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The RB Gene Family in Cell Cycle Control and Cancer The RB Gene

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The RB Gene Family in Cell Cycle Control and Cancer The RB Gene Powered By Docstoc
					                       St. Jude Special Seminar:                                                                          St. Jude Special Seminar:
                             28 April 2005                                                                                      28 April 2005



              The RB Gene Family in Cell                                                                   The RB Gene Family in Cell
               Cycle Control and Cancer                                                                     Cycle Control and Cancer




                                                                                                   Julien Sage, PhD
                              Julien Sage, PhD                                                     Pediatrics and Genetics
                                                                                                   Stanford Medical School




                                                                                                         Mouse model for RB mutation in
                                                                                                                  humans
                                                                                        mice with Rb+/- and Rb-/- cells
                                                                                                                                      pituitary tumors: 100%
                                                                                                                                         LOH at Rb locus


        Why do RB patients develop retinoblastoma




                                                                                                    thyroid tumors: ~50%
 How does RB normally prevent tumorigenesis?                                                          LOH at Rb locus


  Why do RB patients develop retinoblastoma?
                                                                                                                                                0% retinoblastoma




                                                                                        Complexity of mammalian cell cycle regulation

                                                                                                                           p15, p16, p18, p19
                                                        QuickTime™ and a
                                                    TIFF (LZW) decompressor
                                                 are needed to see this picture.




                                                                                                                          CycD1,D2,D3/CDK4,6
                                                                                                     M


        Why do RB patients develop retinoblastoma
 Why do RB patients develop     Why do RB mutant mice develop                                                              pRB, p107, p130
                                                                                                                                pRB                       CycE1,E2/CDK2
                                                                                   G2                        G1
      retinoblastomas?                pituitary tumors?
                                 and why not retinoblastomas?

                                                                                                                             E2F1, 2, 3, 4, 5       ARF       p53    p21
                                                                                               S
Differences in cell cycle regulatory networks around Rb?

                                                                                                                                    APOPTOSIS




                                                                                                                                                                           1
                                                                    The RB gene family
                                                                                                                                                                 The pRB family pathway of G1/S regulation

                                                                                      viral oncoproteins binding
                                                                                            domain (LXCXE)                                                                                                             p16

                                                                                                  S
                                                                                            A          B
                                                                                                 E2F                                                                                                           CycD/CDK4
                                                                                  N                            C                                                                             M

                                                                                                pRB
                                                                                                                                                                                                               X X X
                                                                                                                                                                                                              pRB,p107,p130

                    p107                                                                                                                 p130                           G2                          G1

                                             S                                                                               S                                                                                         E2F
                                     A           B                                                                    A            B
                            N             E2F                                     C                           N              E2F         C                                                   S


                          Similarities between pRB, p107, and p130                                           Similarities between p107 and p130




                                    Overlapping properties of pRB family                                                                                             TKO MEFs do not enter quiescence
                                     proteins in cell cycle progression
                                                                                                                                                                                       confluent cells in 0.1% serum
                                                                                                                                                                                                    p107-/-
                                                                                                                                                                   wt                                                                            Rb-/-
                                                                                                                                                                                                    p130-/-
                                                                                 1500
                                                                                                                                                     FACS - PI



                                                                                                wt
                                                                                                wt
                                                                                                p107-/-
                           Mutant ES cells                                                      130/107
                                                       Number of cells (x10-3)




                                                                                 1200           p130-/-
                                                                                                Rb-/-
                                                                                                Rb
                                                                                                TKO
                                                                                 900
Mutant chimeric embryos
                                                                                 600
                                                                                                                                                                    G1 S G2

                                                                                 300
                                                                                                                                                                                      TKO                      TKO (attached)
                      Mutant mouse
                    embryonic fibroblasts
                                                                                      0
                                                                                          day 1 day 2 day 3 day 4 day 5 day 6 day 8



                                                      Increased proliferation in Rb-/-;p107-/-;p130-/- TKO MEFs




                            p107 levels are up-regulated in quiescent Rb-/- MEFs                                                                                    Inhibition of p107 expression in quiescent
                                                                                                                                                                 germline Rb mutant MEFs leads to cell cycle entry

                                                                                                                                                                                     Rb-/-
                                                                                                                      Quiescent Rb-/- MEFs
Number of cells (x10-3)




                          900        wt
                                     wt
                                                 Proliferating MEFs                                                                                                        si:   -      p107                                          Quiescent Rb-/- MEFs
                                     Rb-/-
                                     Rb
                          600                                                                                                                                           p107                                                          80
                                                                                                                                                                                                                 BrdU incorporation
                                                                                                                                                                                                                   GFP+ cells (%)




                                                                                                                                                                        p130
                                                                                                                                                                                                                                      60
                          300
                                                                                                                                                                                                                                      40
                           0                                                                                                                                             GFP                 p107
                                day 1 day 2 day 3 day 4 day 5 day 6                                                                                                                                                                   20

                                                                                                                                                    Lentivirus                                                                         0
                                    Rb-/-        wt                                                                  Rb-/-         wt                                                                                                                     si
                                                                                                                                                      GFP                                                                                   si
                                                                                                                                                                                                                                                         p107
                                                                                                                                             pRB
                                                                                                                                                    Lentivirus                                                                             24 hours BrdU
                                                                                 p107
                                                                                                                                             p107     GFP
                                                                                                                                                     si-p107
                                                                                 ARPP P0                                                     p130
                                                                                                                       Quiescent                                     Quiescent Rb-/- MEFs




                                                                                                                                                                                                                                                                2
                                                                                                                    Inhibition of p107 expression in quiescent
 Acute loss of Rb in quiescent Rb2lox/2lox MEFs
                                                                                                                 germline Rb mutant MEFs leads to cell cycle entry

                                                                                                                                     Rb-/-
                                                                                                                           si:   -      p107                                                            Quiescent Rb-/- MEFs
                             60
                                  Ad                                                                                    p107                                                                            80
      % BrdU incorporation




                                                                                                                                                                                   BrdU incorporation
                             50




                                                                                                                                                                                     GFP+ cells (%)
                                  Ad-Cre
                                                                                                                        p130
                                                                                                                                                                                                        60
                             40
                                                                                                                                                                                                        40
                             30
                                                                                                                         GFP                 p107
                                                                                                                                                                                                        20
                             20
                                                                                   0.1% serum                                                                                                            0
                             10                                                                          Lentivirus
                                                                                   24 hours BrdU                                                                                                                      si
                                                                                                           GFP                                                                                                si
                                                                                   3 days after Ad-Cre                                                                                                               p107
                             0
                                       Rb-/-                  Rb2lox/2lox                                                                                                                                    24 hours BrdU
                                                                                                         Lentivirus
                                                                                                           GFP
                                                       loxP                 loxP                          si-p107
                                                                     3
                                                                                                                      Quiescent Rb-/- MEFs




 Acute loss of Rb in quiescent Rb2lox/2lox MEFs                                                                                                                  Model


                                                                                                           Rb                    adaptation
                                                                                                         mutation                     (p107, …)
                             60
                                  Ad
      % BrdU incorporation




                             50   Ad-Cre                                                                                                                                Quiescence
                             40
                                                                                                                                                                       (Senescence)
                                                                                                                                                                                                             acute loss of Rb
                             30                                                                                                    more
                                                                                                                                 adaptation
                             20                                                                                                                                                  Window of time when
                                                                                   0.1% serum
                             10                                                    24 hours BrdU                                                                                  cells are sensitive
                                                                                   3 days after Ad-Cre                                                                                 to Rb loss
                             0
                                       Rb-/-                  Rb2lox/2lox
                                                                                                                          Cells insensitive
                                                       loxP
                                                                     3
                                                                            loxP                                             to Rb loss




                                          Speculations                                                        p130 is the major Rb family member bound to
                                                                                                                         E2F in non-dividing cells

Germline loss of Rb                            No compensation in human retinal cells




                                               Developmental adaptation in other cell types                                                           QuickTime™ and a
                                                                                                                                                  TIFF (LZW) decompressor
                                                                                                                                               are needed to see this picture.




                                                                                                                                                                                                              p130

                                                                                                                                                                                                             E2F4
 Sporadic cancers                              Selective advantage before compensation
                                               occurs (breast, brain, prostate, lung…)



                                                                                                         (Mulligan et al., MCB 1998)




                                                                                                                                                                                                                                3
        Loss of p130 in quiescent MEFs



                          Rb-/-   p130-/-   wt


                                                 p130                                 Are compensatory mechanisms induced by loss of
              quiescent




                                                                                             Rb function also present in vivo?
                                                 p107


                                                 pRB




Functional compensation in Rb-mutant mouse retina
                                                                                        Conclusions: functional compensation


                                                                                         Extensive functional redundancy within the
                                                         0% retinoblastoma!             Rb family in the control of cell cycle in culture
                                                                                                          and in vivo.


             Rb-/+        Rb-/-                                                           Does it play a role in cancer prevention?




                                                 (MacPherson, et al., G&D 1998)




    Chimeras composed of Rb-/-;p107-/- or                                         Retinoblastoma in Nestin-Cre p130-/-;Rb2lox/2lox mice
   Rb-/-;p130-/- cells develop retinoblastoma

                                        pituitary tumors:
                                              100%
      Rb +/- mice
      Rb-/-;p107-/-:+/+                 LOH at Rb locus
      Rb -/- chimera
      Rb-/-;p130-/-:+/+




     thyroid tumors: ~50%
       LOH at Rb locus




                                                                                                                        (MacPherson, et al., G&D 1998)




                                                                                                                                                         4
                               Retinoblastoma: conclusions                                         Analysis of MMTV-Cre Rb2lox/2lox mice


Loss of Rb function is sufficient to cause delays in cell                                                     100

 cycle exit and ectopic proliferation, but not cancer in                                                       90

                                                                                                               80
                   the mouse retina.                                                                           70




                                                                                                    % alive
                                                                                                               60

p107 and p130 compensate for loss of Rb in the mouse                                                           50

   retina and prevent retinoblastoma development.                                                              40
                                                                                                                              Rb2lox2/lox
                                                                                                               30
                                                                                                                              Rb2lox/2lox;p130-/-
                                                                                                               20             Rb2lox/2lox;p107-/-
                                                                                                               10
 Do p107 and p130 prevent cancer development after                                                             0
       loss of Rb function in other cell types?                                                                     0    50       100       150      200   250   300   350   400


                                                                                                                                                    days


                                                                                                          Cause of death = pituitary and thyroid tumors




      Analysis of MMTV-Cre Rb2lox/2lox mice                                                    Testis tumors in MMTV-Cre Rb2lox/2lox;p130-/- mice



                               100
                                90                                        Rb
        % animals with tumor




                                80                                        Rb/p107
                                70
                                                                          Rb/p130
                                60
                                50
                                40
                                30
                                20
                                10
                                 0
                                     Pit.   Thyr. Test. Thym. Hist.
                                                            sarcoma

                                                                                                                                                           Leydig cells




Histiocytic sarcoma in MMTV-Cre Rb2lox/2lox;p107-/- mice                                            Functional overlap in tumorigenesis

                                                                                              Rb-/- :                                                pituitary, thyroid
                                                                                              Rb-/-;p107-/- or Rb-/-;p130-/- :                       retina, thymus
                                                                                              Rb-/-;p107-/- :                                        uroepithelium, histiocytes, skin
                                                                                              Rb-/-;p130-/- :                                        Leydig cells




                                                                                                                        pRB                            p107
                                                                                                                                                        p107       p130
                                                                                                                                                                   p130




                                                       Thickened skin
                                                                        Histiocytic sarcoma                   Proliferation Differentiation Apoptosis




                                                                                                                                                                                        5
            Cancer: conclusions                                                End

 Extensive functional redundancy within the Rb
         family in cancer development.                                Julien Sage, PhD


       Triple knock-out mice in progress.

                                                   More medical education materials are available at:
Anti-cancer therapy in RB-deficient human tumors
   by increasing compensatory mechanisms?

       p107 and p130 in human cancers?
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