Application Note 32
Analysis of Tyrosine Kinase Signaling in Human Cancer by Stable Isotope Labeling with Heavy Amino Acids in Mouse Xenografts Utilizing Mouse Express® Lysine 13C6 Mouse Feed
Audrey Sirvent,1 Serge Urbach,2 Serge Roche1
1. CNRS UMR5237, University of Montpelier 1 and 2, CRBM, 34000 Montpelier, France
2. CNRS UMR5203 INSERM U661, University of Montpelier 1 and 2, IGF, 34000 Montpelier, France
Tyrosine kinases (TK) play important roles in the induction of cell growth, survival and migration. They also have oncogenic activity when deregulated, a role originally described for the constitutively active v-SRC1 and since then, observed with most TK in human cancer.2 A large body of evidence indicates that aberrant TK activities contributes to cancer cell growth, survival, angiogenesis and cell dissemination leading to metastasis. This has been illustrated by the capacity of cancer cells transformed by oncogenic TK to induce tumor growth and metastasis formation when injected in nude mice. Since then, they have been considered as attractive therapeutic targets and several inhibitors are currently used in the clinic.2 However, our knowledge of the TK-dependent oncogenic signaling in human tumors is largely incomplete, mostly because the majority of data has been obtained in two-dimensional cell culture models. Moreover, the standard culture conditions of transformed cells do not allow recapitulating all the kinase-dependent signaling cascades that are activated during tumorigenesis to promote tumor growth, angiogenesis and interactions with the microenvironment.
MS-based quantitative phosphoproteomic technology has been a valuable tool to decipher signaling pathways initiated by a given TK.3 Particularly, the stable isotope labeling with amino acids in cell culture (SILAC) method has been employed for the characterization of oncogenic TK signaling pathways in cell culture.4,5 We recently used this powerful approach to investigate oncogenic signaling dependent upon the activity of the TK SRC in colon cancer cells6 and identified the first SRC-dependent tyrosine “phosphoproteome” in these cancer cells. Oncogenic signaling induced by TK could be investigated in vivo using similar MS-based quantitative phosphoproteomic approaches in mouse models or tumor biopsies. However, the application of the SILAC method in vivo has been challenging until recently because it requires efficient protein labeling in different tissues, which is conditioned by the rate of de novo protein synthesis. Recently, Mann et al. described the successful development of a SILAC approach for labeling mice that is based on the addition of L-lysine·2HCl (13C6, 99%) (CLM-2247-H) into their food.7 They reported complete labeling from the F2 generation.
In this note, we describe a novel proteomic approach to label tumors in nude mice xenografted with human cancer cells using Mouse Express® L-lysine (13C6, 99%) mouse feed (MF-LYS-C).8 We reasoned that the high rate of de novo protein synthesis occurring in tumors may induce an efficient labeling of xenografted tumors within a short period of time. We observed a consistent >88% labeling of the tumor proteome by feeding engrafted mice with the SILAC mouse diet for only 30 days. We then used this approach to compare the tyrosine phosphoproteome of SRC positive tumors (labeled with heavy amino acids) and of control tumors (labeled with light amino acids).
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