title: Complementary cell-based high-throughput screens identify novel modulators of the unfolded protein response.
abstract: Despite advances toward understanding the prevention and treatment of many cancers, patients who suffer from oral squamous cell carcinoma (OSCC) confront a survival rate that has remained unimproved for more than 2 decades, indicating our ability to treat them pharmacologically has reached a plateau. In an ongoing effort to improve the clinical outlook for this disease, we previously reported that an essential component of the mechanism by which the proteasome inhibitor bortezomib (PS-341, Velcade) induced apoptosis in OSCC required the activation of a terminal unfolded protein response (UPR). Predicated on these studies, the authors hypothesized that high-throughput screening (HTS) of large diverse chemical libraries might identify more potent or selective small-molecule activators of the apoptotic arm of the UPR to control or kill OSCC. They have developed complementary cell-based assays using stably transfected CHO-K1 cell lines that individually assess the PERK/eIF2α/CHOP (apoptotic) or the IRE1/XBP1 (adaptive) UPR subpathways. An 66 K compound collection was screened at the University of Michigan Center for Chemical Genomics that included a unique library of prefractionated natural product extracts. The mycotoxin methoxycitrinin was isolated from a natural extract and found to selectively activate the CHOP-luciferase reporter at 80 µM. A series of citrinin derivatives was isolated from these extracts, including a unique congener that has not been previously described. In an effort to identify more potent compounds, the authors examined the ability of citrinin and the structurally related mycotoxins ochratoxin A and patulin to activate the UPR. Strikingly, it was found that patulin at 2.5 to 10 µM induced a terminal UPR in a panel of OSCC cells that was characterized by an increase in CHOP, GADD34, and ATF3 gene expression and XBP1 splicing. A luminescent caspase assay and the induction of several BH3-only genes indicated that patulin could induce apoptosis in OSCC cells. These data support the use of this complementary HTS strategy to identify novel modulators of UPR signaling and tumor cell death.
authors.affiliation: Carmen and Ann Adams Department of Pediatrics, Division of Hematology/Oncology, Wayne State University, 421 E. Canfield, Detroit, MI 48201, USA.
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Fribley
authors.forename: Patricia G
authors.initials: PG
authors.lastname: Cruz
authors.forename: Justin R
authors.initials: JR
authors.lastname: Miller
authors.forename: Michael U
authors.initials: MU
authors.lastname: Callaghan
authors.forename: Peter
authors.initials: P
authors.lastname: Cai
authors.forename: Neha
authors.initials: N
authors.lastname: Narula
authors.forename: Richard R
authors.initials: RR
authors.lastname: Neubig
authors.forename: Hollis D
authors.initials: HD
authors.lastname: Showalter
authors.forename: Scott D
authors.initials: SD
authors.lastname: Larsen
authors.forename: Paul D
authors.initials: PD
authors.lastname: Kirchhoff
authors.forename: Martha J
authors.initials: MJ
authors.lastname: Larsen
authors.forename: Douglas A
authors.initials: DA
authors.lastname: Burr
authors.forename: Pamela J
authors.initials: PJ
authors.lastname: Schultz
authors.forename: Renju R
authors.initials: RR
authors.lastname: Jacobs
authors.forename: Giselle
authors.initials: G
authors.lastname: Tamayo-Castillo
authors.forename: David
authors.initials: D
authors.lastname: Ron
authors.forename: David H
authors.initials: DH
authors.lastname: Sherman
authors.forename: Randal J
authors.initials: RJ
authors.lastname: Kaufman
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis; D001897:Boronic Acids / Q000494:pharmacology; D000069286:Bortezomib; D016466:CHO Cells; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy* / Q000473:pathology; D020169:Caspases / Q000235:genetics / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D006224:Cricetinae; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D057166:High-Throughput Screening Assays; D006801:Humans; D008156:Luciferases / Q000032:analysis; D009062:Mouth Neoplasms / Q000188:drug therapy* / Q000473:pathology; D009183:Mycotoxins / Q000494:pharmacology*; D011719:Pyrazines / Q000494:pharmacology; D015398:Signal Transduction; D051742:Transcription Factor CHOP / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic; D056811:Unfolded Protein Response / Q000187:drug effects; D019892:eIF-2 Kinase / Q000235:genetics / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D001897:Boronic Acids; D000069286:Bortezomib; D020169:Caspases; D015852:Eukaryotic Initiation Factor-2; D008156:Luciferases; D009183:Mycotoxins; D011719:Pyrazines; D051742:Transcription Factor CHOP; D019892:eIF-2 Kinase