As the Wnt/β-catenin signaling pathway is linked to melanoma pathogenesis and to patient survival, we conducted a kinome siRNA screen in melanoma cells to expand our understanding of kinases that regulate this pathway, and to illuminate potential therapeutic directions. We found that BRAF signaling, which is constitutively activated in many melanomas by the BRAFV600E mutation, negatively regulates Wnt/β-catenin signaling in human melanoma cells. As inhibitors of BRAFV600E show promise in ongoing clinical trials we investigated whether altering Wnt/β-catenin signaling might enhance the efficacy of the BRAFV600E inhibitor, PLX4720. Surprisingly, endogenous β-catenin is required for PLX4720 to induce apoptosis in melanoma cells, while activation of Wnt/β-catenin signaling strongly synergizes with PLX4720 to decrease tumor growth in vivo and to increase apoptosis in vitro. This synergistic enhancement of apoptosis correlates with a reduction in the abundance of a β-catenin antagonist, AXIN1. In support of the hypothesis that AXIN1 is a mediator rather than a marker of apoptosis, melanoma cell lines that are resistant to apoptosis after treatment with a BRAFV600E inhibitor become susceptible, and undergo apoptosis, when AXIN1 is reduced by siRNA. These findings point to a role for Wnt/β-catenin signaling and AXIN1 in regulating the efficacy of inhibitors of BRAFV600E, and may stimulate consideration of potential combination therapies and biomarkers for use in conjunction with targeted BRAF therapy.

About half of all melanomas harbor a mutation that results in a constitutively active BRAF kinase mutant (BRAF V600E/K ) that can be selectively inhibited by targeted BRAF inhibitors (BRAFis). While patients treated with BRAFis initially exhibit measurable clinical improvement, the majority of patients eventually develop drug resistance and relapse. Here, we observed marked elevation of WNT5A in a subset of tumors from patients exhibiting disease progression on BRAFi therapy. WNT5A transcript and protein were also elevated in BRAFiresistant melanoma cell lines generated by long-term in vitro treatment with BRAFi. RNAi-mediated reduction of endogenous WNT5A in melanoma decreased cell growth, increased apoptosis in response to BRAFi challenge, and decreased the activity of prosurvival AKT signaling. Conversely, overexpression of WNT5A promoted melanoma growth, tumorigenesis, and activation of AKT signaling. Similarly to WNT5A knockdown, knockdown of the WNT receptors FZD7 and RYK inhibited growth, sensitized melanoma cells to BRAFi, and reduced AKT activation. Together, these findings suggest that chronic BRAF inhibition elevates WNT5A expression, which promotes AKT signaling through FZD7 and RYK, leading to increased growth and therapeutic resistance. Furthermore, increased WNT5A expression in BRAFi-resistant melanomas correlates with a specific transcriptional signature, which identifies potential therapeutic targets to reduce clinical BRAFi resistance.

In cancer, Wnt/β-catenin signaling is ubiquitously referred to as an “oncogenic” pathway that promotes tumor progression. This review examines how the regulation and downstream effects of Wnt/β-catenin signaling in cancer varies depending on cellular context, with a focus on malignant melanoma. We emphasize that the cellular homeostasis of Wnt/β-catenin signaling may represent a more appropriate concept than the simplified view of the Wnt/β-catenin pathway as either oncogenic or tumor-suppressing. Ultimately, a more refined understanding of the contextual regulation of Wnt/β-catenin signaling will be essential for addressing if and how therapeutic targeting of this pathway could be leveraged for patient benefit.

Several distinct melanoma syndromes have been defined, and genetic tests are available for the associated causative genes. Guidelines for melanoma genetic testing have been published as an informal “rule of twos and threes,” but these guidelines apply to CDKN2A testing and are not intended for the more recently described non-CDKN2A melanoma syndromes. In order to develop an approach for the full spectrum of hereditary melanoma patients, we have separated melanoma syndromes into two types: “melanoma dominant” and “melanoma subordinate.” Syndromes in which melanoma is a predominant cancer type are considered melanoma dominant, although other cancers, such as mesothelioma or pancreatic cancers, may also be observed. These syndromes are associated with defects in CDKN2A, CDK4, BAP1, MITF, and POT1. Melanoma-subordinate syndromes have an increased but lower risk of melanoma than that of other cancer(s) seen in the syndrome, such as breast and ovarian cancer or Cowden syndrome. Many of these melanoma-subordinate syndromes are associated with well-established predisposition genes (e.g., BRCA1/2, PTEN). It is likely that these predisposition genes are responsible for the increased susceptibility to melanoma as well but with lower penetrance than that observed for the dominant cancer(s) in those syndromes. In this review, we describe our extension of the “rule of twos and threes” for melanoma genetic testing. This algorithm incorporates an understanding of the spectrum of cancers and genes seen in association with melanoma to create a more comprehensive and tailored approach to genetic testing.

The collecting duct of the kidney is composed of two morphologically and physiologically distinct cell types, principal and intercalated cells. To better understand intercalated cell function we generated a transgenic mouse expressing Cre recombinase under the control of a cell type-specific promoter. We used 7 kb of the ATP6V1B1 5′ untranslated region (B1 promoter), a gene found in the intercalated cells of the kidney and the male reproductive tract. We first crossed these B1-Cre transgenic mice with the ROSA26-loxP-stop-loxP-yellow fluorescent protein reporter mice to assess the specificity of Cre expression. Immunohistochemistry and confocal fluorescence microscopy showed that Cre is selectively active in all intercalated cells (type A, type B, and non-A/B cells) within the collecting duct and most cells of the connecting segment. About half of the principal cells of the connecting segment also expressed Cre, a pattern also seen in B1-driven enhanced green fluorescent protein transgenic mice. Cre was found to be active in the male reproductive tract and at a low level in limited non-ATP6V1B1 expressing tissues. The B1-Cre transgenic mice are healthy, breed normally, produce regular sized litters, and transmit the transgene in Mendelian fashion. This new cell-specific Cre expressing mouse should prove useful for the study of intercalated cell physiology and development.

No abstract

No abstract

No abstract