(2010) 29, 150–155 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 $32.00 www.nature.com/onc SHORT COMMUNICATION AKT1 mutations in bladder cancer: identification of a novel oncogenic mutation that can co-operate with E17K

JM Askham1, F Platt1, PA Chambers2, H Snowden2, CF Taylor2 and MA Knowles1

1Cancer Research UK Clinical Centre, Leeds Institute of Molecular Medicine, St James’s University Hospital, Leeds, UK and 2Cancer Research UK Genome Variation Laboratory, St James’s University Hospital, Leeds, UK

The phosphatidylinositol-3- (PI3 kinase)-AKT (Bozulic et al., 2008). AKT is an evolutionarily conserved pathway is frequently activated in cancer. Recent reports kinase, also known as . There are three have identified a transforming mutation of AKT1 in members of the AKT family (AKT1-3), encoded by breast, colorectal, ovarian and lung cancers. We report separate , but with over 80% amino-acid sequence here the occurrence of this mutation in bladder tumours. identity. AKT occupies a key regulatory node in the PI3K The AKT1 G49A (E17K) mutation was found in 2/44 pathway, below which the pathway branches significantly (4.8%) bladder cancer cell lines and 5/184 (2.7%) bladder to influence a wide range of cellular processes that pro- tumours. Cell lines expressing mutant AKT1 show mote cell cycle progression, cell growth, cellular energy constitutive AKT1 activation under conditions of growth metabolism and resistance to . factor withdrawal. We also detected a novel AKT1 Genes encoding many of the components of the PI3 mutation G145A (E49K). This mutation also enhances kinase pathway are targeted by germline and somatic AKT activation and shows transforming activity in mutations, amplifications, rearrangements, over-expres- NIH3T3 cells, though activity is weaker than that of sion, methylation and aberrant splicing (Hennessy et al., E17K. Enhanced activation of AKT1 when E17K and E49K 2005; Kumar and Hung, 2005; Manning and Cantley, mutations are in tandem suggests that they can co-operate. 2007), which generally result in increased AKT activity Oncogene (2010) 29, 150–155; doi:10.1038/onc.2009.315; and aberrantly elevated downstream signalling. In published online 5 October 2009 bladder cancer, mutations have been identified in PIK3CA (Lopez-Knowles et al., 2006) and TSC1 (which Keywords: AKT1; mutation; bladder cancer lies downstream of AKT in the PI3 kinase-AKT-mTOR branch of the PI3 kinase pathway) (Knowles et al., 2003; Pymar et al., 2008), and loss of heterozygosity, homo- zygous deletion and inactivating mutations of PTEN Introduction have been found (Cappellen et al., 1997; Cairns et al., 1998; Aveyard et al., 1999; Wang et al., 2000). The phosphatidylinositol-3-kinase (PI3 kinase)-AKT Given the central function of activated AKT1 in pathway promotes cell growth, proliferation and survi- tumorigenesis, it was somewhat surprising that muta- val, and is the most frequently mutated pathway in tions were not detected in this until recently. human cancer (Brugge et al., 2007). Activated receptor A single activating point mutation in AKT1 has now tyrosine recruit the p85/p110 PI3 kinase complex been described in breast, colorectal and ovarian cancers to the membrane through the p85 regulatory subunit (Carpten et al., 2007; Bleeker et al., 2008; Kim et al., either directly or through insulin receptor substrate 2008) and squamous cell carcinoma of the lung adapter proteins. The active p110 (catalytic) subunit (Malanga et al., 2008). This mutation, G49A (E17K), then phosphorylates phosphatidylinositol-4,5-bisphos- in the pleckstrin homology (PH) domain of the protein phate (PIP2) to phosphatidylinositol-3,4,5-trisphosphate results in its recruitment to the plasma membrane in the (PIP3). PTEN (phosphatase and tensin homologue dele- absence of PI3 kinase signalling and confers transform- ted on 10) catalyses the reverse reaction. ing activity in vitro and in vivo (Carpten et al., 2007). PIP3 recruits protein-dependent kinase 1 (PDK1) As mutations in other components of the PI3 kinase and AKT to the plasma membrane where AKT is pathway are found in bladder cancer, we screened phosphorylated at Thr308 by PDK1 and at Ser473 by bladder tumours and cell lines for this mutation. mTORC2 (Sarbassov et al., 2005). AKT1 can also be phosphorylated at Ser473 by DNA-PK in the nucleus Results and discussion

Correspondence: Professor MA Knowles, Cancer Research UK Clinical Initially, we screened a panel of 42 bladder cell lines by Centre, Leeds Institute of Molecular Medicine, St James’s University PCR and sequencing of AKT1 exon 4 and found that Hospital, Beckett Street, Leeds LS9 7TF, UK. E-mail: [email protected] 2/42 (4.8%) cell lines (KU19-19 and MGH-U3) contained Received 25 July 2008; revised 24 June 2009; accepted 31 July 2009; the G49A (E17K) mutation (Supplementary Figure 1). published online 5 October 2009 In both cell lines, the mutation was heterozygous. AKT1 mutations in bladder cancer JM Askham et al 151 During sequencing of exon 4, we identified a second heterozygous mutation, G145A (E49K), in KU19-19 (Supplementary Figure 2), suggesting the possibility of other activating mutations in the PH domain. The mutation screen was then extended to a panel of 184 well-characterized bladder tumours. These were ana- lysed by a combination of high-resolution melting curve analysis to screen for mutations in AKT1 exon 4 and pyrosequencing as a specific assay for the E17K mutation (Supplementary Methods). Positive results were confirmed by PCR and sequencing (Supplementary Figure 1). E17K was the only mutation detected (5/184; 2.7%). This frequency is within the range published for breast, ovarian, colorectal and lung cancers (Carpten et al., 2007; Bleeker et al., 2008; Kim et al., 2008; Malanga et al., 2008). The mutation was not detected in DNA extracted from blood samples from the corresponding patients. Four of the five tumours were heterozygous for the mutation and one homo- zygous. Mutant tumours included low-grade, non- invasive (Ta grade 2) and muscle invasive (T2 grade 3) tumours. Mutation and loss of heterozygosity status Figure 1 AKT1 is constitutively active in cells with an AKT1 mutation. (a) Immunoblot analysis of lysates of sub-confluent cells for several genes that are commonly mutated in bladder grown in the presence or absence of growth factors with antibodies cancer is known for this tumour series (Platt et al., to AKT1 and phospho-AKT Thr308. In TERT-NHUC cells, 2009). AKT1 mutation was mutually exclusive with phospho-AKT levels are reduced under conditions of respect to PIK3CA mutation and PTEN loss of deprivation, but remain elevated in the cell lines with mutant heterozygosity, but not FGFR3 and TSC1 mutation. AKT1. (b) Immunoblot analysis of AKT1 immunoprecipitates from lysates of sub-confluent growth factor-deprived cells with Analysis of a much larger series of tumours will be antibodies to phospho-AKT Ser473 and Thr308. No phospho- needed to allow conclusions to be drawn about these AKT signal was detected after immunoprecipitation with the non- relationships. specific antibody. Phospho-AKT Ser473/Thr308 signals are greatly The E17K substitution in the PH domain of AKT1 elevated in KU19-19 and MGH-U3 cells compared with TERT- NHUC showing increased AKT1 activation in these cell lines in the allows localization of the protein to the plasma absence of growth factors. membrane in the absence of upstream signalling (Carpten et al., 2007). Membrane-associated AKT is activated by phosphorylation at Thr308 by PDK1 and at Ser473 by mTORC2. Consistent with this, while Thr308 (Figures 2a and b). Levels of phosphorylated phospho-AKT levels (Thr308) were markedly reduced Thr308 were modestly increased in cells expressing both by growth factor withdrawal in telomerase-immorta- AKT1-E17K and AKT1-E49K under growth factor- lized normal human urothelial cells (TERT-NHUC), starved conditions. Under the same conditions, cells levels remained the same in KU19-19 and MGH-U3 expressing AKT1-E17K and AKT1-E49K showed (Figure 1a). Specific examination of AKT1 showed that markedly increased levels of phosphorylated Ser473, phosphorylation at both Ser473 and Thr308 was AKT1-E17K having the greatest effect. Growth factor- elevated in growth factor-deprived KU19-19 and starved cells expressing AKT1-E17K þ E49K showed MGH-U3 compared with TERT-NHUC (Figure 1b), increases in phosphorylation of both Thr308 and Ser473 showing complete activation of AKT1. Although this to levels greater than those produced by either AKT1- experiment does not exclude the possibility of AKT1 E17K or AKT1-E49K alone, suggesting possible co- activation through other means in these tumour cell operation or synergy of the two mutations in AKT lines, the results are entirely consistent with the activation. Notably, cells expressing AKT1-E17K þ published effects of the E17K mutation. E49K showed the highest levels of phosphorylated To investigate the possible functional importance of Thr308 and Ser473 on growth factor reintroduction. the novel E49K mutation, AKT1 cDNA was amplified The AKT substrate GSK3b also showed elevated from KU19-19 cells by RT–PCR and cloned. During phosphorylation during starvation in AKT1-E17K and this process, it became apparent that KU19-19 cells AKT1-E17K þ E49K expressing cells compared with express three AKT1 transcripts, one wild type, one controls, although constitutive activation was lower in E17K and one E17K þ E49K (data not shown). Con- AKT1-E49K cells. sistent with this, KU19-19 contains three copies of To determine the effects of the two mutations on chromosome arm 14q (data not shown). AKT1 activation more clearly, immunoprecipitates of driving expression of wild-type AKT1, AKT1-E17K, lysates of NIH3T3 cells expressing AKT1, AKT1-E17K, AKT1-E49K and AKT1-E17K þ E49K were created and AKT1-E49K and AKT1-E17K þ E49K were analysed used to transduce primary NHUC. AKT activation was for AKT Thr308 and Ser473 phosphorylation determined by assessing phosphorylation at Ser473 and during serum starvation and after subsequent serum

Oncogene AKT1 mutations in bladder cancer JM Askham et al 152

Figure 2 AKT1 mutations cause constitutive and enhanced AKT activation in primary normal human urothelial cells and NIH3T3 cells. Immunoblot analysis (a, c) of AKT1, P-AKT Ser473 and Thr308, GSK3b, P-GSK3b Ser9 in lysates of sub-confluent NHUC (a, b) or immunoprecipitates of lysates of NIH3T3 cells (c, d) transduced with empty virus or virus expressing wild-type AKT1 or AKT1 mutants under conditions of growth factor starvation and subsequent stimulation. Phospho-AKT band intensities were normalized to the corresponding AKT1 expression level and are represented graphically relative to values obtained from cells expressing only endogenous AKT1 (pFB) (b, d). (a, b) AKT Ser473 phosphorylation is barely detectable in growth factor- deprived NHUC with empty virus or expressing wild-type AKT1. Serum-starved cells expressing AKT1-E17K and AKT1-E49K show constitutive phosphorylation at Ser473 and to a more limited extent at Thr308 compared with wild-type AKT1-expressing cells. Cells expressing AKT1-E17K þ E49K show enhanced levels of Ser473 and Thr308 phosphorylation. GSK3b Ser9 phosphorylation is also constitutively elevated in serum-starved cells expressing AKT1-E17K and AKT1-E17K þ E49K, and to a limited extent AKT1-E49K compared with wild-type AKT1-expressing cells. (c, d) Serum-starved NIH3T3 cells expressing AKT1-E17K show increased phosphorylation at both Thr308 and Ser473. Cells expressing AKT1-E49K show a modest increase in Ser473 phosphorylation with no increase at Thr308. Ser473 and Thr308 phosphorylation is greatest in cells expressing AKT1-E17K þ E49K.

reintroduction (Figures 2c and d). Serum-starved cells no increase at Thr308. Serum-starved cells expressing expressing AKT1-E17K showed a dramatic increase in AKT1-E17K þ E49K showed increases in phosphoryla- phosphorylation at both Thr308 and Ser473. Under tion at Thr308 and Ser473 greater than those produced similar conditions, cells expressing AKT1-E49K showed by either AKT1-E17K or AKT1-E49K alone, again a more modest increase in Ser473 phosphorylation with suggesting possible synergy in AKT1 activation. Again,

Oncogene AKT1 mutations in bladder cancer JM Askham et al 153 cells expressing AKT1-E17K þ E49K showed the high- est levels of phosphorylated Thr308 and Ser473 on growth factor reintroduction. NIH3T3 cells transduced with the AKT1 and control viruses were used to assess transforming ability. In no case was morphological transformation induced (data not shown). This is compatible with earlier studies of activated AKT in NIH3T3 cells in which membrane- targeted AKT1 or an activated mutant (E40K) induced anchorage-independent growth and tumourigenicity in nude mice, but with minimal morphological changes (Sun et al., 2001). Growth curve analysis showed that cells expressing AKT1-E49K, AKT1-E17K and AKT1- E17K þ E49K have a significant proliferative advantage over controls (Figures 3a). AKT1-E49K cells were able to form colonies in soft agar, although these were smaller and fewer than those of AKT1-E17K and AKT1-E17K þ E49K cells (Figure 3b and c). This indicates that the E49K mutation is not as strongly transforming as the E17K mutation. There was no evidence for synergism between the two mutations in these assays. This may reflect the use of optimum growth conditions in these assays, in which differences in AKT1 activation were less striking than in growth factor-depleted conditions (Figures 2c and d). Our data show that the E17K mutation results in constitutive activation of AKT1 as shown by increased phosphorylation at Thr308 and Ser473. This is in contrast with the E49K mutation, which results only in constitutively elevated Ser473 phosphorylation. It has been shown that the E17K mutation results in enhanced translocation of AKT1 to the membrane in which phosphorylation by PDK1 occurs (Carpten et al., 2007). Recent results indicate that the effect of E17K is to broaden the lipid-binding specificity of AKT such that it can bind physiological concentrations PIP2 found in the plasma membrane (Landgraf et al., 2008). It has also been shown that in the inactive state, AKT is bound to PDK1, but activation is suppressed through interaction of the PH domain with the kinase domain, preventing PDK1 access to Thr308 and preventing phosphorylation (Calleja et al., 2007). Structural studies of the AKT PH domain, both uncomplexed (Milburn et al., 2003) and complexed to PIP3 (Thomas et al., 2002) have been reported. Comparison of these structures indicates a large conformational change on binding of PIP3 such that the variable loop 2 region, which seems flexible Figure 3 AKT1 mutations are transforming. (a) NIH3T3 cells transduced with empty virus or virus containing wild-type AKT1 or when uncomplexed, shows clustering of 3–4 negative AKT1 mutants were seeded in triplicate in six-well dishes at 15 000 charges (contributed by Asp44, Asp46, Glu49 and to a cells per well and counted at days 1, 3, 6 and 8. AKT1 mutant lesser extent Glu40) when complexed with PIP3 to form expressing cells show increased proliferation. Experiments were an acidic patch facing the solvent. It was suggested that repeated in triplicate. (b) 3T3 cells expressing AKT1 mutants show increased anchorage-independent growth. Cells were seeded in mutation of residues within this patch may allow triplicate in agar at 50 000 per well and allowed to grow for 14 days activation of AKT in the presence of lower concentra- before staining with p-iodotetrazolium violet. AKT1 mutant- tions of PIP3 (Milburn et al., 2003). Indeed, it has been expressing cells formed more large colonies than controls, although shown that the mutation G40L results in increased AKT1-E49K formed fewer colonies than AKT1-E17K and AKT1- activity of AKT in cells (Bellacosa et al., 1998). We E17K þ E49K. Experiments were repeated in triplicate. (c) Typical colonies from each experiment. Bar ¼ 250 mM. hypothesize that the E49K mutation may have a similar effect, but that when present in isolation, this mutation does not facilitate membrane translocation and phos- cytoplasm. Thus, synergy of E49K with E17K could phorylation of Thr308. However, Ser473 phosphoryla- result from enhanced membrane localization combined tion is accomplished by mTORC2, present in the with a reduced ability of the PH domain of the protein

Oncogene AKT1 mutations in bladder cancer JM Askham et al 154 to interact with the kinase domain and increased ability frequencies of involvement of PIK3CA, PTEN, TSC1 to adopt an active conformation. The reduced AKT and AKT1 implicate this pathway in the majority of activating capacity of E49K compared with E17K bladder tumours. Importantly, we have identified a and the ability of E49K to augment the activating novel mutation in this gene that confers increased activity of E17K may explain why the E49K mutation activation, transforming ability in rodent fibroblasts was not detected independently of the E17K mutation. and shows evidence of co-operation with a known Indeed, the identification of alleles with E17K and both activating mutation. Examination of the structural E17K and E49K in the cell line KU-19-19 indicates consequences of this alteration and its effect on that the E17K mutation probably preceded the E49K protein–protein interactions may improve understand- mutation. As larger numbers of tumours with E17K are ing of the mechanism of action of this critical identified, additional mutations involving residues signalling protein. Asp44, Asp46, Glu49 and Glu40 in the ‘acidic patch’ may be identified. The PH domain of AKT1 also interacts with a number of proteins and these interactions impact on Conflict of interest activation. These include Ca2 þ calmodulin (Dong et al., 2007), CKIP-1 (Tokuda et al., 2007) and TCL1 (Laine The authors declare no conflict of interest. et al., 2000). Thus, it is possible that the E49K mutation may also modulate these interactions to enhance AKT1 activity. Acknowledgements In conclusion, we have identified an additional mechanism by which PI3 kinase pathway activation This work was funded by a Programme grant from Cancer can occur in bladder cancer. Taken together, the Research UK (C6228/A5433).

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Oncogene