Molecular Characterization of the Na /H Exchanger in Human Disease

Transcription

Molecular Characterization of the Na /H Exchanger in Human Disease
Molecular Characterization of the
Na+/H+ Exchanger in Human
Disease
Dr. Larry Fliegel
Department Biochemistry,
Faculty Medicine
University of Alberta
[email protected]
780 492-1848
The Na+/H+ Exchanger
Plasma Membrane Glycoprotein
1 Intracellular H+ for 1 Extracellular Na+
NHE1 ubiquitous,
NHE2-10 restricted
H+ Na+
NHE
H+ Na+
Mammals (All tissues)
Model of NHE1
Extracellular
EL1
H+
1 2 3 4
Nterminal
EL4
EL3
EL2
IL1
5 6 7 8
IL2
IL3
501
EL6
EL5
9
10 11 12
IL4
IL5
Na+
PIP2
CHP
P
CAII
Tescalcin
637-815
633-815
684
P
P
CaM
ERK1/2 p90rsk
NIK
p160ROCK
700-815
F-actin
510-530
CaM
636
P
P
Intracellular
503ERM670
513-564
C-terminal
815
Characterization of the Na+/H+
Exchanger in Two Human Diseases
One common
One rare
Common Disease
Breast Cancer
1 in 9 (or 1 in 8) women
Metastatic Triple negative breast cancer
< 30% survive 5 years
Our Study
Role of
the Na+/H+ Exchanger in Breast Cancer
NHE1 and Breast Cancer and Metastasis
NHE1 is more critical in pH regulation in breast
cancer cells
Becomes Hyperactive
Acidifies extracellular microenvironment
Promotes Metastasis
NHE1 in the Tumor Microenvironment
pHe = 7.2 – 7.4
Extracellular
pHe = 6.2 – 6.8
Na+
Na+
NHE
1
H+
NHE
1
CYTOPLASM
H+
Normal Cell
Transformed/Tumor Cell
• Resting pHi: 6.9 – 7.1
• Proton gradient low; outwards
• NHE1 activated by acidification
• Resting pHi: 7.2 – 7.7
• Proton gradient high: Inwards
• Constitutively active NHE1
Reshkin and others work
Hypothesis, NHE1 contributes to
breast cancer tumor growth and
metastasis
Tested the effect of NHE1 knockout
and inhibition on various parameters
Cells and Experimental Conditions
 MDA-MB-231
• Triple negative
• Estrogen Receptor
• Progesterone Receptor
• Her2/Neu Receptor
• Highly invasive
• Endogenous NHE1
 Experimental Culture Conditions
• Normal serum (10%): Resting (UNSTIM)
• Low serum (0.2%): Hyperactive NHE1 (STIM)
Generation of 231-KO Cells
WB: Loss of NHE1 expression
Unstim (10%)
*
Stim (0.2%)
* P < 0.001
Does loss or inhibition of NHE1 change
paclitaxel effects on MDA-MB-231 cells?
Paclitaxel
 Chemotherapy drug for breast and ovarian cancer
• IC50 2.4 nM in MDA-MB-231 cells [Nakayama et al, 2009]
• Used: 1 nM
Cell Viability:
Effect of Paclitaxel ± EMD or HMA
*
Control
*
Paclitaxel
EMD
EMD + Paclitaxel
HMA
HMA + Paclitaxel
* P < 0.001
• NHE1 inhibition increases susceptibility to paclitaxel-mediated
cell death
Wound-Healing Assay: Migration
Wild type vs. KO
UNSTIM
STIM
0h
Percent Gap Closure 18h post-scratch in
UNSTIM and STIM 231-WT vs. 231-KO
[N=4; #fields=10; 4-5mea/field; 29-09-12;25-04-13]
231-KO
231-WT
100
Unstim
(10%)
Stim (0.2%
80
* P < 0.01
Gap Closure (%)
*
60
18 h
40
231-KO
231-WT
20
0
WT
Adapted from Moreno-Bueno et al, 2009 (Nature Protocols)
KO
Migration at 18 h:
Effect of Paclitaxel ± EMD or HMA
Control
Paclitaxel
EMD
EMD + Paclitaxel
HMA
HMA + Paclitaxel
* P < 0.001
*
*
• NHE1 inhibition with EMD or HMA plus paclitaxel greatly
decreases the rate of migration of stimulated 231-WT cells
Boyden Chamber Method: Cell Invasion
 Invasion Assay:
24-well inserts
➛Measure of metastatic
potential
➛Invasive cells move through
pores in Matrigel-coated
0.2% Serum+Media
Extracellular Matrix
10% Serum+Media
24 h
Invasion: Unstim vs Stim 231 WT and KO cells over 24 h
into media containing 10% serum (10X magnification)
[5+13+20+28-06-13, N=4, R=16-18]
Invasion at 24 h: Effect of NHE1 deletion
UNSTIM
STIM
No. of Cells/Field (10X Maginification)
500
*
Unstim (10%)
Stim (0.2%)
KO-STIM
* P < 0.001
400
300
WT-STIM
200
100
0
WT
KO
• Loss of NHE1 greatly reduces the invasive potential of 231-KO cells
compared to the wild-type
Invasion: Effect of Paclitaxel ± EMD or HMA
Control
Paclitaxel
EMD
EMD + Paclitaxel
HMA
HMA + Paclitaxel
* P < 0.001
*
*
• NHE1 inhibition synergizes paclitaxel responses
Effect of NHE1 Knockout on Xenograft tumor
growth in mice
______
B
Summary (Part I NHE1 in a common disease)
 Loss of NHE1 decreases tumor growth in vivo
 Inhibition of NHE1 in tumor mimetic conditions
 Increases TNBC susceptibility to Taxol
 Decreases metastatic potential
 NHE1 inhibitors: Potential adjuvant to chemotherapy?
Future Directions
 NHE1 inhibitors + Taxol: Does it work in vivo?
 How is NHE1 regulated in TNBCs?
Characterization of the Na+/H+
Exchanger in a Rare Human Disease
Mutations in SLC9A1, Encoding
NHE1, Cause Ataxia–Deafness
Lichtenstein–Knorr Syndrome
Collaboration, Claire Guissart, Michel Koenig, Montpellier, France
Xiuju Li
((2015) Human Molecular Genetics 24(2):463-70.)
Mouse NHE1 Knockout Models
Spontaneous mouse
mutant p.Lys442*
Cox et al.
77 aa
deletion
Bell et al.
Lichtenstein–Knorr Syndrome
-autosomal recessive
-1930 by H. Lichtenstein and A. Knorr
-sensorineural hearing loss and cerebellar ataxia
-cause unknown
Identified
Consanguineous family
-recessive cerebellar ataxia/deafness
-exome sequencing
-bioinformatics ranking of genetic variants
CLINICAL Summary
Lichtenstein-Knorr
syndrome
Ataxia
Deafness
22
21
years
17
• Turkish origin, 3 affected
• 1st degree consanguinity
• Cerebellar and posterior column
ataxia
• delayed walking at ages ranging
from 18 months to 5 years
• Deafness (profound in the sisters,
moderate in the younger brother)
• Retarded language in the oldest
sister
• Growth retardation and
microcephaly (brother only)
Localization of the defect to the NHE1
(SLC9A1) Gene
SNP (single nucleotide polymorphism) Mapping
Exon (Exome) Sequencing
Glycine 305 of NHE1
Gly 305 Arg
Putative Location of Gly305
Gly305
Extracellular
EL1
H+
1 2 3 4
Nterminal
EL4
EL3
EL2
IL1
5 6 7 8
IL2
IL3
EL6
EL5
9
10 11 12
IL4
IL5
Na+
PIP2
CHP
P
CAII
Tescalcin
637-815
633-815
684
P
P
CaM
ERK1/2 p90rsk
NIK
p160ROCK
700-815
F-actin
510-530
CaM
636
P
P
Intracellular
503ERM670
513-564
C-terminal
815
Inheritance, One bp Change,
I.2
I.1
I.2
I.1
G305R/N
G305R/N
II.1
II.2
G305R/G305R
II.3
G305R/G305R
II.1
G305R/G305R
II.2
Predicted
deleterious by
SIFT
(score=0.02)
II.3
Gly305Arg
Gly GGG
Arg AGG
Conservation of Gly305 in Na/H p.Gly305Arg
Exchangers
transmembrane
Western blot of Wild Type and Mutant
NHE1 Expressed in AP-1 Cells
Fully Glycosylated
de- or partially glycosylated
AP-1
WT
G305R
NHE1 Activity
NHE1
ACTIVITY
Mutant G305R Activity
Control
Wild
G305R
Cell Surface Targeting is defective
Wild Type
G305R
Immunocytochemistry
WTNHE1
G305R
Summary
-The disease Lichtenstein–Knorr
Syndrome is due to a defect in NHE1
gene (SLC9A1)
-Gly305Arg mutation, results in
defective activity and targeting
Experiments are underway to correct the
defect
Acknowledgements
Xiuju Li
Collaborators
Jodi Wilkinson
Sharaiz Baksh U of AB
Ray Amith
Yongsheng Liu
Claire Guissart,
Michel Koenig, Montpellier, France
Supported by the CIHR, CBCF
Cell Surface Targeting is defective
Wild Type
% On Cell Surface
G305R

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