Isoprenylcysteine carboxylmethyltransferase is required for the impact of mutant KRAS on TAZ protein level and cancer cell self-renewal.

Cancer stem cells possess the capacity for self-renewal and resistance to chemotherapy. It is therefore crucial to understand the molecular regulators of stemness in the quest to develop effective cancer therapies. TAZ is a transcription activator that promotes stem cell functions in post-development mammalian cells; suppression of TAZ activity reduces or eliminates cancer stemness in select cancers. Isoprenylcysteine carboxylmethyltransferase (ICMT) is the unique enzyme of the last step of posttranslational prenylation processing pathway that modifies several oncogenic proteins, including RAS. We found that suppression of ICMT results in reduced self-renewal/stemness in KRAS-driven pancreatic and breast cancer cells. Silencing of ICMT led to significant reduction of TAZ protein levels and loss of self-renewal ability, which could be reversed by overexpressing mutant KRAS, demonstrating the functional impact of ICMT modification on the ability of KRAS to control TAZ stability and function. Contrary to expectation, YAP protein levels appear to be much less susceptible than TAZ to the regulation by ICMT and KRAS, and YAP is less consequential in regulating stemness characteristics in these cells. Further, we found that the ICMT-dependent KRAS regulation of TAZ was mediated through RAF, but not PI3K, signaling. Functionally, we demonstrate that a signaling cascade from ICMT modification of KRAS to TAZ protein stability supports cancer cell self-renewal abilities in both in vitro and in vivo settings. In addition, studies using the proof-of-concept small molecule inhibitors of ICMT confirmed its role in regulating TAZ and self-renewal, demonstrating the potential utility of targeting ICMT to control aggressive KRAS-driven cancers.

p21cip1/waf1 Coordinate Autophagy, Proliferation and Apoptosis in Response to Metabolic Stress.

Cancer cells possess metabolic properties that are different from benign cells. These unique characteristics have become attractive targets that are being actively investigated for cancer therapy. p21cip1/waf1, also known as Cyclin-Dependent Kinase inhibitor 1A, is encoded by the CDKN1A gene. It is a major p53 target gene involved in cell cycle progression that has been extensively evaluated. To date, p21 has been reported to regulate various cell functions, both dependent and independent of p53. Besides regulating the cell cycle, p21 also modulates apoptosis, induces senescence, and maintains cellular quiescence in response to various stimuli. p21 transcription is induced in response to stresses, including those from oxidative and chemotherapeutic treatment. A recent study has shown that in response to metabolic stresses such as nutrient and energy depletion, p21 expression is induced to regulate various cell functions. Despite the biological significance, the mechanism of p21 regulation in cancer adaptation to metabolic stress is underexplored and thus represents an exciting field. This review focuses on the recent development of p21 regulation in response to metabolic stress and its impact in inducing cell cycle arrest and death in cancer cells.

Inhibition of Isoprenylcysteine Carboxylmethyltransferase Induces Cell-Cycle Arrest and Apoptosis through p21 and p21-Regulated BNIP3 Induction in Pancreatic Cancer.

Pancreatic cancer remains one of the most difficult to treat human cancers despite recent advances in targeted therapy. Inhibition of isoprenylcysteine carboxylmethyltransferase (ICMT), an enzyme that posttranslationally modifies a group of proteins including several small GTPases, suppresses proliferation of some human cancer cells. However, the efficacy of ICMT inhibition on human pancreatic cancer has not been evaluated. In this study, we have evaluated a panel of human pancreatic cancer cell lines and identified those that are sensitive to ICMT inhibition. In these cells, ICMT suppression inhibited proliferation and induced apoptosis. This responsiveness to ICMT inhibition was confirmed in in vivo xenograft tumor mouse models using both a small-molecule inhibitor and shRNA-targeting ICMT. Mechanistically, we found that, in sensitive pancreatic cancer cells, ICMT inhibition induced mitochondrial respiratory deficiency and cellular energy depletion, leading to significant upregulation of p21. Furthermore, we characterized the role of p21 as a regulator and coordinator of cell signaling that responds to cell energy depletion. Apoptosis, but not autophagy, that is induced via p21-activated BNIP3 expression accounts for the efficacy of ICMT inhibition in sensitive pancreatic cancer cells in both in vitro and in vivo models. In contrast, cells resistant to ICMT inhibition demonstrated no mitochondria dysfunction or p21 signaling changes under ICMT suppression. These findings not only identify pancreatic cancers as potential therapeutic targets for ICMT suppression but also provide an avenue for identifying those subtypes that would be most responsive to agents targeting this critical enzyme. Mol Cancer Ther; 16(5); 914-23. ©2017 AACR.

A potential mechanism of metformin-mediated regulation of glucose homeostasis: inhibition of Thioredoxin-interacting protein (Txnip) gene expression.

Metformin (dimethylbiguanide) is widely used among diabetic patients to lower the blood sugar level. Although several mechanisms have been proposed, its mode of action in enhancing peripheral glucose uptake and inhibiting hepatic glucose production is not fully understood. Thioredoxin-interacting protein (Txnip) is known to play important roles in glucose metabolism by inhibiting cellular glucose uptake and metabolism and promoting hepatic gluconeogenesis. The expression of the gene encoding Txnip is regulated in a glucose dependent manner via the Mondo:MLX transcription factor complex. In the present study, we report that Txnip mRNA as well as protein expression in cultured cells is markedly reduced upon metformin administration. The binding of Mondo:MLX to the Txnip gene promoter is reduced, suggesting that the transcription of the Txnip gene is repressed by metformin. Moreover, we show that the effect of metformin on Txnip gene transcription is due to the inhibition of mitochondrial complex I and increased glycolysis, and is partially mediated by the AMP activated kinase (AMPK). These observations prompt us to propose that the novel action of metformin on the Txnip gene expression may contribute to its therapeutic effects in the treatment of type II diabetes.

Hypoxia-inducible factor independent down-regulation of thioredoxin-interacting protein in hypoxia.

Thioredoxin-Interacting Protein (Txnip) is an important regulator of glucose metabolism and functions by inhibiting cellular glucose uptake. The expression of the Txnip gene is sensitive to glucose availability and is negatively correlated with the glycolytic rate. Here we show that hypoxia induces a rapid decrease in Txnip mRNA and protein expression in a Hypoxia-Inducible Factor independent manner. Hypoxia caused reduced binding of the glucose responsive MondoA:Mlx transcription factor to the carbohydrate response elements (ChoREs) in the Txnip promoter. Our data suggest that hypoxia decreases MondoA:Mlx activity by increasing glycolytic flux, leading to the depletion of glycolytic intermediates which normally activate MondoA:Mlx. Hypoxia dependent Txnip down-regulation may be an important compensatory mechanism through which cancer cells adapt their metabolism to low oxygen concentrations.

Thioredoxin-interacting protein (Txnip) gene expression: sensing oxidative phosphorylation status and glycolytic rate.

Thioredoxin-interacting protein (Txnip) has important functions in regulating cellular metabolism including glucose utilization; the expression of the Txnip gene is sensitive to the availability of glucose and other fuels. Here, we show that Txnip expression is down-regulated at the transcriptional level by diverse inhibitors of mitochondrial oxidative phosphorylation (OXPHOS). The effect of these OXPHOS inhibitors is mediated by earlier identified carbohydrate-response elements (ChoREs) on the Txnip promoter and the ChoRE-associated transcription factors Max-like protein X (MLX) and MondoA (or carbohydrate-response element-binding protein (ChREBP)) involved in glucose-induced Txnip expression, suggesting that inhibited oxidative phosphorylation compromises glucose-induced effects on Txnip expression. We also show that the OXPHOS inhibitors repress the Txnip transcription most likely by inducing the glycolytic rate, and increased glycolytic flux decreases the levels of glycolytic intermediates important for the function of MLX and MondoA (or ChREBP). Our findings suggest that the Txnip expression is tightly correlated with glycolytic flux, which is regulated by oxidative phosphorylation status. The identified link between the Txnip expression and glycolytic activity implies a mechanism by which the cellular glucose uptake/homeostasis is regulated in response to various metabolic cues, oxidative phosphorylation status, and other physiological signals, and this may facilitate our efforts toward understanding metabolism in normal or cancer cells.