a functional genomics approach to autophagic cell death gene discovery marco marra canada’s...
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A Functional Genomics Approach to Autophagic Cell Death Gene
Discovery
Marco MarraCanada’s Michael Smith Genome Sciences Centre
British Columbia Cancer Agency
CATGGCGTGGGGAT
CATGGCTAATAAAT
CATGGCTCAAGGAG
CATGGCTGGACTCC
CATGGCTGTGGCCA
CATGGCTTTCGTGT
CATGGCTTTTTGGC
CATGGGAACCGACA
CATGGGACCGCCCC
CATGGGACCGCTCA
CATGGGATCACAAT
CATGGGCAACGATC
CATGGGCAGCAAGC
CATGGGCAGCAATT
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 = apoptosisType II = autophagic cell deathType III = non-lysosomal
Types of Programmed Cell Death (PCD)
(adapted from Baehrecke, 2002)
I. Apoptosis
II. Autophagic PCD
Autophagy
Autophagosome
Autophagolysosome
www.uni-marburg.de/cyto/elsaesse/auto.htmThe Cell, A Molecular Approach, G.M. Cooper, Ed., 2000
• Housekeeping• Starvation• PCD
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!
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 mouseBeclin1, summary
J. Mol. Recognit. 2003; 16: 337–348
Autophagic PCD pathways
• Molecular machinery?
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.
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
The Drosophila Salivary Glands
Cell types: duct cells & secretory cells Cell number: 100 cells eachSize 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)
Drosophila salivary gland PCD
(adapted from Jiang et al., 1997)
•autophagic •stage-specific•synchronous
20 hr 24 hr 26 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
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
known or predicted genes
genomic DNA and EST(but no predicted gene)
genomic DNA and/orreverse strand of gene
no match
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)
SAGELibrary
Tags analyzed Transcripts
Total transcripts
16 hr 34,989 3,126
4,62820 hr 31,215 3,034
23 hr 30,823 2,963
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
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 geneswere not annotated (GadFly Release 2)
48 correspond solely to salivary
gland ESTs
Update this slide!! ; Release 3 data
SAGE Identifies Genes Associated Previously With Salivary Gland Death
0
0.0001
0.0002
0.0003
0.0004
0.0005
0.0006
BR-C E74 E75 E93 rpr ark dronc iap2 crq
SG16
SG20
SG23
Tag Frequency
BFTZ-F1
EcR/USP
BR-CE74E93
rprhidark
dronccrq
iap2
CellDeath
E75
Genes associated with autophagic PCD
Expressionfold-difference(16 hr vs 23 hr)
Proteinsynthesis Hormone
related
Trans-cription*
Signal transduction Apoptosis
Immune response/TNF-related
Autophagy
0
10
20
30
40
50
60
70
80
90
100E
f1g
amm
aC
G5
605
CG
384
5e
IF-4
EC
G9
769
CG
101
92C
G7
439
eIF
-5A
Hr7
8C
G1
5505
CG
759
2E
ip63
F-1
Eip
71C
D
bu
nE
P22
37
CG
995
4C
G3
350
Aka
p2
00D
oa
sktl
Ptp
me
gP
R2
CG
167
08C
G8
655
Th
orC
ecA
1C
ecC
Ce
cB De
fD
rsP
GR
P-L
AB
ES
T:G
H0
2 Dif
cact
Myd
88T
raf1
CG
409
1
Dcp
-1em
pC
G1
2789
CG
382
9bu
ffysi
ckle
CG
619
4C
G1
643
CG
542
9C
G1
0861
Ra
b-7
CG
111
59C
G3
132
CG
109
92ca
thD
CG
172
83C
G1
2163
Cp
1
CG
148
30C
G1
908
CG
540
2C
G1
5239
CG
109
65C
G1
8811
AE
003
826
AE
003
481
AE
003
446
AE
003
503
reve
rse
ES
Tre
vers
e E
ST
Unknowns
0
10
20
30
40
50
60
70
*Ce
cB
*CG
40
91
*Ce
cA
1
Do
a
CG
48
59
CG
14
99
5
CG
52
54
CG
38
45
Ptp
me
g
CG
93
21
CG
12
78
9
CG
12
16
larp
CG
78
60
Cy
p1
So
x1
4
CG
13
44
8
CG
81
49
ark
OreR
E93
Gene expression is reduced in a salivary gland death-defective mutant
Fold-differencein 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)
Function-based strategies for characterizing
differentially expressed genes
Mutants available
Overexpression andloss-of-function
in vivo
Prioritization•midgut PCD
•human ortholog/cancer•l(2)mbn cells
RNAi inmammalian cells
RNAi in Drosophila l(2)mbn cells
Phenotype analyses•salivary glands, midguts,
retinas, embryos
Mutants unavailable
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!!!
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
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
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
Percentage of Live l(2)mbn Cells After 10uM 20HE Treament
0
20
40
60
80
100
0 24 48 72
Hours of Treatment
% L
ive
Cel
ls
Control Cells
20HE Treated Cells
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
RNAi screen design
Prepare dsRNA usingT7-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 treatment Ecdysone treatment
Diap1-RNAitreatment
24 hrs
PCD pathways in Drosophila
(Meier et al., Nature 2000)
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)
CG4091 expression profile
0
5
10
15
20
25
30
SG16
SG20
SG23
16 A
PF
20 A
PF
23 A
PF
3rd
insta
r
0 APF
4 APF
6 APF
Fo
ld e
xpre
ssio
n
SAGEQRT-PCR in salivary glands
QRT-PCR in mid-gut
CG4091-RNAi partially blocks PCD induced by Diap1-RNAi
0.00E+00
5.00E+05
1.00E+06
1.50E+06
2.00E+06
2.50E+06
3.00E+06
3.50E+06
4.00E+06
4.50E+06
RNAi construct
No
. of
live
cells
0 after dsRNA treatment (AVE)
24 hrs after dsRNA treatment: (AVE)
5 days after dsRNA treatment: (AVE)
PCD pathways in Drosophila
(Meier et al., Nature 2000)
CG4091
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)
Acknowledgements
BC Cancer AgencyBC Cancer Foundation
National Cancer Institute of CanadaMichael Smith Foundation
for Health ResearchNSERC
BC Cancer Research CentreVictor Ling
GSC PCD group Suganthi Chittaranjan
Doug FreemanSharon Gorski
Melissa McConechyJennifer Kouwenberg
BioinformaticsSteven Jones
Erin PleasanceRichard Varhol
Scott Zuyderduyn
GSC Sequencing Group
University of Maryland Biotech InstituteEric Baehrecke
www.bcgsc.cahttp://sage.bcgsc.ca/tagmapping/http://www.bcgsc.ca/lab/fg/dsage/
Comparison of SAGE and real-time quantitative RT-PCR
Fold-difference by SAGE
Fo
ld-d
iffe
ren
ce b
y Q
RT
-PC
R
1.0
10.0
100.0
1000.0
1.0 10.0 100.0 1000.0
Correlation coefficient = 0.5
II. Correlation coefficient between fold-difference values (64 samples):
I. Direction of Change:
91/96 samples = 95% concordance
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)
KeywordData
base
SG
16
SG
23Gene
Sim
(%)Score
Length
(aa)Swiss
Prot idDatabase Description
death
apoptosis
apoptosis
apoptosis
apoptosis
apoptosis
apoptosis
death
death
survivalautophagy
hormone
hormone
cancer
cancer
tumor
tumor
tumor
apoptosis
TNF
BH3
SP
SP
SP
SP
SP
SP
SP
SP
SP
SP
SP
SP
SP
SP
SP
SP
SP
SP
FB
FB
PCD
0
4
0
0
0
0
1
1
0
1
0
2
0
1
1
45
10
65
1
1EST only
15
0
6
5
4
4
7
61
7
7
7
73
12
7
15
1
0
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
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-foldin 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
Mining Expression Data in Drosophila
I. Keyword-based Data Mining
Q: How many differentially expressed Drosophila geneshave a mammalian homolog that has been associated with
cancer?
A: 33 genes
GadFly – Swissprot Homology Table
Extract entries based on keyword search
Mining Expression Data in Drosophila
II. Cross-species Gene Expression Comparisons
CG4091 upregulated 102-foldin 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)
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
RNAi screen results
Genes tested with RNAi:CG40228CG11051
Sgs7Talin
CG8785CG14214
Diap1CG9911CG7220
TimpCG7059Sema-5cCG4798
Possible phenotype:CG14214
Cell Death Phenotype:diap1
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
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
WST-1 Assay Results
WST-1 Assay with overnight incubation
0.000
0.050
0.100
0.150
0.200
0.250
0.300
48hr control 48hr 20HE
Cell Type
Ab
so
rba
nc
e (
A4
50
-A6
50
nm
)
100uL cell plated
50uLcells plated
Decrease in cell respiration of 20HE treated cells –Directly represents the amount of viable cells
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)
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:
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)
PCD and disease
Function Dysfunction
Deleting damaged cellsCancer
Culling cell number
Deleting structures
Sculpting tissues
Autoimmune diseases
Neurodegenerative diseases
Developmental abnormalities
Genome Sciences CentreProgrammed Cell Death Group
Apoptotic Cell Death Autophagic Cell Death Autophagy
inxs(Doug Freeman)
echinus(Ian Bosdet)
Cloning and Characterization
Mammalian cell linetranscription 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 GeneDiscovery
(Brent Mansfield)
RNAi screenin Drosophila
cell line(Suganthi, Melissa
McConechy, Jennifer Kouwenberg, Amy Leung)
RNAi screen inmammalian cell line
(M. Qadir)
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
Microscopy Cell Death Assays Autophagic 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)
Specific Aim
Identify and characterize genes that are necessary for mammalian autophagic cell death.
Experimental Approach
RNAi analysis of candidate autophagic cell death genes inhuman breast carcinoma cell line MCF-7.
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
GSC Programmed Cell Death Group
Ian Bosdet Amy LeungSuganthi Chittaranjan Melissa McConechyDoug Freeman Erin PleasanceClaire Hou Mohammed QadirJennifer Kouwenberg
Marco MarraVictor LingSharon Gorski
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)