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Supplementary Figure 2 from Genome-Wide Study of Hypomethylated and Induced Genes in Patients with Liver Cancer Unravels Novel Anticancer Targets

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posted on 2023-03-31, 17:52 authored by Barbara Stefanska, David Cheishvili, Matthew Suderman, Ani Arakelian, Jian Huang, Michael Hallett, Ze-Guang Han, Mamun Al-Mahtab, Sheikh Mohammad Fazle Akbar, Wasif Ali Khan, Rubhana Raqib, Imrana Tanvir, Haseeb Ahmed Khan, Shafaat A. Rabbani, Moshe Szyf

PDF file - 492KB, Supplementary Figure S2. (A) Effect of 5-azaCdR at 1.0 microM concentration on non-transformed NorHep cell growth throughout 20 day-treatment. NorHep cells were seeded at a density of 3 x 105 per a 10-cm tissue culture dish. The medium was replaced with fresh medium containing 1.0 microM 5-azaCdR every 24 h for 3 days and every 48 h for the following 17 days (20 days in total). During the experiment the cells were split every 4-5 days after reaching 80 percent confluence and counted on day 3, 6, 9, 12, 14, and 20. (B-D) Expression and methylation levels of RASAL2 and NENF in NorHep after 1 microM 5-azaCdR treatment for 3 (3d) or 20 days (20d). Relative expression is expressed as a percentage of expression in control non-treated cells. Average methylation state of CpG sites was determined by pyrosequencing in control and treated cells for NENF after 3 days of exposure and for RASAL2 after 20 days (no changes were observed after 3 days as shown in Supplementary Figure S1). All results represent mean � S.D. of two or three independent experiments - ***P < 0.001, **P < 0.01, *P < 0.05.

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ARTICLE ABSTRACT

Purpose: We utilized whole-genome mapping of promoters that are activated by DNA hypomethylation in hepatocellular carcinoma (HCC) clinical samples to shortlist novel targets for anticancer therapeutics. We provide a proof of principle of this approach by testing six genes short-listed in our screen for their essential role in cancer growth and invasiveness.Experimental Design: We used siRNA- or shRNA-mediated depletion to determine whether inhibition of these genes would reduce human tumor xenograft growth in mice as well as cell viability, anchorage-independent growth, invasive capacities, and state of activity of nodal signaling pathways in liver, breast, and bladder cancer cell lines.Results: Depletion of EXOSC4, RNMT, SENP6, WBSCR22, RASAL2, and NENF effectively and specifically inhibits cancer cell growth and cell invasive capacities in different types of cancer, but, remarkably, there is no effect on normal cell growth, suggesting a ubiquitous causal role for these genes in driving cancer growth and metastasis. Depletion of RASAL2 and NENF in vitro reduces their growth as explants in vivo in mice. RASAL2 and NENF depletion interferes with AKT, WNT, and MAPK signaling pathways as well as regulation of epigenetic proteins that were previously demonstrated to drive cancer growth and metastasis.Conclusion: Our results prove that genes that are hypomethylated and induced in tumors are candidate targets for anticancer therapeutics in multiple cancer cell types. Because these genes are particularly activated in cancer, they constitute a group of targets for specific pharmacologic inhibitors of cancer and cancer metastasis. Clin Cancer Res; 20(12); 3118–32. ©2014 AACR.