1. 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

2. Overview Background Research Aims Experimental Approach
Preliminary Data

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

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

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

6. 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.

7. 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.

8. 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

9. 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)

10. 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

11. 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)

12. 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:
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

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

14. Autophagic Cell Death Candidate Genes

15. 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
BC Cancer Agency
BC Cancer Foundation
NCIC
NSERC

17. Keyword Gene Sim (%) (aa) Swiss Prot id Database DescriptionSG 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'

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

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