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Abstract
  E-cadherin is a tumor suppressor protein and the loss of E-cadherin expression
promotes tumor progression and metastasis. However, in many cases cells of epithelial
tumors and metastases still express E-cadherin. We have found that E-cadherin exists in
different activity states at the cell surface, and we hypothesize loss of cadherin activity
regulation, rather than expression, may promote cancer progression.
To study whether adhesion activation can inhibit metastasis, we tested the effects
of mAbs that activate E-cadherin at the cell surface. Animals with orthotopic tumors
formed by 4T1 cells expressing human E-cadherin were injected either with adhesion-
activating mAb or neutral nonactivating E-cadherin-specific mAb. We found that 
activating Ab treatment significantly reduced the number of 4T1 mammary tumor cells 
that metastasized to lung after 4 weeks.
We also asked whether naturally occurring missense mutations in the E-cadherin
ectodomain known to promote hereditary diffuse gastric cancer (HDGC) in humans
specifically affect the regulation of adhesive activity at the cell surface. To examine basic
adhesive function several HDGC E-cadherin mutants were expressed in cadherin-free
CHO cells. We observed that some mutants exhibited normal adhesion strength while 
others exhibited substantially reduced adhesion strength, although they still had 
greater adhesion than the totally inactive W2A cadherin mutant. To test whether the
mutant E-cadherins can be activated at the cell surface they were expressed in Colo 205
cells after depletion of endogenous E-cadherin via shRNA. All mutants tested so far 
showed impaired activation upon outside and/or inside initiation (activating mAb or
nocodazole treatment respectively) even though they exhibited basic adhesion
functionality in the CHO adhesion assay. One mutant was strongly activated from the
outside by the activating mAbs, but could not be activated from the inside by nocodazole
or by activating p120-catenin mutations.
These findings show that regulation of E-cadherin activity on cell surface is a 
mechanism that contributes to cancer progression, in addition to the known mechanism
of loss of E-cadherin expression
Acknowledgements
 This work was supported by NIH grant R01 GM52717 and GM52717-14S to BMG
Role of E-cadherin cell surface activation in cancer progression
Yuliya I. Petrova and Barry M. Gumbiner
Department of Cell Biology University of Virginia School of Medicine, Charlottesville, Virginia
G239R
D244G
P172R
A298T
S270A
T340A
A634V
W409R
P429S
A592T
P377R
I415L
V487A
P373L
R224C
L583R
A617T
F626V
T599S
Whether adhesion activation can inhibit metastasis?
 
Method
Group Mice Cells # of cells
per
mouse
# of injection
places
Treatment
Control Balb/c
16 mice
4T1Luc_
hEcad
10000 in
50ul PBS
1, mammary
fat pad
Each mouse injected IP 2 weekly with 5mg/kg body weight 46H7 neutral 
mAb (E-cadherin-specific, EC3 domain)
Treate
d
Balb/c
16 mice
4T1Luc_
hEcad
10000 in
50ul PBS
1, mammary
fat pad
Each mouse injected IP 2 weekly with 5mg/kg body weight 19A11 activating 
mAb mAb (E-cadherin-specific, EC1 domain)
Animals were sacrificed at day 27 and lungs were collected. Lung DNA was purified and used to
analyze metastatic content
4T1-Luc2_hEcadh 
human E-cadherin 
staining 
Whole lung qRT PCR analysis revealed difference between Control and Treated groups
DNA samples purified from whole lung lysate were analyzed for Luciferase 2 expression. To count
metastatic cell number a calibration curve with different amount of 4T1Luc2_hEcadh cells mixed with
lung homogenate was built. DNA sample corresponding to 10,000 4T1 cells was used as a reference in
each qRT PCR run. GAPDH was used as housekeeping gene.
Because data in both groups did not have a Gaussian distribution according to Kolmogorov-Smirnov
normality test, the Mann-Whitney U-test was used to confirm statistical difference between groups.
Alternatively, data transformed as Log(10) showed normal distribution in Kolmogorov-Smirnov test
and was analyzed by Student’s t-test (insert)
Human E-cadherin hereditary diffuse gastric cancer 
(HDGC) Germline  Missense Mutations (19) are 
evenly distributed through the ectodomain. Most 
of them belongs to free loops
Individual mice, # of metastatic cells
Model: 4T1-Luc2 commercial mouse mammary epithelial cell line
that is highly metastatic and still expresses E-cadherin. Human E-
cadherin was introduced by Lentiviral infection to enable human E-
cadherin-specific activating antibody use
Whether adhesion activation can inhibit metastasis?
Results
Whether naturally occurring missense mutations in the E-cadherin 
ectodomain known to promote hereditary diffuse gastric cancer (HDGC) 
in humans specifically affect the regulation of adhesive activity at the 
cell surface?
Whether missense gastric cancer mutations in the E-cadherin ectodomain 
specifically affect the regulation of adhesive activity at the cell surface?
Mutant E-cadherin was introduced in CHO cell by Lentiviral
infection. Expression level was verified by flow cytometry.
E-cadherin mutants showed different adhesion strength as 
evaluated using increasing laminar flow to determine the 
force required to detach cells. Error bars = standard 
deviation of several independent experiments (N=4)
Are those E-cadherin mutants adhesive?
Endogenous E-cadherin in Colo205 cells was
knocked down by shRNA. Cells were
subcloned and lowest-expressing clon 7
was used to introduce mutant E-cadherin
by Lentiviral infection. Expression level of
introduced proteins on cell surface was
verified by flow cytometry.
E-cadherin mutants showed different 
activation abilities as evaluated using 
single cell count after activating Fab-
fragment treatment. Error bars = standard 
deviation
Are those E-cadherin mutants “activatable”?
50mM LiClUntreated Act Fab 19A11 Nocodazole Staurosporine Trypsin
Colo hE WTColo hE 
G239R
G239R E-cadherin mutant showed “normal” activation by activating Fab but no 
activation that requires inside-out signaling
Colo parental Colo hE WT Colo hE G239R
6S,T>A p120 
mutant
Mock (no virus)
G239R E-cadherin mutant has impaired inside-out 
signaling because it can not be activated by phospho-
deficient 6S,T>A p120 catenin mutant 
Average in groups
Experimental outline
Human E-cadherin staining
Human E-cadherin staining
Conclusion:
regulation of E-cadherin 
activity on cell surface is a 
mechanism that 
contributes to cancer 
progression, in addition to
the known mechanism of
loss of E-cadherin
Mutations located in free loops are shown as spheres, mutations that 
belong to structure are shown as cartoon

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Petrova_Poster_12 09 2014_mod

  • 1. Abstract   E-cadherin is a tumor suppressor protein and the loss of E-cadherin expression promotes tumor progression and metastasis. However, in many cases cells of epithelial tumors and metastases still express E-cadherin. We have found that E-cadherin exists in different activity states at the cell surface, and we hypothesize loss of cadherin activity regulation, rather than expression, may promote cancer progression. To study whether adhesion activation can inhibit metastasis, we tested the effects of mAbs that activate E-cadherin at the cell surface. Animals with orthotopic tumors formed by 4T1 cells expressing human E-cadherin were injected either with adhesion- activating mAb or neutral nonactivating E-cadherin-specific mAb. We found that  activating Ab treatment significantly reduced the number of 4T1 mammary tumor cells  that metastasized to lung after 4 weeks. We also asked whether naturally occurring missense mutations in the E-cadherin ectodomain known to promote hereditary diffuse gastric cancer (HDGC) in humans specifically affect the regulation of adhesive activity at the cell surface. To examine basic adhesive function several HDGC E-cadherin mutants were expressed in cadherin-free CHO cells. We observed that some mutants exhibited normal adhesion strength while  others exhibited substantially reduced adhesion strength, although they still had  greater adhesion than the totally inactive W2A cadherin mutant. To test whether the mutant E-cadherins can be activated at the cell surface they were expressed in Colo 205 cells after depletion of endogenous E-cadherin via shRNA. All mutants tested so far  showed impaired activation upon outside and/or inside initiation (activating mAb or nocodazole treatment respectively) even though they exhibited basic adhesion functionality in the CHO adhesion assay. One mutant was strongly activated from the outside by the activating mAbs, but could not be activated from the inside by nocodazole or by activating p120-catenin mutations. These findings show that regulation of E-cadherin activity on cell surface is a  mechanism that contributes to cancer progression, in addition to the known mechanism of loss of E-cadherin expression Acknowledgements  This work was supported by NIH grant R01 GM52717 and GM52717-14S to BMG Role of E-cadherin cell surface activation in cancer progression Yuliya I. Petrova and Barry M. Gumbiner Department of Cell Biology University of Virginia School of Medicine, Charlottesville, Virginia G239R D244G P172R A298T S270A T340A A634V W409R P429S A592T P377R I415L V487A P373L R224C L583R A617T F626V T599S Whether adhesion activation can inhibit metastasis?   Method Group Mice Cells # of cells per mouse # of injection places Treatment Control Balb/c 16 mice 4T1Luc_ hEcad 10000 in 50ul PBS 1, mammary fat pad Each mouse injected IP 2 weekly with 5mg/kg body weight 46H7 neutral  mAb (E-cadherin-specific, EC3 domain) Treate d Balb/c 16 mice 4T1Luc_ hEcad 10000 in 50ul PBS 1, mammary fat pad Each mouse injected IP 2 weekly with 5mg/kg body weight 19A11 activating  mAb mAb (E-cadherin-specific, EC1 domain) Animals were sacrificed at day 27 and lungs were collected. Lung DNA was purified and used to analyze metastatic content 4T1-Luc2_hEcadh  human E-cadherin  staining  Whole lung qRT PCR analysis revealed difference between Control and Treated groups DNA samples purified from whole lung lysate were analyzed for Luciferase 2 expression. To count metastatic cell number a calibration curve with different amount of 4T1Luc2_hEcadh cells mixed with lung homogenate was built. DNA sample corresponding to 10,000 4T1 cells was used as a reference in each qRT PCR run. GAPDH was used as housekeeping gene. Because data in both groups did not have a Gaussian distribution according to Kolmogorov-Smirnov normality test, the Mann-Whitney U-test was used to confirm statistical difference between groups. Alternatively, data transformed as Log(10) showed normal distribution in Kolmogorov-Smirnov test and was analyzed by Student’s t-test (insert) Human E-cadherin hereditary diffuse gastric cancer  (HDGC) Germline  Missense Mutations (19) are  evenly distributed through the ectodomain. Most  of them belongs to free loops Individual mice, # of metastatic cells Model: 4T1-Luc2 commercial mouse mammary epithelial cell line that is highly metastatic and still expresses E-cadherin. Human E- cadherin was introduced by Lentiviral infection to enable human E- cadherin-specific activating antibody use Whether adhesion activation can inhibit metastasis? Results Whether naturally occurring missense mutations in the E-cadherin  ectodomain known to promote hereditary diffuse gastric cancer (HDGC)  in humans specifically affect the regulation of adhesive activity at the  cell surface? Whether missense gastric cancer mutations in the E-cadherin ectodomain  specifically affect the regulation of adhesive activity at the cell surface? Mutant E-cadherin was introduced in CHO cell by Lentiviral infection. Expression level was verified by flow cytometry. E-cadherin mutants showed different adhesion strength as  evaluated using increasing laminar flow to determine the  force required to detach cells. Error bars = standard  deviation of several independent experiments (N=4) Are those E-cadherin mutants adhesive? Endogenous E-cadherin in Colo205 cells was knocked down by shRNA. Cells were subcloned and lowest-expressing clon 7 was used to introduce mutant E-cadherin by Lentiviral infection. Expression level of introduced proteins on cell surface was verified by flow cytometry. E-cadherin mutants showed different  activation abilities as evaluated using  single cell count after activating Fab- fragment treatment. Error bars = standard  deviation Are those E-cadherin mutants “activatable”? 50mM LiClUntreated Act Fab 19A11 Nocodazole Staurosporine Trypsin Colo hE WTColo hE  G239R G239R E-cadherin mutant showed “normal” activation by activating Fab but no  activation that requires inside-out signaling Colo parental Colo hE WT Colo hE G239R 6S,T>A p120  mutant Mock (no virus) G239R E-cadherin mutant has impaired inside-out  signaling because it can not be activated by phospho- deficient 6S,T>A p120 catenin mutant  Average in groups Experimental outline Human E-cadherin staining Human E-cadherin staining Conclusion: regulation of E-cadherin  activity on cell surface is a  mechanism that  contributes to cancer  progression, in addition to the known mechanism of loss of E-cadherin Mutations located in free loops are shown as spheres, mutations that  belong to structure are shown as cartoon