Analysis of SRC Oncogenic Signaling in Colorectal Cancer by Stable Isotope Labeling with Heavy Amino Acids in Mouse Xenografts

The non-receptor tyrosine kinase (TK)1 SRC mediates cellular signaling induced by growth factors and integrins (1) leading to cell growth, survival, and migration. It also has oncogenic activity when deregulated, a role originally described for the constitutively active v-SRC (2) that has since been observed in a large variety of human cancers (3). Remarkably, elevated SRC kinase activity has been found in more than 80% of colorectal cancers (CRCs) to levels (5- to 10-fold) consistent with oncogenic properties (4). Moreover, SRC deregulation has been associated with poor clinical outcomes (3), suggesting an additional function of SRC during late tumorigenesis. SRC deregulation largely occurs in the absence of mutations in the SRC gene. Instead, it primarily involves protein over-expression (2) and inhibition of SRC negative regulators, such as the transmembrane protein Cbp/PAG (5, 6). A large body of evidence indicates that SRC deregulation is an important event in colon tumorigenesis (3, 6). Indeed, SRC controls growth, survival, and invasion of some CRC cell lines in vitro (4). Moreover, it contributes to tumor growth, angiogenesis, and metastasis formation in mouse colon tumor xenograft models (7–11). However, our knowledge of the SRC-dependent oncogenic signaling pathway in CRC is largely incomplete, mostly because the majority of data have been obtained in two-dimensional cell culture models. Moreover, the standard culture conditions of CRC cells do not allow the recapitulation of all the SRC-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 for deciphering signaling pathways initiated by a given TK (12). Particularly, the method of stable isotope labeling with amino acids in cell culture (SILAC) has been employed for the characterization of oncogenic TK signaling pathways in cell culture, including HER2 (13) and BCR-ABL (14). We recently used this powerful approach to investigate SRC-dependent oncogenic signaling in CRC cells (15) and identified the first SRC-dependent tyrosine “phosphoproteome” in these cells. Additionally, we found that SRC phosphorylated a small cluster of TKs that mediate its oncogenic activity, thus uncovering a TK network that is important for the induction of CRC cell growth (15). Whether these signaling processes also operate in vivo is, however, currently unknown. SRC oncogenic signaling could be investigated in vivo using similar MS-based quantitative phosphoproteomic approaches in animal 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 [13C6]-lysine to their food (16). They reported complete labeling from the F2 generation. Similar SILAC approaches were then described for additional multicellular organisms, such as worms (17), flies (18), and zebrafish (19). Here, we report a similar SILAC approach in which we labeled tumors in nude mice xenografted with human CRC cells. We reasoned that the high rate of de novo protein synthesis occurring in tumors should allow efficient tumor labeling in a short period of time. Indeed, we obtained consistent (>88%) labeling of the tumor proteome by feeding xenografted mice with the SILAC mouse diet for only 30 days. We then used this approach to compare the tyrosine phosphoproteome of SRC over-expressing tumors (labeled with heavy amino acids) and of control tumors (labeled with light amino acids) and report the first SRC-dependent tyrosine phosphoproteome of CRC in vivo. Finally, comparison of the in vivo and in vitro SRC-dependent tyrosine phosphoproteomes showed that some of the SRC substrates were specifically activated only in CRC xenograft tumors, and not in cultured CRC cells.