Functional Genomic Analysis of Autophagic Cell Death using RNA interference S. Gorski, Genome Sciences Centre, BC Cancer Agency CATGGCGATATTGT CATGGCGCCAATAT CATGGCGCGCATTT CATGGCGTGGGGAT CATGGCTAATAAAT CATGGCTCAAGGAG CATGGCTGGACTCC CATGGCTGTGGCCA CATGGCTTTCGTGT CATGGCTTTTTGGC CATGGGAACCGACA CATGGGACCGCCCC CATGGGACCGCTCA CATGGGATCACAAT CATGGGCAACGATC CATGGGCAGCAAGC CATGGGCAGCAATT

Overview Background Research Aims Experimental Approach Preliminary Data

Programmed Cell Death (PCD) Function Dysfunction Cancer Deleting damaged cells Autoimmune diseases Culling cell number Neurodegenerative diseases Deleting structures Developmental abnormalities Sculpting tissues

Types of Programmed Cell Death (PCD) I. Apoptosis II. Autophagic PCD (adapted from Baehrecke, 2002)

Autophagic cell death in disease human neurodegenerative diseases (Alzheimer and Parkinson) cardiomyocyte degeneration spontaneous regression of human neuroblastoma tamoxifen-treated mammary carcinoma cells (MCF-7) bcl-2 antisense treatment of human leukemic HL60 cells beclin-1 (apg6) promotes autophagy and inhibits tumorigenesis; expressed at decreased levels in human breast carcinoma

Research Aims Identify the genes involved in autophagic cell death in vivo. Determine which genes are necessary and sufficient for autophagic cell death. Identify the autophagic cell death genes/pathways associated with human disease and investigate potential as molecular markers and/or therapeutic agents.

Experimental Approach Specific Aim Identify and characterize genes that are necessary for mammalian autophagic cell death. Experimental Approach RNAi analysis of candidate autophagic cell death genes in human breast carcinoma cell line MCF-7.

Test candidate genes for expression in MCF-7 by RT-PCR siRNA analysis of autophagic cell death genes in human breast carcinoma cells Overview of experimental design Test candidate genes for expression in MCF-7 by RT-PCR Preparation and storage of siRNAs corresponding to selected human candidate autophagic cell death genes MCF-7 cells + Reporter +/- Tamoxifen Transfection (siPORT Lipid, Ambion) 3-4 days incubation time to ensure protein depletion Test for RNA depletion by RT-PCR Microscopy Cell Death Assays Autophagic vacuole assays

Small Interfering RNAs (siRNA) 5’ phosphate siRNA siRNAs are incorporated into RISC (RNA Induced Silencing Complex) siRNAs unwind and guide RISC to a substrate mRNA substrate cleavage (From McManus and Sharp, 2002 & Hannon, 2002)

siRNA in mammalian cells a new tool to systematically decipher the functions of genes many types of genes in a variety of mammalian cell lines have been knocked down successfully (85-99% knock-down; in McManus and Sharp, 2002) transfectability of cells is the limiting step Potential Problems: may have to try multiple siRNAs per gene effects are transient

Characteristics of the MCF-7 cell line human breast carcinoma cell line amenable to siRNA analyses treatment with tamoxifen (10-6M) induces autophagic cell death (Bursch et al. 1996, 2000)

Preliminary Data Identification of candidate autophagic cell death genes. “A SAGE Approach to Discovery of Genes Involved in Autophagic Cell Death” Gorski et al., 2003, Current Biology, 13: 358-363. Used gene expression profiling in the Drosophila model system to identify several hundred candidate autophagic cell death genes 2. Bioinformatics analyses to relate gene expression patterns in Drosophila autophagic cell death and human cancers

Cross-species Gene Expression Comparisons Drosophila autophagic cell death Human cancer (CGAP) CG4091 upregulated 102-fold in 16 vs 23 hr salivary glands SCC-S2 downregulated 7-fold in human mammary gland ductal carcinoma vs normal CG11059 upregulated > 12-fold CLSTN3 downregulated >16-fold in neuroblastoma vs normal brain kirre downregulated 11-fold SIGLEC5 upregulated > 26-fold in ductal carcinoma vs normal

Autophagic Cell Death Candidate Genes

Acknowledgements Genome Sciences Centre Marco Marra Victor Ling Drosophila PCD Suganthi Chittaranjan Doug Freeman Carrie Anderson Shaun Coughlin Claire Hou Bioinformatics Steven Jones Erin Pleasance Richard Varhol Scott Zuyderduyn GSC Sequencing Group www.bcgsc.ca http://sage.bcgsc.ca/tagmapping/ http://www.bcgsc.ca/lab/fg/dsage/ BC Cancer Agency BC Cancer Foundation NCIC NSERC

Keyword Gene Sim (%) (aa) Swiss Prot id Database Description SG 16 23 Gene Sim (%) Score Length (aa) Swiss Prot id Database Description death apoptosis survival autophagy hormone cancer tumor TNF BH3 SP FB PCD 4 1 2 45 10 65 EST only 15 6 5 7 61 73 12 102 Nc debcl cact Traf1 CG4719 Ptpmeg CG4859 CG10777 Rpn2 stck chrw ciboulot CG8706 Atet CG13907 botv sp6 CG11335 CG10990 CG4091 CG2023 27.2 25.5 37.1 37.0 39.3 31.3 41.6 18.7 23.7 65.7 35.3 68.6 29.6 39.9 33.2 48.6 28.3 42.7 59.9 59.6 53.9 251 127 322 300 232 101 772 87 160 1276 257 114 1204 469 311 2044 427 423 592 330 247 253 133 203 179 116 80 407 444 267 321 170 34 984 240 838 361 182 188 228 ICE6_HUMAN BCL2_HUMAN IKBA_HUMAN TRA1_HUMAN BAR1_HUMAN PTND_HUMAN MM11_MOUSE WRN_HUMAN PSD2_HUMAN PINC_HUMAN RB24_MOUSE TYB4_HUMAN LRP2_HUMAN ABG2_HUMAN MOT1_HUMAN EXL3_HUMAN MASP_MOUSE LYOX_HUMAN   CASPASE-6 PRECURSOR APOPTOSIS REGULATOR BCL-2 NF-KAPPAB INHIBITOR ALPHA TRAF1 BRCA1-ASSOCIATED RING DOMAIN FAS-ASSOCIATED PTP-1 STROMELYSIN-3 PRECURSOR WERNER SYNDROME HELICASE 26S PROTEASOME S2 PINCH PROTEIN RAS-RELATED PROTEIN RAB-24 THYMOSIN BETA-4 LDL RECEPTOR-RELATED PROT BREAST CANCER RESISTANCE MONOCARBOXYLATE TRANSPORT TUMOUR SUPPRESSOR EXL3-LIKE PROTEASE INHIBITOR 5 PROT-LYSINE 6-OXIDASE PREC. MM 'APOPTOSIS PROTEIN MA-3' HS 'TNF-INDUCED PROTEIN GG2-1'

Autophagic cell death in normal physiology Dictyostelium sorocarp formation insect metamorphosis intersegmental muscle, gut, salivary glands mammalian embryogenesis regression of interdigital webs, sexual anlagen mammalian adulthood intestine, mammary gland post-weaning, ovarian atretic follicles

Questions What is the relationship between Autophagic PCD and Cancer? common mechanism in breast and other cancers? gene mutation What is the therapeutic potential of autophagic PCD in cancer? solid tumours apoptotic-resistant tumours