title: High levels of the AR-V7 Splice Variant and Co-Amplification of the Golgi Protein Coding YIPF6 in AR Amplified Prostate Cancer Bone Metastases.
abstract: BACKGROUND
  The relation between androgen receptor (AR) gene amplification and other mechanisms behind castration-resistant prostate cancer (CRPC), such as expression of constitutively active AR variants and steroid-converting enzymes has been poorly examined. Specific aim was to examine AR amplification in PC bone metastases and to explore molecular and functional consequences of this, with the long-term goal of identifying novel molecular targets for treatment.
  
  
  METHODS
  Gene amplification was assessed by fluorescence in situ hybridization in cryo-sections of clinical PC bone metastases (n = 40) and by PCR-based copy number variation analysis. Whole genome mRNA expression was analyzed using H12 Illumina Beadchip arrays and specific transcript levels were quantified by qRT-PCR. Protein localization was analyzed using immunohistochemistry and confocal microscopy. The YIPF6 mRNA expression was transiently knocked down and stably overexpressed in the 22Rv1 cell line as representative for CRPC, and effects on cell proliferation, colony formation, migration, and invasion were determined in vitro. Extracellular vesicles (EVs) were isolated from cell cultures using size-exclusion chromatography and enumerated by nanoparticle tracking analysis. Protein content was identified by LC-MS/MS analysis. Blood coagulation was measured as activated partial thromboplastin time (APTT). Functional enrichment analysis was performed using the MetaCore software.
  
  
  RESULTS
  AR amplification was detected in 16 (53%) of the bone metastases examined from CRPC patients (n = 30), and in none from the untreated patients (n = 10). Metastases with AR amplification showed high AR and AR-V7 mRNA levels, increased nuclear AR immunostaining, and co-amplification of genes such as YIPF6 in the AR proximity at Xq12. The YIPF6 protein was localized to the Golgi apparatus. YIPF6 overexpression in 22Rv1 cells resulted in reduced cell proliferation and colony formation, and in enhanced EV secretion. EVs from YIPF6 overproducing 22Rv1 cells were enriched for proteins involved in blood coagulation and, accordingly, decreased the APTT in a dose-dependent fashion.
  
  
  CONCLUSIONS
  AR amplified CRPC bone metastases show high AR-V7 expression that probably gives resistance to AR-targeting drugs. Co-amplification of the Golgi protein coding YIPF6 gene with the AR may enhance the secretion of pro-coagulative EVs from cancer cells and thereby stimulate tumor progression and increase the coagulopathy risk in CRPC patients. Prostate 77: 625-638, 2017. © 2017 Wiley Periodicals, Inc.
authors.affiliation: Department of Medical Biosciences, Pathology, Umeå University, Umeå, Sweden.
authors.forename: Erik
authors.initials: E
authors.lastname: Djusberg
authors.affiliation: Department of Medical Biosciences, Pathology, Umeå University, Umeå, Sweden.
authors.forename: Emma
authors.initials: E
authors.lastname: Jernberg
authors.affiliation: Department of Medical Biosciences, Pathology, Umeå University, Umeå, Sweden.
authors.forename: Elin
authors.initials: E
authors.lastname: Thysell
authors.affiliation: Department of Medical Biosciences, Medical and Clinical Genetics, Umeå University, Umeå, Sweden.
authors.forename: Irina
authors.initials: I
authors.lastname: Golovleva
authors.affiliation: Department of Medical Biosciences, Medical and Clinical Genetics, Umeå University, Umeå, Sweden.
authors.forename: Pia
authors.initials: P
authors.lastname: Lundberg
authors.affiliation: Department of Surgical and Perioperative Sciences, Orthopedics, Umeå University, Umeå, Sweden.
authors.forename: Sead
authors.initials: S
authors.lastname: Crnalic
authors.affiliation: Department of Radiation Sciences, Oncology, Umeå University, Umeå, Sweden.
authors.forename: Anders
authors.initials: A
authors.lastname: Widmark
authors.affiliation: Department of Medical Biosciences, Pathology, Umeå University, Umeå, Sweden.
authors.forename: Anders
authors.initials: A
authors.lastname: Bergh
authors.affiliation: Department of Medical Biosciences, Pathology, Umeå University, Umeå, Sweden.
authors.forename: Maria
authors.initials: M
authors.lastname: Brattsand
authors.affiliation: Department of Medical Biosciences, Pathology, Umeå University, Umeå, Sweden.
authors.forename: Pernilla
authors.initials: P
authors.lastname: Wikström
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D001859:Bone Neoplasms / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000502:physiology; D005784:Gene Amplification / Q000502:physiology; D006056:Golgi Apparatus / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D008875:Middle Aged; D064129:Prostatic Neoplasms, Castration-Resistant / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000096:biosynthesis* / Q000235:genetics; D011944:Receptors, Androgen / Q000096:biosynthesis* / Q000235:genetics
chemical_list: C410798:AR protein, human; D008565:Membrane Proteins; D020033:Protein Isoforms; D011944:Receptors, Androgen
keywords: AKR1C3; androgen receptor variant 7; blood coagulation; exosomes; extracellular vesicles