mTORC1 promotes survival through translational control of Mcl-1

Abstract

Activation of the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway is a frequent occurrence in human cancers and a major promoter of chemotherapeutic resistance. Inhibition of one downstream target in this pathway, mTORC1, has shown potential to improve chemosensitivity. However, the mechanisms and genetic modifications that confer sensitivity to mTORC1 inhibitors remain unclear. Here, we demonstrate that loss of TSC2 in the E mu-myc murine lymphoma model leads to mTORC1 activation and accelerated oncogenesis caused by a defective apoptotic program despite compromised AKT phosphorylation. Tumors from Tsc2(+/-)E mu-Myc mice underwent rapid apoptosis upon blockade of mTORC1 by rapamycin. We identified myeloid cell leukemia sequence 1 (Mcl-1), a bcl-2 like family member, as a translationally regulated genetic determinant of mTORC1-dependent survival. Our results indicate that the extent by which rapamycin can modulate expression of Mcl-1 is an important feature of the rapamycin response.

Fig. 1.

Tsc2^LOHEμ-myc tumors display accelerated lymphomagenesis and activated mTORC1 signaling. (A) Kaplan–Meier plot illustrating differences in tumor latencies between Tsc2^+/+Eμ-myc (red, n = 41) and Tsc2^+/−Eμ-myc (blue, n = 29) mice (P < 0.001 as determined by log rank analysis). (B) PCR to detect LOH at the Tsc2 locus in Tsc2^+/−Eμ-myc tumors (wt, wild-type allele; mt, mutant allele). (C) Western blot analysis of Eμ-myc tumors of the indicated genotypes probed for activation of AKT-mTOR signaling (p-AKT, α-tubulin, and phosphorylated and total S6K1, S6, and 4E-BP1). (D) Representative micrographs of Tsc2^+/+Eμ-myc and Tsc2^LOHEμ-myc tumor sections stained with H&E, by TUNEL, and for Ki-67. (E) Loss of TSC2 blocks MYC-induced apoptosis to the same extent as AKT activation. The percentage of cells staining positive for TUNEL from Tsc2^+/+Eμ-myc (n = 4), Tsc2^LOHEμ-myc (n = 3), or Eμ-myc/myr-Akt (n = 3) tumors is shown. Tumor tissue sections from three independent animals were analyzed, with three randomly selected fields (≈500 cells) analyzed for TUNEL signal. Error bars represent the SD. ∗, P < 0.001; ∗∗, P ≈0.4 as determined by Student's t test.

Fig. 2.

Activation of mTORC1 leads to defective AKT phosphorylation in MYC-induced lymphomagenesis. (A) Tumors of the indicated genotype were cultured ex vivo and stimulated with 100 nM insulin, 10 μg/ml LPS, or 10 μg/ml IgM. Extracts were prepared and probed for AKT (total and p-S473) and ACTIN. (B) The distribution of mTOR among mTORC1 and mTORC2 is altered in Tsc2^LOHEμ-myc tumors. Tsc2^+/+Eμ-myc and Tsc2^LOHEμ-myc tumors were analyzed for total levels of mTOR, RICTOR, and RAPTOR. MTOR-containing complexes were immunoprecipitated from Eμ-myc tumors of the indicated genotypes, and RICTOR and RAPTOR levels were assessed by Western blotting. (Top) Total levels of mTOR, RICTOR, and RAPTOR in whole-cell extracts. (Bottom) Amounts of RICTOR and RAPTOR coprecipitating (Co-IP) with mTOR. The quantitation of mTORC2:mTORC1 ratios in three tumors of the indicated genotype is shown and represents the average of three experiments ±SD. (C) Tsc2^+/+Eμ-myc and Tsc2^LOHEμ-myc tumors were analyzed for levels of RICTOR-associated mTOR. (D) Amounts of RICTOR and RAPTOR associating with mTOR in Eμ-myc, Tsc2^LOH, myr-Akt, and Pten^+/−Eμ-myc tumors. (E) AKT (S473) phosphorylation is decreased in Tsc2^LOHEμ-myc tumors. Extracts prepared from MSCV/myr-Akt-infected Tsc2^+/+Eμ-myc or Tsc2^LOHEμ-myc tumors were analyzed by Western blotting for AKT (total and p-S473), S6 (total and p-S235/236), and tubulin. AKT and myr-AKT are denoted by an arrow and arrowhead, respectively.

Fig. 3.

Rapamycin and doxorubicin cooperatively induce apoptosis in Tsc2^LOHEμ-myc tumors. (A) Kaplan–Meier plot detailing the time to relapse after treatment of mice bearing Tsc2^LOHEμ-Myc tumors with Dxr, Rap, or a combination of both (Dxr + Rap). P < 0.001 for significance among all curves as determined by log rank test. (B) Extracts prepared from Tsc2^LOHEμ-myc tumors treated with Rap, Dxr, or combination treatment were probed for cleaved PARP (cPARP), tubulin, and rpS6 (S6 and p-S235/236). (C) Tsc2^LOHEμ-myc tumors are more sensitive to Rap-induced apoptosis than Tsc2^+/+Eμ-myc tumors. Eμ-myc derived tumors (Tsc2^+/+ or Tsc2^LOH) were cultured ex vivo in the presence of vehicle or 1 nM Rap for 32 h. Propidium iodide and annexin V staining was used to quantify induction of apoptosis. ∗, P < 0.0007; ∗∗, P ≈0.2 as determined by Student's t test. (D) Tsc2^LOHEμ-myc tumors are more sensitive to growth inhibition than Tsc2^+/+Eμ-myc tumors. Cells were cultured ex vivo in the presence of the indicated concentrations of Rap for 24 h and viability determined by using an MTS assay (Promega). Viability is standardized to nontreated controls (n = 3). (E) mTORC1, not mTORC2, dictates sensitivity to Rap in Tsc2^LOHEμ-myc lymphomas. (Upper) Tsc2^LOHEμ-myc lymphomas were cultured ex vivo and transduced with the indicated MLP-based shRNA vectors. The percentage viable GFP⁺ cells was measured after a 60-h exposure to 50 pM rapamycin or vehicle alone. The results are expressed relative to percentage GFP⁺ cells present before rapamycin exposure (which was arbitrarily set at 1). Shown are averaged over three experiments, and error bars represent the SEM. (Lower) Immunoblots verifying knockdown of target proteins using the indicated MSCV-based shRNA retroviral vectors in NIH 3T3 cells.

Fig. 4.

Rapamycin decreases expression of MCL-1 in Tsc2^LOHEμ-myc tumors. (A) Eμ-myc-derived tumors (Tsc2^+/+ or Tsc2^LOH) were cultured ex vivo in the presence of vehicle or 10 nM Rap for 2 h followed by [³⁵S]Met labeling for the indicated times. Error bars represent SD (n = 2). (B) Rap treatment of Tsc2^LOHEμ-myc tumors impairs recruitment of eIF4GI into the eIF4F complex. Extracts from Tsc2^LOHEμ-myc and Tsc2^+/+Eμ-myc tumors were applied to m⁷GDP affinity resins and washed, and m⁷GTP eluents were probed for eIF4GI, eIF4E, and 4E-BP1. (C) Expression of Mcl-1 is mTOR-dependent in Tsc2^LOHEμ-myc cells. After 1 h of Rap treatment, MCL-1 levels were determined by Western blotting. (D) Translation of Mcl-1 is inhibited by Rap. Polysome profiles from Tsc2^LOHEμ-myc tumors grown ex vivo and exposed to 10 nM Rap for 2 h are shown. RNA was isolated from polysome fractions and subjected to quantitative RT-PCR analysis. The relative amount of Mcl-1 or β-actin mRNA in each fraction is expressed as a percentage of the total in the polysome gradient. Quantitative RT-PCRs were performed in duplicate, and the error bars represent the SEM. Shown is one of three representative experiments performed on different polysome gradients.

Fig. 5.

Mcl-1 is a genetic modifier of rapamycin sensitivity in vivo. (A) Schematic representation of the in vivo competition experimental design. (B) Freshly isolated Tsc2^LOHEμ-myc tumors were transduced ex vivo with MSCV/gfp or MSCV/Mcl-1/gfp virus and introduced into syngenic mice. Upon tumor formation, the GFP content of tumors was determined by fluorescence whole-body scanning with the GE eXplore Optix. The fluorescence signal in each animal was analyzed before and 12 h after a single dose of Rap. The intensity of the GFP signal is depicted by color according the bar scale at the right. (C) Representative flow cytometry analysis of GFP expression in Tsc2^LOHEμ-myc tumors expressing gfp and Mcl-1 in the viable portion of tumors harvested 12 h after Rap exposure.