Christopher A. Natale

CDKN2B Loss Promotes Progression from Benign Melanocytic Nevus to Melanoma

UNLABELLED Deletion of the entire CDKN2B-CDKN2A gene cluster is among the most common genetic events in cancer. The tumor-promoting effects are generally attributed to loss of CDKN2A-encoded p16 and p14ARF tumor suppressors. The degree to which the associated CDKN2B-encoded p15 loss contributes to human tumorigenesis is unclear. Here, we show that CDKN2B is highly upregulated in benign melanocytic nevi, contributes to maintaining nevus melanocytes in a growth-arrested premalignant state, and is commonly lost in melanoma. Using primary melanocytes isolated directly from freshly excised human nevi naturally expressing the common BRAF(V600E)-activating mutation, nevi progressing to melanoma, and normal melanocytes engineered to inducibly express BRAF(V600E), we show that BRAF activation results in reversible, TGFβ-dependent, p15 induction that halts proliferation. Furthermore, we engineer human skin grafts containing nevus-derived melanocytes to establish a new, architecturally faithful, in vivo melanoma model, and demonstrate that p15 loss promotes the transition from benign nevus to melanoma. SIGNIFICANCE Although BRAF(V600E) mutations cause melanocytes to initially proliferate into benign moles, mechanisms responsible for their eventual growth arrest are unknown. Using melanocytes from human moles, we show that BRAF activation leads to a CDKN2B induction that is critical for restraining BRAF oncogenic effects, and when lost, contributes to melanoma.

Systematic Epigenomic Analysis Reveals Chromatin States Associated with Melanoma Progression

The extent and nature of epigenomic changes associated with melanoma progression is poorly understood. Through systematic epigenomic profiling of 35 epigenetic modifications and transcriptomic analysis, we define chromatin state changes associated with melanomagenesis by using a cell phenotypic model of non-tumorigenic and tumorigenic states. Computation of specific chromatin state transitions showed loss of histone acetylations and H3K4me2/3 on regulatory regions proximal to specific cancer-regulatory genes in important melanoma-driving cell signaling pathways. Importantly, such acetylation changes were also observed between benign nevi and malignant melanoma human tissues. Intriguingly, only a small fraction of chromatin state transitions correlated with expected changes in gene expression patterns. Restoration of acetylation levels on deacetylated loci by histone deacetylase (HDAC) inhibitors selectively blocked excessive proliferation in tumorigenic cells and human melanoma cells, suggesting functional roles of observed chromatin state transitions in driving hyperproliferative phenotype. Through these results, we define functionally relevant chromatin states associated with melanoma progression.

Sex steroids regulate skin pigmentation through nonclassical membrane-bound receptors

The association between pregnancy and altered cutaneous pigmentation has been documented for over two millennia, suggesting that sex hormones play a role in regulating epidermal melanocyte (MC) homeostasis. Here we show that physiologic estrogen (17β-estradiol) and progesterone reciprocally regulate melanin synthesis. This is intriguing given that we also show that normal primary human MCs lack classical estrogen or progesterone receptors (ER or PR). Utilizing both genetic and pharmacologic approaches, we establish that sex steroid effects on human pigment synthesis are mediated by the membrane-bound, steroid hormone receptors G protein-coupled estrogen receptor (GPER), and progestin and adipoQ receptor 7 (PAQR7). Activity of these receptors was activated or inhibited by synthetic estrogen or progesterone analogs that do not bind to ER or PR. As safe and effective treatment options for skin pigmentation disorders are limited, these specific GPER and PAQR7 ligands may represent a novel class of therapeutics. DOI: http://dx.doi.org/10.7554/eLife.15104.001

The integrin αv-TGFβ signaling axis is necessary for epidermal proliferation during cutaneous wound healing

ABSTRACT Proliferation and migration of epidermal keratinocytes are essential for proper cutaneous wound closure after injury. αv integrins and several of their ligands—vitronectin, TGFβ and thrombospondin—are up-regulated in healing wounds. However, the role of αv integrins in wound re-epithelialization is unknown. Here, we show that genetic depletion or antibody-mediated blockade of pan-integrin αv, or the specific heterodimer αvβ6, in keratinocytes limited epidermal proliferation at the wound edge and prevented re-epithelialization of wounded human organotypic skin both in vivo and in vitro. While we did not observe a migration defect upon αv blockade in vivo, αv was necessary for keratinocyte migration over longer distances in organotypic skin. Integrin αv is required for local activation of latent TGFβ, and the wound healing defect in the setting of integrin αv loss was rescued by exogenous, active TGFβ, indicating that the αv-TGFβ signaling axis is a critical component of the normal epidermal wound healing program. As chronic wounds are associated with decreased TGFβ signaling, restoration of TGFβ activity may have therapeutic utility in some clinical settings.

Activation of G protein-coupled estrogen receptor signaling inhibits melanoma and improves response to immune checkpoint blockade

Female sex and history of prior pregnancies are associated with favorable melanoma outcomes. Here, we show that much of the melanoma protective effect likely results from estrogen signaling through the G protein-coupled estrogen receptor (GPER) on melanocytes. Selective GPER activation in primary melanocytes and melanoma cells induced long-term changes that maintained a more differentiated cell state as defined by increased expression of well-established melanocyte differentiation antigens, increased pigment production, decreased proliferative capacity, and decreased expression of the oncodriver and stem cell marker c-Myc. GPER signaling also rendered melanoma cells more vulnerable to immunotherapy. Systemically delivered GPER agonist was well tolerated, and cooperated with immune checkpoint blockade in melanoma-bearing mice to dramatically extend survival, with up to half of mice clearing their tumor. Complete responses were associated with immune memory that protected against tumor rechallenge. GPER may be a useful, pharmacologically accessible target for melanoma.

Pharmacologic activation of G protein-coupled estrogen receptor inhibits pancreatic ductal adenocarcinoma

Female sex is associated with lower incidence and improved clinical outcomes for many cancer types, including melanoma and pancreatic ductal adenocarcinoma (PDAC). Although the mechanisms responsible for this sex difference are still emerging, we recently showed that signaling through the G protein-coupled estrogen receptor (GPER) likely contributes to improved clinical outcomes in women with melanoma. Here we used newly developed murine syngeneic tumor models to test whether GPER signaling inhibits KRas-driven PDAC. Activation of GPER with the specific, small molecule agonist G-1 inhibited PDAC proliferation, depleted c-Myc and programmed death ligand 1 (PD-L1), induced cell cycle block, and increased tumor cell immunogenicity. Systemically delivered G-1 significantly lengthened survival, and increased the efficacy of immune therapy in PDAC bearing mice, such that up to 60% of mice completely cleared their tumors. We detected GPER protein in a majority of human PDAC tumors. This finding, coupled with our previous melanoma data, and the wide tissue distribution of GPER, suggest that GPER agonists may be useful against many different cancer types.

Abstract 1239: CDKN2B loss promotes progression from benign melanocytic nevus to melanoma

The CDKN2A locus on chromosome 9p21 encodes two tumor suppressors: the cyclin dependent kinase inhibitor p16INK4a and the MDM2 regulator p14ARF. Homozygous deletion of this locus is among the most common genetic events in human cancer across tissues, with the resultant selective advantage to cancer cells generally attributed to the loss of CDKN2A proteins, which help maintain functional Rb and p53. However, in greater than 90% of cancer tissues harboring CDKN2A deletion, the adjacent CDKN2B gene encoding the p15 cyclin dependent kinase inhibitor, is also deleted. The degree to which CDKN2B-encoded p15 loss contributes to human tumorigenesis is unclear. Here we show that CDKN2B is highly upregulated in common, benign melanocytic nevi (moles) expressing the constitutively active BRAF(V600E) oncogene. A large percentage of melanomas develop in continuity with these pre-existing nevi, suggesting that nevus melanocytes are not permanently senescent, and can serve as tumor-initiating cells upon acquisition of additional genetic changes. By examining primary melanocytes isolated directly from freshly excised benign human nevi, as well as normal primary melanocytes engineered to inducibly express BRAF(V600E), we link BRAF activation to a TGFβ-dependent p15 induction that arrests nevi in their pre-malignant state. We also use the primary nevus cells to establish a new genetically-defined, human in vivo melanoma xenograft model, which maximizes medical relevance by utilizing the naturally occurring melanoma precursor cells in a native 3-dimensional environment. With this approach, we show that p15 depletion in benign nevi promotes progression to melanoma, establishing the reversibility of nevus melanocyte growth arrest, and the functional importance of CDKN2B loss in human cancer. Citation Format: Andrew McNeal, Kevin Liu, Vihang Nakhate, Christopher Natale, Brian Capell, Tzvete Dentchev, Shelley Berger, Meenhard Herlyn, John Seykora, Todd W. Ridky. CDKN2B loss promotes progression from benign melanocytic nevus to melanoma. [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 1239. doi:10.1158/1538-7445.AM2015-1239