Possible role of stromal-cell-derived factor-1/CXCR4 signaling on lymph node metastasis of oral squamous cell carcinoma

doi:10.1016/S0014-4827(03)00344-6

Experimental Cell Research

Volume 290, Issue 2, 1 November 2003, Pages 289-302

https://doi.org/10.1016/S0014-4827(03)00344-6 Get rights and content

Abstract

We examined the role of chemokine signaling on the lymph node metastasis of oral squamous cell carcinoma (SCC) using lymph node metastatic (HNt and B88) and nonmetastatic oral SCC cells. Of 13 kinds of chemokine receptors examined, only CXCR4 expression was up-regulated in HNt and B88 cells. CXCR4 ligand, stromal-cell-derived factor-1α (SDF-1α; CXCL12), induced characteristic calcium fluxes and chemotaxis only in CXCR4-expressing cells. CXCR4 expression in metastatic cancer tissue was significantly higher than that in nonmetastatic cancer tissue or normal gingiva. Although SDF-1α was undetectable in either oral SCC or normal epithelial cells, submandibular lymph nodes expressed the SDF-1α protein, mainly in the stromal cells, but occasionally in metastatic cancer cells. The conditioned medium from lymphatic stromal cells promoted the chemotaxis of B88 cells, which was blocked by the CXCR4 neutralization. SDF-1α rapidly activated extracellular signal-regulated kinase (ERK)1/2 and Akt/protein kinase B (PKB), and their synthetic inhibitors attenuated the chemotaxis by SDF-1α. SDF-1α also activated Src family kinases (SFKs), and its inhibitor PP1 diminished the SDF-1α-induced chemotaxis and activation of both ERK1/2 and Akt/PKB. These results indicate that SDF-1/CXCR4 signaling may be involved in the establishment of lymph node metastasis in oral SCC via activation of both ERK1/2 and Akt/PKB induced by SFKs.

Introduction

Worldwide, oral cancer, excluding pharyngeal cancer, represents 2.65% of all human malignancies [1]. Most oral cancer is histopathologically diagnosed as squamous cell carcinoma (SCC).¹ Since oral cancer arises in a place where it is comparatively easy to discover, the prognosis is not terribly poor, with the ratio of mortality/incidence being 0.48 [1]. However, the ratio in 1990 was 0.47 [2], [3], and the prognosis has not changed during the past 10 years. One of the reasons for this is that oral SCCs are characterized by a high degree of local invasion and a high rate of metastases to cervical lymph nodes. In particular, lymph node metastasis directly affects the prognosis of patients with oral SCC [4]. Therefore, several investigators have been documenting the factors related to lymph node metastasis of oral SCC [5], [6], [7]. We have also reported that a large amount of matrix-degrading enzymes produced by oral SCC cells contributes to tumor invasion and that the net activity of matrix metalloproteinase (MMP)2 [active-MMP2/tissue inhibitor of MMP2] contributes to lymph node metastasis in a nude-mouse orthotopic inoculation model [8]. Furthermore, by microdissection zymography, we demonstrated that active MMP2 in cancer cell nests or an elevated hepatocyte growth factor (HGF) level in the cancer tissue could be a predictive marker for lymph node metastasis formation in patients with oral SCC [9], [10]. Thus, several molecules expressed or produced in cancer cells are considered the metastatic factors; however, it remains unknown which factors produced by the lymph node affect the lymph node metastasis of cancer cells.

Chemokines are a large family of small (7–15 kDa), structurally related heparin-binding proteins that have been identified as attractants of different types of blood leukocytes to sites of infection and inflammation [11], [12]. They are produced locally in the tissues and act on leukocytes through selective membrane-bound GPCRs whose two major subfamilies are designated CCR and CXCR. Chemokines are known to also function as regulatory molecules in the leukocyte maturation, trafficking, and homing of T and B lymphocytes, in the development of lymphoid tissues, and in dendritic cell maturation [11], [12]. Moreover, recent reports have suggested that several types of cancer, such as breast [13], ovary [14], and prostate [15], kidney [16], brain [17], lung [18], and thyroid [19], expressed the chemokine receptor and used the chemokine(s) to metastasize to the target organ as in the homing of hematopoietic cells. Although it has been shown that several signal transduction pathways regulated by GPCRs including chemokine receptors result in these various biologic actions in hematopoietic cells [11], [12], little information is available on them in cancer cells, especially in oral SCC cells.

In this study, we investigated the possible role of chemokine/chemokine receptor interactions and their signal transduction involved in the lymph node metastasis of oral SCC using four characteristic oral SCC cells on the metastatic behavior in vivo. We demonstrate here the possibilities that the interaction of the chemokine receptor, CXCR4, with its ligand, SDF-1, may be involved in the lymph node metastatic process of oral SCC via activation of both the ERK1/2 and the Akt/PKB cascade induced by SFKs.

Section snippets

Patients

We studied 18 bioptic materials of oral SCC obtained at the Second Department of Oral and Maxillofacial Surgery, Tokushima University School of Dentistry, for semi-quantitative RT-PCR analysis. Before the initial treatments, these bioptic materials were resected from the central part of the tumor mass, possibly to avoid contamination of the normal cells. The absence or presence of lymph node metastases was determined clinically. Six normal gingival tissues, which were obtained from non-cancer

Characterization of newly established B88 cells

When orthotopically inoculated into masseter muscle in nude mice, HNt cells frequently metastasized to cervical lymph nodes [8], but BHY [8] and IH cells (data not shown) did not. B88 cells [21] were newly established oral SCC cells derived from a patient with carcinoma of the tongue. Histopathologically, they showed poorly differentiated oral SCC in sc tissue. When orthotopically inoculated into masseter muscle, they aggressively invaded mandibular bone and frequently metastasized to the

Discussion

In this study, we investigated the mechanism of lymph node metastasis of oral SCC, focusing on the chemokine–receptor system. The findings obtained from the present series of experiments are as follows. First, lymph node metastatic oral SCC cells specifically expressed functional CXCR4 and migrated toward its ligand, SDF-1α (CXCL12). Second, the expression of CXCR4 in primary cancer tissues with lymph node metastasis was significantly higher than in those with nonmetastasis. Third, SDF-1α was

Acknowledgements

We thank Drs. Naozumi Ishimaru and Yoshio Hayashi (Department of Pathology, Tokushima University School of Dentistry) for their valuable histopathologic advice. We also thank Dr. Takashi Bando (Second Department of Oral and Maxillofacial Surgery, Tokushima University School of Dentistry) for providing B88 cells. This study was supported in part by a grant-in-aid from the Ministry of Education, Science, and Culture of Japan.

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Regular articlePossible role of stromal-cell-derived factor-1/CXCR4 signaling on lymph node metastasis of oral squamous cell carcinoma

Abstract

Introduction

Section snippets

Patients

Characterization of newly established B88 cells

Discussion

Acknowledgements

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Regular article
Possible role of stromal-cell-derived factor-1/CXCR4 signaling on lymph node metastasis of oral squamous cell carcinoma