Abstract
Malignant mesothelioma (MM) is a highly aggressive tumor of the serous membranes for which there is currently no effective curative modality. Recent data suggest that hyperactivation of the tyrosine kinase SRC has a key role in MM development and therefore this kinase represents an important molecular target for MM therapy. We tested new pyrazolo[3,4-d]pyrimidine SRC inhibitors on a panel of MM cell lines expressing the active form of SRC. These SRC inhibitors exerted a significant proapoptotic effect on MM cells without affecting the normal mesothelial cell line MET-5A, supporting a possible use of these SRC inhibitors for a safe treatment of MM. We also showed that SRC inhibitor-induced apoptosis occurred concomitantly with an increase in the nuclear stability of the cyclin-dependent kinase inhibitor p27. This finding is remarkable considering that loss of nuclear p27 expression is a well-established adverse prognostic factor in MM, and p27 nuclear localization is crucial for its tumor-suppressive function. Consistently, SRC inhibition seems to promote the increase in p27 nuclear level also by inactivating the AKT kinase and downregulating cyclin D1, which would otherwise delay p27 nuclear import and provoke its cytoplasmic accumulation. To determine whether p27 stabilization has a direct role in apoptosis induced by SRC inhibition, we stably silenced the CDKN1B gene, encoding p27, in MSTO-211H and REN mesothelioma cells by transduction with lentiviral vectors expressing short hairpin RNAs against the CDKN1B transcript. Strikingly, p27 silencing was able to suppress the apoptosis induced by these SRC inhibitors in both MM cell lines, suggesting that p27 has a crucial proapoptotic role in MM cells treated with SRC inhibitors. Our findings reveal a new mechanism, dependent on p27 nuclear stabilization, by which SRC inhibition can induce apoptosis in MM cells and provide a new rationale for the use of SRC inhibitors in MM therapy.
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References
Altomare DA, You H, Xiao GH, Ramos-Nino ME, Skele KL, De Rienzo A et al. (2005). Human and mouse mesotheliomas exhibit elevated AKT/PKB activity, which can be targeted pharmacologically to inhibit tumor cell growth. Oncogene 24: 6080–6089.
Baldi A, Santini D, Vasaturo F, Santini M, Vicidomini G, Di Marino MP et al. (2004). Prognostic Significance of cyclooxygenase-2 (COX-2) and expression of cell cycle inhibitors p21 and p27 in human pleural malignant mesothelioma. Thorax 59: 428–433.
Beer TW, Shepherd P, Pullinger NC . (2001). p27 immunostaining is related to prognosis in malignant mesothelioma. Histopathology 38: 535–541.
Bertino P, Piccardi F, Porta C, Favoni R, Cilli M, Mutti L et al. (2008). Imatinib mesylate enhances therapeutic effects of gemcitabine in human malignant mesothelioma xenografts. Clin Cancer Res 14: 41–48.
Blagosklonny MV . (2002). Are p27 and p21 cytoplasmic oncoproteins? Cell Cycle 1: 391–393.
Bloom J, Pagano M . (2003). Deregulated degradation of the cdk inhibitor p27 and malignant transformation. Semin Cancer Biol 13: 41–47.
Bongiovanni M, Cassoni P, De Giuli P, Viberti L, Cappia S, Ivaldi C et al. (2001). p27(kip1) immunoreactivity correlates with long-term survival in pleural malignant mesothelioma. Cancer 92: 1245–1250.
Bravo SB, Pampín S, Cameselle-Teijeiro J, Carneiro C, Domínguez F, Barreiro F et al. (2003). TGF-beta-induced apoptosis in human thyrocytes is mediated by p27kip1 reduction and is overridden in neoplastic thyrocytes by NF-kappaB activation. Oncogene 22: 7819–7830.
Carraro F, Naldini A, Pucci A, Locatelli GA, Maga G, Schenone et al. (2006). Pyrazolo[3,4-d]pyrimidines as potent antiproliferative and proapoptotic agents toward A431 and 8701-BC cells in culture via inhibition of c-Src phosphorylation. J Med Chem 49: 1549–1561.
Chen R, Kim O, Yang J, Sato K, Eisenmann KM, McCarthy J et al. (2001). Regulation of Akt/PKB activation by tyrosine phosphorylation. J Biol Chem 276: 31858–31862.
Chiappetta G, De Marco C, Quintiero A, Califano D, Gherardi S, Malanga D et al. (2007). Overexpression of the S-phase kinaseassociated protein 2 in thyroid cancer. Endocr Relat Cancer 14: 405–420.
Chu I, Sun J, Arnaout A, Kahn H, Hanna W, Narod S et al. (2007). p27 phosphorylation by Src regulates inhibition of cyclin E-Cdk2 and p27 proteolysis. Cell 128: 281–294.
Chu IM, Hengst L, Slingerland JM . (2008). The Cdk inhibitor p27 in human cancer: prognostic potential and relevance to anticancer therapy. Nat Rev Cancer 8: 253–267.
Coqueret O . (2003). New roles for p21 and p27 cell-cycle inhibitors: a function for each cell compartment? Trend Cell Biol 13: 65–70.
Cursi S, Rufini A, Stagni V, Matafora V, Bachi A, Bonifazi AP et al. (2006). Src kinase phosphorylates Caspase-8 on Tyr380: a novel mechanism of apoptosis suppression. Embo J 25: 1895–1905.
Faraonio R, Vergara P, Di Marzo D, Napolitano M, Russo T, Cimino F . (2006). Transcription regulation in NIH3T3 cell clones resistant to diethylmaleate-induced oxidative stress and apoptosis. Antioxid Redox Signal 8: 365–374.
Fero ML, Rivkin M, Tasch M, Porter P, Carow CE, Firpo E et al. (1996). A syndrome of multiorgan hyperplasia with features of gigantism, tumorigenesis, and female sterility in p27(Kipl)-deficient mice. Cell 85: 733–744.
Karni R, Jove R, Levitzki A . (1999). Inhibition of pp60c-Src reduces Bcl-XL expression and reverses the transformed phenotype of cells overexpressing EGF and HER-2 receptors. Oncogene 18: 4654–4662.
Katayose Y, Kim M, Rakkar AN, Li Z, Cowan KH, Seth P . (1997). Promoting apoptosis: a novel activity associated with the cyclin-dependent kinase inhibitor p27. Cancer Res 57: 5441–5445.
Kettunen E, Nissén AM, Ollikainen T, Taavitsainen M, Tapper J, Mattson K et al. (2001). Gene expression profiling of malignant mesothelioma cell lines: cDNA array study. Int J Cancer 91: 492–496.
Kiyokawa H, Kineman RD, Manova-Todorova KO, Scares VC, Hoffman ES, Ono M et al. (1996). Enhanced growth of mice lacking the cyclin-dependent kinase inhibitor function of p27(Kipl). Cell 85: 721–732.
Kudo Y, Takata T, Ogawa I, Kaneda T, Sato S, Takekoshi T et al. (2000). p27Kip1 accumulation by inhibition of proteasome function induces apoptosis in oral squamous cell carcinoma cells. Clin Cancer Res 6: 916–923.
Lee AY, Raz DJ, He B, Jablons DM . (2007). Update on the molecular biology of malignant mesothelioma. Cancer 109: 1454–1461.
Liang J, Zubovitz J, Petrocelli T, Kotchetkov R, Connor MK, Han K et al. (2002). PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest. Nat Med 8: 1153–1160.
Livak KJ, Schmittgen TD . (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25: 402–408.
Masuda A, Osada H, Yatabe Y, Kozaki K, Tatematsu Y, Takahashi T et al. (2001). Protective function of p27(KIP1) against apoptosis in small cell lung cancer cells in unfavorable microenvironments. Am J Pathol 158: 87–96.
Menges CW, Chen Y, Mossman BT, Chernoff J, Yeung AT, Testa JR . (2010). A Phosphotyrosine Proteomic Screen Identifies Multiple Tyrosine Kinase Signaling Pathways Aberrantly Activated in Malignant Mesothelioma. Genes Cancer 1: 493–505.
Mezquita B, Mezquita J, Pau M, Mezquita C . (2010). A novel intracellular isoform of VEGFR-1 activates Src and promotes cell invasion in MDA-MB-231 breast cancer cells. J Cell Biochem 110: 732–742.
Mikami I, Zhang F, Hirata T, Okamoto J, Koizumi K, Shimizu K et al. (2010). Inhibition of activated phosphatidylinositol 3-kinase/AKT pathway in malignant pleural mesothelioma leads to G1 cell cycle arrest. Oncol Rep 24: 1677–1681.
Motti ML, Califano D, Troncone G, De Marco C, Migliaccio I, Palmieri E et al. (2005). Complex regulation of the cyclin-dependent kinase inhibitor p27kip1 in thyroid cancer cells by the PI3K/AKT pathway: regulation of p27kip1 expression and localization. Am J Pathol 166: 737–749.
Nakayama K, Ishida N, Shirane M, Inomata A, Inoue T, Shishido N et al. (1996). Mice lacking p27(Kipl) display increased body size, multiple organ hyperplasia, retinal dysplasia, and pituitary tumors. Cell 85: 707–720.
Ortega S, Malumbres M, Barbacid M . (2002). Cyclin D-dependent kinases, INK4 inhibitors and cancer. Biochim Biophys Acta 1602: 73–87.
Robinson BW, Musk AW, Lake RA . (2005). Malignant mesothelioma. Lancet 366: 397–408.
Schenone S, Brullo C, Bruno O, Bondavalli F, Mosti L, Maga G et al. (2008). Synthesis, biological evaluation and docking studies of 4-amino substituted 1H-pyrazolo[3,4-d]pyrimidines. Eur J Med Chem 43: 2665–2676.
Schenone S, Brullo C, Musumeci F, Botta M . (2010). Novel dual Src/Abl inhibitors for hematologic and solid malignancies. Expert Opin Investig Drugs 19: 931–945.
Schenone S, Bruno O, Ranise A, Bondavalli F, Brullo C, Fossa P et al. (2004). New pirazolo [3,4-d]pyrimidines endowed with A431 antiproliferative activity and inhibitory properties of Src phosphorylation. Bioorg Med Chem Lett 14: 2511–2517.
Schweppe RE, Kerege AA, French JD, Sharma V, Grzywa RL, Haugen BR . (2009). Inhibition of Src with AZD0530 reveals the Src-Focal Adhesion kinase complex as a novel therapeutic target in papillary and anaplastic thyroid cancer. J Clin Endocrinol Metab 94: 2199–2203.
Sherr CJ, Roberts JM . (1999). CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev 13: 1501–1512.
Shor AC, Keschman EA, Lee FY, Muro-Cacho C, Letson GD, Trent JC et al. (2007). Dasatinib inhibits migration and invasion in diverse human sarcoma cell lines and induces apoptosis in bone sarcoma cells dependent on SRC kinase for survival. Cancer Res 67: 2800–2808.
Sinibaldi D, Wharton W, Turkson J, Bowman T, Pledger WJ, Jove R . (2000). Induction of p21WAF1/CIP1 and cyclin D1 expression by the Src oncoprotein in mouse fibroblasts: role of activated STAT3 signaling. Oncogene 19: 5419–5427.
Spreafico A, Schenone S, Serchi T, Orlandini M, Angelucci A, Magrini D et al. (2008). Antiproliferative and proapoptotic activities of new pyrazolo[3,4-d]pyrimidine derivative Src kinase inhibitors in human osteosarcoma cells. FASEB J 22: 1560–1571.
Tsao AS, He D, Saigal B, Liu S, Lee JJ, Bakkannagari S et al. (2007). Inhibition of c-Src expression and activation in malignant pleural mesothelioma tissues leads to apoptosis, cell cycle arrest, and decreased migration and invasion. Mol Cancer Ther 6: 1962–1972.
Tsao AS, Wistuba I, Roth JA, Kindler HL . (2009). Malignant pleural mesothelioma. J Clin Oncol 27: 2081–2090.
Wang X, Gorospe M, Huang Y, Holbrook NJ . (1997). p27Kip1 overexpression causes apoptotic death of mammalian cells. Oncogene 15: 2991–2997.
Wang Z, Kishimoto H, Bhat-Nakshatri P, Crean C, Nakshatri H . (2005). TNFalpha resistance in MCF-7 breast cancer cells is associated with altered subcellular localization of p21CIP1 and p27KIP1. Cell Death Differ 12: 98–100.
Xiao GH, Gallagher R, Shetler J, Skele K, Altomare DA, Pestell RG et al. (2005). The NF2 tumor suppressor gene product, merlin, inhibits cell proliferation and cell cycle progression by repressing cyclin D1 expression. Mol Cell Biol 25: 2384–2394.
Yamaguchi H, Woods NT, Dorsey JF, Takahashi Y, Gjertsen NR, Yeatman T et al. (2008). SRC directly phosphorylates Bif-1 and prevents its interaction with Bax and the initiation of anoikis. J Biol Chem 283: 19112–19118.
Yang H, Zhang Y, Zhao R, Wen YY, Fournier K, Wu HB et al. (2006). Negative cell cycle regulator 14-3-3sigma stabilizes p27 Kip1 by inhibiting the activity of PKB/Akt. Oncogene 25: 4585–4594.
Yeatman TJ . (2004). A renaissance for SRC. Nat Rev Cancer 4: 470–480.
Yuan JS, Reed A, Chen F, Stewart Jr CN . (2006). Statistical analysis of real-time PCR data. BMC Bioinformatics 7: 85.
Acknowledgements
We thank Giuseppe Viglietto for providing lentiviral vectors and Giovanni Gaudino for providing REN cells. PI, FG and VR are the recipients of Human Health Foundation fellowships. FP is also Adjunct Assistant Professor at Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, Philadelphia, PA, USA. This work was funded by Sbarro Health Research Organization (http://www.shro.org) and Human Health Foundation (http://www.hhfonlus.org).
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Indovina, P., Giorgi, F., Rizzo, V. et al. New pyrazolo[3,4-d]pyrimidine SRC inhibitors induce apoptosis in mesothelioma cell lines through p27 nuclear stabilization. Oncogene 31, 929–938 (2012). https://doi.org/10.1038/onc.2011.286
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DOI: https://doi.org/10.1038/onc.2011.286
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