We set out to determine whether BEZ235 would be a more effective inhibitor of HCC progression than RAD001. Unexpectedly, the two in combination are more potent than either agent alone in inhibiting proliferation of HCC cells in culture and tumors in vivo ( and ). Consistent with earlier findings that rapamycin affects substrate specificity, not kinase activity (38
), recent studies show that the ability of the rapamycins to inhibit mTORC1 signaling is more pronounced for S6K1 than 4E-BP1 (39
). This has led to the suggestion that S6K1, but not 4E-BP1, is excluded from interacting with mTORC1 because of its relative larger size (41
). However, S6K1 is about half the size of ULK1, whose phosphorylation is largely unaffected by rapamycin. It is more likely that mTORC1 activity is dependent on the conformation of a ternary complex that includes the kinase, the substrate, and ATP. In contrast, the new mTOR ATP-binding site competitive drugs inhibit both mTOR complexes and block 4E-BP1 phosphorylation to the same extent as S6K1 (39
). Downstream of mTORC1, we recently showed that the main effects of these inhibitors on cell proliferation are attributed to activation of the 4E-BPs (19
). These findings are consistent with our observations using RAD001 and BEZ235 in HCC cell lines ( and fig. S1
); however, inhibition of 4E-BP1 alone is not sufficient to explain the effects on HCC progression, which eventually led us to examine the role of autophagy.
It has been demonstrated that prolonged treatment with rapamycin affects mTORC2, in addition to mTORC1 (44
). This response is more difficult to discern, because rapamycin also relieves the negative feedback loop from mTORC1/S6K1 to PKB/Akt (). A similar effect was observed with BEZ235 at lower concentrations (), suggesting that mTORC1 may be more readily targeted than mTORC2. As with mTORC1, mTORC2 inhibition by BEZ235 was greatly enhanced by RAD001 (). Moreover, from in vitro studies, these effects appear to be elicited at the level of mTORC1 and mTORC2 (). Although in HCC the major effects on proliferation appear to be through mTORC1, it is clear in other tumor types, including phosphatase and tensin homolog deleted from chromosome 10 (PTEN)–deficient prostate tumors, that the effects on tumor progression are mTORC2- dependent (45
). The therapeutic advantage of combining RAD001 with BEZ235 is that it should be efficacious in either tumor type. Moreover, because of the findings here and those of Nyfeler et al. (41
), one would predict that the rapamycins could be used in combination with any mTOR ATP-binding site competitive inhibitor. Combination treatment should decrease the effective dose of either drug, reducing off target effects of the mTOR ATP-binding site competitive inhibitor.
We tested the efficacy of RAD001 and BEZ235 in HCC with the DEN mouse model, which best represents human HCC with unfavorable outcome (21
). Gene expression profiling showed that the major classes of genes affected in both mouse and human HCCs with poor prognosis were cell proliferation and antiapoptotic genes (21
). We find that DEN induced HCCs treated with RAD001 and BEZ235 have a significant cell cycle inhibition signature. Moreover, the drug combination, unlike either RAD001 or BEZ235 alone, revealed a significant number of genes reverting to roughly baseline expression levels of normal livers, suggesting that the effect of the two drugs together cannot be recapitulated by increasing the dose of either drug alone. Recent data in ovarian cancer cells and non–small cell lung cancer cells in culture and xenografts suggest that c-Myc is a major regulator of the tumor response to rapamycin or RAD001 in combination with a PI3K/mTOR inhibitor (46
). However, we found no evidence of significant alterations in genes transcriptionally regulated by c-Myc in placebo- or drug-treated HCC DEN tumors. Our findings suggest that the mechanisms at play may be unique to a syngeneic tumor confronted with an intact cytokine and immune response arising from a natural history in the endogenous stroma or to HCC itself, rather than cultured cell–initiated xenografts in immunocompromised mice.
It has been known for some time that inhibition of mTOR signaling in hepatocytes is associated with the activation of autophagy (48
). Moreover, recent studies describe the spontaneous induction of liver adenomas in mice with a mosaic deletion of Atg5 or a liver specific deletion of Atg7 (28
). However, in other systems, autophagy supports tumor persistence by maintaining cells under nutrient deprived conditions, thus acting as a survival factor (49
). In our hands, RAD001 and BEZ235 synergize at the level of autophagy as shown by accumulation of the GST-BHMT fragment (). The increase in autophagy is independent of 4E-BP1 and correlates with the dephosphorylation of ULK1 at S757, an mTORC1 phosphorylation site (33
). These findings suggest that activation of autophagy, in a 4E-BP1/2/eIF-4G– independent manner, may be implicated in HCC regression observed in tumors treated with combined RAD001/BEZ235 (). With the exception of Atg3, we did not observe significant changes in the gene expression of autophagy genes in tumors treated with the combination of RAD001 and BEZ235, compared to vehicle-treated tumors. The significant autophagic response to mTOR inhibitors in the absence of major transcriptional changes suggests that transcriptional reprogramming of autophagy genes was not required for the mitophagy response. At this point, it will be important to determine the extent to which the activation of mitophagy in DEN-induced HCCs contributes to tumor regression.
RAD001 has been approved by the U.S. Food and Drug Administration for renal clear cell carcinoma, TSC-associated subependymal giant cell astrocytoma, and neuroendocrine tumors. However, in all cases, RAD001 delayed tumor progression, but there were no complete responses. It may be that such effects are due to partial suppression of mTORC1 signaling by rapamycin derivatives, or resistant mechanisms may develop over time. The combination of an ATP-binding site competitive mTOR inhibitor with a rapamycin derivative may prove more effective in inhibiting additional targets of mTORC1. Our hypothesis is that synergy may arise as a function of the ATP-competitive inhibitors having enhanced access to the active site of the kinase. To our knowledge, there are no other examples where two inhibitors act synergistically on the same target; thus, these studies provide a strategy to increase the specificity of ATP-competitive inhibitors. On the basis of our in vivo data, we have begun an investigator-initiated phase 1B-2 dose escalation study of BEZ235 in combination with RAD001 in patients with HCC or other solid tumors. Since rapamycin and its derivatives have already been approved clinically, their combination with a PI3K/mTOR ATP-competitive inhibitor, such as BEZ235, would be a rapid strategy to test the efficacy of this class of drugs in cancer and to fast-track their approval.