Integration of mTOR and estrogen–ERK2 signaling in lymphangioleiomyomatosis pathogenesis

Abstract

Lymphangioleiomyomatosis (LAM) is a destructive lung disease of women associated with the metastasis of tuberin-null cells with hyperactive mammalian target of rapamycin complex 1 (mTORC1) activity. Clinical trials with the mTORC1 inhibitor rapamycin have revealed partial efficacy but are not curative. Pregnancy appears to exacerbate LAM, suggesting that estrogen (E₂) may play a role in the unique features of LAM. Using a LAM patient-derived cell line (bearing biallelic Tuberin inactivation), we demonstrate that E₂ stimulates a robust and biphasic activation of ERK2 and transcription of the late response-gene Fra1 associated with epithelial-to-mesenchymal transition. In a carefully orchestrated collaboration, activated mTORC1/S6K1 signaling enhances the efficiency of Fra1 translation of Fra1 mRNA transcribed by the E₂–ERK2 pathway, through the phosphorylation of the S6K1-dependent eukaryotic translation initiation factor 4B. Our results indicate that targeting the E₂–ERK pathway in combination with the mTORC1 pathway may be an effective combination therapy for LAM.

Lymphangioleiomyomatosis (LAM) is a destructive, progressive, multisystem disease in which smooth muscle-like “LAM cells” invade the lung (1–6) and is associated with chylous pleural effusions, lymphatic obstruction, and renal angiomyolipomas (7). LAM occurs almost exclusively in women with onset often occurring in the childbearing years (5, 6, 8, 9). A hallmark of LAM is mutational inactivation of both alleles of the TSC2 gene, which encodes tuberin (TSC2) (1, 2, 4, 5, 10, 11). Loss of TSC2 results in constitutively activated mammalian target of rapamycin complex 1 (mTORC1) signaling (5), which is a primary target for LAM therapy because of its dominant role in regulating cellular metabolism and growth (12). In a recent clinical trial, the mTORC1 inhibitor sirolimus (a rapamycin analog) stabilized lung function in LAM patients (12). However, sirolimus treatment was not curative, and the benefits were observed only during the treatment period, because patients regained disease-related symptoms posttreatment (12, 13). We have focused our attention on estrogen (E₂) signaling because the disease is exacerbated during pregnancy. E₂, upon binding to its estrogen receptor (ER), has been reported to regulate transcription-dependent and -independent signaling events (14). Thus, in addition to its ability to promote changes in gene expression (10, 15–18), E₂ can induce the activation of signaling proteins such as Src, Akt, and ERK-MAP kinase (14). The importance of ERK-MAP kinase in LAM was suggested by a recent report showing that E₂ promoted the MEK-dependent invasion of cells derived from Eker rat uterine leiomyoma (ELT3 cells) into the lungs of ovariectomized mice (18). However, the molecular basis for E₂-dependent ERK contribution to the enhanced invasive phenotype in the presence of constitutively activated mTORC1 was not defined, and whether E₂ promoted a similar response in patient-derived cells remained to be determined. Here we used TSC2-null LAM patient-derived angiomyolipoma (AML) cells as a platform to examine the cellular response to E₂ and the potential interaction between the E₂–ERK and activated mTORC1 pathways in establishing the metastatic-like phenotype observed in LAM.

ERK is a versatile signaling molecule capable of mediating distinct cellular fates depending on the strength and duration of activation as well as the cellular localization of the active enzyme (19, 20). For example, the rapid activation of ERK and its nuclear translocation are sufficient to induce immediate-early genes (IEGs) such as c-Fos and c-Jun, which contribute to the expression of activator protein 1 (AP1) transcription factor complexes. Increased AP1 levels lead to the induction of late-response genes (LRGs). However, without sustained nuclear activation of ERK, many of the products of these genes are unstable and therefore are poorly induced. To stabilize these IEG products a strong, sustained ERK activation that promotes AP1 accumulation and LRG induction. The LRG product, Fra1, also is stabilized by sustained nuclear ERK activation (21). Our laboratory recently has found that in MCF-10A human mammary epithelial cells, the Ras-mediated EMT-associated change in morphology as well as the ability to migrate and invade are mediated through sustained ERK2 signaling, which leads to expression of the LRG Fra1. Fra1 expression in turn leads to elevated expression of the E-cadherin transcriptional repressor zinc finger E-box-binding homeobox 1/2 (ZEB1/2), which contributes to increased cell migration and invasion (22).

In addition to regulation of cellular responses by ERK signaling, cells have evolved a very elaborate and sensitive mechanism to adapt to environmental changes by regulating cellular metabolism and protein synthesis, and the mTORC1 pathway is a major orchestrator of these processes (23). mTORC1-mediated cap-dependent translation initiation involves the phosphorylation of 4E-binding protein (4E-BP) and its detachment from the 5′ cap-binding protein eukaryotic translation initiation factor 4E (eIF4E), allowing the recruitment of the scaffolding protein eIF4G and associated proteins such as poly-A binding protein (PABP) and the eIF4A RNA helicase to the 5′ end of a target mRNA. mTORC1 activation also promotes the assembly of the translation preinitiation complex, ribosome scanning, and translation initiation. Another level of translation control occurs for mRNAs with highly structured 5′ UTRs. This relatively small number of mRNAs is enriched for regulators of bioenergetics, cell cycle, and angiogenesis. Phosphorylation of eIF4B by the mTORC1–S6K pathway promotes its recruitment into the translation preinitiation complex, where it is proposed to promote significantly the RNA helicase activity of eIF4A leading to the unwinding of the hairpin structures present in the long 5′ UTRs of these mRNAs and thereby greatly enhancing their translation efficiency (23, 24).

We have identified several unique features of both ERK and mTORC1 signaling in LAM patient-derived cells. We observe that E₂ contributes to migration and invasion by stimulating a critical biphasic ERK2 activation these cells. We also demonstrate that Fra1 plays an important role in regulating E₂-stimulated epithelial-to-mesenchymal transition (EMT)-like features in these cells. We provide evidence indicating that in the presence of E₂ and highly active mTORC1, ERK2, and mTORC1 signaling converge at the level of Fra1 to orchestrate its transcription and translation, respectively, and to stimulate migration and invasion in LAM patient-derived cells. Taken together, these observations suggest that inhibiting E₂ signaling in combination with mTORC1 inhibition may target LAM cell proliferation and migration/invasion selectively. We suggest that combination therapy along the E₂–ERK pathway (for example, with an ER antagonist) and along the mTORC1 pathway with rapamycin analogs [drugs that have been approved by the Food and Drug Administration (FDA)] may offer a highly effective treatment for LAM.

Results

E₂-Stimulated Cell Migration and Invasion Correlate with Robust and Biphasic ERK Activation in LAM patient-derived cells.

Previous results had linked E₂-stimulated ERK activity to increased invasion of rat ELT3 cells into mouse lungs in a xenograft model (5, 9). To test whether LAM patient-derived cells also were E₂ responsive along the ERK pathway, we first measured the migratory and invasive response of E₂-stimulated cells in the presence or absence of the MEK1/2 inhibitor AZD6244 (AZD). E₂ stimulated LAM cell migration and invasion across Transwell supports (Fig. 1 A and B). These cellular behaviors were associated with decreased expression of the epithelial marker E-cadherin and increased expression of the mesenchymal marker N-cadherin upon E₂ stimulation; these effects were reversed by inhibition with AZD (Fig. 1C), indicating a role for ERK signaling in the migration and invasion of LAM cells in vitro.

Fig. 1.

E₂-stimulated cell migration and invasion correlate with robust and biphasic ERK activation in LAM patient-derived cells. (A and B) LAM cells, 1 h after attachment to the upper Transwell membrane (30,000 cells per well), were treated with or without 10 nM E2 (lower well) in the presence or absence of the MEK1/2 inhibitor AZD (5 µM) (upper well) in phenol red-, EGF-, and FBS-free IIA complete medium and were studied for migration and invasion. Cells that migrated/invaded to the other side of the Transwell insert membrane were fixed, stained, and quantified. (C) LAM cells were starved overnight in phenol red-, EGF-, and FBS-free IIA complete medium and were stimulated with 10 nM E2 with or without pretreatment with the MEK1/2 AZD (5 µM) for 1 h, Levels of EMT-associated markers E-cadherin (E-Cad) and N-cadherin (N-Cad) were analyzed at different time points by immunoblot. Tubulin was used as an internal control. (D) LAM cells were starved as described above. The transcription inhibitor ActD (5 µg/mL) was added 1 h before or after 10 nM E2 stimulation, and the MEK1/2 inhibitor AZD (5 µM) was added 1 h before E2 stimulation. Levels of phosphorylated ERK1/2 were analyzed at different time points by immunoblot and were quantified by ImageJ. Total ERK1/2 was used as an internal control. Each experiment was performed in triplicate. Graphs are representative of multiple experiments (n = 5).

To understand better how ERK contributes to cell migration, we examined the kinetics of E₂-dependent ERK activation (Fig. 1D). We found that both ERK isoforms, but most prominently ERK2 (addressed in detail in Fig. 2) displayed a reproducible biphasic activation pattern with an early phase around 1 h and a late phase around 6–8 h after E₂ stimulation (Fig. 1D). Because ERK activation was blocked completely by 1-h pretreatment with AZD (Fig. 1D), it is unlikely that E₂ activates ERK independently of MEK1/2 (e.g., by inhibiting the expression of an ERK phosphatase). To understand how E₂ treatment generates the biphasic ERK activation pattern, we investigated whether both peaks were transcription dependent by inhibiting transcription with Actinomycin D (ActD) (Fig. 1D). Treatment of cells with ActD 1 h before or after E₂ treatment did not affect the first ERK activation peak but reduced the second peak to basal levels. Therefore, the first ERK activation peak is transcription independent, whereas the second peak is transcription dependent.

Fig. 2.

ERK2 is required for mediating EMT in LAM patient-derived cells. (A) Control or ERK isoform KD cells, after 1-h attachment to the upper Transwell membrane (30,000 cells per well), were treated with 10 nM E2 in the presence or absence of AZD (5 µM) and were studied for migration/invasion. Cells that migrated/invaded to the other side of the Transwell insert membrane were fixed, stained, and quantified. (B) Morphology of ERK isoform KD in LAM cells. (Magnification: 20×; scale bar: 50 µm.) (C) Expression of EMT-associated markers E-cadherin and N-cadherin in ERK isoform KD cells as shown in A. ERK knockdowns were completed with two distinct shRNA constructs in A, B, and C (SI Materials and Methods). (D and E) The migration and invasion of LAM cells expressing ERK WT were studied using the method described in A. For rescue experiments in ERK KD cells (ERK2 shRNA #2), T7-tagged ERK1 WT or HA-tagged ERK2 WT was expressed. The levels of ERK1/2 and EMT markers are shown in F. Tubulin was used as an internal control. Each experiment was performed in triplicate. Graphs are representative of multiple experiments (n = 3).

ERK2 Is Required for Mediating E₂-Regulated EMT in LAM patient-derived cells.

We previously had shown a role for ERK2 in cell migration and survival in mammary epithelial cells (22, 25). Using lentiviral-based shRNAi constructs to knock down ERK1 or ERK2, we found that E₂ promoted migration in both control GFP and ERK1-KD cells to almost the same level, whereas ERK2 knockdown significantly reduced cell migration to a level as low as that observed when ERK signaling was inhibited by the MEK1/2 inhibitor AZD (Fig. 2A). Furthermore, ERK2 knockdown was associated with a transition from a mesenchymal spindle-like phenotype to an epithelial cuboidal-like morphology (Fig. 2B). A mesenchymal-to-epithelial transition (MET) was confirmed by decreased expression of the mesenchymal marker N-cadherin and increased expression of the epithelial marker E-cadherin (Fig. 2C).

In single-isoform ERK-KD cells, the ability of the remaining ERK isoform to respond to E₂ stimulation was unaffected (Fig. S1A). To analyze further the specific influence of ERK2 on the EMT-like phenotype of LAM patient-derived cells, we overexpressed T7-tagged WT ERK1 or HA-tagged WT ERK2 in LAM cells. LAM cells overexpressing ERK2 WT but not ERK1 WT promoted migration and invasion (Fig. 2 D and E). In addition, overexpression of functional ERK1 WT at levels greater than endogenous ERK2 did not rescue the effects of ERK2 knockdown, whereas rescue with ERK2 WT did reverse the phenotype (Fig. 2F and Fig. S1B). Together, these data indicate ERK2, rather than ERK1, is the main contributor to the E₂-induced migration and invasion in LAM patient-derived cells.

E₂ Induces Fra1 Transcription to Cell Migration and Invasion in LAM patient-derived cells.

We recently reported that the regulation of Fra1 and its effector ZEB1/2 downstream of ERK2 signaling is important in promoting EMT in mammary epithelial cells (22, 25). Because the second peak of the biphasic ERK activation after E₂ stimulation for 6–8 h was transcription-dependent (Fig. 1D), we hypothesized that this later or sustained ERK activation is associated with transcription-dependent changes in gene expression linked to EMT. Supporting this notion, we detected an increase in the expression of the LRGs Fra1 and ZEB1 in LAM patient-derived cells stimulated with E₂ (Fig. 3A).

Fig. 3.

E₂-induced Fra1 expression contributes to migration and invasion of LAM patient-derived cells. (A) Time-course E₂ stimulation in LAM cells. Cells were starved as previously described, stimulated with 10 nM E₂, and lysed at different time points. Levels of phospho-ERK1/2, Fra1, and ZEB1 were analyzed by immunoblot. Total ERK1/2 and tubulin were used as internal controls. (B) ZEB1 expression correlated with Fra1 expression upon 8-h E2 stimulation in control or Fra1 KD cells. (Two distinct shRNA constructs were used. See SI Materials and Methods.) (C) Morphology of Fra1 KD and Fra1 WT in LAM cells. (Magnification: 20×; scale bar: 50 µm.) (D and E) After 1-h attachment to the upper Transwell membrane (30,000 cells per well), control cells, Fra1 KD cells, or LAM cells expressing Fra1 WT were treated with 10 nM E₂ and ertr studied for migration/invasion. Cells that migrated/invaded to the other side of the Transwell insert membrane were fixed, stained, and quantified. (F) Expression of EMT-associated markers E-cadherin and N-cadherin in Fra1 KD cells or LAM cells expressing Fra1 WT. Tubulin was used as an internal control. Each experiment was performed in triplicate. Graphs are representative of multiple experiments (n = 3).

We next knocked down or overexpressed Fra1 to demonstrate further a role for this gene in the E₂-stimulated invasive phenotype of LAM patient-derived cells. As shown in Fig. 3 B–F, ZEB1 expression was associated with Fra1 expression upon E₂ stimulation. Fra1 knockdown led to a significant decrease in migration and invasion, and this change in behavior was tightly linked to a switch from a mesenchymal-like to an epithelium-like phenotype, with decreased expression of N-cadherin and increased expression of E-cadherin. Upon Fra1 overexpression, these results were reversed (Fig. 3F). Importantly, knockdown of Fra1 did not affect E₂-stimulated ERK activation peaks (Fig. S2A). Combined, our results suggest that in LAM cells cultured under the conditions described, E₂ regulates migration and invasion through ERK2–Fra1–ZEB1/2 signaling.

E₂Stimulates Fra1 Transcription Downstream of the ERK2 Pathway Independent of mTORC1 Inhibition in LAM patient-derived cells.

We have shown that E₂–ERK2 signaling regulates the migration and invasion of LAM cells through the induction of the EMT-associated gene Fra1. We have found that rapamycin also can partially suppress migration/invasion in these cells without preventing the induction of Fra1 mRNA (Fig. 4A and Fig. S2 B and C). We therefore investigated whether Fra1 protein expression is regulated by mTORC1 and whether Fra1 is a point of convergence for the E₂–ERK2 and activated mTORC1 pathways in the LAM cells. Because Fra1 mRNA has a structured 5′ UTR (Fig. S3) and because mTORC1 enhances the translation efficiency of mRNAs with a highly structured 5′ UTR, we hypothesized that E₂–ERK2 signaling regulates Fra1 transcription and Fra1 stability (26), whereas mTORC1 might regulate Fra1 translation efficiency.

Fig. 4.

E₂-ERK2 regulates Fra1 mRNA expression. (A–C) SYBR Green-based real-time PCR analysis of Fra1 and ZEB1/2 mRNA expression at 8 h in LAM patient-derived cells starved overnight and treated with 10 nM E₂ in the presence or absence of the transcription inhibitor ActD (5 µg/mL) or rapamycin (rapa; 20 nM) or their combination 1 h before or after E₂ stimulation. β-Actin was used as an internal control. (D–F) After overnight starvation, LAM cells were treated with 20 nM rapamycin and/or the S6K1 inhibitor PF4708671 (30 µM) for 1 h before E₂ stimulation. mRNA expression of Fra1 (D) and ZEB1/2 (E and F) were analyzed at 8 h. Each experiment was performed in triplicate. Graphs are representative of multiple experiments (n = 3).

We first measured the mRNA levels of Fra1 and its downstream effector ZEB1/2 in E₂-treated LAM cells using real-time PCR. Because Fra1 is an LRG, and based on our data shown in Fig. 1D, the LAM patient-derived cells were stimulated with E₂ for 8 h and were treated with ActD or rapamycin for 1 h before or after E₂ stimulation. Treatment of these cells with ActD or rapamycin for 1 h after E₂ allows the induction of IEGs but will reveal if Fra1 expression depends on its transcription and/or translation, respectively. E₂ promoted an increase in mRNA levels of both Fra1 and ZEB1/2 in a transcription-dependent manner, because pretreatment with the transcription inhibitor ActD (5 µg/mL) reduced their expression (Fig. 4 A–C). The mRNA levels of Fra1, in contrast to ZEB1/2, were not affected dramatically when rapamycin was added 1 h before E₂ stimulation (Fig. 4D), consistent with the role of rapidly induced IEGs, such as c-Fos and c-Jun, in the subsequent expression of LGRs such as Fra1 and the contribution of Fra1 to the induction of ZEB1/2. Combined with the observation that addition of the MEK inhibitor after 1 h also prevented Fra1 induction, these data indicate the importance of the later ERK activation peak at around 6–8 h in mediating events linked to Fra1 and ZEB1/2 expression and EMT-like phenotypes.

Interestingly, although the mRNA expression of Fra1 remained elevated with rapamycin treatment at 8 h after E₂ treatment, we found that the expression of its downstream effector ZEB1/2 was reduced significantly (Fig. 4 E and F). This result suggested that Fra1 protein expression might be suppressed by rapamycin, thus preventing it from inducing ZEB1/2 expression. We next investigated if the mTORC1 effector S6K1 regulates the expression of Fra1 and ZEB1/2 mRNA. To do so, we used the S6K1-specific ATP-competitive inhibitor PF4708671. As shown in Fig. 4 D–F, inhibiting either mTORC1 or S6K1 did not affect Fra1 but did suppress ZEB1/2 mRNA levels induced by E₂ at 8 h, suggesting that mTORC1/S6K1 might mediate the translation of Fra1 downstream of E₂–ERK2 pathway.

mTORC1 and S6K1 Enhance the Translation Efficiency of Fra1 Transcribed Downstream of the E₂–ERK2 Pathway Through eIF4B Phosphorylation.

To test the possibility that constitutively activated mTORC1 might contribute to enhanced translation of Fra1, we performed the same experiment described in Fig. 4, but this time we examined the protein levels of Fra1 and ZEB1. Rapamycin inhibition was confirmed by reduced phosphorylation of its downstream regulator S6K1 at T389, whereas blocking the kinase activity of S6K1 with PF4708671 did not affect the phosphorylation of S6K1 in these TSC2-null LAM patient-derived cells but did antagonize downstream signaling (Fig. 5A). Both Fra1 and ZEB1/2 protein levels were reduced significantly upon either mTORC1 or S6K1 inhibition (Fig. 5 A and B). Given that Fra1 mRNA expression was not inhibited by rapamycin, but ZEB1/2 expression was, that Fra1 is an upstream regulator of ZEB1/2 expression (22), and that the expression of Fra1 and ZEB1 protein was suppressed significantly by rapamycin treatment, we conclude that E₂-activated ERK2 regulates the expression of the Fra1 gene and the stability of Fra1 protein (19, 20) and that mTORC1-S6K1 enhances the efficiency of Fra1 translation. It is worth noting that neither mTORC1 nor S6K1 inhibition affected the activation of ERK by E₂ at 8 h, indicating that hyperactive mTORC1 signaling is independent of E₂–ERK2 signaling.

Fig. 5.

mTORC1 regulates Fra1 mRNA translation efficiency. (A and B) After overnight starvation, LAM cells were treated with 5 µg/mL ActD, 20 nM rapamycin, and/or the S6K1 inhibitor PF4708671 (30 µM) for 1 h before E₂ stimulation. Cells were lysed at 8 h for protein expression analyses. Levels of phosphorylated ERK1/2, phospho-S6K1 T389, phospho-eIF4B (pSer422), eIF4B, Fra1, and ZEB1 were analyzed by immunoblot. Tubulin was used as an internal control. Fra1 expression in A was normalized to the internal control and was quantified in B by ImageJ. (C and D) LAM cells stably expressing eIF4B WT or S422D were starved as described and were treated for 1 h with AZD (5 μM) or PF4708671 (30 µM) in the presence of 10 nM E₂. Levels of phospho-eIF4B, eIF4B, Fra1, and ZEB1 were analyzed by immunoblot. Tubulin was used as an internal control. Fra1 expression in C was normalized to the internal control and quantified in D by ImageJ. Graphs are representative of multiple experiments (n = 3).

Because of the loss of TSC2, LAM cells have a high basal level of eIF4B phosphorylation. Reduced eIF4B phosphorylation by upstream inhibition of mTORC1 or S6K1 inhibition was associated with significantly reduced expression of Fra1 and ZEB1 (Fig. 5 A and B). To support our hypothesis that mTORC1–S6K1 signaling could regulate the translation efficiency of Fra1 mRNA, we predicted that Fra1 is highly structured within its 5′ UTR (Fig. S3). The translation efficiency of mRNAs with highly structured 5′ UTRs is enhanced by the RNA helicase activity of eIF4A, which is proposed to be regulated by S6K1-mediated phosphorylation and recruitment of the eIF4B regulatory subunit into the translation preinitiation complex (23). To confirm further the crucial role of eIF4B in regulating Fra1 translation in LAM cells, we generated LAM cell lines that stably expressed eIF4B WT or a S422D mutant that was constitutively phosphomimetic at S422. E₂ promoted Fra1 expression at 8 h in eIF4B WT and S422D mutant cells. Inhibiting the mTORC1 pathway resulted in a significant drop in Fra1 in eIF4B WT cells, whereas the expression of Fra1 and ZEB1 was rescued in cells expressing the phosphomimetic eIF4B S422D upon S6K1 inhibition (Fig. 5 C and D). Our data suggest that the hyperactive mTORC1–S6K1 pathway works downstream of E₂-ERK2–dependent Fra1 transcription to regulate its translation efficiency through phosphorylation of eIF4B, further confirming the crucial role of eIF4B in regulating Fra1 translation in TSC2-null LAM cells.

Discussion

The development of LAM appears to involve a multistep process, with initiation caused by mutations resulting in loss of function of the tumor-suppressor protein complex and a hypothesized role for E₂ in promoting disease progression (5, 9). Because of its constitutive activation in LAM cells, mTORC1 has been considered the primary therapeutic target for LAM (5, 12). Indeed, sirolimus, a rapamycin analog, recently was shown to stabilize pulmonary function in LAM patients (12). However, the clinical efficacy of rapamycin is not permanent, and cessation of treatment results in reacquisition of most disease symptoms (13, 27). The long-term risks and benefits of continuous treatment with mTORC1 inhibitors in this patient population, and whether drug resistance will emerge, are unknown but are predicted based on several studies. We have focused our attention on other pathways that work upstream of or in parallel with the mTORC1 pathway and which may represent additional targets for LAM therapy. Because E₂ has long been thought to contribute to the development of LAM (6, 10, 15–18), we have investigated the possible connections between these signaling processes. Here, we have identified a mechanism for the coordination and integration of E₂-stimulated ERK activation, Fra1 induction, and mTORC1/S6K1-mediated Fra1 translation in contributing to an EMT-like phenotype in LAM patient-derived cells.

We have found that E₂ causes a biphasic activation of ERK1/2 in LAM patient-derived cells (621-101 cells). The early-phase ERK activation (0.5–1 h) is transcription independent, whereas the later-phase ERK activation (6–8 h) is transcription dependent. We also demonstrate that sustained ERK2 signaling is required for the invasive features of the LAM patient-derived cells. Sustained ERK signaling is important for the LRG and protein-expression profiles and for dictating specific cell fates (19, 20, 28). For example, Fra1 is an LRG that increases cell migration and invasion associated with EMT (22, 25).

We therefore examined the possible connection between the E₂–ERK pathway and the constitutively active mTORC1 pathway at the level of the transcription factor Fra1 in E₂-stimulated LAM patient-derived cells based on (i) our observations that the later transcription-dependent ERK activation mediates induction of the EMT-associated LRG Fra1 in LAM cells; (ii) our previous observations that ERK2 signaling is critical for the acquisition of a mesenchymal-like phenotype in mammary epithelial cells expressing Ras-V12 during EMT; and (iii) the demonstration that TSC2-null LAM cells have constitutively high mTORC1 activity regardless of any stimulation. Given that rapamycin treatment of these LAM cells also affects their migratory and invasive properties, we asked how mTORC1 contributes to these phenotypes: Does it act upstream or downstream of the E₂–ERK2 pathway, and does it act in parallel with another pathway that also regulates migration and invasion (29) or in a separate pathway that converges to modulate a key regulator of the EMT-like phenotype observed in E₂-treated LAM cells? Our data reveal a coordination between these pathways and thus support the latter possibility. We have shown that E₂ acts mainly through the ERK2 isoform and promotes the transcriptional up-regulation of the Fra1 gene. The constitutively activated mTORC1 translational machinery enhances the translation efficiency of Fra1 mRNA, which is predicted to have a secondary structure at its 5′ UTR. The presence of highly phosphorylated eIF4B, which is sensitive to mTORC1 and S6K1 inhibitors in these cells, appears to increase the efficiency of Fra1 mRNA translation. Our findings also demonstrate that the combination of both mTORC1 signaling and E₂–ERK2 signaling is more effective than targeting either pathway alone (Fig. S2 B and C).

In conclusion, the E₂-regulated ERK2 pathway and the constitutive mTORC1 pathway converge on the Fra1–ZEB1/2 transcriptional network to promote migration and invasion in LAM patient-derived cells, as summarized in Fig. 6. These observations reveal the importance of examining the effects of targeting E₂ signaling in combination with mTORC1 pathway in treating LAM, because this combination potently inhibits distinct pathways that converge to control cell growth, proliferation, migration/invasion, and survival. These findings have immediate significance and clinical impact. Both the ER antagonist fulvestrant and rapamycin are FDA approved, and thus preclinical trials now can be designed which we anticipate will reveal the effectiveness of this combination. In addition, further defining these converging pathways and the downstream common pathways will reveal new therapeutic targets and new biomarkers for LAM and other ER-sensitive cancers.

Fig. 6.

Summary of the convergence of the E₂–ERK and mTORC1 signaling pathways in regulating migration and invasion in LAM patient-derived cells. These have TSC2 mutations and therefore are hyperactive in mTORC1 activity (10). E₂ activates the ERK pathway, uses biphasic and sustained ERK2 activation, and stimulates the transcriptional induction of the LRG, Fra1. Concurrently, constitutively activation of mTORC1 in LAM cells enhances the translation of accumulating Fra1 mRNA. Blocking mTORC1/S6K1 with either an mTORC1 inhibitor or an S6K1 inhibitor inhibits the phosphorylation of eIF4B and therefore decreases the translation efficiency of Fra1 and downstream cellular processes such as ZEB1/2 induction, EMT markers, and cell migration and invasion. Inhibitors used in this study are the transcription inhibitor ActD (5 µg/mL), the MEK1/2 inhibitor AZD6244 (AZD; 5 µM), the mTORC1 inhibitor rapamycin (rapa; 20 nM), and the S6K1 inhibitor PF4708671 (30 µM).

Materials and Methods

Cells derived from renal angiomyolipomas from LAM patients (621-101 TSC2-null cells) were used and maintained in IIA complete medium with 10% (vol/vol) FBS as previously described (30). Before stimulant treatment, LAM cells were starved overnight in phenol red-, EGF-, and FBS-free IIA complete medium. For inhibition, cells were treated for 1 h with the MEK1/2 inhibitor AZD 6244 at 5 µM, the S6K1 inhibitor PF4708671 at 30 µM, or the mTORC1 inhibitor rapamycin at 20 nM before stimulation with either EGF at 10–20 ng/mL or 17 β-Estradiol (E₂) at 10 nM. For infection, LAM cells were infected with either retroviruses or lentiviruses to overexpress or knock down specific genes of interest using 2 µg/mL puromycin as selection. LAM cells were lysed at different time points for Western blot. To measure migration and invasion, Transwell-based assays (Life Technologies-Invitrogen) were used. Cells that migrated to or invaded the other side of the insert membranes were fixed and quantified. SYBR Green-based RT-PCR was used to detect mRNA levels in LAM cells under different conditions. Full methods are available in SI Materials and Methods.