Malignant melanoma is one of the most lethal types of cancer and its rate of incidence has been rising for decades (1,2). The major reason for the poor prognosis of melanoma seems to be the rapid progression of the invasive and metastatic properties of the cancer cells. Although extensive studies have been carried out on the mechanisms by which melanoma cells develop the highly malignant phenotype, our knowledge on the properties of melanoma cells is still limited. One of the characteristics of the transformation from normal melanocytes to melanoma cells is that these cancer cells acquire the ability to produce their own growth factors, cytokines, and chemokines to form autocrine and/or paracrine stimulatory loops (3,4). Interleukin (IL)-8, a member of the chemokine superfamily, is one of these mediators, and has a potent chemotactic and activating effect on neutrophils and T cells, respectively, (5,6), as well as strong angiogenic activity (7,8). In normal human melanocytes, similar to other normal cell types (5), the production of IL-8 is not continuous but is induced by stimulation with proinflammatory cytokines such as IL-1 and tumor necrosis factor-α (TNF-α) (9). The 5′-flanking region of the IL-8 gene contains several potential binding sites for known transcription factors such as AP-1, NF-IL6 and NF-κB, and the induction of IL-8 by IL-1 and TNF-α can be ascribed, at least partly, to the activation of these transcription factors (10). In contrast, the continuous production of this chemokine has been observed in many cultured human melanoma cells (9,11–13) and melanoma specimens (14,15). Moreover, the serum IL-8 level is elevated in patients with metastatic melanoma (16). Therefore, many researchers have been investigating the role of IL-8 in melanoma cells. The mRNA level of IL-8 in melanoma cells correlates directly with their metastatic potential as evaluated by implantation in nude mice (17). Ultraviolet B irradiation of cultured SB-2 melanoma cells, which do not secrete a detectable level of IL-8 or form tumors in nude mice, promotes their tumorigenic and metastatic properties through induction of IL-8 (18). Ultraviolet B irradiation also enhances melanoma cell motility via induction of autocrine IL-8 secretion (19). Furthermore, it has been shown that overexpression of IL-8 in melanoma cells enhances their tumorigenicity and metastatic potential in mice (20,21). Thus, it appears that continuously produced IL-8 plays crucial roles in melanoma progression and metastasis. It has been reported that capsaicin, an inhibitor of NF-κB, inhibits, only partially, the continuous production of IL-8 in melanoma cells (22), suggesting that not only NF-κB but also other factor(s) participate in the regulation of the continuous expression of IL-8 in melanoma cells. Detailed mechanisms for continuous IL-8 production in melanoma cells, however, have not been fully elucidated.
Signal transducer and activator of transcription (STAT) 3 was originally identified as a DNA-binding protein that responds to stimulation by epidermal growth factor and IL-6 (23,24), and it has been established that STAT3 has an important role in the signalling of these physiologically active substances (24,25). Similar to other STATs, STAT3 is activated by a phosphorylation reaction at a critical tyrosine residue (Tyr705) by JAK2 in response to cytokine stimulation. This is followed by dimerization via a reciprocal interaction between its Src homology 2 domain and the phosphorylated tyrosine residue (26). The dimeric STAT3 translocates to the nucleus, where it binds to defined DNA element(s) within the promoter region of the target genes to activate their transcription (26–28). In normal cells, the duration of STAT3 activation is transient, and usually lasts from a few minutes to several hours (29). In these cells, STAT3 plays crucial roles in the development of various organs and in cell proliferation (30). In contrast, constitutive activation of STAT3 has been observed in many kinds of tumors (31) including melanoma (32), and this constitutively activated STAT3 is thought to contribute to oncogenesis by modulating the expression of a variety of genes (31).
In this study, we examined the involvement of constitutively activated STAT3 in IL-8 production in human melanoma cells, and found that IL-8 production is directly regulated by STAT3 at the transcription level in melanoma cells. Furthermore, we determined the 5′-flanking promoter region of the human IL-8 gene responsible for its production, which contains a putative STAT3-binding site.