Esophageal Squamous Cell Carcinoma Cells Modulate Chemokine Expression and Hyaluronan Synthesis in Fibroblasts

Inga Kretschmer,Till Freudenberger,Sören Twarock,Yu Yamaguchi,Maria Grandoch,Jens W Fischer

doi:10.1074/jbc.m115.708909

Abstract

The aim of this study was to characterize the interaction of KYSE-410, an esophageal squamous cell carcinoma cell line, and fibroblasts with respect to the extracellular matrix component hyaluronan (HA) and chemokine expression. KYSE-410 cells induced the mRNA expression of HA synthase 2 (Has2) in normal skin fibroblasts (SF) only in direct co-cultures. Parallel to Has2 mRNA, Has2 antisense RNA (Has2os2) was up-regulated in co-cultures. Knockdown of LEF1, a downstream target of Wnt signaling, abrogated Has2 and Has2os2 induction. After knockdown of Has2 in SF, significantly less α-smooth muscle actin expression was detected in co-cultures. Moreover, it was investigated whether the phenotype of KYSE-410 was affected in co-culture with SF and whether Has2 knockdown in SF had an impact on KYSE-410 cells in co-culture. However, no effects on epithelial-mesenchymal transition markers, proliferation, and migration were detected. In addition to Has2 mRNA, the chemokine CCL5 was up-regulated and CCL11 was down-regulated in SF in co-culture. Furthermore, co-cultures of KYSE-410 cells and cancer-associated fibroblasts (CAF) were investigated. Similar to SF, Has2 and Ccl5 were up-regulated and Ccl11 was down-regulated in CAF in co-culture. Importantly and in contrast to SF, inhibiting HA synthesis by 4-methylumbelliferone abrogated the effect of co-culture on Ccl5 in CAF. Moreover, HA was found to promote adhesion of CD4(+) but not CD8(+) cells to xenogaft tumor tissues. In conclusion, direct co-culture of esophageal squamous cell carcinoma and fibroblasts induced stromal HA synthesis via Wnt/LEF1 and altered the chemokine profile of stromal fibroblasts, which in turn may affect the tumor immune response.

Highlights

Tumors are complex tissues that are composed of neoplastic tumor cells, a variety of stromal cell types, and extracellular matrix (ECM).2 The various cell types form a microenviron
HA synthase 2 (Has2) mRNA Expression Was Induced in skin fibroblasts (SF) by Direct Co-culture with KYSE-410 Cells—If KYSE-410 cells were used to induce xenograft tumors in nude mice, an HA-rich matrix was associated with ␣SMA-positive cells (Fig. 1A) and with the parenchyma, as identified by CK-18-positive cells
To investigate the regulation of HA synthesis and chemokine expression in the context of interacting cancer cells and fibroblasts, we established a direct co-culture of primary murine SF and human KYSE-410 cells

Summary

Introduction

Tumors are complex tissues that are composed of neoplastic tumor cells, a variety of stromal cell types, and extracellular matrix (ECM). The various cell types form a microenviron-. Fibroblasts fulfill a variety of homeostatic functions, such as the maintenance of the structural integrity of the connective tissues and of the ECM They interact with other cell types, such as endothelial cells, epithelial cells, and immune cells. CAF promote cancer progression [1] They are known to secrete growth factors that support tumor cell proliferation, metastases, and angiogenesis [1,2,3]. CAF influence cancer cell phenotypes, such as stemness, and they can induce epithelial-mesenchymal transition (EMT) [4]. They modulate the inflammatory response in tumor tissues. The observed increase in Has mRNA and Has2os expression was dependent on lymphoid enhancer binding factor 1 (LEF1) expression

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