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AKT1 Mutations in Bladder Cancer: Identification of a Novel Oncogenic Mutation That Can Co-Operate with E17K

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AKT1 Mutations in Bladder Cancer: Identification of a Novel Oncogenic Mutation That Can Co-Operate with E17K Oncogene (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-kinase (PI3 kinase)-AKT (Bozulic et al., 2008). AKT is an evolutionarily conserved pathway is frequently activated in cancer. Recent reports kinase, also known as protein kinase B. 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 genes, 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 apoptosis. 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 gene until recently. human cancer (Brugge et al., 2007). Activated receptor A single activating point mutation in AKT1 has now tyrosine kinases 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 chromosome 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 growth factor 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-
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