PTU-040 HMGB1 in the pathogenesis of colorectal cancer

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Abstract

Introduction

High Mobility Group Box-1 (HMGB1) is a ubiquitous nuclear protein that regulates gene expression. When phosphorylated, it translocates to the cytoplasm and extracellular space to impact immune responses and epithelial cell behaviour. Our previous data revealed that dynamic subcellular localisation of HMGB1 is associated with colorectal neoplastic progression, with cytoplasmic HMGB1 expression a feature of early carcinogenesis. The leading edge of cancer invasion in polyp cancers (CaP) had strong expression in both nuclear and cytoplasmic compartments. Our aim was to define the biological impact of this expression profile.

Methods

CD4+ helper T cells, CD8+ cytotoxic T cells, FOXP3+ regulatory T cells, CD20+ B cells and CD68+ macrophages were assessed by immunohistochemistry on endoscopically retrieved paraffin embedded CaP lesions sourced from the Grampian Tissue Biorepository (n=25). Ethical approval was granted by the Grampian Biorepository Scientific Access Group. The invasive cancer margin was identified by a gastrointestinal pathologist. The immune cell infiltrate was expressed as number of positive cells per high power field (x20) in the adjacent stroma alongside intensity of nuclear and cytoplasmic HMGB1 expression. The impact of HMGB1 on colonic epithelial cell behaviour was assessed by stimulation of Caco-2 cells (ATCC HTB-37TM) for 48–72 hours with 0, 50 and 100 ng/ml recombinant HMGB1 with (1) analysis of wound healing by scratch assay (images captured at time 0 and 24 hour intervals and analysed with ImageJ), (2) proliferation by incorporation of tritiated thymidine and (3) expression of permeability genes (CLDN2, CLDN4, OCLN, CDH1, TJP1) using TaqMan RT-qPCR, normalised to expression of GAPDH and B2M and analysed by the Livak method.

Results

A robust inflammatory infiltrate was identified adjacent to the invasive cancer margin with a preponderance of CD4+ T cells and CD68+ macrophages. The subcellular localisation of HMGB1 (nuclear versus cytoplasmic) did not result in phenotypically distinct populations. There was no difference in epithelial restitution in response to HMGB1 over 48 hours, although high concentration at 72 hours was associated with reduced wound healing (p<0.01) and reduced proliferation rate (p<0.01). HMGB1 stimulation significantly decreased the expression of CLDN4 (p<0.008).

Conclusions

Cytoplasmic HMGB1 expression did not impact the phenotype of adjacent immune cell infiltrate at the cancer margin. HMGB1 influences growth dynamics of colonic epithelial cells and reduces the expression of the pore closing tight junction gene, CLDN4. Understanding HMGB1 driven biology in CaP could identify an important mechanism for early carcinogenesis.

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