Changes in the expression of genes that regulate apoptosis as a factor in the development of chemoresistance in soft tissue sarcoma

Introduction. The active use of highly toxic chemotherapy in the treatment of soft tissue sarcomas determines the need to search for criteria and markers of chemoresistance of patients to the therapy.
Aim. To study the connection between tumor cell resistance to chemotherapy and expression levels of apoptosis-regulating proteins (PUMA, PMAIP-1, PIDD-1, AIFM-2, Bax, GADD45a) in primary cultures of soft tissue sarcomas.
Materials and methods. Primary cultures of soft tissue sarcomas were obtained using enzymatic digestion, cell death was evaluated using resazurine assay. Gene expression was measured using real-time polymerase chain reaction, protein levels using immunoblotting assay.
Results. 73 primary cultures of soft tissue sarcomas were obtained, for which chemosensitivity to doxorubicin, ifosfamide, docetaxel, gemcitabine, pazopanib and their combinations was determined using a resazurin cytotoxicity test. Associations of AIFM-2 gene expression with resistance to pazopanib, doxorubicin and its combination with ifosfamide were found in liposarcoma, synovial and undifferentiated pleomorphic sarcomas. In addition, associations between the expression of the Bax, PUMA, PMAIP-1, GADD45a and PIDD-1 genes and resistance to the studied drugs in various nosological subgroups of sarcomas were identified. When studying the amount of protein, it was revealed that undifferentiated pleomorphic and synovial sarcomas with a low content of GADD45a are more resistant to the studied drugs. Liposarcomas with high Bax expression are more resistant to docetaxel and gemcitabine, while synovial sarcomas with high Bax expression are more sensitive to doxorubicin and ifosfamide.
Conclusion. The data obtained indicate a relationship between the activity of the studied genes-regulators of apoptosis and resistance to drugs used in the treatment of soft tissue sarcomas.

1. Malignant neoplasms in Russia in 2018 (morbidity and mortality). Ed. by A.D. Kaprin, V.V. Starinsky, G.V. Petrov. Moscow: MNIOI im. P.A. Gertsena – filial FGBU “NMITS radiologii” Minzdrava Rossii, 2019. (In Russ.).

2. Sbaraglia M., Bellan E., Dei Tos A.P. The 2020 WHO classification of soft tissue tumours: news and perspectives. Pathologica 2021;113(2):70–84. DOI: 10.32074/1591-951x-213

3. Judson I., Verweij J., Gelderblom H. et al. Doxorubicin alone versus intensified doxorubicin plus ifosfamide for first-line treatment of advanced or metastatic soft-tissue sarcoma: a randomised controlled phase 3 trial. Lancet Oncol 2014;15(4):415–23. DOI: 10.1016/s1470-2045(14)70063-4

4. Neophytou C.M., Trougakos I.P., Erin N., Papageorgis P. Apoptosis deregulation and the development of cancer multi-drug resistance. Cancers 2021;13(17):4363. DOI: 10.3390/cancers13174363

5. Hanahan D. Hallmarks of cancer: new dimensions. Cancer Discov 2022;12(1):31–46. DOI: 10.1158/2159-8290.Cd-21-1059

6. Kirilin E.M., Fetisov T.I., Moiseeva N.I. et al. Soft tissue sarcoma study: association of genetic alterations in the apoptosis pathways with chemoresistance to doxorubicin. Cancers 2022;14(7):1796. DOI: 10.3390/cancers14071796

7. Comprehensive and integrated genomic characterization of adult soft tissue sarcomas. Cell 2017;171(4):950–65.e928. DOI: 10.1016/j.cell.2017.10.014

8. Peña-Blanco A., García-Sáez A.J. Bax, Bak and beyond – mitochondrial performance in apoptosis. FEBS J 2018;285(3):416–31. DOI: 10.1111/febs.14186

9. Weiler E.S., Szabo T.G., Garcia-Carpio I., Villunger A. PIDD1 in cell cycle control, sterile inflammation and cell death. Biochem Soc Trans 2022;50(2):813–24. DOI: 10.1042/bst20211186

10. Tan J.H., Cao R.C., Zhou L. et al. ATF6 aggravates acinar cell apoptosis and injury by regulating p53/AIFM2 transcription in Severe Acute Pancreatitis. Theranostics 2020;10(18):8298–314. DOI: 10.7150/thno.46934

11. Huang Y., Liu N., Liu J. et al. Mutant p53 drives cancer chemotherapy resistance due to loss of function on activating transcription of PUMA. Cell Cycle 2019;18(24):3442–55. DOI: 10.1080/15384101.2019.1688951

12. Shahbandi A., Rao S.G., Anderson A.Y. et al. BH3 mimetics selectively eliminate chemotherapy-induced senescent cells and improve response in TP53 wild-type breast cancer. Cell Death Differ 2020;27(11):3097–116. DOI: 10.1038/s41418-020-0564-6

13. Ren X., Liu H., Zhang M. et al. Co-expression of ING4 and P53 enhances hypopharyngeal cancer chemosensitivity to cisplatin in vivo. Mol Med Rep 2016;14(3):2431–8. DOI: 10.3892/mmr.2016.5552

14. Fairchild C.K., Floros, K.V., Jacob S. et al. Unmasking BCL-2 addiction in synovial sarcoma by overcoming low NOXA. Cancers 2021;13(1):2310. DOI: 10.3390/cancers13102310

15. Mathews J.C., Pouryahya M., Moosmüller C. et al. Molecular phenotyping using networks, diffusion, and topology: soft tissue sarcoma. Sci Rep 2019;9(1):13982. DOI: 10.1038/s41598-019-50300-2

16. Andreotti P.E., Cree I.A., Kurbacher C.M. et al. Chemosensitivity testing of human tumors using a microplate adenosine triphosphate luminescence assay: clinical correlation for cisplatin resistance of ovarian carcinoma. Cancer Res 1995;55(22):5276–82.

17. Tapias L.F., Gilpin S.E., Ren X. et al. Assessment of proliferation and cytotoxicity in a biomimetic three-dimensional model of lung cancer. Ann Thoracic Surg 2015;100(2):414–21. DOI: 10.1016/j.athoracsur.2015.04.035

18. Tanaka K., Ozaki T. Adjuvant and neoadjuvant chemotherapy for soft tissue sarcomas: JCOG Bone and Soft Tissue Tumor Study Group. Japanese J Clin Oncol 2021;51(2):180–4. DOI: 10.1093/jjco/hyaa231

19. Moiseeva N.I., Laletina L.A., Fetisov T.I. et al. Analysis of multiple drug resistance mechanism in different types of soft tissue sarcomas: assessment of the expression of ABC-transporters, MVP, YB-1, and analysis of their correlation with chemosensitivity of cancer cells. Int J Mol Sci 2022;3(6):3183. DOI: 10.3390/ijms23063183

20. De Graaff M.A., de Rooij M.A., van den Akker B.E. et al. Inhibition of Bcl-2 family members sensitises soft tissue leiomyosarcomas to chemotherapy. Br J Cancer 2016;114(11):1219–26. DOI: 10.1038/bjc.2016.117

21. Win T.T., Yusuf Y., Jaafa H. Apoptotic activities in soft tissue sarcoma: immunohistochemical study and their association with tumour characteristics. Malaysian J Med Sci 2013;20(2):10–6.

22. Muenchow A., Weller S., Hinterleitner C. et al. The BCL-2 selective inhibitor ABT-199 sensitizes soft tissue sarcomas to proteasome inhibition by a concerted mechanism requiring BAX and NOXA. Cell Death Disease 2020;11(8):701. DOI: 10.1038/s41419-020-02910-2

23. De Sousa Abreu R., Penalva L.O., Marcotte E.M., Vogel C. Global signatures of protein and mRNA expression levels. Mol biosyst 2009;5(12):1512–26. DOI: 10.1039/b908315d

24. Vogel C., Marcotte E.M. Insights into the regulation of protein abundance from proteomic and transcriptomic analyses. Nat Rev Genet 2012;13(4):227–32. DOI: 10.1038/nrg3185

25. Nakamura K., Asanuma K., Okamoto T. et al. Combination of everolimus and bortezomib inhibits the growth and metastasis of bone and soft tissue sarcomas via JNK/p38/ERK MAPK and AKT pathways. Cancers 2023;15(9): DOI: 10.3390/cancers15092468

26. Mauro A., Ciccarelli C., De Cesaris P.S. et al. PKCalpha-mediated ERK, JNK and p38 activation regulates the myogenic program in human rhabdomyosarcoma cells. J Cell Sci 2002;115(Pt. 18): 3587–99. DOI: 10.1242/jcs.00037

27. Ambroise G., Portier A., Roders N. et al. Subcellular localization of PUMA regulates its pro-apoptotic activity in Burkitt’s lymphoma B cells. Oncotarget 2015;6(35):38181–94. DOI: 10.18632/oncotarget.5901

28. Damerell V., Pepper M.S., Prince S. Molecular mechanisms underpinning sarcomas and implications for current and future therapy. Signal Transduction targ Ther 2021;6(1):246. DOI: 10.1038/s41392-021-00647-8

29. Guttà C., Rahman A., Aura C. et al. Low expression of pro-apoptotic proteins Bax, Bak and Smac indicates prolonged progression-free survival in chemotherapy-treated metastatic melanoma. Cell Death Dis 2020;11(2):124. DOI: 10.1038/s41419-020-2309-3

30. Köhler T., Schill C., Deininger M.W. et al. High Bad and Bax mRNA expression correlate with negative outcome in acute myeloid leukemia (AML). Leukemia 2002;16(1):22–9. DOI: 10.1038/sj.leu.2402340

31. Bairey O., Zimra Y., Shaklai M. et al. Bcl-2, Bcl-X, Bax, and Bak expression in short- and long-lived patients with diffuse large B-cell lymphomas. Clin Cancer Res 1999;5(10):2860–6.

32. Fecker L.F., Geilen C.C., Tchernev G. et al. Loss of proapoptotic Bcl-2-related multidomain proteins in primary melanomas is associated with poor prognosis. J Inv Dermatol 2006;126(6):1366–71. DOI: 10.1038/sj.jid.5700192