{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,11]],"date-time":"2026-07-11T03:38:04Z","timestamp":1783741084084,"version":"3.55.0"},"reference-count":51,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2025,1,1]],"date-time":"2025-01-01T00:00:00Z","timestamp":1735689600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health (NIH)","doi-asserted-by":"publisher","award":["CA280626"],"award-info":[{"award-number":["CA280626"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health (NIH)","doi-asserted-by":"publisher","award":["CA256104"],"award-info":[{"award-number":["CA256104"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health (NIH)","doi-asserted-by":"publisher","award":["GM072643"],"award-info":[{"award-number":["GM072643"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health (NIH)","doi-asserted-by":"publisher","award":["GM1523862"],"award-info":[{"award-number":["GM1523862"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health (NIH)","doi-asserted-by":"publisher","award":["2019-198009"],"award-info":[{"award-number":["2019-198009"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Chan Zuckerberg Initiative","award":["CA280626"],"award-info":[{"award-number":["CA280626"]}]},{"name":"Chan Zuckerberg Initiative","award":["CA256104"],"award-info":[{"award-number":["CA256104"]}]},{"name":"Chan Zuckerberg Initiative","award":["GM072643"],"award-info":[{"award-number":["GM072643"]}]},{"name":"Chan Zuckerberg Initiative","award":["GM1523862"],"award-info":[{"award-number":["GM1523862"]}]},{"name":"Chan Zuckerberg Initiative","award":["2019-198009"],"award-info":[{"award-number":["2019-198009"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Genes"],"abstract":"<jats:p>Background\/Objectives: All 11 metallothionein protein-coding genes are located on human chromosome 16q13. It is unique among human genetics to have an entire pathway\u2019s genes clustered in a short chromosomal region. Since solid tumors, particularly high-grade serous ovarian cancer (HGSC), exhibit high rates of monoallelic aneuploidy, this region is commonly lost. Studies have not yet been performed to determine what vulnerability may be created in cancer cells with low metallothionein expression. Here, a screen of FDA-approved cancer small molecule drugs for those best targeting low metallothionein ovarian cancer was completed. Methods: Screening methods were tested and compared using vehicle-treated negative controls and cadmium chloride, a positive control for cell loss selective for low metallothionein cells. CAOV3 cells, which are unique in their expression of only two metallothionein isoforms, were used, with or without shRNA knockdown of the predominantly expressed MT2A gene. A library of FDA-approved molecules was then screened. Results: The optimal assay utilized Hoechst 33342 nuclear staining and mechanized fluorescent microscope counting of cell content. Encorafenib, an RAF inhibitor, was identified as the most selective for enhanced cytotoxicity in MT2A knockdown cells compared to scrambled controls. Conclusions: The nuclear stain Hoechst 33342, assessed by fluorescence microscopy, provides a low variance, moderate throughput platform for cancer cell loss screens. Low metallothionein ovarian cancer cells exhibit a vulnerability to the RAF inhibitor encorafenib.<\/jats:p>","DOI":"10.3390\/genes16010042","type":"journal-article","created":{"date-parts":[[2025,1,1]],"date-time":"2025-01-01T11:24:19Z","timestamp":1735730659000},"page":"42","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Screening Methods to Discover the FDA-Approved Cancer Drug Encorafenib as Optimally Selective for Metallothionein Gene Loss Ovarian Cancer"],"prefix":"10.3390","volume":"16","author":[{"given":"Amy","family":"Rees","sequence":"first","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Evan","family":"Villamor","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Della","family":"Evans","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6404-5070","authenticated-orcid":false,"given":"Monika","family":"Gooz","sequence":"additional","affiliation":[{"name":"Department of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, Charleston, SC 29425, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Clare","family":"Fallon","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8651-332X","authenticated-orcid":false,"given":"Mirna","family":"Mina-Abouda","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrew","family":"Disharoon","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Scott T.","family":"Eblen","sequence":"additional","affiliation":[{"name":"Department of Pharmacology and Immunology, Medical University of South Carolina, Charleston, SC 29425, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8978-5961","authenticated-orcid":false,"given":"Joe R.","family":"Delaney","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,1,1]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"3673","DOI":"10.1093\/nar\/gkac200","article-title":"SWAN pathway-network identification of common aneuploidy-based oncogenic drivers","volume":"50","author":"Bowers","year":"2022","journal-title":"Nucleic Acids Res."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1929","DOI":"10.1038\/onc.2009.477","article-title":"p53 suppresses structural chromosome instability after mitotic arrest in human cells","volume":"29","author":"Dalton","year":"2010","journal-title":"Oncogene"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1370","DOI":"10.1016\/j.celrep.2019.07.001","article-title":"Integrated Analysis of TP53 Gene and Pathway Alterations in The Cancer Genome Atlas","volume":"28","author":"Donehower","year":"2019","journal-title":"Cell Rep."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1134","DOI":"10.1038\/ng.2760","article-title":"Pan-cancer patterns of somatic copy number alteration","volume":"45","author":"Zack","year":"2013","journal-title":"Nat. Genet."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"357","DOI":"10.1016\/S0079-6603(00)66034-8","article-title":"Regulation of metallothionein gene expression","volume":"66","author":"Ghoshal","year":"2001","journal-title":"Prog. Nucleic Acid. Res. Mol. Biol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1260419","DOI":"10.1126\/science.1260419","article-title":"Proteomics. Tissue-based map of the human proteome","volume":"347","author":"Uhlen","year":"2015","journal-title":"Science"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"54","DOI":"10.1186\/1477-7819-9-54","article-title":"A review of metallothionein isoforms and their role in pathophysiology","volume":"9","author":"Thirumoorthy","year":"2011","journal-title":"World J. Surg. Oncol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"267","DOI":"10.1146\/annurev.pharmtox.39.1.267","article-title":"Metallothionein: An intracellular protein to protect against cadmium toxicity","volume":"39","author":"Klaassen","year":"1999","journal-title":"Annu. Rev. Pharmacol. Toxicol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"750","DOI":"10.1002\/jcb.22049","article-title":"The important role of the apoptotic effects of zinc in the development of cancers","volume":"106","author":"Franklin","year":"2009","journal-title":"J. Cell. Biochem."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1087","DOI":"10.1007\/s00775-011-0823-6","article-title":"Mammalian metallothionein in toxicology, cancer, and cancer chemotherapy","volume":"16","author":"Namdarghanbari","year":"2011","journal-title":"J. Biol. Inorg. Chem."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"637","DOI":"10.1016\/0022-2836(88)90644-4","article-title":"Three-dimensional structure of rabbit liver [Cd7]metallothionein-2a in aqueous solution determined by nuclear magnetic resonance","volume":"201","author":"Arseniev","year":"1988","journal-title":"J. Mol. Biol."},{"key":"ref_12","first-page":"1955","article-title":"An ecological study of cancer mortality rates including indices for dietary iron and zinc","volume":"28","author":"Grant","year":"2008","journal-title":"Anticancer Res."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"196","DOI":"10.1021\/pr050361j","article-title":"Counting the zinc-proteins encoded in the human genome","volume":"5","author":"Andreini","year":"2006","journal-title":"J. Proteome Res."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"6","DOI":"10.1038\/s41392-023-01679-y","article-title":"Cellular zinc metabolism and zinc signaling: From biological functions to diseases and therapeutic targets","volume":"9","author":"Chen","year":"2024","journal-title":"Signal Transduct. Target. Ther."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1186\/s13045-018-0645-x","article-title":"The roles of metallothioneins in carcinogenesis","volume":"11","author":"Si","year":"2018","journal-title":"J. Hematol. Oncol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"20438","DOI":"10.1074\/jbc.M110631200","article-title":"Regulation of metallothionein transcription by the metal-responsive transcription factor MTF-1: Identification of signal transduction cascades that control metal-inducible transcription","volume":"277","author":"Saydam","year":"2002","journal-title":"J. Biol. Chem."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Delaney, J.R., and Stupack, D.G. (2016). Whole Genome Pathway Analysis Identifies an Association of Cadmium Response Gene Loss with Copy Number Variation in Mutant p53 Bearing Uterine Endometrial Carcinomas. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0159114"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"e265","DOI":"10.1002\/mco2.265","article-title":"Understanding and targeting resistance mechanisms in cancer","volume":"4","author":"Lei","year":"2023","journal-title":"MedComm"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"100796","DOI":"10.1016\/j.drup.2021.100796","article-title":"Anticancer drug resistance: An update and perspective","volume":"59","author":"Nussinov","year":"2021","journal-title":"Drug Resist. Updat."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"14423","DOI":"10.1038\/ncomms14423","article-title":"Haploinsufficiency networks identify targetable patterns of allelic deficiency in low mutation ovarian cancer","volume":"8","author":"Delaney","year":"2017","journal-title":"Nat. Commun."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Bowers, R.R., Andrade, M.F., Jones, C.M., White-Gilbertson, S., Voelkel-Johnson, C., and Delaney, J.R. (2022). Autophagy modulating therapeutics inhibit ovarian cancer colony generation by polyploid giant cancer cells (PGCCs). BMC Cancer, 22.","DOI":"10.1186\/s12885-022-09503-6"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Stirling, D.R., Swain-Bowden, M.J., Lucas, A.M., Carpenter, A.E., Cimini, B.A., and Goodman, A. (2021). CellProfiler 4: Improvements in speed, utility and usability. BMC Bioinform., 22.","DOI":"10.1186\/s12859-021-04344-9"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Ortell, K.K., Switonski, P.M., and Delaney, J.R. (2019). FairSubset: A tool to choose representative subsets of data for use with replicates or groups of different sample sizes. J. Biol. Methods, 6.","DOI":"10.14440\/jbm.2019.299"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"401","DOI":"10.1158\/2159-8290.CD-12-0095","article-title":"The cBio cancer genomics portal: An open platform for exploring multidimensional cancer genomics data","volume":"2","author":"Cerami","year":"2012","journal-title":"Cancer Discov."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"609","DOI":"10.1038\/nature10166","article-title":"Integrated genomic analyses of ovarian carcinoma","volume":"474","year":"2011","journal-title":"Nature"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"321","DOI":"10.1016\/j.cell.2018.03.035","article-title":"Oncogenic Signaling Pathways in The Cancer Genome Atlas","volume":"173","author":"Mina","year":"2018","journal-title":"Cell"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"603","DOI":"10.1038\/nature11003","article-title":"The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity","volume":"483","author":"Barretina","year":"2012","journal-title":"Nature"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"pdb-prot087379","DOI":"10.1101\/pdb.prot087379","article-title":"Crystal Violet Assay for Determining Viability of Cultured Cells","volume":"2016","author":"Feoktistova","year":"2016","journal-title":"Cold Spring Harb. Protoc."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"pdb-prot5557","DOI":"10.1101\/pdb.prot5557","article-title":"Labeling nuclear DNA with hoechst 33342","volume":"2011","author":"Chazotte","year":"2011","journal-title":"Cold Spring Harb. Protoc."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1177\/108705719900400206","article-title":"A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays","volume":"4","author":"Zhang","year":"1999","journal-title":"J. Biomol. Screen."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"649","DOI":"10.1016\/S1470-2045(18)30145-1","article-title":"Tazemetostat, an EZH2 inhibitor, in relapsed or refractory B-cell non-Hodgkin lymphoma and advanced solid tumours: A first-in-human, open-label, phase 1 study","volume":"19","author":"Italiano","year":"2018","journal-title":"Lancet Oncol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"6044","DOI":"10.3390\/ijms14036044","article-title":"The role of metallothionein in oxidative stress","volume":"14","author":"Nejdl","year":"2013","journal-title":"Int. J. Mol. Sci."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"237","DOI":"10.1073\/pnas.91.1.237","article-title":"Oxidative metal release from metallothionein via zinc-thiol\/disulfide interchange","volume":"91","author":"Maret","year":"1994","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"3483","DOI":"10.1073\/pnas.95.7.3483","article-title":"The glutathione redox couple modulates zinc transfer from metallothionein to zinc-depleted sorbitol dehydrogenase","volume":"95","author":"Jiang","year":"1998","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1263","DOI":"10.1016\/j.bbamcr.2006.10.001","article-title":"Roles of the Raf\/MEK\/ERK pathway in cell growth, malignant transformation and drug resistance","volume":"1773","author":"McCubrey","year":"2007","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1318","DOI":"10.1021\/acs.chemrestox.9b00152","article-title":"N-Acetylcysteine: Antioxidant, Aldehyde Scavenger, and More","volume":"32","author":"Zhitkovich","year":"2019","journal-title":"Chem. Res. Toxicol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"2501","DOI":"10.1101\/gad.836800","article-title":"Multiple Ras-dependent phosphorylation pathways regulate Myc protein stability","volume":"14","author":"Sears","year":"2000","journal-title":"Genes Dev."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"7772","DOI":"10.1038\/s41467-024-52176-x","article-title":"Human aneuploid cells depend on the RAF\/MEK\/ERK pathway for overcoming increased DNA damage","volume":"15","author":"Zerbib","year":"2024","journal-title":"Nat. Commun."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"8475","DOI":"10.1158\/0008-5472.CAN-10-0603","article-title":"BRAF inactivation drives aneuploidy by deregulating CRAF","volume":"70","author":"Kamata","year":"2010","journal-title":"Cancer Res."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"323","DOI":"10.1186\/s12935-021-02020-9","article-title":"Myc-associated zinc-finger protein promotes clear cell renal cell carcinoma progression through transcriptional activation of the MAP2K2-dependent ERK pathway","volume":"21","author":"Ren","year":"2021","journal-title":"Cancer Cell Int."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"2390","DOI":"10.1159\/000495098","article-title":"Novel Zinc Finger Transcription Factor ZFP580 Facilitates All-Trans Retinoic Acid -Induced Vascular Smooth Muscle Cells Differentiation by Raralpha-Mediated PI3K\/Akt and ERK Signaling","volume":"50","author":"Wei","year":"2018","journal-title":"Cell. Physiol. Biochem."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"209","DOI":"10.1016\/j.taap.2009.01.029","article-title":"Role of oxidative stress in cadmium toxicity and carcinogenesis","volume":"238","author":"Liu","year":"2009","journal-title":"Toxicol. Appl. Pharmacol."},{"key":"ref_43","first-page":"491","article-title":"Cadmium and cancer","volume":"11","author":"Hartwig","year":"2013","journal-title":"Met. Ions Life Sci."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"3945","DOI":"10.1111\/cas.15068","article-title":"Reactive oxygen species in cancer: Current findings and future directions","volume":"112","author":"Nakamura","year":"2021","journal-title":"Cancer Sci."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"192","DOI":"10.1038\/s12276-020-0384-2","article-title":"ROS in cancer therapy: The bright side of the moon","volume":"52","author":"Perillo","year":"2020","journal-title":"Exp. Mol. Med."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"665","DOI":"10.1038\/nm.2143","article-title":"ERK activation drives intestinal tumorigenesis in Apcmin\/+ mice","volume":"16","author":"Lee","year":"2010","journal-title":"Nat. Med."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"427","DOI":"10.1038\/nature08902","article-title":"RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF","volume":"464","author":"Poulikakos","year":"2010","journal-title":"Nature"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"431","DOI":"10.1038\/nature08833","article-title":"RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth","volume":"464","author":"Hatzivassiliou","year":"2010","journal-title":"Nature"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"2","DOI":"10.1038\/s41419-019-2202-0","article-title":"GSK3beta is a key regulator of the ROS-dependent necrotic death induced by the quinone DMNQ","volume":"11","author":"Ciotti","year":"2020","journal-title":"Cell Death Dis."},{"key":"ref_50","doi-asserted-by":"crossref","unstructured":"Sugiura, R., Satoh, R., and Takasaki, T. (2021). ERK: A Double-Edged Sword in Cancer. ERK-Dependent Apoptosis as a Potential Therapeutic Strategy for Cancer. Cells, 10.","DOI":"10.3390\/cells10102509"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"541","DOI":"10.1016\/S0140-6736(21)02175-9","article-title":"Trametinib versus standard of care in patients with recurrent low-grade serous ovarian cancer (GOG 281\/LOGS): An international, randomised, open-label, multicentre, phase 2\/3 trial","volume":"399","author":"Gershenson","year":"2022","journal-title":"Lancet"}],"container-title":["Genes"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4425\/16\/1\/42\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,7]],"date-time":"2025-10-07T15:22:58Z","timestamp":1759850578000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4425\/16\/1\/42"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,1,1]]},"references-count":51,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2025,1]]}},"alternative-id":["genes16010042"],"URL":"https:\/\/doi.org\/10.3390\/genes16010042","relation":{},"ISSN":["2073-4425"],"issn-type":[{"value":"2073-4425","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,1,1]]}}}