Summer Undergraduate Research Program - Fred Hutchinson ...

Role of Integrin β-4 and Fibroblast Growth Factor Receptor-3 in
α-catenin’s Tumor Suppression Function
Yu Zhu1, 2 , Ewa Stepniak2 , Wen-Hui Lien2 , Olga Klezovitch2 , Liem Nguyen2 and Valeri Vasioukhin2 1 Biology Department, Reed College, Portland, OR
2 Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA
2009 Lee Hartwell Poster Award
Cadherin ? α-catenin
?
Objectives
Introduction
Figure 6. Hypothetical mechanism
of tumor-suppression function of
α-catenin. The work-in-progress
model.
1. Analyze β-4 integrin complexes and signaling in αcatenin
-/- cells.
2. Identify and analyze additional signaling pathways
required for the failure of contact-mediated
α-catenin may attenuate β-4
integrin signaling by promoting β-4
integrin-cadherin complex formation.
Without α-catenin, β-4 integrin may
cooperate with FGFR-3 to hyper
activate tyrosine phosphorylation and
downstream signaling, resulting in
loss of contact-mediated inhibition
and tumor formation.
inhibition of cell proliferation in α-catenin -/- cells.
Results
1. α-catenin is necessary for β-4-integrin-cadherin
complex formation
Adherens Junctions (AJs) are cell-cell adhesion structures
composed of classical cadherins, catenins, and associated
proteins. Proper formation and disassembly of AJs is essential
for maintaining tissue integrity as well as dynamic cell
reorganization during embryogenesis and wound healing.
Aberrant regulation of AJs is a common occurrence in epithelial
cancer. Our lab focuses on α-catenin, which links cadherins
and the actin cytoskeleton. α-catenin is often lost in various
epithelial tumors including squamous cell carcinoma (SCC). Our
lab demonstrated that deletion of α-catenin in hair follicle stem
cell niche results in development of SCC (Fig. 1). In this work we
are trying to identify the mechanisms responsible for the tumor
suppression function of α-catenin.
WT KO
kDa
WT KO WT KO
+ - Input + - Input
+ - Input + - Input
191
Integrin
β-4
97
WT KO WT WT KO KO
2 mo. 4 mo.
Conclusions
64
α-Ecatenin
β-catenin
Actin
51
E-cad αE-cat KO
E-cad αE-cat WT
160
(1)β-4 integrin is upregulated in keratinocytes lacking αcatenin.
(2)α-catenin negatively regulates the level of tyrosine
phosphorylation in cultured keratinocytes.
(3)α-catenin is necessary for complex formation between β-4
integrin and cadherin.
(4) FGFR-3 is required for the loss of contact-mediated
inhibition of cell proliferation in α-catenin-/- cells.
β-
39
actin
IP: anti-cadherin (+) or pre-immune (-) serum IP: anti-cadherin (+) or pre-immune (-) serum
WB: anti-integrin β-4 or anti-β-actin
WB: anti-phospho-tyrosine
Figure 3. Integrin β-4 co-immunoprecipitated with E-cadherin in wild-type (WT)
but not α-catenin -/- (KO) keratinocytes (left). Increased tyrosine phosphorylation
in α-catenin -/- (KO) keratinocytes (right).
2. FGFR-3 is required for the failure of contactmediated
inhibition in α-catenin-/- (KO) cells
Keratin 10 WT Keratin 10 KO
H & E WT H & E KO
WT KO
2
1.8
Filagrin WT Filagrin KO
Keratin 14 WT Keratin 14 KO
1.6
1.4
Cre/aE-catflox/flox 1.2
1
) develop SCC (Klezovitch and Lien, unpublished). 0.8
Future Directions
Figure 1. Hair follicle stem cell niche-specific αE-catenin knockout mice (GFAP-
0.6
0.4
(1)Analyze changes in β-4-integrin signaling in α-catenin-/cells.
(2)Analyze changes in FGFR-3 signaling in α-catenin-/- cells.
(3)Analyze potential complex formation between FGFR-3,
cadherin and in β-4-integrin in wild-type and α-catenin-/- cells.
(4)Determine the role of β-4-integrin and FGFR-3 in the tumorsuppression
function of α-catenin in vivo.
0.2
0
Ctrl Erbb3 Erbb2 Met Mst1r Fgrf2 Fgrf3 Epha2 Epha1 Ephb4 Shc1 Ptpn11 Itga6 Ptk6 Frk
Figure 4. Relative cell numbers upon 4 days in confluent culture after transfection
with indicated siRNA oligos.
3. Decrease in tyrosine phosphorylation of FGFR-3 in
α-catenin-/- cells
kDa
input - +
input - +
ko wt ko wt ko wt kDa
ko wt ko wt ko wt
Integrin β-4 is a laminin receptor prominently expressed in
mouse keratinocytes. In addition to cell adhesion, integrin β-4
is also involved in cell signaling and activation of MAPK and
PI3K pathways. Our lab found that α-catenin-/- keratinocytes
are unable to stop proliferation upon reaching confluency, and
that β-4 is required for this cancer–relevant phenotype (Fig. 2).
These data indicate that β-4 can play an important role in the
tumor-suppression function of α-catenin. However, the
molecular mechanisms remain unknown.
Acknowledgements
191
FGFR-3
191
Phospho-
FGFR-3
97
97
Thanks for the mentorship from Dr. Valeri Vasioukhin, Dr. Ewa
Stepniak and other members of the Vasioukhin lab. My
participation in this project is made possible by Mellon
Foundation Opportunity Grant and Reed College SEEDS
Summer Experience Fellowship.
64
64
51
51
Figure 5. Total protein (input) from wild-type (WT) and α-catenin-/- (KO) cells were
immunoprecipitated with anti-FGFR-3 (+) or irrelevant (-) antibodies and analyzed
by western with anti-phospho-tyrosine (left) and anti-FGFR-3 (right) antibodies.
Figure 2. Contact inhibition is rescued in αEcatenin-/-
keratinocytes with integrin β-4 knocked
down (Stepniak and Lien, unpublished).