A non-catalytic scaffolding activity of hexokinase 2 contributes to EMT and metastasis.

Hexokinase 2 (HK2), which catalyzes the first committed step in glucose metabolism, is induced in cancer cells. HK2's role in tumorigenesis has been attributed to its glucose kinase activity. Here, we describe a kinase independent HK2 activity, which contributes to metastasis. HK2 binds and sequesters glycogen synthase kinase 3 (GSK3) and acts as a scaffold forming a ternary complex with the regulatory subunit of protein kinase A (PRKAR1a) and GSK3ß to facilitate GSK3ß phosphorylation and inhibition by PKA. Thus, HK2 functions as an A-kinase anchoring protein (AKAP). Phosphorylation by GSK3ß targets proteins for degradation. Consistently, HK2 increases the level and stability of GSK3 targets, MCL1, NRF2, and particularly SNAIL. In addition to GSK3 inhibition, HK2 kinase activity mediates SNAIL glycosylation, which prohibits its phosphorylation by GSK3. Finally, in mouse models of breast cancer metastasis, HK2 deficiency decreases SNAIL protein levels and inhibits SNAIL-mediated epithelial mesenchymal transition and metastasis.

Systemic Akt1 Deletion after Tumor Onset in p53(-/-) Mice Increases Lifespan and Regresses Thymic Lymphoma Emulating p53 Restoration.

Akt is frequently activated in human cancers. However, it is unknown whether systemic inhibition of a single Akt isoform could regress cancer progression in cancers that are not driven by Akt activation. We systemically deleted Akt1 after tumor onset in p53(-/-) mice, which develop tumors independently of Akt activation. Systemic Akt1 deletion regresses thymic lymphoma in p53(-/-) mice emulating p53 restoration. Furthermore, pharmacological inhibition of Akt selectively kills thymic lymphoma cells and not primary thymocytes. Mechanistically, Akt1 inhibition in p53(-/-) thymic lymphoma inhibits Skp2 expression and induces FasL, which is the primary cause of cell death. Skp2 exerts resistance to cell death by antagonizing the induction of FasL and reducing FAS expression, which is linked to cyclin D1 expression. The results established a paradigm whereby systemic Akt1 inhibition is sufficient to regress tumors that are not driven by Akt activation and a mechanism of cell survival by Skp2.

Hexokinase 2 is required for tumor initiation and maintenance and its systemic deletion is therapeutic in mouse models of cancer.

Accelerated glucose metabolism is a common feature of cancer cells. Hexokinases catalyze the first committed step of glucose metabolism. Hexokinase 2 (HK2) is expressed at high level in cancer cells, but only in a limited number of normal adult tissues. Using Hk2 conditional knockout mice, we showed that HK2 is required for tumor initiation and maintenance in mouse models of KRas-driven lung cancer, and ErbB2-driven breast cancer, despite continued HK1 expression. Similarly, HK2 ablation inhibits the neoplastic phenotype of human lung and breast cancer cells in vitro and in vivo. Systemic Hk2 deletion is therapeutic in mice bearing lung tumors without adverse physiological consequences. Hk2 deletion in lung cancer cells suppressed glucose-derived ribonucleotides and impaired glutamine-derived carbon utilization in anaplerosis.

FoxOs inhibit mTORC1 and activate Akt by inducing the expression of Sestrin3 and Rictor.

FoxO transcription factors and TORC1 are conserved downstream effectors of Akt. Here, we unraveled regulatory circuits underlying the interplay between Akt, FoxO, and mTOR. Activated FoxO1 inhibits mTORC1 by TSC2-dependent and TSC2-independent mechanisms. First, FoxO1 induces Sestrin3 (Sesn3) gene expression. Sesn3, in turn, inhibits mTORC1 activity in Tsc2-proficient cells. Second, FoxO1 elevates the expression of Rictor, leading to increased mTORC2 activity that consequently activates Akt. In Tsc2-deficient cells, the elevation of Rictor by FoxO increases mTORC2 assembly and activity at the expense of mTORC1, thereby activating Akt while inhibiting mTORC1. FoxO may act as a rheostat that maintains homeostatic balance between Akt and mTOR complexes' activities. In response to physiological stresses, FoxO maintains high Akt activity and low mTORC1 activity. Thus, under stress conditions, FoxO inhibits the anabolic activity of mTORC1, a major consumer of cellular energy, while activating Akt, which increases cellular energy metabolism, thereby maintaining cellular energy homeostasis.

mTORC1 hyperactivity inhibits serum deprivation-induced apoptosis via increased hexokinase II and GLUT1 expression, sustained Mcl-1 expression, and glycogen synthase kinase 3beta inhibition.

The current concept is that Tsc-deficient cells are sensitized to apoptosis due to the inhibition of Akt activity by the negative feedback mechanism induced by the hyperactive mTORC1. Unexpectedly, however, we found that Tsc1/2-deficient cells exhibit increased resistance to serum deprivation-induced apoptosis. mTORC1 hyperactivity contributes to the apoptotic resistance of serum-deprived Tsc1/2-deficient cells in part by increasing the growth factor-independent expression of hexokinase II (HKII) and GLUT1. mTORC1-mediated increase in hypoxia-inducible factor 1alpha (HIF1alpha) abundance, which occurs in the absence of serum in normoxic Tsc2-deficient cells, contributes to these changes. Increased HIF1alpha abundance in these cells is attributed to both an increased level and the sustained translation of HIF1alpha mRNA. Sustained glycogen synthase kinase 3beta inhibition and Mcl-1 expression also contribute to the apoptotic resistance of Tsc2-deficient cells to serum deprivation. The inhibition of mTORC1 activity by either rapamycin or Raptor knockdown cannot resensitize these cells to serum deprivation-induced apoptosis because of elevated Akt activity that is an indirect consequence of mTORC1 inhibition. However, the increased HIF1alpha abundance and the maintenance of Mcl-1 protein expression in serum-deprived Tsc2(-/)(-) cells are dependent largely on the hyperactive eIF4E in these cells. Consistently, the reduction of eIF4E levels abrogates the resistance of Tsc2(-/)(-) cells to serum deprivation-induced apoptosis.

The two TORCs and Akt.

The regulatory circuits that control the activities of the two distinct target of rapamycin (TOR) complexes, TORC1 and TORC2, and of Akt have been a focus of intense research in recent years. It has become increasingly evident that these regulatory circuits control some of the most fundamental aspects of metabolism, cell growth, proliferation, survival, and differentiation at both the cellular and organismal levels. As such, they also play a pivotal role in the genesis of diseases including cancer, diabetes, aging, and degenerative diseases. This review highlights recent developments aimed at deciphering the interplay between Akt and mTORCs as well as their role in embryonic development and in cancer.

Akt deficiency impairs normal cell proliferation and suppresses oncogenesis in a p53-independent and mTORC1-dependent manner.

Akt contributes to tumorigenesis by inhibiting apoptosis. Here we establish that Akt is required for normal cell proliferation and susceptibility to oncogenesis independently of its antiapoptotic activity. Partial ablation of Akt activity by deleting Akt1 inhibits cell proliferation and oncogenesis. These effects are compounded by deleting both Akt1 and Akt2. In vivo, Akt1 null mice are resistant to MMTV-v-H-Ras-induced tumors and to skin carcinogenesis. Thus, partial ablation of Akt activity is sufficient to suppress tumorigenesis in vitro and in vivo. The effect of Akt deficiency on cell proliferation and oncogenesis is p53 independent but mTORC1 dependent. Surprisingly, upon mTORC1 hyperactivation, the reduction in Akt activity does not impair cell proliferation and susceptibility to oncogenic transformation; thus, Akt may mediate these processes exclusively via mTORC1.

Hexokinase-mitochondria interaction mediated by Akt is required to inhibit apoptosis in the presence or absence of Bax and Bak.

The serine/threonine kinase Akt inhibits mitochondrial cytochrome c release and apoptosis induced by a variety of proapoptotic stimuli. The antiapoptotic activity of Akt is coupled, at least in part, to its effects on cellular metabolism. Here, we provide genetic evidence that Akt is required to maintain hexokinase association with mitochondria. Targeted disruption of this association impairs the ability of growth factors and Akt to inhibit cytochrome c release and apoptosis. Targeted disruption of mitochondria-hexokinase (HK) interaction or exposure to proapoptotic stimuli that promote rapid dissociation of hexokinase from mitochondria potently induce cytochrome c release and apoptosis, even in the absence of Bax and Bak. These effects are inhibited by activated Akt, but not by Bcl-2, implying that changes in outer mitochondrial membrane (OMM) permeability leading to apoptosis can occur in the absence of Bax and Bak and that Akt inhibits these changes through maintenance of hexokinase association with mitochondria.