Retinoid-X-Receptors (α/β) in Melanocytes Modulate Innate Immune Responses and Differentially Regulate Cell Survival following UV Irradiation

Understanding the molecular mechanisms of ultraviolet (UV) induced melanoma formation is becoming crucial with more reported cases each year. Expression of type II nuclear receptor Retinoid-X-Receptor α (RXRα) is lost during melanoma progression in humans. Here, we observed that in mice with melanocyte-specific ablation of RXRα and RXRβ, melanocytes attract fewer IFN-γ secreting immune cells than in wild-type mice following acute UVR exposure, via altered expression of several chemoattractive and chemorepulsive chemokines/cytokines. Reduced IFN-γ in the microenvironment alters UVR-induced apoptosis, and due to this, the survival of surrounding dermal fibroblasts is significantly decreased in mice lacking RXRα/β. Interestingly, post-UVR survival of the melanocytes themselves is enhanced in the absence of RXRα/β. Loss of RXRs α/β specifically in the melanocytes results in an endogenous shift in homeostasis of pro- and anti-apoptotic genes in these cells and enhances their survival compared to the wild type melanocytes. Therefore, RXRs modulate post-UVR survival of dermal fibroblasts in a “non-cell autonomous” manner, underscoring their role in immune surveillance, while independently mediating post-UVR melanocyte survival in a “cell autonomous” manner. Our results emphasize a novel immunomodulatory role of melanocytes in controlling survival of neighboring cell types besides controlling their own, and identifies RXRs as potential targets for therapy against UV induced melanoma

CRISPR Screening Identifies BET and mTOR Inhibitor Synergy in Cholangiocarcinoma Through Serine Glycine One Carbon

Patients with cholangiocarcinoma have poor clinical outcomes due to late diagnoses, poor prognoses, and limited treatment strategies. To identify drug combinations for this disease, we have conducted a genome-wide CRISPR screen anchored on the bromodomain and extraterminal domain (BET) PROTAC degrader ARV825, from which we identified anticancer synergy when combined with genetic ablation of members of the mTOR pathway. This combination effect was validated using multiple pharmacological BET and mTOR inhibitors, accompanied by increased levels of apoptosis and cell cycle arrest. In a xenograft model, combined BET degradation and mTOR inhibition induced tumor regression. Mechanistically, the 2 inhibitor classes converged on H3K27ac-marked epigenetic suppression of the serine glycine one carbon (SGOC) metabolism pathway, including the key enzymes PHGDH and PSAT1. Knockdown of PSAT1 was sufficient to replicate synergy with single-agent inhibition of either BET or mTOR. Our results tie together epigenetic regulation, metabolism, and apoptosis induction as key therapeutic targets for further exploration in this underserved disease

Multiplatform Analysis of Intratumoral PTEN Heterogeneity in Melanoma

Loss of protein expression of the tumor suppressor PTEN is associated with increased cancer aggressiveness, decreased tumor immune infiltration, and resistance to immune and targeted therapies in melanoma. We assessed a unique cohort of eight melanoma samples with focal loss of PTEN protein expression to understand the features and mechanisms of PTEN loss in this disease. We compared the PTEN-negative (PTEN[-]) areas to their adjacent PTEN-positive (PTEN[+]) areas using DNA sequencing, DNA methylation, RNA expression, digital spatial profiling, and immunohistochemical platforms. Variations or homozygous deletions of PTEN were identified in PTEN(-) areas that were not detected in the adjacent PTEN(+) areas in three cases (37.5%), but no clear genomic or DNA methylation basis for loss was identified in the remaining PTEN(-) samples. RNA expression data from two independent platforms identified a consistent increase in chromosome segregation gene expression in PTEN(-) versus adjacent PTEN(+) areas. Proteomic analysis showed a relative paucity of tumor-infiltrating lymphocytes in PTEN(-) versus adjacent PTEN(+) areas. The findings add to our understanding of potential molecular intratumoral heterogeneity in melanoma and the features associated with the loss of PTEN protein in this disease

A three-drug nanoscale drug delivery system designed for preferential lymphatic uptake for the treatment of metastatic melanoma

Metastatic melanoma has a high mortality rate due to lymphatic progression of the disease. Current treatment is surgery followed by radiation and intravenous chemotherapy. However, drawbacks for current chemotherapeutics lie in the fact that they develop resistance and do not achieve therapeutic concentrations in the lymphatic system. We hypothesize that a three-drug nanoscale drug delivery system, tailored for lymphatic uptake, administered subcutaneously, will have decreased drug resistance and therefore offer better therapeutic outcomes. We prepared and characterized nanoparticles (NPs) with docetaxel, everolimus, and LY294002 in polyethyleneglycol-block-poly(ε-caprolactone) (PEG-PCL) polymer with different charge distributions by modifying the ratio of anionic and neutral end groups on the PEG block. These NPs are similarly sized (~48nm), with neutral, partially charged, or fully charged surface. The NPs are able to load ~2mg/mL of each drug and are stable for 24h. The NPs are assessed for safety and efficacy in two transgenic metastatic melanoma mouse models. All the NPs were safe in both models based on general appearance, weight changes, death, and blood biochemical analyses. The partially charged NPs are most effective in decreasing the number of melanocytes at both the proximal (sentinel) lymph node (LN) and the distal LN from the injection site. The neutral NPs are efficacious at the proximal LN, while the fully charged NPs have no effect on either LNs. Thus, our data indicates that the NP surface charge and lymphatic efficacy are closely tied to each other and the partially charged NPs have the highest potential in treating metastatic melanoma

Role of Melanocytic Vitamin D Receptor (VDR) in UVB-induced Melanocyte Homeostasisand Keratinocytic Retinoid–X-Receptor α (RXRα) in UVB Induced Melanomagenesis

Cutaneous melanoma remains the deadliest form of skin cancer arising from malignant transformation of pigment-producing melanocytes, with a diagnosis of metastasis indicating a median survival rate of less than a year. Solar ultraviolet (UV) radiation, especially childhood sun exposure, is an important etiological risk factor of melanoma. This DNA damage if incorrectly repaired, can result in incorporation of mutations that cause aberrant cell cycling and/or other functional defects that promote tumorigenesis. Melanoma incidence is on the rise throughout the U.S and thus a better understanding of the molecular mechanisms underlying its formation and progression are needed for the purpose of diagnosis and therapeutic targeting. Previous studies in humans have determined that there is an inverse correlation between Vitamin D Receptor (VDR) expression and melanoma progression, and loss of VDR enhances susceptibility to UV induced epithelial tumor formation in mice. To better understand the relationship between VDR loss and melanoma susceptibility, we have developed a mouse model where VDR is selectively deleted in melanocytes, the cells that transform into melanomas, to determine its role in melanocyte homeostasis and UV-induced DNA damage. In vivo ablation of VDR in melanocytes (Vdr[superscript mel-/-] mice), reduced the percentage of melanocyte precursors and mature differentiated melanocytes, as well as proliferating melanocyte, both pre- and post-UVB irradiation. VDR ablation significantly increased the proportion of UV induced DNA damaged melanocytes in the Vdr[superscript mel-/-] mice. Topical application of vitamin D3, inhibited UV induced DNA damage of melanocytes in the presence of functional VDR and this inhibition was abrogated in the absence of melanocytic VDR. Vdr[superscript mel-/-] mice also exhibited a reduction in melanocyte apoptosis after UVB exposure. Altogether, these results suggest that VDR plays an important role in controlling UV-induced DNA damage and melanocyte homeostasis in-vivo, and the protective effects of Vitamin D3 against the UV-induced DNA damage is mediated by functional VDR. Our previous studies have shown that mice selectively lacking the nuclear hormone receptor Retinoid X Receptor α, a heterodimeric partner of VDR, in epidermal keratinocytes (Rxrα[superscript ep-/-]) developed a higher number of aggressive melanocytic tumors compared to wild type mice after two-step chemical carcinogenesis, suggesting a novel role of keratinocytic nuclear receptor signaling during melanoma progression. In the present study, we have generated a mouse model to establish the role for RXRα in spontaneous and acute UVB-induced melanocyte homeostasis and melanomagenesis. Combining Rxrαep-/-mice with activated CDK4 (R24C) and oncogenic NRAS (Q61K) mutations in a trigenic model [Rxrα[superscript ep-/-]| NRas[superscript Q61K]| CDK4[superscript R24C/R24C]] results in enhanced UVR-induced melanomagenesis compared to control mice. These melanomas show increased expression of markers of malignant progression, proliferation and tumor vascularization. Melanomas from trigenic mice display increased metastases of pigment-producing cells to draining lymph nodes. Interestingly, the tumor adjacent normal skin of these mice have reduced expression of pro-apoptotic Bax and upregulation of cyclin D1 and p21 in the presence and absence of UVB treatment suggesting that these changes are primarily driven by loss of keratinocytic RXRα in the tumor microenvironment. These, effects were exacerbated after exposure to a single neonatal UVB treatment and we observed additional activation of AKT and reduction in expression of pro-Caspase 3, suggesting that these changes are likely UV effects in combination with loss of RXRα protein in the keratinocytes. Besides enhancing melanomagenesis, keratinocytic RXRα loss results in a microenvironment favorable to primary tumor formation. Therefore, VDR in melanocytes has a “cell-autonomous” role to protect against UVB induced DNA damage and a pivotal role to control melanocyte homeostasis in vivo, while RXRα in keratinocytes modulate post-UVB survival of melanocytes in a “non-cell autonomous” manner. Altogether these results establish a multitude of roles for type II nuclear receptors in melanocyte homeostasis and melanomagenesis, and identifies them as potential targets for melanoma diagnosis and therapeutics

Ablation of epidermal RXRα in cooperation with activated CDK4 and oncogenic NRAS generates spontaneous and acute neonatal UVB induced malignant metastatic melanomas

Abstract Background Understanding the underlying molecular mechanisms involved in the formation of cutaneous malignant melanoma is critical for improved diagnosis and treatment. Keratinocytic nuclear receptor Retinoid X Receptor α (RXRα) has a protective role against melanomagenesis and is involved in the regulation of keratinocyte and melanocyte homeostasis subsequent acute ultraviolet (UV) irradiation. Methods We generated a trigenic mouse model system (RXRα ep−/− | Tyr-NRAS Q61K | CDK4 R24C/R24C ) harboring an epidermal knockout of Retinoid X Receptor α (RXRα ep−/− ), combined with oncogenic NRAS Q61K (constitutively active RAS) and activated CDK4 R24C/R24C (constitutively active CDK4). Those mice were subjected to a single neonatal dose of UVB treatment and the role of RXR α was evaluated by characterizing the molecular and cellular changes that took place in the untreated and UVB treated trigenic RXRα ep−/− mice compared to the control mice with functional RXRα. Results Here we report that the trigenic mice develops spontaneous melanoma and exposure to a single neonatal UVB treatment reduces the tumor latency in those mice compared to control mice with functional RXRα. Melanomas from the trigenic RXRα ep−/− mice are substantial in size, show increased proliferation, exhibit increased expression of malignant melanoma markers and exhibit enhanced vascularization. Altered expression of several biomarkers including increased expression of activated AKT, p21 and cyclin D1 and reduced expression of pro-apoptotic marker BAX was observed in the tumor adjacent normal (TAN) skin of acute ultraviolet B treated trigenic RXRα ep−/− mice. Interestingly, we observed a significant increase in p21 and Cyclin D1 in the TAN skin of un-irradiated trigenic RXRα ep−/− mice, suggesting that those changes might be consequences of loss of functional RXRα in the melanoma microenvironment. Loss of RXRα in the epidermal keratinocytes in combination with oncogenic NRAS Q61K and CDK4 R24C/R24C mutations in trigenic mice led to significant melanoma invasion into the draining lymph nodes as compared to controls with functional RXRα. Conclusions Our study demonstrates the protective role of keratinocytic RxRα in (1) suppressing spontaneous and acute UVB-induced melanoma, and (2) preventing progression of the melanoma to malignancy in the presence of driver mutations like activated CDK4 R24C/R24C and oncogenic NRAS Q61K

Additional file 1: Figure S1. of Ablation of epidermal RXRα in cooperation with activated CDK4 and oncogenic NRAS generates spontaneous and acute neonatal UVB induced malignant metastatic melanomas

Breeding of mouse lines and UV scheme used in this study. (a) Breeding crossings used. K14-Cretg/O I RxrαL2/L2 is also known as Rxrαep−/−. (b) Scheme for single neonatal UVB treatment of mice. (c) H&E stained section showing poorly differentiated melanoma with abnormally large nuclei compared to the normal range (inset). (TIFF 1999 kb

Additional file 2: of Ablation of epidermal RXRα in cooperation with activated CDK4 and oncogenic NRAS generates spontaneous and acute neonatal UVB induced malignant metastatic melanomas

Table S1. Antibodies used for immuno-histochemical staining and immuno-blotting with details including the host, detection for-, the source, application and the dilution used. Table S2. PCR primers used for genotyping of mouse lines with the forward and reverse primers for K14-Cre, RXRα and CDK4, their sequences and the expected band sizes. Additional Methods. (DOCX 19 kb

La Charente

25 janvier 18861886/01/25 (A15,N5462)-1886/01/25.Appartient à l’ensemble documentaire : PoitouCh

ColemanDanielPharmacyRetinoid-X-Receptors.pdf

Understanding the molecular mechanisms of ultraviolet (UV) induced melanoma formation is becoming crucial with more reported cases each year. Expression of type II nuclear receptor Retinoid-X-Receptor α (RXRα) is lost during melanoma progression in humans. Here, we observed that in mice with melanocyte-specific ablation of RXRα and RXRβ, melanocytes attract fewer IFN-γ secreting immune cells than in wild-type mice following acute UVR exposure, via altered expression of several chemoattractive and chemorepulsive chemokines/cytokines. Reduced IFN-γ in the microenvironment alters UVR-induced apoptosis, and due to this, the survival of surrounding dermal fibroblasts is significantly decreased in mice lacking RXRα/β. Interestingly, post-UVR survival of the melanocytes themselves is enhanced in the absence of RXRα/β. Loss of RXRs α/β specifically in the melanocytes results in an endogenous shift in homeostasis of pro- and anti-apoptotic genes in these cells and enhances their survival compared to the wild type melanocytes. Therefore, RXRs modulate post-UVR survival of dermal fibroblasts in a ‘‘non-cell autonomous’’ manner, underscoring their role in immune surveillance, while independently mediating post-UVR melanocyte survival in a ‘‘cell autonomous’’ manner. Our results emphasize a novel immunomodulatory role of melanocytes in controlling survival of neighboring cell types besides controlling their own, and identifies RXRs as potential targets for therapy against UV induced melanoma