Relative ratio PC(16:0/16:1) (  M) p-Akt Akt PC(16:0/16:1) (  M) kDa p-Erk Erk PC(16:0/16:1) (  M) /44 kDa Relative ratio PC(16:0/16:1) (  M) PC(16:0/16:1) Stage II Stage III + IV Non-neoplastic mucosa Stage I Averaged normalized intensity of PC(16:0/16:1) #1601 Human colorectal cancer marker PC(16:0/16:1) induce cell growth by activating Akt and Erk pathways. Nobuya Kurabe, Toshio Nakamura, Kiyotaka Kurachi, Tomoaki Kahyo, Kazuya Shinmura, Mitsutoshi Setou, and Haruhiko Sugimura Department of Tumor Pathology, Hamamatsu University School of Medicine, Handayama, Higashi Ward, Hamamatsu, Shizuoka, , Japan Background The identification of cancer markers is critical for target-linked cancer therapy. To investigate which species of phosphatidylcholine (PC) is overexpressed in colorectal cancer, an imaging mass spectrometry (MS) was performed using a panel of non-neoplastic mucosal and colorectal cancer (CRC) tissues (Cancer Science vol. 104 no ). Using imaging MS, the elevated levels of PC(16:0/16:1) expression had been observed in a more advanced stage of CRC. Furthermore, the overexpression of lyso-PC acyltransferase 4 (LPCAT4) was shown to be involved in the increased level of PC(16:0/16:1) in CRC. Aim Methods and Results Conclusions To investigate the molecular mechanism on the upregulation of PC(16:0/16:1) in CRC, we focused on the responses of culture cells when PC(16:0/16:1) was administrated in vitro. To analyze the effects of PC(16:0/16:1) on the growth of culture cells, we added PC(16:0/16:1) to the culture media of HCT116 (colorectal cancer cell line) and NIH3T3 (non-cancerous cell line). PC(16:0/16:1) induced the increase in the growth rate of NIH3T3 cell line by about two fold but not HCT116 cell line (data not shown). For the analysis of the signaling pathway in the NIH3T3 cells, western blotting were carried out using various phosphorylation specific antibodies when the cells were subjected to PC(16:0/16:1). Phosphorylations of Akt and Erk were increased by the administration of PC(16:0/16:1). PC(16:0/16:1) induce the growth of NIH3T3 cells by activating Akt and Erk pathways. The identification of cancer biomarkers is critical for target-linked cancer therapy. The overall level of phosphatidylcholine (PC) is elevated in colorectal cancer (CRC), buy the exact nature of PC change in CRC remains to be elucidated. In our previous literature, to investigate which species of PC is overexpressed in colorectal cancer, an imaging mass spectrometry (MS) was performed using a panel of non-neoplastic mucosal and CRC tissues. Using principal component analysis (PCA), a novel biomarker, phosphatidylcholine (PC)(16:0/16:1), had been identified in CRC. Specifically, elevated levels of PC(16:0/16:1) expression had been observed in a more advanced stage of CRC. In an in vitro analysis, we had shown that lyso-PC acyltransferase 4 (LPCAT4) was involved in the deregulation of PC(16:0/16:1) in CRC. To investigate the molecular mechanism on the upregulation of PC(16:0/16:1) in CRC, we next focused on the responses of cells when PC(16:0/16:1) was administrated in vitro. To analyze the effects of PC(16:0/16:1) on the growth of several culture cells, we added PC(16:0/16:1) to the culture media of HCT116 colorectal cancer cell line and NIH3T3 non- cancerous cell line. PC(16:0/16:1) induced the increase in the growth rate of NIH3T3 cell line but not HCT116 cell. For the analysis of the signaling pathway of the cells, western blotting were carried out using various phosphorylation specific antibodies when NIH3T3 cells were subjected to PC(16:0/16:1). Phosphorylations of Akt and Erk were increased by the administration of PC(16:0/16:1). PC(16:0/16:1) induce cell growth by activating Akt and Erk pathways. Abstract Adrenal gland Brain Esophagus StomachIntestineColonRectumLungLiverSpleenKidneyTestisOvaryPancreasHeart LPCAT4 57 kDa  -tubulin 55 kDa LPCAT4 64 kDa Empty 6 x Myc- LPCAT4  -tubulin 55 kDa p-p38 p-S6K  -tubulin PC(16:0/16:1) (  M) kDa 70 kDa 55 kDa p-Src p-EGFR  -tubulin PC(16:0/16:1) (  M) kDa 60 kDa 55 kDa p-Erk Erk Relative ratio PC(16:0/16:1) Ki16425 PF /44 kDa PC(16:0/16:1) LPC(16:0) LPCAT4 PLPA 2 acylCoA fatty acid Lands’ cycle Akt Erk Cell survival and proliferation Autotaxin PF8380 LPA LPA receptor Akt Erk Cell survival and proliferation Ki16425 Fig. 1 Expression pattern of LPCAT4 protein among human tissues and the effect of LPCAT4 on the growth of NIH3T3 cells. Fig. 2 Phosphorylation status of Akt and Erk protein in response to the increasing amount of the administration of PC(16:0/16:1) in NIH3T3 cells. Fig. 3 Phosphorylation status of S6K, p38, EGFR and Src protein when NIH3T3 cells were stimulated with the increasing amount of PC(16:0/16:1), and the effect of PC(16:0/16:1) on the growth of NIH3T3 cells Fig. 4 Phosphorylation status of Akt and Erk protein when NIH3T3 cells were treated to PC(16:0/16:1) in combination with Ki16425 (lysophosphatidic acid receptor inhibitor) or PF8380 (autotaxin inhibitor). Fig. 5 Schematic overview about the role of PC(16:0/16:1) in Akt and Erk signaling. LPA (lysophosphatidic acid); LPC (lyso- phosphatidylcholine); PLPA 2 (phospholipase A 2 ) Colorectal cancer HE PC(16:0/18:1) PC(16:0/16:1) Non-neoplastic mucosa HEPC(16:0/18:1) PC(16:0/16:1) x 20 x 2.5 x 20 x 2.5 Cell number (x10 4 ) Empty 6 x Myc- LPCAT4 NIH3T3 * *p < PC(16:0/16:1) (  M) Cell number (x10 5 ) NIH3T3 60 * *p < 0.01 p-Akt Akt Relative ratio PC(16:0/16:1) Ki16425 PF kDa 10 5