Crosstalk Between Klotho And Wnt5a Drives Age-Related Melanoma Progression

Background: Klotho, an emerging tumor suppressor, was originally identified as an anti-aging gene. The klotho expression level declines with the progression of age and is associated with several pathologies related to human aging. Mortality rates due to melanoma are continuously increasing. Specifically, in older patients, melanoma is very aggressive and has a very poor prognosis, compared to the young. Therefore, there is an urgent need to understand the underlying molecular mechanisms behind this age-related aggression. 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 a driver of melanoma metastasis. Therefore, we hypothesize that the loss of Klotho in aging microenvironment contributes to enhanced Wnt5A signaling, ultimately resulting 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) from the Baltimore Longitudinal Study of Aging (BLSA). 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 and Wnt5A expression is gained 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 our results confirm that Klotho is decreased in aged fibroblasts, furthermore, we also found that the loss of Klotho in the aged microenvironment also gets translated to a loss of Klotho in melanoma cells, and a gain of Wnt5A. Our immunohistochemistry results with the patient samples further extend our in vitro findings and support our hypothesis that Klotho-Wnt5A cross talk 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 and gain in Wnt5A expression levels in the aging tumor microenvironment. In addition, these findings provide a molecular explanation of age-related melanoma aggression. Citation Format: Reeti Behera, Katie Marchbank, Amanpreet Kaur, Vanessa Dang, Marie Webster, Michael O9Connell, Xiaowei Xu, Ashani Weeraratna. Crosstalk between klotho and wnt5A drives age-related melanoma progression. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr A11.