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We have previously shown that Wnt5A drives invasion in melanoma. We have also shown that Wnt5A promotes resistance to therapy designed to target the BRAF(V600E) mutation in melanoma. Here, we show that melanomas characterized by high levels of Wnt5A respond to therapeutic stress by increasing p21 and expressing classical markers of senescence, including positivity for senescence-associated β-galactosidase (SA-β-gal), senescence-associated heterochromatic foci (SAHF), H3K9Me chromatin marks, and PML bodies. We find that despite this, these cells retain their ability to migrate and invade. Further, despite the expression of classic markers of senescence such as SA-β-gal and SAHF, these Wnt5A-high cells are able to colonize the lungs in in vivo tail vein colony-forming assays. This clearly underscores the fact that these markers do not indicate true senescence in these cells, but instead an adaptive stress response that allows the cells to evade therapy and invade. Notably, silencing Wnt5A reduces expression of these markers and decreases invasiveness. The combined data point to Wnt5A as a master regulator of an adaptive stress response in melanoma, which may contribute to therapy resistance.

Cutaneous malignant melanoma is an etiologically heterogeneous disease with genetic, host, and environmental factors, as well as their interactions contributing to its development (Tucker and Goldstein, 2003). Approximately 10% of melanoma cases occur in a familial setting (Goldstein and Tucker, 2001). To date, two high-risk melanoma susceptibility genes, CDKN2A on chromosome 9p21 and CDK4 on 12q14, have been identified. Germline mutations of these two genes account for only a small proportion of familial melanoma susceptibility, suggesting the existence of other high-risk genes. Recently, copy number variants (CNVs) have been shown to contribute substantially to disease susceptibility in several inherited diseases including cancer (Kuiper et al., 2010). Specifically, a recent genome-wide CNV mapping study reported the identification of a major susceptibility gene for a familial cancer, chordoma (Yang et al., 2009), suggesting that screening for complex genomic rearrangements that co-segregate with disease in families may provide a powerful alternative to traditional gene-mapping approaches. We conducted a genome-wide search for CNVs in 30 high-risk melanoma-prone families without known segregating mutations using a whole-genome human array–comparative genomic hybridization (array-CGH) chip (Nimblegen 385K; average probe spacing, 7 kb). The families were from the United States and ascertained through health care professionals or self referrals. The families included at least two living first degree relatives with a history of invasive melanoma. All family members who were willing to participate in the study provided written informed consent under an NCI IRB-approved protocol. Each underwent a full-body skin examination and completed risk factor questionnaires for sun-related exposures. All diagnoses of melanoma were confirmed by histologic review of pathologic material or by pathology reports. We analyzed blood-derived genomic DNA from 79 individuals including 62 melanoma patients (1–4 patients per family) and 17 spouses. We used the Nexus Copy Number built-in Rank Segmentation algorithm to identify significant CNVs (P=1×10−6; number of probes per segment ≥10; log2 ratio>0.25 for gains and

Recent observations suggest that melanoma cells drive disease progression by switching back and forth between phenotypic states of proliferation and invasion. Phenotype switching has been linked to changes in Wnt signalling, and we therefore looked for cell phenotype-specific differences in the levels and activity of β-catenin and its LEF/TCF co-factors. We found that while cytosolic β-catenin distribution is phenotype-specific (membrane-associated in proliferative cells and cytosolic in invasive cells), its nuclear distribution and activity is not. Instead, the expression patterns of two β-catenin co-factors, LEF1 and TCF4, are both phenotype-specific and inversely correlated. LEF1 is preferentially expressed by differentiated/proliferative phenotype cells and TCF4 by dedifferentiated/invasive phenotype cells. Knock-down experiments confirmed that these co-factors are important for the phenotype-specific expression of M-MITF, WNT5A and other genes and that LEF1 suppresses TCF4 expression independently of β-catenin. Our data show that melanoma cell phenotype switching behaviour is regulated by differential LEF1/TCF4 activity.

We have previously shown that Wnt5A-mediated signaling can promote melanoma metastasis. It has been shown that Wnt signaling is antagonized by the protein Klotho, which has been implicated in aging. We show here that in melanoma cells, expressions of Wnt5A and Klotho are inversely correlated. In the presence of recombinant Klotho (rKlotho), we show that Wnt5A internalization and signaling is decreased in high Wnt5A-expressing cells. Moreover, in the presence of rKlotho, we observe an increase in Wnt5A remaining in the medium, coincident with an increase in sialidase activity, and decrease in syndecan expression. These effects can be inhibited using a sialidase inhibitor. In addition to its effects on Wnt5A internalization, we also demonstrate that Klotho decreases melanoma cell invasive potential by a second mechanism that involves the inhibition of calpain and a resultant decrease in filamin cleavage, which we demonstrate is critical for melanoma cell motility.

Neuregulin (NRG) signaling through the receptor tyrosine kinase, ERBB3, is required for embryonic development, and dysregulated signaling has been associated with cancer progression. Here, we show that NRG1/ERBB3 signaling inhibits melanocyte (MC) maturation and promotes undifferentiated, migratory and proliferative cellular characteristics. Embryonic analyses demonstrated that initial MC specification and distribution were not dependent on ERBB3 signaling. However NRG1/ERBB3 signaling was both necessary and sufficient to inhibit differentiation of later stages of MC development in culture. Analysis of tissue arrays of human melanoma samples suggests that ERBB3 signaling may also contribute to metastatic progression of melanoma as ERBB3 was phosphorylated in primary tumors compared with nevi or metastatic lesions. Neuregulin 1-treated MCs demonstrated increased proliferation and invasion and altered morphology concomitant with decreased levels of differentiation genes, increased levels of proliferation genes and altered levels of melanoma progression and metastases genes. ERBB3 activation in primary melanomas suggests that NRG1/ERBB3 signaling may contribute to the progression of melanoma from benign nevi to malignancies. We propose that targeting ERBB3 activation and downstream genes identified in this study may provide novel therapeutic interventions for malignant melanoma.