Tuesday, November 8, 2011

Glucose restriction and TSC

Recently, zooko asked me about my opinion on a recent study just published (1).

Background

TSC1 and TSC2 are a pair of tumor supressor genes, which relevance lies in the inhibition of mTORC1 activity. mTOR (the mammalian target of rapamycin) is a master regulator of cell proliferation, cell growth, cell motility, cell survival, protein synthesis and transcription. Because of this, dysregulation of the mTOR pathway is seen in many cancers (2).

mTOR forms two complexes, mTORC1 and mTORC2 ("rapamycin-insensitive"), which respond to different stimuli. TSC2 has a GAP (GTPase activating protein) domain that stimulates the GTPase activity of Rheb. GDP-Rheb is inactive, while GTP-Rheb is active. By this mechanism, TSC2 accelerates the hydrolysis of GTP, inactivating Rheb. Active Rheb is a potent activator of mTORC1. The interplay between these proteins is shown below (3):


In response to growth factors, Akt phosphorylates TSC2 directly on four or five residues (Ser939, Ser981, Ser1130, Ser1132 and Thr1462). Phosphorylation of TSC2 by Akt impairs its ability to inhibit Rheb, thereby blocking the inhibitory effect of Rheb on mTORC1. Other mechanisms proposed to explain contradictory experimental results include the action of PRAS40 and binding of 14-3-3 to phosphorylated TSC2.

The most conserved pathway for Akt activation is the PI3K/Akt pathway. Insulin/IGF-1 binding to the insulin receptor produces phosphorylation of its cytosolic domain, promoting the binding of IRS (insulin receptor substrate) by its PTB (phosphotyrosine binding) domain. This promotes the association (by SH2 domains in the p85 regulatory subunit) and activation of PI3K. PI3K phosphorylates phosphatidylinositol-4,5-biphosphate (PtdIns(4,5)P2), producing PtdIns (3,4,5)P3. PtdIns (3,4,5)P3 binds to the PH domain of Akt and promotes its translocation to the plasma membrane. PI3K-dependent kinase 1 (PDK1) then phosphorylates Akt on Thr308 and PDK2 phosphorylates Ser473. Both phosphorylations activate Akt. Phosphorylated Akt, as previously mentioned, phosphorylates and inactivates TSC2 and PRAS40 promoting mTORC1 activation. The activation of the PI3K/Akt pathway has many downstream cellular events promoting cell survival and proliferation, which include inactivation of several proapoptotic factors (BAD, procaspase-9 and Forkhead transcription factors), activation of antiapoptotic factors (CREB), activation of IKK, inactivation of p53, among other. The net result is promoting proliferation and cell survival, hallmarks of cellular malignancy development and progression.

Discussion of the study

The basis for the utilization of glucose restriction for treating TSC related tumors can be easily inferred from the above explanation. By restricting glucose, insulin signaling is reduced, so is activation of mTORC1. mTORC1 also upregulates HIF1a, promoting aerobic glycolysis and lactate production (4). Thus, glucose restriction should promote apoptosis, specially in TSC1/TSC2-null cells.

What the authors basically did was treating Tsc2-/- mice with a carbohydrate-free diet (CF) and a Western Diet, alone or combined with 2-deoxyglucose (2-DG). Preliminary in vitro studies showed that Tsc2-/- cells were sensitive to glucose restriction and 2-DG in an additive manner. Contrary to what was expected, in vivo experiments showed that tumor size and growth rate were highest in the CF group and 2-DG supressed tumor growth independently of diet. These results also contradicted the observed standard uptake values (SUV) during the FDG-PET scan (presented as the maximum SUV within each tumor). Theoretically, as these tumors are sensitive to glucose deprivation, there should be a correlation between glucose uptake (measured by uptake values of FDG) and tumor size (increased tumor size should show increased SUV). However, there was no correlation between these parameters, as the CF+2-DG group showed the minimum mean SUV but the largest tumor size. What can be fueling tumor growth? Ketone bodies? An in vitro assay showed that addition of either acetate or beta-hydroxybutyrate to Tsc2-/- cells increased cell confluence and reduced the number of non-viable cells (assessed by trypan blue), compared to glucose alone. To further complicate things, ketonemia was not developed in CF mice, but beta-hydroxybutyrate levels were higher with the Western +2-DG diet. Testing the effects of fatty acids in vitro showed that palmitic acid induced necrosis and oleic acid induced proliferation. This correlated with the histologic analysis of CF mice. Addition of rapamycin reduced cell-size, in contrast with 2-DG, which decreased proliferation. Finally, there was increased activation of mTORC1 (measured by phospho-S6) and low levels of phosphorylated Akt (secondary to feedback inhibition) in all groups, with no differences between groups.

Interpretation

First, the results confirm the potent anti-tumor activity of 2-DG. Second, the CF group failed to establish ketosis, and the Western group had increased levels of beta-hydroxybutyrate, as well as reduced tumor size. This (despite the observed growth-promoting properties of acetate and beta-hydroxybutyrate in vitro, see below) can be interpreted as an inhibitory effect of ketonemia on cancer growth. The comparison of glucose and beta-hydroxybutyrate levels is shown below:


The diet which resulted in lower glucose levels and higher ketone bodies was associated with reduced tumor size, and the diet which produced greater glucose levels and lower ketone bodies was associated with increased tumor size. The results observed in vitro with Tsc2-/- cells and ketone bodies suggest that in this cell line, it is necessary an additional anti-glycolytic factor to control tumor growth (2-DG), because these (and other) cancer cells seem to be capable of metabolizing ketone bodies (5, 6). This underscores the importance of the phenotype of the tumor being treated, an important factor that is not taken into account by some "low-carb" advocates who think that restricting dietary glucose will magically cure all cancers.

Another important factor to take into consideration is that mice were not calorie restricted, and more importantly, that the CF diet was high in protein. Glutamine is a major substrate that can fuel cancer cells (7, 8). On a more general level, aminoacids are potent stimulators of mTORC1 (9, 10). This is specially relevant for this model, because excess aminoacids can by-pass the inhibition of the PI3K/Akt pathway by promoting Rheb co-localization with mTORC1 (11), activating mTORC1 in the abscence of TSC2 (12). Ketone bodies increased ATP levels in Tsc2-/-in vitro. This reduces AMPK activity. AMPK inhibits mTORC1 activity by TSC2 dependent and independent mechanisms (possibly by phosphorylation of Raptor) (12). 2-DG also increases intracellular AMP levels (activating AMPK), which would explain the benefits of its utilization observed in this model (13). Supporting the role of AMPK as a target for cancer treatment, the combination of metformin and 2-DG seems to be more toxic to cancer cells than either by itself (14). Interestingly, AMPK activity was not changed in response to 2-DG in this model, which suggests that there are other mechanisms mediating the anti-proliferative effect of 2-DG.

Summing up

Cancer is a very complex disease which treatment has to be personalized depending on the phenotype. With the increase knowledge in cancer molecular biology and genetics, therapies should be designed depending on specific markers evaluated. This complexity explains why not all cancers can be treated just by restricting glucose and making such statement is ludicrous. Besides calorie, glucose and protein restriction, compounds such as 2-DG and metformin show promising effects for controlling most types of cancer.



ResearchBlogging.orgJiang X, Kenerson HL, & Yeung RS (2011). Glucose deprivation in Tuberous Sclerosis Complex-related tumors. Cell & bioscience, 1 (1) PMID: 22018000

3 comments:

  1. Cancer sure is something we cannot laugh about and it's gladdening to see that there are people or blogger such as yourself, who puts effort in discussing stuff like this extensively so that the public could read and learn more about it. The main issue on this subject is to know what to eat and how to moderate the intake of foods with too much glucose content. Also it's a one good reminder for everybody that, as a person the only one held responsible for their own health condition is wholly themselves and nobody else.

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