1. Phosphoproteomics and Cancer Scott A. Gerber, PhD
Departments of Genetics and Biochemistry
Norris Cotton Cancer Center
Geisel School of Medicine at Dartmouth

2. Kinases, cell division and cancer
CDK1
Plk1
Aurora A
Aurora A/B
Aurora B
PP2A/PP1
PP2A
PP1
Prophase
Prometaphase
Interphase
Metaphase
Cytokinesis
Anaphase
Telophase

3. Kinases, cell division and cancer
multipolar
spindle
chromosome
missegregation
aberrant
cytokinesis
defects
Prophase
Prometaphase
Interphase
Metaphase
Cytokinesis
Anaphase
Telophase

4. chromosome instability & aneuploidy
Kinases, cell division and cancer
chromosome
instability
&
aneuploidy
multipolar
spindle
chromosome
missegregation
aberrant
cytokinesis
defects
Prophase
Prometaphase
Interphase
Metaphase
Cytokinesis
Anaphase
Telophase

5. Genomics-based targeted treatments:
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

6. Phosphoproteomics and cancer: opportunities, challenges & progress
Plk1
AurkA
Plk1
AurkA
Plk1
* Locano 2011, J Trans Med; Wolf 1997, Oncogene
PLK1 mRNA expression N T
N – normal tissue
T – tumor tissue
8,300 tumors
61 (0.7 %) mutations
COSMIC*
9,200 tumors
35 (0.4 %) mutations
Drug(s)
BI6727 (volasertib)
Phase III
MLN8237 (alisertib)

7. 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

8. Phosphoproteomics and cancer: opportunities, challenges & progress
Polo-like kinase 1 (Plk1): protein vs mRNA vs chemosensitivity
α-Plk1
H23
H1650
H1838
H1975
H2170
H1395
H522
relative Plk1 mRNA abundance
relative Plk1 protein abundance
r2 = 0.04
relative Plk1 protein abundance
relative LD50 Plk1 inhibitor
r2 = 0.17
Kettenbach & Gerber, Nature Protocols (2011)

9. Tumor phosphoproteomes represent the balance of opposing activities
Phosphoproteomics and cancer: opportunities, challenges & progress P
ATP
ADP
kinase
phosphatase
cdk1
PP1 P
“active”
Cell cycle progression G1 S G2 M
Cdk1 activity
PP1 activity
Tumor phosphoproteomes represent the balance of opposing activities

10. Stable Isotope Labeling by Amino acids in Cell culture
Quantitative Proteomics: SILAC
Condition 1
Condition 2 P P P A P P
anti-A antibody, etc. P A’ OH
Biology P B C P C’ B
Experiment
12C & 14N Lys & Arg
(light)
13C & 15N Lys & Arg
(heavy)
each tryptic peptide
ends in Lys or Arg
& can be quantified
- mix conditions
- lyse
IP A/A’
SDS-PAGE & digest
mixing cells allows
multi-step, subcellular
fractionation &
accurate quantitation
Information
100%
50%
m/z
condition (1): light
condition (2): heavy
Quantities are ratios:
e.g. heavy / light
Stable Isotope Labeling by Amino acids in Cell culture
Ong et al., Molecular & Cellular Proteomics 2002

11. Spike-in SILAC analysis of human lung cancer tumors
Schweppe, Rigas & Gerber, Journal of Proteomics (2013)

12. Spike-in SILAC analysis of human lung cancer tumors
Two non-small cell lung cancer (NSCLC) tumors
Schweppe, Rigas & Gerber, Journal of Proteomics (2013)

13. Spike-in SILAC analysis of human lung cancer tumors
Target discovery
Dimensionality
reduction
Motif-based
biomarkers
Motif-X –

14. Spike-in SILAC analysis of human lung cancer tumors
Next we wanted to establish what specific pathways and networks might be represented within these data. Here you can see that a number of important oncogenic pathways are indeed present from the distal membrane bound signal integrators like Erbb2 and integrins, down through networks hubs like the mek and erk kinases, through to transcription factors like elk1 and mef2.
Whats more, using phospho-specific antibodies, we were able to validate that those super-silac based ratios we observed in the mass spectrometer were consistant with immuno-staining.

15. phosphopeptide enrichment
Spike-in SILAC analysis of human lung cancer tumors
previously:
control
12C14N
combine
lyse
trypsin digestion
TiO2 +
LC-MS/MS
inhibited
13C15N
phosphopeptide enrichment
SCX chromatography
Plk1 inhibitor
> 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)

16. 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)

17. 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

18. Acknowledgements
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 The Kettenbach Lab Dr. Arminja Kettenbach Adam Petrone Scott Rusin Kate Schlosser NCCC Thoracic Oncology Dr. James Rigas Dr. Konstantin Dragnev Funding American Cancer Society NIH P20-GM R01-CA S10-OD016212
Cambridge Isotopes
ThermoFisher Scientific
GL Sciences