North Shore Long Island Jewish Research Institute and Department of Otolaryngology and Communicative Disorders, Long Island Jewish Medical Center, 270-05 76th Avenue, New Hyde Park, New York 11040, USA1
Author for correspondence: Bettie Steinberg. Fax +1 718 347 2320. e-mail bsteinbe{at}lij.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Point mutations in the PTEN/MMAC1/TEP1 gene (Li & Sun, 1997 ; Li et al., 1997
; Steck et al., 1997
) have been linked to Cowdens disease (Nelen et al., 1997
) and have been implied in the development of various tumours (Cairns et al., 1997
; Liu et al., 1997
; Risinger et al., 1997
; Teng et al., 1997
). PTEN is a dual protein phosphatase capable of dephosphorylating phosphotyrosine and phosphoserine/phosphothreonine in vitro (Li & Sun, 1997
; Li et al., 1997
; Myers et al., 1997
). PTEN is also a lipid phosphatase that dephosphorylates phosphoinositol 3,4,5-triphosphate [PIns(3,4,5)P3] (Maehama & Dixon, 1998
), thereby counteracting the effects elicited by PI3 kinase. The tumour suppression activity of PTEN has been attributed largely to its phospholipid phosphatase activity. A G129E mutation in PTEN that abolished its lipid phosphatase activity while retaining the protein phosphatase activity was identified in a cancer patient (Myers et al., 1998
). The ability of PTEN to function as a tumour suppressor is consistent with its ability to inhibit Akt activation (Cantley & Neel, 1999
). Akt was shown recently to activate NF-
B (Ozes et al., 1999
; Romashkova & Makarov, 1999
) and inhibit Forkhead transcription factors (Brunet et al., 1999
). Both were shown to be involved in cell cycle regulation (Guttridge et al., 1999
; Kaltschmidt et al., 1999
; Medema et al., 2000
; Romashkova & Makarov, 1999
) and programmed cell death (Barkett & Gilmore, 1999
; Brunet et al., 1999
). The mechanism by which PTEN negatively regulates proliferation is not well understood. PTEN expression has been associated with elevated levels of p27kip1 (Cheney et al., 1999
) and reduced expression of c-myc (Ghosh et al., 1999
). Recent observation that overexpressing PTEN had no effect on cell cycle progression in pRb-/- cells underscored the role of the retinoblastoma protein (pRb) in mediating the function of PTEN (Paramio et al., 1999
). The increase in the steady-state level of PTEN in laryngeal papilloma tissues predicts that HPV-infected papilloma cells will grow more slowly than normal laryngeal epithelial cells and that HPV-infected cells will be prone to apoptosis.
STAT3, a member of the signal transducer and activator of transcription (STAT) family, is activated as a result of EGFR activation (Grandis et al., 1998 ; Zhong et al., 1994
). Furthermore, STAT3 has recently been classified as a proto-oncogene (Bromberg et al., 1999
) and has been shown to play a pivotal role in EGF-induced proliferation in head and neck squamous carcinoma cells (Grandis et al., 1998
). Activation of STAT is induced by activation of receptor tyrosine kinase, for example EGFR, or by non-receptor tyrosine kinases, for example JAK family kinases, c-Src (Cao et al., 1996
; Schaefer et al., 1999
) and c-Fes kinase (Nelson et al., 1998
). Tyrosine phosphorylation of STAT leads to its nuclear translocation, binding to a specific promoter-proximal element and subsequent transcription activation. An increased STAT3 activation would be expected in HPV-infected papilloma cells due to constitutive activation of the EGFR.
While intensive research has been focused on the lipid phosphatase activity of PTEN and its effect on Akt, little is known about the protein phosphatase activity of PTEN. One obstacle obstructs the identification of its downstream effectors. To date, only the focal adhesion kinase (FAK) (Tamura et al., 1998 , 1999
) and the adaptor protein Shc (Gu et al., 1999
) have been shown to be potential in vivo substrates for PTEN. In contrast with an ever-increasing number of STAT3-activating kinases, there are very few negative regulators of STAT that have been identified. In this study, we report the identification of PTEN as a negative regulator of STAT3 activation in HPV-infected laryngeal papilloma cells.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Plasmids and antibodies.
pGCN-PTEN and pGCN-G129E mammalian expression constructs (gifts of Michael Myers, Cold Spring Harbor Laboratory) contained, respectively, wild-type and the lipid phosphatase-deficient G129E mutant of PTEN cDNA fused to an N-terminal haemagglutinin (HA) tag (Myers et al., 1998 ). The fusion proteins were expressed under the control of the cytomegalovirus promiscuous promoter. pTATA-4XM67-Luc and control plasmid pTATA-Luc were gifts of Danile Besser (The Rockefeller University, New York). pHygEGFP was from Clontech. Anti-PTEN polyclonal antibody (N-19), anti-PTEN mouse monoclonal antibody (A2B1) and anti-HA mouse monoclonal antibody (F-7) were purchased from Santa Cruz Biotech. Anti-PTyr(705)STAT3 polyclonal and anti-STAT3(pan) were purchased from Cell Signaling Technology. Horseradish peroxidase-conjugated antibodies were purchased from Pierce. All detection of phosphorylated STAT3 used the phospho-specific antibody.
Transfection.
HeLa cells at 6070% confluency were transfected with various amounts of plasmid DNA using Lipofectamine Plus reagents (Life Technologies) according to the manufacturers instructions. Two days after transfection, cells were harvested and lysates were prepared for Western blot analysis. In our hands, this method resulted in 5070% transfection efficiency for HeLa cells. For EGF stimulation experiments, cells were serum-starved for 12 h following transfection. EGF stimulation was done at a concentration of 100 ng/ml. At indicated times after stimulation, cells were harvested and lysates prepared for Western blot analysis.
Immunoprecipitation and Western blot analysis.
Cells were lysed on ice with lysis buffer (20 mM HEPES, pH 7·6, 1% NP-40, 0·4 M NaCl, 10% glycerol, 1 mM DTT) in the presence of Complete protease inhibitor cocktail (Roche Molecular Biochemicals) and phosphatase inhibitors (20 mM -glycerophosphate, 1 mM sodium orthovanadate, 30 mM sodium fluoride). After spinning at top speed in a microcentrifuge, the supernatant was transferred to a new Eppendorf tube. The protein concentration of the lysates was determined using Micro BCA reagents (Pierce). For immunoprecipitation, the NaCl concentration of the lysates was adjusted to 150 mM. Antibody (2 µg) was preadsorbed to protein A or protein G beads (Santa Cruz Biotech). The antibody-coated beads were incubated then with 200 µg lysate for 2 h at 4 °C with rocking. The resultant lysates were tested in phosphatase assays. For Western blot analysis, 40 µg protein was electrophoresed on an SDSpolyacrylamide gel. Separated polypeptides were transferred to a nitrocellulose membrane (Schleicher & Schull). The membrane was blocked with 5% (w/v) non-fat milk and was allowed to react with the primary antibody overnight at 4 °C with rocking. After incubation with the appropriate secondary antibody, the immunoreactive species were detected using SuperSignal West Pico chemiluminescent substrates (Pierce). A commercially available IFN-
-stimulated HeLa cell lysate was used as a positive control for PTyr(705)STAT3, following the suppliers instructions (Cell Signaling Technology). Nitrocellulose membranes were stripped to allow detection of additional proteins through the use of a different primary antibody. Thus, membranes were incubated in stripping buffer (62·5 mM TrisHCl, pH 6·8, 100 mM
-mercaptoethanol, 2% SDS) at 50 °C for 30 min with occasional shaking. Membranes were washed extensively with Tris-buffered saline (TBS) (10 mM TrisHCl, pH 7·5, 1·5 mM MgCl2, 140 mM NaCl) and with TBST (TBS plus 0·05% Tween 20). Membranes were then blocked with TBST plus 5% (w/v) non-fat milk for 1 h, followed by incubation with the primary antibody.
In vitro phosphatase assay.
Lysates were dialysed against phosphatase buffer (50 mM HEPES, pH 7·6, 10 mM MgCl2, 10 mM DTT) for 4 h with one change of buffer before performing the phosphatase assay. Lysates were incubated in phosphatase buffer at 37 °C for 30 min. Reactions were stopped by adding 5x SDS gel loading buffer. Dephosphorylation of STAT3 was determined by Western blot analysis.
Luciferase assay.
HeLa cells were grown in six-well plates and transfection was performed in triplicate using 1·5 µg pGCN-PTEN or pGCN-G129E or pCDNA3 (Invitrogen), plus 0·5 µg pHygEGFP and 0·5 µg of either pTATA-4XM67-Luc or control pTATA-Luc. Forty-eight hours after transfection, cell lysates were made and luciferase assays were performed using commercial luciferase assay reagents (Promega) and a TD-20/20 Luminometer (Turner Designs). STAT3-driven luciferase expression was determined by subtracting the background activity obtained in pTATA-Luc transfections after normalization against GFP expression. The final luciferase activity was expressed as a percentage relative to the control pCDNA3 transfection.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
To determine whether the observed reduction in tyrosine-phosphorylated STAT3, as a result of the overexpression of PTEN, truly reflects STAT3 transactivation capacity in vivo, we performed STAT3 reporter assays. As indicated in Fig. 4(B), overexpressing PTEN reduced STAT3-dependent transactivation, similar to that observed for tyrosine-phosphorylated STAT3, and the lipid phosphatase mutant of PTEN further impaired STAT3 activation. These results not only confirmed the STAT3 phosphorylation data in HeLa cells, but also further supported the link between PTEN overexpression and reduction of STAT3 activation in HPV 6/11-induced papilloma cells.
PTEN does not inhibit STAT3 kinase activity
The effect of PTEN on STAT3 phosphorylation in vivo could either be due to up-regulation of a phosphatase, as suggested above, or result from inactivation of a STAT3 kinase, or both. To determine whether inactivation of a STAT3 kinase was involved, HeLa cells were serum-starved for 12 h following transfection. Subsequent stimulation with 100 ng/ml EGF resulted in a 2-fold increase in tyrosine-phosphorylated STAT3 in the control (Fig. 5). In cells transfected with either wild-type PTEN or the lipid phosphatase mutant, a more robust stimulation (34-fold) was observed. Thus, we concluded that tyrosine phosphorylation of STAT3 is functional in cells overexpressing PTEN, and that the primary effect of PTEN overexpression is a dephosphorylation of STAT3.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
STAT3 has recently been classified as a proto-oncogene (Bromberg et al., 1999 ). STAT3, but not STAT1, is required for EGF-induced growth in head and neck cancer cells (Grandis et al., 1998
). Overexpression of PTEN in laryngeal papillomas, with a resultant reduction in activated STAT3, is thus in accord with the reduced rate of proliferation associated with the papilloma cells (Steinberg et al., 1990
). The anti-apoptotic activity of STAT3 is also well documented (Fukada et al., 1996
; Grandis et al., 2000
; Takeda et al., 1998
). In this regard, the ability of PTEN to negatively regulate STAT3 activation is consistent with its reported tumour suppressor activity, and with the fact that the HPV 6/11-induced papillomas have a low probability of malignant conversion. It is interesting that HPV 16 (a high-risk HPV)-induced low grade tumour cells display elevated PTEN as well, whereas HPV 16-induced high grade tumour cells contain reduced levels of PTEN (K. Auborn & B. M. Steinberg, unpublished results). This observation implies malignant conversion of HPV-induced lesions upon loss of PTEN. It is also interesting to note that STAT3 was reported to be constitutively active in a plethora of prostate cancer cell lines (Ni et al., 2000
), where mutations in PTEN have been frequently detected. It is interesting to note that overexpression of the lipid phosphatase-deficient form of PTEN resulted in greater reduction of PTyr(705)STAT3 (Fig. 4
). It remains to be seen whether eliminating PTENs lipid phosphatase activity augments its protein phosphatase activity in vitro, as our data implied. It is also possible that additional regulatory factors, either activators or repressors of PTEN, are involved in the augmentation of activity. In this case, activation of Akt might activate the activators or inactivate the repressors. Experiments to clarify these possibilities are under way.
It is surprising to learn that both Akt and STAT3 are negatively regulated in HPV-infected laryngeal papilloma cells. Aside from their oncogenic potential, both Akt and STAT3 are known anti-apoptotic regulators. Inhibition of activation of Akt and STAT3 in HPV-infected papilloma cells implies a skew of survival/death signal transduction to the apoptotic side, and one must wonder what advantage would accrue to the virus by inducing such an effect in the target cells. To this end, the observed increase in PTEN levels might be interpreted as a host defence mechanism to reduce cell proliferation and induce cell death in virus-infected cells. However, HPV-infected laryngeal papilloma cells do not die. On the contrary, cultured papilloma cells display a survival advantage, compared with uninfected cells, against the anti-cancer drug adriamycin (unpublished results). It is perhaps relevant that nuclear extracts of laryngeal papilloma showed elevated NF-B DNA-binding activity (Vancurova et al., 2002
), and NF-
B can enhance cell survival (Mayo & Baldwin, 2000
). Another possible explanation involves the fact that HPV productively replicates in differentiated cells, but not in basal proliferating cells (Flores & Lambert, 1997
; Stoler et al., 1989
). To this end, induction of a cell cycle-arresting molecule may promote differentiation and ultimately viral replication.
Fig. 6 illustrates our model for the dual activity of PTEN in controlling two proliferative/survival pathways activated by EGF. STAT3 is tyrosine-phosphoylated through activation of EGFR. This may involve c-Src and/or Jak. PTEN that has been recruited to the plasma membrane is responsible for inactivating tyrosine-phosphorylated STAT3. Note that the model proposes coordinated down-regulation of the Akt and STAT3 pathways by PTEN. Testing this model, and determining the signal transduction pathway(s) that counteract the death-promoting effect of PTEN in HPV-induced papillomas, is currently under investigation.
|
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bromberg, J. F., Wrzeszczynska, M. H., Devgan, G., Zhao, Y., Pestell, R. G., Albanese, C. & Darnell, J. E., Jr (1999). Stat3 as an oncogene. Cell 98, 295303 [published erratum appears in Cell 99, 239].[Medline]
Brunet, A., Bonni, A., Zigmond, M. J., Lin, M. Z., Juo, P., Hu, L. S., Anderson, M. J., Arden, K. C., Blenis, J. & Greenberg, M. E. (1999). Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96, 857-868.[Medline]
Cairns, P., Okami, K., Halachmi, S., Halachmi, N., Esteller, M., Herman, J. G., Jen, J., Isaacs, W. B., Bova, G. S. & Sidransky, D. (1997). Frequent inactivation of PTEN/MMAC1 in primary prostate cancer. Cancer Research 57, 4997-5000.[Abstract]
Cantley, L. C. & Neel, B. G. (1999). New insights into tumor suppression: PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/AKT pathway. Proceedings of the National Academy of Sciences, USA 96, 4240-4245.
Cao, X., Tay, A., Guy, G. R. & Tan, Y. H. (1996). Activation and association of Stat3 with Src in v-Src-transformed cell lines. Molecular and Cellular Biology 16, 1595-1603.[Abstract]
Cheney, I. W., Neuteboom, S. T., Vaillancourt, M. T., Ramachandra, M. & Bookstein, R. (1999). Adenovirus-mediated gene transfer of MMAC1/PTEN to glioblastoma cells inhibits S phase entry by the recruitment of p27Kip1 into cyclin E/CDK2 complexes. Cancer Research 59, 2318-2323.
Flores, E. R. & Lambert, P. F. (1997). Evidence for a switch in the mode of human papillomavirus type 16 DNA replication during the viral life cycle. Journal of Virology 71, 7167-7179.[Abstract]
Fukada, T., Hibi, M., Yamanaka, Y., Takahashi-Tezuka, M., Fujitani, Y., Yamaguchi, T., Nakajima, K. & Hirano, T. (1996). Two signals are necessary for cell proliferation induced by a cytokine receptor gp 130: involvement of STAT3 in anti-apoptosis. Immunity 5, 449-460.[Medline]
Ghosh, A. K., Grigorieva, I., Steele, R., Hoover, R. G. & Ray, R. B. (1999). PTEN transcriptionally modulates c-myc gene expression in human breast carcinoma cells and is involved in cell growth regulation. Gene 235, 85-91.[Medline]
Grandis, J. R., Drenning, S. D., Chakraborty, A., Zhou, M. Y., Zeng, Q., Pitt, A. S. & Tweardy, D. J. (1998). Requirement of Stat3 but not Stat1 activation for epidermal growth factor receptor-mediated cell growth in vitro. Journal of Clinical Investigation 102, 1385-1392.
Grandis, J. R., Drenning, S. D., Zeng, Q., Watkins, S. C., Melhem, M. F., Endo, S., Johnson, D. E., Huang, L., He, Y. & Kim, J. D. (2000). Constitutive activation of Stat3 signaling abrogates apoptosis in squamous cell carcinogenesis in vivo. Proceedings of the National Academy of Sciences, USA 97, 4227-4232.
Gu, J., Tamura, M., Pankov, R., Danen, E. H., Takino, T., Matsumoto, K. & Yamada, K. M. (1999). Shc and FAK differentially regulate cell motility and directionality modulated by PTEN. Journal of Cell Biology 146, 389-403.
Guttridge, D. C., Albanese, C., Reuther, J. Y., Pestell, R. G. & Baldwin, A. S.Jr (1999). NF-kappaB controls cell growth and differentiation through transcriptional regulation of cyclin D1. Molecular and Cellular Biology 19, 5785-5799.
Johnston, D., Hall, H., DiLorenzo, T. P. & Steinberg, B. M. (1999). Elevation of the epidermal growth factor receptor and dependent signaling in human papillomavirus-infected laryngeal papillomas. Cancer Research 59, 968-974.
Kaltschmidt, B., Kaltschmidt, C., Hehner, S. P., Droge, W. & Schmitz, M. L. (1999). Repression of NF-kappaB impairs HeLa cell proliferation by functional interference with cell cycle checkpoint regulators. Oncogene 18, 3213-3225.[Medline]
Lee, J. O., Yang, H., Georgescu, M. M., Di Cristofano, A., Maehama, T., Shi, Y., Dixon, J. E., Pandolfi, P. & Pavletich, N. P. (1999). Crystal structure of the PTEN tumor suppressor: implications for its phosphoinositide phosphatase activity and membrane association. Cell 99, 323-334.[Medline]
Li, D. M. & Sun, H. (1997). TEP 1, encoded by a candidate tumor suppressor locus, is a novel protein tyrosine phosphatase regulated by transforming growth factor beta. Cancer Research 57, 2124-2129.[Abstract]
Li, J., Yen, C., Liaw, D., Podsypanina, K., Bose, S., Wang, S. I., Puc, J., Miliaresis, C., Rodgers, L., McCombie, R., Bigner, S. H., Giovanella, B. C., Ittmann, M., Tycko, B., Mbshoosh, H., Wigler, M. H. & Parsons, R. (1997). PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer (see comments). Science 275, 1943-1947.
Liu, W., James, C. D., Frederick, L., Alderete, B. E. & Jenkins, R. B. (1997). PTEN/MMAC I mutations and EG-FR amplification in glioblastomas. Cancer Research 57, 5254-5257.[Abstract]
Maehama, T. & Dixon, J. E. (1998). The tumor suppressor, PTEN/MMAC 1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate. Journal of Biological Chemistry 273, 13375-13378.
Mayo, M. W. & Baldwin, A. S. (2000). The transcription factor NF-kappaB: control of oncogenesis and cancer therapy resistance. Biochimica et Biophysica Acta 1470, M55-62.[Medline]
Medema, R. H., Kops, G. J., Bos, J. L. & Burgering, B. M. (2000). AFX-like Forkhead transcription factors mediate cell-cycle regulation by Ras and PKB through p27kip1. Nature 404, 782-787.[Medline]
Myers, M. P., Stolarov, J. P., Eng, C., Li, J., Wang, S. I., Wigler, M. H., Parsons, R. & Tonks, N. K. (1997). P-TEN, the tumor suppressor from human chromosome l0q23, is a dual-specificity phosphatase. Proceedings of the National Academy of Sciences, USA 94, 9052-9057.
Myers, M. P., Pass, I., Batty, I. H., van der Kaay, J., Stolarov, J. P., Hemmings, B. A., Wigler, M. H., Downes, C. P. & Tonks, N. K. (1998). The lipid phosphatase activity of PTEN is critical for its tumor supressor function. Proceedings of the National Academy of Sciences, USA 95, 13513-13518.
Nelen, M. R., van Staveren, W. C., Peeters, E. A., Hassel, M. B., Gorlin, R. J., Hamm, H., Lindboe, C. F., Fryns, J. P., Sijmons, R. H., Woods, D. G., Mariman, E. C., Padberg, G. W. & Kremer, H. (1997). Germline mutations in the PTEN/MXIAC I gene in patients with Cowden disease. Human Molecular Genetics 6, 1383-1387.
Nelson, K. L., Rogers, J. A., Bowman, T. L., Jove, R. & Smithgall, T. E. (1998). Activation of STAT3 by the c-Fes protein-tyrosine kinase. Journal of Biological Chemistry 273, 7072-7077.
Ni, Z., Lou, W., Leman, E. S. & Gao, A. C. (2000). Inhibition of constitutively activated Stat3 signaling pathway suppresses growth of prostate cancer cells. Cancer Research 60, 1225-1228.
Ozes, O. N., Mayo, L. D., Gustin, J. A., Pfeffer, S. R., Pfeffer, L. M. & Donner, D. B. (1999). NF-kappaB activation by tumour necrosis factor requires the Akt serinethreonine kinase (see comments). Nature 401, 82-85.[Medline]
Paramio, J. M., Navarro, M., Segrelles, C., Gomez-Casero, E. & Jorcano, J. L. (1999). PTEN tumour suppressor is linked to the cell cycle control through the retinoblastoma protein. Oncogene 18, 7462-7468.[Medline]
Risinger, J. I., Hayes, A. K., Berchuck, A. & Barrett, J. C. (1997). PTEN/MMAAC1 mutations in endometrial cancers. Cancer Research 57, 4736-4738.[Abstract]
Romashkova, J. A. & Makarov, S. S. (1999). NF-kappaB is a target of AKT in antiapoptotic PDGF signalling (see comments). Nature 401, 86-90.[Medline]
Schaefer, L. K., Wang, S. & Schaefer, T. S. (1999). c-Src activates the DNA binding and transcriptional activity of Stat3 molecules: serine 727 is not required for transcriptional activation under certain circumstances. Biochemical and Biophysical Research Communications 266, 481-487.[Medline]
Steck, P. A., Pershouse, M. A., Jasser, S. A., Yung, W. K., Lin, H., Ligon, A. H., Langford, L. A., Baumgard, M. L., Hattier, T., Davis, T., Frye, C., Hu, R., Swedlund, B., Teng, D. H. & Tavtigian, S. V. (1997). Identification of a candidate tumour suppressor gene, MMAC 1, at chromosome 10q23·3 that is mutated in multiple advanced cancers. Nature Genetics 15, 356-362.[Medline]
Steinberg, B. M., Meade, R., Kalinowski, S. & Abramson, A. L. (1990). Abnormal differentiation of human papillomavirus-induced laryngeal papillomas. Archives of Otolaryngology Head and Neck Surgery 116, 1167-1171.[Medline]
Stoler, M. H., Wolinsky, S. M., Whitbeck, A., Broker, T. R. & Chow, L. T. (1989). Differentiation-linked human papillomavirus types 6 and 11 transcription in genital condylomata revealed by in situ hybridization with message-specific RNA probes. Virology 172, 331-340.[Medline]
Takeda, K., Kaisho, T., Yoshida, N., Takeda, J., Kishimoto, T. & Akira, S. (1998). Stat3 activation is responsible for IL-6-dependent T cell proliferation through preventing apoptosis: generation and characterization of T cell-specific Stat3-deficient mice. Journal of Immunology 161, 4652-4660.
Tamura, M., Gu, J., Matsumoto, K., Aota, S., Parsons, R. & Yamada, K. M. (1998). Inhibition of cell migration, spreading, and focal adhesions by tumor suppressor PTEN. Science 280, 1614-1617.
Tamura, M., Gu, J., Takino, T. & Yamada, K. M. (1999). Tumor suppressor PTEN inhibition of cell invasion, migration, and growth: differential involvement of focal adhesion kinase and p130Cas. Cancer Research 59, 442-449.
Teng, D. H., Hu, R., Lin, H., Davis, T., Iliev, D., Frye, C., Swedlund, B., Hansen, K. L., Vinson, V. L., Gumpper, K. L., Ellis, L., El-Naggar, A., Frazier, M., Jasser, S., Langford, L. A., Lee, J., Mills, G. B., Pershouse, M. A., Pollack, R. E., Tornos, C., Troncoso, P., Yung, W. K., Fujii, G., Berson, A. & Steck, P. A. (1997). MMAC1/PTEN mutations in primary tumor specimens and tumor cell lines. Cancer Research 57, 5221-5225.[Abstract]
Vambutas, A., Di Lorenzo, T. P. & Steinberg, B. M. (1993). Laryngeal papilloma cells have high levels of epidermal growth factor receptor and respond to epidermal growth factor by a decrease in epithelial differentiation. Cancer Research 53, 910-914.[Abstract]
Vancurova, I., Wu, R., Miskolci, V. & Sun, S. (2002). Increased p50/p50 NF-kappaB activation in human papillomavirus type 6- or type 11-induced laryngeal papilloma tissue. Journal of Virology 76, 1533-1536.
Wu, X., Hepner, K., Castelino-Prabhu, S., Do, D., Kaye, M. B., Yuan, X. J., Wood, J., Ross, C., Sawyers, C. L. & Whang, Y. E. (2000a). Evidence for regulation of the PTEN tumor suppressor by a membrane-localized multi-PDZ domain containing scaffold protein MAGI-2. Proceedings of the National Academy of Sciences, USA 97, 4233-4238.
Wu, Y., Dowbenko, D., Spencer, S., Laura, R., Lee, J., Gu, Q. & Lasky, L. A. (2000b). Interaction of the tumor suppressor PTEN/MMAC with a PDZ domain of MAGI3, a novel membrane-associated guanylate kinase. Journal of Biological Chemistry 275, 21477-21485.
Zhang, P. & Steinberg, B. M. (2000). Overexpression of PTEN/MMAC1 and decreased activation of Akt in human papillomavirus-infected laryngeal papillomas. Cancer Research 60, 1457-1462.
Zhong, Z., Wen, Z. & Darnell, J. E.Jr (1994). Stat3: a STAT family member activated by tyrosine phosphorylation in response to epidermal growth factor and interleukin-6. Science 264, 95-98.[Medline]
Received 3 December 2001;
accepted 21 February 2002.