Potential therapeutic targets for chordoma: PI3K/AKT/TSC1/TSC2/mTOR pathway

British Journal of Cancer (2009) 100, 1406 – 1414 & 2009 Cancer Research UK All rights reserved 0007 – 0920/09 $32.00 www.bjcancer.com Potential therapeutic targets for chordoma: PI3K/AKT/TSC1/ TSC2/mTOR pathway N Presneau1,2,7, A Shalaby1,2,7, B Idowu2,3, P Gikas4, SR Cannon4, I Gout5, T Diss6, R Tirabosco3 and AM Flanagan*,1,2,3,5 1 UCL Cancer Institute, University College London, 72 Huntley Street, London WC1E 6BT, UK; 2Institute of Orthopaedics and Musculoskeletal Science, University College London, Stanmore, Middlesex HA7 4LP, UK; 3Department of Histopathology, Royal National Orthopaedic Hospital NHS Trust, Stanmore, Middlesex HA7 4LP, UK; 4The Sarcoma Unit, Royal National Orthopaedic Hospital NHS Trust, Stanmore, Middlesex, HA7 4LP, UK; 5 Department of Structural and Molecular Biology, University College London, Darwin Building, Gower Street, London WC1E 6BT, UK; 6Department of Histopathology University College Hospital, University Street, London WC1E 6JJ, UK Translational Therapeutics Chordomas are radio- and chemo-resistant tumours and metastasise in as many as 40% of patients. The aim of this study was to identify potential molecular targets for the treatment of chordoma. In view of the reported association of chordoma and tuberous sclerosis complex syndrome, and the available therapeutic agents against molecules in the PI3K/AKT/TSC1/TSC2/mTOR pathway, a tissue microarray of 50 chordoma cases was analysed for expression of active molecules involved in this signalling pathway by immunohistochemistry and a selected number by western blot analysis. Chordomas were positive for p-AKT (92%), p-TSC2 (96%), p-mTOR (27%), total mTOR (75%), p-p70S6K (62%), p-RPS6 (22%), p-4E-BP1 (96%) and eIF-4E (98%). Phosphatase and tensin homologue deleted on chromosome 10 expression was lost in 16% of cases. Mutations failed to be identified in PI3KCA and RHEB1 in the 23 cases for which genomic DNA was available. Fluorescence in situ hybridisation analysis for mTOR and RPS6 loci showed that 11 of 33 and 21 of 44 tumours had loss of one copy of the respective genes, results which correlated with the loss of the relevant total proteins. Fluorescence in situ hybridisation analysis for loci containing TSC1 and TSC2 revealed that all cases analysed harboured two copies of the respective genes. On the basis of p-mTOR and or p-p70S6K expression there is evidence indicating that 65% of the chordomas studied may be responsive to mTOR inhibitors, rapamycin or its analogues, and that patients may benefit from combined therapy including drugs that inhibit AKT. British Journal of Cancer (2009) 100, 1406 – 1414. doi:10.1038/sj.bjc.6605019 www.bjcancer.com & 2009 Cancer Research UK Keywords: chordoma; mTOR; brachyury; AKT; rapamycin Chordoma is a rare malignant locally destructive tumour with a characteristic morphology and immunohistochemical profile (cytokeratin 19 and brachyury positive) that occurs, in the majority of cases, in vertebral bones and presents mostly with a large mass making wide excision rarely possible. Recurrent disease is a common event and metastases occur in as many as 40% of cases (Henderson et al, 2005; Romeo and Hogendoorn, 2006; Vujovic et al, 2006; Tirabosco et al, 2008; Hallor et al, 2008). Surgery, often ablative, is the main treatment option as the tumours are largely resistant to chemotherapy and radiotherapy, although proton/photon-beam radiotherapy is recommended for local control if available (Park et al, 2006) (for review see Casali et al, 2007). Little is known about the genetic events that account for the development and progression of chordoma although there are reports that this tumour has developed in five patients with tuberous sclerosis complex. This is a tumour suppressor generelated syndrome occurring on a background of germline *Correspondence: Professor AM Flanagan; E-mail: 7 These authors contributed equally to this work Revised 25 February 2009; accepted 10 March 2009 mutations in TSC1 and TSC2 (encoding hamartin and tuberin, respectively). One of the cases harbouring a TSC2 mutation had a clear loss of heterozygosity of the wild-type allele. The second case with a TSC1 mutation revealed a reduced signal corresponding to the wild-type allele (allelic imbalance), and this was interpreted as loss of heterozygosity (Lee-Jones et al, 2004). Inactivation of TSC1/TSC2 function results in the phosphorylation of mTOR and its downstream effector molecules, the end result of which is initiation of translation, cell growth and proliferation (for review see Rowinsky (2004); MacKenzie and von Mehren (2007)). Inhibitors to mTOR are available and therefore the possibility exists of directed therapy for chordomas. mTOR exists in two molecular complexes, (mTOR complex mTORC1 and mTORC2), which exhibit different sensitivities to rapamycin and its analogues (Kim et al, 2002; Sarbassov et al, 2004; Yang et al, 2006b). However, investigation into mTOR activity in chordomas is required before offering such treatments as evidence also exists that the locus harbouring mTOR in chordomas is a region where there is frequent genomic loss (frequency of 0.57) (Scheil et al, 2001; Hallor et al, 2008). The regulation of mTOR is complex, but in summary, AKT, a serine – threonine kinase, can be activated through a number of mechanisms, including the binding of a variety of growth factors to Potential therapeutic targets for chordoma N Presneau et al 1407 from 6 to 120 months, 46% of patients had one local recurrence, 12% had two and the remainder had no recurrences. Metastases involving bone, skin and lung occurred in 22% of patients. Frozen tumour was available from 23 primary tumours and paraffin embedded from all 50 cases (metastatic disease was not included). Five of the tumours had a dedifferentiated component but this material was only available from two cases and therefore the dedifferentiated component was not studied. Tissue microarray The pathology was reviewed by AMF, AS and RT. A tissue microarray (TMA) of all 50 chordomas was constructed using a manual tissue arrayer (Beecher Instruments Inc., Sun Prairie, WI, USA). At least two representative 0.6 mm cores from each case were taken for the array. An additional 120 cores were used as control material and orientation markers and included human salivary gland, kidney, thyroid, tonsil, lymph node, placenta, testis, GIST, thyroid, renal cell and breast carcinomas. Antibodies and analysis of immunohistochemistry MATERIALS AND METHODS Clinical samples This study complies with Central Office for Research Ethics Committees standards. The material, snap-frozen and paraffinembedded tissue, was obtained from the histopathology department of The Royal National Orthopaedic Hospital. The clinical data were retrieved from the clinical notes. The cohort studied consisted of chordomas from 50 patients, 31 males and 19 females, with a median age of 65 years (range 19 – 84). The tumours were characterised by typical chordoma (...truncated)