Download Integrin αvβ3 suppresses expression of the pro

Integrin αvβ3 suppresses expression of the pro-apoptotic mediator PUMA in
Breast Cancer cells
Charlie Michael, Isha Bagga, Qi Sun, Jay Desgrosellier
The Department of Pathology, University of California San Diego, 9500 Gilman Drive, La Jolla, California
Relative expression of Slug dependent genes
Immunoblot Analysis
Slug-dependent Genes
LM2-4
HS578T
PUMA
HD
AC
MU 1
CL C1
DN
1
RB
CD L1
KN
1A
MD
M2
BM
I1
G
CT FI1
NN
NA B1
NO
G
BC
L2
BB
C3
shCtrl
Survival Self-Renewal Cell Cycle
EMT
Among all Slug dependent genes, PUMA appears to be the most
upregulated when β3 is knocked down
qPCR results
HS578T
LM2-4
2.5
2
1.5
1
0.5
0
Relative levels of PUMA
mRNA
Relative PUMA mRNA levels
Integrin αvβ3’s pathway
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
MDA468
Repative PUMA mRNA levels
Metastasis is the number one cause of mortality in
Breast Cancer patients. Mammary gland stem cells have
been linked to more aggressive properties in Breast
Cancer, including metastasis. We have shown that the
Integrin αvβ3 increases expression of the transcription
factor Slug, enhancing stem cell properties in both the
mammary gland and aggressive Breast Cancers. This
suggests to us that there may be Slug dependent genes
that are important for αvβ3's role in Breast Cancer
stemness and metastasis.
To identify Slug dependent genes that are regulated by
avb3, we screened Breast Cancer cells +/- genetic
knockdown of β3 and identified PUMA (p53 upgregulated
modulator of apoptosis) as the most upregulated gene in β3
knockdown cells. In a panel of Breast Cancer cell lines
expressing ectopic αvβ3 or αvβ3 knockdown, we found that
αvβ3 is necessary and sufficient to suppress both PUMA
mRNA and protein expression, using qPCR and
immunoblot analysis, respectively. This suggests that the
αvβ3-Slug pathway may enhance Breast Cancer cell
survival by suppressing PUMA, leading to more aggressive
metastatic tumors.
Relative mRNA Levels
(Fold change β3 knockdown vs control)
Abstract
1.2
1
0.8
0.6
0.4
0.2
0
Ectopic GFP
Ctrl
Ectopic β3
7
6
5
4
3
2
1
0
Using qPCR, we analyzed a
panel of Breast Cancer cell lines
for relative levels of PUMA
mRNA. We found that cell lines
with β3-knockdown (above)
tended to show increased levels
of PUMA mRNA, whereas in cell
lines without endogenous β3 but
with ectopic β3 expression,
PUMA mRNA levels were
decreased.
shCtrl
shβ3
PUMA
PUMA
β-actin
β-actin
shβ3
Immunoblot analysis was used to confirm the
differences in PUMA protein levels between
control cells and ones with β3 knocked down.
This is necessary to confirm that the PUMA
mRNA concentrations correlate to the protein
concentrations. β-actin was used as a loading
control.
PUMA’s role in Pathway
“Stem Cell Activation” Pathway
αvβ3
c-Src
Slug
?
PUMA
Stemness/Metastasis
Conclusions
Our findings show that Integrin αvβ3 is necessary and sufficient to
suppress both PUMA mRNA and protein expression.
Future Research
Further research will continue to investigate a functional role for
PUMA in Breast Cancer metastasis and mammary stem cells
downstream of αvβ3/Slug signaling.