title: HE4 promotes collateral resistance to cisplatin and paclitaxel in ovarian cancer cells.
abstract: BACKGROUND
  Chemotherapy resistance presents a difficult challenge in treating epithelial ovarian cancer patients, particularly when tumors exhibit resistance to multiple chemotherapeutic agents. A few studies have shown that elevated serum levels of the ovarian cancer biomarker HE4 correlate with tumor chemoresistance, response to treatment, and survival. Here, we sought to confirm our previous results that HE4 contributes to collateral resistance to cisplatin and paclitaxel in vitro and uncover factors that may contribute to HE4-mediated chemoresistance.
  
  
  METHODS
  MTS assays and western blots for cleaved PARP were used to assess resistance of HE4-overexpressing SKOV3 and OVCAR8 clones to cisplatin and paclitaxel. CRISPR/Cas technology was used to knockdown HE4 in HE4-overexpressing SKOV3 cells. A microarray was conducted to determine differential gene expression between SKOV3 null vector-transfected and HE4-overexpressing clones upon cisplatin exposure, and results were validated by quantitative RT-PCR. Regulation of mitogen activated protein kinases (MAPKs) and tubulins were assessed by western blot.
  
  
  RESULTS
  HE4-overexpressing SKOV3 and OVCAR8 clones displayed increased resistance to cisplatin and paclitaxel. Knockdown of HE4 in HE4-overexpressing SKOV3 cells partially reversed chemoresistance. Microarray analysis revealed that HE4 overexpression resulted in suppression of cisplatin-mediated upregulation of EGR1, a MAPK-regulated gene involved in promoting apoptosis. Upregulation of p38, a MAPK activated in response to cisplatin, was suppressed in HE4-overexpressing clones. No differences in extracellular signal-regulated kinase (ERK) activation were noted in HE4-overexpressing clones treated with 25 μM cisplatin, but ERK activation was partially suppressed in HE4-overexpressing clones treated with 80 μM cisplatin. Furthermore, treatment of cells with recombinant HE4 dramatically affected ERK activation in SKOV3 and OVCAR8 wild type cells. Recombinant HE4 also upregulated α-tubulin and β-tubulin levels in SKOV3 and OVCAR8 cells, and microtubule associated protein tau (MAPT) gene expression was increased in SKOV3 HE4-overexpressing clones.
  
  
  CONCLUSIONS
  Overexpression of HE4 promotes collateral resistance to cisplatin and paclitaxel, and downregulation of HE4 partially reverses this chemoresistance. Multiple factors could be involved in HE4-mediated chemoresistance, including deregulation of MAPK signaling, as well as alterations in tubulin levels or stability.
authors.affiliation: Women and Infants Hospital, Department of Obstetrics and Gynecology, Program in Women's Oncology, Molecular Therapeutics Laboratory, 200 Chestnut Street, Providence, RI, 02903, USA. Jrribeiro@wihri.org.
authors.forename: J R
authors.initials: JR
authors.lastname: Ribeiro
authors.affiliation: Center for Genomics and Proteomics, Genomics Core Facility, Brown University, 70 Ship Street, Providence, RI, 02903, USA.
authors.forename: C
authors.initials: C
authors.lastname: Schorl
authors.affiliation: Women and Infants Hospital, Department of Obstetrics and Gynecology, Program in Women's Oncology, Molecular Therapeutics Laboratory, 200 Chestnut Street, Providence, RI, 02903, USA.
authors.forename: N
authors.initials: N
authors.lastname: Yano
authors.affiliation: Women and Infants Hospital, Department of Obstetrics and Gynecology, Program in Women's Oncology, Molecular Therapeutics Laboratory, 200 Chestnut Street, Providence, RI, 02903, USA.
authors.forename: N
authors.initials: N
authors.lastname: Romano
authors.affiliation: Wilmot Cancer Institute, Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, University of Rochester Medical Center, Rochester, NY, USA.
authors.forename: K K
authors.initials: KK
authors.lastname: Kim
authors.affiliation: Women and Infants Hospital, Department of Obstetrics and Gynecology, Program in Women's Oncology, Molecular Therapeutics Laboratory, 200 Chestnut Street, Providence, RI, 02903, USA.|Wilmot Cancer Institute, Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, University of Rochester Medical Center, Rochester, NY, USA.
authors.forename: R K
authors.initials: RK
authors.lastname: Singh
authors.affiliation: Women and Infants Hospital, Department of Obstetrics and Gynecology, Program in Women's Oncology, Molecular Therapeutics Laboratory, 200 Chestnut Street, Providence, RI, 02903, USA.|Wilmot Cancer Institute, Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, University of Rochester Medical Center, Rochester, NY, USA.
authors.forename: R G
authors.initials: RG
authors.lastname: Moore
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D064113:CRISPR-Cas Systems; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002945:Cisplatin / Q000494:pharmacology*; D015536:Down-Regulation; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D005260:Female; D015870:Gene Expression; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D020935:MAP Kinase Signaling System; D010051:Ovarian Neoplasms / Q000235:genetics* / Q000378:metabolism; D017239:Paclitaxel / Q000494:pharmacology*; D011506:Proteins / Q000235:genetics*; D014404:Tubulin / Q000378:metabolism; D000081102:WAP Four-Disulfide Core Domain Protein 2; D016875:tau Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; C054369:MAPT protein, human; D011506:Proteins; D014404:Tubulin; D000081102:WAP Four-Disulfide Core Domain Protein 2; C569698:WFDC2 protein, human; D016875:tau Proteins; D017239:Paclitaxel; D002945:Cisplatin
keywords: Chemoresistance; EGR1; Epithelial ovarian cancer; HE4; MAPK signaling; Paclitaxel resistance; Platinum resistance; SKOV3 cells; Tubulin