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RESEARCH COMMUNICATION
1 Department of Genetics and Complex Diseases, Harvard School of Public Health; 2 Department of Systems Biology, Harvard Medical School; Division of Signal Transduction, Department of Medicine, Beth Israel Deaconess Medical Center; 3 Department of Pathology, 4 Hematology Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA
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Abstract |
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[Keywords: Cowden disease; FOXO; PKB; forkhead; hamartoma syndromes; tuberous sclerosis complex]
Received March 11, 2005; revised version accepted June 7, 2005.
). The primary mechanism by which Akt activates mTOR signaling appears to be through direct phosphorylation and inhibition of TSC2 (also known as tuberin) (Inoki et al. 2002; Manning et al. 2002; Potter et al. 2002; McManus et al. 2004). Interestingly, within this PI3K-Akt-mTOR pathway are two different tumor suppressors, PTEN and TSC2, that function to block growth factor-mediated activation of mTOR (Supplementary Fig. 1A).
The PTEN and TSC2 tumor suppressor genes are both mutated in autosomal dominant hamartomatous tumor syndromes (Cantley and Neel 1999; Kwiatkowski 2003). Germline mutations in PTEN cause Cowden disease and related hamartoma syndromes, and PTEN loss is also common in a large number of human cancers (Li et al. 1997; Cantley and Neel 1999; Eng 2003). PTEN reduces intracellular levels of PI3K lipid products, thereby inhibiting Akt activation. In cells lacking PTEN, Akt is constitutively active (Stambolic et al. 1998; Sun et al. 1999), and there is a corresponding increase in TSC2 phosphorylation and mTOR signaling, even in the absence of growth factors (Manning et al. 2002). Therefore, it appears that loss of the PTEN tumor suppressor can lead to inactivation of the TSC2 tumor suppressor and subsequent elevation of basal mTOR signaling, suggesting that this might contribute to tumorigenesis resulting from PTEN mutations. Consistent with this notion, PTEN-/- tumors and cell lines, like those expressing oncogenic PI3K or Akt (Aoki et al. 2001; Wendel et al. 2004), are unusually sensitive to mTOR inhibitors (Neshat et al. 2001; Podsypanina et al. 2001). Inactivating mutations in the TSC2 gene or the TSC1 gene, which encodes a TSC2-binding protein, cause the hamartoma syndrome tuberous sclerosis complex (TSC) (Kwiatkowski 2003). Like loss of PTEN, disruption of TSC1 or TSC2 gives rise to cells and tumors with elevated mTOR signaling (Goncharova et al. 2002; Kwiatkowski et al. 2002; El-Hashemite et al. 2003), and rodent models of TSC develop tumors that are highly sensitive to rapamycin and its analogs (Kenerson et al. 2002; Lee et al. 2005). However, unlike PTEN, TSC2 mutations have not been found in sporadic cancers, and while Cowden patients are predisposed to a large number of cancers (Li et al. 1997; Cantley and Neel 1999; Eng 2003), the development of malignancy is very rare in TSC (Kwiatkowski 2003). Therefore, despite these two tumor suppressors acting to inhibit the same pathway and their loss similarly leading to mTOR-dependent tumorigenesis, the malignancy potential of tumors lacking either PTEN or TSC2 differs greatly.
In this study, we examine the genetic relationship between the mammalian Tsc2 and Pten tumor suppressor genes by crossing Tsc2+/- mice with Pten+/- mice. We find that these genes act synergistically to suppress a subset of phenotypes specific to each single-heterozygous line. However, the effects on underlying signaling pathways vary between different tumor types. Our data strongly support the hypothesis that attenuation of Akt signaling in TSC-related tumors limits their severity. These findings help explain the large phenotypic differences between Cowden and TSC patients and the apparent absence of TSC2 mutations in sporadic cancers.
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Results and Discussion |
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; Zhang et al. 2003; Harrington et al. 2004; Shah et al. 2004; for review, see Manning 2004). We find that MEFs lacking Tsc2, unlike their wild-type counterparts, are unresponsive to serum, IGF1, and PDGF for Akt activation and are, likewise, defective in downstream signaling to the Akt targets FOXO1 and FOXO3a (Supplementary Fig. 1B). Furthermore, FOXO3a protein levels are greatly increased in the Tsc2-/- MEFs, perhaps indicative of a lack of proteasomal degradation previously shown to be stimulated by Akt-mediated phosphorylation (Plas and Thompson 2003). These cell culture experiments raise the intriguing possibility that the benign nature and limited proliferative capacity of TSC tumors, relative to those displaying loss of PTEN, might result from attenuation of Akt signaling. However, whether this negative-feedback loop is active in vivo and affects the behavior of tumors lacking TSC gene function has not been explored previously.
Homozygous loss of either Tsc2 or Pten in mice leads to embryonic lethality (DiCristofano et al. 1998; Suzuki et al. 1998; Kobayashi et al. 1999; Onda et al. 1999; Podsypanina et al. 1999). However, the corresponding heterozygous mice are viable and develop a variety of different neoplasias with nonoverlapping spectra. We assessed the survival of a cohort of 248 offspring (29 litters) from crosses between Tsc2+/- and Pten+/- mice over the course of 1 yr (Fig. 1a). No wild-type or Tsc2+/- mice from this group died before 1 yr of age. However, there was a reduced survival rate in both the Pten+/- and Tsc2+/- Pten+/- mice, with 58% (40 of 69) and 86% (43 of 50), respectively, dying within 1 yr, and Tsc2+/- Pten+/- mice exhibited significantly reduced survival compared with Pten+/- mice (P = 0.0003). The reduced survival of Tsc2+/- Pten+/- mice relative to Pten+/- mice is predominantly due to a decrease in male viability during this interval (Supplementary Fig. 2).
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, 1999, 2001; Suzuki et al. 1998; Podsypanina et al. 1999). However, the overall penetrance and severity of these various Pten+/- phenotypes were similar in the single- and double-heterozygous mice from this cohort.
As lymph node hyperplasia preceding severe lymphadenopathy is a highly penetrant phenotype in Pten+/- mice (DiCristofano et al. 1999; Podsypanina et al. 1999), we palpated all of the mice in our cohort weekly for 40 wk, starting at weaning (4 wk), to detect this phenotype. Interestingly, hyperplastic lymph nodes were detected, primarily in the axillary and submandibular stations, with an earlier onset in the Tsc2+/- Pten+/- double-heterozygous group (mean ± SEM = 14.6 ± 0.9 wk) relative to the Pten+/- group (19.2 ± 0.8; P < 0.0001) (Fig. 1b). Lymph node hyperplasia was not detected in either the wild-type or Tsc2+/- mice within this cohort through 1 yr of age. Consistent with the findings of previous studies (DiCristofano et al. 1999; Podsypanina et al. 1999), the enlarged Pten+/- and Tsc2+/- Pten+/- lymph nodes contained expanded populations of both B and T lymphocytes, as determined by flow cytometric analyses and immunohistochemistry (IHC) (data not shown), and therefore, appear to be polyclonal in nature. Furthermore, as previously found for Pten+/- mice (DiCristofano et al. 1999), PCR (Supplementary Fig. 3A) and immunoblot (Fig. 1c) analyses demonstrate that the lymph node hyperplasia in Tsc2+/- Pten+/- mice is due to haploinsufficiency at both the Pten and Tsc2 loci.
In order to determine the potential mechanism of this enhanced phenotype, signaling downstream of mTOR, including phospho-S6K1 and 4E-BP1 (Fig. 1c) and phospho-S6 (Supplementary Fig. 3B), was examined in lymph node extracts from 15-wk-old mice of each genotype. Relative to other genotoypes, all hyperplastic Tsc2+/- Pten+/- lymph nodes exhibited very high levels of phosphorylated S6K1, S6, and 4E-BP1, while there was only a slight, but reproducible, increase in the Pten+/- samples. Therefore, loss of one copy of each of the Pten and Tsc2 tumor suppressors causes an early onset of lymphoid hyperplasia that coincides with enhanced signaling downstream of mTOR. As both PTEN and TSC2 function to inhibit pathways leading from growth factor receptor stimulation to mTOR signaling (Supplementary Fig. 1A), a combined decrease in their dosage is likely to explain the elevated signaling through this pathway and the corresponding enhanced phenotype. Interestingly, the elevated levels of phospho-Akt observed in the Pten+/- lymph nodes (DiCristofano et al. 1999) are restored to wild-type levels in the Tsc2+/- Pten+/- lymph nodes (Fig. 1c). This suggests that functional inactivation of the remaining TSC2 protein in these lymphocytes by Pten haploinsufficiency partially activates the feedback inhibition mechanism affecting Akt signaling (Supplementary Fig. 1B). This finding further supports that enhanced mTOR signaling, rather than Akt signaling per se, is responsible for this hyperplasia.
In addition to phenotypes common to Pten+/- mice, we also detected the major tumor types previously described for Tsc2+/- mice (Onda et al. 1999) in the Tsc2+/- Pten+/- double-heterozygous mice, including kidney adenomas and cysts, liver hemangiomas, and angiosarcomas (Supplementary Table 1). These tumors were not detected in any wild-type or Pten+/- mice. In the survival study cohort, angiosarcomas on the extremities (e.g., paw or tail) were detected earlier and with a threefold higher incidence in the Tsc2+/- Pten+/- mice (mean ± SEM = 31 ± 1.0 wk; 36%, 18 of 50) compared with the Tsc2+/- mice (45 ± 1.3 wk; 11.9%, seven of 59; P = 0.003 in comparison of overall incidence) (Fig. 2a-c). Necropsies also identified an additional three Tsc2+/- Pten+/- mice with internal angiosarcomas (e.g., Fig. 2c). Post-mortem histological analyses of the Tsc2+/- Pten+/- mice from our survival cohort revealed kidney tumors (Fig. 2e) and liver hemangiomas (Fig. 2g) even in mice <6 mo of age (Supplementary Table 1). Therefore, in order to compare the onset and occurrence of Tsc2+/- phenotypes between single- and double-heterozygous mice, we generated a second cohort from crosses between Tsc2+/- and Pten+/- mice. We sacrificed 10 animals of each genotype at both 6 mo and 1 yr and analyzed the presence of kidney tumors and liver hemangiomas (Fig. 2a). We found no significant difference in the occurrence of kidney adenomas and/or cysts between Tsc2+/- mice (six of 10) and Tsc2+/- Pten+/- mice (seven of 10) at 6 mo of age, and this phenotype was 100% penetrant in both genotypes at 1 yr. The number, size, and severity of these tumors were also indistinguishable in the two genotypes. In contrast, liver hemangiomas were detected with an earlier onset and higher penetrance in the Tsc2+/- Pten+/- mice (five of 10 at 6 mo; nine of 10 at 1 yr) relative to the Tsc2+/- mice (zero of 10 at 6 mo, P = 0.03; four of 10 at 1 yr, P = 0.06). In addition, the overall severity and area of liver affected by hemangiomas in double-heterozygous mice were greatly increased over those of Tsc2+/- mice (e.g., Fig. 2, cf. g and f). Consistent with this observation, 34% (17 of 50) of Tsc2+/- Pten+/- mice from our initial survival study died as the result of massive bleeding from large, highly vascular liver hemangiomas (mean age ± SEM = 37 ± 1.1 wk). However, no Tsc2+/- mice in this cohort died before 1 yr of age.
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), loss of TSC2 expression in Tsc2+/- Pten+/- hemangiomas was detected specifically in the aberrant endothelial cells that comprise these tumors (N = 5 livers examined and >15 total tumors) (Fig. 2h,i). In contrast, PTEN expression is maintained in these tumors (Figs. 2j, 3f; Supplementary Fig. 4A). Therefore, the aggressive liver hemangiomas arising in Tsc2+/- Pten+/- mice appear to be due to loss of Tsc2 in the context of Pten haploinsufficiency. We found high levels of pS6 staining in the endothelial cells of liver hemangiomas from both Tsc2+/- (N = 3 livers and 7 total tumors) (Fig. 3a) and Tsc2+/- Pten+/- mice (N = 3 livers and >9 total tumors analyzed) (Fig. 3b). However, the intensity of staining and percentage of pS6-positive cells within any given area of the liver lesions were indistinguishable between these two lines. This finding is consistent with TSC2 being downstream of PTEN for mTOR regulation (Supplementary Fig. 1A), and therefore, the complete loss of TSC2 expression in both genotypes leads to the same high levels of mTOR signaling. Hence, it is unlikely that differences in mTOR activity account for the striking increase in severity of the liver hemangiomas in the double-heterozygous mice relative to the Tsc2+/- mice.
As described above, MEFs lacking Tsc gene function exhibit strong attenuation of PI3K and Akt (Manning 2004) and a loss of FOXO regulation downstream of Akt (Supplementary Fig. 1B). In order to determine whether this feedback inhibition exists in TSC tumors, we examined Akt signaling in liver hemangiomas. It is difficult to detect quantitative differences between various tumors using IHC with phospho-specific antibodies, and we were unable to get reproducible IHC staining of activated/phosphorylated Akt in any of our tissue samples. We therefore took advantage of the fact that the FOXO proteins are well characterized to shuttle between the nucleus, when Akt is inactive, and the cytoplasm, when Akt is active (Tran et al. 2003). Interestingly, FOXO1 localization in the aberrant endothelial cells of Tsc2+/- liver hemangiomas was found to be predominantly nuclear (Fig. 3c). This localization was different from nontumor endothelial cells within the same livers, where FOXO1 was localized to the cytoplasm and, to a lesser extent, the nucleus (Supplementary Fig. 4B). In stark contrast to Tsc2+/- tumors, the endothelial cells within Tsc2+/- Pten+/- hemangiomas exhibited primarily cytoplasmic FOXO1 localization (Fig. 3d). Quantification of the percentage of cells exhibiting greater cytoplasmic than nuclear FOXO1 staining within hemangiomas from each genotype demonstrated significant differences between Tsc2+/- (mean percent = 14.2 ± 1.8 and 19.6 ± 3.1) and Tsc2+/- Pten+/- mice (80.8 ± 5.4 and 83 ± 2.4; P < 0.0001 for all data set comparisons between the two genotypes) (Fig. 3e). As a control for the ability to detect cytoplasmic localization in Tsc2+/- tumors, cytoplasmic pS6 staining was quantified in adjacent sections from the same tumors and found to be nearly identical in the two genotypes (Tsc2+/-, 94.8 ± 1.4; Tsc2+/- Pten+/-, 95.6 ± 2). Finally, we have also observed attenuation of Akt phosphorylation in crude liver hemangioma lysates prepared from Tsc2+/- but not Tsc2+/- Pten+/- mice (Fig. 3f). Therefore, while mTOR signaling appears to be the same in liver hemangiomas from single- and double-heterozygous mice, signaling downstream of Akt is attenuated in the benign tumors of Tsc2+/- mice but not in the more aggressive tumors arising in Tsc2+/- Pten+/- mice.
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; Harrington et al. 2004; Shah et al. 2004). Furthermore, heterozygous loss of Pten has been shown to be sufficient to activate Akt in the absence of receptor stimulation (DiCristofano et al. 1999; Li et al. 2002). Therefore, it seems likely that the TSC-related tumors that develop in the Tsc2+/- Pten+/- mice are more severe due to reactivation of the many proliferation and survival pathways downstream of Akt, which are normally attenuated in absence of Tsc gene function.
In general, our findings are in agreement with a recently completed study by Ma et al. (2005), who also find that Tsc2+/- Pten+/- mice exhibit reduced survival and that double haploinsufficiency leads to enhanced-Pten+/- phenotypes. However, they find an increase in prostate cancer, which we do not observe, and report no difference in TSC-related phenotypes in their Tsc2+/- Pten+/- mice. These discrepancies are very likely due to strain differences, as studies on both Pten+/- mice and Tsc2+/- mice have detected strain-dependent variations in tumor spectra. This is underscored by the fact that the two TSC-related tumor types that we find to be enhanced in our double-heterozygous mice, angiosarcomas and liver hemangiomas, are not detected at all in the Tsc2+/- mice analyzed in this other study. The initial Tsc2+/- strains used in the two studies are from different sources (Kobayashi et al. 1999; Onda et al. 1999), and those in the Ma et al. (2005) study, unlike those described here, have not been reported to develop angiosarcomas and require substantial aging (>18 mo) to develop liver hemangiomas (Kobayashi et al. 1999). However, the kidney adenomas that develop in their Tsc2+/- mice, as in ours, are not accelerated by Pten haploinsufficiency. It should be noted that, while these kidney adenomas share a location with human TSC tumors, they are not related to the kidney angiomyolipomas (AMLs) most common in TSC patients (>80%) (Henske 2005). AMLs more closely resemble the hemangiomas found in our Tsc2+/- mice, as both are highly vascular lesions that contain a smooth muscle component and express elevated levels of the estrogen receptor and the HMB45 antigen (El-Hashemite et al. 2005).
Our data demonstrate that feedback inhibition of Akt signaling can limit the growth of these TSC-related tumors, and this is likely to explain the benign nature of tumors arising in TSC patients, relative to those caused by loss of PTEN. These findings also suggest that genetic or physiological events leading to reactivation of Akt, or specific signaling events downstream of Akt, might contribute to the large variability in tumor burden and severity observed in TSC patients.
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Materials and methods |
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The Pten+/- and Tsc2+/- mice used in this study were characterized previously (DiCristofano et al. 1998, 1999, 2001; Onda et al. 1999). The two starting strains were both on a mixed C57BL/6j-129/SvJae background and were expanded by crosses with wild-type 129/SvJae mice, and the offspring were mated to generate the study groups described. PCR analyses of genomic tail DNA were used for genotyping as described previously (DiCristofano et al. 1998; Onda et al. 1999).
Survival curves
The Kaplan-Meier plot in Figure 1a includes mice that were found dead or were sacrificed for humane reasons due to morbidity. The plot in Figure 1c was generated by weekly examination of mice in the survival cohort for palpable hyperplastic lymph nodes (estimated to be 2-4 mm in diameter upon detection).
PCR analyses and immunoblotting
PCR analyses of DNA isolated from normal and hyperplastic lymph nodes, as well as corresponding tail DNA from the same mice, were performed using previously described methods for Tsc2 and Pten genotyping (DiCristofano et al. 1998; Onda et al. 1999). Protein extracts from these lymph nodes and from liver hemangiomas were prepared and analyzed by immunoblotting as described previously (Manning et al. 2002; Shaw et al. 2004). Antibodies used for these studies include the C-20 tuberin (TSC2) antibody from Santa Cruz Biotechnology and PTEN, phospho-Akt (T308), phospho-Akt (S473), Akt, phospho-S6K1 (T389), 4E-BP1, phospho-ribosomal S6 (Ser235/236), and eIF4E antibodies from Cell Signaling Technologies.
Histology and IHC
For full necropsies, mice were submerged in Bouin's fixative solution following dissection to expose internal organs and brain tissue. To determine pathological status, all tissues were examined macroscopically and microscopically following paraffin embedding, sectioning, and H&E staining. IHC using the N-19 tuberin (TSC2) antibody from Santa Cruz Biotechnology was done on Bouin's-fixed, paraffin-embedded tissue sections. All other IHCs, including PTEN/MMAC1 Ab-2 from NeoMarkers, Inc., and FKHR (FOXO1) and phospho-ribosomal S6 (Ser235/236) antibodies from Cell Signaling Technologies, were done on 10% formalin-fixed, paraffin-embedded tissue sections. IHC slides were prepared according to the antibody manufacturer's instructions following heat-induced antigen retrieval and were detected using Evision+ (DakoCytomation).
Statistical analyses
P-values for Kaplan-Meier plots were determined by Mantel-Cox log-rank tests. The rates of occurrence of specific tumor types were compared using the two-sample t-test with a two-tailed distribution for samples where N > 20 and the Fisher's exact test for those where N < 20.
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Acknowledgments |
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Footnotes |
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Article published online ahead of print. Article and publication date are at http://www.genesdev.org/cgi/doi/10.1101/gad.1314605.
5 Corresponding author.
E-MAIL bmanning{at}hsph.harvard.edu; FAX (617) 432-5236. 
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