UC Berkeley UC Berkeley Electronic Theses and Dissertations Title Development of a Breast Cancer Stem Cell Model and the Inhibitory Regulation of Small Molecule Phytochemicals on Various Stages of Human Breast Cancer Cells Permalink https://escholarship.org/uc/item/0fw3v9x3 Author Tin, Antony Shen Publication Date 2013 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California Development  of  a  Breast  Cancer  Stem  Cell  Model  and  the   Inhibitory  Regulation  of  Small  Molecule  Phytochemicals   on  Various  Stages  of  Human  Breast  Cancer  Cells     By       Antony  S.  Tin  II         A  dissertation  submitted  in  partial  satisfaction  of  the     Requirements  for  the  degree  of     Doctor  of  Philosophy     In     Endocrinology       In  the       Graduate  Division       Of  the     University  of  California,  Berkeley     Committee  in  charge:     Professor  Gary  L.  Firestone,  Chair   Professor  Jen-­‐Chywan  (Wally)  Wang   Professor  Iswar  K.  Hariharan       Spring  2013 ABSTRACT     Development  of  a  Breast  Cancer  Stem  Cell  Model  and  the  Inhibitory  Regulation  of   Small  Molecule  Phytochemicals  on  Various  Stages  of  Human  Breast  Cancer  Cells       By     Antony  S.  Tin  II     Doctor  of  Philosophy  in  Endocrinology     University  of  California,  Berkeley     Professor  Gary  L  Firestone,  Chair         The  development  of  clinical  breast  cancer  is  a  multistep  process  that  manifests  itself  as  a   disease  with  various  distinct  phenotypes.    At  present,  it  is  believed  that  a  neoplastically-­‐ transformed  cell  undergoes  many  heterogeneous  changes  and  mutations  before  evolving   into  a  tumorous  or  invasive  breast  cancer.      Due  to  the  heterogeneity  of  breast  cancer,  a   single   therapeutic   strategy   is   rarely   completely   effective   for   all   patients.     Current   therapeutic  options  such  as  hormone  antagonists,  radiation  and  chemotherapy  have  many   deleterious   side   effects   which   demonstrates   a   need   for   a   more   efficacious   therapy   alternative  that  can  not  only  target  varying  phenotypes  but  also  ameliorates  harsh  side   effects.    In  addition,  it  is  essential  to  identify  compounds  that  can  slow  the  promotion  of  the   disease   from   preneoplastic   lesions   to   invasive   breast   cancer.     This   thesis   details   the   generation  of  a  novel  breast  cancer  stem  cell  model  and  the  molecular  mechanism  of  two   small   molecule   phytochemicals,   indole-­‐3-­‐carbinol   (I3C),   and   artemisinin,   target   breast   cancer  stem  cells  and  luminal  A,  estrogen  sensitive  breast  cancer,  respectively.      We  show   that  ectopic  expression  of  HER2,  a  member  of  the  epidermal  growth  factor  receptor  family,   in   MCF-­‐10AT   preneoplastic   mammary   epithelial   cells   induces   a   phenotype   with   the   molecular  markers  of  breast  cancer  stem  cells  expression:  CD44+/CD24-­‐/ALDH-­‐1+  with   highly  expressing  levels  of  stem  cell  marker,  nucleostemin.      These  cells  are  capable  of   forming  tumorspheres  in  3-­‐D  cultures,  indicative  of  a  population  highly  enriched  with  stem   cells.    Furthermore,  as  few  as  30,000  cells  are  able  to  form  viable  mammary  tumors  in   athymic  mouse  xenograft  models.    In  breast  cancer  stem  cells,  I3C  induces  apoptosis  by   selectively  targeting  nucleostemin  and  activating  the  p53  apoptotic  pathway.  I3C  induces   the  proteolytic  degradation  of  Akt1  and  thereby  functionally  inactivates  MDM2.    Loss  of   MDM2  phosphorylation  and  its  subsequent  inactivation  frees  p53  to  induce  apoptosis.     Coupled  with  the  activation  of  nucleostemin  by  I3C,  nucleostemin  sequesters  MDM2  away   from  p53  further  enhancing  the  apoptotic  effect.    Knockdown  of  nucleostemin  prevents  the   I3C  apoptotic  effects,  suggesting  the  selective  role  of  I3C  on  breast  cancer  stem  cells.    Also,   expressing  constitutively  active  Akt1  or  a  dominant  negative  form  of  p53  overrides  the  I3C   induced  apoptotic  effect,  highlighting  the  specific  Akt1/MDM2/p53  pathway  modulated  by   I3C.    The  preclinical  results  implicate  I3C  as  a  novel  anti-­‐cancer  agent  that  can  selectively   1 target   cancer   stem   cells   especially   given   that   I3C   also   increases   MDM2-­‐nucleostemin   interactions  and  can  reduce  tumors  volumes  in  vivo.  We  also  show  that  artemisinin,  derived   from  the  sweet  wormwood,  Artemisia  annua,  ablates  key  G1  cell  cycle  regulators  to  induce   growth  arrest  in  luminal  A,  estrogen  sensitive  breast  cancer  as  well  as  inhibit  in  vivo   xenograft  growth  in  athymic  mice.    Artemisinin  is  selective  towards  malignant  cells,  such  as   the   MCF-­‐7   breast   cancer   cell   line   since   it   is   ineffective   in   arresting   growth   in   nontumorigenic  breast  cell  lines.    Artemisinin  exposed  MCF-­‐7  cells  displayed  ablated  levels   of  cyclin-­‐dependent  kinases  2  and  4  (CDK),  cyclin  E,  cyclin  D  as  well  as  E2F1  at  the  protein   1 and   mRNA   level.     Promoter   deletion   mapping   and   subsequent   chromatin   immunoprecipitation  analyses  revealed  that  downregulation  of  E2F1  resulted  in  inhibition   of  CDK2  of  promoter  activity.    Additionally,  constitutive  expression  of  E2F1  reversed  the   growth  arrest,  CDK2  and  cyclin  E  downregulation  induced  by  artemisinin.               2 ACKNOWLEDGEMENTS       I  would  like  to  thank  Professor  Gary  Firestone  for  mentoring  me  on  so  many  different   levels  throughout  my  tenure  in  graduate  school.    I  will  never  forget  that  you  took  me  in   without  hesitation  after  Professor  Timiras  passed  away  and  willingly  gave  me  a  lab  to  call   home.    Gary,  you  have  been  more  than  a  mentor  to  me,  you  have  been  a  friend  and  a  guide   who  has  always  encouraged  me  to  think  critically.    You  have  provided  me  with  the  perfect   balance  of  independence,  while  still  given  me  direction  and  support.    I  am  still  learning   valuable  lessons  from  you  about  research  and  life  and  am  looking  forward  to  using  all  the   skills  you  have  taught  me  for  the  rest  of  academic  and  professional  career.    Thank  you  for   always  being  there  for  me.           I  would  especially  like  to  thank  my  loving  family  for  always  supporting  me  and  being  there   through  everything.    Daddy,  you  have  always  encouraged  me  to  pursue  my  dreams  and   have  given  me  the  world.    You  showed  me  the  true  meaning  of  life  and  exposed  me  to  so   many  different  things.    I  can  honestly  say  you  provided  me  with  all  the  creature  comforts  in   life.    After  all  the  talks  and  advice  you  have  given  me,  I  am  just  starting  to  appreciate  and   understand  the  meaning  of  it  all.    Mommy,  there  are  no  words  to  express  how  much  I  love   you  and  just  knowing  how  much  you  believe  in  me  has  helped  me  get  to  where  I  am  today.     Ever  since  I  was  a  baby,  you  have  taught  me  to  stand  up  for  myself  and  cared  for  me  in  a   way  nobody  else  ever  could.    I  know  you  always  put  me  first  and  I  cannot  begin  to  express   the  way  I  feel  for  your  tireless  efforts.    Pearl,  I  want  to  especially  thank  you  for  always   calling  me  and  being  there  for  me  on  a  moments  notice.    You  have  been  my  emotional   support  and  have  given  me  so  much  perspective  by  teaching  me  the  bigger  picture  in  life.    I   cannot  imagine  having  a  better  older  sister  knowing  how  you  paved  the  way  for  me  in  all   aspects  of  my  life.    I  want  to  say  thank  you  from  the  bottom  of  my  heart  and  I  cannot  stop   thinking  about  how  you  used  to  save  me  your  notes  from  high  school  so  I  could  use  it  two   years  later  or  making  sure  that  I  kept  my  social  life  in  check  during  college  and  helping  me   in  my  professional  life  after  graduate  school.    You  are  the  strongest  person  I  know  and   knowing  that  you  are  there  for  me  helps  me  sleep  at  night.    You  truly  are  an  amazing   person  and  I  will  never  forget  that.    Julia,  I  want  to  thank  you  for  taking  care  of  me  and   raising  me  as  your  own  son.      I  will  always  be  your  son  and  will  always  remember  all  things   you  did  when  raising  me  to  keep  me  happy  like  take  me  to  IHOP  on  my  birthday  and   making  me  cucumbers  to  eat  and  our  walks  at  lake  Chabot.               Shyam  –  I  can’t  express  how  much  I  appreciate  everything  you  have  done  for  me.    Without   you,  I  literally  would  not  know  what  to  do.    You  gave  me  my  projects  that  have  led  me  to  my   thesis  and  PhD  and  it  all  started  because  of  you.    I  know  I  will  use  all  the  skills  you  have   taught  me  and  despite  the  nonstop  drama,  I  wanted  to  thank  you  for  being  the  voice  inside   my  head  telling  me  what  to  do  but  also  keeping  me  sane  and  motivated  to  work.     Kevin  –  You  are  the  one  that  got  me  through  grad  school.    I  can  never  thank  you  enough  for   keeping  me  sane  from  team  estrogen!    I  will  never  forget  the  time  you  helped  me  with  all   the  PCRs  right  before  the  poster  session  or  the  crazy  “adventures”  at  Granlibakken.    It  was   great  being  with  you  in  lab  from  the  very  start  and  you  are  one  of  the  most  thoughtful   i people  I  know.    I  am  so  grateful  to  be  able  to  call  you  my  friend  knowing  you  always  have   my  back.    I  don’t  know  what  to  do  now  that  I  am  leaving  and  won’t  have  you  around  to  help   me  with  all  my  math  questions.       Kalvin  –  We  started  together  and  now  we  are  ending  together!      You  are  my  brother  from   another  mother.    After  all  those  late  nights  complaining  about  the  work  Shyam  put  on  us   has  finally  paid  off  for  the  both  of  us.    Thank  you  for  all  your  help  and  tireless  effort  to  make   the  Ishikawa  and  MCF-­‐7  Art  project  come  to  fruition.  You  are  truly  an  amazing  person  and  I   know  that  you  will  be  rewarded  in  all  your  endeavors.     Anna  –  This  is  for  you.    You  have  been  everything  to  me  and  to  my  life.    I  cannot  imagine  a   world  without  you  being  there  to  support  me  mentally,  emotionally,  and  physically.    I  will   always  think  back  to  the  first  time  I  met  you  at  USC  and  how  I  immediately  knew  that  this   would  be  the  start  of  something  epic.    AT&T  PARK.    (ANNA  TONY  TIN  PARK)!    Without  you,   this  thesis,  my  PhD  would  not  be  possible.    You  have  played  an  integral  part  of  my  academic   career  and  somehow  everything  always  goes  back  to  science.    I  will  miss  just  getting  a  meal   with  you  where  we  end  up  talking  about  the  science  behind  every  signaling  pathway  or   how  cells  differentiate  or  what  genes  do  what.    I  don’t  think  anyone  else  knows  me  better   than  you  and  can  relate  to  me  the  way  you  have.    You  really  changed  my  life  for  the  better   and  helped  keep  me  motivated  in  all  aspects  of  my  life.    Everything  from  exercising  and   doing  a  marathon  and  triathlon,  to  turning  me  into  a  kimchi  connoisseur  where  I  now  love   Korean  food  more  than  Chinese  food…this  would  not  have  happened  without  you.    You  are   the  most  amazing  person  that  I  have  ever  met  and  I  want  you  to  know  that  you  are  the   smartest,  most  caring,  and  selfless  person  I  know.    I  was  so  proud  of  you  graduated  with   honors  and  then  when  you  got  accepted  into  graduate  school.    You  are  a  winner  and  I  know   that  you  will  succeed  in  anything  you  put  your  mind  to  and  that  your  dedication  and   sincerity  will  get  you  very  far  in  life.    You  deserve  to  have  it  all  and  I  have  no  doubt  that  you   will  be  rewarded  for  all  your  hard  work.    I  want  to  thank  you  for  staying  by  my  side  for  so   long  and  enduring  all  the  late  nights,  inconsistencies,  contradictions  and  difficulties  that   you  handle  with  grace  and  poise.         In  addition,  I  would  also  like  to  thank  all  the  colleagues  in  the  Firestone  lab  that  has  been   some  of  my  best  of  friends  I  have  ever  had.    Besides  all  the  help  and  input,  you  guys  kept   my  life  fun  and  I  will  never  forget  all  our  happy  hour  adventures  and  lunches.    You  were  all   sources  of  my  inspiration.                         ii This  work  is  dedicated  to  my  uncle         Pang  Pen  Sheng         From  the  fond  memories  of  playing  with  you  in  the  park  and  riding  the  steam  trains  or   hiking  up  the  mountain  to  watch  the  view,  I  am  constantly  thinking  about  you.    I  miss  you   so  much.    You  raised  me  as  your  own  son  and  the  unconditional  love  and  support  will   forever  inspire  me.    Thank  you  for  always  believing  in  me  and  even  now  still  watching  over   me.                                   ii  i TABLE  OF  CONTENTS       List  of  Figures  ...….…………….….............……….………….………………………………………………………....v   General  Introduction  ………………………………………………………..............…………………………...vii         Chapter  I   Development  of  a  novel  model  system  to  study  breast  cancer  stem  cells...……..................1             Abstract  ……………………….......………………........................................…….......…………........2         Introduction…………………………….………………………………........……………………........  3     Materials  and  Methods……………….………………………………….......………………….......5     Results.…………….….……………………………………………………………………………...........7         Discussion………………………………………………………………………………………...........  30     References…………………………………………………………………………………….........….  32     Chapter  II   Essential  role  of  nucleostemin  in  indole-­‐3-­‐carbinol  anti-­‐proliferative  targeting  of   breast  cancer  stem  cells…………………………………………………………………………………................  35     Abstract………………………………………………………………………………………….............36     Introduction…………………………………………………………………………………...............37     Materials  and  Methods……………………………………………………………………............39       Results……………………………………………………………………………………………...........42               Discussion………………………………………………………………………………………............78     References………………………………………………………………………………………...........80   Chapter  III   Artemisinin  antiproliferative  response  in  human  breast  cancer  cells  requires  the   down-­‐regulated  expression  of  the  E2F1  transcription  factor  and  loss  of  E2F1-­‐target   cell  cycle  genes………………………………………………………………………………........................................82       Abstract………………………………………………………………………………………….............83     Introduction…………………………………………………………………………………...............84     Materials  and  Methods……………………………………………………………………............86           Results……………………………………………………………………………………………...........90               Discussion……………………………………………………………………………………….........125         References………………………………………………………………………………………........127             Chapter  IV   Conclusion  and  Future  Directions...........................................................................................................131     Conclusion  and  Future  Directions...............................................................................132                           iv LIST  OF  FIGURES       General  Introduction  Figures     Figure  1:     Classical  model  of  tumor  progression……………………………………..…....................ix           Figure  2:     The  cancer  stem  cell  model……………………………………..………………......................xii   Figure  3:     Phosphotyrosine  interactome  of  the  epidermal  growth  factor  receptor   kinase  family…………………………………….……………………....................................…...xvi           Figure  4:     The  HER2  signaling  pathway………………………………………..……...….....................xix             Figure  5:     Indole-­‐3-­‐Carbinol  (I3C)………………………….............……………..……...…..................xxii         Figure  6:     Postulated  anticancer  mechanism  of  action  by  artemisinin………….….......xxvi               Chapter  I  Figures     Figure  7:     Characterization  of  CSC  markers  by  HER2  overexpression  in  MCF-­‐10AT   cells……………………………………………..……………………………………..............................9                   Figure  8:     Quantification  of  cell  surface  marker  CD44  and  CD24…………..………..........…..12         Figure 9 Quantification  of  ALDH-­‐1  activity  in  10AT-­‐Her2  and  10AT-­‐Neo  cells.............14             Figure  10:     Formation  of  tumorspheres  by  10AT-­‐Her2  cells  in  vitro………………….............17         Figure  11:     Comparing  10AT-­‐Her2  tumorsphere  forming  efficiency..…..………….............…29   Figure  12:     I3C  inhibition  of  tumorsphere  formation  in  10AT-­‐Her2  cells.…….............……  22   Figure  13:     I3C and 1-benzyl-I3C selectively disrupts cancer stem cell tumorsphere formation  …………………………………………………………………………………............……24     Figure  14:     Tumor-­‐forming  efficiency  between  10AT-­‐Her2  and  10AT-­‐Neo  cells  .............27             Figure  15:     I3C  inhibition  of  10AT-­‐Her2  Tumor  Xenografts….…………..…………................…29             Chapter  II  Figures     Figure  16:     I3C  inhibits  10AT-­‐Her2  cell  proliferation.…………………………............…………....44           Figure  17:     Flow  cytometry  analysis  of  the  I3C  induced  apoptotic  effects  on       10AT-­‐Her2  cells..........…………………..……………………......................................…………46     Figure  18:     I3C  induces  PARP  cleavage  in  10AT-­‐Her2  breast  cancer  stem  cells….............48             Figure  19:     I3C  induced  apoptotic  response  is  mediated  by  p53…………………….................51           Figure  20:     I3C  induces  nucleostemin-­‐mdm2  interactions  in  cancer  stem  cells….............54           Figure  21:     I3C  releases  p53  from  its  E3  ligase,  MDM2  in  cancer  stem  cells…….................56   Figure  22:     I3C  induced  MDM2  translocation  into  the  nucleus  requires  nucleostemin   expression……………………………….………………….................................................………59   Figure  23:     MDM2  translocates  into  the  nucleus  and  colocalizes  with  nucleostemin  foci   upon  I3C  treatment…………………………........................................................………….....62       Figure  24:     Nucleostemin  knockdown  diminishes  I3C  induced  release  of  p53  from   MDM2………………………………………………......................................................………….....65   Figure  25:     Nucleostemin  siRNA  overrides  the  I3C  induced  apoptosis  in  cancer  stem   cells  ………………………………………………………………………………….............................67         Figure  26:     I3C  downregulates  Akt1  expression  at  the  protein  level  ………...................……70           v