Expression profile of HIP1R in B-cell subsets and in silico prediction of its functions in diffuse large B-cell lymphoma

Abstract

Huntingtin-interacting protein 1 (HIP1R) is an endocytic protein involved in endocytosis of surface receptors by regulating actin polymerization. We have previously shown that HIP1R was expressed in lymphoid B cells and diffuse large B-cell lymphoma (DLBCL) associated with better survival. Herein, we examined the expression profile of HIP1R in different immune cell populations and its potential functions in DLBCL. By utilizing a validated anti-HIP1R monoclonal antibody (clone 44), we examined whether the following immune cells in human reactive tonsils expressed HIP1R through double immunostaining: T cells (CD3+), macrophages (CD68+), mantle zone (MZ) B cells (PAX5+), germinal centre (GC) B cells (BCL6+) and plasma cells (IRF4/MUM1+). HIP1R was strongly expressed in PAX5+ MZ B cells, moderately expressed in BCL6+ GC B cells, but absent in CD3+ T cells, CD68+ macrophages and IRF4/MUM1+ plasma cells. In particular, we observed that HIP1R was absent in IRF4/MUM1+ plasma cells residing within the GC or non-GC interfollicular regions, suggesting that IRF4/MUM1 might downregulate HIP1R expression in activated B cells. We have previously shown that HIP1R expression is directly suppressed by the transcription factor FOXP1 in activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL) cells, however FOXP1 is absent in normal plasma cells, suggesting the presence of other regulators. Our previous immunostaining results in a series of DLBCL patient cases (n=155) showed a significant inverse correlation between HIP1R and IRF4/MUM1 (Pearson r = −0.495; p < 0.001). Indeed, knockdown of IRF4/MUM1 expression in the ABC-DLBCL cell line OCI-LY3 by two independent IRF4 siRNA constructs increased HIP1R expression at both transcript and protein levels. In terms of functional relevance, the bioinformatics approach Gene Set Enrichment Analysis (GSEA) was adopted to examine gene sets positively-associated with HIP1R transcript expression profile in three independent gene expression profiling (GEP) datasets of DLBCL patient cases derived from Gene Expression Omnibus database i.e. GSE10846 (n=233), GSE23501 (n=63), and GSE19246 (n=59). Our GSEA results showed that the gene set �Rho GTPase Activator Activity� (GO ID:0005100) was significantly positively-associated with HIP1R expression profile across all three GEP datasets GSE10846 (p = 0.0016), GSE23501 (p < 0.0001) and GSE19246 (p = 0.0167). These results suggest that HIP1R is involved in the activation of Rho GTPase signaling pathway, which has been documented to inhibit migration of DLBCL cells, and HIP1R expression is suppressed by transcription factors involved in B-cell activation including FOXP1 and IRF4/MUM1.