Immunosuppressive Effects and Potent Anti-tumor Efficacy of mTOR Inhibitor Everolimus in Breast Tumor-bearing Mice
-
Davoud Rostamzadeh
,
Mohammad Reza Haghshenas
,
Mahdi Samadi
,
Zahra Mojtahedi
,
Zohreh Babaloo
,
Abbas Ghaderi
Abstract
To investigate the effects of everolimus, a mechanistic/mammalian target of rapamycin (mTOR) inhibitor, on tumor growth and immune response in a mouse model of breast cancer.
Human hormone receptor-positive (HR+)/human epidermal growth receptor 2-negative (HER2-) MC4-L2 cell line was used to establish a mouse model of breast cancer. The inhibitory effects of high (10 mg/kg) and low (5 mg/kg) doses of everolimus were investigated on tumor growth. Additionally, the frequency of CD4+Foxp3+ regulatory T cells (Tregs), CD8+Foxp3+ Tregs, and CD4+ and CD8+ T cells expressing cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) was explored by flow cytometry in bone marrow, lymph nodes, and spleen.
Our results showed that both 10 mg/kg and 5 mg/kg doses of everolimus efficiently inhibited tumor growth, resulting in reduced breast tumor volume. In addition, it was revealed that everolimus-treated mice induced a higher frequency of CD4+Foxp3+ Tregs, CD8+Foxp3+ Tregs, and CD4+Foxp3+CTLA-4+ Tregs as well as CD4+ and CD8+ T cells expressing CTLA-4 in their bone marrow, lymph nodes, and spleen compared with standard control (vehicle-treated) in a dose-dependent manner. Furthermore, we found that everolimus treatment with 10 mg/kg and 5 mg/kg increased the frequency of Helios+Foxp3+ Tregs in the bone marrow of treated mice compared with the control group.
Our results indicate that treatment with everolimus not only inhibits tumor growth but also exerts an immunomodulatory effect by inducing Tregs in the lymphoid organs of breast cancer-bearing mice. The combination of therapy with other anti-cancer agents may negate immune suppression and improve the efficacy of mTOR-targeted breast cancer therapy.
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2. Dancey J. mTOR signaling and drug development in cancer. Nat Rev Clin Oncol. 2010;7(4):209-19.
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4. Kim LC, Cook RS, Chen J. mTORC1 and mTORC2 in cancer and the tumor microenvironment. Oncogene. 2017;36(16):2191-201.
5. Bollard J, Couderc C, Blanc M, Poncet G, Lepinasse F, Hervieu V, et al. Antitumor effect of everolimus in preclinical models of high-grade gastroenteropancreatic neuroendocrine carcinomas. Neuroendocrinology. 2013;97(4):331-40.
6. Du L, Li X, Zhen L, Chen W, Mu L, Zhang Y, et al. Everolimus inhibits breast cancer cell growth through PI3K/AKT/mTOR signaling pathway. Mol Med Rep. 2018;17(5):7163-9.
7. Boulay A, Zumstein-Mecker S, Stephan C, Beuvink I, Zilbermann F, Haller R, et al. Antitumor efficacy of intermittent treatment schedules with the rapamycin derivative RAD001 correlates with prolonged inactivation of ribosomal protein S6 kinase 1 in peripheral blood mononuclear cells. Cancer Res. 2004;64(1):252-61.
8. Yoshida-Ichikawa Y, Tanabe M, Tokuda E, Shimizu H, Horimoto Y, Miura K, et al. Overcoming the adverse effects of everolimus to achieve maximum efficacy in the treatment of inoperable breast cancer: a review of 11 cases at our hospital. Case Rep Oncol. 2018;11(2):511-20.
9. Powell JD, Pollizzi KN, Heikamp EB, Horton MR. Regulation of immune responses by mTOR. Annu Rev Immunol. 2012;30(3):39-68.
10. Nazari N, Jafari F, Ghalamfarsa G, Hadinia A, Atapour A, Ahmadi M, et al. The emerging role of microRNA in regulating the mTOR signaling pathway in immune and inflammatory responses. Immunol Cell Biol. 2021;1(2):19-21.
11. Delgoffe GM, Pollizzi KN, Waickman AT, Heikamp E, Meyers DJ, Horton MR, et al. The kinase mTOR regulates the differentiation of helper T cells through the selective activation of signaling by mTORC1 and mTORC2. Nat Immunol. 2011;12(4):295-303.
12. Rostamzadeh D, Yousefi M, Haghshenas MR, Ahmadi M, Dolati S, Babaloo Z. mTOR Signaling pathway as a master regulator of memory CD8(+) T-cells, Th17, and NK cells development and their functional properties. J Cell Physiol. 2019.
13. Delgoffe GM, Kole TP, Zheng Y, Zarek PE, Matthews KL, Xiao B, et al. The mTOR kinase differentially regulates effector and regulatory T cell lineage commitment. Immunity. 2009;30(6):832-44.
14. Battaglia M, Stabilini A, Roncarolo MG. Rapamycin selectively expands CD4+CD25+FoxP3+ regulatory T cells. Blood. 2005;105(12):4743-8.
15. Noris M, Casiraghi F, Todeschini M, Cravedi P, Cugini D, Monteferrante G, et al. Regulatory T cells and T cell depletion: role of immunosuppressive drugs. J Am Soc Nephrol. 2007;18(3):1007-18.
16. Kruisbeek AM. Isolation of mouse mononuclear cells. Curr Protoc Immunol. 2001;Chapter 3:Unit 3 1.
17. Rostamzadeh D, Haghshenas MR, Daryanoosh F, Samadi M, Hosseini A, Ghaderi A, et al. Altered frequency of CD8(+) CD11c(+) T cells and expression of immunosuppressive molecules in lymphoid organs of mouse model of colorectal cancer. J Cell Physiol. 2019;234(7):11986-98.
18. Swamydas M, Lionakis MS. Isolation, purification and labeling of mouse bone marrow neutrophils for functional studies and adoptive transfer experiments. J Vis Exp. 2013(77):e50586.
19. Zhu Y, Zhang X, Liu Y, Zhang S, Liu J, Ma Y, et al. Antitumor effect of the mTOR inhibitor everolimus in combination with trastuzumab on human breast cancer stem cells in vitro and in vivo. Tumor Biol. 2012;33(5):1349-62.
20. Browne AJ, Kubasch ML, Göbel A, Hadji P, Chen D, Rauner M, et al. Concurrent antitumor and bone-protective effects of everolimus in osteotropic breast cancer. Breast Cancer Res. 2017;19(1):1-15.
21. Ariaans G, Jalving M, De Vries EGE, De Jong S. Anti-tumor effects of everolimus and metformin are complementary and glucose-dependent in breast cancer cells. BMC cancer. 2017;17(1):232.
22. Gorshtein A, Rubinfeld H, Kendler E, Theodoropoulou M, Cerovac V, Stalla GK, et al. Mammalian target of rapamycin inhibitors rapamycin and RAD001 (everolimus) induce anti-proliferative effects in GH-secreting pituitary tumor cells in vitro. Endocrine-related cancer. 2009;16(3):1017-27.
23. Guo H, Zhong Y, Jackson AL, Clark LH, Kilgore J, Zhang L, et al. Everolimus exhibits anti-tumorigenic activity in obesity-induced ovarian cancer. Oncotarget. 2016;7(15):20338.
24. Owonikoko TK, Zhang G, Lallani SB, Chen Z, Martinson DE, Khuri FR, et al. Evaluation of preclinical efficacy of everolimus and pasireotide in thyroid cancer cell lines and xenograft models. PLoS One. 2019;14(2):e0206309.
25. Lui A, New J, Ogony J, Thomas S, Lewis-Wambi J. Everolimus downregulates estrogen receptor and induces autophagy in aromatase inhibitor-resistant breast cancer cells. BMC cancer. 2016;16(1):1-15.
26. Battaglia M, Stabilini A, Migliavacca B, Horejs-Hoeck J, Kaupper T, Roncarolo MG. Rapamycin promotes expansion of functional CD4+CD25+FOXP3+ regulatory T cells of both healthy subjects and type 1 diabetic patients. J Immunol. 2006;177(12):8338-47.
27. Strauss L, Whiteside TL, Knights A, Bergmann C, Knuth A, Zippelius A. Selective survival of naturally occurring human CD4+CD25+Foxp3+ regulatory T cells cultured with rapamycin. J Immunol. 2007;178(1):320-9.
28. Sugimoto N, Oida T, Hirota K, Nakamura K, Nomura T, Uchiyama T, et al. Foxp3-dependent and -independent molecules specific for CD25+CD4+ natural regulatory T cells revealed by DNA microarray analysis. Int Immunol. 2006;18(8):1197-209.
29. Akimova T, Beier UH, Wang L, Levine MH, Hancock WW. Helios expression is a marker of T cell activation and proliferation. PloS one. 2011;6(8):e24226.
30. Kim H-J, Barnitz RA, Kreslavsky T, Brown FD, Moffett H, Lemieux ME, et al. Stable inhibitory activity of regulatory T cells requires the transcription factor Helios. Science. 2015;350(6258):334-9.
31. Thornton AM, Korty PE, Tran DQ, Wohlfert EA, Murray PE, Belkaid Y, et al. Expression of Helios, an Ikaros transcription factor family member, differentiates thymic-derived from peripherally induced Foxp3+ T regulatory cells. J Immunol. 2010;184(7):3433-41.
32. Zabransky DJ, Nirschl CJ, Durham NM, Park BV, Ceccato CM, Bruno TC, et al. Phenotypic and functional properties of Helios+ regulatory T cells. PloS one. 2012;7(3):e34547.
33. Huijts CM, Santegoets SJ, Quiles Del Rey M, de Haas RR, Verheul HM, de Gruijl TD, et al. Differential effects of inhibitors of the PI3K/mTOR pathway on the expansion and functionality of regulatory T cells. Clin Immunol. 2016;168:47-54.
34. Li X, Li D, Shi Q, Huang X, Ju X. Umbilical cord bloodderived Heliospositive regulatory T cells promote angiogenesis in acute lymphoblastic leukemia in mice via CCL22 and the VEGFAVEGFR2 pathway. Mol Med Rep. 2019;19(5):4195-204.
35. Serre K, Benezech C, Desanti G, Bobat S, Toellner KM, Bird R, et al. Helios is associated with CD4 T cells differentiating to T helper 2 and follicular helper T cells in vivo independently of Foxp3 expression. PLoS One. 2011;6(6):e20731.
2. Dancey J. mTOR signaling and drug development in cancer. Nat Rev Clin Oncol. 2010;7(4):209-19.
3. Yang J, Nie J, Ma X, Wei Y, Peng Y, Wei X. Targeting PI3K in cancer: mechanisms and advances in clinical trials. Mol Cancer. 2019;18(1):1-28.
4. Kim LC, Cook RS, Chen J. mTORC1 and mTORC2 in cancer and the tumor microenvironment. Oncogene. 2017;36(16):2191-201.
5. Bollard J, Couderc C, Blanc M, Poncet G, Lepinasse F, Hervieu V, et al. Antitumor effect of everolimus in preclinical models of high-grade gastroenteropancreatic neuroendocrine carcinomas. Neuroendocrinology. 2013;97(4):331-40.
6. Du L, Li X, Zhen L, Chen W, Mu L, Zhang Y, et al. Everolimus inhibits breast cancer cell growth through PI3K/AKT/mTOR signaling pathway. Mol Med Rep. 2018;17(5):7163-9.
7. Boulay A, Zumstein-Mecker S, Stephan C, Beuvink I, Zilbermann F, Haller R, et al. Antitumor efficacy of intermittent treatment schedules with the rapamycin derivative RAD001 correlates with prolonged inactivation of ribosomal protein S6 kinase 1 in peripheral blood mononuclear cells. Cancer Res. 2004;64(1):252-61.
8. Yoshida-Ichikawa Y, Tanabe M, Tokuda E, Shimizu H, Horimoto Y, Miura K, et al. Overcoming the adverse effects of everolimus to achieve maximum efficacy in the treatment of inoperable breast cancer: a review of 11 cases at our hospital. Case Rep Oncol. 2018;11(2):511-20.
9. Powell JD, Pollizzi KN, Heikamp EB, Horton MR. Regulation of immune responses by mTOR. Annu Rev Immunol. 2012;30(3):39-68.
10. Nazari N, Jafari F, Ghalamfarsa G, Hadinia A, Atapour A, Ahmadi M, et al. The emerging role of microRNA in regulating the mTOR signaling pathway in immune and inflammatory responses. Immunol Cell Biol. 2021;1(2):19-21.
11. Delgoffe GM, Pollizzi KN, Waickman AT, Heikamp E, Meyers DJ, Horton MR, et al. The kinase mTOR regulates the differentiation of helper T cells through the selective activation of signaling by mTORC1 and mTORC2. Nat Immunol. 2011;12(4):295-303.
12. Rostamzadeh D, Yousefi M, Haghshenas MR, Ahmadi M, Dolati S, Babaloo Z. mTOR Signaling pathway as a master regulator of memory CD8(+) T-cells, Th17, and NK cells development and their functional properties. J Cell Physiol. 2019.
13. Delgoffe GM, Kole TP, Zheng Y, Zarek PE, Matthews KL, Xiao B, et al. The mTOR kinase differentially regulates effector and regulatory T cell lineage commitment. Immunity. 2009;30(6):832-44.
14. Battaglia M, Stabilini A, Roncarolo MG. Rapamycin selectively expands CD4+CD25+FoxP3+ regulatory T cells. Blood. 2005;105(12):4743-8.
15. Noris M, Casiraghi F, Todeschini M, Cravedi P, Cugini D, Monteferrante G, et al. Regulatory T cells and T cell depletion: role of immunosuppressive drugs. J Am Soc Nephrol. 2007;18(3):1007-18.
16. Kruisbeek AM. Isolation of mouse mononuclear cells. Curr Protoc Immunol. 2001;Chapter 3:Unit 3 1.
17. Rostamzadeh D, Haghshenas MR, Daryanoosh F, Samadi M, Hosseini A, Ghaderi A, et al. Altered frequency of CD8(+) CD11c(+) T cells and expression of immunosuppressive molecules in lymphoid organs of mouse model of colorectal cancer. J Cell Physiol. 2019;234(7):11986-98.
18. Swamydas M, Lionakis MS. Isolation, purification and labeling of mouse bone marrow neutrophils for functional studies and adoptive transfer experiments. J Vis Exp. 2013(77):e50586.
19. Zhu Y, Zhang X, Liu Y, Zhang S, Liu J, Ma Y, et al. Antitumor effect of the mTOR inhibitor everolimus in combination with trastuzumab on human breast cancer stem cells in vitro and in vivo. Tumor Biol. 2012;33(5):1349-62.
20. Browne AJ, Kubasch ML, Göbel A, Hadji P, Chen D, Rauner M, et al. Concurrent antitumor and bone-protective effects of everolimus in osteotropic breast cancer. Breast Cancer Res. 2017;19(1):1-15.
21. Ariaans G, Jalving M, De Vries EGE, De Jong S. Anti-tumor effects of everolimus and metformin are complementary and glucose-dependent in breast cancer cells. BMC cancer. 2017;17(1):232.
22. Gorshtein A, Rubinfeld H, Kendler E, Theodoropoulou M, Cerovac V, Stalla GK, et al. Mammalian target of rapamycin inhibitors rapamycin and RAD001 (everolimus) induce anti-proliferative effects in GH-secreting pituitary tumor cells in vitro. Endocrine-related cancer. 2009;16(3):1017-27.
23. Guo H, Zhong Y, Jackson AL, Clark LH, Kilgore J, Zhang L, et al. Everolimus exhibits anti-tumorigenic activity in obesity-induced ovarian cancer. Oncotarget. 2016;7(15):20338.
24. Owonikoko TK, Zhang G, Lallani SB, Chen Z, Martinson DE, Khuri FR, et al. Evaluation of preclinical efficacy of everolimus and pasireotide in thyroid cancer cell lines and xenograft models. PLoS One. 2019;14(2):e0206309.
25. Lui A, New J, Ogony J, Thomas S, Lewis-Wambi J. Everolimus downregulates estrogen receptor and induces autophagy in aromatase inhibitor-resistant breast cancer cells. BMC cancer. 2016;16(1):1-15.
26. Battaglia M, Stabilini A, Migliavacca B, Horejs-Hoeck J, Kaupper T, Roncarolo MG. Rapamycin promotes expansion of functional CD4+CD25+FOXP3+ regulatory T cells of both healthy subjects and type 1 diabetic patients. J Immunol. 2006;177(12):8338-47.
27. Strauss L, Whiteside TL, Knights A, Bergmann C, Knuth A, Zippelius A. Selective survival of naturally occurring human CD4+CD25+Foxp3+ regulatory T cells cultured with rapamycin. J Immunol. 2007;178(1):320-9.
28. Sugimoto N, Oida T, Hirota K, Nakamura K, Nomura T, Uchiyama T, et al. Foxp3-dependent and -independent molecules specific for CD25+CD4+ natural regulatory T cells revealed by DNA microarray analysis. Int Immunol. 2006;18(8):1197-209.
29. Akimova T, Beier UH, Wang L, Levine MH, Hancock WW. Helios expression is a marker of T cell activation and proliferation. PloS one. 2011;6(8):e24226.
30. Kim H-J, Barnitz RA, Kreslavsky T, Brown FD, Moffett H, Lemieux ME, et al. Stable inhibitory activity of regulatory T cells requires the transcription factor Helios. Science. 2015;350(6258):334-9.
31. Thornton AM, Korty PE, Tran DQ, Wohlfert EA, Murray PE, Belkaid Y, et al. Expression of Helios, an Ikaros transcription factor family member, differentiates thymic-derived from peripherally induced Foxp3+ T regulatory cells. J Immunol. 2010;184(7):3433-41.
32. Zabransky DJ, Nirschl CJ, Durham NM, Park BV, Ceccato CM, Bruno TC, et al. Phenotypic and functional properties of Helios+ regulatory T cells. PloS one. 2012;7(3):e34547.
33. Huijts CM, Santegoets SJ, Quiles Del Rey M, de Haas RR, Verheul HM, de Gruijl TD, et al. Differential effects of inhibitors of the PI3K/mTOR pathway on the expansion and functionality of regulatory T cells. Clin Immunol. 2016;168:47-54.
34. Li X, Li D, Shi Q, Huang X, Ju X. Umbilical cord bloodderived Heliospositive regulatory T cells promote angiogenesis in acute lymphoblastic leukemia in mice via CCL22 and the VEGFAVEGFR2 pathway. Mol Med Rep. 2019;19(5):4195-204.
35. Serre K, Benezech C, Desanti G, Bobat S, Toellner KM, Bird R, et al. Helios is associated with CD4 T cells differentiating to T helper 2 and follicular helper T cells in vivo independently of Foxp3 expression. PLoS One. 2011;6(6):e20731.
| Files | ||
| Issue | Vol 21 No 3 (2022) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijaai.v21i3.9802 | |
| Keywords | ||
| CTLA-4 antigen Everolimus Regulatory T-lymphocytes | ||
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How to Cite
1.
Rostamzadeh D, Haghshenas MR, Samadi M, Mojtahedi Z, Babaloo Z, Ghaderi A. Immunosuppressive Effects and Potent Anti-tumor Efficacy of mTOR Inhibitor Everolimus in Breast Tumor-bearing Mice. Iran J Allergy Asthma Immunol. 2022;21(3):287-299.
