To our knowledge, this study is the first to demonstrate that mTORC1 inhibition can activate MAPK in vitro, in vivo using mouse models, and, most importantly, in cancer patients. Our findings have important implications for cancer therapy because they provide a further explanation for the limited benefit observed in clinical trials utilizing rapamycin analogs as single agents and exemplify how the efficacy of mTORC1 inhibitors could be improved in the treatment of human cancer. It was predicted that tumors with hyperactive mTORC1 would be extremely sensitive to mTOR inhibition. However, the discovery of an mTORC1-PI3K feedback loop (15
), and now the identification of what is to our knowledge a previously undescribed negative feedback loop regulating MAPK signaling raises the question of whether this dual feedback loop may be detrimental to the efficacy of rapamycin and its analogs in cancer therapy. In line with this idea, our results suggest that tumor cells may be more prone to activate the MAPK pathway upon mTORC1 inhibition compared with normal cells, probably due to a higher basal mTORC1 activity. Hence, the development of clinical trials with such targeted therapeutic agents should proceed with caution and be mindful of the impact on other signaling cascades owing to the complex nature of the mTOR signaling network. Moreover, an important implication of our findings is that appropriate patient stratification based on the altered oncogenic pathways is very much needed for more effective antitumoral use of these compounds.
Recently the consequence of mTORC1 inhibition on PI3K pathway was unraveled as an extremely relevant and novel signaling circuit by which mTORC1 activity affects growth, nutrient uptake, and cancer progression (2
). Numerous links have been reported to connect the targets and regulators of mTORC1 with PI3K activation status. First, mTORC1 inhibition increases the levels and activity of the adaptor protein IRS-1 (18
). The mTORC1-mediated IRS-1–negative regulation relies on its target S6K1. This negative feedback loop has been directly related to the indolence of some types of cancers by ourselves (23
) and others (24
), suggesting that tumors with aberrant mTORC1 activation (such as those harboring heterozygous Tsc2
mutations) may in turn display reduced PI3K-AKT activity. Second, Zhang and collaborators have shown that the activation of mTORC1 regulates the expression of PDGFRs and that rapamycin treatment restores PDGFR levels and therefore PI3K signaling (14
). Third, the proximal mTORC1 activator Rheb has been implicated in the direct interaction and inhibition of B-Raf and Raf1 (25
), which highlights the complexity of the connections between mTORC1, PI3K, and MAPK pathways.
Our data demonstrate that, in conditions of mTORC1 inhibition, Ras is activated and signals to MAPK. This activation relies on the relief of the brake on PI3K triggered by S6K1, but not on mTORC2, AKT, or its downstream components nor on PDGFR upregulation. Similarly, acute PI3K activation induced by insulin/IGF-1 also promotes robust ERK activation, an effect that, interestingly, is enhanced by rapamycin. Several reports have suggested the existence of a cross-talk between Ras-MAPK and PI3K-AKT pathways. Ras has been reported to bind to and activate PI3K (28
), and recent studies from our lab have shown that the MAPK pathway regulates the activation of mTORC1 through TSC2 phosphorylation (5
). On the other hand, low concentrations of EGF (31
) can drive Ras activation through PI3K, which may result from the ability of PIP3
to recruit GAP/Shp2 (32
). Furthermore, PI3K inhibitors such as the cytokine β-galactosidase binding protein (βGBP) can suppress MAPK signaling (33
). Through our identification of yet another feedback loop stemming from mTORC1, we now integrate this PI3K/MAPK cross-talk into the mTOR signaling network. We propose that mTORC1 inhibition increases RTK/IRS-1/PI3K activity toward Ras/MAPK, therefore promoting both AKT activation and ERK phosphorylation in what constitutes a dual feedback mechanism (Figure ).
Schematic representation of the pathway described in the study.
In turn, these findings provide the rationale for a combination of MAPK and mTORC1 inhibitors in the treatment of cancer and show that these agents cooperate to enhance the effectiveness of each compound alone (Figure ). In theory, RTK, IRS-1, or PI3K inhibition in combination with rapamycin analogs should prove to be very effective because it would abrogate the activation of the dual feedback (Figure ). In fact, PI3K and mTORC1 inhibition has been shown to act synergistically in different cancer cell types such as T cell leukemia (34
), acute myeloid leukemia (36
), and glioma (37
). Unfortunately, pharmacological blockade of PI3K in the clinic has been ineffective thus far, while novel and more specific inhibitors (derivatives of LY294002 and wortmannin) are being evaluated in preclinical trials (38
). Moreover, several recent studies have shown that RTK (39
) or IRS-1 signaling inhibition (42
) enhances the effect of rapamycin. This strategy is being translated into diverse clinical trials exploiting the combination of RTK inhibitors (trastuzumab, erlotinib) and rapamycin analogs (sirolimus) in breast and renal cell carcinoma and glioblastoma. On the other hand, MEK inhibitors such as PD0325901 and ARRY-142886 are currently being tested in the clinic with promising results (21
). Thus, the combination of MEK and mTORC1 inhibitors could prove very effective, as it would allow the inhibition of mTORC1 without MAPK feedback activation. In line with this idea, our data demonstrate that MEK1/2 inhibition enhances the antitumoral activity of rapamycin in vitro and, more importantly, in vivo. Whether the 2 drug agents interact to inhibit cell proliferation or to induce apoptosis (or both) seems to vary depending on cues differentially present in vitro and in vivo. Further analysis will provide insights into the molecular nature of the observed context-dependent outcomes. In addition, an accompanying study by Kinkade and colleagues demonstrated that the combinatorial use of this class of compounds exerts a potent antitumoral effect in a preclinical mouse model of prostate cancer (43
). The dual feedback loop identified here further unravels the complex pathways involved in the resistance to mTORC1-blocking drugs and provides a rationale for using combinatorial therapy with MAPK inhibitors.