title: Expression of an estrogen-regulated variant transcript of the peroxisomal branched chain fatty acid oxidase ACOX2 in breast carcinomas.
abstract: BACKGROUND
  Alternate transcripts from a single gene locus greatly enhance the combinatorial flexibility of the human transcriptome. Different patterns of exon usage have been observed when comparing normal tissue to cancers, suggesting that variant transcripts may play a role in the tumor phenotype.
  
  
  METHODS
  Ribonucleic acid-sequencing (RNA-seq) data from breast cancer samples was used to identify an intronic start variant transcript of Acyl-CoA oxidase 2, ACOX2 (ACOX2-i9). Difference in expression between Estrogen Receptor (ER) positive and ER negative patients was assessed by the Wilcoxon rank sum test, and the findings validated in The Cancer Genome Atlas (TCGA) breast cancer dataset (BRCA). ACOX2-i9 expression was also assessed in cell lines using both quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and Western blot analysis. Knock down by short hairpin RNA (shRNA) and colony formation assays were used to determine whether ACOX2-i9 expression would influence cellular fitness. The effect of ACOX2-i9 expression on patient survival was assessed by the Kaplan-Meier survival function, and association to clinical parameters was analyzed using a Fisher exact test.
  
  
  RESULTS
  The expression and translation of ACOX2-i9 into a 25 kDa protein was demonstrated in HepG2 cells as well as in several breast cancer cell lines. shRNA knock down of the ACOX2-i9 variant resulted in decreased cell viability of T47D and MDA-MB 436 cells. Moreover, expression of ACOX2-i9 was shown to be estrogen regulated, being induced by propyl pyrazoletriol and inhibited by tamoxifen and fulvestrant in ER+ T47D and Mcf-7 cells, but not in the ER- MDA-MB 436 cell line. This variant transcript showed expression predominantly in ER-positive breast tumors as assessed in our initial set of 53 breast cancers and further validated in 87 tumor/normal pairs from the TCGA breast cancer dataset, and expression was associated with better outcome in ER positive patients.
  
  
  CONCLUSIONS
  ACOX2-i9 is specifically enriched in ER+ breast cancers where expression of the variant is associated with improved outcome. These data identify variant ACOX2 as a potential novel therapeutic biomarker in ER+ breast tumors.
authors.affiliation: Department of Genetics, Institute for Cancer Research, Oslo University Hospital Radiumospitalet, Montebello, 0310, Oslo, Norway. sunniva.bjorklund@gmail.com.|Rutgers Cancer Institute of New Jersey, 195 Little Albany Street, New Brunswick, NJ, , 08903, USA. sunniva.bjorklund@gmail.com.|The K.G. Jebsen Center for Breast Cancer Research, Institute for Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway. sunniva.bjorklund@gmail.com.
authors.forename: Sunniva Stordal
authors.initials: SS
authors.lastname: Bjørklund
authors.affiliation: Department of Genetics, Institute for Cancer Research, Oslo University Hospital Radiumospitalet, Montebello, 0310, Oslo, Norway. v.n.kristensen@medisin.uio.no.|The K.G. Jebsen Center for Breast Cancer Research, Institute for Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway. v.n.kristensen@medisin.uio.no.|Department of Clinical Molecular Biology and Laboratory Science (EpiGen), Akershus University hospital, Division of Medicine, 1476, Lørenskog, Norway. v.n.kristensen@medisin.uio.no.
authors.forename: Vessela N
authors.initials: VN
authors.lastname: Kristensen
authors.affiliation: Center for Advanced Genomic Technology, Boston University, Boston, MA, 02215, USA. miseiler@gmail.com.
authors.forename: Michael
authors.initials: M
authors.lastname: Seiler
authors.affiliation: Department of Genetics, Institute for Cancer Research, Oslo University Hospital Radiumospitalet, Montebello, 0310, Oslo, Norway. surendra.kumar@ibv.uio.no.|Department of Clinical Molecular Biology and Laboratory Science (EpiGen), Akershus University hospital, Division of Medicine, 1476, Lørenskog, Norway. surendra.kumar@ibv.uio.no.
authors.forename: Surendra
authors.initials: S
authors.lastname: Kumar
authors.affiliation: Department of Genetics, Institute for Cancer Research, Oslo University Hospital Radiumospitalet, Montebello, 0310, Oslo, Norway. Grethe.I.Grenaker.Alnas@rr-research.no.
authors.forename: Grethe I Grenaker
authors.initials: GI
authors.lastname: Alnæs
authors.affiliation: Rutgers Cancer Institute of New Jersey, 195 Little Albany Street, New Brunswick, NJ, , 08903, USA. yaomi@cinj.rutgers.edu.
authors.forename: Yao
authors.initials: Y
authors.lastname: Ming
authors.affiliation: Rutgers Cancer Institute of New Jersey, 195 Little Albany Street, New Brunswick, NJ, , 08903, USA. kerrigje@cinj.rutgers.edu.
authors.forename: John
authors.initials: J
authors.lastname: Kerrigan
authors.affiliation: The K.G. Jebsen Center for Breast Cancer Research, Institute for Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway. bna@ous-hf.no.|Department of Oncology, Oslo University Hospital, Radiumhospitalet, Oslo, Norway. bna@ous-hf.no.
authors.forename: Bjørn
authors.initials: B
authors.lastname: Naume
authors.affiliation: Department of Oncological Sciences, Mount Sinai School of Medicine, New York, NY, , 10029, USA. ravi.mssm@gmail.com.
authors.forename: Ravi
authors.initials: R
authors.lastname: Sachidanandam
authors.affiliation: Rutgers Cancer Institute of New Jersey, 195 Little Albany Street, New Brunswick, NJ, , 08903, USA. gyanbhanot@gmail.com.|Department of Molecular Biology & Biochemistry, Rutgers University, Piscataway, NJ, , 08854, USA. gyanbhanot@gmail.com.
authors.forename: Gyan
authors.initials: G
authors.lastname: Bhanot
authors.affiliation: Department of Genetics, Institute for Cancer Research, Oslo University Hospital Radiumospitalet, Montebello, 0310, Oslo, Norway. a.l.borresen-dale@medisin.uio.no.|The K.G. Jebsen Center for Breast Cancer Research, Institute for Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway. a.l.borresen-dale@medisin.uio.no.
authors.forename: Anne-Lise
authors.initials: AL
authors.lastname: Børresen-Dale
authors.affiliation: Rutgers Cancer Institute of New Jersey, 195 Little Albany Street, New Brunswick, NJ, , 08903, USA. ganesash@cinj.rutgers.edu.
authors.forename: Shridar
authors.initials: S
authors.lastname: Ganesan
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D045744:Cell Line, Tumor; D002470:Cell Survival; D018387:Codon, Initiator; D004958:Estradiol / Q000031:analogs & derivatives / Q000494:pharmacology; D005260:Female; D000077267:Fulvestrant; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D014644:Genetic Variation; D057809:HEK293 Cells; D006367:HeLa Cells; D056945:Hep G2 Cells; D006801:Humans; D061986:MCF-7 Cells; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D010636:Phenols / Q000494:pharmacology; D011379:Prognosis; D011720:Pyrazoles / Q000494:pharmacology; D011960:Receptors, Estrogen / Q000378:metabolism*; D017423:Sequence Analysis, RNA / Q000379:methods*; D016019:Survival Analysis; D013629:Tamoxifen / Q000494:pharmacology
chemical_list: D018387:Codon, Initiator; D010636:Phenols; D011720:Pyrazoles; D011960:Receptors, Estrogen; D013629:Tamoxifen; C486184:4,4',4''-(4-propyl-((1)H)-pyrazole-1,3,5-triyl) tris-phenol; D000077267:Fulvestrant; D004958:Estradiol; D010088:Oxidoreductases; C576470:ACOX2 protein, human