ABSTRACT
BACKGROUND: Arterial stiffness is a cardiovascular risk factor and dramatically increases as women transition through menopause. The current study assessed whether a mouse model of menopause increases arterial stiffness in a similar manner to aging and whether activation of the G-protein-coupled estrogen receptor could reverse stiffness. METHODS: Female C57Bl/6J mice were ovariectomized at 10 weeks of age or aged to 52 weeks, and some mice were treated with G-protein-coupled estrogen receptor agonists. RESULTS: Ovariectomy and aging increased pulse wave velocity to a similar extent independent of changes in blood pressure. Aging increased carotid wall thickness, while ovariectomy increased material stiffness without altering vascular geometry. RNA-sequencing analysis revealed that ovariectomy downregulated smooth muscle contractile genes. The enantiomerically pure G-protein-coupled estrogen receptor agonist, LNS8801, reversed stiffness in ovariectomy mice to a greater degree than the racemic agonist G-1. In summary, ovariectomy and aging induced arterial stiffening via potentially different mechanisms. Aging was associated with inward remodeling, while ovariectomy-induced material stiffness independent of geometry and a loss of the contractile phenotype. CONCLUSIONS: This study enhances our understanding of the impact of estrogen loss on vascular health in a murine model and warrants further studies to examine the ability of LNS8801 to improve vascular health in menopausal women.
Subject(s)
Ovariectomy , Receptors, G-Protein-Coupled , Vascular Stiffness , Animals , Female , Mice , Aging/physiology , Carotid Arteries , Estrogens/pharmacology , GTP-Binding Proteins , Ovariectomy/adverse effects , Pulse Wave Analysis , Receptors, Estrogen/metabolism , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Vascular Stiffness/drug effects , Vascular Stiffness/physiologyABSTRACT
Despite recent therapeutic advances, the 5-year survival rate for adults with acute myeloid leukemia (AML) is poor and standard-of-care chemotherapy is associated with significant toxicity, highlighting the need for new therapeutic approaches. Recent work from our group and others established that the G protein-coupled estrogen receptor (GPER) is tumor suppressive in melanoma and other solid tumors. We performed a preliminary screen of human cancer cell lines from multiple malignancies and found that LNS8801, a synthetic pharmacologic agonist of GPER currently in early phase clinical trials, promoted apoptosis in human AML cells. Using human AML cell lines and primary cells, we show that LNS8801 inhibits human AML in preclinical in vitro models, while not affecting normal mononuclear cells. Although GPER is broadly expressed in normal and malignant myeloid cells, this cancer-specific LNS8801-induced inhibition appeared to be independent of GPER signaling. LNS8801 induced AML cell death primarily through a caspase-dependent apoptosis pathway. This was independent of secreted classical death receptor ligands, and instead required induction of reactive oxygen species (ROS) and activation of endoplasmic reticulum (ER) stress response pathways including IRE1α. These studies demonstrate a novel activity of LNS8801 in AML cells and show that targeting ER stress with LNS8801 may be a useful therapeutic approach for AML. Significance: Previous work demonstrated that LNS8801 inhibits cancer via GPER activation, especially in solid tumors. Here we show that LNS8801 inhibits AML via GPER-independent mechanisms that include ROS induction and ER activation.
Subject(s)
Endoribonucleases , Leukemia, Myeloid, Acute , Adult , Humans , Reactive Oxygen Species , Protein Serine-Threonine Kinases , Leukemia, Myeloid, Acute/drug therapy , Estrogens , Endoplasmic Reticulum StressABSTRACT
Arterial stiffness is a cardiovascular risk factor and dramatically increases as women transition through menopause. The current study assessed whether a mouse model of menopause increases arterial stiffness in a similar manner to aging, and whether activation of the G protein-coupled estrogen receptor (GPER) could reverse stiffness. Female C57Bl/6J mice were ovariectomized (OVX) at 10 weeks of age or aged to 52 weeks, and some mice were treated with GPER agonists. OVX and aging increased pulse wave velocity to a similar extent independent of changes in blood pressure. Aging increased carotid wall thickness, while OVX increased material stiffness without altering vascular geometry. RNA-Seq analysis revealed that OVX downregulated smooth muscle contractile genes. The enantiomerically pure GPER agonist, LNS8801, reversed stiffness in OVX mice to a greater degree than the racemic agonist G-1. In summary, OVX and aging induced arterial stiffening via potentially different mechanisms. Aging was associated with inward remodeling while OVX induced material stiffness independent of geometry and a loss of the contractile phenotype. This study helps to further our understanding of the impact of menopause on vascular health and identifies LNS8801 as a potential therapy to counteract this detrimental process in women.
ABSTRACT
Uveal melanoma is the most common primary intraocular malignancy in adults and has a high incidence of metastatic disease. Current treatments have shown limited clinical activity in patients with uveal melanoma with metastasis and there is an urgent need for new effective therapies. Recent findings have shown that women with uveal melanoma have better survival rates than men. The G protein-coupled estrogen receptor-1 (GPER) has distinct functions from those of the classic estrogen receptors ERα/ß and its activation by specific agonists has tumor-suppressive roles in several cancers. However, the role of GPER had not previously been investigated in uveal melanoma. We demonstrated that downregulation of GPER in uveal melanoma cells decreased expression of p53 and stimulated cell growth. In contrast, the clinical GPER agonist, LNS8801, upregulated p53 and p21, induced melanocytic differentiation markers, inhibited cell proliferation and cell migration, and induced apoptosis. Furthermore, LNS8801 treatment arrested the cells in G2-M-phase of the cell cycle with concomitant activation of mitotic markers and disruption of the mitotic spindle apparatus. LNS8801 significantly inhibited tumor growth of uveal melanoma xenografts in vivo, suggesting that GPER agonists may be a novel treatment for uveal melanoma. Significance: Current treatments against metastatic uveal melanoma have shown limited clinical activity and there is an urgent need for effective therapies. Here, we demonstrate that the GPER agonist LNS8801 induced both GPER-dependent and GPER-independent effects and elicited potent anticancer activities in vitro and in vivo. Our results complement and support the ongoing clinical trial of LNS8801 in advanced uveal melanoma.
Subject(s)
Melanoma , Tumor Suppressor Protein p53 , Male , Adult , Humans , Female , Tumor Suppressor Protein p53/pharmacology , Melanoma/drug therapy , Estrogens/metabolism , Apoptosis , Cell Line, TumorABSTRACT
GPER (G protein-coupled estrogen receptor) has been reported to play roles in several areas of physiology including cancer, metabolic disorders, and cardiovascular disease. However, the understanding of where this receptor is expressed in human tissue is limited due to limited available tools and methodologies that can reliably detect GPER protein. Recently, a highly specific monoclonal antibody against GPER (20H15L21) was developed and is suitable for immunohistochemistry. Using this antibody, we show that GPER protein expression varies markedly between normal human tissue, and also among cancer tissue. As GPER is an emerging therapeutic target for cancer and other diseases, this new understanding of GPER distribution will likely be helpful in design and interpretation of ongoing and future GPER research.
ABSTRACT
Here, we provide a protocol to model the effects of changes to a small number of cells, such as those arising from a mutation or a virus infection, in stratified epithelia. We describe steps for diluting engineered human keratinocytes into a larger population of unmodified cells and using these cells to grow three-dimensional organotypic cultures. We detail steps to observe effects that are not apparent in homogenous organotypic epithelial cultures by visualizing the localization of modified keratinocytes in epithelial layers. For complete details on the use and execution of this protocol, please refer to Hatterschide et al. (2022).1.
Subject(s)
Carcinoma, Squamous Cell , Keratinocytes , Humans , Epithelium , Epithelial Cells , Cells, CulturedABSTRACT
Melanoma risk is 30 times higher in people with lightly pigmented skin versus darkly pigmented skin. Using primary human melanocytes representing the full human skin pigment continuum and preclinical melanoma models, we show that cell-intrinsic differences between dark and light melanocytes regulate melanocyte proliferative capacity and susceptibility to malignant transformation, independent of melanin and ultraviolet exposure. These differences result from dihydroxyphenylalanine (DOPA), a melanin precursor synthesized at higher levels in melanocytes from darkly pigmented skin. We used both high-throughput pharmacologic and genetic in vivo CRISPR screens to determine that DOPA limits melanocyte and melanoma cell proliferation by inhibiting the muscarinic acetylcholine receptor M1 (CHRM1) signaling. Pharmacologic CHRM1 antagonism in melanoma leads to depletion of c-Myc and FOXM1, both of which are proliferation drivers associated with aggressive melanoma. In preclinical mouse melanoma models, pharmacologic inhibition of CHRM1 or FOXM1 inhibited tumor growth. CHRM1 and FOXM1 may be new therapeutic targets for melanoma.
ABSTRACT
Melanoma and most other cancers occur more frequently and have worse prognosis in males compared with females. Although sex steroids are thought to be involved, classical androgen and estrogen receptors are not detectable in most melanomas. Here we show that testosterone promotes melanoma proliferation by activating ZIP9 (SLC39A9), a zinc transporter that is widely expressed in human melanoma but not intentionally targeted by available therapeutics. This testosterone activity required an influx of zinc, activation of MAPK, and nuclear translocation of YAP. FDA-approved inhibitors of the classical androgen receptor also inhibited ZIP9, thereby antagonizing the protumorigenic effects of testosterone in melanoma. In male mice, androgen receptor inhibitors suppressed growth of ZIP9-expressing melanomas but had no effect on isogenic melanomas lacking ZIP9 or on melanomas in females. These data suggest that ZIP9 might be effectively targeted in melanoma and other cancers by repurposing androgen receptor inhibitors that are currently approved only for prostate cancer. SIGNIFICANCE: Testosterone signaling through ZIP9 mediates some of the sex differences in melanoma, and drugs that target AR can be repurposed to block ZIP9 and inhibit melanoma in males.
Subject(s)
Androgen Receptor Antagonists/pharmacology , Cation Transport Proteins/antagonists & inhibitors , Melanoma/drug therapy , Receptors, Androgen/chemistry , Testosterone/pharmacology , Androgens/pharmacology , Animals , Apoptosis , Cation Transport Proteins/genetics , Cation Transport Proteins/metabolism , Cell Movement , Cell Proliferation , Humans , Male , Melanoma/genetics , Melanoma/metabolism , Melanoma/pathology , Mice , Mice, Inbred C57BL , Mice, SCID , Receptors, Androgen/genetics , Receptors, Androgen/metabolism , Sex Factors , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND & AIMS: Female sex is associated with lower incidence and improved clinical outcomes for most cancer types including pancreatic ductal adenocarcinoma (PDAC). The mechanistic basis for this sex difference is unknown. We hypothesized that estrogen signaling may be responsible, despite the fact that PDAC lacks classic nuclear estrogen receptors. METHODS: Here we used murine syngeneic tumor models and human xenografts to determine that signaling through the nonclassic estrogen receptor G protein-coupled estrogen receptor (GPER) on tumor cells inhibits PDAC. RESULTS: Activation of GPER with the specific, small molecule, synthetic agonist G-1 inhibited PDAC proliferation, depleted c-Myc and programmed death ligand 1 (PD-L1), and increased tumor cell immunogenicity. Systemically administered G-1 was well-tolerated in PDAC bearing mice, induced tumor regression, significantly prolonged survival, and markedly increased the efficacy of PD-1 targeted immune therapy. We detected GPER protein in a majority of spontaneous human PDAC tumors, independent of tumor stage. CONCLUSIONS: These data, coupled with the wide tissue distribution of GPER and our previous work showing that G-1 inhibits melanoma, suggest that GPER agonists may be useful against a range of cancers that are not classically considered sex hormone responsive and that arise in tissues outside of the reproductive system.
Subject(s)
Antineoplastic Agents/pharmacology , Carcinoma, Pancreatic Ductal/drug therapy , Pancreatic Neoplasms/drug therapy , Receptors, G-Protein-Coupled/agonists , Animals , Antineoplastic Agents/therapeutic use , Carcinoma, Pancreatic Ductal/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Female , Humans , Male , Mice , Mice, Inbred C57BL , Pancreatic Neoplasms/metabolism , Receptors, Estrogen/metabolism , Receptors, G-Protein-Coupled/metabolism , Small Molecule Libraries/pharmacology , Small Molecule Libraries/therapeutic useABSTRACT
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.
Subject(s)
Antineoplastic Agents/therapeutic use , Melanoma/drug therapy , Melanoma/pathology , Receptors, G-Protein-Coupled/agonists , Signal Transduction , Animals , Disease Models, Animal , Humans , Mice , Pigments, Biological , Receptors, Estrogen , Survival Analysis , Treatment Outcome , Tumor Cells, CulturedABSTRACT
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.
Subject(s)
Chromatin/metabolism , Epigenomics , Histones/metabolism , Acetylation , Cell Line , Cell Proliferation/drug effects , Chromatin Immunoprecipitation , Disease-Free Survival , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylases/chemistry , Histone Deacetylases/metabolism , Humans , Hydroxamic Acids/pharmacology , Kaplan-Meier Estimate , Melanoma/metabolism , Melanoma/mortality , Melanoma/pathology , PTEN Phosphohydrolase/antagonists & inhibitors , PTEN Phosphohydrolase/genetics , PTEN Phosphohydrolase/metabolism , Principal Component Analysis , RNA Interference , RNA, Small Interfering/metabolism , Signal Transduction , VorinostatABSTRACT
Proliferation and migration of epidermal keratinocytes are essential for proper cutaneous wound closure after injury. αv integrins and several of their ligands-vitronectin, TGFß 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 αvß6, 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 TGFß, and the wound healing defect in the setting of integrin αv loss was rescued by exogenous, active TGFß, indicating that the αv-TGFß signaling axis is a critical component of the normal epidermal wound healing program. As chronic wounds are associated with decreased TGFß signaling, restoration of TGFß activity may have therapeutic utility in some clinical settings.
Subject(s)
Epidermis/pathology , Integrin alphaV/metabolism , Signal Transduction , Transforming Growth Factor beta/metabolism , Wound Healing , Animals , Antibodies, Blocking/pharmacology , Cell Movement , Cell Proliferation/drug effects , Epithelium/drug effects , Epithelium/metabolism , Epithelium/pathology , HEK293 Cells , Humans , Infant, Newborn , Male , Mice, SCID , Signal Transduction/drug effects , Wound Healing/drug effectsABSTRACT
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 (17ß-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.
Subject(s)
Estrogens/metabolism , Melanins/metabolism , Progesterone/metabolism , Receptors, Estrogen/metabolism , Receptors, G-Protein-Coupled/metabolism , Receptors, Progesterone/metabolism , Skin Pigmentation , Cells, Cultured , Humans , Melanocytes/metabolismABSTRACT
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, TGFß-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.