Membrane Morphology and Function
Signalling specificity in the Akt pathway in breast cancer Abbe R. Clark* and Alex Toker*1 *Department of Pathology and Cancer Center, Beth Israel Deaconess Medical Center, Boston, MA 02215, U.S.A.
Biochemical Society Transactions
www.biochemsoctrans.org
Abstract Aberrant activation of fundamental cellular processes, such as proliferation, migration and survival, underlies the development of numerous human pathophysiologies, including cancer. One of the most frequently hyperactivated pathways in cancer is the phosphoinositide 3-kinase (PI3K)/Akt signalling cascade. Three isoforms of the serine/threonine protein kinase Akt (Akt1, Akt2 and Akt3) function to regulate cell survival, growth, proliferation and metabolism. Strikingly, non-redundant and even opposing functions of Akt isoforms in the regulation of phenotypes associated with malignancy in humans have been described. However, the mechanisms by which Akt isoform-specificity is conferred are largely unknown. In the present review, we highlight recent findings that have contributed to our understanding of the complexity of Akt isoform-specific signalling and discussed potential mechanisms by which this isoform-specificity is conferred. An understanding of the mechanisms of Akt isoform-specificity has important implications for the development of isoform-specific Akt inhibitors and will be critical to finding novel targets to treat disease.
Introduction The serine/threonine protein kinase Akt, also known as protein kinase B (PKB), plays a key role in signalling downstream of growth factors and other stimuli, regulating critical cellular functions, including proliferation and survival [1]. Aberrant hyperactivation of Akt, due to signalling pathway activation, gene amplification or somatic mutation, is frequently associated with human pathophysiologies, including cancer. In physiological conditions, Akt activation is initiated by receptor tyrosine kinase-induced activation of phosphoinositide 3-kinase (PI3K) leading to the synthesis of the second messengers phosphatidylinositol 3,4,5-trisphosphate (PIP3) and phosphatidylinositol 3,4bisphosphate (PIP2) [2,3]. Both PIP3 and PIP2 bind to the pleckstrin homology (PH) domain of Akt, recruiting the inactive enzyme to the plasma membrane to initiate activation via phosphorylation of two highly conserved residues: Thr308 in the activation loop mediated by the phosphoinositide-dependent kinase 1 (PDK-1) [4,5] and Ser473 in the hydrophobic motif mediated by the mammalian target of rapamycin complex 2 (mTORC2) [6]. Termination of Akt signalling is tightly controlled and accomplished in part by the 3 lipid phosphatase phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and the Key words: Akt, breast cancer, cancer, phosphoinositide 3-kinase (PI3K), phosphorylation, signalling, substrate. Abbreviations: EMT, epithelial to mesenchymal transition; FOXO, forkhead box O; GSK3, glycogen synthase kinase 3; IGF-1, insulin-like growth factor 1; INPP4B, inositol polyphosphate 4phosphatase type II; MEF, mouse embryonic fibroblast; mTORC2, mammalian target of rapamycin complex 2; PDK-1, phosphoinositide-dependent kinase 1; PH, pleckstrin homology; PHLPP, PH domain leucine-rich repeat protein phosphatase; PI3K, phosphoinositide 3-kinase; PIP2, phosphatidylinositol 3,4-bisphosphate; PIP3, phosphatidylinositol 3,4,5-trisphosphate, protein kinase B; PP2A, protein phosphatase 2A; PTEN, phosphatase and tensin homologue deleted on chromosome 10; TNBC, triple-negative breast cancer; TSC2, tuberous sclerosis complex 2. 1 To whom correspondence should be addressed (email [email protected]).
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4 lipid phosphatase inositol polyphosphate 4-phosphatase type II (INPP4B), which dephosphorylate PIP3 and PIP2 respectively [7,8] (Figure 1). In addition, Akt is directly dephosphorylated at Thr308 and Ser473 by protein phosphatase 2A (PP2A) [9] and the PH domain leucine-rich repeat protein phosphatases 1 and 2 (PHLPP1/2) respectively [9,10] Activating mutations in PIK3CA, the gene encoding the p110α subunit of PI3K, and loss-of-function mutations or loss of heterozygosity (LOH) of PTEN occur with high frequency in breast cancer across all molecular subtypes [11]. In addition, INPP4B has previously been identified as a tumour suppressor in basal-like breast tumours [8,12]. In humans, the three Akt isoforms (Akt1, Akt2 and Akt3) are derived from unique genes on separate chromosomes. The canonical Akt primary structure comprises the following four basic modules or domains: PH domain, linker region, catalytic domain and C-terminal regulatory region (Figure 2). Although the PH, catalytic and regulatory domains share a high degree of homology among Akt isoforms, the linker domain diverges significantly across Akt1, Akt2 and Akt3. Importantly, mouse studies using individual knockout of the three isoforms originally suggested non-redundant functions of Akt proteins [13–19]. In support of specificity as opposed to redundancy, studies in cell culture have revealed isoform-specific and even opposing functions of Akt isoforms in cellular phenotypes commonly deregulated in malignancies, in particular cell migration and metastatic dissemination [20–22]. These studies have been corroborated by genetically engineered mouse models, revealing that whereas Akt1 can function as a suppressor of invasive migration leading to metastasis, at least in the context of breast cancer, Akt2 functions to enhance these phenotypes [23,24]. C The
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Figure 1 Regulation of Akt activation Receptor tyrosine kinase-induced PI3K activation leads to synthesis of the second messengers PIP3 and PIP2. Via binding to the PH domain, PIP3 and PIP2 recruit inactive Akt to the plasma membrane. Here, activation is initiated by phosphorylation of two highly conserved residues, Thr308 and Ser473 , mediated by PDK-1 and mTORC2 respectively. Phosphatases terminate Akt signalling both by removing PIP3 and PIP2 from the plasma membrane, which is accomplished by PTEN and INPP4B respectively, and by directly dephosphorylating Akt at Thr308 (PP2A) and Ser473 (PHLPP1/2). Three Akt isoforms exist in mammals, Akt1, Akt2 and Akt3, and upon activation transduce the signal by directly phosphorylating substrates such as FOXO transcription factors, murine double minute 2 (MDM2), GSK3β and others. Phosphorylation of these substrates by Akt serves to either activate or inactivate their function, leading to alterations in the phenotypes depicted.
Figure 2 Homology between Akt isoforms Akt isoforms can be divided into the following four domains: PH domain, linker region, catalytic domain and C-terminal regulatory region. The homology between domains of Akt isoforms is depicted below the primary structure comparisons of Akt1/2, Akt1/3 and Akt2/3.
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The majority of the studies investigating isoform-specific functions of Akt signalling in the context of cancer progression have largely been limited to Akt1 and Akt2. These studies have been reviewed extensively, and we direct readers to several previous reviews [25–27]. By contrast, specific roles for Akt3 in modulating phenotypes associated with malignancy have thus far been largely ignored. Furthermore, the majority of studies describing isoform-specific functions of Akt in either cell culture models or mouse models have been observational, and few studies have provided a mechanistic basis by which Akt isoformspecificity is achieved. Although Akt isoform-specific inhibitors have yet to be developed for clinical evaluation, a number of catalytic as well as allosteric pan-Akt inhibitors have been developed and are currently in clinical trials. Recent work describing isoformspecific roles of Akt in breast cancer suggests that targeting all Akt isoforms may result in unwanted on-target effects. Therefore, in order for isoform-specific Akt inhibitors to be a viable therapeutic approach, a comprehensive understanding of distinct isoform-specific roles of Akt proteins is essential. In the present article, we discuss recent studies that have contributed to a greater appreciation of the complexities of isoform-specific Akt signalling in breast cancer, including isoform-specific roles of Akt3 in breast cancer models and discussed potential mechanisms by which Akt isoformspecificity may be conferred.
Isoform-specific roles of Akt in breast cancer As introduced above, the opposing functions of Akt1 and Akt2 in the context of breast cancer have been reviewed extensively elsewhere [25–27]. More recently, however, isoform-specific functions of Akt3 in breast cancer have been described. In the present article, we summarize new findings of Akt isoform-specificity in breast cancer and described recent efforts that have examined isoform-specific functions of Akt in breast cancer that are contributing to our growing awareness of the complexities of isoform-specific Akt signalling.
Akt1 and Akt2 in breast cancer Irie et al. [20] initially demonstrated that although Akt1 knockdown enhances migration and induction of an epithelial to mesenchymal transition (EMT) in human mammary epithelial cells expressing insulin-like growth factor 1 (IGF1) receptor, Akt2 knockdown reverses hyperproliferation of the same cells in growing 3D culture. Our own laboratory confirmed that Akt1 suppresses breast cancer cell invasion and migration, as measured by invasion through Matrigel or migration towards 3T3-conditioned medium [22,28,29]. In addition, Akt2 enhances breast cancer cell migration, since knockdown with isoform-specific siRNA inhibits migration [22]. Similar findings were subsequently made by the Bissell laboratory [30].
In support of these initial in vitro findings, in vivo models of ErbB2-mediated mammary tumorigenesis confirmed the opposing function of Akt1 and Akt2 [23,31,32]. Although co-expression of activated Akt1 and ErbB2 in mammary epithelium of mice accelerates tumour formation, it prevents metastasis when compared with expression of activated ErbB2 alone [23]. However, although co-expression of activated Akt2 and ErbB2 does not affect induction of tumour formation, it significantly increases lung metastases compared with expression of Akt1 and ErbB2 or ErbB2 alone [32]. Moreover, it has recently been appreciated that specific PI3K/Akt pathway mutations signal through individual Akt isoforms. Our laboratory recently described a unique role for Akt2 in mediating survival and maintenance of PTENdeficient cancers, including prostate, glioblastoma and breast [33]. Using a doxycycline-inducible Akt isoform-specific shRNA system, we have evaluated isoform-specific roles of Akt in PTEN-deficient tumour lines and found that whereas silencing all three isoforms prevents 3D spheroid formation in vitro, Akt2 is specifically required for 3D spheroid maintenance in MDA-MB468 cells and BT-549 cells. Moreover, mechanistic studies indicate that the cell cycle regulator p21 is at least one critical downstream effector of Akt2 in the context of PTEN inactivation.
A novel role for Akt3 in breast cancer Although non-redundant functions of Akt1 and Akt2 have been appreciated for some time, an isoform-specific role for Akt3 has only recently been described. We have recently identified a function of Akt3 in basal-like triple-negative breast cancer (TNBC) [22]. Analysis of The Cancer Genome Atlas reveals that Akt3 is preferentially amplified and overexpressed in TNBC tumours and cell lines. Therefore, to assess the function of individual Akt isoforms, we have used a doxycycline-inducible Akt isoform-specific shRNA system. Similar to Akt1 silencing, Akt3 silencing enhances breast cancer cell migration in MCF10DCIS, MDA-MB468 and BT-549 cells. We have also found that silencing of Akt3 in MDA-MB231 cells inhibits 3D spheroid growth, whereas neither Akt1 nor Akt2 had significant effects. Furthermore, Akt3 silencing in two xenograft models attenuates tumour growth in vivo. An isoform-specific function of Akt3 in Balb-neuT mice, a transgenic model of ErbB2-breast cancer, has also recently been proposed [34]. Grabinski et al. [34] evaluated the in vitro kinase activity of Akt isoforms in BalbneuT breast tumours and found that Akt3 has higher in vitro kinase activity than Akt1 or Akt2. In addition, Akt1 and Akt3 silencing more dramatically reduces proliferation of Balb-neuT mammary tumour-derived cells compared with Akt2 silencing. Furthermore, Akt3 knockdown, but not Akt1 or Akt2 knockdown, leads to decreased ErbB2 and ErbB3 expression; increased ERα expression and Akt3 knockdown also sensitizes Balb-neuT mammary tumour-derived cells to tamoxifen treatment. These studies highlight a previously unappreciated role for Akt3 in mediating breast cancer progression, especially in certain molecular subtypes, such as TNBC, and indicate that Akt3-specific inhibitors may be C The
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particularly beneficial for these patient populations for which there is an unmet need in terms of targeted therapy.
of Akt phosphorylation may confer isoform-specificity. However, the potential for this mechanism to contribute to Akt isoform-specific signalling in cancer pathophysiology remains to be determined.
Mechanisms of isoform-specificity Despite the discovery that Akt isoforms have nonredundant functions, mechanisms by which this isoformspecificity is conferred remain unclear. Isoform-specific signalling can be conveyed through various mechanisms: regulation of kinase activity, post-translational modifications, substrate specificity or subcellular localization. Below we have proposed mechanisms by which Akt isoform-specific functions may be achieved and discussed recent advances made in this area.
Isoform-specific activation/deactivation One mechanism by which isoform-specific signalling could be mediated is by differential activation or deactivation of Akt isoforms. In the canonical pathway, both Thr308 and Ser473 are required for full activation [2,35,36]. However, both Thr308 and Ser473 phosphorylation may not be required for productive phosphorylation of all Akt substrates. For example, using a pan-Akt consensus motif substrate antibody, Jacinto et al. [37] reported that deletion of the mTORC2 subunit Sin1 in mouse embryonic fibroblasts (MEFs), which abolishes Ser473 phosphorylation, does not affect the phosphorylation of the majority of Akt substrates. Specifically, Ser473 phosphorylation is required for forkhead box O (FOXO) 1/3a phosphorylation, but dispensable for glycogen synthase kinase 3 (GSK3) α/β or tuberous sclerosis complex 2 (TSC2) phosphorylation [37]. There is also in vivo evidence suggesting that phosphorylation of one, but not both, conserved Akt phosphorylation site is important for the downstream phosphorylation of only a subset of Akt substrates. Vincent et al. [38] demonstrated that phosphorylation of proline-rich Akt substrate of 40 kDa (PRAS40), TBC (Tre-2/Bub2/Cdc16) 1 domain family, member 4 (TBC1D4) and TSC2 correlates with phosphorylation of Akt on Thr308 , but not Ser473 , in human non-small-cell lung tumours. Differential phosphorylation of Thr308 and Ser473 of Akt isoforms may thus influence phosphorylation of Akt substrates, thereby conferring isoform-specificity. The isoform-selectivity of this potential mechanism has yet to be explored. Termination of Akt activity is in part regulated by the phosphatases PP2A [9] and PHLPP1/2 [9,10]. Differential termination of Akt activity by PHLPP isoforms has been suggested [10], such that silencing of PHLPP1 leads to an increase in both Akt2 and Akt3 phosphorylation, whereas silencing of PHLPP2 leads to an increase in both Akt1 and Akt3 phosphorylation. Furthermore, silencing of PHLPP isoforms leads to differential activation of Akt substrates. Although silencing of PHLPP1, but not PHLPP2, leads to an increase in human double minute 2 (HDM2) and GSK3α phosphorylation, and silencing of PHLPP2, but not PHLPP1, leads an increase in FOXO1 phosphorylation at Thr24 . These results indicate that differential termination C The
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Post-translational modifications Post-translational modifications that contribute differentially to Akt isoform-specific activation, localization or substrate phosphorylation constitute one potential mechanism that accounts for specificity. Numerous Akt post-translational modifications have been identified in global MS screens, although the functional significance of many of these has not been determined [39]. In addition to Thr308 and Ser473 phosphorylation, ubiquitination of Akt is required for full activation [40–42]. Tumour-necrosis-factor-receptorassociated factor 6 (TRAF6) and Skp2 directly ubiquitinate Akt at Lys8 and Lys14 in response to IGF-1 and epidermal growth factor (EGF) respectively [40,42]. Although isoformspecific ubiquitination of Akt has not been demonstrated, isoform-specific oxidation of Akt2 has been suggested. Wani et al. [43] reported that platelet-derived growth factor (PDGF) stimulation specifically induces Akt2, but not Akt1, oxidation. Cys124 , located in the linker region of Akt2, is not conserved in Akt1 or Akt3 and serves as the major site of redox-sensitivity in Akt2. This oxidation results in decreased Akt2, but not Akt1, activity. Furthermore, this regulation of Akt2 activity contributes to a cell migration phenotype [44]. These results highlight the potential for isoform-specific post-translational modifications to play an important role in Akt isoform-specific signalling. Additional sites for posttranslational modifications are likely to exist and will have to be evaluated as a mechanism for achieving selectivity in Akt signalling.
Isoform-specific substrates As suggested above, Akt isoform-specific phosphorylation of substrates represents a key mechanism by which selectivity is achieved in the context of pathophysiology. A recent evaluation of the phosphoproteomes of Akt triple-knockout lung fibroblasts and their single Akt isoform-expressing derivatives revealed significant isoform-specific differences [45]. This study also identified a large number of novel, putative isoform-specific substrates to be validated in future studies. The majority of Akt substrates described in the literature are phosphorylated by all Akt isoforms [27]. Although Akt3-specific substrates have not been described, Akt1 and Akt2 isoform-specific substrates have recently been identified (Table 1). Palladin was identified by our laboratory as a novel Akt1-specific substrate [29]. We have demonstrated that Akt1, but not Akt2, phosphorylates palladin in vitro and that Akt1, but not Akt2, knockdown results in reduced palladin phosphorylation in cell culture. Furthermore, Akt1-specific palladin phosphorylation regulates breast cell migration, connecting isoform-specific substrate phosphorylation with an isoform-specific cancer cell phenotype. Isoform-specificity for many of the proposed substrates has not been rigorously
Membrane Morphology and Function
Table 1 Proposed isoform-specific substrates of Akt Akt1- and Akt2-specific substrates have been identified in published studies. Akt3-specific substrates have yet to be described. Ankrd2, ankyrin repeat domain 2; ARRP, ankyrin repeat domain 2 protein; AS160, Akt substrate of 160 kDa; HDM2, human double minute 2.
Substrate
Isoform-specific Akt isoform Assays used to demonstrate isoform-specificity phosphorylation responsible for site phosphorylation In vitro Cell culture Site-directed mutagenesis? Reference(s)
p21
Thr145
Akt1
Akt1/2 kinase assays Myr-Akt1
Yes, in vitro and in vivo
[51,52]
Skp2 Palladin AS160
Ser72 Ser507 Ser588 , Thr642
Akt1 Akt1 Akt2
Akt1/2 kinase assays Myr-Akt1/2 Akt1/2 kinase assays Akt1/2 shRNA None Membrane-targeted
Yes, in vivo Yes, in vivo No
[53,54] [29] [49,55]
No
[10]
Yes, in vitro Yes, in vitro
[56] [57]
HDM2
Ser166
Myosin5a Ser1650 Ankrd2/ARRP Ser99
Akt2 construct; Akt1/2 siRNA Akt1/2/3 siRNA
Akt2
None
Akt2 Akt2
Akt2 kinase assay Akt1/2 siRNA Akt1/2 kinase assays Akt1/2 siRNA
tested with in vivo analyses. In addition, Akt2- or Akt3specific substrates that account for specific cancer phenotypes have yet to be described.
miRNA expression Numerous studies in recent years have demonstrated the important roles of non-coding RNAs in cancer [46]. A recent study from the Tsichlis laboratory has demonstrated that differential regulation of miRNA expression by Akt isoforms constitutes one mechanism of Akt isoform-specificity in cancer [47]. Iliopoulos et al. [47] screened the miRNA expression signatures of lung fibroblasts, expressing single Akt isoforms using a 365 miRNA array. IGF-1 stimulation induced notable differences in the miRNA expression signatures of the single-Akt isoform expressing cells. miR200 family members miR-200a and miR-200c were downregulated in Akt2-expressing MEFs, but not those expressing Akt1 or Akt3. Knockdown of Akt1, but not Akt2, with isoform-specific siRNA promoted EMT and decreased the abundance of miR-200a and miR-200c; overexpression of miR-200a or miR-200c inhibited cell migration in Akt1silenced MCF10A cells. Furthermore, analysis of Akt1 and Akt2 mRNA levels and miR-200a and miR-200c levels in a small subset of human primary and metastatic breast cancer tumour samples revealed a correlation between lower Akt1/Akt2 ratios and decreased miR-200a and miR-200c levels. It remains to be determined whether these findings can be extended to analysis of a larger group of breast cancer samples. In addition, it will be of interest to determine the specific contexts in which Akt isoform-specificity is conferred by regulation of non-coding RNA expression.
Localization Differential subcellular localization of Akt isoforms would enable isoform-specific access to the plasma membrane for activation and interactions with substrates or chaperone proteins. One study has shown that Akt isoforms have distinct localization patterns when comparing non-transformed
and transformed breast cancer cell lines [48]. Subcellular localization of Akt2 has been proposed as a mechanism for Akt2-specific regulation of trafficking the glucose transporter type 4 (GLUT4) [49]. However, the precise contribution of subcellular localization to Akt isoform-specific mechanisms of breast cancer progression remains to be elucidated.
Molecular determinants of Akt isoform-specificity Implicit in the mechanisms of isoform-specificity suggested above is the notion that Akt isoforms contain intrinsic molecular differences in the primary structure. Recent studies using Akt chimaeric constructs comprising swapped Akt1 and Akt2 domains demonstrated that the PH and/or linker domains distinguish Akt1 from Akt2, at least with respect to the phenotype of cell migration [29,50]. In this context, it has been shown that Akt1-specific palladin phosphorylation requires the linker domain of Akt1. The lack of an Akt3specific phenotype, until recently, or Akt3-specific substrates has limited the use of chimaeras to examining the domains of Akt1 and Akt2 required for isoform-specific functions.
Perspectives Although much is known regarding the mechanisms of PI3K and Akt activation and in turn how signal relay through this pathway modulates phenotypes associated with malignancy, there is a considerable paucity of knowledge regarding the elements that distinguish Akt isoform signalling. It is likely that context-specificity contributes significantly to Akt isoform-specific signalling that have been reported in cellbased experiments as well as in mouse studies. This notion is supported by the addiction to specific Akt isoforms in pathway-mutant driven cancers, particularly breast cancer. However, the precise basis by which individual isoforms of Akt are differentially required remains obscure. Furthermore, the identification of Akt isoform-specific substrates suggests C The
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that there exist molecular determinants that distinguish Akt isoforms and that contribute to isoform-specific functions in vivo. A major challenge in the field remains the identification of the molecular determinants of Akt isoform-specificity, which is predicted to have far-reaching implications especially with respect to the development of isoform-selective inhibitors. This would not only provide much needed tool compounds to more precisely study isoform-specific Akt signalling and biology, but also provide a potential therapeutic basis to regulate Akt pathway activation.
Acknowledgements We thank all past and present members of the Toker laboratory.
Funding Research in our laboratory is supported in part by the National Institutes of Health, the Susan G. Komen Foundation for the Cure and the Department of Defense Breast Cancer Research Program.
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11 Miller, T.W., Rexer, B.N., Garrett, J.T. and Arteaga, C.L. (2011) Mutations in the phosphatidylinositol 3-kinase pathway: role in tumor progression and therapeutic implications in breast cancer. Breast Cancer Res. 13, 224 CrossRef PubMed 12 Fedele, C.G., Ooms, L.M., Ho, M., Vieusseux, J., O’Toole, S.A., Millar, E.K., Lopez-Knowles, E., Sriratana, A., Gurung, R., Baglietto, L. et al. (2010) Inositol polyphosphate 4-phosphatase II regulates PI3K/Akt signaling and is lost in human basal-like breast cancers. Proc. Natl. Acad. Sci. U.S.A. 107, 22231–22236 CrossRef PubMed 13 Chen, W.S., Xu, P.Z., Gottlob, K., Chen, M.L., Sokol, K., Shiyanova, T., Roninson, I., Weng, W., Suzuki, R., Tobe, K. et al. (2001) Growth retardation and increased apoptosis in mice with homozygous disruption of the Akt1 gene. Genes Dev. 15, 2203–2208 CrossRef PubMed 14 Cho, H., Thorvaldsen, J.L., Chu, Q., Feng, F. and Birnbaum, M.J. (2001) Akt1/PKBα is required for normal growth but dispensable for maintenance of glucose homeostasis in mice. J. Biol. Chem. 276, 38349–38352 CrossRef PubMed 15 Cho, H., Mu, J., Kim, J.K., Thorvaldsen, J.L., Chu, Q., Crenshaw, III, E.B., Kaestner, K.H., Bartolomei, M.S., Shulman, G.I. and Birnbaum, M.J. (2001) Insulin resistance and a diabetes mellitus-like syndrome in mice lacking the protein kinase Akt2 (PKBβ). Science 292, 1728–1731 CrossRef PubMed 16 Garofalo, R.S., Orena, S.J., Rafidi, K., Torchia, A.J., Stock, J.L., Hildebrandt, A.L., Coskran, T., Black, S.C., Brees, D.J., Wicks, J.R. et al. (2003) Severe diabetes, age-dependent loss of adipose tissue, and mild growth deficiency in mice lacking Akt2/PKBβ. J. Clin. Invest. 112, 197–208 CrossRef PubMed 17 Easton, R.M., Cho, H., Roovers, K., Shineman, D.W., Mizrahi, M., Forman, M.S., Lee, V.M., Szabolcs, M., de Jong, R., Oltersdorf, T. et al. (2005) Role for Akt3/protein kinase Bγ in attainment of normal brain size. Mol. Cell. Biol. 25, 1869–1878 CrossRef PubMed 18 Schultze, S.M., Jensen, J., Hemmings, B.A., Tschopp, O. and Niessen, M. (2011) Promiscuous affairs of PKB/AKT isoforms in metabolism. Arch. Physiol. Biochem. 117, 70–77 CrossRef PubMed 19 Dummler, B. and Hemmings, B.A. (2007) Physiological roles of PKB/Akt isoforms in development and disease. Biochem. Soc. Trans. 35, 231–235 CrossRef PubMed 20 Irie, H.Y., Pearline, R.V., Grueneberg, D., Hsia, M., Ravichandran, P., Kothari, N., Natesan, S. and Brugge, J.S. (2005) Distinct roles of Akt1 and Akt2 in regulating cell migration and epithelial–mesenchymal transition. J. Cell Biol. 171, 1023–1034 CrossRef PubMed 21 Chin, Y.R. and Toker, A. (2009) Function of Akt/PKB signaling to cell motility, invasion and the tumor stroma in cancer. Cell. Signal. 21, 470–476 CrossRef PubMed 22 Chin, Y.R., Yoshida, T., Marusyk, A., Beck, A.H., Polyak, K. and Toker, A. (2014) Targeting Akt3 signaling in triple-negative breast cancer. Cancer Res. 74, 964–973 CrossRef PubMed 23 Hutchinson, J.N., Jin, J., Cardiff, R.D., Woodgett, J.R. and Muller, W.J. (2004) Activation of Akt-1 (PKB-α) can accelerate ErbB-2-mediated mammary tumorigenesis but suppresses tumor invasion. Cancer Res. 64, 3171–3178 CrossRef PubMed 24 Arboleda, M.J., Lyons, J.F., Kabbinavar, F.F., Bray, M.R., Snow, B.E., Ayala, R., Danino, M., Karlan, B.Y. and Slamon, D.J. (2003) Overexpression of AKT2/protein kinase Bβ leads to up-regulation of β1 integrins, increased invasion, and metastasis of human breast and ovarian cancer cells. Cancer Res. 63, 196–206 PubMed 25 Gonzalez, E. and McGraw, T.E. (2009) The Akt kinases: isoform specificity in metabolism and cancer. Cell Cycle 8, 2502–2508 CrossRef PubMed 26 Chin, Y.R. and Toker, A. (2011) Akt isoform-specific signaling in breast cancer: uncovering an anti-migratory role for palladin. Cell Adh. Migr. 5, 211–214 CrossRef PubMed 27 Toker, A. (2012) Achieving specificity in Akt signaling in cancer. Adv. Biol. Regul. 52, 78–87 CrossRef PubMed 28 Yoeli-Lerner, M. and Toker, A. (2006) Akt/PKB signaling in cancer: a function in cell motility and invasion. Cell Cycle 5, 603–605 CrossRef PubMed 29 Chin, Y.R. and Toker, A. (2010) The actin-bundling protein palladin is an Akt1-specific substrate that regulates breast cancer cell migration. Mol. Cell 38, 333–344 CrossRef PubMed 30 Liu, H., Radisky, D.C., Nelson, C.M., Zhang, H., Fata, J.E., Roth, R.A. and Bissell, M.J. (2006) Mechanism of Akt1 inhibition of breast cancer cell invasion reveals a protumorigenic role for TSC2. Proc. Natl. Acad. Sci. U.S.A. 103, 4134–4139 CrossRef PubMed
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31 Maroulakou, I.G., Oemler, W., Naber, S.P. and Tsichlis, P.N. (2007) Akt1 ablation inhibits, whereas Akt2 ablation accelerates, the development of mammary adenocarcinomas in mouse mammary tumor virus (MMTV)-ErbB2/neu and MMTV-polyoma middle T transgenic mice. Cancer Res. 67, 167–177 CrossRef PubMed 32 Dillon, R.L., Marcotte, R., Hennessy, B.T., Woodgett, J.R., Mills, G.B. and Muller, W.J. (2009) Akt1 and Akt2 play distinct roles in the initiation and metastatic phases of mammary tumor progression. Cancer Res. 69, 5057–5064 CrossRef PubMed 33 Chin, Y.M., Yuan, X., Balk, S.P. and Toker, A. (2014) PTEN-deficient tumors depend on AKT2 for maintenance and survival. Cancer Discov. 4, 942–955 CrossRef PubMed 34 Grabinski, N., Mollmann, K., Milde-Langosch, K., Muller, V., Schumacher, U., Brandt, B., Pantel, K. and Jucker, M. (2014) AKT3 regulates ErbB2, ErbB3 and estrogen receptor α expression and contributes to endocrine therapy resistance of ErbB2 + breast tumor cells from Balb-neuT mice. Cell. Signal. 26, 1021–1029 CrossRef PubMed 35 Hart, J.R. and Vogt, P.K. (2011) Phosphorylation of AKT: a mutational analysis. Oncotarget 2, 467–476 PubMed 36 Alessi, D.R., Andjelkovic, M., Caudwell, B., Cron, P., Morrice, N., Cohen, P. and Hemmings, B.A. (1996) Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J. 15, 6541–6551 PubMed 37 Jacinto, E., Facchinetti, V., Liu, D., Soto, N., Wei, S., Jung, S.Y., Huang, Q., Qin, J. and Su, B. (2006) SIN1/MIP1 maintains rictor–mTOR complex integrity and regulates Akt phosphorylation and substrate specificity. Cell 127, 125–137 CrossRef PubMed 38 Vincent, E.E., Elder, D.J., Thomas, E.C., Phillips, L., Morgan, C., Pawade, J., Sohail, M., May, M.T., Hetzel, M.R. and Tavare, J.M. (2011) Akt phosphorylation on Thr308 but not on Ser473 correlates with Akt protein kinase activity in human non-small cell lung cancer. Br. J. Cancer 104, 1755–1761 CrossRef PubMed 39 Moritz, A., Li, Y., Guo, A., Villen, J., Wang, Y., MacNeill, J., Kornhauser, J., Sprott, K., Zhou, J., Possemato, A. et al. (2010) Akt–RSK–S6 kinase signaling networks activated by oncogenic receptor tyrosine kinases. Sci. Signal. 3, ra64 CrossRef PubMed 40 Yang, W.L., Wang, J., Chan, C.H., Lee, S.W., Campos, A.D., Lamothe, B., Hur, L., Grabiner, B.C., Lin, X., Darnay, B.G. and Lin, H.K. (2009) The E3 ligase TRAF6 regulates Akt ubiquitination and activation. Science 325, 1134–1138 CrossRef PubMed 41 Yang, W.L., Wu, C.Y., Wu, J. and Lin, H.K. (2010) Regulation of Akt signaling activation by ubiquitination. Cell Cycle 9, 487–497 PubMed 42 Chan, C.H., Li, C.F., Yang, W.L., Gao, Y., Lee, S.W., Feng, Z., Huang, H.Y., Tsai, K.K., Flores, L.G., Shao, Y. et al. (2012) The Skp2-SCF E3 ligase regulates Akt ubiquitination, glycolysis, herceptin sensitivity, and tumorigenesis. Cell 149, 1098–1111 CrossRef PubMed 43 Wani, R., Qian, J., Yin, L., Bechtold, E., King, S.B., Poole, L.B., Paek, E., Tsang, A.W. and Furdui, C.M. (2011) Isoform-specific regulation of Akt by PDGF-induced reactive oxygen species. Proc. Natl. Acad. Sci. U.S.A. 108, 10550–10555 CrossRef PubMed 44 Wani, R., Bharathi, N.S., Field, J., Tsang, A.W. and Furdui, C.M. (2011) Oxidation of Akt2 kinase promotes cell migration and regulates G1 –S transition in the cell cycle. Cell Cycle 10, 3263–3268 CrossRef PubMed
45 Sanidas, I., Polytarchou, C., Hatziapostolou, M., Ezell, S.A., Kottakis, F., Hu, L., Guo, A., Xie, J., Comb, M.J., Iliopoulos, D. and Tsichlis, P.N. (2014) Phosphoproteomics screen reveals akt isoform-specific signals linking RNA processing to lung cancer. Mol. Cell 53, 577–590 CrossRef PubMed 46 Sana, J., Faltejskova, P., Svoboda, M. and Slaby, O. (2012) Novel classes of non-coding RNAs and cancer. J. Transl. Med. 10, 103 CrossRef PubMed 47 Iliopoulos, D., Polytarchou, C., Hatziapostolou, M., Kottakis, F., Maroulakou, I.G., Struhl, K. and Tsichlis, P.N. (2009) MicroRNAs differentially regulated by Akt isoforms control EMT and stem cell renewal in cancer cells. Sci. Signal. 2, ra62 CrossRef PubMed 48 Santi, S.A. and Lee, H. (2010) The Akt isoforms are present at distinct subcellular locations. Am. J. Physiol. Cell Physiol. 298, C580–C591 CrossRef PubMed 49 Gonzalez, E. and McGraw, T.E. (2009) Insulin-modulated Akt subcellular localization determines Akt isoform-specific signaling. Proc. Natl. Acad. Sci. U.S.A. 106, 7004–7009 CrossRef PubMed 50 Zhou, G.L., Tucker, D.F., Bae, S.S., Bhatheja, K., Birnbaum, M.J. and Field, J. (2006) Opposing roles for Akt1 and Akt2 in Rac/Pak signaling and cell migration. J. Biol. Chem. 281, 36443–36453 CrossRef PubMed 51 Rossig, L., Jadidi, A.S., Urbich, C., Badorff, C., Zeiher, A.M. and Dimmeler, S. (2001) Akt-dependent phosphorylation of p21Cip1 regulates PCNA binding and proliferation of endothelial cells. Mol. Cell. Biol. 21, 5644–5657 CrossRef PubMed 52 Heron-Milhavet, L., Franckhauser, C., Rana, V., Berthenet, C., Fisher, D., Hemmings, B.A., Fernandez, A. and Lamb, N.J. (2006) Only Akt1 is required for proliferation, while Akt2 promotes cell cycle exit through p21 binding. Mol. Cell. Biol. 26, 8267–8280 CrossRef PubMed 53 Gao, D., Inuzuka, H., Tseng, A., Chin, R.Y., Toker, A. and Wei, W. (2009) Phosphorylation by Akt1 promotes cytoplasmic localization of Skp2 and impairs APCCdh1-mediated Skp2 destruction. Nat. Cell Biol. 11, 397–408 CrossRef PubMed 54 Lin, H.K., Wang, G., Chen, Z., Teruya-Feldstein, J., Liu, Y., Chan, C.H., Yang, W.L., Erdjument-Bromage, H., Nakayama, K.I., Nimer, S. et al. (2009) Phosphorylation-dependent regulation of cytosolic localization and oncogenic function of Skp2 by Akt/PKB. Nat. Cell Biol. 11, 420–432 CrossRef PubMed 55 Ng, Y., Ramm, G., Lopez, J.A. and James, D.E. (2008) Rapid activation of Akt2 is sufficient to stimulate GLUT4 translocation in 3T3-L1 adipocytes. Cell Metab. 7, 348–356 CrossRef PubMed 56 Yoshizaki, T., Imamura, T., Babendure, J.L., Lu, J.C., Sonoda, N. and Olefsky, J.M. (2007) Myosin 5a is an insulin-stimulated Akt2 (protein kinase Bβ) substrate modulating GLUT4 vesicle translocation. Mol. Cell. Biol. 27, 5172–5183 CrossRef PubMed 57 Cenni, V., Bavelloni, A., Beretti, F., Tagliavini, F., Manzoli, L., Lattanzi, G., Maraldi, N.M., Cocco, L. and Marmiroli, S. (2011) Ankrd2/ARPP is a novel Akt2 specific substrate and regulates myogenic differentiation upon cellular exposure to H2 O2 . Mol. Biol. Cell 22, 2946–2956 CrossRef PubMed
Received 30 May 2014 doi:10.1042/BST20140160
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Signalling specificity in the Akt pathway in breast cancer Abbe R. Clark* and Alex Toker*1 *Department of Pathology and Cancer Center, Beth Israel Deaconess Medical Center, Boston, MA 02215, U.S.A.
Biochemical Society Transactions
www.biochemsoctrans.org
Abstract Aberrant activation of fundamental cellular processes, such as proliferation, migration and survival, underlies the development of numerous human pathophysiologies, including cancer. One of the most frequently hyperactivated pathways in cancer is the phosphoinositide 3-kinase (PI3K)/Akt signalling cascade. Three isoforms of the serine/threonine protein kinase Akt (Akt1, Akt2 and Akt3) function to regulate cell survival, growth, proliferation and metabolism. Strikingly, non-redundant and even opposing functions of Akt isoforms in the regulation of phenotypes associated with malignancy in humans have been described. However, the mechanisms by which Akt isoform-specificity is conferred are largely unknown. In the present review, we highlight recent findings that have contributed to our understanding of the complexity of Akt isoform-specific signalling and discussed potential mechanisms by which this isoform-specificity is conferred. An understanding of the mechanisms of Akt isoform-specificity has important implications for the development of isoform-specific Akt inhibitors and will be critical to finding novel targets to treat disease.
Introduction The serine/threonine protein kinase Akt, also known as protein kinase B (PKB), plays a key role in signalling downstream of growth factors and other stimuli, regulating critical cellular functions, including proliferation and survival [1]. Aberrant hyperactivation of Akt, due to signalling pathway activation, gene amplification or somatic mutation, is frequently associated with human pathophysiologies, including cancer. In physiological conditions, Akt activation is initiated by receptor tyrosine kinase-induced activation of phosphoinositide 3-kinase (PI3K) leading to the synthesis of the second messengers phosphatidylinositol 3,4,5-trisphosphate (PIP3) and phosphatidylinositol 3,4bisphosphate (PIP2) [2,3]. Both PIP3 and PIP2 bind to the pleckstrin homology (PH) domain of Akt, recruiting the inactive enzyme to the plasma membrane to initiate activation via phosphorylation of two highly conserved residues: Thr308 in the activation loop mediated by the phosphoinositide-dependent kinase 1 (PDK-1) [4,5] and Ser473 in the hydrophobic motif mediated by the mammalian target of rapamycin complex 2 (mTORC2) [6]. Termination of Akt signalling is tightly controlled and accomplished in part by the 3 lipid phosphatase phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and the Key words: Akt, breast cancer, cancer, phosphoinositide 3-kinase (PI3K), phosphorylation, signalling, substrate. Abbreviations: EMT, epithelial to mesenchymal transition; FOXO, forkhead box O; GSK3, glycogen synthase kinase 3; IGF-1, insulin-like growth factor 1; INPP4B, inositol polyphosphate 4phosphatase type II; MEF, mouse embryonic fibroblast; mTORC2, mammalian target of rapamycin complex 2; PDK-1, phosphoinositide-dependent kinase 1; PH, pleckstrin homology; PHLPP, PH domain leucine-rich repeat protein phosphatase; PI3K, phosphoinositide 3-kinase; PIP2, phosphatidylinositol 3,4-bisphosphate; PIP3, phosphatidylinositol 3,4,5-trisphosphate, protein kinase B; PP2A, protein phosphatase 2A; PTEN, phosphatase and tensin homologue deleted on chromosome 10; TNBC, triple-negative breast cancer; TSC2, tuberous sclerosis complex 2. 1 To whom correspondence should be addressed (email [email protected]).
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4 lipid phosphatase inositol polyphosphate 4-phosphatase type II (INPP4B), which dephosphorylate PIP3 and PIP2 respectively [7,8] (Figure 1). In addition, Akt is directly dephosphorylated at Thr308 and Ser473 by protein phosphatase 2A (PP2A) [9] and the PH domain leucine-rich repeat protein phosphatases 1 and 2 (PHLPP1/2) respectively [9,10] Activating mutations in PIK3CA, the gene encoding the p110α subunit of PI3K, and loss-of-function mutations or loss of heterozygosity (LOH) of PTEN occur with high frequency in breast cancer across all molecular subtypes [11]. In addition, INPP4B has previously been identified as a tumour suppressor in basal-like breast tumours [8,12]. In humans, the three Akt isoforms (Akt1, Akt2 and Akt3) are derived from unique genes on separate chromosomes. The canonical Akt primary structure comprises the following four basic modules or domains: PH domain, linker region, catalytic domain and C-terminal regulatory region (Figure 2). Although the PH, catalytic and regulatory domains share a high degree of homology among Akt isoforms, the linker domain diverges significantly across Akt1, Akt2 and Akt3. Importantly, mouse studies using individual knockout of the three isoforms originally suggested non-redundant functions of Akt proteins [13–19]. In support of specificity as opposed to redundancy, studies in cell culture have revealed isoform-specific and even opposing functions of Akt isoforms in cellular phenotypes commonly deregulated in malignancies, in particular cell migration and metastatic dissemination [20–22]. These studies have been corroborated by genetically engineered mouse models, revealing that whereas Akt1 can function as a suppressor of invasive migration leading to metastasis, at least in the context of breast cancer, Akt2 functions to enhance these phenotypes [23,24]. C The
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Figure 1 Regulation of Akt activation Receptor tyrosine kinase-induced PI3K activation leads to synthesis of the second messengers PIP3 and PIP2. Via binding to the PH domain, PIP3 and PIP2 recruit inactive Akt to the plasma membrane. Here, activation is initiated by phosphorylation of two highly conserved residues, Thr308 and Ser473 , mediated by PDK-1 and mTORC2 respectively. Phosphatases terminate Akt signalling both by removing PIP3 and PIP2 from the plasma membrane, which is accomplished by PTEN and INPP4B respectively, and by directly dephosphorylating Akt at Thr308 (PP2A) and Ser473 (PHLPP1/2). Three Akt isoforms exist in mammals, Akt1, Akt2 and Akt3, and upon activation transduce the signal by directly phosphorylating substrates such as FOXO transcription factors, murine double minute 2 (MDM2), GSK3β and others. Phosphorylation of these substrates by Akt serves to either activate or inactivate their function, leading to alterations in the phenotypes depicted.
Figure 2 Homology between Akt isoforms Akt isoforms can be divided into the following four domains: PH domain, linker region, catalytic domain and C-terminal regulatory region. The homology between domains of Akt isoforms is depicted below the primary structure comparisons of Akt1/2, Akt1/3 and Akt2/3.
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The majority of the studies investigating isoform-specific functions of Akt signalling in the context of cancer progression have largely been limited to Akt1 and Akt2. These studies have been reviewed extensively, and we direct readers to several previous reviews [25–27]. By contrast, specific roles for Akt3 in modulating phenotypes associated with malignancy have thus far been largely ignored. Furthermore, the majority of studies describing isoform-specific functions of Akt in either cell culture models or mouse models have been observational, and few studies have provided a mechanistic basis by which Akt isoformspecificity is achieved. Although Akt isoform-specific inhibitors have yet to be developed for clinical evaluation, a number of catalytic as well as allosteric pan-Akt inhibitors have been developed and are currently in clinical trials. Recent work describing isoformspecific roles of Akt in breast cancer suggests that targeting all Akt isoforms may result in unwanted on-target effects. Therefore, in order for isoform-specific Akt inhibitors to be a viable therapeutic approach, a comprehensive understanding of distinct isoform-specific roles of Akt proteins is essential. In the present article, we discuss recent studies that have contributed to a greater appreciation of the complexities of isoform-specific Akt signalling in breast cancer, including isoform-specific roles of Akt3 in breast cancer models and discussed potential mechanisms by which Akt isoformspecificity may be conferred.
Isoform-specific roles of Akt in breast cancer As introduced above, the opposing functions of Akt1 and Akt2 in the context of breast cancer have been reviewed extensively elsewhere [25–27]. More recently, however, isoform-specific functions of Akt3 in breast cancer have been described. In the present article, we summarize new findings of Akt isoform-specificity in breast cancer and described recent efforts that have examined isoform-specific functions of Akt in breast cancer that are contributing to our growing awareness of the complexities of isoform-specific Akt signalling.
Akt1 and Akt2 in breast cancer Irie et al. [20] initially demonstrated that although Akt1 knockdown enhances migration and induction of an epithelial to mesenchymal transition (EMT) in human mammary epithelial cells expressing insulin-like growth factor 1 (IGF1) receptor, Akt2 knockdown reverses hyperproliferation of the same cells in growing 3D culture. Our own laboratory confirmed that Akt1 suppresses breast cancer cell invasion and migration, as measured by invasion through Matrigel or migration towards 3T3-conditioned medium [22,28,29]. In addition, Akt2 enhances breast cancer cell migration, since knockdown with isoform-specific siRNA inhibits migration [22]. Similar findings were subsequently made by the Bissell laboratory [30].
In support of these initial in vitro findings, in vivo models of ErbB2-mediated mammary tumorigenesis confirmed the opposing function of Akt1 and Akt2 [23,31,32]. Although co-expression of activated Akt1 and ErbB2 in mammary epithelium of mice accelerates tumour formation, it prevents metastasis when compared with expression of activated ErbB2 alone [23]. However, although co-expression of activated Akt2 and ErbB2 does not affect induction of tumour formation, it significantly increases lung metastases compared with expression of Akt1 and ErbB2 or ErbB2 alone [32]. Moreover, it has recently been appreciated that specific PI3K/Akt pathway mutations signal through individual Akt isoforms. Our laboratory recently described a unique role for Akt2 in mediating survival and maintenance of PTENdeficient cancers, including prostate, glioblastoma and breast [33]. Using a doxycycline-inducible Akt isoform-specific shRNA system, we have evaluated isoform-specific roles of Akt in PTEN-deficient tumour lines and found that whereas silencing all three isoforms prevents 3D spheroid formation in vitro, Akt2 is specifically required for 3D spheroid maintenance in MDA-MB468 cells and BT-549 cells. Moreover, mechanistic studies indicate that the cell cycle regulator p21 is at least one critical downstream effector of Akt2 in the context of PTEN inactivation.
A novel role for Akt3 in breast cancer Although non-redundant functions of Akt1 and Akt2 have been appreciated for some time, an isoform-specific role for Akt3 has only recently been described. We have recently identified a function of Akt3 in basal-like triple-negative breast cancer (TNBC) [22]. Analysis of The Cancer Genome Atlas reveals that Akt3 is preferentially amplified and overexpressed in TNBC tumours and cell lines. Therefore, to assess the function of individual Akt isoforms, we have used a doxycycline-inducible Akt isoform-specific shRNA system. Similar to Akt1 silencing, Akt3 silencing enhances breast cancer cell migration in MCF10DCIS, MDA-MB468 and BT-549 cells. We have also found that silencing of Akt3 in MDA-MB231 cells inhibits 3D spheroid growth, whereas neither Akt1 nor Akt2 had significant effects. Furthermore, Akt3 silencing in two xenograft models attenuates tumour growth in vivo. An isoform-specific function of Akt3 in Balb-neuT mice, a transgenic model of ErbB2-breast cancer, has also recently been proposed [34]. Grabinski et al. [34] evaluated the in vitro kinase activity of Akt isoforms in BalbneuT breast tumours and found that Akt3 has higher in vitro kinase activity than Akt1 or Akt2. In addition, Akt1 and Akt3 silencing more dramatically reduces proliferation of Balb-neuT mammary tumour-derived cells compared with Akt2 silencing. Furthermore, Akt3 knockdown, but not Akt1 or Akt2 knockdown, leads to decreased ErbB2 and ErbB3 expression; increased ERα expression and Akt3 knockdown also sensitizes Balb-neuT mammary tumour-derived cells to tamoxifen treatment. These studies highlight a previously unappreciated role for Akt3 in mediating breast cancer progression, especially in certain molecular subtypes, such as TNBC, and indicate that Akt3-specific inhibitors may be C The
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particularly beneficial for these patient populations for which there is an unmet need in terms of targeted therapy.
of Akt phosphorylation may confer isoform-specificity. However, the potential for this mechanism to contribute to Akt isoform-specific signalling in cancer pathophysiology remains to be determined.
Mechanisms of isoform-specificity Despite the discovery that Akt isoforms have nonredundant functions, mechanisms by which this isoformspecificity is conferred remain unclear. Isoform-specific signalling can be conveyed through various mechanisms: regulation of kinase activity, post-translational modifications, substrate specificity or subcellular localization. Below we have proposed mechanisms by which Akt isoform-specific functions may be achieved and discussed recent advances made in this area.
Isoform-specific activation/deactivation One mechanism by which isoform-specific signalling could be mediated is by differential activation or deactivation of Akt isoforms. In the canonical pathway, both Thr308 and Ser473 are required for full activation [2,35,36]. However, both Thr308 and Ser473 phosphorylation may not be required for productive phosphorylation of all Akt substrates. For example, using a pan-Akt consensus motif substrate antibody, Jacinto et al. [37] reported that deletion of the mTORC2 subunit Sin1 in mouse embryonic fibroblasts (MEFs), which abolishes Ser473 phosphorylation, does not affect the phosphorylation of the majority of Akt substrates. Specifically, Ser473 phosphorylation is required for forkhead box O (FOXO) 1/3a phosphorylation, but dispensable for glycogen synthase kinase 3 (GSK3) α/β or tuberous sclerosis complex 2 (TSC2) phosphorylation [37]. There is also in vivo evidence suggesting that phosphorylation of one, but not both, conserved Akt phosphorylation site is important for the downstream phosphorylation of only a subset of Akt substrates. Vincent et al. [38] demonstrated that phosphorylation of proline-rich Akt substrate of 40 kDa (PRAS40), TBC (Tre-2/Bub2/Cdc16) 1 domain family, member 4 (TBC1D4) and TSC2 correlates with phosphorylation of Akt on Thr308 , but not Ser473 , in human non-small-cell lung tumours. Differential phosphorylation of Thr308 and Ser473 of Akt isoforms may thus influence phosphorylation of Akt substrates, thereby conferring isoform-specificity. The isoform-selectivity of this potential mechanism has yet to be explored. Termination of Akt activity is in part regulated by the phosphatases PP2A [9] and PHLPP1/2 [9,10]. Differential termination of Akt activity by PHLPP isoforms has been suggested [10], such that silencing of PHLPP1 leads to an increase in both Akt2 and Akt3 phosphorylation, whereas silencing of PHLPP2 leads to an increase in both Akt1 and Akt3 phosphorylation. Furthermore, silencing of PHLPP isoforms leads to differential activation of Akt substrates. Although silencing of PHLPP1, but not PHLPP2, leads to an increase in human double minute 2 (HDM2) and GSK3α phosphorylation, and silencing of PHLPP2, but not PHLPP1, leads an increase in FOXO1 phosphorylation at Thr24 . These results indicate that differential termination C The
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Post-translational modifications Post-translational modifications that contribute differentially to Akt isoform-specific activation, localization or substrate phosphorylation constitute one potential mechanism that accounts for specificity. Numerous Akt post-translational modifications have been identified in global MS screens, although the functional significance of many of these has not been determined [39]. In addition to Thr308 and Ser473 phosphorylation, ubiquitination of Akt is required for full activation [40–42]. Tumour-necrosis-factor-receptorassociated factor 6 (TRAF6) and Skp2 directly ubiquitinate Akt at Lys8 and Lys14 in response to IGF-1 and epidermal growth factor (EGF) respectively [40,42]. Although isoformspecific ubiquitination of Akt has not been demonstrated, isoform-specific oxidation of Akt2 has been suggested. Wani et al. [43] reported that platelet-derived growth factor (PDGF) stimulation specifically induces Akt2, but not Akt1, oxidation. Cys124 , located in the linker region of Akt2, is not conserved in Akt1 or Akt3 and serves as the major site of redox-sensitivity in Akt2. This oxidation results in decreased Akt2, but not Akt1, activity. Furthermore, this regulation of Akt2 activity contributes to a cell migration phenotype [44]. These results highlight the potential for isoform-specific post-translational modifications to play an important role in Akt isoform-specific signalling. Additional sites for posttranslational modifications are likely to exist and will have to be evaluated as a mechanism for achieving selectivity in Akt signalling.
Isoform-specific substrates As suggested above, Akt isoform-specific phosphorylation of substrates represents a key mechanism by which selectivity is achieved in the context of pathophysiology. A recent evaluation of the phosphoproteomes of Akt triple-knockout lung fibroblasts and their single Akt isoform-expressing derivatives revealed significant isoform-specific differences [45]. This study also identified a large number of novel, putative isoform-specific substrates to be validated in future studies. The majority of Akt substrates described in the literature are phosphorylated by all Akt isoforms [27]. Although Akt3-specific substrates have not been described, Akt1 and Akt2 isoform-specific substrates have recently been identified (Table 1). Palladin was identified by our laboratory as a novel Akt1-specific substrate [29]. We have demonstrated that Akt1, but not Akt2, phosphorylates palladin in vitro and that Akt1, but not Akt2, knockdown results in reduced palladin phosphorylation in cell culture. Furthermore, Akt1-specific palladin phosphorylation regulates breast cell migration, connecting isoform-specific substrate phosphorylation with an isoform-specific cancer cell phenotype. Isoform-specificity for many of the proposed substrates has not been rigorously
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Table 1 Proposed isoform-specific substrates of Akt Akt1- and Akt2-specific substrates have been identified in published studies. Akt3-specific substrates have yet to be described. Ankrd2, ankyrin repeat domain 2; ARRP, ankyrin repeat domain 2 protein; AS160, Akt substrate of 160 kDa; HDM2, human double minute 2.
Substrate
Isoform-specific Akt isoform Assays used to demonstrate isoform-specificity phosphorylation responsible for site phosphorylation In vitro Cell culture Site-directed mutagenesis? Reference(s)
p21
Thr145
Akt1
Akt1/2 kinase assays Myr-Akt1
Yes, in vitro and in vivo
[51,52]
Skp2 Palladin AS160
Ser72 Ser507 Ser588 , Thr642
Akt1 Akt1 Akt2
Akt1/2 kinase assays Myr-Akt1/2 Akt1/2 kinase assays Akt1/2 shRNA None Membrane-targeted
Yes, in vivo Yes, in vivo No
[53,54] [29] [49,55]
No
[10]
Yes, in vitro Yes, in vitro
[56] [57]
HDM2
Ser166
Myosin5a Ser1650 Ankrd2/ARRP Ser99
Akt2 construct; Akt1/2 siRNA Akt1/2/3 siRNA
Akt2
None
Akt2 Akt2
Akt2 kinase assay Akt1/2 siRNA Akt1/2 kinase assays Akt1/2 siRNA
tested with in vivo analyses. In addition, Akt2- or Akt3specific substrates that account for specific cancer phenotypes have yet to be described.
miRNA expression Numerous studies in recent years have demonstrated the important roles of non-coding RNAs in cancer [46]. A recent study from the Tsichlis laboratory has demonstrated that differential regulation of miRNA expression by Akt isoforms constitutes one mechanism of Akt isoform-specificity in cancer [47]. Iliopoulos et al. [47] screened the miRNA expression signatures of lung fibroblasts, expressing single Akt isoforms using a 365 miRNA array. IGF-1 stimulation induced notable differences in the miRNA expression signatures of the single-Akt isoform expressing cells. miR200 family members miR-200a and miR-200c were downregulated in Akt2-expressing MEFs, but not those expressing Akt1 or Akt3. Knockdown of Akt1, but not Akt2, with isoform-specific siRNA promoted EMT and decreased the abundance of miR-200a and miR-200c; overexpression of miR-200a or miR-200c inhibited cell migration in Akt1silenced MCF10A cells. Furthermore, analysis of Akt1 and Akt2 mRNA levels and miR-200a and miR-200c levels in a small subset of human primary and metastatic breast cancer tumour samples revealed a correlation between lower Akt1/Akt2 ratios and decreased miR-200a and miR-200c levels. It remains to be determined whether these findings can be extended to analysis of a larger group of breast cancer samples. In addition, it will be of interest to determine the specific contexts in which Akt isoform-specificity is conferred by regulation of non-coding RNA expression.
Localization Differential subcellular localization of Akt isoforms would enable isoform-specific access to the plasma membrane for activation and interactions with substrates or chaperone proteins. One study has shown that Akt isoforms have distinct localization patterns when comparing non-transformed
and transformed breast cancer cell lines [48]. Subcellular localization of Akt2 has been proposed as a mechanism for Akt2-specific regulation of trafficking the glucose transporter type 4 (GLUT4) [49]. However, the precise contribution of subcellular localization to Akt isoform-specific mechanisms of breast cancer progression remains to be elucidated.
Molecular determinants of Akt isoform-specificity Implicit in the mechanisms of isoform-specificity suggested above is the notion that Akt isoforms contain intrinsic molecular differences in the primary structure. Recent studies using Akt chimaeric constructs comprising swapped Akt1 and Akt2 domains demonstrated that the PH and/or linker domains distinguish Akt1 from Akt2, at least with respect to the phenotype of cell migration [29,50]. In this context, it has been shown that Akt1-specific palladin phosphorylation requires the linker domain of Akt1. The lack of an Akt3specific phenotype, until recently, or Akt3-specific substrates has limited the use of chimaeras to examining the domains of Akt1 and Akt2 required for isoform-specific functions.
Perspectives Although much is known regarding the mechanisms of PI3K and Akt activation and in turn how signal relay through this pathway modulates phenotypes associated with malignancy, there is a considerable paucity of knowledge regarding the elements that distinguish Akt isoform signalling. It is likely that context-specificity contributes significantly to Akt isoform-specific signalling that have been reported in cellbased experiments as well as in mouse studies. This notion is supported by the addiction to specific Akt isoforms in pathway-mutant driven cancers, particularly breast cancer. However, the precise basis by which individual isoforms of Akt are differentially required remains obscure. Furthermore, the identification of Akt isoform-specific substrates suggests C The
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that there exist molecular determinants that distinguish Akt isoforms and that contribute to isoform-specific functions in vivo. A major challenge in the field remains the identification of the molecular determinants of Akt isoform-specificity, which is predicted to have far-reaching implications especially with respect to the development of isoform-selective inhibitors. This would not only provide much needed tool compounds to more precisely study isoform-specific Akt signalling and biology, but also provide a potential therapeutic basis to regulate Akt pathway activation.
Acknowledgements We thank all past and present members of the Toker laboratory.
Funding Research in our laboratory is supported in part by the National Institutes of Health, the Susan G. Komen Foundation for the Cure and the Department of Defense Breast Cancer Research Program.
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Received 30 May 2014 doi:10.1042/BST20140160
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