{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,28]],"date-time":"2026-09-28T05:33:25Z","timestamp":1790573605189,"version":"4.1.0"},"reference-count":70,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2025,1,30]],"date-time":"2025-01-30T00:00:00Z","timestamp":1738195200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Institutes of Health","award":["T32CA047888"],"award-info":[{"award-number":["T32CA047888"]}]},{"name":"National Institutes of Health","award":["NCI R01 CA178441"],"award-info":[{"award-number":["NCI R01 CA178441"]}]},{"name":"National Cancer Institute, National Institutes of Health","award":["T32CA047888"],"award-info":[{"award-number":["T32CA047888"]}]},{"name":"National Cancer Institute, National Institutes of Health","award":["NCI R01 CA178441"],"award-info":[{"award-number":["NCI R01 CA178441"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Breast cancer (BC) is a widespread malignancy that affects the lives of millions of women each year, and its resulting financial and healthcare hardships cannot be overstated. These issues, in combination with side effects and obstacles associated with the current standard of care, generate considerable interest in new potential targets for treatment as well as means for BC prevention. One potential preventive compound is Withaferin A (WFA), a traditional medicinal compound found in winter cherries. WFA has shown promise as an anticancer agent and is thought to act primarily through its effects on the epigenome, including, in particular, the methylome. However, the relative importance of specific genes\u2019 methylation states to WFA function remains unclear. To address this, we utilized human BC cell lines in combination with CRISPR-dCas9 fused to DNA methylation modifiers (i.e., epigenetic editors) to elucidate the importance of specific genes\u2019 promoter methylation states to WFA function and cancer cell viability. We found that targeted demethylation of promoters of the tumor suppressors p21 and p53 within MDA-MB-231\/MCF7 cells resulted in around 1.7\u00d7\/1.5\u00d7 and 1.2\u00d7\/1.3\u00d7 increases in expression, respectively. Targeted methylation of the promoter of the oncogene CCND1 within MDA-MB-231\/MCF7 cells resulted in 0.5\u00d7\/0.8\u00d7 decreases in gene expression. These changes to p21, p53, and CCND1 were also associated with decreases in cell viability of around 25%\/50%, 5%\/35%, and 12%\/16%, respectively, for MDA-MB-231\/MCF7 cells. When given in combination with WFA in both p53 mutant and wild type cells, we discovered that targeted methylation of the p21 promoter was able to modulate the anticancer effects of WFA, while targeted methylation or demethylation of the promoters of p53 and CCND1 had no significant effect on viability decreases from WFA treatment. Taken together, these results indicate that p21, p53, and CCND1 may be important targets for future in vivo studies that may lead to epigenetic editing therapies and that WFA may have utility in the prevention of BC through its effect on p21 promoter methylation independent of p53 function.<\/jats:p>","DOI":"10.3390\/ijms26031210","type":"journal-article","created":{"date-parts":[[2025,1,30]],"date-time":"2025-01-30T11:08:23Z","timestamp":1738235303000},"page":"1210","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["p21 Promoter Methylation Is Vital for the Anticancer Activity of Withaferin A"],"prefix":"10.3390","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8766-1033","authenticated-orcid":false,"given":"Andrew","family":"Brane","sequence":"first","affiliation":[{"name":"Department of Biology, University of Alabama at Birmingham, 3100 Science & Engineering Complex\u2014East Science Hall, 902 14th Street South, Birmingham, AL 35205, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Madeline","family":"Sutko","sequence":"additional","affiliation":[{"name":"Department of Biology, University of Alabama at Birmingham, 3100 Science & Engineering Complex\u2014East Science Hall, 902 14th Street South, Birmingham, AL 35205, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0284-1511","authenticated-orcid":false,"given":"Trygve O.","family":"Tollefsbol","sequence":"additional","affiliation":[{"name":"Department of Biology, University of Alabama at Birmingham, 3100 Science & Engineering Complex\u2014East Science Hall, 902 14th Street South, Birmingham, AL 35205, USA"},{"name":"Comprehensive Cancer Center, University of Alabama at Birmingham, 1802 6th Avenue South, Birmingham, AL 35294, USA"},{"name":"Integrative Center for Aging Research, University of Alabama at Birmingham, 933 19th Street South, Birmingham, AL 35294, USA"},{"name":"Nutrition Obesity Research Center, University of Alabama at Birmingham, 1675 University Blvd, Birmingham, AL 35294, USA"},{"name":"Comprehensive Diabetes Center, University of Alabama at Birmingham, 1825 University Blvd, Birmingham, AL 35294, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,1,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"17","DOI":"10.3322\/caac.21763","article-title":"Cancer statistics, 2023","volume":"73","author":"Siegel","year":"2023","journal-title":"CA A Cancer J. 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