/0008-5472.CAN-11-3985PubMedView ArticleGoogle Scholar
- Dery KJ, Gusti V, Gaur S, Shively JE, Yen Y, Gaur RK: Alternative splicing as a therapeutic target for human diseases. Methods Mol Biol 2009, 555: 127–144. 10.1007/978-1-60327-295-7_10PubMed CentralPubMedView ArticleGoogle Scholar
- Krishna SS, Majumdar I, Grishin NV: Structural classification of zinc fingers: survey and summary. Nucleic Acids Res 2003, 31: 532–550. 10.1093/nar/gkg161PubMed CentralPubMedView ArticleGoogle Scholar
- Friedman JR, Fredericks WJ, Jensen DE, Speicher DW, Huang XP, Neilson EG, Rauscher FJ 3rd: KAP-1, a novel corepressor for the highly conserved KRAB repression domain. Genes Dev 1996, 10: 2067–2078. 10.1101/gad.10.16.2067PubMedView ArticleGoogle Scholar
- Vissing H, Meyer WK, Aagaard L, Tommerup N, Thiesen HJ: Repression of transcriptional activity by heterologous KRAB domains present in zinc finger proteins. FEBS Lett 1995, 369: 153–157. 10.1016/0014-5793(95)00728-RPubMedView ArticleGoogle Scholar
- Pengue G, Calabro V, Bartoli PC, Pagliuca A, Lania L: Repression of transcriptional activity at a distance by the evolutionarily conserved KRAB domain present in a subfamily of zinc finger proteins. Nucleic Acids Res 1994, 22: 2908–2914. 10.1093/nar/22.15.2908PubMed CentralPubMedView ArticleGoogle Scholar
- Agata Y, Matsuda E, Shimizu A: Two novel Kruppel-associated box-containing zinc-finger proteins, KRAZ1 and KRAZ2, repress transcription through functional interaction with the corepressor KAP-1 (TIF1beta/KRIP-1). J Biol Chem 1999, 274: 16412–16422. 10.1074/jbc.274.23.16412PubMedView ArticleGoogle Scholar
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