Glycoproteomic identification of novel plasma biomarkers for oral cancer

Shu-Chieh Chang ^a, Wei-Ling Lin ^b, Yin-Fan Chang ^c, Chih-Ting Lee ^c, Jin-Shang Wu ^c, Pang-Hung Hsu ^d,**, Chuan-Fa Chang ^a,e,*

^a Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University,

Tainan 70101, Taiwan

^b Department of Pathology, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung

University, Tainan 70101, Taiwan

^c Department of Family Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng

Kung University, Tainan 70101, Taiwan

^d Department of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung 20224, Taiwan

^e Department of Medical Laboratory Science and Biotechnology, College of Health Sciences, Kaohsiung Medical University, Kaohsiung 80708, Taiwan

Oral cancer with high incidence rates is occurring in many countries including in India, Pakistan, Bangladesh, Sri Lanka and Taiwan. Smoking, alcoholism, and betel nut chewing are considered to be the main risk factors for oral cancer. Further, deaths from oral cancer have increased year by year. Although several oral cancer-associated biomarkers have been reported, very few useful biomarkers have been applied for early diagnosis. Therefore, the investigation of oral cancer-specific biomarkers is urgently needed. We previously investigated N-glycomes of oral cancer cells and patient plasma. We found that both mRNA levels of FUT8 and core-fucosylated glycoproteins increase in cases of oral cancer relative to normal cases. In this study we aim to discover novel core-fucosylated glycoprotein biomarkers for oral cancer diagnosis with glycoproteomic approaches. First, forty plasma samples obtained from the Human Bioinformation Bank of NCKUH were subjected to AAL (Aleuria aurantia lectin) affinity chromatography. Core-fucosylated proteins were collected and applied for LC-MS/MS followed by electrophoresis. Fourteen proteins were identified, and expression levels of proteins in plasma were verified by western blot. Expression levels of some glycoproteins were elevated in the oral cancer group, including ceruloplasmin, haptoglobin, and leucin-rich alpha-2-glycoprotein 1 (LRG1). However, levels of some glycoproteins decreased in the cancer group, including apolipoprotein A-I (apo A-I) and apolipoprotein A-IV (apo A-IV). Via ELISA analysis, we found that apo A-IV and apo A-IV/ total protein ratios were decreased in plasma accompanied with cancer stages. The LRG1/total protein ratio was found to increase while plasma levels of LRG1 were not found to differ between the oral cancer plasma and normal groups. An ROC curve analysis reveals strong diagnosis performance when combining apo A-IV levels and LRG1/total protein ratios. Taken together, apo A-IV and LRG1, given their strong performance in detecting oral cancer, can serve as useful biomarkers and may be used as a useful tool for oral cancer screening and early diagnosis.