Inflammation can act as a crucial mediator of epithelial-to-mesenchymal transition (EMT). In this study, we show that oncostatin M (OSM) is expressed in an autocrine/paracrine fashion in invasive breast carcinoma. OSM stimulation promotes spontaneous lung metastasis of MCF-7 xenografts in nude mice. A conspicuous epigenetic transition was induced by OSM stimulation not only in breast cancer cell lines but also in MCF-7 xenografts in nude mice. The expression of miR-200 and let-7 family members in response to OSM stimulation was downregulated in a signal transducer and activator of transcription factor 3 (Stat3)-dependent manner, resulting in comprehensive alterations of the transcription factors and oncoproteins targeted by these microRNAs. Inhibition of Stat3 activation or the ectopic expression of let-7 and miR-200 effectively reversed the mesenchymal phenotype of breast cancer cells. Stat3 promotes the transcription of Lin-28 by directly binding to the Lin-28 promoter, resulting in the repression of let-7 expression and concomitant upregulation of the let-7 target, high-mobility group A protein 2 (HMGA2). Knock down of HMGA2 significantly impairs OSM-driven EMT. Our data indicate that downregulation of let-7 and miR-200 levels initiates and maintains OSM-induced EMT phenotypes, and HMGA2 acts as a master switch of OSM-induced EMT. These findings highlight the importance of Stat3-coordinated Lin-28B-let-7-HMGA2 and miR-200-ZEB1 circuits in the cytokine-mediated phenotypic reprogramming of breast cancer cells.

In-depth analysis and thorough understanding of Nglycosylation is critical for therapeutic proteins. For IgG or IgG-like molecules, the conserved N-glycosylation at Asn297 has critical safety and efficacy implications and therefore is under close scrutiny. Any additional N-glycosylation that increases complexity and heterogeneity of the molecule, should be avoided at the early stage of drug discovery. However, unexpected glycosylation at atypical sites brings unforeseen challenges for antibody drug development process. Here, we observed a two-peak elution profile when developing ion exchange purification process for a novel bispecific VHH-IgG fusion protein. The charge and size properties of the two peaks were characterized by a variety of analytical methods. Two atypical N-glycosylation sites in the VHH domain were identified by means of mass spectrometry and confirmed by site-specific mutagenesis. Further characterization indicated that G0F was the predominant glycan species for both sites with varying occupancy. Interestingly, the addition of neutral glycans changed the charge behaviour of the fusion protein, leading to the unexpected ion exchange profile. These findings suggest that glycosylation may occur at non-canonical sites and may have significant effect on the process development and drug developability.