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 <ul><li>Implicated in development </li></ul><ul><li>Implicated in transformation </li></ul>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 <ul><li>-Sequence-specific DNA-binding </li></ul><ul><li>(5’- GCCNNNGGC -3) </li></ul><ul><li>-AP-2 specific reporter activation </li></ul><ul><ul><li>(3X-AP2-CAT) </li></ul></ul><ul><li>-Target gene activation </li></ul><ul><li>(p21 WAF1/CIP1 ) </li></ul>Ad-LacZ Ad-AP2 SW480 H460 HT1080 A F C D E B
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
<ul><li>Caspase 3 is essential </li></ul><ul><li>Intrinsic pathway </li></ul><ul><li>Caspase 8 and FADD adapter not needed </li></ul><ul><li>Caspase 9 and Apaf1 are essential </li></ul><ul><li>Mitochondrial membrane potential is lost </li></ul><ul><li>Bax translocates to mitochondria </li></ul><ul><li>Cytochrome c is released from mitochondria </li></ul><ul><li>Bax is essential </li></ul><ul><li>AP-2 binds to Bcl-2 promoter and represses </li></ul><ul><li>its transcription </li></ul>
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
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 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 <ul><li>- Control </li></ul><ul><li>- 5aza2dC </li></ul><ul><li>Adria </li></ul><ul><li>Mock + Adria + 5aza2dC </li></ul><ul><li>Lamin siRNA + Adria + 5aza2dC </li></ul><ul><li>AP-2 siRNA + Adria + 5aza2dC </li></ul>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
<ul><li>D-type cyclin-CDK4 complexes can suppress the skeletal muscle differentiation </li></ul>What is the significance? <ul><li>Irreversible cell cycle arrest is a key component of myogenic differentiation </li></ul><ul><li>Cyclin D1, cyclin D2 and to some extent cyclin D3 overexpression can block differentiation of myoblasts to myotubes </li></ul>
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
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
<ul><li>AP-2 is induced during muscle differentiation and </li></ul><ul><li>is needed for differentiation </li></ul><ul><li>Loss of AP-2 expression leads to rhabdosarcoma development </li></ul><ul><li>Can one reverse the AP-2 expression-methylation/ase inhibitors? </li></ul><ul><li>Will it reverse the tumorigenic phenotype of rhabdosarcoma? </li></ul><ul><li>Therapeutic value!!! </li></ul>