LAUSR

The receptor tyrosine kinase c-ErbB2 is amplified in breast and ovarian cancer. The linear pathways through which signals by c-ErbB2 are transduced is well known. However, second generation questions that address spatial aspects of signaling remain. To address this, we have undertaken a mass spectrometry approach to identify phosphoproteins. We have used two tyrosine kinase inhibitors, Lapatinib and CP724714, that inhibit phosphorylation of c-ErbB2 to identify phosphoproteins. SKOV-3, an ovarian cancer cell line that endogenously overexpresses c-ErbB2 was grown in culture without serum for 72 hrs. Cells were then stimulated in the presence or absence of inhibitor with EGF (100ng/ml) as a ligand for 60 mins. Subsequently, cells were lysed and evaluated by western blotting with anti-phosphotyrosine antibody (4G10). Following stimulation of cells with EGF, maximal phosphorylation of c-ErbB2 was observed at 60 minutes. Lapatinib (10μM) and CP724714 (15μM) completely inhibited phosphorylation of c-ErbB2, which was confirmed by immunoprecipitation. This was further confirmed by the inhibition of downstream effectors (Erk1/2, Akt). Lapatinib (10 μM) also completely inhibited phosphorylation of EGFR while CP724714 (15μM) only inhibited partially. Cellular lysates were prepared from quiescent cells (grown without serum), after stimulation with EGF in the presence or absence of inhibitors. Purified phosphoproteins from all three samples following digestion with trypsin were subjected to mass spectrometry (Nano LC ESI MS/MS). We identified totally 62 phosphoproteins. Twenty seven phosphoproteins were observed in all the 3 samples while 17 phosphoproteins were identified both in the EGF stimulated and lapatinib treated samples. Eighteen unique phosphoproteins were observed only in the EGF stimulated sample suggesting that they are specific to signaling by c-ErbB2. The novel phosphoproteins included the proteins that partcipate in carbohydrate metabolism,cytoskeleton, cell migration and proliferation. We have evaluated two phosphoproteins, LASP-1 and Aldose reductase that has not been previously described following phosphorylation of c-ErbB2. LASP-1 is an oncogene and is located as the same arm 17q21 as c-ErbB2. It was not expressed in the normal ovary or fallopian tube. However, it was over-expressed in 17% of tumours (n=85) from patients with ovarian cancer. c-ErbB2 was not expressed in tumours that expressed LASP1. Aldose reductase is a cytosolic NADPH dependent oxidoreductase that catalyzes the reduction of glucose to sorbitol, the first step in polyol pathway of glucose metabolism. The activity of aldose reductase in reducing NADPH as a substrate was significantly higher in lysates from EGF stimulated as compared to the starved cells. Identification of phosphoproteins by using mass spectrometry is promising in identifying novel substrates and pathways following phosphorylation of c-ErbB2. Citation Format: C Sidhanth, Manoj Garg, P Manasa, S Krishna Priya, S Bindhya, S Sneha, R.P. Nagare, S Shirley, M Kanchan, Trivadi S. Ganesan. Identification by mass spectrometry of unique phosphoproteins subsequent to signaling through c-ErbB2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 212. doi:10.1158/1538-7445.AM2017-212