hBDs comprise a class of inflammatory molecules, whose primary role at epithelial surfaces appears to be the initiation of an early host immune response to clear invading microorganisms (27
). They are capable of bridging innate and adaptive immunity through their ability to attract antigen-presenting cells to inflammatory sites and to provide ‘danger signals’ that facilitate the maturation of these immune cells. However, despite our understanding of the role of defensins in host–pathogen interactions and immunity, little is known about their functions within the context of an inflammatory tumor microenvironment. Particularly for squamous mucosal malignancies, which are a source of hBD secretion, even less is known about any potential pro-tumorigenic properties that may result from a dysregulation of inflammatory mediators such as defensins. In this paper, we demonstrate that hBD3 induces CCR7 expression in primary SCCHN tumor cells in an NF-κB dependent manner, providing migratory and pro-survival signals within the tumor microenvironment. Furthermore, the stimulation of tumors with hBD3 appears to confer a survival benefit to these tumors, mediated by PI3K/Akt activation. Our findings suggest that hBD3 overexpression by epithelia can be exploited by developing tumors in autocrine and paracrine fashion to enhance their growth, survival and evolution into a metastatic phenotype. In particular, the hBD3-stimulated induction of CCR7 expression in primary tumors may contribute to the predictable pattern of regional lymph node metastases commonly observed in SCCHN.
Others have begun to characterize the inflammatory effects of hBDs, which may be pro-tumorigenic. In mouse ovarian carcinoma, the expression of β-defensin 29, the murine homologue to human β-defensin 2, was found to chemoattract DCs through CCR6 and enhance tumor vascularization and growth through cooperation with vascular endothelial growth factor A (29
). Human tumors overexpressing β-defensins have been found to possess increased populations of infiltrating monocytes, macrophages and DCs suggesting that the pro-angiogenic and pro-tumorigenic functions of β-defensins may play a role in the development of these tumors. Indeed, a recent study by Jin et al.
) reported that hBD3 expression by oral tumors was associated with increased macrophage recruitment via CCR2 and increased secretion of tumor-promoting cytokines. Our studies indicate that SCCHN cell lines secrete low levels of hBD3 into the culture medium. Interestingly, stimulation with pro-inflammatory cytokines such as interferon-γ can stimulate increased secretion of hBD3 by these cell lines (Mburu,Y.K., Ferris,R.L, unpublished results). However, any direct effects of hBD3 on tumor growth and metastasis have not yet been elucidated.
Here, we tested the ability of hBD3 to directly affect the survival and migratory ability of human SCCHN tumors. We previously identified a distinct pattern of chemokine expression in SCCHN tumors whereby CCR7 was upregulated in patient-matched metastatic tumors (15
). The expression of CCR7 by metastatic SCCHN tumors may contribute to the frequent lymph node metastases that are reported in this and other malignancies. However, since CCR7 expression has generally been identified on tumors that are already present at the metastatic lymph node sites, the induction signals responsible for, and significance of, CCR7 expression on primary tumor cells emigrating from the mucosal tumor site are still unclear. Our work suggests that pro-inflammatory and cellular mechanisms cooperate to induce CCR7 expression and anti-apoptotic effects on the parental non-metastatic SCCHN tumors.
We show here that hBD3 is an inflammatory signal that stimulates the expression of CCR7 in tumors. Interestingly, hBD3 treatment had no significant effects on the expression of CCR6 and CXCR4, which are also expressed by head and neck cancers. Furthermore, we report that the induction of CCR7 by hBD3 was dependent on NF-κB activation. Our data is consistent with the notion that CCR7 is an NF-κB target gene (18
) since the inhibition of NF-κB activation significantly abolished the observable hBD3-induced CCR7 upregulation. The tumorigenic effects of deregulated NF-κB activation in oral squamous cell carcinoma have been well documented since this transcription factor has been shown to regulate various oncogenes, including CCR7 (31
). Indeed, we have examined the CCR7 promoter and found the existence of functional NF-κB-binding sites, which are important in the regulation of CCR7 expression (Mburu,Y.K., Walker, W.H., and Ferris, R.L, unpublished results). In SCCHN, various signals are known to induce constitutive NF-κB activation (32
). Our studies suggest that the exposure to hBD3 within an inflammatory tumor microenvironment results in upregulation of CCR7 expression via NF-κB-dependent mechanisms.
In previous reports, the expression of CCR7 in immune cells and tumors has been shown to provide a survival advantage to cells and is correlated with tumor aggressiveness (20
). This increased survival has been linked to the activation of the PI3K/Akt signaling pathway. In this study, we found that hBD3 stimulation activates PI3K-dependent Akt signaling. This activation and phosphorylation of Akt was biologically significant as it enhanced the survival of SCCHN tumor cells in the presence of cisplatin. Moreover, since the cells were pretreated with hBD3 for only 1 h prior to cisplatin treatment, we can attribute the observed pro-survival benefit directly to hBD3 stimulation of PI3K/Akt.
We also examined the potential extracellular receptors involved in the observed hBD3 effects on primary SCCHN cells. Remarkably, even though our primary SCCHN tumor cells express various GPCRs and TLRs (22
), the hBD3-mediated CCR7 upregulation was not blocked by the Gαi
inhibitor pertussis toxin or by TLR peptide inhibitors, both of which inhibit β-defensin effects on immune cells (3
). Interestingly, we found that hBD3 is taken up through endocytic mechanisms and accumulates in the perinuclear regions in tumor cells. It is also valuable to note that although other hBDs (such as hBD2) do not have similar effects on tumor CCR7 expression as hBD3, it appears that they penetrate tumor cells in a similar manner to that described for hBD3 (data not shown). Indeed, evidence in the literature suggests that the small cationic nature of these peptides allows them to associate with cellular membranes and translocate through the membrane using temperature and energy-dependent processes (34
). Our findings show that incubation at low temperatures or the inhibition of microtubule polymerization using nocodazole significantly reduced the observed internalization of hBD3 (P
< 0.05). It is notable, however, that over an extended incubation period, hBD3 overcomes nocodazole inhibition and penetrates cells. This observation, along with the toxicity of nocodazole at higher concentrations made it difficult to directly assess the effects of nocodazole treatment on hBD3-induced CCR7 expression. Nevertheless, while we cannot exclude receptor-mediated internalization, our data suggest that hBD3 signaling can also occur through direct binding to intracellular receptors/targets as has been described for other antimicrobial peptides (25
). Indeed, hBD3 could be binding to yet unidentified receptors on tumors.
To our knowledge, this is the first study directly linking defensin expression to tumor chemokine expression. Our data show that hBD3 stimulation induces pro-inflammatory, NF-κB-mediated functional CCR7 expression in SCCHN cells derived from primary, non-metastatic tumors. This gain in CCR7 expression may be important in facilitating the chemotactic migration of the tumor cells toward CCL19. Furthermore, we found that hBD3 stimulation provides pro-survival signals to primary SCCHN tumor cells through the activation of PI3K/Akt signaling. Future investigations into the receptor(s) involved in hBD3 stimulation on tumors could provide potential therapeutic avenues for clinical intervention.