Ischemia in tumors induces early and sustained phosphorylation changes in stress kinase pathways but does not affect global protein levels.
Protein abundance and phosphorylation convey important information about pathway activity and molecular pathophysiology in diseases including cancer, providing biological insight, informing drug and diagnostic development, and guiding therapeutic intervention. Analyzed tissues are usually collected without tight regulation or documentation of ischemic time. To evaluate the impact of ischemia, we collected human ovarian tumor and breast cancer xenograft tissue without vascular interruption and performed quantitative proteomics and phosphoproteomics after defined ischemic intervals. Although the global expressed proteome and most of the >25,000 quantified phosphosites were unchanged after 60 min, rapid phosphorylation changes were observed in up to 24% of the phosphoproteome, representing activation of critical cancer pathways related to stress response, transcriptional regulation, and cell death. Both pan-tumor and tissue-specific changes were observed. The demonstrated impact of pre-analytical tissue ischemia on tumor biology mandates caution in interpreting stress-pathway activation in such samples and motivates reexamination of collection protocols for phosphoprotein analysis.
From the ‡Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142; scarr@broad.mit.edu pmertins@broadinstitute.org.
Philipp
P
Mertins
‖Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington 99352;
Feng
F
Yang
‖Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington 99352;
Tao
T
Liu
From the ‡Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142;
D R
DR
Mani
‖Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington 99352;
Vladislav A
VA
Petyuk
From the ‡Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142;
Michael A
MA
Gillette
From the ‡Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142;
Karl R
KR
Clauser
From the ‡Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142;
Jana W
JW
Qiao
‖Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington 99352;
Marina A
MA
Gritsenko
‖Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington 99352;
Ronald J
RJ
Moore
**Gynecology Service/Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York 10065;
Douglas A
DA
Levine
‡‡Department of Medicine, Washington University, St. Louis, Missouri 63110;
Reid
R
Townsend
‡‡Department of Medicine, Washington University, St. Louis, Missouri 63110;
Petra
P
Erdmann-Gilmore
‡‡Department of Medicine, Washington University, St. Louis, Missouri 63110;
Jacqueline E
JE
Snider
‡‡Department of Medicine, Washington University, St. Louis, Missouri 63110;
Sherri R
SR
Davies
§§Department of Biochemistry, New York University Langone Medical Center, New York, New York 10016;
Kelly V
KV
Ruggles
§§Department of Biochemistry, New York University Langone Medical Center, New York, New York 10016;
David
D
Fenyo
‡‡Department of Medicine, Washington University, St. Louis, Missouri 63110;
R Thomas
RT
Kitchens
‡‡Department of Medicine, Washington University, St. Louis, Missouri 63110;
Shunqiang
S
Li
**Gynecology Service/Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York 10065;
Narciso
N
Olvera
**Gynecology Service/Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York 10065;
Fanny
F
Dao
¶¶National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892;
Henry
H
Rodriguez
‖‖Department of Pathology, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21287;
Daniel W
DW
Chan
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232;
Daniel
D
Liebler
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139;
Forest
F
White
‖Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington 99352;
Karin D
KD
Rodland
Department of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030;
Gordon B
GB
Mills
‖Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington 99352;
Richard D
RD
Smith
Fred Hutchinson Cancer Research Center, Seattle, Washington 98109.
Amanda G
AG
Paulovich
‡‡Department of Medicine, Washington University, St. Louis, Missouri 63110;
Matthew
M
Ellis
From the ‡Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142; scarr@broad.mit.edu pmertins@broadinstitute.org.
Steven A
SA
Carr
D000818:Animals; D001943:Breast Neoplasms / Q000378:metabolism*; D050377:Cold Ischemia; D005260:Female; D020869:Gene Expression Profiling; D006801:Humans; D051379:Mice; D016688:Mice, Inbred NOD; D009368:Neoplasm Transplantation; D010051:Ovarian Neoplasms / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics; D014183:Transplantation, Heterologous
D010750:Phosphoproteins; D020543:Proteome