Carbonic Anhydrase IX: regulation and role in cancer
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Lecture by Prof. Silvia Pastorekova in the course: "Tumour Hypoxia: From Biology to Therapy III". For the complete e-Course see http://www.myhaikuclass.com/MaastroClinic/metoxia
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Carbonic Anhydrase IX: regulation and role in cancer
- 1. METOXIA training course Maastricht 2012 Carbonic anhydrase IX: regulation and role in cancer S. Pastorekova Department of Molecular Medicine, Institute of Virology Slovak Academy of Sciences Bratislava, Slovakia
- 2. Carbonic anhydrases M75 Pastorekova et al, Virology, 1992 CO2 + H2O HCO3- + H+ Zavada et al, Int J Cancer, 1993 Pastorek et al,, Oncogene, 1994 Opavsky et al, Genomics, 1996
- 3. Carbonic anhydrase IX PG Domain PG Domain TM TM I I C C Hilvo et al, J Biol Chem, 2008 Alterio et al: PNAS 2009
- 4. CA IX expression CAIX Expression CAIX Expression Broad range of solid tumors in Normal tissues in Solid tumors Oosterwijk et al, 1986- Parkkila et al, 1997- Harris et al, 2000- >400 papers
- 5. CA IX expression Transcription activated by hypoxia (+ VHL inactivation, high cell density, modulating factors e.g. acidosis) Alternative splicing (hypoxia-related) Shedding of ectodomain (hypoxia-related, ADAM17-driven) Kaluz et al, JBC, 1999 Wykoff et al,, Cancer Res, 2000 Kopacek et al, BBA, 2005 Zatovicova et al, Br J Cancer, 2005 Barathova et al, Br J Cancer, 2008
- 6. IHC biomarker of hypoxia Marker for in vivo imaging Indicator of prognosis CA IX expression pattern Target for immunotherapy G250 Circulating marker for non-invasive monitoring
- 7. Hypoxia in tumor tissue • Acid-base imbalance Reduced cell-cell • Activation of pH adhesion regulating machinery • Reversed pH gradient Increased cell migration/invasion >
- 8. Adhesion – motility - invasion Purpose: tumor expansion, escape of cells from hostile microenvironment Pouyssegur, Nature, 2006
- 9. Shift in metabolism Purpose: Maintain biomass and energy production HIF-1 Vander Heiden, Science, 2009
- 10. pH regulation Purpose: Maitain neutral pHi & survive acidify pHe & facilitate invasion
- 11. CA IX role in tumor cells • I EW S REVpH regulation Acidosis not only due tois interesting toprotons in tum It lactate and note that that were treated with compound 1 ( compound 2, the acidic pH e was revers normal pH values, which was undoub Glycolysis-deficient inhibition of the tumour-associated cells form acidic drases28,45,46. Compound 1 (n=1), with a CA9, was shown via non-invasive fluo tumors in vivo to bind to cells only when the CA9 prote and when cells were hypoxic, in an i cancer 46. Fluorescently labelled sulph Shift (rather than therefore provide a powerful tool for vis responses in in metabolism could switch) solid tumours and patients for CA9-directed therapies46. An i nteresti ng study repor ted t LS CO2 – important overexpres 174Tr tumour cells that CA12, it was possible to genetically sile component of carbonic anhydrase isoforms47. Genetic β β acidosis alone caused a 40% reduction in xen volume and led to the overexpression of these cells were kept at an artificially low Not just proton tration, and this observation has not been Genetic silencing of both CA9 and CA extrusion, but also reduction in tumour growth, which su bicarbonate import and CA12 are major tumour prosurviv enzymes47. Membrane-impermeable sulphonam (compounds 4 and 5), which are based o Figure 1 | Proteins involved in pH regulation within a tumour cell. The figure shows various proteins that are involved in regulating pH within tumours, including: mide scaffold to which either fluorescein monocarboxylate transporters (MCT Discovery, 2011 lactic acid and other Neri & Supuran, Nature Rev Drugs), which transport or albumin-binding moieties were attac monocarboxylates formed by the glycolytic degradation of glucose; Na+/H+ exchangers tumour growth in mice with xenograf
- 12. CA IX role in tumor cells pH regulation CA IX activity = inhibited by bicarbonate but not lactate Bicarbonate metabolon
- 13. CA IX & extracellular acidification A B N H N H lactate CA IX mock CA IX MDCK C Normoxia Hypoxia CA IX mock Svastova e al, FEBS Letters, 2004
- 14. CA IX & intracellular neutralization Swietach, Vaughan-Jones, Harris, Cancer Mestast Rev, 2007
- 15. CA IX role in tumor cells
- 16. CA IX role in tumor cells • Reduced E cadherin-mediated CA IX cell-cell adhesion E cadherin Svastova et al, Exp Cell Res, 2003
- 17. CA IX & cell dissociation neo CA IX Svastova et al, Exp Cell Res, 2003
- 18. CA IX domains Cell de-adhesion pH regulation cell de-adhesion ? Removal leads to loss of membrane localization
- 19. „Inside-out signaling“ from IC Hulikova et al, FEBS Letters, 2009
- 20. ! ! ! ! ! CA IX phosphorylation at Thr443 regulates ! ! ! ! the acidification of pHe ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !! Diite et al., Cancer Res, 2011 ! ! !
- 21. ! ! ! CA IX is a PKA substrate ! Diite et al., Cancer Res, 2011 !
- 22. ! ! ! ! PKA is activated by hypoxia ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Diite et al., Cancer Res, 2011 !
- 23. PKA activation in hypoxia
- 24. CA IX & PKA CA IX & PKA SUBSTRATE
- 25. CA IX role in tumor cells • pH regulation - survival in hypoxia/acidosis pH~6.8 pH~7.4
- 26. CA IX role in tumor cells • pH regulation and cell migration MDCK-CA IX, 4h HGF Stock & Schwab, Pflugers Arch, 2009
- 27. CA IX improves migration MDCK-neo MDCK-CA IX start 24 h Svastova et al., J Biol Chem, 2012
- 28. A549 Hy 48h, wound healing with HGF in hypoxia CA IX actin merge 15 min 1h
- 29. CA IX role in tumor cells • Increased scattering (EMT) and cell migration Svastova et al, J Biol Chem 2012
- 30. CA IX co-localizes with bicarbonate transporters CA IX NBC merge Svatova et al., J Biol Chem, 2012 CA IX AE2 merge
- 31. CA IX interacts with bicarbonate transporters Svastova et al., J Biol Chem, 2012
- 32. CA IX phosphorylation affects CA IX is present cell migration in invading tumor cells
- 33. CA IX role in tumor cells • CA IX role in cell migration /invasion /EMT Svastova et al, J Biol Chem, 2012 • Role in focal adhesion Radvak et al, submitted
- 34. Expression pattern & function >>> CA IX as a target for therapy 1. Antibodies (immunotherapy, drug delivery) G250 Girentuximab 2. Inhibitors (blocking enzyme function from outside) 3. Small molecules (blocking enzyme function from inside)
- 35. aidepolcycne eerf eht ,aidepikiW morF edimalozatecA cinobrac a si ,xomaiD eman edart eht rednu dlos ,edimalozatecA ,seruzies citpelipe ,amocualg taert ot desu si taht rotibihni esardyhna ,ssenkcis edutitla ,)irberec romutoduesp( noisnetrepyh lainarcartni ngineb gurd cireneg a sa elbaliava si edimalozatecA .aisatce larud dna ,airunitsyc .citeruid a sa desu osla si dna stnetnoC noitca fo msinahceM 1 sesU 2 amocualG 1.2 cigolorueN 2.2 emordnys nafraM 3.2 aenpa peelS 4.2 eman )CAPUI( citametsyS ssenkcis niatnuom etucA 5.2 edimateca)ly-2-lozaidaiht-4,3,1-lyomaflus-5(-N eruliaF traeH evitsegnoC 6.2 atad lacinilC amedE decudnI-gurD 7.2 )SU(C )UA(3B .tac ycnangerP stceffe-ediS 3 )SU( ylno-℞ )KU( M OP sutats lageL snoitacidniartnoC 4 yrtsimehC 5 VI ,larO setuoR secnerefeR 6 atad citenikocamrahP enoN msilobateM sruoh 9 ot 3 efil-flaH noitca fo msinahceM laneR noitercxE desu eb yam ti yllacidem ;rotibihni esardyhna cinobrac a si edimalozatecA srefiitnedI .sisolakla yrotaripser ro cilobatem ereves ot etaredom fo snoitidnoc taert ot 5-66-95 r e bm u n S A C eht ni noitproser )-3OCH( etanobracib htiw gnirefretni yb siht seod tI 10CE10S edoc CTA .)eniru eht gnizinilakla suht dna( doolb eht gniyfidica-er ybereht ,syendik 6891 DIC mehCbuP gniwollof eht fo trap tsrfi eht sezylatac )AC( esardyhna cinobraC 91100DRPA knaBgurD era )O H( retaw dna )2OC( edixoid nobrac hcihw ni ,noitcaer elbisrever 2 9091 redipSmehC :asrev-eciv dna )3OC2H( dica cinobrac ot detrevnoc I0V569XF3O I INU - 81200D GGEK 3 OCH + +H >--< 3OC2H >--AC--< O2H + 2OC 02LBMEHC L B M Eh C enibmoc yllamron snoi negordyh deterces yllacol ,selubut yendik eht nI atad lacimehC Inhibitors d .)3OC2H( dica cinobrac mrof ot )-3OCH( etanobracib eretl fi htiw alumroF ,esardyhna cinobra c yb nopu detca yllamron si nrut ni dica cinobraC 2 3 S O 4 N 6H 4 C lom /g 542.222 ssam .loM yb selubut eht sevael yldipar 2OC sA .2OC fo noitamrof ot gnidael mehCbuP & seluceloMe SELIMS detfihs snur yllamron noitcaer evoba eht ,senarbmem llec ssorca gnisuffid
- 36. Inhibitors: Opportunities & Challenges • Combination therapy – uptake of drugs, radiosenitization, blocking adaptation to hypoxia induced by anti- angiogenic therapy • Specificity, selectivity – membrane impermeant, bulky • Toxicity, solubility, off-target effects (HIF pathway) • Unexpected inhibition by other drugs (pazopanib, celecoxib, imanitib, nilotinib, steroid sulfatase inhibitiors)
- 37. • Acetazolamide (AAA) – general CA inhibitor • HeLa cervical carcinoma cells – no CA II, no CA XII, but hypoxia-induced CA IX • 3D sferoids to mimic hypoxia in tumors, CA IX in core • Long-term treatments (up to 20 days), cDNA microarray • Transcriptional effects – CA IX down, ADAM17 up > increased CA IX shedding – loss of target?
- 38. CA IX shedding CA IX ectodomain Membrane -bound ? CA IX ? ?
- 39. Antibodies Renal cell carcinoma immunotherapy – ADCC related G250 monoclonal antibody-based 3rd phase of clinical trials Shuch-BJU-08
- 40. Tumor uptake of VII/20 MAb HT-29-Luc cells, Luciferin i.p. MAb-Alexa Fluor 750
- 41. Internalization – yes or not? 1 V II/2 0 M A b C A IX 2 o r re cy pt e cl rec in g P 3 ? 6 ly e n d o s ar o e m e li g n nd 4 a io t d e g ra d a lo w p H ly s osom e 5 nd e osom e lig a n d a t io n ad nd a gr re c e e p to r d n u cle us cyto p la sm VII/20 Zatovicova et al, Curr Pharm Design, 2010
- 42. SUMMARY CA IX – transmembrane CA isoform, high enzyme activity, active site exposed extracellularly Expression associated with tumors Complex regulation, hypoxia as a key factor, HRE near to transcription initiation site Functionally implicated in pH control and cell adhesion, migration, invasion Clinical application as a marker of hypoxia, prognostic indicator , monitoring marker and potential therapeutic target Specific monoclonal antibodies & selective inhibitors as tools for the research and clinical use in cancer detection, prognostication and therapy
- 43. Jaromir Pastorek Eliska Svastova Peter Ditte Miriam Zatovicova Lucia Csaderova Martina Takacova Juraj Kopacek Peter Radvak & other lab members FUNDING Franck Dequiedt EU STRUCTURAL FUNDS University of Liege 6FP EUROXY 7FP METOXIA Claudiu Supuran 6FP CELLCHCECK Univerity of Florence 7FP ENGCABRA Seppo Parkkila COST HypoxiaNet Univerity of Tampere WILEX A.G. BAYER PHARMA METOXIA partners APVV SLOVAK GA
- 44. E. Svastova and S. Pastor ekova N. M asson and P. J. Ratcliffe A. Gör lach T. M . F Connor and P. H. M axwell O. Kr izanova C. M ichiels S. Winning and J. Fandr ey M . Takacova, T. Goliasova K. J. Nytko, D. P. Stiehl, R. H. Wenger and J. Kopacek S. Peeter s, P. Lambin and L. Dubois A. Loboda, H. Was, U. Flor czyk , A. Jozkowicz and J. Dulak B. Reymen and P. Lambin E. O. Petter sen K. J. Williams and S. R. M cKeown E. P. Cummins, S. F. Fitzpatr ick and C. T. Taylor A. Rapisar da, N. Fer and G. M elillo R. Hr stka, P. M üller, P. J. Coates, B. Vojtesek C. T. Supur an T. Kietzmann