role of crumbs2 in tumour progression
TRANSCRIPT
Modulating Crb2 protein levels effect on tumour progression
Baijun KouDepartment of Biomedical Sciences&
School of Medicine
University of Sheffield, U.K.
European Union Crumbs Consortium
June, 2008. Marseille, France
APICAL-BASAL POLARITY IS VITAL FOR NORMAL CELLULAR FUNCTION
Defects in apical-basal polarity are associated with
developmental defects
Cancer et al.
No
rma
l tissue C
arcin
om
a tissu
e
CRB2 expression in human breast cancer
Grade I Grade II Grade III
CRB2 expression in human normal & prostate cancer
CRB2 is a Crumbs family member with at least 3 isoforms (R.Walker)
Located at human chromosome 9q33.3.CRB2 is a type I transmembrane protein with multiple EGF-like domains and Laminin G-like domains.
CRB2
Extracellular
hCrb2s(1-10a exon)
hCrb2f(1-13exon)
hCrb26a(6a-13exon)
Laminin G-like domain EGF-like domain
signal peptidetransmembrane domain
cbEGF-like domain
cytoplasmic domain intracellularextracellular
Patial EGF-like domain
Three protein complexes maintain cells polarity
Alteration CRB2 affects cells proliferation and apoptosis
WT Over KD
Immunocytochemistry(Ki67)
ß-actin
CPP32
sh129
WT6a-1
31-
13 WTsh
128
WB
Overexpression knockdown
n=3 *p<0.05
0
500
1000
1500
2000
2500
3000
3500
4000
4500
K-C K-6a-13 K-1-13 K-sh128 K-sh129
Cel
ls a
popt
osis
leve
l(AU
)*
*
0
10
20
30
40
50
60
K-C K-6a-13 K-1-13 K-128 K-129ki6
7 p
os
itiv
e c
ells
nu
mb
er
pe
r 1
00
ce
lls
**
******
n=10,*p<0.05,***p<0.0001
Altering CRB2 expression levels affects….migrationWounding assay:
*** ***
** ****p<0.01,***p<0.001 v K-C
0
5
10
15
20
K-C
K6a-1
3
K1-13
K128
K129
Wid
th o
f w
ou
nd
aft
er
24
ho
urs
(U
)
*** ***
**p<0.01,***p<0.001 v K-C
Alteration of CRB2 affects cells invasion
8 12 16 20 24 28 32 36 40 44 48(hrs)
36hrs 40hrs 44hrs
Mitomycin(1ng/ml)
n=4 p<0.05 ,ANOVA
Ki67 antibody staining with the membrane
Altering CRB2 expression levels affects
…cadherin expression
Pan-cad
K-c 6a-13 1-13 k-c sh128 sh129
γ-tubulin
WB
Summary
Modulating Crb2 protein levels results in:
•A positive correlation with proliferation and negative with apoptosis
•A positive correlation with migration & invasion
•Interfered with cell adhesion and tight junction markers
•Crb2 depletion induces tumor suppressor protein expression
Crb2 protein is localised to the apical surface of the developing neural tube and normal adult
epithelial tissue
Neural tube in chicken embryo
Human ovarian tissue
CRB2 expression in cancer cell lines
1 2 3 4 5 6 7 8 9 10 1112 13
6a 10a
54 human cancer cell lines were screened- by RT-PCR10 cell lines express different exons of CRB2 with variable levels
A way to find the external partner of crumbs (EPOC)?
WB
1-9a cells w
hole lysis
1-9a protein
1-9a culture supernatant
V5 antibody
F-actin staining
1-9a protein was added
3 days in K-C cells
K-C
Altering CRB2 expression levels affects
… ß-Catenin expression
ß-Catenin
γ-tubulin
K-c 6a-13 1-13 K-c sh128 sh129
WB
WT Over-expression Knockdown
ß-Cat
DAPI
Alteration of CRB2 assembles PATJ
WB
K-C 6a-13 1-13 sh128 sh129
PATJ
γ--tublin
WT
PATJ
DAPI
3-D
PATJ
Pan-cad
DAPI
2-DOverexpression Knockdown
CRB2 knockdown induced tumor suppressor proteins expression
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
K-C
K6a-13
K1-13
K-C
K128
K129
SCRIB
-tubulin
LKB1
LGL1
WB
Tight junction
Adherin junction
Altering CRB2 expression levels affects …ability to polarization
K-C
K1-13
K128
-catenin DAPI Merge
5000 cells were cultured in matrigel coated chamber with 10% FBS RPMI 1640 for 6 days, Images were taken by Zeiss 510 confocal microscope
Future questions & work• What are the cells I have been working on?-in progress
• How do different Crb2 splice forms interact with the cell cytoskeleton (Rho
activation?)- in progress
• Can K1-9a cells enable us to identify EPOC? - in progress
• How does knockdown of Crb2 increase/stabilise Lgl1 etc?
• Does Crb2 levels effect tumour formation in mice?- in progress
• Are the results repeatable in other cell lines?-in progress
• Determine role of CRB2 in cancer progression
AcknowledgementsAcknowledgements
Penny RashbassPenny Rashbass( Supervisor)( Supervisor)
Rob WalkerRob WalkerJean IllesJean IllesThorsten BoroviakThorsten BoroviakMariyam MurtazaMariyam Murtaza
Funding: Yorkshire Cancer Research
CollaboratorsCollaboratorsSimon CrossSimon CrossJim CattoJim CattoRichard BryantRichard BryantSteve WinderSteve WinderDuncan BakerDuncan BakerKim ReevesKim ReevesNicky BrownNicky Brown
EU Crumbs consortium EU Crumbs consortium
Ki67 antibody staining with the membrane
Counting 100 cells per field n=4, p>0.05 After adding mitomycin 36hrs
k-c k6a-13 k1-13 k128 k129
Histo- microarray-immunostaining of CRB2
Oesophageal (82ca /39con)
Ovary(58ca/29con)
Lung(84ca/4con)
Prostate(121ca/44con)
Breast(336ca)
Colorectal(112ca)
Brain(43ca/4con)
Baijun Kou
Summary-1 CRB2 expression was profiled in 7 different epithelial tissues - 786 cancer & 116 non-cancer
samples. In normal & benign adenoma - CRB2 is usually apical In breast, colorectal & oesophageal adeno
- CRB2 expression levels In prostate, ovary, brain & oesophageal squamous-
CRB2 expression levels CRB2 tends to become cytoplasmic
In general, within tumour samples, there is a negative correlation between grade &/or stage and CRB2 expression levels.
• Need to be done: Quantification of CRB2 expression in human cancer
tissues by q-PCR.
Western blot analysis of CRB2& F-actin expression of cytoskeleton after alteration of CRB2 level
ß-actin
C 1-9a 6a-13 1-13 C sh128 sh129
overexpression knockdown
CRB2
WT 1-9a 6a-13 1-13 sh128 sh129
Gibson & Perrimon 2003
Complexes maintaining cell polarity
Functions & binding partners Include:•Polarity•Adhesion•Signalling•Vesicular trafficking•Proliferation•Actin cytoskeleton•microtubules
Crumb2 knockdown induces cancer suppressor genes expression and induces cells to epithelial phenotype
LGL1
Scribble
5000 cells were cultured in matrigel coated chamber with 10% FBS RPMI 1640 for 4 days, Images were taken by Zeiss 510 confocal microscope
WT Overexpression Knockdown
β-cat
Crb2
DAPI
γ-tublin
K-c 6a-13 1-13 k-c sh128 sh129
6 days culture
Tissues Cancer/non-ccancer grade stage mislocalization local invasion lymph node vascular /distance Expression ratio metastasis metastasis
Oesophageal squa adeno squa adeno NR NR almost all NR NR NR (including non-cancer) Prostate NR NR Cancer>non-cancer NR all ( ) NR Ovary NR NR(grade) PR
Lung NR almost all cancer NR NR trend NR Breast NR (tumor size)NR apical-cytoplasma-lose with grade increase Colon NR apical-cytoplasma-lose with grade increase Brain all
CRB2 expression in epithelial carcinomas
mCrb2 overexpression in kelly cell lines
1 2 3 4 5 6 7 8 9 10 1112 13
Have used mouse cDNA constructs
1-13
6a-13
1-9a
Robert Walker