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A Functional Genomics Approach to Autophagic Cell Death Gene Discovery

CATGGCGTGGGGAT CATGGCTAATAAAT CATGGCTCAAGGAG CATGGCTGGACTCC CATGGCTGTGGCCA CATGGCTTTCGTGT CATGGCTTTTTGGC CATGGGAACCGACA CATGGGACCGCCCC CATGGGACCGCTCA CATGGGATCACAAT CATGGGCAACGATC CATGGGCAGCAAGC CATGGGCAGCAATT. A Functional Genomics Approach to Autophagic Cell Death Gene Discovery.

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A Functional Genomics Approach to Autophagic Cell Death Gene Discovery

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

  2. Programmed cell death (PCD) • PCD isa 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) I. Apoptosis II. Autophagic PCD (adapted from Baehrecke, 2002)

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

  5. Autophagic PCD in Development UPDATE! • 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

  6. Autophagic PCD in Disease UPDATE! Lurcher mouse Beclin1, summary • 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

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

  8. 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.

  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 scale 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 (from Kucharova-Mahmood et al., 2002)

  11. autophagic • stage-specific • synchronous (@18ºC) 20 hr 24 hr 26 hr (adapted from Jiang et al., 1997) • known cell death genes are highly conserved and • regulated transcriptionally RT - + - + - + - + - + - + 16 18 20 22 23 24 hr (APF, 18°C) diap2 rpr hid Drosophila salivary gland PCD

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

  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) 6.5% known or predicted genes genomic DNA and EST (but no annotated gene) 25.3% 61.9% genomic DNA only 6.2% no match

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

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

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

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

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

  19. Prioritization Scheme Update this!!! • 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)

  20. Human cancer SAGE libraries Drosophila SAGE library Differentially expressed genes Differentially expressed genes Human orthologues Drosophila orthologues Finding PCD genes by orthology and expression 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 • Find common genes: • Downregulated in PCD: 7 Drosophila genes, 8 human orthologs • Upregulated in PCD: 16 Drosophila genes, 18 human orthologs Map to LocusLink, Unigene

  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. 24 hrs No treatment Ecdysone treatment Diap1-RNAi treatment 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)

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

  26. A A “ “ “ “ Double knock Double knock Double knock Double knock - - - - out” strategy out” strategy out” strategy out” strategy Add dsRNA of Add dsRNA of Add dsRNA of Add dsRNA of Add Add Add Add iap1 iap1 iap1 iap1 another gene of another gene of another gene of another gene of dsRNA dsRNA dsRNA dsRNA interest (eg. interest (eg. interest (eg. interest (eg. CG4091 CG4091 CG4091 CG4091 , , , , rpr rpr rpr rpr ) ) ) ) Mbn2 cell line Mbn2 cell line Mbn2 cell line Mbn2 cell line Tryphan blue stain Tryphan blue stain Tryphan blue stain Tryphan blue stain Apoptotic Apoptotic Apoptotic Apoptotic body body body body Cell forming Cell forming Cell forming Cell forming apoptotic apoptotic apoptotic apoptotic body body body body Dead cell Dead cell Dead cell Dead cell 1. 1. 1. 1. Gene knock Gene knock Gene knock Gene knock - - - - out of i out of i out of i out of i ap1 ap1 ap1 ap1 (anti (anti (anti (anti - - - - cell cell cell cell death gene) causes apoptosis death gene) causes apoptosis death gene) causes apoptosis death gene) causes apoptosis 1. 1. 1. 1. Knock Knock Knock Knock - - - - out of genes down out of genes down out of genes down out of genes down - - - - 2. 2. 2. 2. Knock Knock Knock Knock - - - - out of genes upstream of out of genes upstream of out of genes upstream of out of genes upstream of stream of stream of stream of stream of iap1 iap1 iap1 iap1 in the same in the same in the same in the same iap1 iap1 iap1 iap1 in the same pathway and in the same pathway and in the same pathway and in the same pathway and pathway will inhibit pathway will inhibit pathway will inhibit pathway will inhibit iap iap iap iap - - - - 1 1 1 1 the genes that do not interact the genes that do not interact the genes that do not interact the genes that do not interact induced programmed cell death. induced apoptosis induced programmed cell death. induced apoptosis with with with with iap1 iap1 iap1 iap1 will still induce will still induce will still induce will still induce programmed cell death apoptosis programmed cell death apoptosis 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)

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

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

  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 Research Centre Victor Ling GSCPCD 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/ BC Cancer Agency BC Cancer Foundation National Cancer Institute of Canada Michael Smith Foundation for Health Research NSERC

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

  32. CATGAGGAGTGAAT AAAAA Gene X Tag-to-gene Mapping in Drosophila (E. Pleasance, M. Marra and S. Jones, submitted) • 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. GadFly – Swissprot Homology Table Extract entries based on keyword search II. Cross-species Gene Expression Comparisons Drosophila autophagic cell death Human cancer SCC-S2 downregulated 7-fold in human mammary gland ductal carcinoma vs normal CG4091 upregulated 102-fold in 16 vs 23 hr salivary glands Mining Expression Data in Drosophila I. Keyword-based Data Mining e.g. Keyword cancer: 33 associations

  34. GadFly – Swissprot Homology Table Extract entries based on keyword search 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

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

  36. 72hr Untreated Control Cells 72hr 10µM 20-hydroxyecdysone Treated Cells 72hr Untreated Control Cells 72hr 10µM 20-hydroxyecdysone Treated Cells

  37. 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

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

  39. 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

  40. 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

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

  42. 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)

  43. 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:

  44. 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)

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

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

  47. 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

  48. 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.

  49. 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

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