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)