LNS8801: An enantiomerically pure agonist of the G protein-coupled estrogen receptor suitable for clinical development.

Estrogen effects in tissue are mediated in part through activation of the surface estrogen receptor GPER, a broadly expressed G protein-coupled receptor that impacts a wide range of normal and pathologic processes, including metabolism, vascular health, inflammation, and cancer. A commonly used synthetic and specific GPER agonist, named G-1, antagonizes tumors by promoting cellular differentiation and enhancing tumor immunogenicity. G-1 is a racemic compound, and since its discovery, the question of whether both enantiomers display agonist activity or the agonist activity resides primarily in a single enantiomer has never been fully resolved.

An In Vivo Study of LNS8801, a GPER Agonist, in a Spontaneous Melanoma-Prone Mouse Model, TGS.

Melanoma is the most aggressive and deadly form of skin cancer that arises from the transformation of melanocytes, the pigment producing cells of the skin. In the year 2024 there will be approximately 10,000 new cases of melanoma diagnosed and approximately 8,000 deaths attributed to melanoma in the United States. In this study we treated a group of male and female transgenic mice that spontaneously develop metastatic melanoma, TGS, with a G-protein-coupled estrogen receptor agonist LNS8801 to assess the efficacy on disease progression.

LNS8801 inhibits Acute Myeloid Leukemia by Inducing the Production of Reactive Oxygen Species and Activating the Endoplasmic Reticulum Stress Pathway.

Unlabelled: 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.

Analysis of human GPER expression in normal tissues and select cancers using immunohistochemistry.

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.

Monitoring cell fate in 3D organotypic human squamous epithelial cultures.

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.

Targeting anti-apoptotic pathways eliminates senescent melanocytes and leads to nevi regression.

Human melanocytic nevi (moles) result from a brief period of clonal expansion of melanocytes. As a cellular defensive mechanism against oncogene-induced hyperplasia, nevus-resident melanocytes enter a senescent state of stable cell cycle arrest. Senescent melanocytes can persist for months in mice and years in humans with a risk to escape the senescent state and progress to melanoma.

Endogenous DOPA inhibits melanoma through suppression of CHRM1 signaling.

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.

ZIP9 Is a Druggable Determinant of Sex Differences in Melanoma.

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.

Drug Resistant Melanoma May Be Vulnerable to Inhibitors of Serine Synthesis.

NRAS-driven melanomas frequently develop resistance to MAPK/extracellular signal-regulated kinase kinase inhibitors (MEKis), which limits their therapeutic utility. Nguyen et al. (2020) show that MEKi-resistant cells upregulate phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme in serine synthesis.

Pharmacologic Activation of the G Protein-Coupled Estrogen Receptor Inhibits Pancreatic Ductal Adenocarcinoma.

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.

Voriconazole enhances UV-induced DNA damage by inhibiting catalase and promoting oxidative stress.

Cutaneous squamous cell carcinoma (cSCC) is the second most common form of skin cancer and is associated with cumulative UV exposure. Studies have shown that prolonged voriconazole use promotes cSCC formation; however, the biological mechanisms responsible for the increased incidence remain unclear. Here, we show that voriconazole directly increases oxidative stress in human keratinocytes and promotes UV-induced DNA damage as determined by comet assay, 8-oxoguanine immunofluorescence and mass spectrometry.

LSD1 Inhibition Promotes Epithelial Differentiation through Derepression of Fate-Determining Transcription Factors.

Self-renewing somatic tissues depend upon the proper balance of chromatin-modifying enzymes to coordinate progenitor cell maintenance and differentiation, disruption of which can promote carcinogenesis. As a result, drugs targeting the epigenome hold significant therapeutic potential. The histone demethylase, LSD1 (KDM1A), is overexpressed in numerous cancers, including epithelial cancers; however, its role in the skin is virtually unknown.

Expression of p15 in a spectrum of spitzoid melanocytic neoplasms.

Background: Accurate classification of spitzoid melanocytic lesions is difficult due to overlapping clinical and histopathologic features between Spitz nevi, atypical Spitz tumors (ASTs), and spitzoid melanomas. Expression of p16 (CDKN2A) has been used as a marker of spitzoid lesions. However, its expression may be variable.

Lysosomes Support the Degradation, Signaling, and Mitochondrial Metabolism Necessary for Human Epidermal Differentiation.

Keratinocytes undergo significant structural remodeling during epidermal differentiation, including a broad transformation of the proteome coupled with a reduction in total cellular biomass. This suggests that intracellular digestion of proteins and organelles is necessary for keratinocyte differentiation. Here, we use both genetic and pharmacologic approaches to demonstrate that autophagy and lysosomal functions are required for keratinocyte differentiation in organotypic human skin.

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.

p15 Expression Differentiates Nevus from Melanoma.

Most melanomas are driven by BRAF(V600E)-activating mutations, while nevi harboring the same mutations have growth arrest. Although decreased p16 expression has been associated with melanoma formation, in recent work, p15 represented a primary effector of oncogene-induced senescence in nevomelanocytes that was diminished in melanomas. This study determined whether decreased p15 levels represent a general biomarker for the transition from nevus to melanoma.

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

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.

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).

Selective Vulnerability of Cancer Cells by Inhibition of Ca(2+) Transfer from Endoplasmic Reticulum to Mitochondria.

In the absence of low-level ER-to-mitochondrial Ca(2+) transfer, ATP levels fall, and AMPK-dependent, mTOR-independent autophagy is induced as an essential survival mechanism in many cell types. Here, we demonstrate that tumorigenic cancer cell lines, transformed primary human fibroblasts, and tumors in vivo respond similarly but that autophagy is insufficient for survival, and cancer cells die while their normal counterparts are spared. Cancer cell death is due to compromised bioenergetics that can be rescued with metabolic substrates or nucleotides and caused by necrosis associated with mitotic catastrophe during their proliferation.

MLL1 is essential for the senescence-associated secretory phenotype.

Oncogene-induced senescence (OIS) and therapy-induced senescence (TIS), while tumor-suppressive, also promote procarcinogenic effects by activating the DNA damage response (DDR), which in turn induces inflammation. This inflammatory response prominently includes an array of cytokines known as the senescence-associated secretory phenotype (SASP). Previous observations link the transcription-associated methyltransferase and oncoprotein MLL1 to the DDR, leading us to investigate the role of MLL1 in SASP expression.

Focal-adhesion-independent integrin-αv regulation of FAK and c-Myc is necessary for 3D skin formation and tumor invasion.

Integrins play crucial roles in epithelial adhesion, proliferation, wound healing and cancer. In the epidermis, the roles of many integrin subunits are incompletely defined and mechanistic details regarding their functions are lacking. We performed a multiplexed small hairpin (sh)RNA screen to define roles for each subunit in human organotypic skin.

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.

IQGAP1 and IQGAP3 Serve Individually Essential Roles in Normal Epidermal Homeostasis and Tumor Progression.

IQ motif-containing GTPase-activating protein (IQGAP) scaffolding proteins regulate many essential cellular processes including growth factor receptor signaling, cytoskeletal rearrangement, adhesion, and proliferation and are highly expressed in many cancers. Using genetically engineered human skin tissue in vivo, we demonstrate that diminished, sub-physiologic expression of IQGAP1 or IQGAP3 is sufficient to maintain normal epidermal homeostasis, whereas significantly higher levels are required to support tumorigenesis. To target this tumor-specific IQGAP requirement in vivo, we engineered epidermal keratinocytes to express individual IQGAP protein domains designed to compete with endogenous IQGAPs for effector protein binding.