Role of PI3K, mTOR and Akt2 signalling in hepatic tumorigenesis via the control of PKM2 expression.

To sustain increased growth, rapidly proliferating cells, such as tumour cells, undergo metabolic adaptations. In recent years, the mechanisms of glycolysis activation as a key metabolic adaptation in proliferating cells became the topic of intense research. Although this phenomenon was described more than 50 years ago by Otto Warburg, the molecular mechanisms remained elusive. Only recently, it was demonstrated that the expression of specific glycolytic enzymes, namely PKM2 (pyruvate kinase M2) and HK2 (hexokinase 2), occurs simultaneously with the glycolytic addiction of cancer cells. The PI3K (phosphoinositide 3-kinase)/mTOR [mammalian (or mechanistic) target of rapamycin] signalling pathway is a central signalling hub co-ordinating the growth in response to growth factor signalling and nutrient availability. Not surprisingly, it is found to be activated in the majority of the tumour cells. In the present article, we discuss the requirement of different PI3K/mTOR downstream effectors for the metabolic adaptation in liver cancer cells driven by this signalling pathway. We provide evidence for a selective involvement of the mTOR target Akt2 in tumoral growth. In addition, PTEN (phosphatase and tensin homologue deleted on chromosome 10)-negative human hepatocellular carcinoma cell lines display an up-regulation of PKM2 expression in an Akt2-dependent manner, providing an advantage for cell proliferation and anchorage-independent growth. Our data have implications on the link between the metabolic action of insulin signal transduction and tumorigenesis, identifying Akt2 as a potential therapeutical target in liver malignancies depending on cancer genotype.

The role of the mTOR pathway during liver regeneration and tumorigenesis.

It is established that overnutrition is a risk factor for hepatocellular carcinoma. Il has been proposed that hepatic steatosis leads to a subinflammatory response and to the production of mitogenic cytokines. Our team is focused on the role of mammalian Target of Rapamycin (mTOR) in two pathophysiological conditions that modulate liver growth: liver regeneration after partial hepatectomy, and steatosis-associated tumorigenesis. Target kinases of mTOR seem more specifically involved in these processes: while S6K1 contributes to liver regeneration following hepatectomy, Akt2 is implicated in steatosis-associated tumorigenesis. In addition, recent data indicate that the transcription factor PPARγ, through an activation of glycolytic enzymes, could promote liver steatosis, hypertrophy and hyperplasia.

PPARγ contributes to PKM2 and HK2 expression in fatty liver.

Rapidly proliferating cells promote glycolysis in aerobic conditions, to increase growth rate. Expression of specific glycolytic enzymes, namely pyruvate kinase M2 and hexokinase 2, concurs to this metabolic adaptation, as their kinetics and intracellular localization favour biosynthetic processes required for cell proliferation. Intracellular factors regulating their selective expression remain largely unknown. Here we show that the peroxisome proliferator-activated receptor gamma transcription factor and nuclear hormone receptor contributes to selective pyruvate kinase M2 and hexokinase 2 gene expression in PTEN-null fatty liver. Peroxisome proliferator-activated receptor gamma expression, liver steatosis, shift to aerobic glycolysis and tumorigenesis are under the control of the Akt2 kinase in PTEN-null mouse livers. Peroxisome proliferator-activated receptor gamma binds to hexokinase 2 and pyruvate kinase M promoters to activate transcription. In vivo rescue of peroxisome proliferator-activated receptor gamma activity causes liver steatosis, hypertrophy and hyperplasia. Our data suggest that therapies with the insulin-sensitizing agents and peroxisome proliferator-activated receptor gamma agonists, thiazolidinediones, may have opposite outcomes depending on the nutritional or genetic origins of liver steatosis.

S6 kinase 1 is required for rapamycin-sensitive liver proliferation after mouse hepatectomy.

Rapamycin is an antibiotic inhibiting eukaryotic cell growth and proliferation by acting on target of rapamycin (TOR) kinase. Mammalian TOR (mTOR) is thought to work through 2 independent complexes to regulate cell size and cell replication, and these 2 complexes show differential sensitivity to rapamycin. Here we combine functional genetics and pharmacological treatments to analyze rapamycin-sensitive mTOR substrates that are involved in cell proliferation and tissue regeneration after partial hepatectomy in mice. After hepatectomy, hepatocytes proliferated rapidly, correlating with increased S6 kinase phosphorylation, while treatment with rapamycin derivatives impaired regeneration and blocked S6 kinase activation. In addition, genetic deletion of S6 kinase 1 (S6K1) caused a delay in S phase entry in hepatocytes after hepatectomy. The proliferative defect of S6K1-deficient hepatocytes was cell autonomous, as it was also observed in primary cultures and hepatic overexpression of S6K1-rescued proliferation. We found that S6K1 controlled steady-state levels of cyclin D1 (Ccnd1) mRNA in liver, and cyclin D1 expression was required to promote hepatocyte cell cycle. Notably, in vivo overexpression of cyclin D1 was sufficient to restore the proliferative capacity of S6K-null livers. The identification of an S6K1-dependent mechanism participating in cell proliferation in vivo may be relevant for cancer cells displaying high mTOR complex 1 activity and cyclin D1 accumulation.

S6 kinase inactivation impairs growth and translational target phosphorylation in muscle cells maintaining proper regulation of protein turnover.

A defect in protein turnover underlies multiple forms of cell atrophy. Since S6 kinase (S6K)-deficient cells are small and display a blunted response to nutrient and growth factor availability, we have hypothesized that mutant cell atrophy may be triggered by a change in global protein synthesis. By using mouse genetics and pharmacological inhibitors targeting the mammalian target of rapamycin (mTOR)/S6K pathway, here we evaluate the control of translational target phosphorylation and protein turnover by the mTOR/S6K pathway in skeletal muscle and liver tissues. The phosphorylation of ribosomal protein S6 (rpS6), eukaryotic initiation factor-4B (eIF4B), and eukaryotic elongation factor-2 (eEF2) is predominantly regulated by mTOR in muscle cells. Conversely, in liver, the MAPK and phosphatidylinositol 3-kinase pathways also play an important role, suggesting a tissue-specific control. S6K deletion in muscle mimics the effect of the mTOR inhibitor rapamycin on rpS6 and eIF4B phosphorylation without affecting eEF2 phosphorylation. To gain insight on the functional consequences of these modifications, methionine incorporation and polysomal distribution were assessed in muscle cells. Rates and rapamycin sensitivity of global translation initiation are not altered in S6K-deficient muscle cells. In addition, two major pathways of protein degradation, autophagy and expression of the muscle-specific atrophy-related E3 ubiquitin ligases, are not affected by S6K deletion. Our results do not support a role for global translational control in the growth defect due to S6K deletion, suggesting specific modes of growth control and translational target regulation downstream of mTOR.