Role Of Klotho In Age-Related Melanoma Progression

Background: Mortality rates due to melanoma are continuously increasing. Specifically, in older patients, melanoma is very aggressive and has a poor prognosis, compared to the young. Therefore, there is an urgent need to understand the underlying molecular mechanisms behind this age-related aggression. The expression level of klotho, an emerging tumor suppressor, declines with the progression of age and is associated with several pathologies related to human aging. A previous study from our group has demonstrated that loss of klotho in melanoma cells is associated with enhanced melanoma cell motility by decreasing the ability of melanoma cells to internalize Wnt5A, a non-canonical Wnt protein, known to be one of the drivers of melanoma metastasis. Therefore, we hypothesize that the loss of Klotho in aging microenvironment contributes to enhanced Wnt5A signaling and ultimately results in age-related melanoma aggression. Methods: In order to study the effects of aged microenvironment on melanoma cells, we have obtained and cultured normal skin fibroblasts from aged (55-65 years old) and young donors (25-35 years old). The expression levels of klotho and Wnt5A in these fibroblasts were analyzed by immunoblot, immunofluorescence and real time-PCR assays. We examined melanoma cell/ fibroblast co-cultures and artificial skin reconstructs made with these fibroblasts to understand the age-related effects of tumor microenvironment on the disease progression. We also used immunohistochemistry technique to examine the expression levels of Klotho and Wnt5A in these artificial skin reconstructs and in the paraffin-embedded skin sections obtained from young and old melanoma patients. Results: Our western blot and real-time PCR results of the young vs. old fibroblasts show that klotho expression level is lost with progression of age. Analysis of artificial skin reconstructs made using skin fibroblasts isolated from aged donors demonstrate induced invasion of melanoma cells to a much greater extent than those cultured from young donors. We also analyzed these skin reconstructs for the expression levels of Klotho and Wnt5A, using immunohistochemistry technique and the results confirm that Klotho is decreased in aged fibroblasts. Furthermore, we also found that the loss of Klotho in the aged microenvironment translated to a loss of Klotho in melanoma cells. Our western blot results also suggest that loss of Klotho in the aging microenvironment enhances Wnt5A internalization and signaling and this promotes tumor metastasis. Our immunohistochemistry results using patient samples further extend our in vitro findings and support our hypothesis that Klotho plays pivotal role in age-related melanoma progression. Significance: These results for the first time demonstrate a correlation between age-related melanoma progression and the loss of klotho level. In addition, these findings provide a molecular explanation of age-related melanoma aggression. Citation Format: Reeti Behera, Amanpreet Kaur, Katie Marchbank, Vanessa Dang, Marie Webster, Xiaowei Xu, Ashani T. Weeraratna. Role of Klotho in age-related melanoma progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1556. doi:10.1158/1538-7445.AM2015-1556