Protective autophagy by thymidine causes resistance to rapamycin in colorectal cancer cells in vitro

Abstract

Aim: Thynidine phosphorylase (TP) acts as a proangiogenic growth factor which may regulate mammalian Target of Rapamycin (mTOR). We investigated whether the TP substrate thymidine and overexpression of TP affected mTOR signaling by comparing Colo320 (TP deficient) cells and its TP-transfected variant (Colo320TP1).

Methods: Drug resistance was assessed with the sulforhodamine B assay, protein expression with Western blotting, cell cycle distribution and cell death with Fluorescence-activated cell sorting analysis, and autophagy with immunofluorescence.

Results: Colo320 and Colo320TP1 cells had comparable levels of sensitivity to the mTOR inhibitor rapamycin. Thymidine treatment led to 13- and 50-fold resistance to rapamycin in Colo320 and Colo320TP1 cells, respectively. In Colo320TP1 cells, the thymidine phosphorylase inhibitor (TPI) reversed the thymidine induced resistance to rapamycin, but not in Colo320 cells, indicating a role for TP in the protection. Thymidine increased p70/S6k-phosphorylation (downstream of mTOR) in Colo320TP1, but it was not affected in Colo320. As a mechanism behind resistance, we studied the levels of autophagy and found that, in Colo320TP1 cells, autophagy was highly induced by thymidine-rapamycin, which was decreased by TPI. In addition, the autophagy inhibitor 3-methyl-adenine completely inhibited autophagy and its protection.

Conclusion: Rapamycin resistance in TP-expressing cancer cells may therefore be related to thymidine-mediated autophagy activation.

Keywords

Thymidine phosphorylase / mTOR / rapamycin / thymidine / thymidine phosphorylase inhibitor / autophagy

References

[1] Bruin M, Temmink OH, Hoekman K, Pinedo HM, Peters GJ. Role of platelet derived endothelial cell growth factor/thymidine phosphorylase in health and disease.Cancer Therapy2006;4:99-124

[2] Bronckaers A,Balzarini J.The dual role of thymidine phosphorylase in cancer development and chemotherapy.Med Res Rev2009;29:903-53 PMCID:PMC7168469

[3] Ackland SP.Thymidine phosphorylase: its role in sensitivity and resistance to anticancer drugs.Drug Resist Updat1999;2:205-14

[4] Brown NS.Thymidine phosphorylase, 2-deoxy-D-ribose and angiogenesis.Biochem J1998;334:1-8 PMCID:PMC1219653

[5] Liekens S,Pérez-Pérez MJ.Targeting platelet-derived endothelial cell growth factor/thymidine phosphorylase for cancer therapy.Biochem Pharmacol2007;74:1555-67

[6] Matsushita S,Furukawa T.The effect of a thymidine phosphorylase inhibitor on angiogenesis and apoptosis in tumors.Cancer Res1999;59:1911-6

[7] Mayer RJ,Falcone A.Randomized trial of TAS-102 for refractory metastatic colorectal cancer.N Engl J Med2015;372:1909-19

[8] Peters GJ.Therapeutic potential of TAS-102 in the treatment of gastrointestinal malignancies.Ther Adv Med Oncol2015;7;340-56 PMCID:PMC4622302

[9] Peters GJ.TAS-102: more than an antimetabolite.Lancet Oncol2012;13:e518-9

[10] Bijnsdorp IV,Jansen EE.Accumulation of thymidine-derived sugars in thymidine phosphorylase overexpressing cells.Biochem Pharmacol2010;80:786-92

[11] Hotchkiss KA,Schwartz EL.Thymidine phosphorylase and 2-deoxyribose stimulate human endothelial cell migration by specific activation of the integrins alpha 5 beta 1 and alpha V beta 3.J Biol Chem2003;278:19272-9

[12] Sengupta S,Matheson HB.Thymidine phosphorylase induces angiogenesis in vivo and in vitro: An evaluation of possible mechanisms.Br J Pharmacol2003;139:219-31 PMCID:PMC1573835

[13] Stevenson DP,Collins WP.Effects of platelet-derived endothelial cell growth factor/thymidine phosphorylase, substrate, and products in a three-dimensional model of angiogenesis.Am J Pathol1998;152:1641-6 PMCID:PMC1858429

[14] Azam M,Roman S.Addition of 2-deoxy-d-ribose to clinically used alginate dressings stimulates angiogenesis and accelerates wound healing in diabetic rats.J Biomater Appl2019;34:463-75

[15] Vara D,Fortunato TM.Direct Activation of NADPH Oxidase 2 by 2-Deoxyribose-1-Phosphate Triggers Nuclear Factor Kappa B-Dependent Angiogenesis.Antioxid Redox Signal2018;28:110-30 PMCID:PMC5725637

[16] Pula G,Dunn WB.Paracrine stimulation of endothelial cell motility and angiogenesis by platelet-derived deoxyribose-1-phosphate.Arterioscler Thromb Vasc Biol2010;30:2631-8 PMCID:PMC7528120

[17] Bijnsdorp IV,Kruyt FA.Thymidine phosphorylase in cancer cells stimulates human endothelial cell migration and invasion by the secretion of angiogenic factors.Br J Cancer2011;104:1185-92 PMCID:PMC3068508

[18] Brown NS,Fujiyama C,Bicknell R.Thymidine phosphorylase induces carcinoma cell oxidative stress and promotes secretion of angiogenic factors.Cancer Res2000;60:6298-302

[19] Bijnsdorp IV,Laan AC,Peters GJ.The role of platelet-derived endothelial cell growth factor/thymidine phosphorylase in tumor behavior.Nucleosides Nucleotides Nucleic Acids2008;27:681-91

[20] Seeliger H,Koehl GE.Blockage of 2-deoxy-D-ribose-induced angiogenesis with rapamycin counteracts a thymidine phosphorylase-based escape mechanism available for colon cancer under 5-fluorouracil therapy.Clin Cancer Res2004;10:1843-52

[21] Chiang GG.Targeting the mTOR signaling network in cancer.Trends Mol Med2007;13:433-42

[22] Zou Z,Li H.mTOR signaling pathway and mTOR inhibitors in cancer: progress and challenges.Cell Biosci2020;10:31 PMCID:PMC7063815

[23] Klionsky D,Abe A.Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition).Autophagy2016;12:1-222 PMCID:PMC4835977

[24] Bijnsdorp IV,Temmink OH,Kruyt FA.Differential activation of cell death and autophagy results in an increased cytotoxic potential for trifluorothymidine compared to 5-fluorouracil in colon cancer cells.Int J Cancer2010;126:2457-68

[25] Guo XL,Hu F.Targeting autophagy potentiates chemotherapy-induced apoptosis and proliferation inhibition in hepatocarcinoma cells.Cancer Lett2012;320:171-9

[26] de la Cruz-Morcillo MA.P38MAPK is a major determinant of the balance between apoptosis and autophagy triggered by 5-fluorouracil: implication in resistance.Oncogene2012;31:1073-85

[27] Selvakumaran M,Vasilevskaya IA.Autophagy inhibition sensitizes colon cancer cells to antiangiogenic and cytotoxic therapy.Clin Cancer Res2013;19:2995-3007

[28] Karasic TB,Loaiza-Bonilla A.Effect of gemcitabine and nab-paclitaxel with or without hydroxychloroquine on patients with advanced pancreatic cancer: a phase 2 randomized clinical trial.JAMA Oncol2019;5:993-8 PMCID:PMC6547080

[29] Rosenfeld MR,Supko JG.A phase I/II trial of hydroxychloroquine in conjunction with radiation therapy and concurrent and adjuvant temozolomide in patients with newly diagnosed glioblastoma multiforme.Autophagy2014;10:1359-68 PMCID:PMC4203513

[30] de Bruin M,Van der Born K.Role of platelet-derived endothelial cell growth factor/thymidine phosphorylase in fluoropyrimidine sensitivity.Br J Cancer2003;88:957-64 PMCID:PMC2377090

[31] Keepers YP,Peters GJ,Winograd B.Comparison of the sulforhodamine B protein and tetrazolium (MTT) assays for in vitro chemosensitivity testing.Eur J Cancer1991;27:897-900

[32] Bijnsdorp IV,Gokoel S,Peters GJ.Synergistic interaction between trifluorothymidine and docetaxel is sequence dependent.Cancer Sci2008;99:2302-8

[33] Emura T,Fujioka A,Kitazato K.Thymidine kinase and thymidine phosphorylase level as the main predictive parameter for sensitivity to TAS-102 in a mouse model.Oncol Rep2004;11:381-7

[34] Temmink OH,de Bruin M,Peters GJ.Therapeutic potential of the dual-targeted TAS-102 formulation in the treatment of gastrointestinal malignancies.Cancer Sci2007;98:779-89

[35] Rots MG,Kaspers GJL.Differential methotrexate resistance in childhood T- versus Common/PreB-Acute lymphoblastic Leukemia can be measured by an in situ thymidylate synthase inhibition assay, but not by the MTT assay.Blood1999;93:1067-74

[36] White E.The double-edged sword of autophagy modulation in cancer.Clin Cancer Res2009;15:5308-16 PMCID:PMC2737083

[37] Stankov MV,Leverkus M.Autophagy inhibition due to thymidine analogues as novel mechanism leading to hepatocyte dysfunction and lipid accumulation.AIDS2012;26:1995-2006

[38] Yang ZJ,Huang S.The role of autophagy in cancer: therapeutic implications.Mol Cancer Ther2011;10:1533-41 PMCID:PMC3170456

[39] Bijnsdorp IV.Deoxyribose protects against rapamycin induced cytotoxicity in colorectal cancer cells in vitro.Nucleosides Nucleotides Nucleic Acids2011;30:1197-202