DOI: http://dx.doi.org/10.18203/issn.2454-2156.IntJSciRep20211035

Cell-based siRNA screens highlight triple-negative breast cancer cell epigenetic vulnerability

Albane Gaudeau, Coralie Clua Provost, Thierry Dorval, Andrew Walsh, Michael Hannus, Franck Perez, Jacques Camonis, Elaine Del Nery, Jean-Philippe Stephan

Abstract


Background: Triple-negative breast cancer (TNBC) is a heterogeneous disease defined by ER-, PR- and HER2-negative phenotype and in most cases, a relatively aggressive clinical behaviour. The lack of specific targeted therapies and low efficiency of currently available chemotherapies spurred several clinical trials in the last few years. Despite encouraging results, TNBC still remains a major unmet medical need that prompted us to explore the role of 863 epigenetic modulators in TNBC cell survival.

Methods: A comprehensive siRNA library was screened to explore the role of known epigenetic modulators in TNBC cell viability and growth. The knock-down effect was evaluated for 863 epigenetic genes using 4 siRNAs/gene in two TNBC and a non-TNBC cell lines using ATP-based luminescence and nuclei count image-based assays. Considering siRNA off-target effects, four analysis methods including a classical threshold-based analysis and three ranking methods were applied to determine on-target hits for each screen readout. Hit genes common to both phenotypic readouts highlighted strong epigenetic players involved in TNBC cell survival.

Results: Overall, knock-down of many epigenetic modulator genes mitigates cell survival in TNBC and a non-TNBC cell lines depicted from both phenotypic readouts. Interestingly, ranking-based analysis confirmed hit genes identified in threshold-based analysis and also revealed additional hits enabling us to confirm CDK1 and KMT5A as important regulators in TNBC cell viability and growth. Surprisingly, CHAF1A appeared as a new candidate gene involved in TNBC cell survival.

Conclusions: Taken together, siRNA epigenetic screening results identified CHAF1A as a novel regulator of TNBC cell survival.


Keywords


TNBC, Epigenetics, siRNA, High-throughput screening

Full Text:

PDF

References


Harbeck N, Penault-Llorca F, Cortes J, Gnant M, Houssami N, Poortmans P et al. Breast cancer. Nat Rev Dis Primer. 2019;5:66.

Thiery JP. Epithelial-mesenchymal transitions in development and pathologies. Curr Opin Cell Biol. 2003;15:740-6.

Liu S, Dontu G, Wicha MS. Mammary stem cells, self-renewal pathways, and carcinogenesis. Breast Cancer Res. 2005;7:86-95.

Toh TB, Lim JJ, Chow EK-H. Epigenetics in cancer stem cells. Mol Cancer. 2017;16:29.

Heerboth S, Lapinska K, Snyder N, Leary M, Rollinson S, Sarkar S. Use of Epigenetic Drugs in Disease: An Overview. Genet Epigenetics. 2014;6:GEG:S12270.

Ar P, Fernandes GF DS, Jl DS. Clinical Pharmacology: Epigenetic Drugs at a Glance. Biochem Pharmacol. 2018;7:2.

Travaglini L, Vian L, Billi M, Grignani F, Nervi C. Epigenetic reprogramming of breast cancer cells by valproic acid occurs regardless of estrogen receptor status. Int J Biochem Cell Biol. 2009;41:225-34.

Shah P, Gau Y, Sabnis G. Histone deacetylase inhibitor entinostat reverses epithelial to mesenchymal transition of breast cancer cells by reversing the repression of E-cadherin. Breast Cancer Res Treat. 2014;143:99-111.

Schech A, Kazi A, Yu S, Shah P, Sabnis G. Histone Deacetylase Inhibitor Entinostat Inhibits Tumor-Initiating Cells in Triple-Negative Breast Cancer Cells. Mol Cancer Ther. 2015;14:1848-57.

Ma F, Li H, Wang H, Shi X, Fan Y, Ding X et al. Enriched CD44+/CD24-population drives the aggressive phenotypes presented in triple-negative breast cancer (TNBC). Cancer Lett. 2014;353:153-9.

Idowu MO, Kmieciak M, Dumur C, Burton RS, Grimes MM, Powers CN et al. CD44+/CD24-/low cancer stem/progenitor cells are more abundant in triple-negative invasive breast carcinoma phenotype and are associated with poor outcome. Hum Pathol. 2012;43:364-73.

Garmpis N, Christos D, Garmpi A, Kalampokas E, Kalampokas T, Spartalis E et al. Histone Deacetylases as New Therapeutic Targets in Triple-negative Breast Cancer: Progress and Promises. Cancer Genomics Proteomics. 2017;14:299-313.

Ogier A, Dorval T. HCS-Analyzer: open-source software for high-content screening data correction and analysis. Bioinformatics. 2012;28:1945-6.

Carey LA, Dees EC, Sawyer L, Gatti L, Moore DT, Collichio F et al. The Triple Negative Paradox: Primary Tumor Chemosensitivity of Breast Cancer Subtypes. Clin Cancer Res. 2007;13:2329-34.

Bonotto M, Gerratana L, Poletto E, Driol P, Giangreco M, Russo S et al. Measures of Outcome in Metastatic Breast Cancer: Insights From a Real-World Scenario. The Oncologist. 2014;19:608-15.

Lee A, Djamgoz MBA. Triple negative breast cancer: Emerging therapeutic modalities and novel combination therapies. Cancer Treat Rev. 2018;62:110-22.

Jones PA, Baylin SB. The Epigenomics of Cancer. Cell. 2007;128:683-92.

Rodríguez-Paredes M, Esteller M. Cancer epigenetics reaches mainstream oncology. Nat Med. Nature Publishing Group; 2011;17:330-9.

Horiuchi D, Kusdra L, Huskey NE, Chandriani S, Lenburg ME, Gonzalez-Angulo AM et al. MYC pathway activation in triple-negative breast cancer is synthetic lethal with CDK inhibition. J Exp Med. 2012;209:679-96.

Liu Y, Zhu Y-H, Mao C-Q, Dou S, Shen S, Tan Z-B et al. Triple negative breast cancer therapy with CDK1 siRNA delivered by cationic lipid assisted PEG-PLA nanoparticles. J Controlled Release. 2014;192:114-21.

Klauber-DeMore N, Schulte BA, Wang GY. Targeting MYC for triple-negative breast cancer treatment. Oncoscience. 2018;5:120-1.

Xia Q, Cai Y, Peng R, Wu G, Shi Y, Jiang W. The CDK1 inhibitor RO3306 improves the response of BRCA-proficient breast cancer cells to PARP inhibition. Int J Oncol. Spandidos Publications; 2014;44:735-44.

Yang F, Sun L, Li Q, Han X, Lei L, Zhang H et al. SET8 promotes epithelial-mesenchymal transition and confers TWIST dual transcriptional activities: SET8 promotes TWIST-induced EMT. EMBO J. 2012;31:110-23.

Verreault A, Kaufman PD, Kobayashi R, Stillman B. Nucleosome Assembly by a Complex of CAF-1 and Acetylated Histones H3/H4. Cell. 1996;87:95-104.

Kadyrova LY, Blanko ER, Kadyrov FA. CAF-I-dependent control of degradation of the discontinuous strands during mismatch repair. Proc Natl Acad Sci U S A. 2011;108:2753-8.

Smith CL, Matheson TD, Trombly DJ, Sun X, Campeau E, Han X et al. A separable domain of the p150 subunit of human chromatin assembly factor-1 promotes protein and chromosome associations with nucleoli. Mol Biol Cell. Am Society Cell Biol: 2014;25:2866-81.

Murzina N, Verreault A, Laue E, Stillman B. Heterochromatin dynamics in mouse cells: interaction between chromatin assembly factor 1 and HP1 proteins. Mol Cell. 1999;4:529-40.

Reese BE, Bachman KE, Baylin SB, Rountree MR. The Methyl-CpG Binding Protein MBD1 Interacts with the p150 Subunit of Chromatin Assembly Factor 1. Mol Cell Biol. 2003;23:3226-36.

Xu M, Jia Y, Liu Z, Ding L, Tian R, Gu H et al. Chromatin assembly factor 1, subunit A (P150) facilitates cell proliferation in human hepatocellular carcinoma. OncoTargets Ther. 2016;9:4023-35.

Peng H, Du B, Jiang H, Gao J. Over-expression of CHAF1A promotes cell proliferation and apoptosis resistance in glioblastoma cells via AKT/FOXO3a/Bim pathway. Biochem Biophys Res Commun. 2016;469:1111-6.

Barbieri E, Preter KD, Capasso M, Chen Z, Hsu DM, Tonini GP et al. Histone Chaperone CHAF1A Inhibits Differentiation and Promotes Aggressive Neuroblastoma. Cancer Res. American Association for Cancer Research; 2014;74:765-74.

Glinsky GV. Genomic models of metastatic cancer: functional analysis of death-from-cancer signature genes reveals aneuploid, anoikis-resistant, metastasis-enabling phenotype with altered cell cycle control and activated Polycomb Group (PcG) protein chromatin silencing pathway. Cell Cycle Georget Tex. 2006;5:1208-16.

Jiao R, Bachrati CZ, Pedrazzi G, Kuster P, Petkovic M, Li J-L et al. Physical and Functional Interaction between the Bloom’s Syndrome Gene Product and the Largest Subunit of Chromatin Assembly Factor 1. Mol Cell Biol. 2004;24:4710-9.

Poleshko A, Einarson MB, Shalginskikh N, Zhang R, Adams PD, Skalka AM et al. Identification of a Functional Network of Human Epigenetic Silencing Factors. J Biol Chem. 2010;285:422-33.

Zheng L, Liang X, Li S, Li T, Shang W, Ma L et al. CHAF1A interacts with TCF4 to promote gastric carcinogenesis via upregulation of c-MYC and CCND1 expression. EBio Medicine. 2018;38:69-78.

Xia D, Yang X, Liu W, Shen F, Pan J, Lin Y et al. Over-expression of CHAF1A in Epithelial Ovarian Cancer can promote cell proliferation and inhibit cell apoptosis. Biochem Biophys Res Commun. 2017;486:191-7.

Cai Y, Dong ZY, Wang JY. MiR-520b inhibited metastasis and proliferation of non-small cell lung cancer by targeting CHAF1A. Eur Rev Med Pharmacol Sci. 2018;22(22):7742-9.

Tang Q, Ouyang H, He D, Yu C, Tang G. MicroRNA-based potential diagnostic, prognostic and therapeutic applications in triple-negative breast cancer. Artif Cells Nanomedicine Biotechnol. 2019;47:2800-9.

Montes de Oca R, Gurard-Levin ZA, Berger F, Rehman H, Martel E, Corpet A et al. The histone chaperone HJURP is a new independent prognostic marker for luminal A breast carcinoma. Mol Oncol. 2015;9:657-74.