In the past two decades
In the past two decades, studies of the non-B cell-derived Ig, including cancer cell-derived IgG, have been predominately facilitated by general anti-human Ig antibodies, which do not distinguish the non-B Ig from classical Ig derived from B cells. Due to this caveat, it remains obscure about whether non-B Igs possess structural features that are distinct from B cell Igs. Previous studies have discovered that RP215 is a monoclonal antibody that recognizes aberrant patterns of glycosylation on cancer Ig . However, the molecular identity of the glycosylation is still unknown. In this study, we identified that LSCC cells express high levels of CIgG that is specifically recognized by RP215, and the epitope was determined to be an N-glycan on Asn162 located in the CH1 domain (Fig. 2). Importantly, this is different from the previously known N-glycan on Asn297 located at the consensus N-glycosylation motif (NXT/S) in the CH2 domain of regular IgG. To our knowledge, this non-classical glycosylation is the first unique post-translational modification of cancer-derived Ig molecules. It is important to note that RP215 also recognizes Ig molecules expressed by other cancer types. Therefore, it will be critical to determine whether CIgG of other cancers carry the same glycosylated epitope. The integrin α6β4 complex plays an essential role in tumorigenesis and metastasis of different cancers [, , , , ]. Integrin β4 is considered as an adverse prognostic indicator in breast [, , ], BW 723C86 hydrochloride [, , ], and in squamous cell carcinomas of the head and neck [, , , ]. Moreover, studies of lung cancer revealed that integrin β4 is overexpressed in NSCLC, and the expression is particularly high in LSCC (85%) compared with only 25% in pulmonary adenocarcinomas cases, and was notably absent in small cell carcinomas [39,40]. Interestingly, our study revealed that RP215-CIgG is overexpressed in NSCLC, particularly at high frequency in LSCC (86%) compared with only 34% in lung adenocarcinomas and no expression in small cell carcinomas (Fig. 1 and Table 1). Moreover, similar expression profile of RP215-CIgG and integrin β4 were observed in clinical samples and LSCC cells (Fig. 4C and D). The similar distribution profile of RP215-CIgG and integrin β4 suggest physical and functional connections between them. In support of this, our IP-MS analysis identified RP215-CIgG as a novel interacting protein of integrin β4 (Supplementary Fig. 4). Furthermore, immunolabeling analysis revealed colocalization between RP215-CIgG and integrin β4 at the cell surface of LSCC cells (Fig. 4E). Integrin complexes activate a variety of downstream signaling pathways that are involved in tumor initiation, growth, invasion and metastasis. Silencing of RP215-CIgG hampered phosphorylation of FAK, Src, cortactin and paxilin, which are key oncogenic signaling events downstream of integrin activation . Consistently, knockdown and overexpression experiments indicate that RP215-CIgG is essential for the malignant activity of LSCC cells in clonal formation and migration assays (Fig. 3). These results suggested that RP215-CIgG has import functions in LSCC progression via activation of the integrin α6β4-FAK pathway. Mechanistically, the RP215-specific N-glycan epitope is critically involved in the oncogenic activity of CIgG, as RP215 significantly inhibited the FAK and Src signaling and the CH1mu CIgG mutant without the glycosylation site failed to rescue (Fig. 5, Fig. 6). Interestingly, although the N-glycan epitope plays a significant role in FAK and Src activation (Fig. 5H), it is not essential for the interaction between RP215-CIgG and integrin β4 (Fig. 5I). Therefore, it is likely that this glycosylation epitope facilitates downstream signaling events after RP215-CIgG is engaged with integrin β4. It will be of critical importance to elucidate how the unique N-glycan is involved in the molecular mechanisms underlying the role of RP215-CIgG in LSCC carcinogenesis. Given the activity of RP215 in inhibiting the in vivo growth of LSCC PDX tumors (Fig. 6E–G), a thorough understanding of the function of RP215-CIgG will pave the way to developing additional therapeutic strategies for LSCC.