Ap2 Alpha

Chemoresistance and Transformation -ABC family- membrane transport protein MDR1-P-gp 48 different ABC transporters extrude many types of drugs from cancer cells, thereby conferring multidrug resistance Is there something common? -Oncogenic signal -Defect in apoptotic pathway -Many drugs induce apoptotic pathways that defects in this pathway also results in multidrug resistancc Myc and ras Myc and bcl-2 Myc and mutant p53 E1A and E1B Increasing our knowledge of the components involved in the pathways that mediate cell death and survival….. Gives us the hope is that targeting specific molecules ( Targeted therapy ) will impart sensitivity to Chemotherapy — a combination therapy is possible Chemoresistance Transformation

Cancer chemotherapeutic drugs that induce apoptosis

A ctivator P rotein 2   (AP-2  ) AP-2 gene family: AP-2  , AP-2  , AP-2  , AP-2δ and AP-2  5’- GCCNNNGGC -3’ 1 437 Activation domain DNA Binding domain helix-span-helix motif Dimerization domain N C

Homozygous deletion for any of the AP-2 genes results in lethality either during embryogenesis or shortly after birth
Implicated in development
Implicated in transformation
AP-2  activated p21 , inhibited DNA synthesis and stable colony formation (Zeng Y-X., Somasundaram, K., and El-Deiry WS Nature Genet ,, 1997) No genetic alterations in AP-2  have been reported Epigenetic silencing ? Progressive loss of expression of these genes has been linked to the progression of human cancers Breast carcinoma (Gee et al., 1999) Colon carcinoma (Ropponen et al., 2001) Melanoma (Several papers from * Bar-Eli’s group) Prostate cancer (Ruiz et al., 2001 Glioma (Bar-Eli’s group)

AP-2  adenovirus (Ad-AP2) makes functional AP-2  protein
-Sequence-specific DNA-binding
(5’- GCCNNNGGC -3)
-AP-2 specific reporter activation
(3X-AP2-CAT)
-Target gene activation
(p21 WAF1/CIP1 )
Ad-LacZ Ad-AP2 SW480 H460 HT1080 A F C D E B

AP-2  inhibits cancer cell growth by inducing cell cycle arrest and apoptosis 0 20 40 60 80 100 120 0 20 40 60 - Ad-LacZ - Ad-AP2 % Viability MOI AP-2 PARP Actin Ad-LacZ Ad-AP2 0 24 48 72 Hrs 0 24 48 72 Hrs 24 48 24 48 %A %G1 %S %G2 2.04 57.32 26.19 14.45 1.08 58.52 25.48 14.92 1.71 82.45 3.24 12.60 42.44 32.51 6.30 16.75 Ad-LacZ Ad-AP2 Virus 24 hr 48 hr Ad-LacZ Ad-AP2 G1 A G2 S S PI-DNA content Brdu-DNA synthesis

Are caspases activated ? If yes, how important they are? How important caspase 3 is? Caspase 8 or 9 or both are important! Apoptosis Two broad pathways that lead to apoptosis: Apoptosis Extrinsic Intrinsic Adapter

Caspase 3, 8 and 9 are activated during AP-2  induced apoptosis Ad-LacZ Ad-AP2 Caspase 9 p46 p35 p18 p55 p42 Caspase 8 p32 p17 Caspase 3 Actin AP-2  PARP p12 p10 p20 0 48 72 Hrs 0 48 72

PAN Caspase inhibitor z-VAD-fmk Essential role of Caspases in AP-2  induced apoptosis Ad-AP2 DMSO z-VAD-fmk AP-2  Actin p32 p17 Caspase 3 p20 PARP 48hrs 72hrs 48hrs 72hrs Ad-LacZ Ad-AP2 Control z-VAD-fmk PI-DNA content

Role of Caspase 3 in AP-2  induced apoptosis MCF7/pv MCF7/c3 Caspase 3 Actin 1 2 MCF-7/pv Ad-LacZ Ad-AP2 24hr 48hr MCF-7/c3 Ad-LacZ Ad-AP2 24hr 48hr PARP MCF7/c3 Ad-LacZ Ad-AP2 Ad-LacZ Ad-AP2 AP-2  Actin 0 48 72 MCF7/pv Hrs 0 48 72 0 48 72 0 48 72 DAPI BrdU Ad-LacZ Ad-AP2 DAPI BrdU MCF7/pv MCF7/c3

Extrinsic pathway Caspase 8 inhibitor z-IETD-fmk Intrinsic pathway Caspase 9 inhibitor z-LEHD-fmk Caspase 9, not 8, is needed for AP-2  induced apoptosis PARP p32 p17 Caspase 3 p20 p55 p42 Caspase 8 p12 p10 Caspase 9 p46 p35 AP-2  Actin DMSO z-IETD-fmk Ad-AP2 AP-2  PARP p55 p42 Caspase 8 p12 p10 Caspase 9 p46 p35 p32 p17 Caspase 3 p20 Actin DMSO z-LEHD-fmk Ad-AP2 Channels (FL2-H) 0 30 60 90 120 150 Number 0 60 120 180 240 48hrs 72hrs 48hrs 72hrs Ad-LacZ Ad-AP2 Control z-IETD-fmk z-LEHD-fmk pCEP4/AP-2  pCEP4 pCEP4/AP-2  + z-IETD-fmk pCEP4/AP-2  + z-LEHD-fmk pCEP4/AP-2  + z-VAD-fmk Empty

? ? Apoptosis Extrinsic Intrinsic Adapter

Role of FADD in AP-2  induced apoptosis -Ad-LacZ -Ad-LacZ/Mock -Ad-LacZ/Lamin siRNA -Ad-LacZ/FADD siRNA -Ad-AP-2 -Ad-AP-2/Mock -Ad-AP-2/Lamin siRNA -Ad-AP-2/FADD siRNA 0 20 40 60 80 100 120 0 10 20 30 40 50 60 % Live cells MOI FADD siRNA Mock Lamin siRNA FADD GAPDH 1 2 3 Lamin Actin FADD FADD siRNA Mock Lamin siRNA 1 2 3 4 Control hrs 48 72 48 72 Ad-LacZ Ad-AP2 1.98 1.37 1.54 2.23 33.49 54.68 31.97 48.62 Lamin siRNA FADD siRNA siRNA % Apoptosis Ad-LacZ Lamin siRNA FADD siRNA Ad-AP2 Lamin siRNA FADD siRNA 48 hrs 72 hrs Channels (FL2-H) 0 30 60 90 120 150 Number 0 40 80 120 160

? Apoptosis Extrinsic Intrinsic Adapter

Role of Apaf-1 in AP-2  induced Apoptosis 0 20 40 60 80 100 120 0 10 20 30 40 50 60 -Ad-LacZ -Ad-LacZ/Mock -Ad-LacZ/Lamin siRNA -Ad-LacZ/Apaf1 siRNA -Ad-AP-2  -Ad-AP-2  /Mock -Ad-AP-2  /Lamin siRNA -Ad-AP-2  /Apaf1 siRNA % Live cells MOI hrs 48 72 48 72 Ad-LacZ Ad-AP2 1.44 1.23 1.79 1.39 27.96 49.48 5.98 6.23 Lamin siRNA Apaf1 siRNA siRNA % Apoptosis Ad-LacZ Lamin Apaf1 Ad-AP2 Lamin Apaf1 48 hrs 72 hrs siRNA Apaf1 siRNA Mock Lamin siRNA Apaf1 GAPDH 1 2 3 Lamin Apaf-1 Actin Control Mock Lamin siRNA Apaf-1 siRNA 1 2 3 4

Apoptosis Extrinsic Intrinsic Adapter

Apoptosis Extrinsic Intrinsic Adapter Mitochondria

Mitochondrial membrane potential Mitochondrial Outer Membrane Permeabilization Pro-apoptotic Bcl-2 member Bax translocates to Mitochondria Cytochrome C is released into cytoplasm

0 10 20 30 40 50 60 70 80 90 100 Control -AP-2  % cells positive for J-aggregates +AP-2  Valinomycin 1 2 3 4 Cyt c  -actin Cytosolic Mitochondrial Ad-AP2 Cytosolic Mitochondrial 0 24 36 48 Bax Oxidative complex 1 0 24 36 48 0 24 36 48 0 24 36 48 Ad-LacZ Hrs Measurement of Mitochondrial Membrane Potential using JC-1 Dye Mitochondria Cytoplasm - - - - - - - - - Nucleus Healthy cell Apoptotic cell JC-1 JC-1

Apoptosis Extrinsic Intrinsic Adapter

Role of Bax in AP-2  induced apoptosis HCT116 Bax -/- HCT116 WT Bax Actin 1 2 Ad-LacZ Ad-AP2 HCT116 Bax -/- HCT116 WT PARP Actin AP-2  0 48 72 0 48 72 0 48 72 0 48 72 Ad-LacZ Ad-AP2 24 hr 48 hr 72 hr HCT116 WT HCT116 Bax -/- Ad-LacZ Ad-AP2 Ad-LacZ Ad-AP2 Ad-AP-2 0 24 48 72 Ad-LacZ Bax Actin Con. Bax AP-2  hrs 0 24 48 72

Bcl-2 family Anti-apoptotic - Pro-apoptotic Multi-domain - BH3-only - Bax Bax Bax Bcl-2 Bcl-2 Bcl-X-S Cell death Cell survival Cell death Bax Bax

Role of Bcl-2 in AP-2  induced apoptosis Bcl-2 AP-2 Actin Ad-LacZ Ad-AP2 hrs 0 24 48 0 24 48 -AP-2  -Bcl-2 -1 0 1 2 3 4 5 6 7 8 Normalized Fold change -2 6 12 24 hrs after Tet removal -3 -2 -1 0 H460 SW480 1 2 Normalized Fold change -Bcl-2 Ad-AP2

AP-2  binds to and represses Bcl-2 promoter 0 100 200 300 400 500 600 700 Luciferase activity (CPM X 1000) Bcl-2 prom-Luc pSG5 pSG5/AP-2  + 5 - + - 5 + 10 - + - 10 + 15 - + - 15 P1+1 P2 +610 AP-2 173 bps (-129 to +44) -1291 bs GST-AP-2 Bcl-2 wt SV40 AP2 wt SV40 AP2 m - - - - 1 + - - - 2 + - - 3 + - - 4 + - - 5 + - - 6 + - - 7 + - - 8 + - - 9 + - - 10 + - - 11 5’-CTAATTTTTACTCC CTC TCCC CGC GACTCCTGA-3’ -30 -62 GCC N(3/4) GGC 5’-CTAATTTTTACTCC tat TCCC aaa GACTCCTGA-3’ Bcl-2 wt Bcl-2 m AP-2 consensus binding motif GST-AP-2 Bcl-2 wt Bcl-2 m + - - 1 + - - 2 + - 3 + - 4 + - 5 + - 6 + - 7 + - 8 Ladder Bcl-2 prom Input Anti-Lamin Anti-AP-2  Bcl-2 1 2 3 4 5

How important Bcl-2 down regulation is? 24 hr 48 hr 72 hr SW480/Neo Ad-LacZ Ad-AP2 Ad-LacZ Ad-AP2 SW480/Bcl-2 # 3 Bcl-2 Actin SW480/Neo SW480/Bcl-2 # 3 SW480/Bcl-2 # 15 1 3 4 SW480/Neo 0 48 72 Ad-LacZ Ad-AP2 AP-2  Actin PARP 0 48 72 0 48 72 0 48 72 1 2 3 4 5 6 7 8 9 10 11 12 Ad-LacZ Ad-AP2 SW480/Bcl-2 # 3 Hrs Vector Bcl-2 AP-2  AP-2  + Bcl-2

Caspase 3 is essential
Intrinsic pathway
Caspase 8 and FADD adapter not needed
Caspase 9 and Apaf1 are essential
Mitochondrial membrane potential is lost
Bax translocates to mitochondria
Cytochrome c is released from mitochondria
Bax is essential
AP-2  binds to Bcl-2 promoter and represses
its transcription

Apoptosis Intrinsic AP-2  How does AP-2  induce apoptosis? Bcl-2 Bax Bax Bax

Does AP-2  has any role in cancer cell Chemosensitivity ?

p53 50% of primary tumor have mutated p53 Need for identification of other determinants Major chemosensitivity determinant Gets activated upon DNA damage and induces apoptosis  irradiation Chemotherapy

Chemosensitivity of cancer cells over expressing AP-2  - Chemodrug - Ad-LacZ + Chemodrug - Ad-AP2 + Chemodrug 0 20 40 60 80 100 120 Adriamycin Etoposide Cisplatin Taxol Carboplatin Percent IC 50 Cells O/N Mock/ Ad-LacZ/ Ad-AP-2 6 hrs Add chemo 48 hrs % Live cells

Tet-Off system Transcription is turned off by tet tTA expressing adenovirus tTA – tetracyclin controlled transactivator - Tet AP-2  under Tet-responsive element P min CMV TRE AP-2  tTA active pCMV tetR VP16 tTA Tet bound tTA Tet + Tet inactive

Controlled expression of AP-2  increases the cancer cell chemosensitivity AP-2  tTA + + + + + + SW480-1 SW480-2 SW480-3 1 2 3 4 5 6 Tet + - + - + - -Chemodrug -Chemodrug + tTA + Tet -Chemodrug + tTA + 0.1  g Tet 0 20 40 60 80 100 120 Percent IC 50 Adriamycin Etoposide Cisplatin Taxol Carboplatin Ad-tTA + Tet Ad-tTA % A % S % S % A Hrs 0 12 24 48 40.28 44.85 38.75 37.20 40.28 13.36 5.71 2.35 2.64 1.01 1.40 0.25 2.64 0.54 3.34 36.02 0.001 tTA 1 0.1 0.01 - Tet (  g/ml) + + + + + AP-2  Actin 1 2 3 4 3X-AP2CAT pSG5 pSG5/AP2 tTA Tet + + - - - + - + - - + - - + + + - - + - % CAT Conversion 0 5 10 15 20 25 AP-2 binding sites CAT 3X AP2-CAT

AP-2  expression sensitizes cells to undergo apoptosis upon chemotherapy Adria tTA Tet IC25 IC50 B + - - + + 1  g + + 0.1  g Control/ 0 hr tTA - + Tet - 0.1  g MOCK AP-2  A G1 G2 A S S

AP-2  expression sensitizes cells to undergo apoptosis upon chemotherapy Adria tTA Tet - - - - + 0.1  g 0 5 10 15 20 25 30 35 40 45 1 2 3 4 5 6 7 8 IC 25 - - IC 50 - - IC 25 + 1.0  g IC 50 + 1.0  g IC 25 + 0.1  g IC 50 + 0.1  g AP-2  Adria Adria Adria+ AP-2  - % <G1 - % S % Apoptosis/ % DNA synthesis 0 10 20 30 40 50 60 70 Chemo tTA Tet - - - - + 0.1  g IC 25 - - IC 50 - - IC 25 + 1.0  g IC 50 + 1.0  g IC 25 + 0.1  g IC 50 + 0.1  g AP-2  Chemo Chemo Chemo+ AP-2  - Cisplatin - Taxol - Etoposide 1 2 3 4 5 6 7 8 % Apoptosis

What is the role of endogenous AP-2  in chemosensitivity ? Taxol Cisplatin Adriamycin 0 24 48 72 hrs AP2 Actin Etoposide AP2 Actin AP2 Actin AP2 Actin 48 hrs GPDH AP-2  72 hrs Untreated Adriamycin Cisplatin Etoposide Taxol Marker Untreated Adriamycin Cisplatin Etoposide Taxol

What is the role of Chemotherapy induced AP-2  in Cancer cell chemosensitvity? Chemotherapy induced AP-2  contributes to chemosensitivity - Control - Mock - Lamin siRNA - AP-2  siRNA 0 10 20 30 40 50 60 70 80 90 Etoposide Adria Cisplatin Taxol % Viability GAPDH AP-2 Control Mock siRNA LaminA/C siRNA AP-2 1 2 3 4 Lamin A/C - Mock siRNA Lamin A/C siRNA AP-2  Control AP-2 Actin - + + + + Adria 1 2 3 4 5 AP-2 Actin - + + + + Cisplatin AP-2 Actin - + + + + Taxol

Chemotherapy induced AP-2  contributes to chemosensitivity Plate cells Transfect siRNA Add Adria Stain the colonies O/N 2 days 2 weeks Control Mock Lamin/ siRNA AP-2  siRNA 0 0.1 0.2 0.4 0.8 1 Adriamycin  g/ml

In cell culture, silenced AP-2  is re-expressed by methylation inhibitor (5-aza-2 deoxycytidine) AP-2  expression and Breast Cancer progression Effect of re-expression of silenced AP-2  on chemosensitivity ? AP-2  expression + + - Normal breast epithelium Ductal carcinoma in situ (DCIS) Invasive breast tumors Hypermethylation of AP-2  promoter - + 75% (12/16) (Douglas et al., 2004) + 16% (3/19)

5aza2dC induced re-expression of epigenetically silenced AP-2  in MDA-MB-231 cells increases the chemosensitivity and is AP-2  dependent - + - + SW480 MDA-MB-231 5aza2dC AP-2 Actin 1 2 3 4 MDA-MB-231 - Mock Lamin siRNA AP-2  siRNA Control - + + + + 5aza2dC 1 2 3 4 5 AP-2 Actin 0 10 20 30 40 50 60 70 1 2 3 4 5 Chemotherapy 5aza2dC Mock Lamin siRNA AP-2  siRNA                         % Viability - Adriamycin - Cisplatin

5aza2dC fails to induce apoptosis in AP-2  siRNA transfected MDA- MB-231 cells upon chemotherapy Control Mock Lamin siRNA AP-2  siRNA 2.0 1.4 1.2 0.8 5.4 7.3 5.4 6.3 0.7 0.7 0.2 0.8 64.7 56.4 57.2 13.9 % Apoptosis Adria 5aza2dC - - + - - + + + Control Mock Lamin siRNA AP-2  siRNA AP-2  PARP Actin Control Mock Lamin siRNA AP-2  siRNA Control Mock Lamin siRNA AP-2  siRNA Control Mock Lamin siRNA AP-2  siRNA 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 - adria + adria - adria + adria + 5aza2dC - 5aza2dC -Adria +Adria + 5aza2dC AP-2  siRNA Control Mock Lamin siRNA +Adria - 5aza2dC -Adria Channels (FL2-H) 0 30 60 90 120 150 Number 0 100 200 300 400 500

5aza2dC treatment inhibits the tumorigenicity of MDA-MB-231 cells upon chemotherapy in an AP-2  dependent manner 0 1.0 2.0 3.0 4.0 Tumor volume (1000 X mm 3 ) Days 5.0 6.0 0 5 10 15 20 8.0 7.0 25 30
- Control
- 5aza2dC
Adria
Mock + Adria + 5aza2dC
Lamin siRNA + Adria + 5aza2dC
AP-2  siRNA + Adria + 5aza2dC
MDA-MB-231

5aza2dC treatment inhibits the tumorigenicity of MDA-MB-231 cells upon chemotherapy in an AP-2  dependent manner Treatment No of tumors/ Mean volume (%) No of mice mm 3 ± SE Control 3/3 6426 ± 1118 100 5aza2dC 3/3 5100 ± 291 79 Adria 3/3 2348 ± 1172 37 Mock + 5aza2dC + Adria 0/4 0 ± 0 0 Lamin siRNA + 5aza2dC+ Adria 0/4 0 ± 0 0 AP-2  siRNA + 5aza2dC + Adria 5/5 1832 ± 300 29

AP-2  overexpression increases the chemosensitivity of cancer cells Conclusions 5aza2dC induced re-expression of AP-2  in breast cancer cells increases chemosensitivity and inhibits tumorigenicity upon chemotherapy Chemotherapy induces endogenous AP-2  , which contributes to chemosensitivity AP-2  sensitizes cancer cells undergo apoptosis upon chemotherapy

AP-2  inhibits cancer cell growth by inducing cell cycle arrest and apoptosis Wajapeyee and Somasundaram, 2003 JBC 0 20 40 60 80 100 120 0 20 40 60 - Ad-LacZ - Ad-AP2 % Viability MOI Hrs 24 48 24 48 %A %G1 %S %G2 2.04 57.32 26.19 14.45 1.08 58.52 25.48 14.92 1.71 82.45 3.24 12.60 42.44 32.51 6.30 16.75 Ad-LacZ Ad-AP2 Virus 24 hr 48 hr Ad-LacZ Ad-AP2 G1 A G2 S S PI-DNA content Brdu-DNA synthesis

Apoptosis Intrinsic Apoptosis induction-two pathways Wajapeyee and Somasundaram, 2006 JBC Extrinsic Adapter AP-2  Bcl-2 Bax Bax Bax How does AP-2  induce apoptosis?

How does AP-2  inhibit cell cycle progression?

Normal cells: HEL299 cells – human normal lung fibroblasts 0 24 48 24 48 >G1 G1 S G2/M hr 4.30 4.58 2.17 3.78 1.59 53.10 54.46 54.26 70.90 75.86 18.56 20.36 19.61 9.08 6.34 23.76 19.73 25.02 16.60 16.20 Mock Ad-LacZ Ad-AP2 Virus Cell cycle profile Ad-LacZ Ad-AP2 DAPI AP-2  BrdU a b c d e f Brdu incorporation

Serum Starvation -48hrs Serum Stimulation (Release) 0 hr Virus infection -16 FACS & Western +24 hrs FACS & Western +18 hrs Mock Release 0 hrs Release 18 hrs Release 24 hrs G1: 81.70 S: 9.21 G2: 9.09 G1: 73.68 S: 9.74 G2: 16.58 G1: 42.20 S: 26.42 G2: 31.38 Ad-LacZ Ad-AP2 G1: 87.54 S: 6.11 G2: 6.35 G1: 75.41 S: 8.22 G2: 16.36 G1: 42.06 S: 26.71 G2: 31.23 G1: 89.95 S: 4.54 G2: 5.62 G1: 90.61 S: 3.61 G2: 5.71 G1: 85.57 S: 5.25 G2: 9.18 Channels (FL2-H) 0 50 100 150 200 Number 0 200 400 600 Channels (FL2-H) 0 50 100 150 200 Number 0 100 200 300 400 500 Channels (FL2-H) 0 40 80 120 160 200 Number 0 50 100 150 200 Channels (FL2-H) 0 50 100 150 200 Number 0 300 600 900 1200 Channels (FL2-H) 0 50 100 150 200 Number 0 100 200 300 400 500 Channels (FL2-H) 0 50 100 150 200 Number 0 50 100 150 200 Channels (FL2-H) 0 50 100 150 200 Number 0 200 400 600 800 1000 Channels (FL2-H) 0 50 100 150 200 Number 0 200 400 600 800 Channels (FL2-H) 0 50 100 150 200 Number 0 200 400 600

Regulation of Cell cycle E2F pRB Senescence Serum starvation Cell-to-cell contact Growth inhibition E2F P P P pRB DNA damage p53 CDC2 CDC2

CDC2 CDK4 Ad-LacZ Ad-AP2 CDK6 CDK2 1 2 3 4 5 6 7 8 9 10 Cyclin D2 Cyclin B Cyclin D1 Cyclin E1 Cyclin E2 p21 p27 p57 AP-2  Actin pRB AP-2  represses Cyclin D2 Hrs after serum release Mock Ad-LacZ Ad-AP2 AP-2  Cyclin D2 Actin 0 18 24 0 18 24 0 18 24 Control Control Control 1 2 3 4 5 6 7 8 9 10 11 12 Cyclin E1 Cyclin B Cyclin D1

AP-2  represses Cyclin D2 Hrs after virus infection -10 -5 0 5 10 15 Fold change 0 6 12 24 36 -AP-2  -Cyclin D2

Cell cycle E2F pRB Senescence Serum starvation Cell-to-cell contact Growth inhibition E2F P P P pRB DNA damage p53 CDC2 CDC2

AP-2  inhibits CDK4 and CDK6 100 96 24 12 CDK4 Cyclin D2 32 P-GSTpRB Ad-LacZ Ad-AP2 Control Ad-p21 IP: CDK4 1 2 3 4 0 20 40 60 80 100 120 Intensity 100 86 54 21 Ad-LacZ Ad-AP2 Control Ad-p21 IP: CDK6 1 2 3 4 CDK6 Cyclin D2 32 P-GSTpRB 0 20 40 60 80 100 120 Intensity 32 P-Histone H1 Cdk2 Cyclin E1 Ad-LacZ Ad-AP2 Control Ad-p21 IP: CDK2 1 2 3 4 100 100 90 40 0 20 40 60 80 100 120 Intensity 100 96 84 32 Ad-LacZ Ad-AP2 Control Ad-p53 IP: CDC2 1 2 3 4 0 20 40 60 80 100 120 Intensity 32 P-Histone H1 Cdc2 Cyclin B1 Plate cells Infect with viruses IP: CDK O/N 24 hrs
Kinase
assay
 32 P-ATP
Substrate

AP-2  failed to inhibit transcription from Cyclin D2 promoter

How does AP-2  repress cyclin D2 ? E-box Cyclin D2 Promoter of Cyclin D2 C-myc AP-2 site AP-2  x E-box Cyclin D2 Promoter of Cyclin D2 C-myc
J

C-myc mediated activation of cyclin D2 is inhibited by AP-2  0 20 40 60 80 100 1 2 3 4 5 Luciferase activity (CPM X 1000) Cyc D2 Promoter c-MYC AP-2  + - - + + - + + + + + + 120 LUC Cyc D2 promoter MYC AP-2 Actin Ad-MYC 0 24 36 48 Ad-AP2+ Ad-MYC 0 24 36 48 Hrs Cyclin D2 Ad-LacZ 0 24 36 48 Cyclin D1 0 2 3 4 5 6 7 8 9 10 11 12

AP-2  binds to a overlapping AP-2/c-myc binding sequence -1886 AP-2 E-box -1596 to -1591 AP-2 -1604 to -1595 197 bps -1645 to -1448 +1 5'- GCCcgctGCA CGTG -3 E-box GST-AP-2 Cyc D2 wt SV40 AP2 wt SV40 AP2 m - - - - 1 + - - - 2 + - - 3 + - - 4 + - - 5 + - - 6 + - - 7 + - - 8 + - - 9 + - - 10 + - - 11 Cyc D2 wt 5'-GCCAT GCC CGCT GCA CGTGCC AGCTTGGC-3' GCC N(4) GGC AP-2 consensus binding motif E-box AP-2 site is a bonafide element Marker Genomic DNA Input Lamin AP-2  Myc Ad-Myc Ad-Myc+ Ad-AP2 1 2 3 4 5 6 AP-2 binding interferes with c-myc

How important cyclin D2 repression is? CycD2 Actin SW480/Neo SW480/CycD2 # 15 SW480/CycD2 # 5 SW480/CycD2 # 11 SW480/CycD2 # 26 1 2 3 4 5 SW480/Neo SW480/ CycD2 # 15 % BrdU incorporation 0 10 20 30 40 50 60 70 80 90 -Ad-AP2 -Ad-LacZ 1 2 3 4 G1 S G2/M Hr 0 12 18 24 12 18 24 60.77 58.79 63.13 57.63 74.97 78.91 86.55 36.44 38.63 30.90 36.79 22.51 17.85 9.74 2.79 2.58 5.97 5.59 2.52 3.21 4.71 Ad-AP2 Virus Ad-LacZ Control SW480/Neo 0 12 18 24 12 18 24 56.88 61.40 60.50 65.17 58.38 57.25 58.86 39.19 34.91 33.30 31.02 35.94 35.60 34.90 3.93 3.69 4.07 3.80 5.67 7.15 6.14 G1 S G2/M Hr Ad-AP2 Virus Ad-LacZ Control SW480/Cyc D2 # 15

D-type cyclin-CDK4 complexes can suppress the skeletal muscle differentiation
What is the significance?
Irreversible cell cycle arrest is a key component of myogenic differentiation
Cyclin D1, cyclin D2 and to some extent cyclin D3 overexpression can block differentiation of myoblasts to myotubes

Myoblast Myotube DM C2C12 MUSCLE DIFFERENTATION MODEL SYSTEM

DM DM+ NS siRNA DM+ AP-2  siRNA GM 10X 40X 10X 100X AP-2  is is needed for differentiation

Days in DM AP-2 MYC Cyclin D2 MHC GAPDH C2C12 NS siRNA C2C12 AP-2  siRNA 0 3 5 7 0 3 5 7 1 2 3 4 5 6 7 8 C2C12 NS siRNA C2C12 AP-2  siRNA 0 3 5 7 0 3 5 7 1 2 3 4 5 6 7 8 Days in DM AP-2 MYC Cyclin D2 MHC GAPDH C2C12 NS siRNA C2C12 AP-2  siRNA 1 2 3 4 5 6 7 8 Days in DM AP-2 Cyclin D2 MHC GAPDH 0 3 5 7 0 3 5 7

A possible simple hypothesis !!! Cyclin D2 C-myc AP-2  Myoblast Myotube

GM GM + AP-2  DM DM + c-myc DM + cyc D2 DM + AP-2  + c-myc DM + AP-2  + cyc D2 DM + c-myc + cyc D2 siRNA √ Χ Χ Χ √ √ √ Χ Myoblast Myotube

Forced AP-2  expression induces myogenic differentiation GM GM+ AP-2  10X 100X a b c d

AP-2  downregulates c-Myc mediated induction of cyclin D2 DM DM + Cyclin D2 DM + cMYC 10X 100X a g b h c i DM + MYC + AP-2  DM + Cyclin D2 + AP-2  DM + MYC + Cyclin D2 siRNA d j e k f l 10X 100X

Cancer of striated muscle tissue There are three major forms: alveolar rhabdosarcoma - most often afflicts adolescents, typically develops in the extremities, body or eye cavities. embryonal rhabdosarcoma – occurs in infants and young children, develops in the head, neck, extremities or lower genitourinary tract. pleiomorphic rhabdosarcoma – occurs in adults and typically develops in the extremities Rhabdosarcoma AP-2  is induced during muscle differentiation De-differentiation results in cancer Loss of AP-2  leads to rhabdosarcoma !!!

N P1 P2 P3 P4 AP-2  fold downregulation 0 5 10 15 20 25 30 AP-2  AP-2  is silenced by promoter methylation -1000 +800 1 47 N P1 P2 P3 P4 Bisulfite sequencing

AP-2  is induced during muscle differentiation and
is needed for differentiation
Loss of AP-2  expression leads to rhabdosarcoma development
Can one reverse the AP-2  expression-methylation/ase inhibitors?
Will it reverse the tumorigenic phenotype of rhabdosarcoma?
Therapeutic value!!!

Ap2 Alpha

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