Scott Gerber - Norris Cotton Cancer Center

Report
Phosphoproteomics
and Cancer
Scott A. Gerber, PhD
Departments of Genetics and Biochemistry
Norris Cotton Cancer Center
Geisel School of Medicine at Dartmouth
Kinases, cell division and cancer
CDK1
Plk1
Aurora A
CDK1
Plk1
Aurora A
PP1
Prophase
CDK1
Plk1
Aurora A/B
Prometaphase
PP2A/PP1
Interphase
Metaphase
Cytokinesis
PP2A
CDK1
Plk1
PP1
Plk1
Aurora B
Anaphase
Telophase
Aurora B
Kinases, cell division and cancer
spindle
defects
multipolar
spindle
aberrant
cytokinesis
Prophase
Prometaphase
Interphase
Metaphase
Cytokinesis
chromosome
missegregation
Telophase
Anaphase
Kinases, cell division and cancer
spindle
defects
multipolar
spindle
aberrant
cytokinesis
Prophase
Interphase
Cytokinesis
Prometaphase
chromosome
instability
&
aneuploidy
Metaphase
chromosome
missegregation
Telophase
Anaphase
Phosphoproteomics and cancer: opportunities, challenges & progress
Genomics-based targeted treatments:
•EGFR (~10%) – gefitinib/erlotinib
•ALK (~4%)- crizotinib
•RAS/RAF (~28%) – MEK inhibitors
adapted from Pao and Girard, Lancet 2011
Phosphoproteomics and cancer: opportunities, challenges & progress
Plk1
AurkA
Plk1
AurkA
PLK1 mRNA expression
N
T
N
T
N
T
N – normal tissue
T – tumor tissue
Plk1
COSMIC*
Drug(s)
8,300 tumors
BI6727 (volasertib)
Phase III
61 (0.7 %) mutations
9,200 tumors
35 (0.4 %) mutations
MLN8237 (alisertib)
Phase III
*http://cancer.sanger.ac.uk/cancergenome/projects/cosmic,La Locano 2011, J Trans Med; Wolf 1997, Oncogene
Phosphoproteomics and cancer: opportunities, challenges & progress
NIH3T3
U-2OS
A-432
U-251MG
Schwanhäusser et al. 2011, Nature; Lundberg et al. 2010, Mol. Sys. Biol
Phosphoproteomics and cancer: opportunities, challenges & progress

Polo-like kinase 1 (Plk1): protein vs mRNA vs chemosensitivity
Abundance
HINPVAASLIQK
α-Plk1
AQUA
endogenous
18.0
19.0
20.0
21.0
22.0
r2 = 0.04
1
0.5
relative LD50 Plk1 inhibitor
relative Plk1 protein abundance
Time (min)
r2 = 0.17
1
0.5
0
0
0
0.5
1
relative Plk1 mRNA abundance
0
0.5
1
relative Plk1 protein abundance
Kettenbach & Gerber, Nature Protocols (2011)
Phosphoproteomics and cancer: opportunities, challenges & progress
cdk1
ATP
ADP
P
PP1
PP1
kinase
“active”
P
Cdk1 activity
PP1 activity
P
phosphatase
G1
S
G2
M
Cell cycle progression
Tumor phosphoproteomes represent the balance of opposing activities
G1
Quantitative Proteomics: SILAC
Condition 1
Biology
Experiment
P
P
P
C
B
12C
B
P
P
P
P
A
P
Condition 2
C’
13C & 15N Lys & Arg
& 14N Lys & Arg
(heavy)
(light)
- mix conditions
- lyse
-IP A/A’
-SDS-PAGE & digest
100%
A
A
P
A’
OH
A’
C
mixing cells allows
multi-step, subcellular
fractionation &
accurate quantitation
OH
P
condition (1): light
condition (2): heavy
B
50%
each tryptic peptide
ends in Lys or Arg
& can be quantified
A’
A
Information
anti-A antibody, etc.
OH
A’
C’
P
P
A
A’
Quantities are ratios:
e.g. heavy / light
m/z
Stable Isotope Labeling by Amino acids in Cell culture
Ong et al., Molecular & Cellular Proteomics 2002
Spike-in SILAC analysis of human lung cancer tumors
Schweppe, Rigas & Gerber, Journal of Proteomics (2013)
Spike-in SILAC analysis of human lung cancer tumors
Two non-small cell lung cancer (NSCLC) tumors
Schweppe, Rigas & Gerber, Journal of Proteomics (2013)
Spike-in SILAC analysis of human lung cancer tumors
Target discovery
Dimensionality
reduction
Motif-based
biomarkers
Motif-X – http://motif-x.med.harvard.edu/
Spike-in SILAC analysis of human lung cancer tumors
Spike-in SILAC analysis of human lung cancer tumors
previously:
control
12C14N
combine
lyse
+
inhibited
13C15N
Plk1 inhibitor
trypsin
digestion
TiO2
phosphopeptide
enrichment
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
LC-MS/MS
0
20
40
60
SCX
chromatography
> 700 Plk1 candidate substrate phosphorylation sites
in these lung tumors:
Kinase-specific “substrate-omes” may be reflective of in vivo activity
Kettenbach et al., Science Signaling (2011); Schweppe, Rigas & Gerber, Journal of Proteomics (2013)
Spike-in SILAC analysis of human lung cancer tumors
What about protein abundance differences?
Tumor phosphoproteomes contain greater
dynamic information than proteomes
Schweppe, Rigas & Gerber, Journal of Proteomics (2013)
Translational phosphoproteomics: Status
Currently moving forward with the analysis of 40 NSCLC lung tumors
for phosphoproteomic analysis
• Study population
• Subjects undergoing thoracic surgery for presumed lung
cancer
• Determine if significant correlation exists between Aurora A
and/or Plk1 substrates and progression-free survival
• Correlate global phosphoproteomics patterns with disease-free
survival, time to disease recurrence, lung cancer-specific survival
and overall survival
• New instrumentation affords deeper coverage
• New instrumentation enables multiplexed analyses
The Gerber Lab
proteomics.dartmouth.edu
Dr. Lilian Kabeche
Dr. Devin Schweppe (past)
Jason Gilmore
Jeffrey Milloy
Sierra Cullati
Katelyn Cassidy
Andrew Grassetti
Mark Adamo
Acknowledgements
The Kettenbach Lab
www.kettenbachlab.org
Dr. Arminja Kettenbach
Adam Petrone
Scott Rusin
Kate Schlosser
NCCC Thoracic Oncology
Dr. James Rigas
Dr. Konstantin Dragnev
Funding
American Cancer Society
NIH P20-GM103413
R01-CA155260
S10-OD016212
Cambridge Isotopes
ThermoFisher Scientific
GL Sciences

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