Restoration of CD44S in non-small cell lung cancer cells enhanced their susceptibility to the macrophage cytotoxicity

Abstract

CD44 is a cell surface receptor for osteopontin (OPN) and hyaluronate. Transformation of normal tissue to a variety of cancers has been demonstrated to be associated with alterations of CD44 isoform expression. However, few reports have paid attention on differences in CD44S expression between non-small cell lung cancer (NSCLC) and adjacent normal lung tissue. In this study, we demonstrate that CD44S expression is down-regulated in NSCLC tissue when compared with paired normal lung tissue. To investigate the role of CD44S down-regulation in NSCLC cells, we reintroduced the CD44S back into the NSCLC cell line, H322 cells, which originally lack CD44S expression. The cytotoxicity by activated macrophage (RAW264.7 cells stimulated with lipopolysaccharide and interferon-γ) against the H322 cells transfected with the CD44S gene (H322ΔS) is more prominent than that against the H322 control transfectants (H322Δneo). The enhanced susceptibility of H322ΔS cells to the activated macrophage cytotoxicity appears to be mediated by the interaction between CD44S expression on H322ΔS cells and OPN produced by activated macrophages since it is completely blocked by either anti-OPN or anti-CD44 antibody. Moreover, H322ΔS cells are attracted toward OPN produced by activated RAW264.7 cells to a much greater extent than H322Δneo cells. These findings suggest that CD44S down-regulation in NSCLC cells may confer a protective advantage of allowing escape from tumoricidal effector cells including activated macrophages of the host.

Introduction

CD44 is expressed on a wide variety of normal and malignant tissues [1], [2], [3]. CD44 interacts with its ligands, hyaluronate and osteopontin (OPN) and is involved in not only lymphocyte homing [3] but also T-cell activation [4] and tumor metastasis [5]. Several CD44 isoforms have been demonstrated to arise from mRNA alternative splicing [2], [6]. The predominant CD44 isoform expressed by hematopoietic cells, fibroblasts, melanoma cells and glial cells is CD44S (CD44H), an isoform encoded by a transcript that does not contain any of the central alternatively spliced exons [7], [8]. In contrast, many epithelial cells and epithelial tumor cells express high molecular weight CD44 isoforms containing one or more of the alternatively spliced exons [1]. Numerous studies have revealed that alteration of CD44 isoforms is associated with the transformation of normal cells to cancer tissues [1], [7], [9]. We previously revealed that high molecular weight CD44 isoforms are predominantly expressed on colon carcinoma cells in comparison with normal colonic mucosa, which expresses mainly CD44S isoform [1]. Although functional differences in between CD44S and specific high molecular weight CD44 isoforms have been examined on hematopoietic cells [10], melanoma cells [11], and colon carcinoma cells [1], it is however controversial in non-small cell lung cancer (NSCLC). Several reports have demonstrated that both CD44S and CD44 high molecular weight CD44 isoforms including CD44v6 are overexpressed in NSCLC [12], [13], [14], [15], [16], [17], [18]. The function of CD44S in tumors varies according to the affected organ. For instance, overexpression of CD44S in melanoma and lymphoma cells enhances tumorigenecity [11], while up-regulation of CD44S on colon carcinoma cells reduces tumorigenecity [19], [20]. However, relatively a few number of papers have paid attention on the function of CD44S expressed on NSCLC [12], [16]. Recently, Pirinen et al. [21] revealed that reduction of CD44S expression was associated with not only lymph node metastases but also shortened disease-free survival. On the basis of these findings, we hypothesized that transformation of normal lung tissue to NSCLC is associated with reduced CD44S expression in NSCLC cells. In this study, we analyzed CD44 isoforms expression in several NSCLC tissue as well as the adjacent normal lung tissues with Western blot, and revealed that CD44S isoform is significantly down-regulated in tumors in comparison with that of normal lung tissue. To examine the functional consequences of decreased CD44S expression in NSCLC tissues, we reintroduced CD44S into human NSCLC cells to determine whether the down-regulation of this particular CD44 isoform on NSCLC cells influences on the chemotactic potential to CD44 ligands and susceptibility to cytotoxicity of activated macrophage. We also discuss about the biological significance in the down-regulation of CD44S in NSCLC from the aspect of host defense mechanism against NSCLC cells.