The ability of cancer cells to evade apoptosis is a vital property (31
) that confers resistance to chemotherapeutic agents (32
). Understanding apoptotic resistance may inform strategies to restore apoptotic sensitivity in cancer cells and ultimately to improve cancer therapy (3
). Multicellular spheroids have emerged as a promising tool in cancer research because of their acquired resistance to apoptosis that may provide insights into apoptotic resistance acquired in vivo
). In this study, we have identified, for the first time, that 3D multicellular spheroids acquire a resistance to combinatorial therapy with bortezomib, and show that it can be bypassed by blockade of the Bcl-2 family proteins. To our knowledge, this study represents the first to describe multicellular apoptotic resistance to a bortezomib-containing regimen. We believe that this investigation is significant because it offers insights that may be relevant to understanding the resistance in the clinical setting.
Proteasome inhibition has attracted growing interest in the last decade as a cancer-targeting strategy, and the efficacy of proteasome inhibitors, such as bortezomib and the more recent molecules, carfilzomib and NPI-0052, has been widely validated in several hematological malignancies (35
). In preclinical studies in NSCLC, bortezomib has been very effective, either used alone or combined with other agents (36
). Despite the encouraging in vitro
studies, bortezomib has had disappointing results in clinical trials of patients with NSCLC (17
). The greater effectiveness in hematologic malignancies than in most solid tumors suggests that apoptotic resistance to bortezomib may derive from a 3D setting.
In this study, many possible mechanisms for apoptotic resistance to the combinatorial apoptotic strategy in multicellular spheroids were considered and excluded. Limited diffusion of treatments has been suspected as a possible explanation for the reduced efficacy of treatments in solid tumors and 3D spheroids (39
). However, in our study, bortezomib inhibited the proteasome activity of monolayers and spheroids to similar levels (see
), and TRAIL induced Bid cleavage at similar levels in monolayers and spheroids (see
), showing that these two agents were active in the NSCLC spheroids. Apoptotic cells were evenly distributed throughout the spheroid, suggesting adequate diffusion of reagents (see
). Inhibition of NF-κB survival signaling has been considered to represent a major mechanism by which proteasome inhibitors induce apoptosis in tumors (4
); however, NF-κB was not found to be important in our system. For one reason, NF-κB was not stimulated by TRAIL in our A549 stable reporter cells under the conditions of these experiments, and thus would not have been expected to play a role in survival, unlike in other studies in which TRAIL was found to induce NF-κB signaling in H460 lung cancer cells (10
) or in A549 cells (40
). For another reason, even when activated by TNF, NF-κB was equally stimulated in monolayers and spheroids, and equally inhibited by bortezomib, indicating that NF-κB signaling would not account for the differing apoptotic responses between monolayers and spheroids. Akt/mTOR signaling did not mediate apoptotic resistance in A549 spheroids, unlike the situation found in mesothelioma spheroids (21
). Finally, FLIPS
up-regulation in spheroids was not found to contribute to this acquired resistance, either by diminishing TRAIL signals proximally or by altering apoptotic resistance generally.
Instead, we found a role for the antiapoptotic Bcl-2 family proteins in spheroid survival and apoptotic resistance. Spheroids exhibited a difference in expression of Bcl-2 family proteins compared with that in monolayers, a difference that was generally maintained after exposure to bortezomib. In monolayers, according to previously published reports and also in our study, bortezomib is known to decrease the expression of antiapoptotic Bcl-2 family proteins, such as Bcl-2, and increase the expression of proapoptotic BH3-only proteins, including Bim and Noxa (6
). These proapoptotic changes may be countered by the up-regulation of the antiapoptotic molecule, Mcl-1 (37
), a molecule that may account for resistance to bortezomib (6
). Although this pattern and the potential role of Mcl-1 in resistance have been well described, the effect of spheroid formation on these proteins and the actions of bortezomib on spheroids has not been addressed previously. We found that spheroid formation of A549 and H1299 cells led to an increase in Bcl-2 and a decrease in Mcl-1 compared with monolayers. After bortezomib, this differential pattern was retained, with Bcl-2 higher in spheroids and Mcl-1 lower in spheroids than in monolayers; in addition, in the spheroids, Noxa up-regulation was absent. In observing these changes, we considered that the shift in relative expression of Bcl-2 family members in the spheroids may have altered the relative roles of these antiapoptotic proteins.
Indeed, in A549 cells, blockade with ABT-737, a small molecule inhibitor of Bcl-2, Bcl-xL, and Bcl-w, but not of Mcl-1, revealed that these Bcl-2 proteins had assumed a role in survival not evident in the monolayers. In spheroids, ABT-737 reduced the multicellular resistance to bortezomib plus TRAIL, indicating that these Bcl-2 proteins suppressed the apoptosis induced by this combinatorial therapy. In addition, even in spheroids at baseline, ABT-737 by itself induced apoptosis, indicating that the spheroids had acquired a dependence or an addiction to this apoptotic block for survival (42
). Bcl-2 antiapoptotic proteins are thought to sequester BH3-only proapoptotic proteins, thereby preventing them from interacting with Bax and Bak, the proapoptotic molecules necessary for inducing apoptosis (43
). The apoptotic response to blockade of the Bcl-2 family indicates that BH3-only molecules, such as Bim, are active, and that the cell is primed to undergo apoptosis if the antiapoptotic brakes are removed (44
). The BH3-only molecule Noxa was apparently not required for induction of apoptosis in spheroids by ABT-737, indicating that there were enough other proapoptotic BH3-only molecules, such as Bim, to mediate apoptosis after the removal of the antiapoptotic Bcl-2 block. Thus, we propose that the altered expression of Bcl-2 family proteins in A549 spheroids led to a greater reliance on Bcl-2, as revealed by the response to ABT-737. The reduction of Mcl-1 in the spheroids may have contributed to the shift to Bcl-2; indeed, the addiction to Bcl-2 and the sensitivity to ABT-737 has been shown to become apparent when Mcl-1 is absent or neutralized (45
Several of these findings were confirmed in H1299 cells, although the relative importance of Mcl-1 exceeded that of Bcl-2. In the treated spheroids, Mcl-1, even though its expression decreased in spheroids, was still sufficient to support multicellular apoptotic resistance to bortezomib plus TRAIL. Blockade of Bcl-2/Bcl-xL/Bcl-w by ABT had a partial effect, whereas blockade of Mcl-1 nearly completely removed apoptotic resistance. In untreated spheroids, survival was maintained when either Mcl-1 or Bcl-2 alone was blocked, but blockade of both was deadly, suggesting that the cells were primed for death and restrained by either one of these antiapoptotic proteins.
The causes for the changes in expression of Bcl-2 family proteins were not investigated in this study. Bcl-2 can be up-regulated by an elevated NF-κB (46
); however, we did not find increased activity of NF-κB in spheroids. It is more likely that the up-regulation of Bcl-2 in the 3D spheroids is due to the increased cell adhesion of multicellular spheroids, because Bcl-2 up-regulation has been associated with the engagement of integrins α5β1 (47
) or αvβ3 (48
). Mcl-1 is known to be tightly regulated by multiple pathways and at multiple levels (49
), but, to our knowledge, down-regulation with the transition to 3D has not been reported. Because cells alter their shape, function, and metabolism with aggregation into spheroids, many factors may be involved in the changes in the altered protein expression that we observed.
We have concentrated on the antiapoptotic defenses used by the cancer cell lines in 3D spheroids. Of course, finding an active role for antiapoptotic proteins, such as Bcl-2 or Mcl-1, in maintaining multicellular resistance does not exclude a role for a lack of proapoptotic proteins, such as Noxa, which showed no up-regulation in spheroids after bortezomib. Nonetheless, despite the lack of Noxa, reduction of the antiapoptotic defenses allowed apoptosis, presumably because other proapoptotic BH3-only molecules were active and able to replace the proapoptotic function of Noxa. Inhibition of antiapoptotic molecules is also likely to be a more feasible therapeutic strategy than restoration of proapoptotic molecules. The specific contribution of Bcl-2 family antiapoptotic proteins may differ in different cell lines or in different tumors, but we suggest that antiapoptotic Bcl-2 family proteins may represent a final common denominator of multicellular resistance and one that is amenable to therapeutic manipulation.
In conclusion, this study demonstrates that lung cancer spheroids acquire multicellular resistance to bortezomib plus TRAIL and that Bcl-2 family proteins contribute to this acquired resistance. The multicellular resistance found in 3D spheroids may model the therapeutic resistance found in solid tumors. The antiapoptotic Bcl-2 family proteins might be attractive targets to increase the clinical efficacy of bortezomib against lung cancer.