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 Eμ-myc murine lymphoma model leads to mTORC1 activation and accelerated oncogenesis caused by a defective apoptotic program despite compromised AKT phosphorylation. Tumors from Tsc2+/−Eμ-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.
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An important consequence of phosphatidylinositol 3-kinase (PI3K)/AKT pathway activation is stimulation of mammalian target of rapamycin (mTOR), which is associated with altered cell growth, cell survival, and therapeutic response. AKT signaling leads to disruption of the tuberous sclerosis complex [composed of two gene products TSC1 (Harmartin) and TSC2 (Tuberin)] that in turn no longer attenuates RHEB-GTPase activity and results in mTOR activation. There are two structurally distinct mTOR signaling complexes in mammalian cells: the mTOR complex 1 (mTORC1) and mTORC2 (1). One well characterized function of mTORC1 is to maintain protein synthesis in homeostasis with the cellular environment. It achieves this through phosphorylation of at least two direct targets, eukaryotic initiation factor (eIF) 4E-binding proteins (4E-BPs) and ribosomal protein S6 kinases (S6Ks) (2). 4E-BPs bind to and prevent eIF4E from entering into eIF4F, a heterotrimeric complex required for the cap-dependent ribosome recruitment phase of translation initiation. S6K also regulates translation initiation by controlling the activity of eIF4A, a DEAD box RNA helicase, essential to eIF4F function (3).
Rapamycin (Rap), an mTOR inhibitor, is currently being investigated as a therapy against numerous cancers (4). It targets primarily mTORC1, although in some cell types, long-term exposure to Rap interferes with mTORC2 activity, presumably by sequestering newly synthesized mTOR and reducing nascent mTORC2 complex formation (5). Although many cancers show elevated mTORC1 activity, treatment with Rap (or its analogs) is only effective against a subset of tumors (4). Identifying determinants of Rap sensitivity is therefore critical to understanding when mTOR inhibition could be an effective cancer therapy. Two proposed mediators of the Rap response are: (i) the ability of rapamycin to reactivate AKT signaling, normally suppressed by S6K1 (6), and (ii) elevated levels of eIF4E (7, 8), although the targets directly mediating sensitivity remain unknown.
In this work, we use a preclinical mouse model to investigate the effect of mTORC1 activation (via TSC2 loss) on translation initiation, oncogenesis, and tumor chemosensitivity. We find that Tsc2+/−Eμ-Myc mice developed aggressive drug-resistant lymphomas that depended on mTORC1 activity for survival. We demonstrate that myeloid leukemia cell sequence 1 (Mcl-1), an important antiapoptotic regulator required for the maintenance of mature lymphocytes, is translationally regulated by mTORC1.
Results
Loss of TSC2 Accelerates MYC-Induced Lymphomagenesis.
In the Eμ-myc mouse tumors model, activation of AKT signaling leads to accelerated tumorigenesis and drug resistance (8). Loss of AKT signaling inhibits cell proliferation and oncogenesis, an effect that can be rescued by hyperactivation of mTORC1, suggesting that AKT may mediate its oncogenic effects exclusively through mTORC1 (9). We therefore sought to determine whether a genetic lesion downstream of AKT, and capable of activating mTORC1, could accelerate lymphomagenesis and drug resistance in vivo. Because inactivation of TSC fulfills these requirements (10), we crossed Tsc2+/− mice to Eu-Myc transgenic mice. Eμ-Myc mice developed tumors with a mean onset time of 114 days (Fig. 1A) (8, 11). Tsc2+/− mice did not develop any lymphoid hyperplasia over the course of this work. In contrast, Tsc2+/−Eμ-Myc mice developed tumors at a significantly accelerated rate (Fig. 1A, mean onset time is 47 days, P < 0.001). We found that the majority (66%) of tumors from Tsc2+/−Eμ-Myc mice had lost the remaining wild-type Tsc2 allele (Fig. 1B), an event also confirmed by immunoblotting (J.R.M., unpublished observations). Tumors from Tsc2+/−Eμ-Myc mice did not infiltrate the visceral organs as aggressively as Akt-driven Eμ-Myc tumors (8). They did, however, show a propensity to invade the subarachnoid space of the brain, often accumulating in blood vessels of the cerebellum where they induced infarction leading to overt motor control defects [supporting information (SI) Fig. S1]. Thus, loss of TSC2 greatly accelerated the onset and severity of myc-driven lymphomas.
Fig. 1.
mTORC1 signaling was activated in primary Tsc2+/−Eμ-Myc tumors that underwent loss of heterozygosity (LOH; referred to in this work as Tsc2LOHEμ-Myc), as judged by phosphorylation of S6K1 and 4E-BP1 (Fig. 1C; lanes 7–9). 4E-BP1 and S6K1 phosphorylation were elevated in Eμ-Myc/myr-Akt tumors although not as extensively (lanes 4–6). Tumors lacking lesions in the AKT/mTORC1 pathway showed little phosphorylation of rpS6, S6K1, and 4E-BP1 (lanes 1–3). Taken together, the results indicate robust mTORC1 activation in Tsc2LOHEμ-Myc tumors.
Forced activation of AKT in the context of the Eμ-Myc mouse model results in tumors that show little change in proliferation rates but have suppressed apoptosis (8). Immunohistochemical (IHC) analysis confirmed that both tumor genotypes showed similar levels of proliferation as revealed by Ki67 staining; however, Tsc2LOHEμ-Myc tumors had reduced apoptotic rates, similar to what was observed in AKT-driven tumors (Fig. 1 D and E). IHC also verified that mTORC1 was active because there was elevated p-S6 staining in Tsc2LOHEμ-Myc tumors compared with Tsc2+/+Eμ-Myc tumors (Fig. 1D). This is consistent with activated mTORC1 regulating programmed cell death in the Eμ-Myc mouse model (8). The impact of Tsc2 loss on tumor behavior was reminiscent of AKT-driven lymphomas because Tsc2LOHEμ-Myc tumors maintained functional p53, whereas Eμ-Myc commonly do not (12). p53 alleles were intact and lacked any mutations as determined by sequencing of all 11 p53 exons from a panel of 11 Tsc2LOHEμ-Myc tumors (data not shown). Tsc2LOHEμ-Myc tumors retained p53 protein expression and repressed ARF expression (Fig. S2A). Detection of exon 1β of Arf verified the presence of the Arf locus (Fig. S2B), and Tsc2LOHEμ-Myc tumors responded appropriately to γ-irradiation by up-regulating p53 and p21 expression (Fig. S2C). Knockdown of p53 in Tsc2LOHEμ-Myc tumors blocked induction of p21, whereas p53−/−Eμ-Myc tumors failed to activate p21 (Fig. S2C). Thus, Tsc2LOHEμ-Myc tumors retained a functional p53 pathway.
Tsc2LOHEμ-Myc Lymphomas Have Defective AKT Activation.
Loss of TSC2 in mouse embryonic fibroblasts (MEFs) impairs AKT activation by blocking PI3K-dependent recruitment to the membrane, where it is fully activated by phosphorylation on S473 by mTORC2 (13, 14). We tested the inducibility of AKT in Tsc2LOHEμ-Myc tumors by measuring S473 phosphorylation after treatment with various PI3K stimulators (Fig. 2A). Insulin, LPS, and IgM were incapable of eliciting AKT (S473) phosphorylation in Tsc2LOHEμ-Myc tumors (lanes 1–4). In contrast, Tsc2+/+Eμ-Myc tumors were responsive to these stimuli (lanes 5–8).
Fig. 2.
Another important consequence of loss of TSC2 is an alteration in the distribution of mTOR among its complexes, mTORC1 and mTORC2 (15). Loss of mTORC2 has been shown to stimulate mTORC1 signaling (16). We therefore measured the proportion of mTOR present in mTORC1 (associating with RAPTOR) or mTORC2 (associating with RICTOR) in Tsc2LOHEμ-Myc and Tsc2+/+Eμ-Myc tumors (Fig. 2B). Anti-mTOR antibodies immunoprecipitated less RICTOR from Tsc2LOHEμ-Myc lymphomas compared with Tsc2+/+Eμ-Myc lymphomas (Fig. 2B; ≈2.5-fold decrease). Similarly, anti-RICTOR antibodies immunoprecipitated less mTOR in Tsc2LOHEμ-Myc tumors (Fig. 2C). Tsc2−/−p53−/− MEFs had similar changes in mTORC1 and mTORC2 levels compared with Tsc2+/+p53−/− MEFs (Fig. S3). We also analyzed mTOR complexes in Pten+/−Eμ-Myc and Eμ-Myc/myr-Akt tumors and did not find significant changes in mTORC2:mTORC1 ratios compared with Eμ-Myc tumors (Fig. 2D, compare lanes 3 and 5 with 1). Loss of mTORC2 complexes could explain why we noted only weak phosphorylation of constitutively membrane recruited myr-AKT in Tsc2LOHEμ-Myc tumors (Fig. 2E). Hence, despite loss of AKT activation, Tsc2LOHEμ-Myc tumors develop rapidly with a suppressed apoptotic program.
mTORC1 Mediates Cell Survival in Tsc2LOHEμ-Myc Lymphomas.
Tsc2LOHEμ-Myc derived tumors had significantly lower levels of apoptosis (Fig. 1E), prompting us to assess their drug sensitivity. C57BL/6 mice bearing transplanted primary Tsc2LOHEμ-Myc lymphomas were treated with either doxorubicin (Dxr), rapamycin (Rap), or combination therapy (Dxr + Rap) and monitored for tumor-free survival. Here, animals bearing Arf−/−Eμ-myc lymphomas were used as controls because these lymphomas are also highly aggressive but remain chemosensitive (8, 17). Mice bearing control tumors underwent complete remission for ≈18 days after Dxr treatment (data not shown) as reported in ref. 8. Mice bearing Tsc2LOHEμ-Myc lymphomas showed little response to Dxr, with tumors relapsing by 5 days after treatment (Fig. 3A; P < 0.001 relative to control lymphomas). Mice bearing control lymphomas did not respond to Rap (data not shown), whereas mice harboring Tsc2LOHEμ-Myc lymphomas showed complete remission for ≈10 days after Rap therapy (Fig. 3A; medium dash, P < 0.001 compared with control lymphomas). Tsc2LOHEμ-Myc lymphomas were highly sensitive to the combination of Rap and Dxr, similar to what has been observed for Eμ-Myc/myr-Akt- derived lymphomas (Fig. 3A; P < 0.001 compared with single agents). This enhanced sensitivity was associated with increased apoptosis as assessed by PARP cleavage (Fig. 3B). Tsc2LOHEμ-Myc tumors rapidly underwent apoptosis after a single dose of Rap in vivo (Fig. 3B and Fig. S4 A and B). Arf−/−Eμ-myc or Eμ-Myc tumors were both unresponsive to five consecutive daily doses of Rap in vivo (data not shown). These differences were recapitulated ex vivo (Fig. 3 C and D).
Fig. 3.
An in vitro competition assay revealed that shRNAs directed against RAPTOR were selected against whether Rap was present or not, whereas decreasing RICTOR had no influence on Rap sensitivity (Fig. 3E). shRNA directed against eIF4E, a downstream effector of mTORC1, proved to be moderately selected against under normal growth conditions; however, in the presence of Rap, eIF4E shRNA-expressing tumors were eliminated from the population. Taken together, our results suggest that mTORC1 mediates survival signaling in Tsc2LOHEμ-Myc lymphomas.
Mcl-1 Translation Is mTORC1-Regulated.
Activation of mTORC1 is predicted to stimulate protein synthesis (2). Indeed, Eμ-Myc tumors lacking TSC2 showed a 2-fold increase in protein synthetic rates compared with Tsc2+/+Eμ-Myc tumors (Fig. 4A). This increased translation is mTORC1-dependent because Rap inhibited translation to levels comparable with those observed in Tsc2+/+Eμ-Myc tumors (Fig. 4A). Similar results were obtained with Tsc2−/−p53−/− and Tsc2+/+p53−/− MEFs (Fig. S5A). To determine whether the Rap-mediated decreases in translation were associated with altered levels of eIF4F complex, we measured the relative levels of eIF4E-binding partners, eIF4GI and 4E-BP1, associating with cap-bound eIF4E (Fig. 4B). As expected, the amount of eIF4G associating with eIF4E was reduced, whereas the amount of inhibitory 4E-BP1 increased in Rap-treated samples (compare lane 4 with 3). Thus, Rap reduces eIF4F complexes in Tsc2LOHEμ-Myc tumors, and this correlates with a decrease in translation rates.
Fig. 4.
Given that mTORC1 inhibition in Tsc2LOHEμ-Myc lymphomas leads to a rapid onset of apoptosis, we postulated that activated mTORC1 was promoting drug resistance by increasing the translation of antiapoptotic proteins. Unlike other antiapoptotic bcl-2-like family members, MCL-1 is a highly unstable protein and requires active translation to maintain its expression levels (18). MCL-1 is necessary for early embryonic development and for maintenance of hematopoietic cell lineages (19). In the Eμ-Myc mouse model, eIF4E has been shown to regulate MCL-1 expression (20). Indeed, we observed modest but consistent increases in MCL-1 expression in Tsc2LOHEμ-Myc tumors compared with Eμ-Myc tumors (Fig. 4C) and in Tsc2−/−p53−/− MEFs compared with Tsc2+/+p53−/− MEFs under low serum conditions (Fig. S5B). Rescue of TSC2 expression in Tsc2−/−p53−/− MEFs reduced MCL-1 to levels observed in Tsc2+/+p53−/− MEFs (Fig. S5B). Inhibition of mTORC1 by Rap reduced MCL-1 to basal levels in both Tsc2LOHEμ-Myc tumors and Tsc2−/−p53−/− MEFs (Fig. 4C and Fig. S5 B and C). Interestingly, in both lymphomas and MEFs lacking a lesion activating mTORC1 signaling, Rap neither induced apoptosis nor appreciably decreased MCL-1 expression (Fig. S5 B and C). Hence, loss of MCL-1 expression by mTORC1 inhibition occurs in the context of activated AKT/mTORC1 signaling.
To determine whether this was a consequence of a decrease in Mcl-1 translation, we monitored the polysome distribution of Mcl-1 in untreated or Rap-treated Tsc2LOHEμ-Myc tumors (Fig. 4D). Rap treatment of Tsc2LOHEμ-Myc tumors produced a depression in the distribution of heavy polysomes and increased the amount of lighter polysomes and free ribosomes (Fig. 4D). Quantitative RT-PCR revealed a redistribution of Mcl-1 mRNA from heavy polysomes (fraction 20–22) in Rap-treated cells toward lighter polysomes (fraction 12–14). This contrasts with the behavior of β-actin mRNA from the same polysome fractions, which showed only slight differences in distribution between untreated and Rap-treated samples (Fig. 4D).
Rapamycin-Induced Apoptosis Is Rescued by MCL-1 Overexpression.
These findings raised the question of whether Rap-induced apoptosis in Tsc2LOHEμ-Myc tumors was initiated by the loss of Mcl-1 expression. To address this, we established an in vivo competition assay that allowed us to generate genetically defined mixed tumor populations and examine relative competitiveness of each population in vivo (Fig. 5A). Tsc2LOHEμ-Myc tumors were freshly isolated from donors and partially infected with either MSCV/gfp or MSCV/Mcl-1/gfp. These populations were reintroduced into recipient syngenic animals, and upon tumor manifestation, the mice received either vehicle or Rap. Before therapy, mice were visualized to detect GFP+ tumors in the abdominal region. At 12 h after Rap, the time required to detect appreciable loss of tumor mass in Tsc2LOHEμ-Myc mice, the same mice were again visualized to quantitate the amount of GFP+ tumor cells (Fig. 5B). Rap treatment led to a detectable decrease in the tumor size and correlated with a loss of GFP signal intensity (Fig. 5B Left pair). In contrast, an increase in GFP intensity was observed in mice bearing Tsc2LOHEμ-Myc tumors infected with MSCV/Mcl-1/gfp (Fig. 5B Right pair). The amount of GFP+ cells was quantitated by harvesting the residual tumor cells and subjecting these to flow cytometry (Fig. 5C). Enrichment of GFP+ cells after Rap therapy is clearly seen in MSCV/Mcl-1/gfp-infected cells and indicates resistance to therapy. Thus, Mcl-1 is a genetic modifier of Rap sensitivity.
Fig. 5.
Discussion
Here, we established that mTORC1 acts as a key mediator of AKT prosurvival functions in B cell lymphomas driven by MYC overexpression. Tsc2+/−Eμ-Myc mice rapidly developed aggressive lymphomas with tumors frequently undergoing LOH (Fig. 1 A and B), an event associated with hyperactive mTORC1 signaling (Fig. 1C). The c-myc oncogene promotes apoptosis, such that genetic alterations that disable apoptotic programs often accelerate MYC-driven tumorigenesis (21). Not surprisingly, Tsc2LOHEμ-Myc lymphomas arose with defective apoptotic responses to oncogene activation and chemotherapy. These defects were reversible upon mTORC1 inhibition. We reported that tumors in Pten+/−Eμ-Myc mice arise at a rate comparable to Eμ-Myc mice. Interestingly, Pten+/−Eμ-Myc did not undergo LOH, nor did they arise with robust activation of mTORC1 as seen in Eμ-Myc/myr-Akt lymphomas or Tsc2LOHEμ-Myc lymphomas (8). These findings highlight the importance of mTORC1 activity as a potent antiapoptotic signal and as a critical arm of AKT-driven tumorigenesis in lymphoma development.
One mechanism by which mTORC1 activity can promote cell survival is through increased availability of eIF4E for assembly into the eIF4F, a rate-limiting complex required for the ribosome recruitment phase of translation initiation (2, 22). Increased eIF4E levels can effectively suppress c-myc-induced apoptosis in vivo and alter chemosensitivity in vivo (8). We hypothesized that a sizeable proportion of AKT survival signaling may result from deregulated translation, perhaps through altering the recruitment of antiapoptotic mRNAs to the translation apparatus (7, 8, 20). We find that Tsc2LOHEμ-Myc lymphomas have ≈2-fold higher translation rates than Tsc2+/+Eμ-Myc lymphomas (Fig. 4A). mTORC1 inhibition reduced translation rates to levels that resembled those observed in Tsc2+/+Eμ-Myc cells and decreased eIF4F levels (Fig. 4 A and B). In contrast, translation rates in Eμ-Myc tumors were minimally affected by Rap, consistent with the absence of mTORC1 activation in these tumors (Fig. 4A). By using an in vitro competition assay with shRNAs targeting components of mTOR signaling complexes, we determined that the sensitivity to rapamycin was indeed mediated through the mTORC1/eIF4E arm (Fig. 3E and data not shown). Higher eIF4E levels preferentially increase the translation of mRNAs having lengthy, G+C-rich, highly structured 5′ UTRs and drive cellular transformation and metastatic progression in preclinical models (22). In this fashion, the translation of “weak” mRNAs is preferentially affected when eIF4F levels are modulated by mTORC1 (22, 23). Hence, treatment of Tsc2LOHEμ-Myc tumor cells with rapamycin may reflect translation inhibition of weak mRNAs (Fig. 4A).
One such mRNA is Mcl-1, which has a G+C-rich (>70%) 5′ UTR (see NM_021960). MCL-1 is a short-lived protein with a complex mode of regulation. Our previous studies and work by others have implicated MCL-1 as a potential downstream target of eIF4E (20, 24, 25). Elimination of MCL-1 is required for the initiation of apoptosis after UV irradiation. During DNA damage, Mcl-1 protein synthesis is blocked, and the existing pool of MCL-1 is rapidly degraded by the proteasome (18). Inhibition of Mcl-1 expression is effective at inducing apoptosis in some human cancers (26, 27). We demonstrate that Rap caused a decrease in MCL-1 levels in Tsc2LOHEμ-Myc tumors. This response is associated with a decrease in the translational efficiency of the Mcl-1 mRNA (Fig. 4 C and D) and induction of apoptosis (Fig. 3 B and D and Fig. S4 A and B). Consistent with deregulated Mcl-1 expression modulating drug sensitivity, constitutive expression of Mcl-1 in Tsc2LOHEμ-Myc cells prevented tumor regression upon mTORC1 inhibition (Fig. 5). Our results do not exclude the possibility that additional levels of Mcl-1 regulation contribute to the effects reported herein, but they do indicate that Mcl-1 mRNA translation is an important target of rapamycin in vivo. Tumors arising with activating lesions in the AKT pathway often display Rap sensitivity. Interestingly, we find that Rap only inhibits Mcl-1 expression in the context of activating lesions in the AKT/mTORC1 pathway (Fig. S4C). In lymphomas, lacking activation of this pathway, Rap does not induce tumor regression, nor does it reduce expression of Mcl-1. Our findings highlight the importance of mTORC1 activity as a potent antiapoptotic signal through Mcl-1 and as a critical arm of PI3K/AKT-driven tumorigenesis in lymphoma development.
Materials and Methods
Immunblotting.
For immunoblots, 50 μg of protein extract was typically loaded per lane and electrophoretically separated on SDS/polyacrylamide gels. Proteins were transferred to Immobilon-P membranes (Millipore). Specific proteins were detected with antibodies listed in the SI Materials and Methods.
In Vivo Tumor Monitoring.
Tumors were analyzed for gfp expression by using the GE eXplore Optix system (GE Healthcare) with a 470-nm emitting laser. Photon emission counts were collected and converted to GFP signal intensity. Mice bearing tumors were analyzed before and 12 h after Rap therapy.
Tumor Analyses.
For generating whole-cell protein extracts, tumors were harvested, and single cell suspensions were made. These cells were treated with ACK buffer [0.15 M NH4Cl, 0.1 mM EDTA, 0.01 M KHCO3 (pH 7.3)] to remove red blood cells. Cells were pelleted, washed several times with ice-cold PBS, and resuspended in ice-cold lysis buffer [10 mM Tris·HCl (pH 7.6), 1% Nonidet P-40, 0.5% sodium desoxycholate, 0.1% SDS, 20 mM β-glycerophosphate, 10 mM NaF, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 μg/ml pepstatin, 1 μg/ml leupeptin, and 1 μg/ml aprotinin] and left on ice for 15 min. The resulting lysates were then sonicated on ice for 10 s, and debris was pelleted by centrifugation at 14,000 × g for 15 min at 4°C. The resulting supernatant was quantitated for protein concentration and frozen at −80°C.
Coimmunoprecipitations.
mTOR and RICTOR coimmunprecipitations were performed according to published methods (28) with slight modifications, described in SI Materials and Methods.
Polysome Analysis and Real-Time Quantitative RT-PCR.
For polysome profiling, Tsc2LOHEμ-myc lymphomas were treated for 2 h with vehicle (MeOH) or 4.0 mg/kg Rap. Cells were then processed for polysome analysis as described in SI Materials and Methods.
Acknowledgments.
We thank Dr. David Kwiatkowski (Brigham and Women's Hospital, Boston, MA) for supplying Tsc2−/−p53−/− and Tsc2+/+p53−/− MEFs. We thank Yifei Yan for statistical evaluations, Isabelle Harvey for assistance with the mouse colony, and Kris Pike and Dr. David Munroe for p53 gene sequencing. This work was supported by Canadian Breast Cancer Research Alliance Translational Acceleration Grant 16512 and Canadian Institutes of Health Research Grant MOP-79385 (to J.P.) and National Cancer Institute/National Institutes of Health Grant CA87497 (to S.W.L.).
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© 2008 by The National Academy of Sciences of the USA.
Submission history
Received: May 6, 2008
Published online: August 5, 2008
Published in issue: August 5, 2008
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Acknowledgments
We thank Dr. David Kwiatkowski (Brigham and Women's Hospital, Boston, MA) for supplying Tsc2−/−p53−/− and Tsc2+/+p53−/− MEFs. We thank Yifei Yan for statistical evaluations, Isabelle Harvey for assistance with the mouse colony, and Kris Pike and Dr. David Munroe for p53 gene sequencing. This work was supported by Canadian Breast Cancer Research Alliance Translational Acceleration Grant 16512 and Canadian Institutes of Health Research Grant MOP-79385 (to J.P.) and National Cancer Institute/National Institutes of Health Grant CA87497 (to S.W.L.).
Notes
This article contains supporting information online at www.pnas.org/cgi/content/full/0804821105/DCSupplemental.
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The authors declare no conflict of interest.
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