Regulation of Protein Kinase B activity by PTEN and SHIP2 in human prostate-derived cell lines
Introduction
Cell growth and survival in normal epithelial cells is regulated by signals from growth factors and from cell–cell and cell–matrix contact. Cell dependence upon these signals allows control of tissue growth and maintenance of epithelial tissue architecture. Tumour progression to invasiveness and metastasis characteristically involves loss of dependence upon such signals. The PI3K–PKB pathway, activated in response to growth factors and adhesion to matrix or other cells, is central to signalling pathways that regulate these processes (see Ref. [1], [2] for reviews). Activated PI3K converts PI(4,5)P2 to PI(3,4,5)P3 (PIP3), which recruits PKB to the cell membrane and allows phosphatidylinositol-dependent kinase 1 (PDK1) and a second kinase (termed PDK2, though not yet conclusively identified) to phosphorylate and activate PKB at thr308 and ser473 respectively [3], [4]. Activation of PKB in turn leads to the phosphorylation of a number of downstream targets which control proliferation and survival, including glycogen synthase kinase 3 (GSK3), the proapoptotic protein BAD, mTOR (via TSC2) and MDM2/HDM2. PKB activation also regulates p27-Kip1 and the forkhead family of transcription factors [5]. The PI3K–PKB pathway is deregulated in many tumour types, and constitutively raised levels of activated PKB have been implicated in enhanced proliferation and inappropriate cell survival independently of growth factor stimulation and normal requirements for cell–cell and cell–matrix contact.
The PTEN tumour-suppressor gene is frequently inactivated in a wide variety of tumour types; PTEN activity is characteristically lost (or reduced by haploinsufficiency) in late-stage tumours and has been associated with the development of invasiveness and metastatic capacity ([6]; see also review, [7]). The PTEN gene product is a multiple-specificity phosphatase that antagonises PI3K by degrading PI(3,4,5)P3 back to PI(4,5)P2 [8] in addition to its ability to dephosphorylate protein targets such as focal adhesion kinase (FAK) [9]. Many reports have implicated reduction or loss of PTEN activity as causal in the constitutive activation of PKB in tumour cells. However, a second group of enzymes, the SHIP (SH2-containing inositolphosphatase) family, has been identified as also being potentially important in regulating PKB through degradation of PI(3,4,5)P3 to PI(3,4)P2 [10], [11]. SHIP1 expression is largely confined to the haematopoietic system and sSHIP to stem cells, but SHIP2 is ubiquitously expressed [12], [13] and may act as an alternative or additional mechanism for antagonising PI3K both in the presence and the absence of PTEN. Although the PH domain of PKB can bind to PI(3,4)P2, the demonstration that SHIP2 as well as PTEN can downregulate PKB activation indicates that PI(3,4,5)P3 rather than either PI(3,4)P2 or PI(4,5)P2 is the critical phospholipid involved in assembling the PKB activation complex at the plasma membrane [14].
PTEN inactivation has been widely implicated in progression to metastasis of prostate cancer, with approximately 60% of late-stage prostate tumours showing loss of PTEN function [15], [16]. Haploinsufficiency of the PTEN gene has been shown to promote prostate cancer progression in a transgenic mouse model [17]. In one of the first papers describing PTEN, two of the model cell lines for metastatic prostate disease, LNCaP and PC3, were shown to lack expression of the protein: LNCaP has a two-base pair deletion in codon 6, while PC3 cells have a deletion at the 3′ end of the gene [18], [19]. These cell lines have been widely used as models for the behaviour of PTEN-null cells; in particular, the high level of activated PKB observed in LNCaP and PC3 cells has been ascribed to PIP3 accumulation resulting from failure to degrade this compound due to absence of the lipid phosphatase activity of PTEN [20], [21]. Deregulation of the PI3K–PKB signalling axis can clearly contribute to survival and deregulated proliferation in tumorigenesis and cancer metastasis [1], [2]. However, the significance of loss of PTEN in promoting this deregulation will depend on whether PTEN is the only lipid phosphatase responsible for the negative control of this pathway in a given cell type, or whether additional PIP3-degrading enzymes function to regulate PKB activity alongside PTEN or in its absence.
The aim of the present study is to determine whether PTEN shares the role of antagonist to PI3K in the regulation of PKB phosphorylation with other PIP3-degrading enzymes, including SHIP2, in cell lines derived from human prostatic epithelium. We also investigate the extent to which alternative mechanisms for the breakdown of PIP3 can substitute for PTEN in regulating PKB in the PTEN-null model prostate cancer cell lines LNCaP and PC3.
Section snippets
Cell lines
PNT1a and PNT2 [22], [23] are non-tumorigenic epithelial cell lines derived by SV40 immortalisation of normal prostate epithelial outgrowths. P4E6 is a cell line derived from an early stage carcinoma of prostate by immortalisation with the E6 gene of human papillomavirus 16 [24]. LNCaP and PC3 are established prostatic carcinoma cell lines with androgen-dependent and androgen-independent phenotypes respectively, obtained from American Type Culture Collection. PNT1a, PNT2, P4E6 and LNCaP cells
Sensitivity of prostatic epithelial cell lines to serum deprivation and PI3K inhibition
PNT2 and PNT1a (non-tumour prostate epithelium) and P4E6 (early prostate tumour) cells express wild-type PTEN [19]. The prostate tumour cell lines LNCaP (from lymph node metastasis) and PC3 (from bone metastasis) lack wild-type PTEN [18], [19]. When grown in medium containing serum, all of these cells showed PKB activation as evidenced by ser473 phosphorylation (Fig. 1, lane 1). In PNT2, PNT1a, P4E6 and PC3, treatment with the PI3K inhibitor LY294002 (10 μM) for 15 min resulted in almost
Discussion
The cell lines LNCaP and PC3 have been extensively used as model systems of metastatic prostate cancer and as examples of PTEN-null cells. Indeed, it is often assumed that PKB in these cell lines is constitutively activated as a result of PTEN abrogation and concomitant effects of deregulated synthesis of PIP3. In this paper we show clear differences between LNCaP and PC3 cells in terms of their regulation of the PI3K/PKB pathway. While LNCaP cells show a genuine reduction in ability to degrade
Acknowledgements
This work was supported by Yorkshire Cancer Research (Harrogate, United Kingdom) and the United Kingdom National Cancer Research Institute.
We thank Professor Peter Downes of the University of Dundee for his generous gift of the GRP1PH(ΔNLS)EGFP expression plasmid. We are also indebted to Dr. Martin Rumsby of the University of York for discussion of the results and critical reading of the manuscript.
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