The role of Thioredoxin-interacting protein (TXNIP) in mediating the effects of phenformin and Torin1 on glucose uptake and cell proliferation

Biguanides (e.g.metformin) are commonly used for type 2 diabetes mellitus (T2DM) treatment and have been reported to have anti-neoplastic activity.Systemic effects of biguanides are chiefly mediated by its effects in the liver, leading to decrease in gluconeogenesis and consequent reduction in insulinemia.However, compelling evidence shows that the anti-neoplastic effects of biguanides occur at least in part on a cell-autonomous manner by likely targeting complex I of the mitochondria.This results in inhibition of oxidative phosphorylation and leads to a compensatory increase in cellular glucose uptake.Biguanides also repress the mammalian/mechanistic target of rapamycin (mTOR).We have previously shown that metformin and mTOR inhibitors (PP242 and rapamycin) affect translation of a number of overlapping mRNAs.Unexpectedly, we identified thioredoxin-interacting protein (TXNIP) to be translationally repressed by metformin, but not by direct mTOR inhibitors.Independent of its role in cellular redox homeostasis, TXNIP functions as a negative regulator of glucose uptake in part by directly binding and internalizing GLUT1 and GLUT4.We hypothesize that the mTORindependent suppression of TXNIP expression and subsequent increase in glucose uptake play a major role of adaptation of cancer cells to biguanide-induced energy stress.We show in MCF7 cells that phenformin (a more potent biguanide than metformin) increases glucose uptake and decreases TXNIP expression, while torin1 (active-site mTOR inhibitor) shows the opposite effects.Of note, both drugs decrease cell proliferation.Inhibition of oxidative phosphorylation by phenformin raises AMP/ATP ratio which, in turn, activates AMP-activated protein kinase (AMPK).In human, it has been shown that TXNIP residue Ser-308 is phosphorylated by AMPK, and as a result increases TXNIP degradation which is mediated by E3 ubiquitin ligase ITCH.In iii this study, we aim to link TXNIP expression to the effects of phenformin and torin1 on glucose uptake and cell proliferation.To date, we have generated MCF7 cells that constitutively express wild-type and Ser-308-Ala mutant TXNIP constructs lacking endogenous promoter and 5' and 3' untranslated regions (UTRs).In this, we confirmed that a Ser-308-Ala mutation increases stability of TXNIP protein.Indeed, Ser-308-Ala TXNIP mutant was more stable than TXNIP wild-type in phenformin treated cells.This was paralleled by a ~20% reduction in glucose uptake in cells expressing the mutant vs. wild-type form of the protein.Since the overexpressed TXNIP constructs were devoid of endogenous transcriptional and translational regulatory elements, these finding suggests that phenformin suppression of TXNIP expression occurs at least in part at the level of protein stability, and that TXNIP degradation may mediate phenformin-induced increase in glucose uptake.We also generated CRISPR/Cas9-mediated TXNIP-deficient MCF7 cells that show increase in glucose uptake relative to control cells, confirming previous studies of TXNIP, a suppressor of glucose uptake.Deletion of TXNIP in MCF7 cells attenuates the effects of torin1 on glucose uptake.Since glucose metabolism plays a major role in cancer, these results suggest that TXNIP may play a central role in mediating the effects of biguanides and mTOR inhibitors on cancer metabolism.

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