Melanocyte Inducing Transcription Factor Research Articles

A PAX3 insertion in the Celestial breed and certain feline breeding lines with dominant blue eyes.

During the last 60 years many inherited traits in domestic outbred cats were selected and retained giving birth to new breeds characterised by singular coat or morphological phenotypes. Among them, minimal white spotting associated with blue eyes was selected by feline breeders to create the Altai, Topaz, and Celestial breeds. Various established breeds also introduced this trait in their lineages. The trait, that was confirmed as autosomal dominant by breeding data, was first described in domestic cats from Kazakhstan and Russia, in British shorthair and British longhair from Russia, and in Maine Coon cats from the Netherlands, suggesting different founding effects. Using a genome-wide association study we identified a single region on chromosome C1 that was associated with the minimal white spotting and blue eyes phenotype (also called DBE by breeders for dominant blue eyes) in the French Celestial breed. Within that region we identified Paired Box3 (PAX3) as the strongest candidate gene, since PAX3 is a key regulator of MITF (Melanocyte-Inducing Transcription Factor) and PAX3 variants have been previously identified in various species showing white spotting with or without blue eyes including the mouse and the horse. Whole genome sequencing of a Celestial cat revealed an endogenous retrovirus LTR (long terminal repeat) insertion within PAX3 intron 4 known to contain regulatory sequences (conserved non-coding element [CNE]) involved in PAX3 expression. The insertion is in the vicinity of CNE2 and CNE3. All 52 Celestial and Celestial-mixed cats with a DBE phenotype presented the insertion, that was absent in their 22 non-DBE littermates and in 87 non-DBE cats from various breeds. The outbred Celestial founder was also heterozygous for the insertion. Additionally, the variant was found in nine DBE Maine Coon cats related to the Celestial founder and four DBE Siberian cats with an uncertain origin. Segregation of the variant in the Celestial breed is consistent with dominant inheritance and does not appear to be associated with deafness. We propose that this NC_018730.3:g.206974029_206974030insN[395] variant represents the DBECEL (Celestial Dominant Blue Eyes) allele in the domestic cat.

Contemporary views on the clinical, epidemiological and molecular genetic characteristics of melanoma of the skin and mucous membranes

Melanoma of the skin and mucous membranes remains a global medical problem, which is associated with the increasing prevalence of this disease and the lack of adequate molecular genetic markers for its diagnosis and prognosis. The development of molecular approaches in the treatment of this type of tumor is associated with the identification of mutations, and with the development of immunotherapeutic and targeted drugs that can improve the effectiveness of treatment of patients with this pathology. However, the heterogeneity of the mechanisms of tumor development and the formation of resistance are a problem. It is worth noting the presence of many epigenetic mechanisms that are promising markers of the development, diagnosis and prognosis of the effectiveness of treatment of melanoma of the skin and mucous membranes. This review contains up-to-date information on the molecular mechanisms of the disease associated with the genetic characteristics of the tumor and biological factors of resistance to therapy. Of particular interest is the intersection of signaling pathways associated with melanocyte-inducing transcription factor (MITF), which is associated with transcription and growth factors, and is a target of epigenetic regulation using microRNAs and long non-coding RNAs.

Abstract 1704: Interplay of MITF and NCOA3 in orchestrating oncogenic transcriptional network in uveal melanoma

Abstract Uveal melanoma (UM) originates from melanocytes residing in the uveal tract of an eye. Despite effective local control, 50% of patients develop metastatic disease responding poorly to chemotherapy, targeted therapy, and immunotherapy. The molecular basis of events leading to initiation of UM is related to mutations in G alpha proteins GNAQ or GNA11, rendering them constitutively active and activating downstream PKC, ERK and AKT pathways sustaining cell proliferation. In our previous work, we showed that NCOA3 regulates proliferation of UM cells in vitro and the growth of UM xenografts in vivo downstream of GNAQ or GNA11. The critical role of NCOA3 in UM is manifested by transcriptional regulation of proliferation-related genes that are massively suppressed by NCOA3 knockout or knockdown. Here, we interrogated transcription factors that NCOA3 interacts with in UM to execute oncogenic programs and identified Melanocyte inducing transcription factor (MITF) as its critical partner. Chromatin immunoprecipitation (ChIP) using an NCOA3 recognizing antibody followed by sequencing was performed to determine NCOA3 binding sites in the human genome. The known transcription binding motifs within NCOA3 peaks were discovered using HOMER algorithm. Co-immunoprecipitation was performed to examine direct NCOA3 and MITF interaction. ChIP sequencing for MITF was performed to determine co-localization of NCOA3 and MITF at chromatin. Proliferation of UM cells after inhibition of MITF by either siRNA mediated knockdown or MITF inhibitor ML329 was evaluated using MTT assay. Functional enrichment analysis of transcriptome was used to identify classes of genes regulated by MITF, with specific attention to the subset regulated by MITF partnered with NCOA3. NCOA3 chromatin peaks are highly enriched in MITF binding motifs, and 77% of the NCOA3 peaks co-localizes with the MITF peaks. NCOA3 co-immunoprecipitates with MITF. Among transcriptional programs regulated by MITF in UM are those related to melanocyte biology, differentiation, and melanin production, but also those related to melanoma growth, especially cell division, DNA replication, myc response, purine and pyrimidine metabolism, and mTOR signaling. Strong suppression of these transcriptional signatures by inhibition of MITF using siRNA-mediated knockdown or pharmacological inhibition correlates with dramatic loss of cell viability. Clinical data indicate that higher expression of MITF-positively regulated genes correlates with lower survival rates of UM patients. Importantly, the transcriptional programs regulated by MITF cooperating with NCOA3 were related to TGF beta signaling, cell adhesion, ECM interaction and actin cytoskeleton. Importance of MITF as an oncogene is manifested in uveal melanoma cell lines and clinical data. Oncogenic function of MITF is partially dependent on its interaction with a transcription co-activator NCOA3. Citation Format: Aleksandra Rusin, Maria E. Echartea, Sai P. Manikonda, Darlene G. Skapura, Christel M. Davis, Erik A. Ehli, Sandra L. Grimm, Cristian Coarfa, Salma Kaochar. Interplay of MITF and NCOA3 in orchestrating oncogenic transcriptional network in uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1704.

Skin protective effect of Indian gooseberry and barley sprout complex on skin dryness, wrinkles, and melanogenesis by cell models.

UV radiation is a major factor contributing to DNA damage in skin cells, including stem cells and mesenchymal stem cells, leading to the depletion of these crucial cells. This study examined whether a mixture of Indian gooseberry and barley sprout (IB) could inhibit UVB irradiation and 3-isobutyl-1-methylxanthine (IBMX)-induced photoaging and oxidative stress in the skin using HaCaT, Hs27, and B16F10 cells. The moisturizing-related factors, the collagen synthesis-related c-Jun N-terminal kinase (JNK)/c-Fos/c-Jun/matrix metalloproteinases (MMPs) pathway, and the melanogenesis-related cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/cAMP-responsive binding protein (CREB)/melanocyte inducing transcription factor (MITF)/tyrosinase-related protein (TRP)/tyrosinase activation pathways were analyzed in vitro by an enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and Western blot analysis. The IB complex increased the hyaluronic acid and sphingomyelin levels and the collagenase inhibitory activity, enhanced hydration-related factors, including collagen, hyaluronic acid synthase (HAS), elastin, long chain base subunit 1 (LCB1) (serine palmitoyltransferase; SPT), and delta 4-desaturase sphingolipid 1 (DEGS1), modulated the inflammatory cytokines levels, antioxidant enzyme activities and the NF-κB/MMPs/cyclooxygenase-2 (COX-2) pathway in UVB-irradiated HaCaT cells, and inhibited wrinkle formation by down-regulation of the JNK/c-Fos/c-Jun/MMP pathway and up-regulation of the transforming growth factor-β receptor I (TGFβR1)/small mothers against decapentaplegic homolog (Smad3)/procollagen type І pathway in UVB-irradiated Hs27 cells. Moreover, the IB complex prevented melanin production by down-regulating the PKA/CREB/MITF/TRP-1/TRP-2 pathway in IBMX-induced B16F10 cells. These findings suggest that the IB complex has the potential to serve as a safeguard, shielding the skin from UVB radiation-induced photo-damage.

Silk Fibroin-Based Hydrogel Microneedles Deliver α-MSH to Promote Melanosome Delivery for Vitiligo Treatment.

Microneedles have shown great advantages in subcutaneous drug delivery and skin disease treatment. Vitiligo is a difficult-to-cure skin disease characterized by the depigmentation of the epidermis. Melanosomes produced in melanocytes are transported through dendrites to adjacent keratinocytes, where they accumulate, resulting in skin pigmentation. However, melanocytes in vitiligo patients are functionally disrupted. Silk fibroin (SF) methacrylate hydrogel microneedle can deliver α-MSH to the epidermis directly, where α-MSH helps the protection of melanocytes, extension of melanocytic dendrites, and transfer of melanosomes. In addition, the expression of melanogenesis-related melanocyte-inducing transcription factor and tyrosinase-related protein 1 (TRP1) was up-regulated, and the number of hair follicle stem cells increased with good proliferative activity. This slow release α-MSH SF-based hydrogel microneedles provides a new idea for the treatment of vitiligo.

Abstract 154: Establishment and characterization of a novel clear cell sarcoma organoid model derived from a human biopsy

Abstract Clear Cell Sarcoma (CCS) is an extremely aggressive, rare soft tissue cancer accounting for approximately 1% of all sarcomas. Often diagnosed in young adults with a median age of 25 years, CCS has a strong propensity for local recurrence and metastasis with an overall high mortality rate. Current treatment recommendations include radical surgical extirpation, followed by radiation or chemotherapy. Nonetheless, there is a need for extensive postoperative follow-up as the efficiency of adjuvant therapy in managing this disease remains poor. Consequently, it is imperative to identify clinically relevant models to advance our understanding of CCS tumor biology and facilitate the development of novel therapies. Compared to conventional preclinical tumor models, patient-derived organoids (PDOrgs) better recapitulate the patient tumor biology, genetic heterogeneity, and therapeutic responses. Here, we describe the development and validation of a human biopsy-derived CCS organoid model 1010561-T. 1010561-T was established from a male patient's metastatic paraspinal mass. This PDOrg demonstrates a moderate growth rate (doubling time of ~20 days) and pleomorphic traits, consisting mainly of solid, grape-like clusters, as well as elongated cells of mixed morphology. Furthermore, this model was successfully maintained over 7 months in culture and remained stable more than one year after cryopreservation. 1010561-T exhibits the cytogenetic hallmark of CCS which is the presence of a recurrent t(12;22)(q13;q12) chromosomal translocation, resulting in a fusion between the Ewing sarcoma gene and activating transcription factor 1 (EWS/ATF1 fusion). Reverse-transcription polymerase chain reaction (RT-PCR) confirmed that 1010561-T harbors a dual EWS/ATF1 fusion, namely EWS exon 8 to ATF1 codon 65 fusion (type 1) and EWS exon 7 to ATF1 codon 110 fusion (type 3). Quantitative RT-PCR and immunocytochemistry revealed strong expression of melanocytic differentiation markers typical of CCS including melanocyte inducing transcription factor (MITF), melan-A (MLANA), premelanosome protein (PMEL), and S100 calcium binding protein A11 (S100A11). Additionally, the gene expression profile of 1010561-T is in concordance with a well-established CCS cell line, SU-CCS-1. To our knowledge, 1010561-T is the first human CCS organoid model. It is, therefore, a valuable resource for advancing our understanding of the biology of this very rare disease, in addition to being a useful tool for validating new preclinical therapies that are undoubtedly needed to achieve better clinical outcomes for patients. To this end, 1010561-T will be transferred to the National Cancer Institute’s Patient-Derived Models Repository (https://pdmr.cancer.gov) where it will be made available to the scientific community. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261201800001I. Citation Format: Petreena S. Campbell, Erik D. Harris, Nancy Moore, Ralph E. Parchment, Nathan P. Coussens, Beverly A. Teicher, Alice Chen, James H. Doroshow, Annamaria Rapisarda. Establishment and characterization of a novel clear cell sarcoma organoid model derived from a human biopsy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 154.

Integrative analyses of RNA-seq and ChIP-seq Reveal MITF as a Target Gene of TFPI-2 in MDA231 Cells.

Breast cancer is the most prevalent cancer in female patients worldwide. Tissue factor pathway inhibitor 2 (TFPI-2) is identified as an important tumor suppressor in various cancers. Recent studies have shown that TFPI-2 translocates into the nucleus, where it modulates the transcription of the matrix metalloproteinase-2 (MMP-2) gene. However, its biological role and molecular mechanisms in the progression of breast cancer remain unclear. In this study, we identified 5125 differentially expressed genes (DEGs) from RNA sequencing (RNA-seq) in TFPI-2-overexpressing MDA231 cells compared with control cells. Gene ontology and Kyoto encyclopedia of genes and genomes (KEGG) analysis shown that cell cycle, cell differentiation, proteoglycans in cancer, and pathways associated with cancer were highly enriched in downregulated DEGs. Integration of the RNA-seq and ChIP-sequencing (ChIP-seq) data identified 73 genes directly controlled by TFPI-2 in MDA231 cells. Among them, melanocyte inducing transcription factor (MITF) gene expression was repressed by TFPI-2, which was further verified by a luciferase reporter assay and ChIP-quantitative PCR. Our study provides evidence of a novel role of TFPI-2 in human breast cancer involving targeting of the MITF.

In Silico Molecular Docking Approach of Melanin Against Melanoma Causing MITF Proteins and Anticancer, Oxidation-Reduction, Photoprotection, and Drug-Binding Affinity Properties of Extracted Melanin from Streptomyces sp. strain MR28.

Melanin is a biopolymer reported for diverse biological actions to secure organisms over adverse environmental factors. In the last decade, melanin attributed considerable attention for its use in bioelectronics, photoprotection, environmental bioremediation, and drug discovery. Molecular docking study is the emerging trend in drug discovery for drug designing by targeting proteins. Considering the therapeutic nature of the melanin, we extracted melanin from Streptomyces sp. strain MR28, and it was tested for various biological activities, viz., DPPH free radical scavenging potency, sun protection factor (SPF), drug likeness by SwissADME, molecular docking of melanin on melanocyte-inducing transcription factor (MITF) proteins, cytotoxic activity on A375 malignant melanoma with induction of apoptosis study by flow cytometry, and adsorption study of melanin on doxorubicin and camptothecin drug for drug uptake by melanin. The melanin showed good scavenging potency of DPPH free radicals in a concentration-dependent manner. SPF of 38.64 ± 0.63, 55.53 ± 0.53, and 67.07 ± 0.82 were recorded at 0.06, 0.08, and 0.1µg/mL, concentrations, respectively. SwissADME screening confirms the drug likeness of melanin. Docking of melanin with MITF proteins exhibited a maximum of - 9.2kcal/mol binding affinity for 4ATK protein. Cytotoxicity of the melanin drug exhibited good inhibition of melanoma cells in dose-dependent way with significant IC50 of 65.61µg/mL; apoptotic study reveals melanin showed 64.02% apoptosis for melanin and 33.8% apoptosis for standard drug (doxorubicin). The maximum adsorptions for selected drugs camptothecin and doxorubicin to melanin were recorded at 90min. In conclusion, the extracted melanin showed significant results over many biological applications and it can be used in the pharmaceutical field to avoid chemical-based drugs.

MiR-182 Is Upregulated in Prostate Cancer and Contributes to Tumor Progression by Targeting MITF.

Altered expression of microRNA-182-5p (miR-182) has been consistently linked with many cancers, but its specific role in prostate cancer remains unclear. In particular, its contribution to epithelial-to-mesenchymal transition (EMT) in this setting has not been well studied. Therefore, this paper profiles the expression of miR-182 in prostate cancer and investigates how it may contribute to progression of this disease. In vitro experiments on prostate cancer cell lines and in silico analyses of The Cancer Genome Atlas (TCGA) prostate adenocarcinoma (PRAD) datasets were performed. PCR revealed miR-182 expression was significantly increased in prostate cancer cell lines compared to normal prostate cells. Bioinformatic analysis of TCGA PRAD data similarly showed upregulation of miR-182 was significantly associated with prostate cancer and clinical markers of disease progression. Functional enrichment analysis confirmed a significant association of miR-182 and its target genes with EMT. The EMT-linked gene MITF (melanocyte inducing transcription factor) was subsequently shown to be a novel target of miR-182 in prostate cancer cells. Further TCGA analysis suggested miR-182 expression can be an indicator of patient outcomes and disease progression following therapy. In summary, this is the first study to report that miR-182 over-expression in prostate cancer may contribute to EMT by targeting MITF expression. We propose miR-182 as a potentially useful diagnostic and prognostic biomarker for prostate cancer and other malignancies.

SNP associations and genetic-parameter estimation for nose and hoof pigmentation in Corriedale sheep

Context Quantity and quality of both meat and wool are important for selection programs of the dual-purpose Corriedale sheep. In Corriedales, black pigmentation of nose skin and hooves is preferred as part of the breed standard. However, within the breed, pigmentation can vary from none to complete pigmentation. Aims The aim was to discover single-nucleotide polymorphisms (SNPs) and genes associated with nose and hoof pigmentation and to estimate genetic parameters of nose and hoof pigmentation, wool traits and meat traits. Methods The phenotype and genotype data on Corriedale lambs (n = 764) produced from 44 sires (12 studs) and 300 ewes in 2017–2019 were used in this study. Lambs were slaughtered at 6–7 months of age. Nose pigmentation and hoof pigmentation were scored on a five-point scale, where no pigmentation was scored 0 and complete pigmentation was scored 5. Wool- and meat-quality traits were measured, including greasy fleece weight, fibre diameter, weaning weight, hot standard carcass weight and intramuscular fat percentage. The lambs were genotyped with the GGPOvine50K SNP chip. A genome-wide association study (GWAS) was conducted. Genetic parameters and bivariate analyses were estimated using ASReml-R. Results The lambs had a mean nose-pigmentation score of 3.69 and hoof-pigmentation score of 4.12. The nose pigmentation (h2 = 0.58) and hoof pigmentation (h2 = 0.51) were highly heritable and highly correlated (rG = 0.73). The GWAS identified a genome-wide significant SNP (OAR19_33278780.1) associated with nose pigmentation (q-value = 0.04). This SNP is positioned in a known pigmentation gene, melanocyte-inducing transcription factor (MITF). The same SNP was the sixth-most associated SNP for hoof pigmentation but did not reach significance level. OAR19_33278780.1 also had a significant effect on back conformation and weaning weight. Conclusions The results of these analyses have provided information and an understanding of the genetics for pigmentation of nose and hooves in Corriedale sheep. Implications Nose and hoof pigmentation are highly genetically correlated with each other, but with no negative effects on production traits.

De-novo and genome-wide meta-analyses identify a risk haplotype for congenital sensorineural deafness in Dalmatian dogs

Congenital sensorineural deafness (CSD) has been reported to affect up to 30% of Dalmatian dogs world-wide and while unilaterally deaf dogs can live a close to normal life, dogs suffering bilateral deafness are frequently euthanized. Extreme-white coat patterning as encoded by the gene Melanocyte Inducing Transcription Factor (MITF) has long been postulated as the major risk factor for CSD in the Dalmatian breed. While attempts to identify causative risk variants associated with CSD have been numerous, no genome-wide association study has positively identified MITF as a risk locus for either bilateral or unilateral deafness in the Dalmatian breed to date. In this study, we identified an association with CSD on CFA20 in the vicinity of MITF within Australian Dalmatian dogs. Although not genome-wide significant, the association signal was validated by reanalysing publicly available data and merging the wider data resource with the local data to improve statistical power. The merged data, representing three major global populations of Dalmatian dogs, enabled us to identify a single, well-defined genome-wide significant risk haplotype for CSD. The haplotype was formed by three genome-wide significant associated markers (BICF2G630233852T>C, BICF2G630233861T>C, BICF2G630233888G>A) on CFA20 with 62% of bilaterally deaf dogs homozygous for the risk haplotype (CCA), while 30% of bilaterally deaf and 45% of hearing dogs carried one copy of the risk haplotype. Animals homozygous or heterozygous for the low-risk haplotype were less likely to be unilaterally deaf. While the association between the risk haplotype and deafness is incomplete, animals homozygous for the risk haplotype were 10-times more likely to be bilaterally deaf. Although the underlying causative variants are yet to be discovered, results from this study can now assist with reducing deafness in Dalmatian dogs.

Transcriptional activation of hepcidin by the microphthalmia/transcription factor E family.

Hepcidin negatively regulates the circulating iron levels by inhibiting the intestinal absorption of iron as well as iron release from macrophages. Hepcidin activity is largely determined by its expression, which is regulated at the transcriptional level. Hepcidin transcription is induced not only by the iron status-related bone morphogenetic protein (BMP)-2/6, but also by inflammatory cytokines, such as interleukin (IL)-1β and IL-6. The present study reveals that the microphthalmia (MiT)/transcription factor E (TFE) family members are novel regulators of hepcidin transcription. Melanocyte-inducing transcription factor (MITF)-A, a member of the MiT/TFE family, was identified as a positive regulator of hepcidin transcription via stimulus screening for transcription regulators. An E-box (5'-CATGTG-3') spanning nt-645 to nt-640 of the murine hepcidin promoter was identified as an MITF-A-responsive element. Responsiveness to MITF-A on hepcidin transcription decreased when the cells were stimulated with BMP2 or IL-1β. These results suggest a functional interaction between the MITF pathway and BMP- or IL-1β-mediated signaling. TFEB and TFE3, members of the MiT/TFE family, also stimulated hepcidin transcription, but the main region responsible for hepcidin transcription was distinct from that induced by MITF-A. The region spanning nt-581 to nt-526 was involved in TFEB/TFE3-mediated hepcidin transcription. Considering that members of the MiT/TFE family act as regulators of starvation-induced lysosomal biogenesis, hepcidin expression may be controlled by additional pathways apart from those identified so far.

Enhanced Anti-Melanogenic Effect of Adlay Bran Fermented with Lactobacillus brevis MJM60390

Fermentation is a traditional technique used to increase nutrients, flavonoids, vitamins, minerals, and the flavor of raw materials. In this study, adlay bran was fermented by Lactobacillus brevis MJM60390 (FAB), and the anti-melanogenic effect was investigated. The results demonstrated that FAB significantly suppressed melanin accumulation in mouse melanogenic B16F10 cells, and the activity was higher than non-fermented adlay bran (NFAB). The molecular mechanism study showed that FAB inhibited melanin synthesis by suppressing the gene expression of melanocortin 1 receptor (Mc1r), melanocyte-inducing transcription factor (Mitf), tyrosinase (Tyr), tyrosinase-related protein-1 (Trp-1), and tyrosinase-related protein-2 (Trp-2) genes. Western blotting analysis showed that FAB strongly decreased the expression of Tyr, Trp-1, and Trp-2 compared to NFAB. Furthermore, phenolic compounds such as gallic acid, p-coumaric acid, ferulic acid, and sinapic acid, which are known for their anti-melanogenic effects, were significantly increased in FAB compared with NFAB. These findings suggest that FAB holds great potential as an anti-melanogenic agent and can be used for the development of whitening cosmetics.

Candidate therapeutic agents in a newly established triple wild-type mucosal melanoma cell line.

BackgroundMucosal melanoma has characteristically distinct genetic features and typically poor prognosis. The lack of representative mucosal melanoma models, especially cell lines, has hindered translational research on this melanoma subtype. In this study, we aimed to establish and provide the biological properties, genomic features and the pharmacological profiles of a mucosal melanoma cell line that would contribute to the understanding and treatment optimization of molecularly‐defined mucosal melanoma subtype.MethodsThe sample was collected from a 67‐year‐old mucosal melanoma patient and processed into pieces for the establishment of cell line and patient‐derived xenograft (PDX) model. The proliferation and tumorigenic property of cancer cells from different passages were evaluated, and whole‐genome sequencing (WGS) was performed on the original tumor, PDX, established cell line, and the matched blood to confirm the establishment and define the genomic features of this cell line. AmpliconArchitect was conducted to depict the architecture of amplified regions detected by WGS. High‐throughput drug screening (HTDS) assay including a total of 103 therapeutic agents was implemented on the established cell line, and selected candidate agents were validated in the corresponding PDX model.ResultsA mucosal melanoma cell line, MM9H‐1, was established which exhibited robust proliferation and tumorigenicity after more than 100 serial passages. Genomic analysis of MM9H‐1, corresponding PDX, and the original tumor showed genetic fidelity across genomes, and MM9H‐1 was defined as a triple wild‐type (TWT) melanoma subtype lacking well‐characterized “driver mutations”. Instead, the amplification of several oncogenes, telomerase reverse transcriptase (TERT), v‐Raf murine sarcoma viral oncogene homolog B1 (BRAF), melanocyte Inducing transcription factor (MITF) and INO80 complex ATPase subunit (INO80), via large‐scale genomic rearrangement potentially contributed to oncogenesis of MM9H‐1. Moreover, HTDS identified proteasome inhibitors, especially bortezomib, as promising therapeutic candidates for MM9H‐1, which was verified in the corresponding PDX model in vivo.ConclusionsWe established and characterized a new mucosal melanoma cell line, MM9H‐1, and defined this cell line as a TWT melanoma subtype lacking well‐characterized “driver mutations”. The MM9H‐1 cell line could be adopted as a unique model for the preclinical investigation of mucosal melanoma.

Melanopsin (Opn4) is an oncogene in cutaneous melanoma

The search for new therapeutical targets for cutaneous melanoma and other cancers is an ongoing task. We expanded this knowledge by evaluating whether opsins, light- and thermo-sensing proteins, could display tumor-modulatory effects on melanoma cancer. Using different experimental approaches, we show that melanoma cell proliferation is slower in the absence of Opn4, compared to Opn4WT due to an impaired cell cycle progression and reduced melanocyte inducing transcription factor (Mitf) expression. In vivo tumor progression of Opn4KO cells is remarkably reduced due to slower proliferation, and higher immune system response in Opn4KO tumors. Using pharmacological assays, we demonstrate that guanylyl cyclase activity is impaired in Opn4KO cells. Evaluation of Tumor Cancer Genome Atlas (TCGA) database confirms our experimental data as reduced MITF and OPN4 expression in human melanoma correlates with slower cell cycle progression and presence of immune cells in the tumor microenvironment (TME). Proteomic analyses of tumor bulk show that the reduced growth of Opn4KO tumors is associated with reduced Mitf signaling, higher translation of G2/M proteins, and impaired guanylyl cyclase activity. Conversely, in Opn4WT tumors increased small GTPase and an immune-suppressive TME are found. Such evidence points to OPN4 as an oncogene in melanoma, which could be pharmacologically targeted.

Interferon-gamma induces melanogenesis via post-translational regulation of tyrosinase.

Melanogenesis (melanin pigment production) in melanocytes is canonically stimulated by the alpha melanocyte stimulating hormone (αMSH), which activates the cyclic-AMP-mediated expression of the melanocyte inducing transcription factor (MITF) and its downstream melanogenic genes, including the principal rate-limiting melanogenic enzyme tyrosinase (TYR). Here, we report that interferon-gamma (IFNG; type II interferon), but not interferon-alpha (a type I interferon), induces a noncanonical melanogenic pathway in mouse and human melanocytic cells. Inhibition of IFNG pathway by the JAK1/2 inhibitor ruxolitinib or knocking out Stat1gene abrogated the IFNG-induced melanogenesis. Interestingly, IFNG-induced melanogenesis was independent of MITF. IFNG markedly increased the TYR protein expression but did not affect the mRNA expression, suggesting a post-translational regulatory mechanism. In contrast, IFNG had no effect on the expression of other melanogenesis-related proteins, for example, tyrosinase-related protein 1 (TYRP1) and dopachrome tautomerase (DCT). Glycosidase digestion assays revealed that IFNG treatment increased the mature glycosylated form of TYR, but not its de novo synthesis. Moreover, cycloheximide chase assay showed that degradation of TYR was decreased in IFNG-treated cells. These results suggest that the IFNG-STAT1 pathway regulates melanogenesis via regulation of the post-translational processing and protein stability of TYR.

Lysosomal inhibition sensitizes TMEM16A-expressing cancer cells to chemotherapy

Squamous cell carcinoma of the head and neck (SCCHN) is a devastating disease that continues to have low cure rates despite the recent advances in therapies. Cisplatin is the most used chemotherapy agent, and treatment failure is largely driven by resistance to this drug. Amplification of chromosomal band 11q13 occurs in ∼30% of SCCHN tumors. This region harbors the ANO1 gene that encodes the TMEM16A ion channel, which is responsible for calcium-activated chloride transport in epithelial tissues. TMEM16A overexpression is associated with cisplatin resistance, and high TMEM16A levels correlate with decreased survival. However, the mechanistic underpinning of this effect remains unknown. Lysosomal biogenesis and exocytosis have been implicated in cancer because of their roles in the clearance of damaged organelles and exocytosis of chemotherapeutic drugs and toxins. Here, we show that TMEM16A overexpression promotes lysosomal biogenesis and exocytosis, which is consistent with the expulsion of intracellular cisplatin. Using a combination of genetic and pharmacologic approaches, we find that TMEM16A promotes lysosomal flux in a manner that requires reactive oxygen species, TRPML1, and the activation of the β-catenin–melanocyte-inducing transcription factor pathway. The lysosomal inhibitor hydroxychloroquine (HCQ) synergizes with cisplatin in killing SCCHN cells in vitro. Using a murine model of SCCHN, we show that HCQ and cisplatin retard the growth of cisplatin-resistant patient-derived xenografts in vivo. We propose that TMEM16A enables cell survival by the up-regulation of lysosomal sequestration and exocytosis of the cytotoxic drugs. These results uncover a model of treatment for resistance in cancer, its reversal, and a role for TMEM16A.

Dual Skin-Whitening and Anti-wrinkle Function of Low-Molecular-Weight Fish Collagen.

In this study, we investigated the protective effects of low-molecular-weight fish collagen from tilapia against melanogenesis in melanocytes, ultraviolet B (UVB)-irradiated Hs27 skin fibroblasts, and hairless mice. We observed collagen production-related pathways in UVB-irradiated Hs27 skin fibroblasts and hairless mice, and the melanogenesis-related pathways in melanocyte and UVB-irradiated hairless mice. The collagen production-related pathways were activated in the UVB-irradiated Hs27 skin fibroblasts and hairless mice. In addition, UVB exposure stimulated the melanogenesis-related pathways in melanocytes and hairless mice. However, treatment with low-molecular-weight fish collagen significantly increased the messenger RNA expressions of collagen production-related factors and significantly decreased the production of cytokines. Furthermore, treatment with low-molecular-weight fish collagen suppressed melanogenesis by inhibiting glutathione synthesis and downregulating melanocyte-inducing transcription factor expression through the suppression of cyclic AMP/protein kinase A/cAMP-responsive binding protein signaling and nitric oxide production. Low-molecular-weight fish collagen exerts protective effects against UVB-induced photoaging, through anti-inflammatory, antioxidant, and anti-melanogenesis activities and could be used for developing effective natural anti-photoaging products.

Vitamin D3 and carbamazepine protect against Clostridioides difficile infection in mice by restoring macrophage lysosome acidification

ABSTRACT Clostridioides difficile infection (CDI) is a common cause of nosocomial diarrhea. TcdB is a major C. difficile exotoxin that activates macrophages to promote inflammation and epithelial damage. Lysosome impairment is a known trigger for inflammation. Herein, we hypothesize that TcdB could impair macrophage lysosomal function to mediate inflammation during CDI. Effects of TcdB on lysosomal function and the downstream pro-inflammatory SQSTM1/p62-NFKB (nuclear factor kappa B) signaling were assessed in cultured macrophages and in a murine CDI model. Protective effects of two lysosome activators (i.e., vitamin D3 and carbamazepine) were assessed. Results showed that TcdB inhibited CTNNB1/β-catenin activity to downregulate MITF (melanocyte inducing transcription factor) and its direct target genes encoding components of lysosomal membrane vacuolar-type ATPase, thereby suppressing lysosome acidification in macrophages. The resulting lysosomal dysfunction then impaired autophagic flux and activated SQSTM1-NFKB signaling to drive the expression of IL1B/IL-1β (interleukin 1 beta), IL8 and CXCL2 (chemokine (C-X-C motif) ligand 2). Restoring MITF function by enforced MITF expression or restoring lysosome acidification with 1α,25-dihydroxyvitamin D3 or carbamazepine suppressed pro-inflammatory cytokine expression in vitro. In mice, gavage with TcdB-hyperproducing C. difficile or injection of TcdB into ligated colon segments caused prominent MITF downregulation in macrophages. Vitamin D3 and carbamazepine lessened TcdB-induced lysosomal dysfunction, inflammation and histological damage. In conclusion, TcdB inhibits the CTNNB1-MITF axis to suppress lysosome acidification and activates the downstream SQSTM1-NFKB signaling in macrophages during CDI. Vitamin D3 and carbamazepine protect against CDI by restoring MITF expression and lysosomal function in mice. Abbreviations: ATP6V0B: ATPase H+ transporting V0 subunit b; ATP6V0C: ATPase H+ transporting V0 subunit c; ATP6V0E1: ATPase H+ transporting V0 subunit e1; ATP6V1H: ATPase H+ transporting V1 subunit H; CBZ: carbamazepine; CDI: C. difficile infection; CXCL: chemokine C-X-X motif ligand; IL: interleukin; LAMP1: lysosomal-associated membrane protein 1; LC3: microtubule-associated protein 1 light chain 3; LEF: lymphoid enhancer binding factor 1; MITF: melanocyte inducing transcription factor; NFKB: nuclear factor kappa B; PMA: phorbol 12-myristate 13-acetate; TcdA: Clostridial toxin A; TcdB: Clostridial toxin B; TFE3: transcription factor E3; TFEB: transcription factor EB.

The Tietz syndrome associated with cardiac malformation: a case report with literature review

BackgroundTietz syndrome is a very rare clinical entity characterized by the association of profound bilateral congenital sensorineural deafness and generalized hypopigmentation of skin, eyes, and integuments (snow white appearance). It is an autosomal dominant syndrome due to a mutation in the melanocyte inducing transcription factor (MITF) gene. The association of a heart malformation has never been reported in this syndrome.Case presentationWe report two cases of two cousins aged 5 years and 20 months respectively with a history of first degree consanguineous parents. Both girls presented with diffuse hypopigmentation of the skin, blond hair, blue eyes, and bilateral diffuse retinal hypopigmentation at ocular fundus exam. Bilateral profound sensorineural hearing loss was confirmed by auditory brainstem response in both cases. Echocardiography revealed a cardiac malformation such as interventricular communication in the older cousin and interatrial communication in the younger cousin. The family investigation did not reveal a similar case among ancestors. The diagnosis of Tietz syndrome was based on clinical criteria and pedigree. The older cousin underwent a total optical correction and a right unilateral cochlear implantation followed by speech therapy with a satisfactory result after a follow-up of two years. Unfortunately, the little cousin died following a head trauma.ConclusionsTietz syndrome is a rare autosomal dominant genetic disorder, characterized by generalized albinism with bilateral profound hearing loss. It results from a nontruncating mutation in the basic domain of in the MITF gene. Its management must include, in addition to hearing and ophthalmic rehabilitation, the research and treatment of cardiac malformations which may be life-threatening.