(447c) Phospho-Proteomics Reveals Oncogenic Phospho-Tyrosine Signaling Networks in Cancers Lacking Mutated Or Amplified Tyrosine Kinases

Introduction: Many human tumors exhibit aberrant activation of tyrosine kinase
signaling due to mutation or DNA amplification of tyrosine kinases (eg, EGFR). In contrast, prostate cancer and some subtypes
of glioblastoma (GBM) lack mutated or amplified tyrosine
kinases. To investigate the regulation of phospho-tyrosine
signaling in the absence of tyrosine kinase mutations or amplifications, we applied
quantitative, label-free mass spectrometry to a GBM cell line, a mouse model of
prostate cancer and human metastatic castration resistant prostate cancer
(CRPC) biopsies.

Materials and Methods: Human GBM cells (U87) were treated with
glucose starvation, epidermal growth factor (EGF), hydrogen peroxide (H₂O₂)
or the phosphatase inhibitor vanadate. Mouse models of prostate cancer were
generated by expressing combinations of non-tyrosine kinase oncogenes commonly
found in prostate cancer (constitutively active AKT, androgen receptor (AR),
ERG and K-Ras G12V) in the prostate in vivo regeneration model system
(Lawson et al, 2009). Human treatment naïve and metastatic castration resistant prostate cancer (CRPC) samples were
obtained from the UCLA Translational Pathology Core Laboratory and the
University of Michigan Rapid Autopsy Program. Quantitative, label-free mass
spectrometry was performed on peptides immunoprecipitated
using a pan-specific anti-phospho-tyrosine antibody (Drake et al, 2012; Graham et al,
2012).

Results
and Discussion:
U87 cells, which lack mutated or amplified tyrosine kinases, were treated with
glucose starvation, EGF, H₂O₂ or vanadate. Each stimulus induced
a unique signature of phospho-tyrosine signaling
(Fig. 1a, n = 46 peptides). Unexpectedly, glucose starvation induced phosphorylation
of proteins associated with focal adhesions, which suggested additional
experiments that revealed a positive feedback loop between tyrosine kinase
signaling and NADPH oxidase activity within focal adhesions (Graham et al,
2012). Next, analysis of mouse models of prostate cancer expressing non-tyrosine
kinase oncogenes (eg, activated Akt
and overexpressed ERG) (Drake et al, 2012) revealed oncogene-specific
signatures of phospho-tyrosine signaling (Fig. 1b, n
= 147 peptides), including an enrichment of EGFR substrates in Akt-ERG tumors. Finally, analysis of human prostate cancer
patient samples demonstrated differential activation of tyrosine kinase
signaling in treatment naïve and metastatic CRPC tissues (Fig. 1c, n = 126
peptides), including the presence of Src kinase
substrates in metastatic tumors.

Figure 1.
Hierarchical clustering of phospho-tyrosine peptides in a) U87 cells, b) mouse models
of prostate cancer c) human CRPC patient (Pt) samples (LN, lymph node; Met,
metastasis). Rows represent phospho-tyrosine peptides
and columns represent samples run in duplicate. Red and green denote high and
low phosphorylation, respectively.

Conclusions: We applied quantitative, label-free mass spectrometry to investigate
regulation of phospho-tyrosine signaling in systems
without mutated or amplified tyrosine kinases. This analysis revealed that a) metabolic
perturbation (ie, glucose starvation) induces a
positive feedback loop leading to focal adhesion-related tyrosine kinase
signaling in U87 cells, b) the non-tyrosine kinase oncogenes Akt and ERG activate EGFR signaling in a mouse model of
prostate cancer, and c) metastatic human CRPC biopsies exhibit elevated Src signaling compared to treatment naïve biopsies. Together, these data demonstrate that cell
lines and tumors lacking tyrosine kinase mutations or DNA amplifications can
exhibit elevated tyrosine kinase signaling, suggesting that tyrosine kinase
inhibitors may be effective therapeutics even in these cancers.

Acknowledgements: N.A.G. is supported by the UCLA Scholars in Oncologic Molecular Imaging
(SOMI) program, NIH grant R25T CA098010. J.M.D. is supported by the Department of Defense
Prostate Cancer Research Program W81XWH-11-1-0504.

References: Drake, J.M., PNAS (2012),
109(5), 1643-1648; Graham, N.A., et al, Mol Syst Biol
(2012), 8:589; Lawson, D.A., et al, PNAS
(2009).