Abstract
Purpose
Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with poor survival outcomes. Metformin has been shown to have antitumor effects by lowering serum levels of the mitogen insulin and having pleiotropic effects on cancer cell signaling pathways. BMS-754807 is a potent and reversible inhibitor of both insulin-like growth factor 1 receptor (IGF-1R) and insulin receptor (IR). Both drugs have been reported to have some efficacy in TNBC. However, it is unclear whether the combination of the two drugs is more effective than single drug treatment in TNBC.
Methods
We treated a panel of TNBC cell lines with metformin and BMS-754807 alone and in combination and tested cell viability using MTS assays. We used the CompuSyn software to analyze for additivity, synergism, or antagonism. We also examined the molecular mechanism by performing reverse phase protein assay (RPPA) to detect the candidate pathways altered by single drugs and the drug combination and used Western blotting to verify and expand the findings.
Results
The combination of metformin and BMS-754807 showed synergy in 11 out of 13 TNBC cell lines tested (85%). RPPA analysis detected significant alterations by the drug combination of multiple proteins known to regulate cell cycle and tumor growth. In particular, the drug combination significantly increased levels of total and phosphorylated forms of the cell cycle inhibitor p27Kip1 and decreased the level of the p27Kip1 E3 ligase SCFSkp2.
Conclusions
We conclude that the combination of metformin and BMS-754807 is more effective than either drug alone in inhibiting cell proliferation in the majority of TNBC cell lines, and that one important mechanism may be suppression of SCFSkp2 and subsequent stabilization of the cell cycle inhibitor p27Kip1. This combination treatment may represent an effective targeted therapy for a significant subset of TNBC cases and should be further evaluated.
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Abbreviations
- TNBC:
-
Triple-negative breast cancer
- IGF-1R:
-
Insulin-like growth factor 1 receptor
- IGF:
-
Insulin-like growth factor
- IR:
-
Insulin receptor
- ER:
-
Estrogen receptor
- PR:
-
Progesterone receptor
- HER2:
-
Human epidermal growth factor receptor 2
- RPPA:
-
Reverse phase protein array
References
Miller KD, Nogueira L, Mariotto AB, Rowland JH, Yabroff KR, Alfano CM, Jemal A, Kramer JL, Siegel RL (2019) Cancer treatment and survivorship statistics, 2019. CA Cancer J Clin 69(5):363–385. https://doi.org/10.3322/caac.21565
Foulkes WD, Smith IE, Reis-Filho JS (2010) Triple-negative breast cancer. N Engl J Med 363(20):1938–1948. https://doi.org/10.1056/NEJMra1001389
Yee D (2018) Anti-insulin-like growth factor therapy in breast cancer. J Mol Endocrinol 61(1):T61–T68. https://doi.org/10.1530/JME-17-0261
Lee AV, Yee D (2011) Targeting IGF-1R: at a crossroad. Oncology 25(6):535–536
Belfiore A, Frasca F (2008) IGF and insulin receptor signaling in breast cancer. J Mammary Gland Biol Neoplasia 13(4):381–406. https://doi.org/10.1007/s10911-008-9099-z
Goodwin PJ, Ennis M, Pritchard KI, Trudeau ME, Koo J, Madarnas Y, Hartwick W, Hoffman B, Hood N (2002) Fasting insulin and outcome in early-stage breast cancer: results of a prospective cohort study. J Clin Oncol 20(1):42–51. https://doi.org/10.1200/JCO.2002.20.1.42
Papa V, Gliozzo B, Clark GM, McGuire WL, Moore D, Fujita-Yamaguchi Y, Vigneri R, Goldfine ID, Pezzino V (1993) Insulin-like growth factor-I receptors are overexpressed and predict a low risk in human breast cancer. Cancer Res 53(16):3736–3740
Osborne CK, Bolan G, Monaco ME, Lippman ME (1976) Hormone responsive human breast cancer in long-term tissue culture: effect of insulin. Proc Natl Acad Sci USA 73(12):4536–4540. https://doi.org/10.1073/pnas.73.12.4536
Pylayeva-Gupta Y, Grabocka E, Bar-Sagi D (2011) RAS oncogenes: weaving a tumorigenic web. Nat Rev Cancer 11(11):761–774. https://doi.org/10.1038/nrc3106
Vivanco I, Sawyers CL (2002) The phosphatidylinositol 3-Kinase AKT pathway in human cancer. Nat Rev Cancer 2(7):489–501. https://doi.org/10.1038/nrc839
Wittman MD, Carboni JM, Yang Z, Lee FY, Antman M, Attar R, Balimane P, Chang C, Chen C, Discenza L, Frennesson D, Gottardis MM, Greer A, Hurlburt W, Johnson W, Langley DR, Li A, Li J, Liu P, Mastalerz H, Mathur A, Menard K, Patel K, Sack J, Sang X, Saulnier M, Smith D, Stefanski K, Trainor G, Velaparthi U, Zhang G, Zimmermann K, Vyas DM (2009) Discovery of a 2,4-disubstituted pyrrolo[1,2-f][1,2,4]triazine inhibitor (BMS-754807) of insulin-like growth factor receptor (IGF-1R) kinase in clinical development. J Med Chem 52(23):7360–7363. https://doi.org/10.1021/jm900786r
Carboni JM, Wittman M, Yang Z, Lee F, Greer A, Hurlburt W, Hillerman S, Cao C, Cantor GH, Dell-John J, Chen C, Discenza L, Menard K, Li A, Trainor G, Vyas D, Kramer R, Attar RM, Gottardis MM (2009) BMS-754807, a small molecule inhibitor of insulin-like growth factor-1R/IR. Mol Cancer Ther 8(12):3341–3349. https://doi.org/10.1158/1535-7163.MCT-09-0499
Awasthi N, Zhang C, Ruan W, Schwarz MA, Schwarz RE (2012) BMS-754807, a small-molecule inhibitor of insulin-like growth factor-1 receptor/insulin receptor, enhances gemcitabine response in pancreatic cancer. Mol Cancer Ther 11(12):2644–2653. https://doi.org/10.1158/1535-7163.MCT-12-0447
Dayyani F, Parikh NU, Varkaris AS, Song JH, Moorthy S, Chatterji T, Maity SN, Wolfe AR, Carboni JM, Gottardis MM, Logothetis CJ, Gallick GE (2012) Combined Inhibition of IGF-1R/IR and Src family kinases enhances antitumor effects in prostate cancer by decreasing activated survival pathways. PLoS ONE 7(12):e51189. https://doi.org/10.1371/journal.pone.0051189
Franks SE, Jones RA, Briah R, Murray P, Moorehead RA (2016) BMS-754807 is cytotoxic to non-small cell lung cancer cells and enhances the effects of platinum chemotherapeutics in the human lung cancer cell line A549. BMC Res Notes 9:134. https://doi.org/10.1186/s13104-016-1919-4
Huang F, Hurlburt W, Greer A, Reeves KA, Hillerman S, Chang H, Fargnoli J, Graf Finckenstein F, Gottardis MM, Carboni JM (2010) Differential mechanisms of acquired resistance to insulin-like growth factor-i receptor antibody therapy or to a small-molecule inhibitor, BMS-754807, in a human rhabdomyosarcoma model. Cancer Res 70(18):7221–7231. https://doi.org/10.1158/0008-5472.CAN-10-0391
Litzenburger BC, Creighton CJ, Tsimelzon A, Chan BT, Hilsenbeck SG, Wang T, Carboni JM, Gottardis MM, Huang F, Chang JC, Lewis MT, Rimawi MF, Lee AV (2011) High IGF-IR activity in triple-negative breast cancer cell lines and tumorgrafts correlates with sensitivity to anti-IGF-IR therapy. Clin Cancer Res 17(8):2314–2327. https://doi.org/10.1158/1078-0432.CCR-10-1903
Desai J, Solomon BJ, Davis ID, Lipton LR, Hicks R, Scott AM, Park J, Clemens PL, Gestone TA, Finckenstein FG (2010) Phase I dose-escalation study of daily BMS-754807, an oral, dual IGF-1R/insulin receptor (IR) inhibitor in subjects with solid tumors. J Clin Oncol 28:3104. https://doi.org/10.1200/jco.2010.28.15_suppl.3104
Kolb EA, Gorlick R, Lock R, Carol H, Morton CL, Keir ST, Reynolds CP, Kang MH, Maris JM, Billups C, Smith MA, Houghton PJ (2011) Initial testing (stage 1) of the IGF-1 receptor inhibitor BMS-754807 by the pediatric preclinical testing program. Pediatr Blood Cancer 56(4):595–603. https://doi.org/10.1002/pbc.22741
Grunt TW, Mariani GL (2013) Novel approaches for molecular targeted therapy of breast cancer: interfering with PI3K/AKT/mTOR signaling. Curr Cancer Drug Targets 13(2):188–204. https://doi.org/10.2174/1568009611313020008
Zardavas D, Baselga J, Piccart M (2013) Emerging targeted agents in metastatic breast cancer. Nat Rev Clin Oncol 10(4):191–210. https://doi.org/10.1038/nrclinonc.2013.29
Bailey CJ, Turner RC (1996) Metformin. N Engl J Med 334(9):574–579. https://doi.org/10.1056/NEJM199602293340906
Salpeter SR, Buckley NS, Kahn JA, Salpeter EE (2008) Meta-analysis: metformin treatment in persons at risk for diabetes mellitus. Am J Med 121(2):149–157. https://doi.org/10.1016/j.amjmed.2007.09.016
Pernicova I, Korbonits M (2014) Metformin–mode of action and clinical implications for diabetes and cancer. Nat Rev Endocrinol 10(3):143–156. https://doi.org/10.1038/nrendo.2013.256
Kahn BB, Alquier T, Carling D, Hardie DG (2005) AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism. Cell Metab 1(1):15–25. https://doi.org/10.1016/j.cmet.2004.12.003
Rattan R, Giri S, Singh AK, Singh I (2005) 5-Aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside inhibits cancer cell proliferation in vitro and in vivo via AMP-activated protein kinase. J Biol Chem 280(47):39582–39593. https://doi.org/10.1074/jbc.M507443200
Evans JM, Donnelly LA, Emslie-Smith AM, Alessi DR, Morris AD (2005) Metformin and reduced risk of cancer in diabetic patients. BMJ 330(7503):1304–1305. https://doi.org/10.1136/bmj.38415.708634.F7
Giovannucci E, Harlan DM, Archer MC, Bergenstal RM, Gapstur SM, Habel LA, Pollak M, Regensteiner JG, Yee D (2010) Diabetes and cancer: a consensus report. Diabetes Care 33(7):1674–1685. https://doi.org/10.2337/dc10-0666
Bonanni B, Puntoni M, Cazzaniga M, Pruneri G, Serrano D, Guerrieri-Gonzaga A, Gennari A, Trabacca MS, Galimberti V, Veronesi P, Johansson H, Aristarco V, Bassi F, Luini A, Lazzeroni M, Varricchio C, Viale G, Bruzzi P, Decensi A (2012) Dual effect of metformin on breast cancer proliferation in a randomized presurgical trial. J Clin Oncol 30(21):2593–2600. https://doi.org/10.1200/JCO.2011.39.3769
Bayraktar S, Hernadez-Aya LF, Lei X, Meric-Bernstam F, Litton JK, Hsu L, Hortobagyi GN, Gonzalez-Angulo AM (2012) Effect of metformin on survival outcomes in diabetic patients with triple receptor-negative breast cancer. Cancer 118(5):1202–1211. https://doi.org/10.1002/cncr.26439
Jiralerspong S, Palla SL, Giordano SH, Meric-Bernstam F, Liedtke C, Barnett CM, Hsu L, Hung MC, Hortobagyi GN, Gonzalez-Angulo AM (2009) Metformin and pathologic complete responses to neoadjuvant chemotherapy in diabetic patients with breast cancer. J Clin Oncol 27(20):3297–3302. https://doi.org/10.1200/JCO.2009.19.6410
Camacho L, Dasgupta A, Jiralerspong S (2015) Metformin in breast cancer - an evolving mystery. Breast Cancer Res 17:88. https://doi.org/10.1186/s13058-015-0598-8
Hadad S, Iwamoto T, Jordan L, Purdie C, Bray S, Baker L, Jellema G, Deharo S, Hardie DG, Pusztai L, Moulder-Thompson S, Dewar JA, Thompson AM (2011) Evidence for biological effects of metformin in operable breast cancer: a pre-operative, window-of-opportunity, randomized trial. Breast Cancer Res Treat 128(3):783–794. https://doi.org/10.1007/s10549-011-1612-1
Liu B, Fan Z, Edgerton SM, Deng XS, Alimova IN, Lind SE, Thor AD (2009) Metformin induces unique biological and molecular responses in triple negative breast cancer cells. Cell Cycle 8(13):2031–2040. https://doi.org/10.4161/cc.8.13.8814
Lee JO, Kang MJ, Byun WS, Kim SA, Seo IH, Han JA, Moon JW, Kim JH, Kim SJ, Lee EJ, In Park S, Park SH, Kim HS (2019) Metformin overcomes resistance to cisplatin in triple-negative breast cancer (TNBC) cells by targeting RAD51. Breast Cancer Res 21(1):115. https://doi.org/10.1186/s13058-019-1204-2
Shi P, Liu W, Tala WH, Li F, Zhang H, Wu Y, Kong Y, Zhou Z, Wang C, Chen W, Liu R, Chen C (2017) Metformin suppresses triple-negative breast cancer stem cells by targeting KLF5 for degradation. Cell Discov 3:17010. https://doi.org/10.1038/celldisc.2017.10
Strekalova E, Malin D, Rajanala H, Cryns VL (2017) Metformin sensitizes triple-negative breast cancer to proapoptotic TRAIL receptor agonists by suppressing XIAP expression. Breast Cancer Res Treat 163(3):435–447. https://doi.org/10.1007/s10549-017-4201-0
Dowling RJ, Zakikhani M, Fantus IG, Pollak M, Sonenberg N (2007) Metformin inhibits mammalian target of rapamycin-dependent translation initiation in breast cancer cells. Cancer Res 67(22):10804–10812. https://doi.org/10.1158/0008-5472.CAN-07-2310
Zakikhani M, Dowling R, Fantus IG, Sonenberg N, Pollak M (2006) Metformin is an AMP kinase-dependent growth inhibitor for breast cancer cells. Cancer Res 66(21):10269–10273. https://doi.org/10.1158/0008-5472.CAN-06-1500
Creighton CJ, Huang S (2015) Reverse phase protein arrays in signaling pathways: a data integration perspective. Drug Des Dev Ther 9:3519–3527. https://doi.org/10.2147/DDDT.S38375
Chou TC (2010) Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Res 70(2):440–446. https://doi.org/10.1158/0008-5472.CAN-09-1947
Bu W, Liu Z, Jiang W, Nagi C, Huang S, Edwards DP, Jo E, Mo Q, Creighton CJ, Hilsenbeck SG, Leavitt AD, Lewis MT, Wong STC, Li Y (2019) Mammary precancerous stem and non-stem cells evolve into cancers of distinct subtypes. Cancer Res 79(1):61–71. https://doi.org/10.1158/0008-5472.CAN-18-1087
Lehmann BD, Bauer JA, Chen X, Sanders ME, Chakravarthy AB, Shyr Y, Pietenpol JA (2011) Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. J Clin Investig 121(7):2750–2767. https://doi.org/10.1172/JCI45014
Podust VN, Brownell JE, Gladysheva TB, Luo RS, Wang C, Coggins MB, Pierce JW, Lightcap ES, Chau V (2000) A Nedd8 conjugation pathway is essential for proteolytic targeting of p27Kip1 by ubiquitination. Proc Natl Acad Sci USA 97(9):4579–4584. https://doi.org/10.1073/pnas.090465597
Vlach J, Hennecke S, Amati B (1997) Phosphorylation-dependent degradation of the cyclin-dependent kinase inhibitor p27. EMBO J 16(17):5334–5344. https://doi.org/10.1093/emboj/16.17.5334
Carrano AC, Eytan E, Hershko A, Pagano M (1999) SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27. Nat Cell Biol 1(4):193–199
Tsvetkov LM, Yeh KH, Lee SJ, Sun H, Zhang H (1999) p27(Kip1) ubiquitination and degradation is regulated by the SCF(Skp2) complex through phosphorylated Thr187 in p27. Curr Biol 9(12):661–664. https://doi.org/10.1016/s0960-9822(99)80290-5
Nakayama KI, Nakayama K (2006) Ubiquitin ligases: cell-cycle control and cancer. Nat Rev Cancer 6(5):369–381. https://doi.org/10.1038/nrc1881
Chu IM, Hengst L, Slingerland JM (2008) The Cdk inhibitor p27 in human cancer: prognostic potential and relevance to anticancer therapy. Nat Rev Cancer 8(4):253–267. https://doi.org/10.1038/nrc2347
Fero ML, Randel E, Gurley KE, Roberts JM, Kemp CJ (1998) The murine gene p27Kip1 is haplo-insufficient for tumour suppression. Nature 396(6707):177–180. https://doi.org/10.1038/24179
Blain SW, Scher HI, Cordon-Cardo C, Koff A (2003) p27 as a target for cancer therapeutics. Cancer Cell 3(2):111–115. https://doi.org/10.1016/s1535-6108(03)00026-6
Kossatz U, Dietrich N, Zender L, Buer J, Manns MP, Malek NP (2004) Skp2-dependent degradation of p27kip1 is essential for cell cycle progression. Genes Dev 18(21):2602–2607. https://doi.org/10.1101/gad.321004
Bashir T, Dorrello NV, Amador V, Guardavaccaro D, Pagano M (2004) Control of the SCF(Skp2-Cks1) ubiquitin ligase by the APC/C(Cdh1) ubiquitin ligase. Nature 428(6979):190–193. https://doi.org/10.1038/nature02330
Pagano M, Benmaamar R (2003) When protein destruction runs amok, malignancy is on the loose. Cancer Cell 4(4):251–256. https://doi.org/10.1016/s1535-6108(03)00243-5
Liang J, Zubovitz J, Petrocelli T, Kotchetkov R, Connor MK, Han K, Lee JH, Ciarallo S, Catzavelos C, Beniston R, Franssen E, Slingerland JM (2002) PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest. Nat Med 8(10):1153–1160
Shin I, Yakes FM, Rojo F, Shin NY, Bakin AV, Baselga J, Arteaga CL (2002) PKB/Akt mediates cell-cycle progression by phosphorylation of p27(Kip1) at threonine 157 and modulation of its cellular localization. Nat Med 8(10):1145–1152
Viglietto G, Motti ML, Bruni P, Melillo RM, D'Alessio A, Califano D, Vinci F, Chiappetta G, Tsichlis P, Bellacosa A, Fusco A, Santoro M (2002) Cytoplasmic relocalization and inhibition of the cyclin-dependent kinase inhibitor p27(Kip1) by PKB/Akt-mediated phosphorylation in breast cancer. Nat Med 8(10):1136–1144
Grimmler M, Wang Y, Mund T, Cilensek Z, Keidel EM, Waddell MB, Jakel H, Kullmann M, Kriwacki RW, Hengst L (2007) Cdk-inhibitory activity and stability of p27Kip1 are directly regulated by oncogenic tyrosine kinases. Cell 128(2):269–280. https://doi.org/10.1016/j.cell.2006.11.047
Chu I, Sun J, Arnaout A, Kahn H, Hanna W, Narod S, Sun P, Tan CK, Hengst L, Slingerland J (2007) p27 phosphorylation by Src regulates inhibition of cyclin E-Cdk2. Cell 128(2):281–294. https://doi.org/10.1016/j.cell.2006.11.049
Murphree AL, Benedict WF (1984) Retinoblastoma: clues to human oncogenesis. Science 223(4640):1028–1033. https://doi.org/10.1126/science.6320372
Lindvall C, Bu W, Williams BO, Li Y (2007) Wnt signaling, stem cells, and the cellular origin of breast cancer. Stem Cell Rev 3(2):157–168
Nakai K, Hung MC, Yamaguchi H (2016) A perspective on anti-EGFR therapies targeting triple-negative breast cancer. Am J Cancer Res 6(8):1609–1623
Crane R, Gadea B, Littlepage L, Wu H, Ruderman JV (2004) Aurora A, meiosis and mitosis. Biol Cell 96(3):215–229. https://doi.org/10.1016/j.biolcel.2003.09.008
Xu J, Wu X, Zhou WH, Liu AW, Wu JB, Deng JY, Yue CF, Yang SB, Wang J, Yuan ZY, Liu Q (2013) Aurora-A identifies early recurrence and poor prognosis and promises a potential therapeutic target in triple negative breast cancer. PLoS ONE 8(2):e56919. https://doi.org/10.1371/journal.pone.0056919
Zhang H, Liu X, Warden CD, Huang Y, Loera S, Xue L, Zhang S, Chu P, Zheng S, Yen Y (2014) Prognostic and therapeutic significance of ribonucleotide reductase small subunit M2 in estrogen-negative breast cancers. BMC Cancer 14:664. https://doi.org/10.1186/1471-2407-14-664
Robson M, Im SA, Senkus E, Xu B, Domchek SM, Masuda N, Delaloge S, Li W, Tung N, Armstrong A, Wu W, Goessl C, Runswick S, Conte P (2017) Olaparib for metastatic breast cancer in patients with a germline BRCA mutation. N Engl J Med 377(6):523–533. https://doi.org/10.1056/NEJMoa1706450
Litton JK, Rugo HS, Ettl J, Hurvitz SA, Goncalves A, Lee KH, Fehrenbacher L, Yerushalmi R, Mina LA, Martin M, Roche H, Im YH, Quek RGW, Markova D, Tudor IC, Hannah AL, Eiermann W, Blum JL (2018) Talazoparib in patients with advanced breast cancer and a germline BRCA mutation. N Engl J Med 379(8):753–763. https://doi.org/10.1056/NEJMoa1802905
Schmid P, Adams S, Rugo HS, Schneeweiss A, Barrios CH, Iwata H, Dieras V, Hegg R, Im SA, Shaw Wright G, Henschel V, Molinero L, Chui SY, Funke R, Husain A, Winer EP, Loi S, Emens LA (2018) Atezolizumab and Nab-paclitaxel in advanced triple-negative breast cancer. N Engl J Med 379(22):2108–2121. https://doi.org/10.1056/NEJMoa1809615
Acknowledgements
This work was supported in part by the following grants: Susan G. Komen Foundation CCR13263802 (S.J.), National Institutes of Health R01CA204926 (Y.L.) and P50CA186784 Project 3 (Y.L.), Cancer Prevention & Research Institute of Texas Proteomics & Metabolomics Core Facility Support Award (Grant No. RP170005) (S.H.), and NCI Cancer Center Support Grant to Antibody-based Proteomics Core/Shared Resource (Grant No. P30CA125123) (S.H.). L.X. was supported in part by a scholarship from the Chinese Scholarship Council (CSC) (Grant Number: 201406860002). We thank Drs. Kimal Rajapakshe and Cristian Coarfa for RPPA data processing and normalization, and Ms. Fuli Jia and Dr. Danli Wu from the Antibody-based Proteomics Core/Shared Resource for their excellent technical assistant in performing RPPA experiments.
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Xue, L., Chen, F., Yue, F. et al. Metformin and an insulin/IGF-1 receptor inhibitor are synergistic in blocking growth of triple-negative breast cancer. Breast Cancer Res Treat 185, 73–84 (2021). https://doi.org/10.1007/s10549-020-05927-5
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DOI: https://doi.org/10.1007/s10549-020-05927-5