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RESEARCH PAPER
1 Cancer Biology and Genetics Program, 2 Department of Pathology, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA; 3 Graduate Program in Physiology, Biophysics and Systems Biology, Weill Graduate School of Medical Sciences, Cornell University, New York, New York 10021, USA; 4 Department of Cell Biology and 5 Department of Experimental Pathology, Cancer Institute, Japanese Foundation for Cancer Research, Tokyo 170-8455, Japan
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Abstract |
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[Keywords: PTEN; TSC2; prostate cancer; haploinsufficiency]
Received March 11, 2005; revised version accepted June 8, 2005.
; Di Cristofano and Pandolfi 2000). PTEN alterations have been robustly implicated in human prostate cancer (CaP), with PTEN deletions and/or mutations of at least one allele in up to 60% of primary prostate cancers, while homozygous PTEN inactivation is more frequently associated with metastatic prostate tissues (Gray et al. 1995; Cairns et al. 1997; Suzuki et al. 1998b). Tuberous sclerosis (TSC), an autosomal dominant tumor syndrome caused by mutations in TSC genes, is characterized by a wide spectrum of hamartomas, but rare progression to malignancy with the exception of renal cancer (Gomez et al. 1999). Biochemical studies have demonstrated that the PTEN tumor suppressor gene encodes a phosphatase that dephosphorylates phosphatidylinositol 3,4,5-triphosphate (PIP3) and therefore antagonizes phosphatidylinositol 3-kinase (PI3K) activity (Cantley and Neel 1999; Di Cristofano and Pandolfi 2000), while mammalian TSC2 is an inhibitory phosphorylation substrate of Akt (Inoki et al. 2002; Manning et al. 2002). Genetic disruption of the Pten or Tsc2 gene in mice leads to tumorigenesis in distinct tissues. Pten+/- mice display hyperplastic-dysplastic features and are highly susceptible to developing epithelial tumors (Di Cristofano et al. 1998, 2001; Suzuki et al. 1998a; Podsypanina et al. 1999). These mice often die from a lethal autoimmune disorder, with a high penetrance of prominent lymphoadenopathy and splenomegaly showing marked lymphoid hyperplasia (Di Cristofano et al. 1999). In addition, Pten+/- mice represent a faithful mouse model of CaP, as they develop prostatic intraepithelial neoplasia (PIN) in the dorsolateral prostate (DLP) and the anterior prostate (AP), but not in the ventral prostate (VP), which progresses to invasive CaP upon additional loss of the Cdkn1b gene (encoding the p27kip1 Cdk inhibitor) (Di Cristofano et al. 2001). On the other hand, Tsc2+/- mice specifically develop renal carcinoma within a 6-10-mo latency and hepatic hemangioma with a later onset (12-18 mo; Kobayashi et al. 1999; Onda et al. 1999).
The recently identified biochemical relationship between mammalian TSC2 and Akt (Inoki et al. 2002; Manning et al. 2002) suggests that concomitant loss of PTEN and TSC2 may cooperate in tumorigenesis. However, the picture is further complicated by the negative feedback response resulting in impaired Akt activation which has been observed in Tsc2-deficient cells (Jaeschke et al. 2002). In addition, TSC2 appears to integrate signals from multiple pathways, including the Ras-Erk and AMPK pathways (Inoki et al. 2003; Ma et al. 2005). In this study, we focused on the PI3K pathway and sought to determine the genetic relationship between mammalian Pten and Tsc2 in tumor suppression by studying tumorigenesis in the Pten+/-Tsc2+/- compound mouse mutants.
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; Suzuki et al. 1998a; Kobayashi et al. 1999; Onda et al. 1999; Podsypanina et al. 1999). We therefore crossed Pten+/- with Tsc2+/- mutants in an identical 129Sv/C57BL/6 background. Four viable genotypes were obtained from this cross at approximately 1:1:1:1 ratio. Loss of one Tsc2 allele had no effect on survival on its own, but led to a significant reduction of lifespan in Pten+/- mice (Fig. 1A). Similar to Pten+/- mice, Pten+/-Tsc2+/- compound mutants often died from a severe lymphoadenopathy and splenomegaly. The Pten+/- and Pten+/-Tsc2+/- lymph nodes were indistinguishable in composition, as they both exhibited an expansion of B and T lymphocytes and appeared polymorphous in nature (Supplementary Fig. 1A). However, the lymph nodes from Pten+/-Tsc2+/- mice displayed increased staining of Ki-67, a cell cycle-specific marker, compared with those from Pten+/- mice (Fig. 1B), demonstrating that Tsc2 opposes the augmented cell proliferation triggered by Pten heterozygous loss. Although we did not observe an earlier onset of cervical lymphoadenopathy compared with Pten+/- mice, several systemic signs distinctive of this disorder were exacerbated in the compound mutants: Acute inflammation, characterized by infiltration of polymorphonuclear cells, was observed in the liver of some of the compound mutants, but was never found in either single mutant (Supplementary Fig. 1B). Chronic inflammation, characterized by lymphoid infiltration, was observed in the kidney and liver with a 100% incidence in females older than 5 mo, and appeared much more severe than in Pten+/- mice (Supplementary Fig. 1C). Additionally, the spleens in the majority of Pten+/-Tsc2+/- mice displayed red pulp expansion and a marked extramedullary hematopoiesis with megakarocytes and myeloid and erythroid elements, which was also massively exacerbated compared with Pten+/- mutants (Supplementary Fig. 1D). Interestingly, associated with severe lymphoadenopathy and splenomegaly, 
35% of Pten+/-Tsc2+/- female mice (eight of 23) had ulcerated pelvic abscess filled with pus and necrotic cells, a phenotype never observed in the single mutants. Histological analysis revealed a necrotic center lined by neutrophils, foamy histiocytes, and a fibrotic wall (Supplementary Fig. 1E).
Loss of heterozygosity (LOH) and mutations of PTEN have been found in human patients with squamous cell carcinomas (SCC) (Shao et al. 1998). Pten complete inactivation in keratinocyte-specific Pten conditional mutants (k5Ptenflox/flox mice) leads to skin tumorigenesis, as evidenced by the development of epidermal hyperplasia, squamous papillomas, and SCC (Suzuki et al. 2003). In contrast, Pten+/- mice do not develop skin malignancies, while SCC has been reported in Pten+/-Ink4a/Arf-/- compound mutants (two of 46) (You et al. 2002). We observed that 10% of the Pten+/-Tsc2+/- double-heterozygous mutants (four of 41; with a 7-9-mo latency) showed large ulcerated SCCs, characterized by a number of typical features such as detached squamous islands surrounded by extensive keratinization, nests of squamous pearls with central keratinization, mitotic figures, disorganized basal cells with atypia, and dyskeratosis (Fig. 1C). Thus, compound Pten+/-Tsc2+/- mutants exhibit an expanded tumor spectrum.
Pten+/- mice are highly prone to epithelial tumors in a variety of organs. However, some of the tumor types observed in Pten+/- mutants do in fact occur after a long latency. For instance, PIN, which occurs at an 50% penetrance, is never observed prior to 8 mo of age and never found to progress to CaP (Di Cristofano et al. 2001). Tsc2+/- mice were not susceptible to PIN or CaP (Fig. 2A,C). The majority of prostate glands in the Tsc2+/- mutants analyzed were normal, with only a few glands becoming mildly hyperplastic in elder mutants (in mice older than 8 mo; n = 4; Fig. 2C). In contrast, Pten+/-Tsc2+/- compound mutants developed PIN at 100% penetrance, and more strikingly, CaP at a high penetrance. The onset of CaP in double-heterozygous males was 5 mo of age, and by 9 mo virtually all males developed CaP (Fig. 2A,B). Moreover, these cancers exhibited manifest invasion, as evidenced by disruption of the basal membrane of the epithelium (Fig. 2C). By 8 mo of age, invasive CaP was observed in all three lobes (AP, VP, and DLP), while the earliest occurrence of invasive CaP was observed in the AP of Pten+/-Tsc2+/- compound mutants at 5 mo of age (Supplementary Fig. 2). Importantly, Pten immunostaining was retained in both normal tissues and neoplastic lesions in both Pten+/- mice and Pten+/-Tsc2+/- compound mutants (n = 5 mice and >15 neoplastic lesions examined for each genotype) (Fig. 2D).
Next, we performed laser capture microdissection of PIN cells from Pten+/- mice and prostate carcinoma cells from Pten+/-Tsc2+/- mice on paraffin-embedded sections, followed by LOH analysis of Pten and Tsc2. As shown in Figure 2E, the wild-type alleles of both genes were retained in prostate tumor cells in each case. These findings provide strong support to the notion that Pten is haploinsufficient for suppression of pathogenesis of PIN and, importantly, that Tsc2 is haploinsufficient for repression of carcinogenesis initiated by Pten heterozygosity.
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; Onda et al. 1999). Consistent with these reports, Tsc2+/- mice developed renal cystadenomas and/or renal carcinomas. However, Tsc2+/- and Pten+/-Tsc2+/- mutants developed these tumors with similar onset and incidence (Fig. 3A-C). Kidneys from both Tsc2+/- and Pten+/-Tsc2+/- mice often showed macroscopically visible cysts on the surface. The cysts could be simple cysts, but mostly were associated with adenomas or carcinomas in the cortical region. Conversely, renal carcinomas could be cystic or unassociated with cysts, with the majority displaying a papillary feature (Fig. 3D). The other tumor phenotype observed in Tsc2+/- mice in the mixed 129Sv/C57BL/6 background was unexpected hepatic hemangiomas after a long latency (12-18 mo), which are not seen in either TSC patients or the Eker rat model of TSC (Kobayashi et al. 1999). No hepatic hemangiomas were found in a 12-mo follow-up in either Tsc2+/- or Pten+/-Tsc2+/- mice in the genetic background we studied. To explain why renal carcinogenesis in Tsc2+/- mice is not accelerated upon Pten heterozygous loss, two possibilities can be entertained: (1) The tumor-promoting effect of Pten deficiency might be counterbalanced by less frequent Tsc2 LOH in the compound mutant; (2) more likely, tumorigenesis initiated by Tsc2 heterozygosity must be followed by Tsc2 LOH in order to give rise to full-blown malignancy. To investigate this latter possibility, we performed LOH analysis on microdissected renal carcinomas from both Tsc2+/- mice and Pten+/-Tsc2+/- compound mutants. While no Pten LOH was detected, the wild-type Tsc2 allele was found invariably lost in all renal cancers from these mice (Fig. 3E). Thus, in contrast to what was observed in the prostate, Tsc2 is not haploinsufficient for repression of renal carcinogenesis even in a Pten+/- genetic background.
Next, we sought to determine the biochemical and biological consequences of concomitant heterozygous loss of Pten and Tsc2, both in vivo as well as in primary mouse embryonic fibroblasts (MEFs). As predicted by the specific role of Pten in the PI3-kinase pathway, the phospho-Akt level was found increased in the prostate of either Pten+/- or Pten+/-Tsc2+/- mice (n = 4 mice examined for each genotype; representative results shown in Fig. 4A). In contrast, phosphorylation at S2448 of the key regulator of mRNA translation mTOR (mammalian target of rapamycin; phosphorylation at this residue occurs via activation of the PI3K/Akt pathway, and can be suppressed by TSC1-TSC2 overexpression and augmented by TSC2 RNA interference [RNAi]) (Inoki et al. 2002) was found markedly enhanced only in the prostates (in both normal epithelium and carcinomas) of Pten+/-Tsc2+/- mice (n = 4 mice examined for each genotype; representative results shown in Fig. 4B). To date, it remains in question whether mTOR S2448 phosphorylation always leads to increased downstream signaling (Hay and Sonenberg 2004). We therefore examined the phospho-S6 level in prostate homogenates from three to five 6-mo-old mice of each genotype by immunoblot analysis (representative results shown in Fig. 4C), and found that signaling downstream of mTOR was also manifestly enhanced in all Pten+/-Tsc2+/- prostate tissues. These results are therefore in agreement with the fact that TSC2 functions downstream of Akt and upstream of TOR (Potter et al. 2001; Tapon et al. 2001). In Pten+/- mice, expression of the Ki-67 proliferation marker was mostly found increased in the PIN lesions, whereas in double-heterozygous mice a marked increase in the proliferative index was observed not only in the tumor lesions, but also in the preneoplastic tissues (Fig. 4D).
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-gal staining), which could be almost completely overcome by compound Pten and Tsc2 heterozygous inactivation, whereas only a partial reduction in cellular senescence was observed in MEFs from single mutants (Fig. 5B).
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). We found that phosphorylation of S6K and 4E-BP1 at the mTOR-dependent site was comparable to that of wild-type cells (or only slightly increased) in both Pten+/- and Tsc2+/- single-heterozygous MEFs. In contrast, compound inactivation of Pten and Tsc2 led to enhanced S6K and 4E-BP1 phosphorylation (Fig. 5C), further demonstrating that concomitant loss of Pten and Tsc2 has a cooperative effect in the activation of mTOR. |
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). They also report that the development of renal cystadenomas and/or renal carcinomas is not accelerated in Pten+/-Tsc2+/- mutants. Furthermore, even though hepatic hemangiomatosis displays an earlier onset in Pten+/-Tsc2+/- compound mice than in single Tsc2+/- mutants in their genetic background, Manning et al. (2005) report that these tumors are nevertheless invariably characterized by loss of Tsc2 (but not Pten) expression, consistent with the frequent Tsc2 LOH previously reported in these tumors from Tsc2 single-heterozygous mice. In contrast, they find no Pten LOH in the hyperplastic lymph nodes from either Pten+/- or Pten+/-Tsc2+/- mice (Manning et al. 2005). These results further reinforce our finding of both asymmetrical haploinsufficiency and a nonreciprocal relationship between Pten and Tsc2. The possible underlying mechanisms for this enhanced phenotype that Manning et al. (2005) report could be accelerated Tsc2 LOH induced by heterozygous loss of Pten in hepatic tumors in their genetic background and/or, as Manning et al. propose, reactivation of pathways downstream of Akt other than the mTOR pathway.
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It should be noted that loss of Tsc1 or Tsc2 was recently found to result in impaired PI3K-Akt activation (Jaeschke et al. 2002; Zhang et al. 2003). On the basis of this observation, one might predict that the phenotype of Pten+/- mice would be suppressed rather than accelerated by loss of Tsc2. However, this negative feedback loop has been shown to inhibit the PI3K-Akt pathway by down-regulating upstream activators of PI3K such as PDGF receptor and insulin receptor substrate (IRS) proteins (Zhang et al. 2003; Harrington et al. 2004; Shah et al. 2004). Upon Pten deficiency, PIP3 accumulates and Akt is activated even in the absence of receptor tyrosine kinase signaling. Indeed, we demonstrate here that in the compound mutants, heterozygous Pten inactivation is sufficient to overcome the negative feedback and activate Akt in vivo (Fig. 4A) and in MEFs (Fig. 5C), while accompanying loss of Tsc2 leads to elevated mTOR activation (Figs. 4B,C, 5C). Therefore, concomitant reduction in the dosage of two tumor suppressors acting in the same pathway, which gives rise to enhanced signaling through this pathway, can explain the accelerated phenotype. This, in turn, has important therapeutic implications, as based on our report that combination treatment with agents that concomitantly block PI3-kinase/Akt and mTOR activation could be beneficial. Furthermore, our findings prompt the analysis of the status of the TSC complex/genes in human cancers that suffer from PTEN functional loss (e.g., prostate cancer).
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Materials and methods |
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Pten+/- or Tsc2+/- single-heterozygous mice in the identical 129Sv/C57BL/6 background were crossed to obtain Pten+/-Tsc2+/- compound mutants. Genotyping by PCR on tail DNA was performed as previously described (Di Cristofano et al. 1998; Kobayashi et al. 1999). All mice from the above cross were divided into two groups. One group of mice was monitored throughout their lives to determine the overall survival. In a second group, two to four mice (males and females) from each genotype were sacrificed every month between 4 and 9 mo for gross and microscopic examination of all organs—the prostate, kidney, and liver in particular—to determine the disease onset and incidence.
Autopsy, histopathology, and immunohistochemistry
Mice were autopsied and all tissues were analyzed regardless of their pathological condition. Tissue samples were fixed in 10% buffered formalin for 12 h, followed by wash with PBS and transfer to 70% ethanol, and the following histological procedures (paraffin embedding, sectioning, and hematoxylin and eosin [H&E] staining) were provided by Histoserv, Inc. The IHC detection using anti-B220 (Pharmingen), anti-CD3 (Dako), anti-PTEN (Neomarkers), anti-phospho-Akt and anti-phospho-mTOR (Cell Signaling Technology), and anti-Ki-67 (Novocastra Laboratories Ltd.) was performed with automated IHC procedures at the Molecular Cytology Core Facility of Memorial Sloan-Kettering Cancer Center.
Laser capture microdissection and DNA extraction
Formalin-fixed, paraffin-embedded blocks were cut into 7-µm-thick sections on uncharged glass slides. Sections were slightly stained with hematoxylin and microdissected using a PALM laser microdissection and pressure catapulting (LMPC) system. A 0.5-mL clean microcentrifuge tube was inserted in the tube holder with 20 µL nuclease-free water in the cap. The marked cells were catapulted into the cap. The cap was then closed and the tube was centrifuged at 13,000 rpm for 5-10 min. Twenty microliters of ATL buffer (a component of QIAamp DNA Micro Kit; QIAGEN) and 10 µL of Proteinase K solution (20 mg/mL) were added. The samples were vortexed for 10-15 sec, followed by incubation for 20-24 h at 55°C and then for 10 min at 95°C. The samples were then processed for DNA extraction using the QIAamp DNA Micro Kit, according to the manufacturer's instructions.
LOH analysis
Genomic DNA from formalin-fixed, paraffin-embedded tissues was prepared as described above. Primers used for Pten LOH analysis were P1 (5'-TGGGAAGAACCTAGCTTGGAGG-3' in the intron 5 region), P2 (5'-TTCCATTTGTCACGTCCTGCAC-3' in the neo region), and P3 (5'-ACTCTACCAGCCCAAGGCC-3' in the intron 5 region). P1 and P3 were used for the wild-type allele, and P2 and P3 were used for the mutated allele. Primers used for Tsc2 LOH analysis and PCR conditions were previously described (Kobayashi et al. 1999).
MEF isolation, growth, and senescence
MEFs were derived from 13.5-d-old embryos using a previously described protocol (Kamijo et al. 1997) and maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, and 100 U/mL penicillin and streptomycin (GIBCO). Growth curves were determined using a crystal violet method as previously described (Serrano et al. 1997). SA--gal staining was performed using the Senescence Detection Kit (Oncogene) according to the manufacturer's instructions.
Protein analysis
Prostates from dissected mice were minced and incubated in RIPA lysis buffer (150 mM NaCl, 10 mM Tris at pH 7.5, 1% NP40, 1% deoxycholate, 0.1% SDS, protease inhibitor cocktail [Roche], phosphatase inhibitor cocktail [Sigma]), and homogenized using a tissue homogenizer. MEF cells were harvested and directly lysed in RIPA lysis buffer. Proteins from total lysates were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, blocked in 5% nonfat milk or BSA, and blotted with the following antibodies: anti-PTEN (Cascade Bioscience), anti-TSC2 (Santa Cruz), anti-actin (Sigma), anti-phospho-pAkt (S473), anti-Akt, anti-phospho-S6K (T389), anti-S6K, anti-phospho-4EBP1 (S65), anti-4EBP1, anti-phospho-S6 (S235/S236), and anti-S6 (Cell Signaling Technology).
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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.1314405.
6 These authors contributed equally to this work. 
7 Corresponding author.
E-MAIL p-pandolfi{at}ski.mskcc.org; FAX (212) 717-3102.
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