Our study provides evidence that integrin-specific adhesion promotes mTORC1 signaling through inactivation of the tumor suppressor merlin. In addition, we link the loss of merlin to the activation of mTORC1 and sensitivity to rapamycin in malignant mesothelioma. These findings reveal that merlin is a negative regulator of mTORC1 signaling and suggest that loss-of-function mutations in NF2 contribute to tumor initiation and maintenance through activation of mTORC1.
Although it has long been known that matrix adhesion is necessary for mRNA translation (1
), it has remained unclear whether integrin signaling exerts specific control on mRNA translation and, if so, through which mechanism and with what consequences. Our results indicate that the α5β1 integrin promotes, through PAK, inactivation of merlin, thereby controlling mTORC1 signaling and cap-dependent mRNA translation, whereas the α2β1 integrin is unable to activate this signaling pathway. In addition, we have observed that integrin-specific signaling is required to sustain mRNA translation during the mid- to late G1
phase, when the effect of growth factor stimulation on the activation of AKT has subsided. If integrin-specific signaling is not activated during the mid- to late G1
phase, several mRNAs are not translated and total protein biosynthesis declines. Under these conditions, endothelial cells undergo cell cycle arrest and eventually apoptosis. Presumably, other cell types in which cyclin D is under translational control (17
) may undergo a similar demise if plated onto an inappropriate matrix. These results confirm and extend those of Maeshima and colleagues, who have found that the angiogenesis inhibitor tumstatin induces apoptosis in endothelial cells that are stably adherent to a complex matrix by interfering with the ability of the αvβ3 integrin to activate mTORC1 signaling (26
). Together, the findings of these studies highlight the impact of integrin-specific signaling through mTORC1 on cell survival and proliferation.
It is known that RTK activation controls mTORC1 signaling through AKT- and possibly ERK-dependent phosphorylation of TSC1-TSC2 (15
). In this report, we show that integrin-specific adhesion controls mTORC1 through PAK, which phosphorylates and inactivates merlin. In addition, depletion or ablation of merlin activates mTORC1 in several cell types, and this effect appears to occur independently of AKT or ERK. Since the biochemical function and direct target-effectors of merlin are not known, we have not investigated in this study the mechanism through which inactivation of merlin activates mTORC1. We have, instead, focused on the biological consequences of this new signaling connection.
Extracellular stimuli converge on cyclin D to control cell cycle progression (28
). Our study suggests that, although joint integrin-RTK signaling controls the transcription of cyclin D (63
), integrin-specific activation of mTORC1 is necessary for the translation of cyclin D mRNA and hence progression through the G1
phase of the cell cycle. This observation suggests the possibility that matrix adhesion coordinately regulates cell growth, i.e., increase in cell size, and cell cycle progression through the activation of mTORC1. Prior studies have suggested that cell cycle progression is dependent on cell growth, leading to the hypothesis of a cell growth checkpoint for cell cycle progression (20
). Our findings suggest that cell growth and cell cycle progression are coupled via the translational control of cyclin D1. In this model, cyclin D1 would function as a sensor of cap-dependent translation and, consequently, of the growth rate and as a cell cycle regulator. The regulation of the budding yeast G1
cyclin CLN3 provides additional support for this model. In this system, a short upstream open reading frame in the CLN3
mRNA 5′ leader sequence attenuates the translation of the full-length CLN3 coding region under suboptimal growth conditions, when the ribosome content is limiting (45
Our results indicate that integrin-specific signaling through mTORC1 also contributes to sustain cell survival. It is well established that lack of proper attachment to the matrix results in apoptosis, a phenomenon that has been named anoikis, and earlier studies have identified a variety of mechanisms underlying the induction of anoikis (12
). We have observed that endothelial cells adhering to laminin 1 undergo apoptosis even if they are exposed to otherwise mitogenic concentrations of growth factors. It is unlikely that defective activation of AKT or ERK contributes to cell death on laminin 1, as these signaling effectors are activated to comparable levels on fibronectin and laminin 1. Two lines of evidence suggest instead that endothelial cell survival is dependent on mTORC1 signaling. First, knockdown of merlin activates mTORC1 signaling and rescues endothelial cells plated onto laminin 1 from apoptosis and treatment with rapamycin reverses this effect. Second, overexpression of 4EBP1 induces apoptosis in cells plated onto fibronectin. These results are consistent with the findings of prior studies showing that a constitutively active form of mTOR prevents apoptosis in normal cells deprived of growth factors (9
) and that inhibition of mTORC1 by rapamycin induces apoptosis in tumor cells (2
mTORC1 is abnormally activated in several tumor types, owing to activation of Ras or PI-3K or inactivation of NF1, PTEN, TSC, or LKB1 (15
). Our results provide strong evidence that loss-of-function mutations in NF2
activate mTORC1 signaling in malignant mesothelioma. In the accompanying paper, James and colleagues show that loss of merlin activates mTORC1 signaling in meningiomas and that this occurs, as we have also shown here, independently of AKT or ERK (18
). Together, the results of these studies indicate that the activation of mTORC1 signaling is a key feature of NF2
mutant tumors. Heterozygous NF2
mutations cause type 2 neurofibromatosis, which belongs to a group of cancer predisposition syndromes characterized by neurocutaneous manifestations, the phakomatoses (62
). Interestingly, several other phakomatoses are caused by heterozygous mutations in tumor suppressor genes (NF1
for type 1 neurofibromatosis, PTEN
for Cowden disease, LKB1
for Peutz-Jeghers syndrome, and TSC1
for tuberous scleroses 1 and 2) that negatively regulate mTORC1 signaling. These observations suggest the possibility that activated mTORC1 signaling contributes to the neurocutaneous manifestations of phakomatoses.
Experiments with mouse models and the remarkable efficacy of certain oncogene-targeted therapies have led to the belief that cancer cells are addicted to oncogene signaling (66
). Accordingly, recent studies have employed drug sensitivity to infer oncogene pathway activation in tumor cells (30
). Our observation that merlin-deficient malignant mesothelioma cells are selectively sensitive to the growth-inhibitory effect of rapamycin suggests that mTORC1 signaling contributes to the expansion of NF2
mutant tumors in vivo. Although future studies using mouse models will be required to validate this model, the observation that blockage of mTORC1 inhibits in vitro two distinct tumor types caused by NF2
mutations corroborates the hypothesis that a major function of merlin is to suppress mTORC1 signaling.
Malignant mesothelioma is an aggressive tumor type that responds poorly to standard chemotherapy or radiation therapy (61
). In fact, most patients die within 4 to 12 months from the onset of the first symptoms. The observation that loss of merlin predicts sensitivity to rapamycin identifies mTORC1 as a therapeutic target in the large fraction of malignant mesotheliomas that carry NF2
mutations. We suggest that clinical trials employing mTORC1 inhibitors for malignant mesothelioma are warranted and that patients should be stratified according to NF2 status in preparation for such trials.