Understanding the mechanisms by which tumors resist apoptosis may open new doors for bypassing chemoresistance and designing more effective therapy (1
). Nonetheless, investigating apoptosis using monolayer cultures may be limited because tumor cells in a two-dimensional environment may not exhibit the multicellular resistance seen in human solid tumors (3
). Compared with cells grown on two-dimensional plastic dishes, tumor cells in three-dimensional structures acquire a high level of resistance that may relate better to the clinical setting (23
). One putative survival factor that may contribute to acquired resistance in solid tumors is the Akt/mTOR pathway (7
). In a recent study, we have also shown that mesothelioma cell lines allowed to form into three-dimensional aggregates called multicellular spheroids acquired a multicellular resistance in part due to mTOR (20
). Thus, in this and in other studies (20
), the survival function of mTOR may be exhibited best in the three-dimensional setting, suggesting that findings in three dimensions may offer novel insights not necessarily predicted from their two-dimensional counterparts.
To study the role of the Akt/mTOR pathway in a clinically relevant three-dimensional setting, we used tumor fragment spheroids generated from mesothelioma tumor tissue. Malignant mesothelioma, a tumor extremely resistant to chemotherapy, seems well suited for this approach: surgical resection generally provides a large amount of tumor that can be studied ex vivo
, and mesothelioma is known to form spheroidal aggregates in vivo
). Novel approaches in preclinical studies are also attractive for mesothelioma because it is a relatively uncommon tumor, thereby slowing accrual into clinical trials. Thus, we turned to an ex vivo
model of mesothelioma to explore its defenses against apoptosis. We chose to stimulate apoptosis with the strategy that we have found to be most effective in resistant mesothelioma cells in monolayer culture, the activation of the extrinsic death receptor apoptotic pathway using TRAIL combined with the activation of the intrinsic damage pathway (12
In striking contrast to cell lines in monolayer culture, mesothelioma cells in the tumor fragment spheroids are resistant to the combination of TRAIL together with either of its sensitizers, cycloheximide or gemcitabine, even when used at higher concentrations and at multiple dosing over longer times. In this setting of high resistance, rapamycin-induced blockade of mTOR was able to sensitize the cells to apoptosis from TRAIL combinatorial therapy. Interestingly, LY294002, a broad PI3K inhibitor, had no significant effect on apoptosis, despite suggestions of benefit in a small number of tumors in our previous study (15
) and a high concentration (100 μM), perhaps because LY294002 did not fully inhibit mTOR (see
). This speculation is supported in our recent study of multicellular spheroids (20
), in which LY294002 alone did inhibit mTOR more effectively than it did in the tumor fragment spheroids used in this study, and was able to inhibit apoptotic resistance. However, in both studies, whether using multicellular spheroids or tumor fragment spheroids, the combination of LY294002 with rapamycin was no more effective than rapamycin alone, suggesting that mTOR, not Akt, was the important effector of survival in mesothelioma cells. Indeed, in some studies of the tumorigenic function of Akt, both the survival and the oncogenic effects of Akt have been attributed to mTOR (7
). Thus, great interest has arisen in the potential benefits of mTOR inhibition in tumors (6
). Our study supports a therapeutic role for mTOR inhibition in mesothelioma. Indeed, one potential drawback of mTOR inhibition, a possible feedback activation of Akt (9
), was not seen in our study, at least during short-term inhibition.
mTOR sits at an intersection of inputs to the cell, integrating signals from both growth factors and nutrients and mediating its effects via two known effectors with roles in protein translation, S6K and 4E-BP1/eIF4E (5
). The mTOR pathway has several known effects on survival, including phosphorylation and sequestration of the pro-apoptotic Bad (28
), up-regulation of FLIPs (29
), translation of anti-apoptotic proteins, or sustaining the energy of the cell and the mitochondrial potential difference (5
). Hyperactive mTOR signaling may also signal the presence of dependency or “addiction” to this oncogenic kinase pathway (31
), so that inhibition of mTOR could promote cell death by interrupting this lifeline or by attenuating survival signals (32
). In many cases, however, the specific mechanism by which mTOR mediates survival has been elusive (1
). It is worth noting that the inhibition of mTOR did not itself induce apoptosis, but sensitized to the apoptotic effect of other agents. Thus, for the rapamycin-responsive tumors, it appears that mTOR is acting as a brake on apoptosis, and its inhibition could serve as an adjunct to other apoptotic therapies. The nonresponsive tumors, with a lower mTOR activity, may instead rely on other mechanisms for survival.
In support of mTOR as a mediator of survival, its downstream target S6K appeared to play an important role. S6K knockdown in the tumor fragments enhanced sensitivity to apoptosis in the same way as did rapamycin. In addition, the staining for p-S6K, and not p-Akt, in the original tumors correlated with the responsiveness of the spheroids to rapamycin. Indeed, when analyzed in 37 mesotheliomas, the two phosphorylated kinases did not strongly correlate with each other, suggesting that mTOR has inputs and regulation in addition to that of Akt in this tumor. It is now known, for example, that mTOR, in its role as a sensor of nutrients as well as of growth factors, can be activated by factors other than Akt, such as by elevated levels of amino acids or ATP (33
) and by the ERK pathway (34
). mTOR may also be inhibited in ways that do not involve Akt signals, such as by heat shock or osmotic stress (35
). Thus, compared with p-Akt, it should not be surprising that p-S6K would be a better marker for mTOR pathway activation and for those tumors that could respond to mTOR inhibition.
mTOR activity in the tumors, as evidenced by p-S6K staining, correlated with the response to rapamycin but did not correlate with histologic subtype. We do not have access to clinical information to know whether p-S6K staining or the responsiveness to rapamycin correlated with patient outcome or other clinical variables. In recent studies of other tumors, however, mTOR activation has been associated with a poor prognosis (36
). Thus, activation of the mTOR pathway in mesothelioma may portend a poor prognosis even as it signals a possible therapeutic intervention. If confirmed in further studies to be a useful biomarker, p-S6K has the potential for serving as a window into tumor biology and providing an independent assessment of mesothelioma prognosis and anticipated response to therapies, separate from pathologic appearance.
The tumor fragment spheroid model attempts to retain the microenvironment of the actual human tumor. We had previously shown that the spheroids grown from mesothelioma contained viable tumor cells for weeks to months, whereas the cells disaggregated from these same tumors failed to proliferate (15
). In this study, we also attempted to grow mesothelioma cells disaggregated from these tumors but found that the cells showed an unacceptably high background level of apoptosis. Thus, tumor fragments can be a useful way to maintain human tumor cells in culture. Although diffusion may be a factor in any three-dimensional model, we did not see evidence of a limitation of diffusion in our study; the homogeneous reduction of p-S6K and p-Akt with inhibitors and the homogeneous location of apoptotic cells after treatment suggested that diffusion was adequate. Moreover, diffusion is a factor that is relevant to actual tumors, where the avascular unit that relies on diffusion is thought to be between 1 and 2 mm in diameter, similar to the size of our fragments (38
). Thus, the issues of diffusion will be relevant to treatment of tumors clinically (39
mTOR inhibitors are now under investigation in the treatment of many tumors (10
), including mesothelioma. This ex vivo
study suggests that mTOR inhibition would be a useful adjunct for enhancing apoptotic strategies in mesothelioma. Furthermore, p-S6K could be an appropriate biomarker for identifying those who would respond to this approach. The mesothelioma tumors also expressed high levels of DR5, the TRAIL receptor, suggesting that they have the capacity to respond to TRAIL therapy. Indeed, clinical trials with TRAIL or with agonist antibodies to the TRAIL receptor have begun for many tumors, including mesothelioma. It is our hope that our studies will help identify ways to enhance the efficacy of TRAIL cancer therapy.
In summary, we have used an ex vivo model to investigate the apoptotic resistance of actual human tumor cells. The findings suggest a role for the mTOR pathway in a majority of mesothelioma tumors and propose a biomarker for identifying those who might benefit. Such findings will need to be confirmed in patients with this disease. The ex vivo approach of using tumor fragment spheroids may lend itself to studies of other novel therapies considered for mesothelioma or other solid tumors.