Identification of binding molecules for HMGB1 in HCT116 and evaluation of HMGB1 as a novel diagnostic biomarker of colorectal cancer

Abstract

HMGB1, a nuclear protein, is known to be overexpressed and secreted in cancer cells. However, little is known about the roles of HMGB1 in the cytoplasm and secretion pathway in cancer cells. Previously, I have reported that PKC-mediated serine phosphorylations of HMGB1 are critical to HMGB1 translocation. In this study, I have identified specific PKC family critical for HMGB1 phosphorylation, and showed PKC-ζ is critical for HMGB1 secretion in HCT116 cell line. To clarify the roles in the cytoplasm and secretory pathway of HMGB1, I fractionated each location of one colon cancer cell line, HCT116, and analysed the binding proteins. Pull-down experiment with recombinant HMGB1 protein as a bait, followed by mass spectrometry analysis identified 162 interacting proteins. Among them, 74 proteins are known to be localized exclusively in the extra-nuclear region and 60 proteins are known to be localized both in the nucleus and extra-nuclear region. The known functions of these binding proteins include cell cycle progression, cell proliferation, anti-apoptosis, and angiogenesis. In addition, 9 proteins are related to protein translocation and secretion. These include annexin A2, myosin IC isoform a, myosin-9, and Ras-related protein Rab10, which are involved in non-conventional protein secretion. In the cytoplasmic protein fraction, HMGB1 expression is mostly correlated to the lysosomal fraction of cytosol. I further demonstrated that, LAMP1, a lysosomal marker is co-localized with HMGB1. I also verified that secreted HMGB1 as a colorectal carcinoma diagnostic biomarker, and showed that HMGB1 is more sensitive than CEA, especially in early stage of colon cancers. All of these findings suggest that HMGB1 is translocated and secreted in colon cancers by PKC-ζ through lysosomal compartments and translocated HMGB1 has functions for carcinogenesis. Furthermore, secreted HMGB1 might be useful for the early cancer diagnosis.