Several studies have focused on Klotho and its role in the aging phenotype. In particular, its expression has been shown to decrease with advancing age, and Klotho knockout mice exhibit age-related degeneration, including muscle degeneration, increased artherosclerosis, and loss of skin integrity (Kuro-o et al., 1997
). In contrast, Wnt5A, the increased expression of which can promote melanoma metastasis, is increased in the skin of aged mice (data not shown). An interaction between the Wnt pathway and Klotho was described in a study by Liu et al
., in which the authors report the suppression of Wnt biological activity in the presence of Klotho, when both of these molecules are artificially expressed (Liu et al., 2007
). In this study, we show that Klotho and Wnt5A, a member of the non-canonical Wnt pathway, are endogenously expressed in melanoma cells of different invasive capacity, and can regulate each other. Interestingly, Wnt5A regulates Klotho at the transcriptional level. Over expression of Wnt5A has been previously shown to be associated with the downregulation of a large number of genes (Dissanayake et al., 2007
), probably through epigenetic silencing, although the mechanism in melanoma cells has not yet been described. A very recent paper indicates that Klotho is epigenetically silenced in late-stage cervical cancer largely via histone deacetylation activity (Lee et al, 2010
). Whether or not this is a mechanism via which Wnt5A can transcriptionally regulate Klotho is under investigation in our laboratory.
Our results clearly indicate that expression of Klotho is lost as cells become more metastatic, and describe a mechanism whereby this loss may contribute to Wnt5A-mediated metastasis. Coincident with this, others have suggested a tumor suppressor function for Klotho, in particular based on its decreased expression in more advanced lung cancer (Chen et al, 2010
), cervical cancer (Lee et al, 2010
), and breast cancer (Wolf et al., 2008
). The loss of Klotho expression in more metastatic and higher Wnt5A expressing melanoma cells suggests one of two things: 1) in less metastatic cells, high expression of Klotho inhibits Wnt5A expression, but as the cells become more metastatic, loss of Klotho results in increased levels of Wnt5A, or 2) low levels of Wnt5A in less metastatic cells permit Klotho expression, but as the cells become more metastatic, higher levels of Wnt5A inhibit Klotho expression. These two possibilities are not mutually exclusive, and indeed, these two molecules may exist in a regulatory feedback loop.
The inhibition of Wnt signaling by Klotho was previously reported (Liu et al., 2007
) and hypothesized to be the result of reduced ability of Wnt to bind to its receptor. In a recent study, we showed a role for heparan sulfate proteoglycans (HSPGs) in Wnt5A signaling in melanoma metastasis (O'Connell et al., 2009b
). In particular, we showed that the expression of syndecans 1 and 4 was increased in more metastatic melanoma cell lines and promoted the binding and internalization of Wnt5A, and thus, an increase in Wnt5A signaling (O'Connell et al., 2009b
). Knockout of syndecans 1 and 4 decreased Wnt5A internalization, signaling, and effects on motility. This could be overcome by adding back excess recombinant Wnt5A. Klotho has been shown to affect the glycosylation status of glycoproteins by modification of N-linked glycans (Chang et al., 2005
). It has also been shown to have both glucuronidase (Tohyama et al., 2004
), (Chang et al., 2005
) and sialidase activity (Cha et al., 2008
), which in turn have been shown to affect the glycosylation and expression of heparan sulfate proteoglycans. We hypothesized that Klotho may affect the glycosylation and sialylation of syndecans and limit their ability to promote Wnt5A internalization, resulting in reduced Wnt5A signaling. We show here that the presence of Klotho increases the release of Wnt5A in the medium, suggesting a decrease in receptor-mediated internalization, and this effect correlates with a decrease in syndecan-1 and syndecan-4 protein expression. We also show that this decrease in syndecan and Wnt5A internalization in the presence of Klotho correlates with an increase in sialidase activity that can be specifically attributed to Klotho, and the use of a sialidase inhibitor can promote the co-localization and internalization of syndecan and Wnt5A. Hence, we clearly implicate Klotho sialidase activity in reducing syndecan 1 and 4-mediated Wnt5A signaling. These results are particularly interesting when considering the correlation between excessive sialic acid expression and increased metastatic ability (reviewed in Miyagi et al., 2004
, Miyagi et al., 2008
). The sialidase NEU1 for example, can act as a metastasis suppressor in colon cancer. Increases in sialic acid residues have been shown to augment melanoma metastasis (Uemura et al., 2009
). It is possible that, in melanoma, the sialidase activity of Klotho plays a similar role to NEU1, one of the effects of which is a decrease in the internalization and subsequent signaling of Wnt5A.
In addition to inhibiting the internalization of Wnt5A, we show here that Klotho has a second effect on melanoma metastasis, related to, but not dependent upon Wnt5A signaling. We have previously shown that Wnt5A signaling induced Filamin A cleavage, which resulted in increased melanoma cell motility (O'Connell et al., 2009a
) and that this Wnt5A-induced cleavage of Filamin A was dependent on the activation of calpain-1. Several studies have shown increased calpain activity during aging (Baudry et al., 1986
, Lynch et al., 1986
), consistent with an increased intracellular or calcium influx in aging tissues (Besse et al., 1994
, Blalock et al., 1999
, Ouanounou et al., 1999
and Romero et al., 2002
). In turn, deficiency in Klotho protein was reported by others to lead to the overactivation of calpain-1 (Manya et al., 2002
). We show here that Klotho expression results in a decrease in calpain-1 expression in melanoma cells. Although this decrease in expression is not a direct measure of activity, in the presence of Klotho we also observe a decrease in the cleavage of Filamin A, an effect that we have previously attributed to calpain activity (O'Connell et al., 2009a
). Treatment with excess rWnt5A, which we have shown can overcome decreases in syndecan-mediated internalization (O'Connell et al., 2009b
), simply by the increase in ligand availability, cannot rescue the effect of Klotho pre-treatment on Filamin A in this case, and more importantly cannot restore motility to the melanoma cells (). These data suggest that the effect of Klotho on Filamin A has less to do with its effects on Wnt5A than its direct effects on calpain, providing a second impediment to melanoma metastasis. This has huge implications for the potential use of Klotho as an anti-metastatic agent.
Several lines of evidence indicate a link between advanced age and tumor onset and progression, and melanoma is no exception (Lachiewicz et al, 2008
). In particular, a number of factors secreted by senescent cells in the direct microenvironment of melanoma cells can contribute to the acquisition of a more invasive phenotype (reviewed in (Campisi and d'Adda di Fagagna, 2007
)). Loss of Klotho is associated with an aging phenotype, and older animals have less secreted Klotho in their serum (Xiao et al., 2004
). It could be that in a “younger” microenvironment, Klotho is present and acts as a check on Wnt5A signaling, Filamin cleavage and metastatic progression. In a scenario where the microenvironment of the tumor has significant effects on its outcome, the progressive loss of Klotho in an aging microenvironment could potentially allow for an increase in Wnt5A expression, and calpain activity and subsequent Filamin cleavage, thus increasing melanoma progression. These data suggest that reconstitution of Klotho may have beneficial effects, not only for reversal of aging-related phenotypes, but also for the successful management of metastatic melanoma.