Breast Cancer Cells and Impairs the Tumor-Killing Effect of the Vaccinia Virus

Mariam Nafea¹, Aamna Basheer¹, Ohud Alkhaibry¹ and Alanoud Alsaedan^1,

¹Regional Laboratory, Ministry of Health, Riyadh, Saudi Arabia

Cite this paper:
Mariam Nafea, Aamna Basheer, Ohud Alkhaibry and Alanoud Alsaedan. Breast Cancer Cells and Impairs the Tumor-Killing Effect of the Vaccinia Virus. American Journal of Medical and Biological Research. 2024; 12(2):36-44. doi: 10.12691/ajmbr-12-2-1

Abstract

Abstract Purpose Vaccinia virus is widely used as an oncolytic agent for human cancer therapy, and several versions of vaccinia virus have demonstrated robust antitumor effects in breast cancer. Most vaccinia viruses are modified by thymidine kinase (TK) deletion. The function of the cyclin-dependent kinase inhibitor p21 in breast cancer remains controversial. We explored the impact of p21 gene knockdown (KD) on breast cancer cells and whether p21 KD interferes with the antitumor effect of TK-negative vaccinia virus. Methods p21 KD MDA-MB-231 and p21 KD MCF-7 cells were prepared, and cell proliferation and migration rates were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and scratch healing assays. The tumor growth of xenografts originating from p21KD MDA-MB-231 cells and control cells was compared in a mouse model. The colony formation and sphere-forming abilities of p21 KD breast cancer cells were also determined using low-melting agarose and serum-free culture. The tumor-killing effect of the vaccinia virus was determined in breast cancer cells and mouse models using an MTT assay and tumor cell xenografts. Results p21 KD increased the growth and migration of MDA-MB-231 and MCF-7 cells and promoted the cell growth of MDA-MB-231 cells in mice, while decreasing the colony formation and sphere formation abilities. Expression of TK was reduced in p21 KD MDA-MB-231 cells. Oncolytic effects of both wild-type and TK-deleted vaccinia viruses were attenuated in p21KD MDA-MB-231 cells. The tumor-killing effect of TK-deleted vaccinia virus was also weakened in xenografted mice bearing p21 KD MDA-MB-231 cells. Conclusion Targeted inhibition of p21 accelerates the proliferation and migration of breast cancer cells and impairs the tumor-killing effect of vaccinia virus, suggesting that p21 levels in cancer cells interfere with vaccinia virus oncolytic therapy.

Keywords:
Keywords: Breast Neoplasms p21 Vaccinia Virus

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References:

[1]	Harbeck N, Penault-Llorca F, Cortes J, Gnant M, Houssami N, Poortmans P, et al. Breast cancer. Nat Rev Dis Primers 2019; 5: 66.
[2]	Chavez KJ, Garimella SV, Lipkowitz S. Triple negative breast cancer cell lines: one tool in the search for better treatment of triple negative breast cancer. Breast Dis 2010; 32: 35–48.
[3]	Simu S, Marcovici I, Dobrescu A, Malita D, Dehelean CA, Coricovac D, et al. Insights into the behavior of triple-negative MDA-MB-231 breast carcinoma cells following the treatment with 17β-ethinylestradiol and levonorgestrel. Molecules 2021; 6: 2776.
[4]	Treeck O, Schüler-Toprak S, Ortmann O. Estrogen actions in triple-negative breast cancer. Cells 2020; 9: 2358.
[5]	Yin L, Duan JJ, Bian XW, Yu SC. Triple-negative breast cancer molecular subtyping and treatment progress. Breast Cancer Res 2020; 22: 61.
[6]	Harper JW, Adami GR, Wei N, Keyomarsi K, Elledge SJ. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 1993; 75: 805–816.
[7]	Shamloo B, Usluer S. p21 in cancer research. Cancers (Basel) 2019; 11: 1178.
[8]	Kreis NN, Louwen F, Yuan J. The multifaceted p21 (Cip1/Waf1/CDKN1A) in cell differentiation, migration and cancer therapy. Cancers (Basel) 2019; 11: 1220.
[9]	Zohny SF, Al-Malki AL, Zamzami MA, Choudhry H. p21Waf1/Cip1: its paradoxical effect in the regulation of breast cancer. Breast Cancer 2019; 26: 131–137.
[10]	Wei CY, Tan QX, Zhu X, Qin QH, Zhu FB, Mo QG, et al. Expression of CDKN1A/p21 and TGFBR2 in breast cancer and their prognostic significance. Int J Clin Exp Pathol 2015; 8: 14619–14629.
[11]	Kirn DH, Thorne SH. Targeted and armed oncolytic poxviruses: a novel multi-mechanistic therapeutic class for cancer. Nat Rev Cancer 2009; 9: 64–71.
[12]	Chan WM, McFadden G. Oncolytic poxviruses. Annu Rev Virol 2014; 1: 119–141.
[13]	Breitbach CJ, Burke J, Jonker D, Stephenson J, Haas AR, Chow LQ, et al. Intravenous delivery of a multi-mechanistic cancer-targeted oncolytic poxvirus in humans. Nature 2011; 477: 99–102.
[14]	Huang DY, Chang ZF. Interaction of human thymidine kinase 1 with p21(Waf1). Biochem J 2001; 356: 829–834.
[15]	Zhu X, Shi C, Peng Y, Yin L, Tu M, Chen Q, et al. Thymidine kinase 1 silencing retards proliferative activity of pancreatic cancer cell via E2F1-TK1-P21 axis. Cell Prolif 2018;51:e12428.
[16]	Chen YL, Eriksson S, Chang ZF. Regulation and functional contribution of thymidine kinase 1 in repair of DNA damage. J Biol Chem 2010; 285: 27327–27335.
[17]	Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method. Methods 2001; 25: 402–408.
[18]	Mansilla SF, de la Vega MB, Calzetta NL, Siri SO, Gottifredi V. CDK-independent and PCNA-dependent functions of p21 in DNA replication. Genes (Basel) 2020; 11: 593.
[19]	González-Magaña A, Blanco FJ. Human PCNA structure, function and interactions. Biomolecules 2020; 10: 570.
[20]	Juríková M, Danihel Ľ, Polák Š, Varga I. Ki67, PCNA, and MCM proteins: markers of proliferation in the diagnosis of breast cancer. Acta Histochem 2016; 118: 544–552.
[21]	Arrindell J, Desnues B. Vimentin: from a cytoskeletal protein to a critical modulator of immune response and a target for infection. Front Immunol 2023; 14: 1224352
[22]	Satelli A, Li S. Vimentin in cancer and its potential as a molecular target for cancer therapy. Cell Mol Life Sci 2011; 68: 3033–3046.
[23]	Na TY, Schecterson L, Mendonsa AM, Gumbiner BM. The functional activity of E-cadherin controls tumor cell metastasis at multiple steps. Proc Natl Acad Sci U S A 2020; 117: 5931–5937.
[24]	Horne HN, Oh H, Sherman ME, Palakal M, Hewitt SM, Schmidt MK, et al. E-cadherin breast tumor expression, risk factors and survival: Pooled analysis of 5,933 cases from 12 studies in the Breast Cancer Association Consortium. Sci Rep 2018; 8: 6574.
[25]	Brix N, Samaga D, Belka C, Zitzelsberger H, Lauber K. Analysis of clonogenic growth in vitro. Nat Protoc 2021; 16: 4963–4991.
[26]	Braun SE, Mantel C, Rosenthal M, Cooper S, Liu L, Robertson KA, et al. A positive effect of p21cip1/waf1 in the colony formation from murine myeloid progenitor cells as assessed by retroviral-mediated gene transfer. Blood Cells Mol Dis 1998; 24: 138–148.
[27]	Lüönd F, Tiede S, Christofori G. Breast cancer as an example of tumour heterogeneity and tumour cell plasticity during malignant progression. Br J Cancer 2021; 125: 164–175.
[28]	Weiss RH, Marshall D, Howard L, Corbacho AM, Cheung AT, Sawai ET. Suppression of breast cancer growth and angiogenesis by an antisense oligodeoxynucleotide to p21 (Waf1/Cip1). Cancer Lett 2003; 189: 39–48.
[29]	Fan Y, Borowsky AD, Weiss RH. An antisense oligodeoxynucleotide to p21 (Waf1/Cip1) causes apoptosis in human breast cancer cells. Mol Cancer Ther 2003; 2: 773–782.
[30]	Gutu N, Binish N, Keilholz U, Herzel H, Granada AE. p53 and p21 dynamics encode single-cell DNA damage levels, fine-tuning proliferation and shaping population heterogeneity. Commun Biol 2023; 6: 1196.
[31]	Benard O, Qian X, Liang H, Ren Z, Suyama K, Norton L, et al. p21CIP1 promotes mammary cancer-initiating cells via activation of Wnt/TCF1/CyclinD1 signaling. Mol Cancer Res 2019; 17: 1571–1581.
[32]	Maheshwari M, Yadav N, Hasanain M, Pandey P, Sahai R, Choyal K, et al. Inhibition of p21 activates Akt kinase to trigger ROS-induced autophagy and impacts on tumor growth rate. Cell Death Dis 2022; 13: 1045.
[33]	O’Connor K. A cautionary tale about the use of colony-forming efficiency as a proxy for the survival of mesenchymal stem cells. Stem Cell Res Ther 2020; 11: 292.
[34]	Ottestad L, Tveit KM, Hannisdal E, Skrede M, Nesland JM, Gundersen S, et al. Colony forming ability of human breast carcinomas: lack of prognostic significance. Br J Cancer 1989; 60: 216–219.
[35]	Smart CE, Morrison BJ, Saunus JM, Vargas AC, Keith P, Reid L, et al. In vitro analysis of breast cancer cell line tumourspheres and primary human breast epithelia mammospheres demonstrates inter- and intrasphere heterogeneity. PLoS One 2013; 8: e64388
[36]	Bahmad HF, Cheaito K, Chalhoub RM, Hadadeh O, Monzer A, Ballout F, et al. Sphere-formation assay: three-dimensional in vitro culturing of prostate cancer stem/progenitor sphere-forming cells. Front Oncol 2018; 8: 347.
[37]	Deng L, Fan J, Ding Y, Yang X, Huang B, Hu Z. Target therapy with vaccinia virus harboring IL-24 for human breast cancer. J Cancer 2020; 11: 1017–1026.
[38]	Gholami S, Chen CH, Lou E, De Brot M, Fujisawa S, Chen NG, et al. Vaccinia virus GLV-1h153 is effective in treating and preventing metastatic triple-negative breast cancer. Ann Surg 2012; 256: 437–445.
[39]	Wang H, Chen NG, Minev BR, Szalay AA. Oncolytic vaccinia virus GLV-1h68 strain shows enhanced replication in human breast cancer stem-like cells in comparison to breast cancer cells. J Transl Med 2012; 10: 167.