Functional Genomic Analysis of Autophagic Cell Death using RNA
interference CATGGCGATATTGT CATGGCGCCAATAT CATGGCGCGCATTT
CATGGCGTGGGGAT CATGGCTAATAAAT CATGGCTCAAGGAG CATGGCTGGACTCC
CATGGCTGTGGCCA CATGGCTTTCGTGT CATGGCTTTTTGGC CATGGGAACCGACA
CATGGGACCGCCCC CATGGGACCGCTCA CATGGGATCACAAT CATGGGCAACGATC
CATGGGCAGCAAGC CATGGGCAGCAATT S. Gorski, Genome Sciences Centre, BC
Cancer Agency Slide 2 Overview Background Research Aims
Experimental Approach Preliminary Data Slide 3 Programmed Cell
Death (PCD) Function Dysfunction Deleting damaged cells Cancer
Culling cell number Deleting structures Sculpting tissues
Autoimmune diseases Neurodegenerative diseases Developmental
abnormalities Slide 4 Types of Programmed Cell Death (PCD) (adapted
from Baehrecke, 2002) I. Apoptosis II. Autophagic PCD Slide 5
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 Slide 6 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. Slide 7 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. Slide 8 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) Slide 9 Small Interfering RNAs (siRNA) (From McManus and
Sharp, 2002 & Hannon, 2002) siRNA siRNAs unwind and guide RISC
to a substrate siRNAs are incorporated into RISC (RNA Induced
Silencing Complex) mRNA substrate cleavage 5 phosphate Slide 10
siRNA in mammalian cells Potential Problems: may have to try
multiple siRNAs per gene effects are transient 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 Slide 11
Characteristics of the MCF-7 cell line human breast carcinoma cell
line amenable to siRNA analyses treatment with tamoxifen (10 -6 M)
induces autophagic cell death (Bursch et al. 1996, 2000) Slide 12
Preliminary Data 1.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 Slide 13
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 Slide 14 Autophagic Cell Death Candidate
Genes Slide 15 Acknowledgements BC Cancer Agency BC Cancer
Foundation NCIC NSERC 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/ Slide 16 Slide 17 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 Slide 18 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 Slide 19 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
