title: A functional proteogenomic analysis of endometrioid and clear cell carcinomas using reverse phase protein array and mutation analysis: protein expression is histotype-specific and loss of ARID1A/BAF250a is associated with AKT phosphorylation.
abstract: BACKGROUND
  Ovarian cancer is now recognized as a number of distinct diseases primarily defined by histological subtype. Both clear cell ovarian carcinomas (CCC) and ovarian endometrioid carcinomas (EC) may arise from endometriosis and frequently harbor mutations in the ARID1A tumor suppressor gene. We studied the influence of histological subtype on protein expression with reverse phase protein array (RPPA) and assessed proteomic changes associated with ARID1A mutation/BAF250a expression in EC and CCC.
  
  
  METHODS
  Immunohistochemistry (IHC) for BAF250a expression was performed on 127 chemotherapy-naive ovarian carcinomas (33 CCC, 29 EC, and 65 high-grade serous ovarian carcinomas (HGSC)). Whole tumor lysates were prepared from frozen banked tumor samples and profiled by RPPA using 116 antibodies. ARID1A mutations were identified by exome sequencing, and PIK3CA mutations were characterized by MALDI-TOF mass spectrometry. SAM (Significance Analysis of Microarrays) was performed to determine differential protein expression by histological subtype and ARID1A mutation status. Multivariate logistic regression was used to assess the impact of ARID1A mutation status/BAF250a expression on AKT phosphorylation (pAKT). PIK3CA mutation type and PTEN expression were included in the model. BAF250a knockdown was performed in 3 clear cell lines using siRNA to ARID1A.
  
  
  RESULTS
  Marked differences in protein expression were observed that are driven by histotype. Compared to HGSC, SAM identified over 50 proteins that are differentially expressed in CCC and EC. These included PI3K/AKT pathway proteins, those regulating the cell cycle, apoptosis, transcription, and other signaling pathways including steroid hormone signaling. Multivariate models showed that tumors with loss of BAF250a expression showed significantly higher levels of AKT-Thr308 and AKT-Ser473 phosphorylation (p < 0.05). In 31 CCC cases, pAKT was similarly significantly increased in tumors with BAF250a loss on IHC. Knockdown of BAF250a by siRNA in three CCC cell lines wild type for ARID1A showed no increase in either pAKT-Thr308 or pAKT-S473 suggesting that pAKT in tumor tissues is indirectly regulated by BAF250a expression.
  
  
  CONCLUSIONS
  Proteomic assessment of CCC and EC demonstrates remarkable differences in protein expression that are dependent on histotype, thereby further characterizing these cancers. AKT phosphorylation is associated with ARID1A/BAF250a deficient tumors, however in ovarian cancers the mechanism remains to be elucidated.
authors.forename: Kimberly C
authors.initials: KC
authors.lastname: Wiegand
authors.forename: Bryan T
authors.initials: BT
authors.lastname: Hennessy
authors.forename: Samuel
authors.initials: S
authors.lastname: Leung
authors.forename: Yemin
authors.initials: Y
authors.lastname: Wang
authors.forename: Zhenlin
authors.initials: Z
authors.lastname: Ju
authors.forename: Mollianne
authors.initials: M
authors.lastname: McGahren
authors.forename: Steve E
authors.initials: SE
authors.lastname: Kalloger
authors.forename: Sarah
authors.initials: S
authors.lastname: Finlayson
authors.forename: Katherine
authors.initials: K
authors.lastname: Stemke-Hale
authors.forename: Yiling
authors.initials: Y
authors.lastname: Lu
authors.forename: Fan
authors.initials: F
authors.lastname: Zhang
authors.forename: Michael S
authors.initials: MS
authors.lastname: Anglesio
authors.forename: Blake
authors.initials: B
authors.lastname: Gilks
authors.forename: Gordon B
authors.initials: GB
authors.lastname: Mills
authors.forename: David G
authors.initials: DG
authors.lastname: Huntsman
authors.affiliation: Department of Surgical Oncology, British Columbia, Cancer Agency, Vancouver, BC, Canada. mark.carey@vch.ca.
authors.forename: Mark S
authors.initials: MS
authors.lastname: Carey
mesh_terms: D018262:Adenocarcinoma, Clear Cell / Q000235:genetics* / Q000378:metabolism* / Q000401:mortality / Q000473:pathology; D018269:Carcinoma, Endometrioid / Q000235:genetics* / Q000378:metabolism* / Q000401:mortality / Q000473:pathology; D058534:Class I Phosphatidylinositol 3-Kinases; D016000:Cluster Analysis; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins; D005260:Female; D055785:Gene Knockdown Techniques; D006801:Humans; D007150:Immunohistochemistry; D009154:Mutation; D060787:Neoplasm Grading; D009367:Neoplasm Staging; D009687:Nuclear Proteins / Q000235:genetics; D051059:PTEN Phosphohydrolase / Q000235:genetics / Q000378:metabolism; D063868:Patient Outcome Assessment; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011379:Prognosis; D020543:Proteome; D040901:Proteomics / Q000379:methods; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D014157:Transcription Factors / Q000235:genetics
chemical_list: D058534:Class I Phosphatidylinositol 3-Kinases; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D051059:PTEN Phosphohydrolase; D019869:Phosphatidylinositol 3-Kinases; D020543:Proteome; D051057:Proto-Oncogene Proteins c-akt; D014157:Transcription Factors; C112476:ARID1A protein, human; C484760:PIK3CA protein, human