title: Downregulation of membrane complement inhibitors CD55 and CD59 by siRNA sensitises uterine serous carcinoma overexpressing Her2/neu to complement and antibody-dependent cell cytotoxicity in vitro: implications for trastuzumab-based immunotherapy.
abstract: BACKGROUND
  We evaluated the expression of CD46, CD55 and CD59 membrane-bound complement-regulatory proteins (mCRPs) in primary uterine serous carcinoma (USC) and the ability of small interfering RNA (siRNA) against these mCRPs to sensitise USC to complement-dependent cytotoxicity (CDC) and antibody (trastuzumab)-dependent cellular cytotoxicity (ADCC) in vitro.
  
  
  METHODS
  Membrane-bound complement-regulatory proteins expression was evaluated using real-time PCR (RT-PCR) and flow cytometry, whereas Her2/neu expression and c-erbB2 gene amplification were assessed using immunohistochemistry, flow cytometry and fluorescent in-situ hybridisation. The biological effect of siRNA-mediated knockdown of mCRPs on HER2/neu-overexpressing USC cell lines was evaluated in CDC and ADCC 4-h chromium-release assays.
  
  
  RESULTS
  High expression of mCRPs was found in USC cell lines when compared with normal endometrial cells (P<0.05). RT-PCR and FACS analyses demonstrated that anti-mCRP siRNAs were effective in reducing CD46, CD55 and CD59 expression on USC (P<0.05). Baseline complement-dependent cytotoxicity (CDC) against USC cell lines was low (mean ± s.e.m.=6.8 ± 0.9%) but significantly increased upon CD55 and CD59 knockdown (11.6 ± 0.8% and 10.7 ± 0.9%, respectively, P<0.05). Importantly, in the absence of complement, both CD55 and CD59, but not CD46, knockdowns significantly augmented ADCC against USC overexpressing Her2/neu.
  
  
  CONCLUSIONS
  Uterine serous carcinoma express high levels of the mCRPs CD46, CD55 and CD59. Small interfering RNA inhibition of CD55 and CD59, but not CD46, sensitises USC to both CDC and ADCC in vitro, and if specifically targeted to tumour cells, may significantly increase trastuzumab-mediated therapeutic effect in vivo.
authors.affiliation: Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University School of Medicine, New Haven, CT, USA.
authors.forename: S
authors.initials: S
authors.lastname: Bellone
authors.forename: D
authors.initials: D
authors.lastname: Roque
authors.forename: E
authors.initials: E
authors.lastname: Cocco
authors.forename: S
authors.initials: S
authors.lastname: Gasparrini
authors.forename: I
authors.initials: I
authors.lastname: Bortolomai
authors.forename: N
authors.initials: N
authors.lastname: Buza
authors.forename: M
authors.initials: M
authors.lastname: Abu-Khalaf
authors.forename: D-A
authors.initials: DA
authors.lastname: Silasi
authors.forename: E
authors.initials: E
authors.lastname: Ratner
authors.forename: M
authors.initials: M
authors.lastname: Azodi
authors.forename: P E
authors.initials: PE
authors.lastname: Schwartz
authors.forename: T J
authors.initials: TJ
authors.lastname: Rutherford
authors.forename: S
authors.initials: S
authors.lastname: Pecorelli
authors.forename: A D
authors.initials: AD
authors.lastname: Santin
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D061067:Antibodies, Monoclonal, Humanized / Q000627:therapeutic use*; D000920:Antibody-Dependent Cell Cytotoxicity; D018958:CD55 Antigens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018957:CD59 Antigens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003167:Complement Activation; D018284:Cystadenocarcinoma, Serous / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D003602:Cytotoxicity, Immunologic; D015536:Down-Regulation; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D051916:Membrane Cofactor Protein / Q000235:genetics / Q000378:metabolism; D008875:Middle Aged; D011379:Prognosis; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D000068878:Trastuzumab; D002583:Uterine Cervical Neoplasms / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D018958:CD55 Antigens; D018957:CD59 Antigens; D051916:Membrane Cofactor Protein; D034741:RNA, Small Interfering; D018719:Erb-b2 Receptor Tyrosine Kinases; D000068878:Trastuzumab; C508053:ERBB2 protein, human