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
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
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
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
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,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
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eman )CAPUI( citametsyS
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eruliaF traeH evitsegnoC 6.2
atad lacinilC amedE decudnI-gurD 7.2
)SU(C )UA(3B .tac ycnangerP stceffe-ediS 3
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yrtsimehC 5
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secnerefeR 6
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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
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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