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Characterization of the mechanisms by which Carbonic Anhydrase IX facilitates tumour growth and metastasis Vallejo Espi, Maria de Lourdes 2015

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CHARACTERIZATION	  OF	  THE	  MECHANISMS	  BY	  WHICH	  CARBONIC	  ANHYDRASE	  IX	  FACILITATES	  TUMOUR	  GROWTH	  AND	  METASTASIS	  	  by	  	  Maria	  de	  Lourdes	  Vallejo	  Espi	  	  MSc,	  UNAM,	  2010	  BSc,	  BUAP,	  2008	  	  	  A	  THESIS	  SUBMITTED	  IN	  PARTIAL	  FULFILLMENT	  OF	  THE	  REQUIREMENTS	  FOR	  THE	  DEGREE	  OF	  	  DOCTOR	  OF	  PHILOSOPHY	  	  in	  	  The	  Faculty	  of	  Graduate	  and	  Postdoctoral	  Studies	  	  (Biochemistry	  and	  Molecular	  Biology)	  	  	  THE	  UNIVERSITY	  OF	  BRITISH	  COLUMBIA	  (Vancouver)	  	  April	  2015	  	  	  ©	  Maria	  de	  Lourdes	  Vallejo	  Espi,	  2015	  	  	   ii	  Abstract	  The	   presence	   of	   hypoxic	   microenvironments	   in	   solid	   tumours	   is	   a	   marker	   of	   poor	  prognosis	  in	  numerous	  cancer	  types,	  including	  breast	  cancer;	  the	  second	  leading	  cause	  of	  cancer-­‐related	  death.	  Hypoxia	   results	   in	  an	  adaptive	   response	   in	   tumour	  cells	   through	   the	  activation	  of	   the	  transcription	   factor	   Hypoxia	   Inducible	   Factor-­‐1α	   (HIF-­‐1α),	   which	   stimulates	   the	  expression	  of	  a	  large	  number	  of	  genes	  that	  contribute	  to	  tumour	  progression.	  One	  of	  the	  most	  prominently	  activated	  genes	  is	  Carbonic	  Anhydrase	  IX	  (CAIX),	  which	  facilitates	  the	  acidification	   of	   the	   extracellular	   space	   and	   cell	   invasion	   by	   producing	   protons.	  Moreover,	  it	  assists	  in	  keeping	  the	  intracellular	  space	  neutral	  through	  the	  generation	  of	  bicarbonate,	   which	   is	   shuttled	   into	   the	   cytoplasm	   by	   bicarbonate	   transporters,	  ultimately	  favouring	  cell	  survival.	  CAIX	  facilitates	  breast	  tumour	  growth	  and	  metastasis;	  however	   the	   exact	   mechanism	   remains	   unknown.	   The	   overexpression	   of	   CAIX	   in	  hypoxic	   solid	   tumours,	   its	   limited	  expression	   in	  normal	   tissue	  and	   the	  presence	  of	  an	  extracellular	  catalytic	  domain	  makes	  this	  protein	  an	  excellent	  therapeutic	  target.	  	  The	  intent	  of	  this	  thesis	  was	  to	  unveil	  the	  mechanisms	  by	  which	  CAIX	  facilitates	  tumour	  progression,	   and	   to	   characterize	   novel	   small	   molecule	   inhibitors	   and	   antibodies	  targeting	  CAIX.	  	  It	  was	   found	   that	   the	   intracellular	   (IC)	  domain	  of	  CAIX	   regulates	   its	   catalytic	   activity,	  which	   is	   required	   for	   cell	   survival,	   cell	   invasion	   and	   metastasis.	   The	   extracellular	  proteoglycan-­‐like	   (PG-­‐like)	   domain	   of	   CAIX	   does	   not	   regulate	   CAIX	   catalytic	   activity;	  however	  it	  does	  modulate	  cell	  migration,	  invasion	  and	  metastasis.	  I	  identified	  a	  role	  of	  CAIX	   in	   promoting	   tumour	   cell	   invasion	   through	   interaction	   with	   membrane-­‐bound	  matrix	  metalloprotease-­‐14	  (MMP-­‐14)	  and	  localization	  in	  invadopodia.	  The	  IC	  domain	  of	  CAIX	   mediates	   this	   interaction	   and	   CAIX	   enzymatic	   activity	   appears	   to	   regulate	   the	  ability	  of	  MMP-­‐14	  to	  degrade	  type	  I	  collagen	  during	  cell	  invasion.	  	  From	   the	   pool	   of	   anti-­‐CAIX	   inhibitors	   and	   antibodies	   characterized	   in	   this	   thesis,	   the	  inhibitor	   U-­‐104	   was	   excellent	   at	   blocking	   CAIX	   enzymatic	   activity	   and	   has	   entered	  	   iii	  phase	  I	  clinical	  trials.	  Likewise,	  anti-­‐CAIX	  antibody	  MM-­‐26	  blocked	  50%	  of	  CAIX	  activity	  and	  induced	  cell	  death	  in	  vitro.	  The	  work	  described	  here	  provides	  new	   insight	   into	   the	  mechanism	  of	  CAIX-­‐mediated	  tumour	   invasion	  and	  metastasis	  and	  has	   identified	  two	  new	  therapeutic	  strategies	   for	  targeting	  CAIX.	  	   iv	  Preface	  This	   thesis	   is	  presented	   in	  six	  chapters.	  All	  of	   the	  experiments	  described	   in	  Chapter	  3	  were	  designed	  by	  Dr.	  Shoukat	  Dedhar,	  Dr.	  Paul	  McDonald	  and	  myself	  and	  carried	  out	  by	  myself.	  	  The	  flow	  enrichment	  experiments	  described	  in	  Chapter	  4	  were	  performed	  by	  the	  Flow	  Cytometry	  Core	  Facility	  (FCCF)	  housed	  in	  the	  Terry	  Fox	  Laboratory	  of	   the	  B.C.	  Cancer	  Research	  Center	  (BCCRC).	  Dr.	  Shawn	  Chafe	  and	  Dr.	  Eiko	  Kawamura	  helped	  me	  with	  the	  set	   up	   of	   the	   Flow	   Cytometer	   for	   membrane-­‐bound	   CAIX	   analysis	   and	   Cell	   Cycle	  analysis,	  respectively	  (Chapter	  4,	  Figures	  4.4	  and	  4.13).	  Dr.	  Shawn	  Chafe	  generated	  the	  4T1	   knockdown	   cell	   lines	   (4T1shCAIX,	   Chapter	   4,	   Figure	   4.2)	   that	   I	   utilized	   for	   the	  overexpression	   of	   WT	   huCAIX	   and	   mutant	   forms.	   Dr.	   Mykola	   Maydan	   generated	   the	  CAIX	   constructs	   (Chapter	   4,	   Figure	   4.1).	   Dr.	   Shawn	   Chafe	   also	   generated	   the	   CAIX	  knockdown	  in	  MDA-­‐MB-­‐231	  LM2-­‐4,	  BxPC-­‐3	  and	  Pk-­‐8	  cell	  lines	  (Chapter	  5,	  Figure	  5.1).	  Mass	  spectrometry	  experiments	  described	  in	  Chapter	  4	  were	  performed	  in	  Dr.	  Leonard	  Foster’s	  laboratory	  at	  the	  Centre	  for	  High-­‐Throughput	  Biology	  (CHiBi)	  in	  UBC.	  	  Dr.	   Yuanmei	   Lou,	   Jordan	   Gillespie	   and	   Christina	   Ostlund	   performed	   the	   in	   vivo	  experiments	  described	  in	  Chapter	  5.	  All	  animal	  studies	  and	  procedures	  were	  performed	  in	  accordance	  with	  protocols	  approved	  by	  the	  Institution	  Animal	  Care	  Committee	  at	  the	  BC	  Cancer	  Research	  Centre	  and	  University	  of	  British	  Columbia	  (Vancouver,	  BC,	  Canada)	  as	   per	   protocol	   number:	   A14-­‐0058	   and	   under	   the	   project’s	   title:	   “Targeting	   Carbonic	  Anhydrase	  IX	  and	  hypoxia	  for	  the	  diagnosis	  and	  treatment	  of	  aggressive	  breast	  cancer”.	  	  Dr.	  Kevin	  Bennewith	  and	  Nancy	  E.	  LePard	  carried	  out	  the	  clonogenic	  experiments	  from	  the	   spontaneous	   metastasis	   assay	   (Figure	   5.1)	   and	   Dr.	   Shawn	   Chafe	   and	   myself	  performed	  the	  clonogenic	  experiments	  from	  the	  experimental	  metastasis	  assay	  (Figure	  5.2).	  	  The	   real	   names	   of	   the	   novel	   antibodies	   developed	   by	   SignalChem	   Lifesciences	  Corporation	   and	   described	   in	   Chapter	   3	   have	   been	   hidden	   for	   patenting	   reasons	   and	  alternative	  names	  are	  used	  instead.	  	   v	  All	  other	  experiments	  were	  designed	  by	  Dr.	  Shoukat	  Dedhar	  and	  myself	  and	  carried	  out	  by	  me.	  	  	   	  	   vi	  Table	  of	  Contents	  Abstract	  .................................................................................................................................................................	  ii	  Preface	  ..................................................................................................................................................................	  iv	  Table	  of	  Contents	  ..............................................................................................................................................	  vi	  List	  of	  Tables	  ........................................................................................................................................................	  x	  List	  of	  Figures	  ....................................................................................................................................................	  xi	  List	  of	  Abbreviations	  .....................................................................................................................................	  xiv	  Acknowledgements	  ....................................................................................................................................	  xviii	  Dedication	  .........................................................................................................................................................	  xix	  Chapter	  1.	  Introduction	  ..................................................................................................................................	  1	  1.1	  Breast	  cancer	  ..........................................................................................................................................	  1	  1.1.1	  Classification	  ..................................................................................................................................	  1	  1.1.2	  Risk	  factors	  ......................................................................................................................................	  2	  1.1.3	  Epidemiology	  .................................................................................................................................	  2	  1.1.4	  Models	  for	  studying	  breast	  cancer	  ........................................................................................	  3	  1.2	  Pancreatic	  cancer	  ..................................................................................................................................	  4	  1.2.1	  Classification	  ..................................................................................................................................	  4	  1.2.2	  Epidemiology	  .................................................................................................................................	  4	  1.2.3	  Models	  for	  studying	  pancreatic	  cancer	  ...............................................................................	  5	  1.3	  Hallmarks	  of	  cancer	  .............................................................................................................................	  5	  1.3.1	  Cell	  migration,	  invasion	  and	  metastasis	  .............................................................................	  6	  1.3.1.1	  EMT	  ............................................................................................................................................	  7	  1.3.1.2	  Different	  types	  of	  cell	  migration	  ...................................................................................	  8	  1.3.1.2.1	  Collective	  migration	  ...................................................................................................	  8	  1.3.1.2.2	  Individual	  migration	  ..................................................................................................	  8	  1.3.1.3	  pH	  and	  migration	  ..............................................................................................................	  13	  1.3.1.4	  The	  role	  of	  MMPs	  in	  tumour	  cell	  invasion	  .............................................................	  14	  1.3.1.5	  pH	  and	  invasion	  .................................................................................................................	  15	  1.3.1.6	  Invadopodia	  and	  tumour	  cell	  invasion	  ....................................................................	  16	  1.4	  Hypoxia,	  HIF-­‐1α	  and	  pH	  homeostasis	  .......................................................................................	  21	  1.4.1	  Hypoxia	  and	  the	  hypoxia-­‐inducible	  factor-­‐1α	  (HIF-­‐1α)	  ...........................................	  21	  1.4.2	  pH	  homeostasis	  in	  the	  tumour	  cell	  ....................................................................................	  24	  1.5	  CAIX	  .........................................................................................................................................................	  27	  1.5.1	  CAIX	  structure	  ............................................................................................................................	  27	  1.5.2	  CAIX	  expression	  and	  tissue	  distribution	  .........................................................................	  30	  1.5.3	  CAIX	  function	  ..............................................................................................................................	  31	  1.5.4	  Proposed	  role	  of	  each	  domain	  in	  the	  regulation	  of	  CAIX	  enzymatic	  activity	  ..	  33	  1.5.5	  CAIX	  role	  in	  the	  regulation	  of	  intracellular	  pH	  .............................................................	  34	  1.5.6	  CAIX	  role	  in	  cell	  survival	  and	  proliferation	  ....................................................................	  34	  1.5.7	  CAIX	  role	  in	  cell	  adhesion,	  migration	  and	  invasion	  ....................................................	  35	  1.5.8	  CAIX	  and	  cell	  signalling	  ...........................................................................................................	  36	  1.5.9	  CAIX	  binding	  partners	  .............................................................................................................	  36	  1.5.10	  CAIX	  role	  in	  tumour	  progression	  and	  metastasis	  .....................................................	  37	  1.5.11	  CAIX	  as	  a	  therapeutic	  target	  ..............................................................................................	  38	  	   vii	  1.6	  CAIX-­‐specific	  small	  molecule	  inhibitors	  ..................................................................................	  38	  1.6.1	  CAIX	  inhibitors	  currently	  available	  ...................................................................................	  38	  1.6.1.1	  Previous	  characterization	  of	  U-­‐104,	  GC-­‐205	  and	  SLC-­‐148,	  -­‐149	  and	  -­‐150	  .................................................................................................................................................................	  40	  1.7	  Anti-­‐CAIX	  monoclonal	  antibodies	  ...............................................................................................	  41	  1.7.1	  Mechanisms	  of	  tumour	  cell	  killing	  by	  antibodies	  ........................................................	  41	  1.7.2	  Pros	  and	  cons	  of	  antibodies	  as	  cancer	  therapy	  ............................................................	  42	  1.7.3	  Anti-­‐CAIX	  antibodies	  currently	  available	  .......................................................................	  43	  1.7.4	  Novel	  anti-­‐CAIX	  antibodies	  ...................................................................................................	  44	  1.8	  Objectives	  and	  hypothesis	  .............................................................................................................	  45	  1.8.1.	  Determine	  the	  mechanism	  by	  which	  CAIX	  is	  facilitating	  tumour	  growth	  and	  metastasis	  ................................................................................................................................................	  45	  1.8.2.	  Characterize	  novel	  CAIX	  small	  molecule	  inhibitors	  and	  antibodies	  ..................	  46	  1.8.3	  Hypothesis	  ....................................................................................................................................	  46	  Chapter	  2.	  Materials	  and	  Methods	  ..........................................................................................................	  47	  2.1	  Cell	  culture,	  antibodies	  and	  reagents	  ........................................................................................	  47	  2.2	  Generation	  of	  stable	  cell	  lines	  .......................................................................................................	  48	  2.3	  Analysis	  of	  protein	  expression	  .....................................................................................................	  49	  2.4	  Analysis	  of	  CAIX	  membrane	  localization	  .................................................................................	  50	  2.5	  Immunofluorescence	  ........................................................................................................................	  50	  2.6	  CAIX	  activity	  assay	  ............................................................................................................................	  51	  2.6.1	  In	  vitro	  activity	  assay	  (recombinant	  CAIX)	  ....................................................................	  51	  2.6.2	  CAIX	  “In-­‐cell”	  activity	  assay	  ...................................................................................................	  52	  2.6.3	  Calculation	  of	  extent	  of	  inhibition	  ......................................................................................	  52	  2.7	  Binding	  of	  FITC-­‐CAI	  ..........................................................................................................................	  53	  2.8	  Migration	  and	  invasion	  assays	  .....................................................................................................	  53	  2.9	  Cell	  cycle	  analysis	  ..............................................................................................................................	  54	  2.10	  Growth	  curves	  ..................................................................................................................................	  54	  2.11	  MTT	  assays	  .........................................................................................................................................	  55	  2.12	  Immunoaffinity	  purification	  followed	  by	  protein	  identification	  by	  mass	  spectrometry	  ...............................................................................................................................................	  55	  2.12.1	  Immunoprecipitation	  using	  CNBr	  beads	  ......................................................................	  55	  2.12.2	  Immunoprecipitation	  using	  A/G	  sepharose	  beads	  ..................................................	  56	  2.13	  Apoptosis	  assays	  .............................................................................................................................	  57	  2.14	  Statistical	  analysis	  ...........................................................................................................................	  57	  Chapter	  3.	  Characterization	  of	  Novel	  CAIX	  Inhibitors	  and	  Antibodies	  ...................................	  58	  3.1	  Synopsis	  .................................................................................................................................................	  58	  3.2	  Inhibitory	  capacities	  of	  small	  molecules	  U-­‐104	  and	  GC-­‐205	  ..........................................	  60	  3.3	  Inhibitory	  capacities	  of	  U-­‐104	  derivatives	  SLC-­‐148,	  SLC-­‐149	  and	  SLC-­‐150	  ............	  64	  3.4	  Specificity	  and	  function-­‐blocking	  capacities	  of	  novel	  anti-­‐CAIX	  polyclonal	  antibody	  359-­‐1	  and	  its	  derivatives	  ....................................................................................................	  70	  3.5	  Specificity	  and	  function-­‐blocking	  capacities	  of	  novel	  anti-­‐CAIX	  monoclonal	  antibodies	  538-­‐542	  ..................................................................................................................................	  81	  3.6	  Specificity	  and	  function-­‐blocking	  capacities	  of	  novel	  anti-­‐CAIX	  monoclonal	  antibody	  MM-­‐26	  .........................................................................................................................................	  90	  3.7	  Discussion	  .............................................................................................................................................	  98	  	   viii	  3.7.1	  Novel	  CAIX	  small	  molecule	  inhibitors	  ..............................................................................	  98	  3.7.2	  Novel	  CAIX	  antibodies	  ...........................................................................................................	  100	  3.7.2.1	  KalGene	  Pharmaceuticals	  anti-­‐CAIX	  antibodies	  ................................................	  100	  3.7.2.2	  Deeley-­‐UVic	  anti-­‐CAIX	  antibodies	  ...........................................................................	  103	  3.7.2.3	  SignalChem	  Lifesciences	  anti-­‐CAIX	  antibody	  .....................................................	  104	  Chapter	  4.	  Characterization	  of	  CAIX	  Mutants	  ..................................................................................	  107	  4.1	  Synopsis	  ...............................................................................................................................................	  107	  4.2	  Expression	  and	  localization	  of	  human	  CAIX	  mutants	  ......................................................	  108	  4.3	  Activity	  of	  human	  CAIX	  mutants	  ...............................................................................................	  118	  4.4	  Cell	  survival	  and	  proliferation	  of	  human	  CAIX	  mutants	  .................................................	  124	  4.5	  Cell	  migration	  of	  human	  CAIX	  mutants	  ..................................................................................	  131	  4.6	  Intracellular	  binding	  partners	  of	  human	  CAIX.	  ...................................................................	  133	  4.7	  Discussion	  ...........................................................................................................................................	  138	  4.7.1	  Expression	  and	  localization	  ................................................................................................	  138	  4.7.2	  Activity	  .........................................................................................................................................	  140	  4.7.3	  Cell	  survival	  and	  proliferation	  ...........................................................................................	  142	  4.7.4	  Migration	  .....................................................................................................................................	  144	  4.7.5	  Identification	  of	  binding	  proteins	  by	  immunoaffinity	  purification	  followed	  by	  mass	  spectrometry.	  ...........................................................................................................................	  145	  Chapter	  5.	  Hypoxia-­‐induced	  Carbonic	  Anhydrase	  IX	  Regulates	  Invasion	  and	  Metastasis	  by	  Interacting	  with	  and	  Activating	  MMP-­‐14	  within	  Invadopodia	  ..........................................	  148	  5.1	  Synopsis	  ...............................................................................................................................................	  148	  5.2	  Introduction	  .......................................................................................................................................	  148	  5.3	  Materials	  and	  methods	  ..................................................................................................................	  150	  5.3.1	  Cell	  culture	  and	  antibodies	  .................................................................................................	  150	  5.3.2	  Cloning	  of	  CAIX	  truncation	  and	  point	  mutants	  ...........................................................	  151	  5.3.3	  Generation	  of	  stable	  cell	  lines	  ............................................................................................	  151	  5.3.4	  Analysis	  of	  protein	  expression	  ..........................................................................................	  152	  5.3.5	  Invasion	  assays	  .........................................................................................................................	  152	  5.3.6	  Mouse	  tumour	  models	  ..........................................................................................................	  153	  5.3.7	  Gelatin	  zymography	  ...............................................................................................................	  154	  5.3.8	  Collagen	  degradation	  .............................................................................................................	  154	  5.3.9	  Invadopodia	  formation	  and	  imaging	  ..............................................................................	  154	  5.3.10	  Co-­‐immunoprecipitation	  ...................................................................................................	  155	  5.3.11	  Statistical	  analysis	  ................................................................................................................	  156	  5.4	  Results	  ..................................................................................................................................................	  156	  5.4.1	  CAIX	  is	  required	  for	  breast	  and	  pancreatic	  tumour	  cell	  invasion	  and	  metastasis	  ..............................................................................................................................................	  156	  5.4.2	  The	  intracellular	  and	  proteoglycan-­‐like	  domains	  of	  CAIX	  are	  required	  for	  tumour	  invasion	  and	  metastasis	  ..................................................................................................	  158	  5.4.3	  CAIX	  regulates	  MMP-­‐14-­‐mediated	  type	  I	  collagen	  invasion	  and	  degradation	  ....................................................................................................................................................................	  160	  5.4.4	  CAIX	  co-­‐localizes	  with	  MMP-­‐14	  in	  mature	  invadopodia	  ........................................	  163	  5.4.5	  CAIX	  and	  MMP-­‐14	  interact	  through	  the	  CAIX	  intracellular	  domain	  .................	  166	  5.5	  Discussion	  ...........................................................................................................................................	  170	  Chapter	  6.	  Conclusions	  and	  Future	  Directions	  ................................................................................	  175	  	   ix	  6.1	  Novel	  CAIX	  antibodies	  and	  small	  molecules	  inhibitors	  ...................................................	  175	  6.2	  Characterization	  of	  CAIX	  mutants	  ............................................................................................	  177	  6.3	  Role	  of	  CAIX	  in	  cell	  invasion	  ........................................................................................................	  181	  References	  .......................................................................................................................................................	  184	  	   	  	   x	  List	  of	  Tables	  Table	  3.1	  Inhibitory	  constants	  of	  CAIX	  inhibitors	  U-­‐104	  and	  GC-­‐205	  ...................................	  60	  Table	  3.2	  Inhibitory	  constants	  of	  CAIX	  inhibitors	  SLC148-­‐150	  ................................................	  64	  Table	  4.1	  List	  of	  proteins	  identified	  by	  mass	  spectrometry	  as	  binding	  partners	  of	  human	  CAIX	  ..................................................................................................................................................................	  136	  	   	  	   xi	  List	  of	  Figures	  Figure	  1.1	  Epithelial-­‐mesenchymal	  transition	  ....................................................................................	  7	  Figure	  1.2	  Different	  types	  of	  cell	  migration	  .......................................................................................	  10	  Figure	  1.3	  The	  five-­‐step	  model	  of	  individual	  cell	  migration	  and	  the	  role	  of	  pH	  ................	  12	  Figure	  1.4	  The	  steps	  of	  invadopodia	  formation	  ...............................................................................	  19	  Figure	  1.5	  Intracellular	  pH	  regulation	  in	  the	  tumour	  cell	  ...........................................................	  26	  Figure	  1.6	  Representation	  of	  CAIX	  in	  the	  cell	  membrane	  ............................................................	  28	  Figure	  3.1	  Function-­‐blocking	  abilities	  of	  novel	  CAIX	  inhibitors	  U-­‐104	  and	  GC-­‐205	  ........	  63	  Figure	  3.2	  Function-­‐blocking	  abilities	  of	  novel	  CAIX	  inhibitors	  SLC-­‐148,	  -­‐149	  and	  -­‐150	  ...............................................................................................................................................................................	  66	  Figure	  3.3	  Dose-­‐response	  of	  U-­‐104	  and	  SLC-­‐149	  ...........................................................................	  68	  Figure	  3.4	  Percent	  inhibition	  of	  CAIX	  activity	  by	  U-­‐104	  and	  SLC-­‐149	  ...................................	  69	  Figure	  3.5	  Specificity	  of	  novel	  anti-­‐CAIX	  antibodies	  359-­‐1	  and	  360-­‐1	  ..................................	  72	  Figure	  3.6	  Immunoprecipitation	  abilities	  of	  novel	  anti-­‐CAIX	  antibody	  359-­‐1	  ...................	  74	  Figure	  3.7	  Immunofluorescence	  staining	  abilities	  of	  novel	  anti-­‐CAIX	  antibody	  359-­‐1	  .......	  	  ...............................................................................................................................................................................	  76	  Figure	  3.8	  Function-­‐blocking	  capacity	  of	  novel	  anti-­‐CAIX	  antibody	  359-­‐1	  .........................	  78	  Figure	  3.9	  Specificity	  of	  novel	  anti-­‐CAIX	  antibodies	  4H1-­‐4L1	  and	  16H1	  .............................	  80	  Figure	  3.10	  Specificity	  of	  novel	  anti-­‐CAIX	  antibodies	  538-­‐542	  ................................................	  82	  Figure	  3.11	  Immunofluorescence	  staining	  abilities	  of	  novel	  anti-­‐CAIX	  antibodies	  538-­‐542	  .......................................................................................................................................................................	  84	  Figure	  3.12	  Specificity	  of	  antibodies	  539	  and	  541	  towards	  CAIX	  ............................................	  86	  Figure	  3.13	  Specificity	  of	  antibodies	  539	  and	  541	  towards	  the	  catalytic	  domain	  of	  CAIX	  ...............................................................................................................................................................................	  87	  Figure	  3.14	  Immunoprecipitation	  abilities	  of	  novel	  anti-­‐CAIX	  antibodies	  539	  and	  541	  ....	  	  ...............................................................................................................................................................................	  88	  Figure	  3.15	  Function-­‐blocking	  abilities	  of	  novel	  anti-­‐CAIX	  antibodies	  538-­‐542	  ..............	  90	  Figure	  3.16	  Epitope	  determination	  of	  anti-­‐CAIX	  antibodies	  MM-­‐26	  and	  MM-­‐04	  .............	  92	  Figure	  3.17	  Function-­‐blocking	  ability	  of	  novel	  anti-­‐CAIX	  antibody	  MM-­‐26	  ........................	  94	  	   xii	  Figure	  3.18	  In-­‐cell	  CAIX	  function-­‐blocking	  ability	  of	  novel	  anti-­‐CAIX	  antibody	  MM-­‐26	  .....	  	  ...............................................................................................................................................................................	  96	  Figure	  3.19	  Anti-­‐CAIX	  antibody	  MM-­‐26-­‐mediated	  induction	  of	  cell	  death	  ..........................	  98	  Figure	  4.1	  Diagram	  of	  human	  CAIX	  mutants	  ..................................................................................	  109	  Figure	  4.2	  Knockdown	  of	  mouse	  CAIX	  in	  4T1	  cells	  .....................................................................	  110	  Figure	  4.3	  Expression,	  dimerization	  and	  localization	  of	  human	  CAIX	  mutants	  ..............	  113	  Figure	  4.4	  Percent	  of	  human	  CAIX	  present	  at	  the	  cell	  membrane	  ........................................	  115	  Figure	  4.5	  Co-­‐staining	  of	  human	  CAIX	  and	  the	  ER	  .......................................................................	  117	  Figure	  4.6	  Contribution	  of	  each	  domain	  to	  the	  activity	  of	  CAIX:	  in	  vitro	  assay	  ...............	  119	  Figure	  4.7	  Contribution	  of	  each	  domain	  to	  the	  activity	  of	  CAIX:	  In-­‐cell	  assay	  .................	  121	  Figure	  4.8	  CAIX	  activity	  in	  4T1shNS	  and	  4T1shCAIX	  cell	  lines:	  binding	  of	  FITC-­‐CAI	  ...........	  	  ............................................................................................................................................................................	  122	  Figure	  4.9	  Contribution	  of	  each	  domain	  to	  the	  activity	  of	  CAIX:	  binding	  of	  FITC-­‐CAI	  .........	  	  ............................................................................................................................................................................	  123	  Figure	  4.10	  The	  intracellular	  domain	  of	  CAIX	  regulates	  cell	  survival	  .................................	  125	  Figure	  4.11	  Cell	  viability	  and	  proliferation	  of	  4T1	  cells	  expressing	  human	  CAIX	  mutants	  ............................................................................................................................................................................	  127	  Figure	  4.12	  Growth	  curves	  of	  4T1	  cells	  expressing	  human	  CAIX	  mutants	  .......................	  128	  Figure	  4.13	  Cell	  cycle	  analysis	  of	  4T1	  cells	  expressing	  human	  CAIX	  mutants	  ................	  130	  Figure	  4.14	  Contribution	  of	  each	  domain	  of	  CAIX	  to	  the	  regulation	  of	  cell	  migration	  .........	  	  ............................................................................................................................................................................	  132	  Figure	  4.15	  Immunoprecipitation	  of	  human	  CAIX	  from	  4T1sh/WT	  huCAIX	  and	  4T1sh/ΔIC	  cell	  lines	  ..................................................................................................................................	  134	  Figure	  5.1	  CAIX	  is	  required	  for	  cell	  invasion	  and	  metastasis	  ..................................................	  157	  Figure	  5.2	  Intracellular	  (IC)	  and	  proteoglycan-­‐like	  (PG-­‐like)	  domains	  of	  CAIX	  regulate	  cell	  invasion	  and	  metastasis	  	  .................................................................................................................	  159	  Figure	  5.3	  CAIX	  regulates	  MMP-­