TLRs play a pivotal role in the recognition of bacteria and viruses. Members of the family recognize specific pathogen sequences to trigger both MyD88 and TRIF-dependent pathways to stimulate a plethora of cells. Aberrant activation of these pathways is known to play a critical role in the development of autoimmunity and cancer. However, how these pathways are entirely regulated is not fully understood. In these studies, we have identified Annexin-A1 (ANXA1) as a novel regulator of TLR-induced IFN-β and CXCL10 production. We demonstrate that in the absence of ANXA1, mice produce significantly less IFN-β and CXCL10, and macrophages and plasmacytoid dendritic cells have a deficiency in activation following polyinosinic:polycytidylic acid administration in vivo. Furthermore, a deficiency in activation is observed in macrophages after LPS and polyinosinic:polycytidylic acid in vitro. In keeping with these findings, overexpression of ANXA1 resulted in enhanced IFN-β and IFN-stimulated responsive element promoter activity, whereas silencing of ANXA1 impaired TLR3- and TLR4-induced IFN-β and IFN-stimulated responsive element activation. In addition, we show that the C terminus of ANXA1 directly associates with TANK-binding kinase 1 to regulate IFN regulatory factor 3 translocation and phosphorylation. Our findings demonstrate that ANXA1 plays an important role in TLR activation, leading to an augmentation in the type 1 IFN antiviral cytokine response.

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Febrile temperatures can induce stress responses which protect cells from damage and can reduce inflammation during infections and sepsis. However, the mechanisms behind the protective functions of heat in response to the bacterial endotoxin LPS are unclear. We have recently shown that Annexin-1 (ANXA1)-deficient macrophages exhibited higher TNFα levels after LPS stimulation. Moreover, we have previously reported that ANXA1 can function as a stress protein.Therefore in this study, we determined if ANXA1 is involved in the protective effects of heat on cytokine levels in macrophages after heat and LPS. Exposure of macrophages to 42°C for 1 h prior to LPS results in an inhibition of TNFα production, which was not evident in ANXA1 −/− macrophages. We show that this regulation involves primarily MYD88-independent pathways. ANXA1 regulates TNFα mRNA stability after heat and LPS, and this is dependent on endogenous ANXA1 expression and not exogenously secreted factors. Further mechanistic studies revealed the possible involvement of the heat shock protein HSP70 and JNK in the heat and inflammatory stress response regulated by ANXA1. This study shows that ANXA1, an immunomodulatory protein, is critical in the heat stress response induced after heat and endotoxin stimulation.

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Metastasis is responsible for the majority of cancer related deaths and remains a global medical challenge in cancer treatment. The Y-box binding protein-1 (YB-1), a DNA and RNA binding protein, is known to regulate a plethora of fundamental cellular processes, including cell proliferation, chemoresistance and DNA repair. There is also cumulative evidence that YB-1 enhances breast cancer spread although the exact mechanism of YB-1-mediated metastasis is still not clear. Hence, we aim to determine the role of YB-1 overexpression in breast cancer progression. We first stably overexpressed YB-1 in non-aggressive MCF7 breast cancer cells (MCF7-YB-1) using a turboGFP tagged plasmid and performed phenotypic assays. We observed a significant increase in cell migration in MCF7-YB-1 in the Boyden chamber migration assay, which was substantiated by performing live imaging of the cells in a wound healing assay. Furthermore, a significant decrease in cell proliferation with G1 phase arrest was observed by the alamarBlue assay and flow cytometry analysis of propidium iodide staining, respectively for MCF7-YB-1 cells. We then performed a whole proteome analysis by stable isotope labelling with amino acids in cell culture (SILAC), a powerful proteomics platform. The whole proteome analysis identified 122 upregulated proteins and 120 downregulated proteins in the MCF-7-YB-1 cells. Bioinformatics analysis of the differentially regulated proteins using the Metacore software revealed the involvement of proteins that promote cell migration and invasion, such as those belonging to the S100 family which are enriched in the extracellular vesicles and exosome compartment, providing some mechanistic insights into the metastasis promoting role of YB-1. Taken together, our results showed that YB-1 could enhance the migratory potential of breast cancer cells, an important step in the metastatic cascade and therefore warrants a more in-depth study. Acknowledgement: Jia Pei Lim is a recipient of Ong Hin Tiang Scholarship in Cancer Research. This research was supported by Ministry of Education Grant (MOE2013-T2-1-129). Citation Format: Jia Pei Lim, Sunitha Nair, Sukanya Shyamasundar, Jayantha Gunaratne, Boon Huat Bay. Advanced quantitative mass-spectrometry-based SILAC proteome profiling of Y-box binding protein-1 (YB-1) overexpressing breast cancer cells unravels proteins involved in metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1061.