Oncogenic miRNAs and the Perils of Losing Control of a Stem Cell’s Epigenetic Identity

One of the fundamental traits of malignant tumors is  their  capacity  to  grow  indefi- nitely,  beyond the  natural  limits  usually observed  for most  normal,  differentiated cells.  This property, often  referred to  as immortality,  was  among the  very  first to be   recognized  by   cellular   oncologists and  is  regarded as  a  fundamental hall- mark    of   the    transformed   phenotype (Hanahan  and   Weinberg,  2011).  In  the eye  of the  stem cell  biologist,  however, the  capacity  for  extensive  growth   and expansion does not constitute, in and  of itself, a  pathological trait.  By  definition, stem cells  are  selectively endowed with the capacity to self-renew: they preserve intact their ability for long-term expansion over    multiple rounds of sequential divisions,  and serve  as a constant source of mature cells throughout the lifetime of an   organism.  If   considered  from   this perspective, therefore,  immortality  could be  interpreted as  a  pathological form  of self-renewal,  in  which  the  normal   con- straints that regulate stem cell expansion and  ensure homeostatic control  of tissue size  have  been disabled as  the  result  of oncogenic   mutations   (Dalerba   et   al.,

2007). Our ability to test  this concept has  been  limited  by  an  incomplete  under- standing of the  basic molecular circuitry that   defines  the   epigenetic  identity   of normal stem cells and the degree to which this circuitry is either mirrored  or hijacked by cancer cells.

In  an  impressive  tour   de   force  that combines evidence from  two  indepen- dent  studies (Song et al., 2013a, 2013b), Song  and colleagues have  now identified miR-22 to be both  a new regulator of the self-renewal machinery and  a  powerful oncogene that  directly  targets multiple members  of  the   ‘‘ten-eleven-transloca- tion’’   (TET) protein   family,  a   group   of enzymes involved in DNA demethylation. In their  first  study  (Song et  al.,  2013b), the   authors   examined  the   effects   of constitutive  miR-22   overexpression  on the   breast  epithelium   using   transgenic mice  engineered to  achieve constitutive expression of miR-22 in

mammary epithe- lial cells. Their results indicate that miR-22 overexpression causes epithelial   cells to    acquire   biochemical    features   of the  epithelial-to-mesenchymal  transition (EMT), such  as downregulation of E-cad- herin  and  upregulation of Zeb1.  In  addi- tion, miR-22 overexpression is associated with upregulation  of  the  Bmi1 oncogene and   an   increase  in  the   frequency   of normal  mammary stem/progenitor  cells, which   can   reconstitute   full   mammary epithelial  trees in transplantation assays. These  changes are  followed  by  sponta- neous                neoplastic                transformation         of normal  mouse breast epithelia  into meta- static   breast  carcinomas.   When   com- bined  with  additional  oncogenic insults, such   as  when  miR-22  transgenic mice are crossed with MMTV-PyVT or MMTV- neu transgenic mice, constitutive miR-22 overexpression  accelerates both   tumor progression and  metastasis. Finally, high levels  of miR-22  expression are  associ- ated with high-grade tumors and reduced survival in breast cancer patients.

In   their   second  study   (Song   et   al.,

2013a), the authors used a similar trans- genic  approach to investigate the effects of constitutive miR-22 overexpression on mouse hematopoietic cells. In this second case,  however,  miR-22  overexpression was   not  specific  to  the   hematopoietic system and  the  authors decided  to test  the  effects of increased  miR-22  dosage using transplantation assays. Constitutive miR-22  overexpression  augmented  the proliferative   capacity  of  hematopoietic stem/progenitor  cells  (HSPCs),  causing them  to  progressively  outcompete their wild-type  counterparts in cotransplanta- tion   experiments.  When   observed   for longer    periods   of   time,    transplanted HSPCs overexpressing miR-22 gave  rise to  a  disease reminiscent of a  myelodis- plastic   syndrome  (MDS),  which  subse- quently  progressed to  full-blown  acute myeloid  leukemia  (AML). As  observed in the  case of breast cancer, high levels  of miR-22 expression were  associated with reduced survival in human  MDS patients. In both  the  mammary  epithelium   and the  hematopoietic system, the  biological effects  of  miR-22  were  mediated by  its capacity to  suppress  expression  of  TET family members. TET  proteins are  DNA hydroxylases  that convert 5-methylcyto- sine    into  5-hydroxymethylcytosine to initiate    DNA   demethylation   (Wu    and Zhang, 2011). Indeed, genetic inactivation of   TET  proteins   is   known   to   disrupt the  epigenetic  remodeling that  accom- panies  normal  differentiation processes, and     TET   mutations   are    commonly observed in human hematological malig- nancies.  Similar  to  constitutive  miR-22 overexpression,  genetic   inactivation   of Tet2 in mice is associated with a numeri-  cal  expansion of HSPCs and  neoplastic transformation (Cimmino et al., 2011). 

In  a  fascinating  set   of   experiments, Song    and   collaborators  also   showed that  constitutive miR-22  overexpression is associated  with  hypermethylation and epigenetic   silencing    of   the   miR-200c promoter. This is accompanied  by upre- gulation  of Bmi1,  a  key  member of the Polycomb  group   (PcG)   protein    family and  a  core  element of  the  self-renewal machinery  in  both   hematopoietic   and mammary   epithelial    stem   cells   (Park et al., 2003; Pietersen et al., 2008). These findings  are  consistent  with  previous re- ports  that  identified human  miR-200c as a  direct  repressor of  BMI1, limiting the expansion and   tumorigenicity  of  breast cancer cells (Shimono et al., 2009). Impor- tantly,   the   effects   of   miR-22   on   the expression  of  miR-200c  and   Bmi1  are mediated through a  direct  interaction of miR-22  with  TET  mRNAs  and   can   be reproduced   in  a   line   of   immortalized mammary  epithelial    cells   by   shRNA- mediated knockdown of TET2 and  TET3. These observations provide  fundamental mechanistic insights  into  developmental biology in that  they explain  how different arms   of  the  molecular  machinery  that shapes  the  epigenetic identity  of  stem cells  work together in an integrated sys- tem to control  the capacity to self-renew. Members of the  TET family act  as  initia- tors of DNA demethylation while Bmi1, a member  of   the    Polycomb    repressor complex 1  (PRC1),  regulates  chromatin remodeling through specific histone mod- ifications  such  as ubiquitination of lysine-

119 of histone-2A. Both systems oversee the   coordinated   regulation  of  multiple gene  expression programs during  differ- entiation. Learning  how these epigenetic pathways interact is a  fundamental step toward understanding how even relatively subtle  genetic  manipulations   (e.g.   the constitutive  expression  of  one   miRNA) can  ‘‘ripple’’ into  profound perturbations of   stem  cell   homeostasis  and   cause cancer.

In   our   opinion,   however,  the   most compelling  finding   that   emerges  from the  aggregate work  of Song  and  collab-  orators   is    that   chromatin-remodeling systems with  opposing  effects  on  cell identity  (self-renewal  versus  differentia- tion) appear to  directly  antagonize each other  through opposing sets of miRNAs (e.g.  miR-22  versus miR-200c).  A  series of  theoretical questions  thus  arises.  If chromatin-remodeling systems   directly antagonize each other  as  part  of a  dy- namic equilibrium  between  self-renewal and differentiation, what  tilts the balance toward one fate or the other? Under phys-  iological conditions, what makes changes in  stem cell  identity  (i.e.,  differentiation) irreversible? The  answer to  these ques- tions  lies  in  a more  advanced, systems- level  understanding of  these  molecular circuitries  and  in a  deeper characteriza- tion  of their  positive  and  negative feed- back  loops.  For  example, are  members of the  Polycomb family able  to  regulate miR-22  expression? If  so,  do  they  posi-  tively   affect   miR-22   expression,  thus

‘‘locking’’ the  stem cell identity in a self- reinforcing  loop,  or do  they  suppress it, thus  ‘‘limiting’’ the stem cell identity  in a cell-autonomous manner? The challenge for  the   future   will be  to  develop  new experimental   approaches,  and   mathematical   algorithms,  to  model   the  inte- grated action  of these  complex relation-  ships    and   their   impact  on   cell   fate (Sahoo, 2012).