1. A Functional Genomics Approach to Autophagic Cell Death Gene Discovery Marco Marra Canada’s Michael Smith Genome Sciences Centre British Columbia Cancer Agency CATGGCGTGGGGAT CATGGCTAATAAAT CATGGCTCAAGGAG CATGGCTGGACTCC CATGGCTGTGGCCA CATGGCTTTCGTGT CATGGCTTTTTGGC CATGGGAACCGACA CATGGGACCGCCCC CATGGGACCGCTCA CATGGGATCACAAT CATGGGCAACGATC CATGGGCAGCAAGC CATGGGCAGCAATT

2. Programmed cell death (PCD) PCD is a genetically regulated type of cell death in which the cell uses specialized cellular machinery to kill itself; it is a cell suicide mechanism that enables metazoans to control cell number and eliminate cells that threaten the animal's survival Types (Schweichel & Merker, 1973): Type I = apoptosis Type II = autophagic cell death Type III = non-lysosomal

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

4. Autophagy Autophagosome Autophagolysosome www.uni-marburg.de/cyto/elsaesse/auto.htm The Cell, A Molecular Approach, G.M. Cooper, Ed., 2000 Housekeeping Starvation PCD

5. Autophagic PCD in Development 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 UPDATE!

6. Autophagic PCD 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 UPDATE! Lurcher mouse Beclin1, summary

7. J. Mol. Recognit. 2003; 16: 337–348 Autophagic PCD pathways Molecular machinery?

8. Research Aims 1.Identify the genes involved in autophagic cell death in vivo. 2.Determine which genes are necessary and sufficient for autophagic cell death. 3.Identify the autophagic cell death genes/pathways associated with human disease and investigate potential as molecular markers and/or therapeutic agents.

9. Experimental Approach Gene expression profiling (SAGE) and RNAi: Comprehensive Gene Discovery Drosophila model system: Known cell death genes/pathways are conserved Genetic and molecular tools Sequence resources FlyBase and GadFly databases Multiple tissues undergo PCD; well-characterized morphologically

10. The Drosophila Salivary Glands Cell types: duct cells & secretory cells Cell number: 100 cells each Size of gland: Total RNA/pair of glands: 0.6 µg (20 pairs/microSAGE library) Development: ectodermally-derived during late embryogenesis; during metamorphosis, a pulse of ecdysone triggers larval salivary gland PCD; adult salivary glands arise from a pair of imaginal rings scale (from Kucharova-Mahmood et al., 2002)

11. Drosophila salivary gland PCD (adapted from Jiang et al., 1997) autophagicstage-specificsynchronous 20 hr24 hr26 hr (@18ºC) known cell death genes are highly conserved and regulated transcriptionally hr (APF, 18°C) RT - + - + - + 16 18 20 22 23 24 diap2 rpr hid

12. Serial Analysis of Gene Expression (SAGE) (Velculescu et al., 1995) quantitative sequence based method to generate global gene expression profiles potential for new transcript discovery yields 14 bp tags that can be compared against transcript and genome sequences to identify genes

13. Salivary gland SAGE library and tag mapping summary (S. Gorski et al., Curr Biol 13: 358-363, 2003) (E. Pleasance et al., Genome Res 13: 1203-15, 2003) SAGE Library Tags analyzed Transcripts Total transcripts 16 hr34,9893,126 4,628 20 hr31,2153,034 23 hr30,8232,963 61.9% 6.2% 6.5% 25.3% known or predicted genes genomic DNA and EST (but no annotated gene) genomic DNA only no match

14. 1244 transcripts are expressed differentially (p<.05) prior to salivary gland PCD 512 genes have associated biological annotations (Gene Ontology in Flybase) 732 genes have unknown functions 377 of these genes were not annotated (GadFly Release 2) 48 correspond solely to salivary gland ESTs Update this slide!! ; Release 3 data

15. SAGE Identifies Genes Associated Previously With Salivary Gland Death Tag Frequency BFTZ-F1 EcR/USP BR-C E74 E93 rpr hid ark dronc crq iap2 Cell Death E75

16. Genes associated with autophagic PCD Expression fold- difference (16 hr vs 23 hr) Protein synthesis Hormone related Trans- cription* Signal transduction Apoptosis Immune response/ TNF-related Autophagy Unknowns

17. Gene expression is reduced in a salivary gland death-defective mutant Fold-difference in expression (16 hr vs 23 hr) E93 is an ecdysone-induced gene that encodes a DNA binding protein required for salivary gland cell death (Lee et al., 2000, 2001)

18. Function-based strategies for characterizing differentially expressed genes Mutants available Overexpression and loss-of-function in vivo Prioritization midgut PCD human ortholog/cancer l(2)mbn cells RNAi in mammalian cells RNAi in Drosophila l(2)mbn cells Phenotype analyses salivary glands, midguts, retinas, embryos Mutants unavailable

19. Prioritization Scheme Differentially expressed (p < 0.05) tags corresponding to known/predicted genes (= 361 + ___) and showing at least 5-fold difference in expression (____) similar differential expression prior to midgut PCD (Li & White, Dev Cell, 2003, & in-house QRT-PCR) mammalian ortholog (give numbers) (% with ortholog) mammalian ortholog differentially expressed in cancer present in vitro (Affymetrix analysis of mbn2 cells - % present) Update this!!!

20. Finding PCD genes by orthology and expression Human cancer SAGE libraries Differentially expressed genes Drosophila orthologues Drosophila SAGE library Differentially expressed genes Human orthologues Set of Drosophila/human orthologues perturbed in both cancer and Drosophila PCD

21. Determining orthology Find orthologs using InParanoid (Remm et al, 2001) –Based on BLAST similarity –Groups genes arising from duplications – eg. one Drosophila gene may be orthologous to multiple human genes Find orthologs between Drosophila genes (from GadFly/FlyBase) and human RefSeq sequences

22. Differentially expressed gene sets Genes differentially expressed between 16h and 23h in Drosophila salivary gland 361 genes upregulated 203 genes downregulated Genes differentially expressed between normal and cancer SAGE libraries from CGAP (Unigene) 2277 genes Find human RefSeq orthologs 197/361 upregulated 99/203 downregulated Map to LocusLink, Unigene Find common genes: Downregulated in PCD: 7 Drosophila genes, 8 human orthologs Upregulated in PCD: 16 Drosophila genes, 18 human orthologs

23. Drosophila l(2)mbn cell line established in 1978 by Gateff consists of tumorous haemocytes isolated from a larva of the Drosophila mutant lethal (2) malignant blood neoplasm. form vacuoles and die in response to 20-hydroxyecdysone (20HE) treatment die in response to treatment with Diap1-RNAi (apoptotic-like) morphology and gene expression changes currently under investigation

24. RNAi screen design Prepare dsRNA using T7-tailed gene specific primers (average product size = 500 bp) Add approx 50nM dsRNA directly to Drosophila l(2)mbn cells under serum-free conditions & incubate 1 hr. Add serum. Incubate 4-5 days Cell counts/WST-1 colorimetric assay (cell viability) Microscopic observation (cell morphology) No treatmentEcdysone treatment Diap1-RNAi treatment 24 hrs

25. PCD pathways in Drosophila (Meier et al., Nature 2000)

26. CG4091 Met all criteria: Upregulated prior to salivary gland PCD (X 105 in SAGE) Upregulated prior to midgut PCD (X 9) Expressed in mbn2 cells Human ortholog (TNF-induced protein GG2-1/SCC-S2) GG2-1/SCC-S2 possibly associated with human cancer: SCC-S2 amplified in a metastatic head and neck carcinoma-derived cell line compared to matched primary tumor-derived cell line (Kumar et al., JBC, 2000) A “Double knock-out” strategy Mbn2 cell line Addiap1 dsRNA Add dsRNA of another gene of interest (eg.CG4091, rpr) 1.Gene knock-out of iap1(anti-cell death gene) causes apoptosis 2.Knock-out of genes upstream of iap1in the same pathway and the genes that do not interact withiap1will still induce apoptosis Apoptotic body Dead cell Cell forming apoptotic body Tryphan blue stain 1.Knock-out of genes down- stream ofiap1in the same pathway will inhibitiap-1 induced apoptosis “Double knock-out” strategy Mbn2 cell line Addiap1 dsRNA Add dsRNA of another gene of interest (eg.CG4091, rpr) 1.Gene knock-out of iap1(anti-cell death gene) causes apoptosis 2.Knock-out of genes upstream of iap1in the same pathway and the genes that do not interact withiap1will still induce programmed cell death Apoptotic body Dead cell Cell forming apoptotic body Tryphan blue stain 1.Knock-out of genes down- stream ofiap1in the same pathway will inhibitiap-1 induced programmed cell death. A “Double knock-out” strategy Mbn2 cell line Addiap1 dsRNA Add dsRNA of another gene of interest (eg.CG4091, rpr) 1.Gene knock-out of iap1(anti-cell death gene) causes apoptosis 2.Knock-out of genes upstream of iap1in the same pathway and the genes that do not interact withiap1will still induce apoptosis Apoptotic body Dead cell Cell forming apoptotic body Tryphan blue stain 1.Knock-out of genes down- stream ofiap1in the same pathway will inhibitiap-1 induced apoptosis “Double knock-out” strategy Mbn2 cell line Addiap1 dsRNA Add dsRNA of another gene of interest (eg.CG4091, rpr) 1.Gene knock-out of iap1(anti-cell death gene) causes apoptosis 2.Knock-out of genes upstream of iap1in the same pathway and the genes that do not interact withiap1will still induce programmed cell death Apoptotic body Dead cell Cell forming apoptotic body Tryphan blue stain 1.Knock-out of genes down- stream ofiap1in the same pathway will inhibitiap-1 induced programmed cell death.

27. CG4091-RNAi partially blocks PCD induced by Diap1-RNAi

28. PCD pathways in Drosophila (Meier et al., Nature 2000) CG4091

29. CG4091: work in progress Overexpression in vitro and in vivo P element excision to generate loss-of-function mutations for in vivo analysis P-element (flybase.bio.indiana.edu)

30. Acknowledgements BC Cancer Agency BC Cancer Foundation National Cancer Institute of Canada Michael Smith Foundation for Health Research NSERC BC Cancer Research Centre Victor Ling GSC PCD group Suganthi Chittaranjan Doug Freeman Sharon Gorski Melissa McConechy Jennifer Kouwenberg Bioinformatics Steven Jones Erin Pleasance Richard Varhol Scott Zuyderduyn GSC Sequencing Group University of Maryland Biotech Institute Eric Baehrecke www.bcgsc.ca http://sage.bcgsc.ca/tagmapping/ http://www.bcgsc.ca/lab/fg/dsage/

31. Comparison of SAGE and real-time quantitative RT-PCR Fold-difference by SAGE Fold-difference by QRT-PCR Correlation coefficient = 0.5 II. Correlation coefficient between fold-difference values (64 samples): I. Direction of Change: 91/96 samples = 95% concordance

32. Tag-to-gene Mapping in Drosophila (E. Pleasance, M. Marra and S. Jones, submitted) AAAAA CATGAGGAGTGAAT Gene X Platform: Queryable ACEDB database Resources: Drosophila genomic sequence and annotation (GadFly Release 2) predicted UTRs 259,620 ESTs and full-length cDNAs (BDGP) 5,181 salivary gland 3’ ESTs (GSC)

33. Keyword Data base SG 16 SG 23 Gene Sim (%) Score Length (aa) Swiss Prot id Database Description death apoptosis death survival autophagy hormone cancer tumor apoptosis TNF BH3 SP FB PCD 0 4 0 1 0 1 0 2 0 1 45 10 65 1 EST only 15 0 6 5 4 7 61 7 73 12 7 15 1 0 5 102 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 34 984 240 203 838 361 182 469 188 228 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' BH3

34. Mining Expression Data in Drosophila I. Keyword-based Data Mining e.g. Keyword cancer: 33 associations GadFly – Swissprot Homology Table Extract entries based on keyword search II. Cross-species Gene Expression Comparisons CG4091 upregulated 102-fold in 16 vs 23 hr salivary glands SCC-S2 downregulated 7-fold in human mammary gland ductal carcinoma vs normal Drosophila autophagic cell death Human cancer

35. Mining Expression Data in Drosophila I. Keyword-based Data Mining Q: How many differentially expressed Drosophila genes have a mammalian homolog that has been associated with cancer? A: 33 genes GadFly – Swissprot Homology Table Extract entries based on keyword search

36. Mining Expression Data in Drosophila II. Cross-species Gene Expression Comparisons CG4091 upregulated 102-fold in 16 vs 23 hr salivary glands SCC-S2 downregulated 7-fold in human mammary gland ductal carcinoma vs normal Drosophila autophagic cell death Human cancer (CGAP)

37. 72hr Untreated Control Cells 72hr 10µM 20-hydroxyecdysone Treated Cells 72hr Untreated Control Cells 72hr 10µM 20-hydroxyecdysone Treated Cells

38. What are l(2)mbn cells? Tumorous haemocyte derived from a Drosophila mutant lethal malignant blood neoplasm. Established in 1978, isolated from mutant larvae Non-homogenous mixture of cells: 3 types 1) plasmatocyes, 2) lamellocytes, 3) podocytes

39. RNAi screen results Genes tested with RNAi: CG40228 CG11051 Sgs7 Talin CG8785 CG14214 Diap1 CG9911 CG7220 Timp CG7059 Sema-5c CG4798 Possible phenotype: CG14214 Cell Death Phenotype: diap1

40. WST-1 Assay Colourmetric assay, absorption at 450nm Viable cells with mitochondrial dehydrogenase activity causes and increase in formazan production This correlates directly with the amount of metabolically active cells

41. WST-1 Assay Use to determine the amount of live cells present in non-treated control cells and 20HE treated cells Expect a difference in absorption readings between 20HE treated and non-treated cells Fast, easy screening assay for RNAi screen

42. WST-1 Assay Results Decrease in cell respiration of 20HE treated cells – Directly represents the amount of viable cells

43. Autophagic PCD and cancer Autophagic PCD is associated with the following: spontaneous regression of human neuroblastoma (Kitanaka et al. 2002) tamoxifen-treated human breast cancer cells, MCF-7 (Bursch et al. 1996) tamoxifen-treated murine breast cancer cells, FM3A (Bilir et al. 2001) TNFa-treated T lymphoblastic leukaemic cells (Jia et al. 1997) bcl-2 antisense treated human leukemic HL60 cells (Saeki et al. 2000) oncogenic Ras-expressing human glioma and gastric cancer cells (Chi et al. 1999)

44. PCD and Cancer The role of apoptosis in cancer is well established: “resistance toward apoptosis is a hallmark of most and perhaps all types of cancer” (Hanahan & Weinberg, Cell, 2000) Autophagic PCD and autophagy have been associated with cancer recently:

45. Autophagy and cancer Beclin-1 (apg6) is an autophagy gene: Monoallelically deleted and expressed at reduced levels in human breast and ovarian cancers beclin1 knockout mouse indicated that beclin1 is a haploinsufficient tumor suppressor gene; heterozygotes displayed an increase in the incidence of lymphoma, lung carcinoma and liver carcinoma (Qu et al. 2003; Yue et al. 2003) Current notion: “Defective autophagy can lead to cancer” (Edinger & Thompson, 2003)

46. PCD and disease Function Dysfunction Deleting damaged cells Cancer Culling cell number Deleting structures Sculpting tissues Autoimmune diseases Neurodegenerative diseases Developmental abnormalities

47. Genome Sciences Centre Programmed Cell Death Group Apoptotic Cell DeathAutophagic Cell DeathAutophagy inxs (Doug Freeman) echinus (Ian Bosdet) Cloning and Characterization Mammalian cell line transcription profiling and RNAi (M. Qadir) Gene expression profiling (SAGE) of autophagic PCD in Drosophila salivary glands Role of Akap200 (Claire Hou) Role of CG4091 (Suganthi Chittaranjan) Bioinformatic analyses: associations between autophagic PCD, apoptosis, autophagy, and cancer (Erin Pleasance) Novel Gene Discovery (Brent Mansfield) RNAi screen in Drosophila cell line (Suganthi, Melissa McConechy, Jennifer Kouwenberg, Amy Leung) RNAi screen in mammalian cell line (M. Qadir)

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

49. 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.

50. 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

51. GSC Programmed Cell Death Group Ian BosdetAmy Leung Suganthi ChittaranjanMelissa McConechy Doug FreemanErin Pleasance Claire HouMohammed Qadir Jennifer Kouwenberg Marco Marra Victor Ling Sharon Gorski

53. Drosophila expression analysis data SAGE libraries for PCD gene identification from salivary gland (Sharon Gorski, Suganthi Chittaranjan, Doug Freeman) 3 SAGE libraries (16h, 20h, 23h) hr (APF, 18°C) RT - + - + - + 16 18 20 22 23 24 diap2 rpr hid (S. Gorski et al. Curr. Biol. 13: 358-363, 2003)