Cancer Research Annual Meeting 2010 Genetics and Biology of Brain Cancer
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Cancer Research 67, 7106, August 1, 2007. doi: 10.1158/0008-5472.CAN-06-4798
© 2007 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

Identification of S664 TSC2 Phosphorylation as a Marker for Extracellular Signal-Regulated Kinase–Mediated mTOR Activation in Tuberous Sclerosis and Human Cancer

Li Ma1,2,3, Julie Teruya-Feldstein2, Pauline Bonner2, Rosa Bernardi1,2, David Neal Franz4,5, David Witte4,6, Carlos Cordon-Cardo2 and Pier Paolo Pandolfi1,2

1 Cancer Biology and Genetics Program and 2 Department of Pathology, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center; 3 Graduate Program in Physiology, Biophysics and Systems Biology, Weill Graduate School of Medical Sciences, Cornell University, New York, New York; and Departments of 4 Pediatrics, 5 Neurology, and 6 Pathology, University of Cincinnati College of Medicine, Cincinnati, Ohio

Requests for reprints: Pier Paolo Pandolfi, Cancer Biology and Genetics Program, Department of Pathology, Memorial Sloan-Kettering Cancer Center, Box 110, 1275 York Avenue, New York, NY 10021. Phone: 212-639-6168; Fax: 212-717-3102; E-mail: p-pandolfi{at}ski.mskcc.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Constitutive activation of extracellular signal-regulated kinases (Erk1/2) is frequently implicated in human cancers. Recently, aberrantly activated Erk was also found in brain lesions associated with tuberous sclerosis (TSC). We reported previously that Erk might contribute to tumorigenesis by phosphorylating TSC2 at specific residues, particularly S664. In our present study, 25 TSC-related cortical tubers or subependymal giant cell astrocytomas, as well as tissue microarrays of six types of human cancers, were analyzed for the expression of phospho-Erk (pErk) 1/2, S664-phospho-TSC2 (pTSC2), and phospho-S6 (pS6) by immunohistochemistry. We found that Erk-mediated TSC2 phosphorylation occurred at a high incidence and positively correlated with mitogen-activated protein kinase (MAPK) and mammalian target of rapamycin (mTOR) activation in TSC-associated brain lesions as well as in various cancers. Interestingly, in certain types of cancers (e.g., breast carcinoma and colon carcinoma), S664-pTSC2 seemed to be a more sensitive marker than pErk. Furthermore, most of the pTSC2-positive samples (~75%) were positive for pS6, but only 40% to 55% of the pS6-positive tumors exhibited TSC2 phosphorylation. Our results show that S664 TSC2 phosphorylation is a marker for Erk-mediated (as opposed to Akt-mediated) mTOR activation in TSC and human cancer. On the basis of these findings, TSC2 phosphorylation at S664 can be used to identify patients that may benefit from antitumor therapy with MAPK and mTOR inhibitors. Importantly, our results indicate that Erk-mediated phosphorylation and inactivation of TSC2 can be critical in development of hamartomatous lesions in TSC and cancer pathogenesis. [Cancer Res 2007;67(15):7106–12]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Extracellular signal-regulated kinases (Erk) 1 and 2, the first characterized members of the mitogen-activated protein kinase (MAPK) family, are activated by MAPK kinase (MEK) 1/2–mediated phosphorylation on both Thr202 and Tyr204 residues, through a cascade of phosphorylation events downstream of the Ras proto-oncogene (1). Activation of the Erk pathway may critically contribute to tumorigenesis or cancer growth, as evidenced by the presence of activated Erk1/2 in a variety of human tumor cell lines (2) and cancer tissues, with high frequency in tumors derived from colon, lung, kidney, ovary, and breast (2–4). Although Erk is not a Food and Drug Administration–approved cancer biomarker in prognosis (like prostate-specific antigen in prostate cancer and HER2/neu in breast cancer), studies showed that Erk activity has potentially prognostic value for patient outcomes in several types of cancers, including breast, ovarian, and non–small-cell lung cancer (5–7). These led to the hypothesis that the Ras-Erk pathway may be a good target for anticancer therapy. In fact, a small-molecule MEK inhibitor could inhibit tumor growth as much as 80% in mice with colon carcinomas (8).

In addition, it has been shown that hyperactivation of Erk is frequently observed in cortical tubers (9, 10), the predominant hamartomatous lesion in tuberous sclerosis (TSC), which are rarely associated with biallelic inactivation of TSC2 (11). Moreover, the in vivo tumorigenicity of the Tsc2ang1 cell line (a tumor cell line derived from a cutaneous sarcoma of a Tsc2+/– mouse) can be blocked by dominant-negative MEK1, suggesting that Erk may play a causal role in development of TSC-related tumor lesions (9). The mechanistic link was provided when our group reported that TSC2 is a target of the Ras-Erk signaling and that TSC2 direct phosphorylation by Erk leads to suppression of its biochemical and biological tumor-suppressive functions (12).

In an attempt to assess the relevance of Erk-mediated TSC2 phosphorylation in TSC and, more general, in human cancer, we generated a novel phospho-specific antibody and analyzed the levels of Erk-phospho-TSC2 (pTSC2) using tumor specimens from patients with TSC and various cancers compared with MAPK and mammalian target of rapamycin (mTOR) activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmids, antibodies, and chemicals. The HA-MEK1S218D/S222D, Xpress-TSC2, FLAG-TSC2 constructs, and various TSC2 mutants were described previously (12). The commercial antibodies used in this study were as follows: anti-FLAG (Sigma); anti–phospho-Erk (pErk; Thr202/Tyr204) and anti–phospho-S6 (pS6; Ser240/244; Cell Signaling Technology); anti-TSC2, polyclonal (for immunoblotting and immunohistochemistry; Santa Cruz Biotechnology); and anti-TSC2, monoclonal (for immunoprecipitation; Zymed). U0126 was purchased from Calbiochem. Phorbol 12-myristate 13-acetate (PMA) was from Sigma.

Generation of the phospho-specific TSC2 antibody. pTSC2 (S664) polyclonal antibody was produced by immunizing rabbits with a synthetic keyhole limpet hemocyanin (KLH)–coupled phosphopeptide encompassing Ser664 of human TSC2:KKTSGPLS(p)PPTGPP, with an additional cysteine at the NH2 terminus (for KLH conjugation). The titers were determined by ELISA and reached plateau after the third bleed. Therefore, affinity purification of the antibody was done with the third bleed against the unphosphorylated peptide by affinity chromatography.

Cell culture and transfections. The HEK293T and various cancer cell lines [American Type Culture Collection (ATCC)] were maintained in appropriate medium (as suggested by ATCC) supplemented with 10% fetal bovine serum, 2 mmol/L glutamine, and 100 units/mL penicillin and streptomycin (Life Technologies). Transfections were done using the LipofectAMINE 2000 reagent (Invitrogen) according to manufacturer's instructions. Four hours after transfection, cells were recovered in full serum for 24 h, then serum starved for 16 h, treated as indicated, and harvested.

Immunoblotting and immunoprecipitation. Cells were harvested in radioimmunoprecipitation assay lysis buffer [150 mmol/L NaCl, 10 mmol/L Tris, 1% NP40, 1% deoxycholate, 0.1% SDS, protease inhibitor cocktail (Roche), phosphatase inhibitor cocktail (Sigma; pH 7.5)]. For immunoprecipitation, lysates were precleared with IgG and then incubated with the precipitating antibody overnight followed by 1-h incubation with protein A/G beads (Santa Cruz Biotechnology). Immune complexes were then washed thrice in lysis buffer and boiled in gel loading buffer. Proteins from total cell lysates or immunoprecipitates were resolved by SDS-PAGE, transferred to nitrocellulose membrane, blocked in 5% nonfat milk in PBS/Tween 20, and blotted with the appropriate antibody.

Patient samples and immunohistochemistry. Seventeen cortical tubers and 8 subependymal giant cell astrocytomas (SEGA) were collected from Tuberous Sclerosis Clinic at Cincinnati Children's Hospital Medical Center. All cases were clinically diagnosed as TSC based on established criteria. Tissue microarrays (TMA) were constructed as published previously (13), however, using a fully automated Beecher Instrument, ATA-27. The study cohort comprised breast carcinoma, colon carcinoma, endometrial carcinoma, renal cell carcinoma (RCC; papillary type), prostate carcinoma, and diffuse large B-cell lymphoma, consecutively ascertained at the Memorial Sloan-Kettering Cancer Center (MSKCC) between 1993 and 2005. Use of tissue samples was approved with an Institutional Review Board waiver. All cancer biopsies were evaluated at MSKCC, and the histologic diagnosis was based on H&E staining. Each TMA also included one to several normal tissue samples correlating with the specific tumor type. The pTSC2 (S664) rabbit polyclonal antibody was used at a 1:50 dilution, after enzyme-induced epitope retrieval was done with 0.05% trypsin at 37°C for 30 min. Secondary biotinylated antirabbit IgG (H+L, Vector Laboratories, Inc.) was used at a 1:500 dilution, and tertiary strepavidin-horseradish peroxidase (DAKO) was used at a 1:500 dilution. The pErk rabbit monoclonal antibody (immunohistochemistry preferred; Cell Signaling Technology) was used at a 1:100 dilution, after heat-induced epitope retrieval with citrate buffer (pH 6.0) and steam pretreatment for 30 min. Secondary biotinylated antirabbit IgG (H+L) was used at a 1:500 dilution, and tertiary streptavidin-peroxidase conjugate (Roche Diagnostic GmbH) was used at a 1:500 dilution. The TSC2 and pS6 (S240/S244) antibodies were used according to the manufacturer's immunohistochemistry protocol.

Statistical analysis. Fisher's exact probability test for 2 x 2 tables was used to assess correlation between TSC2 S664 phosphorylation and Erk phosphorylation. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of the S664 TSC2 phospho-specific antibody. Our previous work indicated that S664 TSC2 phosphorylation might correlate with Erk activation in TSC and, more in general, in human cancer (12). Therefore, to assess the relevance of our findings in human tumor specimens, we generated a phospho-specific antibody against S664 of TSC2, anti-pTSC2 (S664). To test its specificity, we first immunoprecipitated FLAG-tagged TSC2 from HEK293T cells with or without MEK1 cotransfection. Only samples in the presence of MEK1 coexpression showed strong reactivity (Fig. 1A ). We next overexpressed wild-type (WT) TSC2 and various alanine mutants together with an excess of active MEK1. As expected, the pTSC2 (S664) antibody had high affinity with WT TSC2 as well as the TSC2S540A mutant but did not react with the TSC2S664A or TSC2S664A/S540A mutant (Fig. 1B). Furthermore, this antibody could recognize endogenous TSC2 from HEK293T immunoprecipitates in a PMA- and Erk-dependent manner (Fig. 1C and D). Additionally, the specificity of anti-pTSC2 (S664) against endogenous TSC2 was further shown by immunohistochemical staining (Fig. 1E). These results are the first direct proof that the endogenous TSC2 protein can be phosphorylated by Erk.


Figure 1
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  		Figure 1. Characterization of the pTSC2 (S664) antibody and proof of Erk-dependent phosphorylation of endogenous TSC2. A, FLAG-tagged TSC2 was immunoprecipitated (IP) from 293T cells with or without cotransfection of HA-MEK1S218D/S222D. The immunoprecipitates were blotted with anti-pTSC2 (S664) followed by anti-TSC2. B, 293T cells were transfected with empty vector, HA-MEK1S218D/S222D, and/or Xpress-tagged WT TSC2 or S664A, S540A, or S664A/S540A mutants. Lysates were blotted with anti-pTSC2 (S664) followed by anti-TSC2. C, 293T cells were serum starved, then left untreated, stimulated with 1 µg/mL PMA for 40 min, or pretreated with 25 µmol/L U0126 for 30 min. Lysates were immunoprecipitated with anti-TSC2, probed with anti-pTSC2 (S664), and reprobed with anti-TSC2. D, endogenous TSC2 was immunoprecipitated from 293T cells with or without transfection of HA-MEK1S218D/S222D. The immunoprecipitates were blotted with anti-pTSC2 (S664) followed by anti-TSC2. E, 293T cells were serum starved, then left untreated, or stimulated with 1 µg/mL PMA for 40 min. Cells were then trypsinized, fixed in formalin, and embedded in paraffin. Sections were processed for immunohistochemical staining with the anti-pTSC2 (S664) antibody. Magnification, x400. Magnification, x600 (inset).

 
We also characterized the pErk antibody for immunohistochemistry, using a series of formalin-fixed, paraffin-embedded cell lines derived from human tumors. The staining was in complete agreement with the parallel Western blot analysis: high levels of pErk1/2 in MDA-MB-231 (breast), H1650 (lung), and SK-MEL28 (melanoma) cell lines, whereas very weak or undetectable pErk1/2 in MCF7 (breast), H1978 (lung), and PC3 (prostate) cell lines (data not shown).

TSC2 is phosphorylated in TSC brain lesions, which correlates with Erk and mTOR activation. The central nervous system (CNS) is most commonly and seriously affected by TSC. Cortical tubers are heterogeneous zones of cortical dysplasia and represent the hallmark of this disease. They result in epileptic seizures in over 90% of affected individuals, 20% to 30% of which can be medically intractable. Fifty percent to 60% of the patients have cognitive impairments, and it is the most common medical diagnosis associated with autism. Five percent to 15% develop SEGAs or other types of CNS neoplasia, which can result in hydrocephalus, optic neuropathy, and dementia (14, 15). Loss of heterozygosity or other somatic mutations in the TSC2 gene was rarely detected in cortical tubers or SEGAs, and expression of the TSC2 protein in these lesions is consistently present (10, 11, 16). These findings are highly suggestive that post-translational inactivation of TSC2 is responsible for the cortical dysplasia and neoplastic disease seen in TSC. Notably, aberrant activation of Erk has been found recently in CNS lesions from patients with TSC (9, 10). Treatment of CNS disease associated with TSC has largely been palliative and symptomatic, involving anticonvulsant drugs, surgical resection, and rehabilitative therapies. Only when a better understanding of the mechanisms of the disease is obtained will the development of other therapies be possible. Rapamycin, an inhibitor of mTOR, has been reported recently to induce regression of SEGAs, raising the possibility of targeted therapy for this disorder (17).

We therefore determined the status of Erk phosphorylation and S664 TSC2 phosphorylation in surgically removed brain lesions, including 17 cortical tubers and 8 SEGAs (all cases proved to be TSC clinically, and mutational analysis revealed that most of these patients carry exon deletion or point mutation of TSC2; data not shown), along with one normal brain autopsy (cortical region). Whereas the normal cortex seemed essentially negative for both phospho-antibodies, in all 25 cases of brain lesions, Erk was found activated, in agreement with previous findings (9, 10). Furthermore, pErk immunoreactivity was more pronounced in SEGAs than in tubers, indicating that aberrant Erk activation is not only sustained but also augmented in more advanced brain tumors in TSC (Fig. 2 ). Strikingly, S664-pTSC2 was also detected at a much higher level in SEGAs compared with that in cortical tubers (Fig. 2). In contrast, expression levels of total TSC2 protein in normal brain tissues, tubers, and SEGAs were similar (Fig. 2). These results suggest that in TSC-related brain tumorigenesis, it is Erk-mediated inactivation of the TSC2 protein rather than complete loss of TSC2 that causes neoplastic transformation.


Figure 2
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  		Figure 2. Expression of pErk, pTSC2, total TSC2, and pS6 in cortical tubers and SEGAs from TSC patients. Left, normal brain; middle, cortical tuber; right, SEGA. Arrows, calcifications that are typically associated with epileptogenic tubers. Magnification, x400.

 
To find out whether phosphorylation of Erk and TSC2 in these brain lesions is coupled with mTOR activation, we did immunostaining of pS6, which has been proven to be a potential marker for both mTOR pathway activation and mTOR pharmacologic inhibition (18). We found marked S6 phosphorylation in both tubers and SEGAs, indicating activation of mTOR (Fig. 2). Moreover, compared with tubers, SEGAs exhibited more uniform pS6 expression, which is consistent with the clonal nature of SEGA, whereas tubers are heterogeneous dysplastic lesions (Fig. 2).

Expression of Erk-pTSC2 and its correlation with MAPK activation in human cancer. Having at hand this new powerful tool, we attempted to address the intriguing question on whether Erk-dependent phosphorylation of TSC2 is restricted to TSC (especially in TSC2+/– cells in which Erk is constitutively activated). We hypothesized that this phosphorylation might be a relatively common event in various cancers, considering that Erk is frequently activated in human malignancies, often through gain-of-function mutations of Ras and Raf family members.

Staining of various cancer cell lines indeed showed a positive association between pTSC2 and pErk (data not shown). To analyze cancer tissues in a relatively large scale, we constructed TMAs of six types of tumors from cancer patients and subjected them to immunohistochemical staining with the pErk and pTSC2 (S664) antibodies, respectively. We observed that four types of tumors, breast carcinoma, colon carcinoma, endometrial carcinoma, and RCC (papillary type), were often associated with high degree of Erk phosphorylation (33–60%), whereas the remaining two types of tumors, prostate carcinoma and diffuse large B-cell lymphoma, showed extremely low incidence of activated Erk (<5%). Consistent with these results, the vast majority of prostate carcinoma and diffuse large B-cell lymphoma specimens did not show immunoreactivity with pTSC2, whereas S664 TSC2 phosphorylation was detected at a high incidence (range, 40–79%) in the four types of carcinomas with frequent Erk activation (Fig. 3 ; Table 1 ). As expected, the localization of pTSC2 was predominantly cytoplasmic, with a few cells also showing nuclear staining, whereas pErk was present in both cytoplasm and nucleus, sometimes showing strong nuclear staining (Fig. 3A–D). Furthermore, a large proportion of tumor specimens (>60%) were either double positive or double negative for the two phosphorylations (Fig. 3; Table 1), suggesting that these two events are closely associated.


Figure 3
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  		Figure 3. Expression of pTSC2 and pErk in human cancer. A, endometrial carcinoma. B, renal cell carcinoma, papillary type. C, breast carcinoma. D, colon carcinoma. Top, normal epithelial tissue; middle, an example of double-positive tumors; bottom, an example of double-negative tumors. Magnification, x400.

 

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  		Table 1. Correlation of pTSC2 and pErk in human cancer

 
Hence, we scored these two phosphorylations for each TMA (a tumor was scored positive only if definite staining was present, otherwise it was scored negative) and did the statistical analysis. A clear correlation was seen in all four types of cancers (P = 0.02–0.05), with subtle variations (Table 1). Based on the manner of the correlation, these tumors can be grouped into two categories. (a) Symmetrical correlation (endometrial carcinoma and papillary RCC): the majorities of the pErk-positive specimens were also positive for pTSC2 and vice versa. For instance, in 22 of 30 (73%) pErk-positive endometrial carcinomas, pTSC2 was positive; on the other hand, in 22 of 31 (71%) pTSC2-positive tumors, pErk was positive (Table 1). (b) Asymmetrical correlation (breast carcinoma and colon carcinoma): on one hand, the majorities of the pErk-positive specimens were also pTSC2 positive; conversely, however, a considerable number of the pTSC2-positive specimens were in fact negative for pErk. For example, 24 of 26 (92%) pErk-positive breast carcinomas were also pTSC2 positive; however, in 18 of 42 (43%) pTSC2-positive tumors, pErk was negative (Table 1). The similar relationship was also observed in colon carcinoma (Table 1).

Comparison of TSC2 phosphorylation and mTOR activation in human cancer. We next determined the status of mTOR activation by assessing phosphorylation of the S6 ribosomal protein. We found frequently pS6 in these cancers (>70%), and pS6 was predominantly present in the cytoplasm (Fig. 4 ). When compared with S664 TSC2 phosphorylation, most of the pTSC2-positive samples (~75%) were indeed positive for pS6. Conversely, however, pS6-positive tumors did not always show TSC2 phosphorylation. For instance, 43% (12 of 28) of the pS6-positive RCCs and 60% (29 of 48) of the pS6-positive colon carcinomas were pTSC2 negative (Table 2 ). These findings agree with the notion that both Akt and Erk are frequently activated in human cancer and that mTOR can be activated through either axis. Indeed, activated Erk and Akt have been found in ~48% and 38% of RCCs (3, 19). In colon cancer, ~50% of the tumors show activated Erk (2), and Akt overexpression also occurs frequently (57%) during colon carcinogenesis (20).


Figure 4
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  		Figure 4. Examples of pS6-positive and pS6-negative renal cell carcinoma and colon carcinoma. Magnification, x400.

 

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  		Table 2. Comparison of pTSC2 and pS6 in human cancer

 
In addition, prostate tumors, which showed extremely low frequency of pErk and pTSC2, also exhibited a high incidence of S6 phosphorylation (32 of 38; data not shown), which is consistent with frequent Akt activation in these tumors (mainly due to loss of PTEN).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Results of this study show that the TSC2 protein expressed in the CNS lesions from TSC patients is phosphorylated by Erk and therefore incapable of repressing mTOR, resulting in hyper-pS6. These findings corroborate our original report (12) and highlight the crucial role of Erk-mediated mTOR activation in TSC.

In addition to TSC, S664 TSC2 phosphorylation seems to be a frequent and significant event in a variety of human cancers, particularly in epithelial cancers where Erk is found frequently activated, indicating that Erk-dependent inactivation of TSC2 may be critical in a large fraction of human cancers.

A good, although not absolute, association between TSC2 phosphorylation and Erk phosphorylation is observed in all of the four tumor types analyzed. In certain types of cancers, such as endometrial carcinoma and papillary RCC, this correlation is reciprocal, suggesting that S664-pTSC2 can serve as a good marker for activation of the Erk pathway. Interestingly, however, in some other types of cancers, such as breast carcinoma and colon carcinoma, the correlation is asymmetrical, whereby pErk positivity is predictive for pTSC2 positivity, but a large percentage of pTSC2-positive tumors show negativity for pErk. This indicates that in certain circumstances, S664-pTSC2 might be an even more sensitive marker than pErk, taking into account that Erk is the only kinase known to phosphorylate S664. Furthermore, most of the pTSC2-positive samples exhibited mTOR activation (reflected by S6 hyperphosphorylation), but pS6-positive tumors did not always show TSC2 phosphorylation.

Taken together, these findings are of high clinical interest as S664 TSC2 phosphorylation can from now on be used for the stratification of cancer patients in clinical trials with combinatorial targeted therapies (e.g., MAPK and mTOR inhibitors or Akt and mTOR inhibitors), allowing to define in which cohort of patients mTOR activation is the consequence of the activation of the Ras-Erk or the phosphatidylinositol 3-kinase-Akt pathway or both.


   Acknowledgments
 
Grant support: NIH/National Cancer Institute grants CA82328 and CA84292 (P.P. Pandolfi).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank San San Yi and Dr. Paul Tempst for phosphopeptide synthesis; Louis Lopez and Emma Iskidarova for technical assistance in TMA construction; Marina Asher and Maria Socorro Jiao for technical expertise in antibody characterization and immunohistochemistry; and Drs. George Thomas and Brett Carver for discussion and comments.


   Footnotes
 
Note: Current address for L. Ma: Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142.

Received 12/29/06. Revised 4/12/07. Accepted 5/ 2/07.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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W. Feng, R. E. Brown, C. D. Trung, W. Li, L. Wang, T. Khoury, S. Alrawi, J. Yao, K. Xia, and D. Tan
Morphoproteomic Profile of mTOR, Ras/Raf Kinase/ERK, and NF-{kappa}B Pathways in Human Gastric Adenocarcinoma
Ann. Clin. Lab. Sci., January 1, 2008; 38(3): 195 - 209.
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