The long-term objective of this project is to measure phosphoproteins in human cancer tissues. As the first step towards this goal, the objective of this R21 grant is to identify optimal conditions for preparation of tissue samples by testing multiple combinations of tissue fixation and protein extraction buffers. Xenograft tissues are frozen or fixed in a non-crosslinking fixative or in formalin. Proteins are extracted with 4 buffers and analyzed for protein phosphorylation using a dot-blot assay.
In Aim 1, we measure global phosphorylation on tyrosine and threonine. We also use proQ Diamond to detect all phosphorylated proteins.
In Aim 2 we evaluate specific phosphorylation sites in proteins that are of clinical significance as drug targets or predictive biomarkers. We use statistical methods to identify sample preparation protocols that are (1) reproducible, (2) provide a high yield of extracted proteins, and (3) preserve protein phosphorylation during the extraction process. We rank the results and compare the top ranked protocols to a reference sample preparation procedure. We anticipate that by identifying sample preparation protocols for measurement of global and specific protein phosphorylation in cancer tissues, we will improve drug selection and response rates for many patients with solid tumors. ? ? Public Health Relevance Statement: Phosphoproteins represent effective predictive and prognostic biomarkers in human cancers; however, their expression is unstable and their measurement difficult. The objective of this project is to test novel sample preparation protocols that may better preserve protein phosphorylation in human cancer tissues for precise and reproducible measurement. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21CA118592-01A2
Application #
7503182
Study Section
Special Emphasis Panel (ZCA1-SRRB-C (M1))
Program Officer
Chuaqui, Rodrigo F
Project Start
2008-09-08
Project End
2010-08-31
Budget Start
2008-09-08
Budget End
2009-08-31
Support Year
1
Fiscal Year
2008
Total Cost
$237,600
Indirect Cost
Name
Fred Hutchinson Cancer Research Center
Department
Type
DUNS #
078200995
City
Seattle
State
WA
Country
United States
Zip Code
98109
Huang, Fangjin; Ma, Zhaoxuan; Pollan, Sara et al. (2016) Quantitative imaging for development of companion diagnostics to drugs targeting HGF/MET. J Pathol Clin Res 2:210-222
Akfirat, Canan; Zhang, Xiaotun; Ventura, Aviva et al. (2013) Tumour cell survival mechanisms in lethal metastatic prostate cancer differ between bone and soft tissue metastases. J Pathol 230:291-7
Putzke, Aaron P; Ventura, Aviva P; Bailey, Alexander M et al. (2011) Metastatic progression of prostate cancer and e-cadherin regulation by zeb1 and SRC family kinases. Am J Pathol 179:400-10
Ventura, Aviva P; Radhakrishnan, Sabarinath; Green, Ann et al. (2010) Activation of the MEK-S6 pathway in high-grade ovarian cancers. Appl Immunohistochem Mol Morphol 18:499-508