1. HEREDITARY BREAST CANCER IN DEVELOPING COUNTRIES Richard G. Pestell, MD, PhD Kimmel Cancer Center Jefferson University Hospital

2. Major Genetic Defects in Breast Cancer Established Familial Breast Genes (All Tumor Suppressors) Gene Chromosomal Location Disease TP53 (p53) 17p13 (mutated, LOH) Li-Fraumeni syndrome of multiple hereditary cancers PTEN10q23 (mutated, LOH) Cowdens syndrome of multiple hereditary cancers BRCA-117q21 (mutated, LOH)Familial female breast and ovarian cancers BRCA-213q14 (mutated, LOH)Familial female and male breast cancers

3. Established Breast Cancer Progression Genes Gene Chromosomal Location ClassFunction C-ERBB217q12Oncogene (amplified)Growth factor receptor subunit C-MYC8q24Oncogene (amplified) Cell-cycle/cell death regulator; protein synthesis CCND1 (cyclin D1)11q13Oncogene (amplified)Cell-cycle G 1 regulator CDKN2 (p16)9p21 Suppressor gene (methylated, LOH) Cell-cycle G 1 regulator RB-113q14 Suppressor gene (mutated, LOH) Cell-cycle G 1 and G 1 /S regulator TP53 (p53)17p13 Suppressor gene (mutated, LOH) Cell-cycle/cell death/DNA repair regulator CDH1 (E-cadherin)16q22-23 Suppressor gene (methylated, LOH) Cell-cell adhesion protein Major Genetic Defects in Breast Cancer

4. Sex Hormone Regulation of Growth Factor Systems in Breast Cancer EGF FAMILY Growth factors: EGF, TGF-, amphiregulin Receptors: EGFR, c-erbB 2 IGF FAMILY Growth factors: IGF-2 Receptors: IGF-1R, IGF-2R, insulin receptor Binding proteins: BP-2, BP-3, BP-4, BP-5 TGF- FAMILY Growth factors: TGF- 1, TGF- 2, TGF- 3 PDGF FAMILY Growth factors: PDGF-1, PDGF-2 BP, binding protein; EGF (R), epidermal growth factor (receptor); IGF, insulin-like growth factor; PDGF, platelet-derived growth factor; TGF, transforming growth factor.

5. Metastatic Cancer Further phenotypic alterations in cell cycle Further phenotypic alterations in cell death and response to therapy Phenotypic changes in growth factor secretion governing angiogenesis and metastatic spread Mutations in pathways governing invasion CDH1 Defects in mismatch repair of DNA Predisposing genetic risk Mutations affecting DNA repair and apoptosis in BRCA-1 BRCA-2 TP53 PTEN Carcinoma in Situ Mutations in Growth Factor and Sex Steroid pathways governing the cell cycle C-ERBB2 C-MYC CCDN1 CDKN2 RB-1 Mutations in Cell Death Pathways TP53 Overall Chromosomal Instability Hyperplasia Overstimulation of cell cycle and suppression of apoptosis by Estrogen Progesterone Growth Factors Immortalization of cells by expression of Telomerase Genetic Alteration of Mammary Epithelial Cells - Progression of Breast Cancer

6. BRCA1 Protein Function Regulation of cell cycle progression DNA repair: caretaker function Programmed cell death (apoptosis) Regulation of gene transcription

7. BRCA1 Protein: Structural Features -1863 aa -220 kDa nuclear phosphoprotein -N-terminal RING domain (aa 20-64) interacts with BARD1 ubiquitin ligase activity -C-terminal TAD (last 95-100 aa) mediates transactivation BRCT repeats

8. Role in DNA Damage Response Specialized DNA repair processes -Homology-directed repair -Transcription coupled repair -Mismatch repair -Nucleotide excision repair -Fanconi repair DNA damage signaling: ATM, ATR, Chk2 DNA damage responsive cell cycle checkpoints: S & G2/M

9. Regulation of BRCA1 Expression Increased Expression -cancer chemoprevention – indole-3-carbinol, genistein -mammary epithelial cell differentiation -cell cycle - in late G1, early S-phase Decreased Expression -30-40% sporadic breast cancers: promoter methylation -ethanol -polycyclic aromatic hydrocarbons – B(a)P, BPDE -persistent organochlorines (PCBs) -p53 activation -Id4 (bHLH transcriptional regulator)

10. BRCA1 Regulated Transcriptional Pathways -interacts w/ basal transcription factor (RNA helicase A) -C-terminus: chromatin unfolding activity (COBRA1) -associates w/ Brg1 (SWI/SNF chromatin remodelling) -stimulates Gadd45, p21 WAF1, p27 Kip1 expression -coactivator for p53 -inhibits c-Myc activity and TERT expression -interacts with STAT1–interferon -mediated transcription -regulates nuclear receptor function

11. BRCA1 transcriptional regulation

12. BRCA1-interacting proteins

13. BRCA1-transcription repression

14. Science, V284, p1354, 1999 BRCA1 represses ER

15. BRCA1 Domains for ER Inhibition Du-145 T47D 220 200 120 100 80 60 40 20 0 Luciferase Activity (% of pcDNA3 Vector Control) pcDNA3 wt BRCA1 5677-InsA Bam H1 Kpn 1 EcoR 1 185delAG T300G RXRXH 5382InsC C5365G 1 1 1 1 1 1 1 1 1 RING NLS Rad51 TAD 1 500 1000 1500 1863 1852 1313 771 302 38 61 Cys/Gly 358 RXRXH 1863 1755 1749Pro/Arg 1863 wt BRCA1 5677-InsA Bam H1 Kpn 1 EcoR 1 185delAG T300G RXRXH 5382InsC C5365G LXCXE

16. ER Binds Cyclin D1 WesternIP WB E2E2 - + - + AB ER HE74 F 1-530 E/F 1-282 DBD LBD AF-1 AF-2 1-378 ER HE74 F 1-530 E/F 1-282 1-378 *** Cancer Res, Wang et al 2005

17. GST-Cyclin D1 Binds to ER GST GST D1 ER HE74 F 1-530 E/F 1-282 DBD LBD AF-1 AF-2 HE19C *** 1595 1 179595 1554 1530 1388 1282 Cancer Res, Wang et al 2005

18. Cyclin D1 Overcomes Repression by BRCA1 N C BRCA1 NC N C ER Co-activator D1

19. Hereditary Breast Cancer in Developing Countries -Relative contribution to breast cancer ? -Common or distinct mutations ? -Role of polymorphisms in onset,progression and therapy (cyclin D1,Bcl2, other) ? -Role in screening- cultural sensitivity vs treatment triage -Economic priorities ? -Global collaboration in molecular epidemiology ?

20. BRCA1 Inhibits Estrogen-Induced Genes DMEM+10%FCS - + - + E2 (10 -6 M) - - + + wtBRCA1 pS2 Cathepsin-D -Actin DMEM+10%FCS - + - + E2 (10 -6 M) - - + + wtBRCA1 pS2 Cathepsin-D -Actin Serum-Free DMEM