The Role of Galectin-3 in Ceacam6 Enhanced Egfr Signaling and Its Implication in Head and Neck Cancer Therapy

Head and neck squamous cell carcinoma (HNSCC) is one of the major cancers in the world. However, the current treatments of HNSCC are still limited in surgery, radiotherapy and chemotherapy because there is no biomarker for target therapy. Our recent study [Oncogene 2017 (In press)] found that carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), a glycophosphoinositol (GPI)-anchor protein, is a heavily-glycosylated molecule. CEACAM6 promoted EGFR signaling of HNSCC metastasis via the complex N-glycosylation, but it is unclear how glycosylated CEACAM6 interacts with EGFR. Therefore, this proposal aims to dissect the molecular mechanism by which CEACAM6 interacts with EGFR. Galectin-3 (Gal-3), a gaclactoside-binding protein, can form pentamers to cross-link glycoproteins like a “lattice” microdomain in the lipid-rafts of cancer cell membrane. Our results showed that Gal-3 was involved in the CEACAM6/EGFR complex and blocking the N-glycosylation of CEACAM6 inhibited the CEACAM6/Gal-3 interactions. Furthermore, knockdown of Gal-3 suppressed EGFR signaling. Based on the above evidence, we thus hypothesize that the co-clustering of CEACAM6 and EGFR glycans by endogenous Gal-3 increases CEACAM6/EGFR interactions and affect subsequent EGFR endocytosis and intracellular trafficking. Furthermore, because CEACAM6 was robustly increased in cetuximab-resistant HNSCC cells and targeting CEACAM6 by anti-CECAM6 antibodies (TMU sdAb, single-domain antibody) suppressed the EGFR signaling in HNSCC cells. Therefore, we will also investigate whether disrupting the glycosylation-dependent CEACAM6/EGFR interaction by anti-CEACAM6 antibodies or Gal-3 inhibitors overcomes the cetuximab-resistance of HNSCC. Three specific aims are proposed to achieve our goals.Aim 1. To investigate the role of galectin-3 in the glycosylation-dependent interaction of CEACAM6/EGFR in lipid-rafts. Aim 2. To investigate the effect of CEACAM6 on the EGFR endocytosis and intracellular trafficking and whether knockout of Gal-3 reverse the CEACAM6 effect on EGFR. Aim 3. To investigate the therapeutic implications of targeting Gal-3/CEACAM6/EGFR complex in primary and acquired cetuximab-resistant HNSCC and the underlying mechanisms.Significance: The study of molecular mechanisms of CEACAM6/EGFR interactions will be not only a new concept in tumor biology but also provide a new therapy to overcome the cetuximab-resistance of HNSCC.

目前頭頸癌的療法還是侷限在手術，放射線療法和化學治療，沒有顯著的生物標誌做標靶治療，我們最近的研究[Oncogene 2017 (In press)]發現癌胚抗原相關細胞黏附分子6 (CEACAM6)是一個高度醣基化的磷脂醯肌醇錨定蛋白，可藉由N端複雜醣基化來調控表皮生長因子接受器(EGFR)訊息傳遞而調控頭頸癌細胞侵犯、爬行、轉移。但是CEACAM6和EGFR的交互作用的機制還不清楚，因此，這個計畫將進一步剖析EGFR和CEACAM6 交互作用的分子機制以及在頭頸癌治療的應用。之前的文獻指出癌細胞中的半乳糖凝集素-3(Galectin-3, Gal-3)能夠藉由形成五聚體(pentamer)的方式和多個膜接受器醣苷結合，使多個細胞膜接受器交叉結合在癌細胞膜的脂筏。我們的研究也發現Gal-3存在於CEACAM6/EGFR的複合物中，而阻斷CEACAM6複雜醣基化也能夠抑制Gal-3/CEACAM6交互作用，另外降低Gal-3表現量可以抑制EGFR訊息傳遞，因此本計畫假設Gal-3使CEACAM6及EGFR的醣苷共聚而使CEACAM6和EGFR產生交互作用，進而影響EGFR的胞吞作用和細胞內運輸。另外因為CEACAM6大量表現在cetuximab (一個標靶EGFR的單株抗體)產生抗藥性的頭頸癌細胞株，並且之前的研究也發現利用標靶醣基化CEACAM6的單區域抗體能夠有效抑制EGFR的訊息傳遞，因此這個研究也將探討利用anti-CEACAM6抗體及Gal-3抑制劑來阻斷CEACAM6/EGFR交互作用，是否能夠成為克服cetuximab抗藥性頭頸癌的新療法，我們提出三個專一性的目標來達成我們的目的。目標一、探討Gal-3 參與醣基化依賴型CEACAM6/EGFR在脂筏(lipd-raft)的交互作用分子機制。目標二、探討CEACAM6對EGFR胞吞作用(endocytosis)及細胞內運輸(intracellular trafficking)的影響及是否剔除Gal-3基因反轉CEACAM6對EGFR的影響。目標三、探討標靶Gal-3/CEACAM6/EGFR的交互作用在治療原生(primary)或後天(acquired) cetuximab抗藥性(resistantance)頭頸癌的治療應用及參與其中的分子機制。