Mammalian target of rapamycin contributes to the acquired apoptotic resistance of human mesothelioma multicellular spheroids

Barbone D, Yang TM, Morgan JR, Gaudino G, Broaddus VC.
Lung Biology Center, San Francisco General Hospital, University of California, San Francisco, California 94110, USA.

A compelling strategy for cancer therapy is to undermine cancer’s extensive defenses against apoptosis, thereby either killing cancer cells outright or lowering their defenses against standard therapies (48). Understanding the molecular mechanisms of apoptotic resistance thus assumes an important role in counteracting cancer resistance to therapy. It has become apparent in recent years that the standard two-dimensional monolayer in vitro model does not exhibit the full range of resistance to apoptosis seen in human cancers (49). Cancer cells have been found to acquire apoptotic resistance when grown as three-dimensional structures that may model the apoptotic resistance of human solid tumors (3).
In our study, by using a three-dimensional system, we have identified a role for mTOR in cell survival that was not apparent in the same cells grown as monolayers. Indeed, this strategy has allowed us to identify mTOR as a contributor to acquired multicellular resistance to apoptosis of mesothelioma thereby suggesting that mTOR could be a useful target in this highly resistant tumor. It is true that mTOR/S6K inhibition did not induce apoptosis by itself, but it enhanced the response to other apoptotic stimuli, presumably by releasing an inhibitory step at the level of the mitochondria. It is also worth pointing out that mTOR/S6K inhibition did not remove all acquired resistance, but it did account for a significant portion (roughly 40%) that would be amenable to currently available inhibitors. Our results provide a fresh insight into the functions of mTOR/S6K in mesothelioma and perhaps in other refractory solid tumors.
mTOR has been increasingly recognized as a crucial kinase in cancer and metabolic diseases because of its role in the integration of diverse mitogenic and metabolic inputs (41) and its recognition as a mediator of survival (16, 17, 19, 50, 51). In addition, with its own inputs and restraints, mTOR can act independently of the PI3K/Akt pathway (52, 53). Several of our observations point to the importance of mTOR signaling, as apart from Akt signaling, in mesothelioma cells. For one, mTOR/S6K was activated, as judged by an elevated P-S6K, in all our cells, whereas P-Akt was elevated only in M28 and VAMT (see Fig. 4A). For another, despite the increase in P-Akt with rapamycin (see Fig. 3A), rapamycin was as effective as the other inhibitors in reducing apoptotic resistance. This suggests that, within the PI3K/Akt/mTOR pathway, mTOR is the major mediator of the acquired apoptotic resistance in mesothelioma spheroids. Finally, knockdown of S6K reproduced the effect of rapamycin, confirming the role of the mTOR/S6K arm in the survival of spheroids.
By silencing Bid expression and removing an essential amplification step between the death receptor and mitochondrial apoptotic pathways (54), we localized the anti-apoptotic activity of mTOR/S6K to the level of the mitochondria. Indeed, inhibition of apoptosis at the mitochondrial level would account for the observed resistance to a wide array of apoptotic stimuli involving the intrinsic and extrinsic pathways (see Fig. 8). mTOR has also been shown to associate with mitochondria (55) possibly serving as a modulator of stress signals (56) and cell fate (57). Although we were able to localize the resistance to the mitochondria, we were not able to identify a specific molecule that transduced the survival function of mTOR/S6K in spheroids. In particular, with the development of a three-dimensional structure, there was no clear change in the abundance of many pro-/anti-apoptotic proteins thought to be important for mesothelioma apoptotic resistance (45) or for molecules known to be regulated by S6K such as phospho-Bad (supplemental Fig. S4) (18). Anti-apoptotic Bcl-2 and FLIPs, which were up-regulated in the transition from two-dimensional to three-dimensional, were found to be independent of mTOR/S6K activity, suggesting that they may account for resistance that is not controlled by mTOR/S6K, at least in two of the cell lines. mTOR may also contribute to survival in general by its support of metabolism and energy (56) or of protein translation, key functions to which the cells may become reliant or “addicted” (58).
The survival function of mTOR became evident only in the three-dimensional setting. This may represent a redirection of the function of mTOR in three-dimensional, as has been noted for other signaling pathways that, with a transition from two- to three-dimensional, can be spatially reorganized (59, 60), coupled to other pathways (61), or redirected downstream to different functions (59). In fact, mTOR has previously been noted to have a greater effect on survival in three-dimensional than in two-dimensional breast cancer cell cultures (62). The Akt/mTOR pathway was clearly altered by the three-dimensional environment as seen in the reduction in phosphorylation of Akt and S6K (see Fig. 4, A and B). Because the signals can respond to stresses (see supplemental Fig. S2), we consider the reduction in phosphorylation to represent a down-regulation, instead of a suppression, of the pathway. Indeed, down-regulation may provide an improved signal to noise ratio, as can be achieved with modulation from feedback loops (63). An increase in PTEN activity in spheroids, as suggested by our finding of a decrease in the inhibitory phosphorylation at Ser-380 (see supplemental Fig. S3), might account for down-regulation by providing a brake on unrestrained PI3K activity. Such an increase in PTEN may parallel a general increase in phosphatases that has been described in spheroids compared with monolayers (64). Like other pathways, the Akt/mTOR pathway may thus be differently regulated and assume different functions in the three-dimensional setting.
Limited diffusion of macromolecules and the presence of a hypoxic core have been proposed to drive resistance of three-dimensional cell models (35, 36). We did not find evidence that the spheroids utilized in this study were affected by an impaired diffusion of agents. They had no baseline apoptosis, they showed homogeneous TRAIL diffusion and P-Akt/P-S6K staining, and they responded similarly as microspheroids of a different size and shape. Of note, the estimated maximal diffusion distance of the spheroids was 25–75 μm, below the distance of 100–200 μm described as limiting for the diffusion of oxygen in vivo (31). Clearly though, there will be diffusion gradients in three-dimensional models that may be relevant to gradients existing in avascular units of tumor units (36).
In this study of three-dimensional spheroids, we have discovered a role for mTOR in survival that could provide a therapeutic rationale for the use of mTOR inhibitors against mesothelioma, probably as an adjunct to current therapies. Inhibitors of mTOR are currently in use or in clinical trials for several tumors (65). If inhibition of mTOR is found to be useful in mesothelioma, it will underscore the value of three-dimensional studies for revealing underlying causes of acquired multicellular resistance in tumors.

3D Petri Dishes™ used in this paper (please click catalog numbers for detailed product descriptions):
Catalog # 12-256, and 24-96 are the MicroTissues, Inc products that grow small spheroids. Catalog # 12-81 and 24-35 are used to grow larger spheroids.

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