Methods 77–78 (2015) 1–2
Contents lists available at ScienceDirect
Methods journal homepage: www.elsevier.com/locate/ymeth
Guest Editor’s Introduction
A rapid guide to PTEN function
PTEN is a tumor suppressor gene frequently mutated in tumors and in the germ line of patients with clinically heterogeneous tumor-predisposition syndromes (PHTS), as well as in a subset of patients with autism. PTEN is a phosphatidylinositol 3,4,5trisphosphate [PI(3,4,5)P3] phosphatase that counteracts the activity of the PI3K proto-oncogene. In addition, PTEN possesses lipid phosphatase- and phosphatase-independent activities which are also important for its tumor suppressor function, as well as for cell development and cell survival functions. This makes PTEN a multifaceted protein with high relevance in clinics. This issue of Methods is focused on experimental methodologies and up-to-date coverage related to the diversity of PTEN functions, its regulation in health and disease, its relevance as a biomarker and a diagnostic tool in human cancer, and its potential as a direct therapeutic target in human pathologies. The first article is a brief overview on PTEN, with an outline of the duality of this protein in different human diseases [1]. The next papers document the involvement of PTEN gene in PHTS [2] and the use of anti-PTEN antibodies and IHC techniques to assess PTEN expression in human cancers, including vulvar [3], prostate [4], and endometrial cancer [5]. Aitziber et al. also presents methodology for the isolation of RNA and detection of PTEN transcripts from tumor biopsies and the urine from prostate cancer patients [4]. Eritja et al. also illustrates the importance of PTEN in glandular morphogenesis using in vitro 3D endometrial cell cultures [5]. The regulation of PTEN expression by competing endogenous RNA (ceRNA) networks, and its relevance in tumorigenesis, is reviewed by Poliseno et al. [6]. Next, methodologies to analyze PTEN catalytic activity, including enzymatic assays for the measurement of PTEN phosphatase activity [7] and electrophoretic assays of PTEN redox status [8], are presented. The inhibition of PTEN by small molecules, and its therapeutic potential in human disease, together with experimental procedures to measure PTEN inhibition, is presented by Mak et al. [9]. The core of the PTEN protein is formed by a catalytic protein tyrosine phosphatase (PTP) and a lipid-binding C2 domain. Malaney et al. highlight the presence of additional disordered domains, with high regulatory potential, in PTEN protein [10]. PTEN is regulated by a wide variety of post-translational modifications, among which phosphorylation is the more widely studied. A comprehensive review on PTEN phosphorylation is provided in this issue by Fragoso et al. [11], and time-resolved NMR methodology to quantitatively study the dynamics of PTEN phosphorylation is provided by Cordier et al. [12]. Post-translational modifications are important for the control of PTEN subcellular localization, which constitutes a major regulatory mechanism of PTEN http://dx.doi.org/10.1016/j.ymeth.2015.02.006 1046-2023/Ó 2015 Elsevier Inc. All rights reserved.
biological activity. The issue includes an overview and methodological guide on PTEN subcellular localization [13], and a report covering the coordinated role of phosphorylation and sumoylation in PTEN nuclear accumulation [14]. In addition, the use of molecular traps to study PTEN ubiquitylation and sumoylation is presented [15]. The positioning of PTEN at the plasma membrane and its association with lipids is essential to regulate PI(3,4,5)P3 levels and to exert the canonical PTEN tumor suppressor activity. In this issue, papers are presented that document the last advances in the definition of the molecular mechanisms that regulate PTEN membrane-binding [16], as well as biophysical and molecular dynamics methodologies to study PTEN-lipid dynamic interactions [17] and PTEN-membrane complexes [18] at high resolution. Protein-protein interactions are also essential in the modulation of PTEN function. Sotelo et al. review the current knowledge on PDZ/PTEN interaction networks and report the physical association of PTEN and the tyrosine phosphatase PTN13 [19]. The emerging cellnonautonomous roles for PTEN function, by means of its secretion via exosomes [20] or via the secretory pathway (in the case of the novel PTEN-L isoform) [21] is also reviewed in the issue. Hokadoski et al. also presents the analysis in cells of the PTEN negative regulator, P-REX2 [21]. Different experimental models suitable to study PTEN function in vivo are presented. These include the use of the yeast Saccharomyces cerevisiae [22], the mold Dictyostelium discoideum [16], and the worm Caenorhabditis elegans [23] as appropriate model systems for high-throughput genetic and mutational functional screening of PTEN. The zebrafish (Danio rerio) model, and its application to study the function of PTEN isoforms, is presented by Stumpf et al. [24]. Finally, Cho et al. document genetic methods for functional validation of human metastasis drivers using Trp53sensitized, non-metastatic PTEN deficient mice [25]. I wish to thank to all authors for their valuable contributions to this special issue of Methods. It was our intention to provide with these articles a useful reference guide for those interested in the study of PTEN function and regulation, and how this may impact human physiology and pathology. References [1] R. Pulido, Methods 77–78 (2015) 3–10. [2] J. Ngeow, C. Eng, Methods 77–78 (2015) 11–19. [3] A.M. Lavorato-Rocha, L.G. Anjos, I.W. Cunha, J. Vassallo, F.A. Soares, R.M. Rocha, Methods 77–78 (2015) 20–24. [4] U. Aitziber et al., Methods 77–78 (2015) 25–30. [5] N. Eritja, M. Santacana, O. Maiques, X. Gonzalez-Tallada, X. Dolcet, X. MatiasGuiu, Methods 77–78 (2015) 31–40.
2
Guest Editor’s Introduction / Methods 77–78 (2015) 1–2
[6] L. Poliseno, P.P. Pandolfi, Methods 77–78 (2015) 41–50. [7] L. Spinelli, N.R. Leslie, Methods 77–78 (2015) 51–57. [8] S.J. Han, Y. Ahn, I. Park, Y. Zhang, I. Kim, H.W. Kim, C.S. Ku, K.O. Chay, S.Y. Yang, B.W. Ahn, D.I. Jang, S.R. Lee, Methods 77–78 (2015) 58–62. [9] L.H. Mak et al., Methods 77–78 (2015) 63–68. [10] P. Malaney, V.N. Uversky, V. Davé, Methods 77–78 (2015) 69–74. [11] R. Fragoso, J.T. Barata, Methods 77–78 (2015) 75–81. [12] F. Cordier, A. Chaffotte, N. Wolff, Methods 77–78 (2015) 82–91. [13] A. Bononi, P. Pinton, Methods 77–78 (2015) 92–103. [14] J. Ho, C. Bassi, V. Stambolic, Methods 77–78 (2015) 104–111. [15] V. Lang, F. Aillet, E. Da Silva-Ferrada, W. Xolalpa, L. Zabaleta, C. Rivas, M.S. Rodriguez, Methods 77–78 (2015) 112–118. [16] J.M. Yang, H.N. Nguyen, H. Sesaki, P.N. Devreotes, M. Iijima, Methods 77–78 (2015) 119–124. [17] R.K. Harishchandra, B.M. Neumann, A. Gericke, A.H. Ross, Methods 77–78 (2015) 125–135. [18] H. Nanda, F. Heinrich, M. Lösche, Methods 77–78 (2015) 136–146. [19] N.S. Sotelo, J.T.G. Schepens, M. Valiente, W.J.A.J. Hendriks, R. Pulido, Methods 77–78 (2015) 147–156.
[20] U. Putz, S. Mah, C.P. Goh, L.H. Low, J. Howitt, S.S. Tan, Methods 77–78 (2015) 157–163. [21] C. Hodakoski, B. Fine, B. Hopkins, R. Parsons, Methods 77–78 (2015) 164–171. [22] I. Rodríguez-Escudero, T. Fernández-Acero, I. Bravo, N.R. Leslie, R. Pulido, M. Molina, V.J. Cid, Methods 77–78 (2015) 172–179. [23] J. Liu, I.D. Chin-Sang, Methods 77–78 (2015) 180–190. [24] M. Stumpf, S. Choorapoikayil, J. den Hertog, Methods 77–78 (2015) 191–196. [25] H. Cho, T. Herzka, C. Stahlhut, K. Watrud, B.D. Robinson, L.C. Trotman, Methods 77–78 (2015) 197–204.
Rafael Pulido Biocruces Health Research Institute, Barakaldo, Bizkaia, Spain IKERBASQUE, Basque Foundation for Science, Bilbao, Bizkaia, Spain
Contents lists available at ScienceDirect
Methods journal homepage: www.elsevier.com/locate/ymeth
Guest Editor’s Introduction
A rapid guide to PTEN function
PTEN is a tumor suppressor gene frequently mutated in tumors and in the germ line of patients with clinically heterogeneous tumor-predisposition syndromes (PHTS), as well as in a subset of patients with autism. PTEN is a phosphatidylinositol 3,4,5trisphosphate [PI(3,4,5)P3] phosphatase that counteracts the activity of the PI3K proto-oncogene. In addition, PTEN possesses lipid phosphatase- and phosphatase-independent activities which are also important for its tumor suppressor function, as well as for cell development and cell survival functions. This makes PTEN a multifaceted protein with high relevance in clinics. This issue of Methods is focused on experimental methodologies and up-to-date coverage related to the diversity of PTEN functions, its regulation in health and disease, its relevance as a biomarker and a diagnostic tool in human cancer, and its potential as a direct therapeutic target in human pathologies. The first article is a brief overview on PTEN, with an outline of the duality of this protein in different human diseases [1]. The next papers document the involvement of PTEN gene in PHTS [2] and the use of anti-PTEN antibodies and IHC techniques to assess PTEN expression in human cancers, including vulvar [3], prostate [4], and endometrial cancer [5]. Aitziber et al. also presents methodology for the isolation of RNA and detection of PTEN transcripts from tumor biopsies and the urine from prostate cancer patients [4]. Eritja et al. also illustrates the importance of PTEN in glandular morphogenesis using in vitro 3D endometrial cell cultures [5]. The regulation of PTEN expression by competing endogenous RNA (ceRNA) networks, and its relevance in tumorigenesis, is reviewed by Poliseno et al. [6]. Next, methodologies to analyze PTEN catalytic activity, including enzymatic assays for the measurement of PTEN phosphatase activity [7] and electrophoretic assays of PTEN redox status [8], are presented. The inhibition of PTEN by small molecules, and its therapeutic potential in human disease, together with experimental procedures to measure PTEN inhibition, is presented by Mak et al. [9]. The core of the PTEN protein is formed by a catalytic protein tyrosine phosphatase (PTP) and a lipid-binding C2 domain. Malaney et al. highlight the presence of additional disordered domains, with high regulatory potential, in PTEN protein [10]. PTEN is regulated by a wide variety of post-translational modifications, among which phosphorylation is the more widely studied. A comprehensive review on PTEN phosphorylation is provided in this issue by Fragoso et al. [11], and time-resolved NMR methodology to quantitatively study the dynamics of PTEN phosphorylation is provided by Cordier et al. [12]. Post-translational modifications are important for the control of PTEN subcellular localization, which constitutes a major regulatory mechanism of PTEN http://dx.doi.org/10.1016/j.ymeth.2015.02.006 1046-2023/Ó 2015 Elsevier Inc. All rights reserved.
biological activity. The issue includes an overview and methodological guide on PTEN subcellular localization [13], and a report covering the coordinated role of phosphorylation and sumoylation in PTEN nuclear accumulation [14]. In addition, the use of molecular traps to study PTEN ubiquitylation and sumoylation is presented [15]. The positioning of PTEN at the plasma membrane and its association with lipids is essential to regulate PI(3,4,5)P3 levels and to exert the canonical PTEN tumor suppressor activity. In this issue, papers are presented that document the last advances in the definition of the molecular mechanisms that regulate PTEN membrane-binding [16], as well as biophysical and molecular dynamics methodologies to study PTEN-lipid dynamic interactions [17] and PTEN-membrane complexes [18] at high resolution. Protein-protein interactions are also essential in the modulation of PTEN function. Sotelo et al. review the current knowledge on PDZ/PTEN interaction networks and report the physical association of PTEN and the tyrosine phosphatase PTN13 [19]. The emerging cellnonautonomous roles for PTEN function, by means of its secretion via exosomes [20] or via the secretory pathway (in the case of the novel PTEN-L isoform) [21] is also reviewed in the issue. Hokadoski et al. also presents the analysis in cells of the PTEN negative regulator, P-REX2 [21]. Different experimental models suitable to study PTEN function in vivo are presented. These include the use of the yeast Saccharomyces cerevisiae [22], the mold Dictyostelium discoideum [16], and the worm Caenorhabditis elegans [23] as appropriate model systems for high-throughput genetic and mutational functional screening of PTEN. The zebrafish (Danio rerio) model, and its application to study the function of PTEN isoforms, is presented by Stumpf et al. [24]. Finally, Cho et al. document genetic methods for functional validation of human metastasis drivers using Trp53sensitized, non-metastatic PTEN deficient mice [25]. I wish to thank to all authors for their valuable contributions to this special issue of Methods. It was our intention to provide with these articles a useful reference guide for those interested in the study of PTEN function and regulation, and how this may impact human physiology and pathology. References [1] R. Pulido, Methods 77–78 (2015) 3–10. [2] J. Ngeow, C. Eng, Methods 77–78 (2015) 11–19. [3] A.M. Lavorato-Rocha, L.G. Anjos, I.W. Cunha, J. Vassallo, F.A. Soares, R.M. Rocha, Methods 77–78 (2015) 20–24. [4] U. Aitziber et al., Methods 77–78 (2015) 25–30. [5] N. Eritja, M. Santacana, O. Maiques, X. Gonzalez-Tallada, X. Dolcet, X. MatiasGuiu, Methods 77–78 (2015) 31–40.
2
Guest Editor’s Introduction / Methods 77–78 (2015) 1–2
[6] L. Poliseno, P.P. Pandolfi, Methods 77–78 (2015) 41–50. [7] L. Spinelli, N.R. Leslie, Methods 77–78 (2015) 51–57. [8] S.J. Han, Y. Ahn, I. Park, Y. Zhang, I. Kim, H.W. Kim, C.S. Ku, K.O. Chay, S.Y. Yang, B.W. Ahn, D.I. Jang, S.R. Lee, Methods 77–78 (2015) 58–62. [9] L.H. Mak et al., Methods 77–78 (2015) 63–68. [10] P. Malaney, V.N. Uversky, V. Davé, Methods 77–78 (2015) 69–74. [11] R. Fragoso, J.T. Barata, Methods 77–78 (2015) 75–81. [12] F. Cordier, A. Chaffotte, N. Wolff, Methods 77–78 (2015) 82–91. [13] A. Bononi, P. Pinton, Methods 77–78 (2015) 92–103. [14] J. Ho, C. Bassi, V. Stambolic, Methods 77–78 (2015) 104–111. [15] V. Lang, F. Aillet, E. Da Silva-Ferrada, W. Xolalpa, L. Zabaleta, C. Rivas, M.S. Rodriguez, Methods 77–78 (2015) 112–118. [16] J.M. Yang, H.N. Nguyen, H. Sesaki, P.N. Devreotes, M. Iijima, Methods 77–78 (2015) 119–124. [17] R.K. Harishchandra, B.M. Neumann, A. Gericke, A.H. Ross, Methods 77–78 (2015) 125–135. [18] H. Nanda, F. Heinrich, M. Lösche, Methods 77–78 (2015) 136–146. [19] N.S. Sotelo, J.T.G. Schepens, M. Valiente, W.J.A.J. Hendriks, R. Pulido, Methods 77–78 (2015) 147–156.
[20] U. Putz, S. Mah, C.P. Goh, L.H. Low, J. Howitt, S.S. Tan, Methods 77–78 (2015) 157–163. [21] C. Hodakoski, B. Fine, B. Hopkins, R. Parsons, Methods 77–78 (2015) 164–171. [22] I. Rodríguez-Escudero, T. Fernández-Acero, I. Bravo, N.R. Leslie, R. Pulido, M. Molina, V.J. Cid, Methods 77–78 (2015) 172–179. [23] J. Liu, I.D. Chin-Sang, Methods 77–78 (2015) 180–190. [24] M. Stumpf, S. Choorapoikayil, J. den Hertog, Methods 77–78 (2015) 191–196. [25] H. Cho, T. Herzka, C. Stahlhut, K. Watrud, B.D. Robinson, L.C. Trotman, Methods 77–78 (2015) 197–204.
Rafael Pulido Biocruces Health Research Institute, Barakaldo, Bizkaia, Spain IKERBASQUE, Basque Foundation for Science, Bilbao, Bizkaia, Spain
