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Aberrant protein glycosylation could be a distinct surface-marker of cancer cells that influences cancer progression and metastasis because glycosylation can regulate membrane protein folding which alters receptor activation and changes epitope exposure for antibody (Ab) recognition. Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), a glycophosphoinositol-anchored protein, is a heavily glycosylated tumor antigen. However, the clinical significance and biological effect of CEACAM6 glycosylation has not been addressed in cancers. We recently developed an anti-CEACAM6 Ab (TMU) from an immune llama library which can be engineered to a single-domain (sd)Ab or a heavy-chain (HC)Ab. The TMU HCAb specifically recognized glycosylated CEACAM6 compared to the conventional antibodies. Using the TMU HCAb, we found that glycosylated CEACAM6 was a tumor marker associated with recurrence in early-stage OSCC (oral squamous cell carcinoma) patients. CEACAM6 promoted OSCC cell invasion, migration, cytoskeletal rearrangement, and metastasis via interaction with epidermal growth factor (EGF) receptor (EGFR) and enhancing EGFR activation, clustering and intracellular signaling cascades. These functions were modulated by N-acetylglucosaminyltransferase 5 (MGAT5) which mediated N-glycosylation at Asn 256 (N256) of CEACAM6. Finally, the TMU sdAb and HCAb treatment inhibited the migration, invasion and EGF-induced signaling in CEACAM6-overexpressing cells. In conclusion, the complex N-glycosylation of CEACAM6 is critical for EGFR signaling of OSCC invasion and metastasis. Targeting glycosylated CEACAM6 with the TMU sdAb or TMU HCAb could be a feasible therapy for OSCC.

Cellular senescence functions as a tumor suppressor that protects against cancer progression. α-Catulin, an α-catenin-related protein, is reported to have tumorigenic potential because it regulates the nuclear factor-κB (NF-κB) pathway, but little is known about its clinical relevance and the mechanism through which it regulates cancer progression. Here, we found that α-catulin mRNA levels were significantly upregulated in cancer cell lines and clinical oral squamous cell carcinomas, which positively correlated with tumor size (P=0.001) and American Joint Committee on Cancer (AJCC) stage (P=0.004). α-Catulin knockdown in the OC2 and A549 cancer cell lines dramatically decreased cell proliferation and contributed to cellular senescence, and inhibited OC2 xenograft growth. Mechanistic dissection showed that α-catulin depletion strongly induced the DNA-damage response (DDR) in both cell lines, via a p53/p21-dependent pathway in A549 cells, but a p53/p21-independent pathway in OC2 cells carrying mutant p53. Global gene expression analysis revealed that α-catulin knockdown altered cell-cycle regulation and DDR pathways at the presenescent stage as well as significantly downregulate several crucial genes related to mitotic chromosome condensation, DDR and DNA repair systems, which suggests that its depletion-induced cellular senescence might be caused by chromosome condensation failures, severe DNA damage and impaired DNA repair ability. Our study provides evidence that α-catulin promotes tumor growth by preventing cellular senescence and suggests that downregulating α-catulin may be a promising therapeutic approach for cancer treatment.

Galectin-1 (Gal-1), a homodimeric prototype of the galectins with a single carbohydrate-recognition domain, was recently identified as being overexpressed in tumor-associated capillary endothelial cells. The role of Gal-1 in endothelial cellular functions and the mechanism of action of Gal-1 remain unknown. Neuropilin-1 (NRP1) is a neuronal receptor that mediates repulsive growth cone guidance, and NRP1 functions in endothelial cells as a coreceptor (with vascular endothelial growth factor receptors (VEGFRs)) for VEGF(165). In this study, we found that Gal-1 was overexpressed in the tumor-associated endothelial cells of oral squamous cell carcinomas (P<0.001). Gal-1 increased the proliferation and adhesion of endothelial cells, and enhanced cell migration in combination with VEGF(165). Surprisingly, Gal-1 selectively bound NRP1 via the carbohydrate-recognition domain, but did not bind VEGFR-1, VEGFR-2 or VEGFR-3. The Gal-1-NRP1 interaction mediated the migration and adhesion of endothelial cells. The binding of Gal-1 to NRP1 enhanced VEGFR-2 phosphorylation and stimulated the activation of the mitogen activated protein (MAP) kinases SAPK1/JNK (stress activated protein kinase-1/c-Jun NH2-terminal kinase). These findings show, for the first time, that Gal-1 can directly bind to NRP1 on endothelial cells, and can promote the NRP1/VEGFR-2-mediated signaling pathway as well as NRP1-mediated biological activities.