RESUMEN
Extrusion-based bioprinting is an established method in biofabrication. Suitable bioinks have fundamentally different compositions and characteristics, which should be examined, in order to find a perfect model system. Here, we investigate the effect of two alginate-based, yet unalike 3D-printed bioinks, pre-crosslinked alginate-dialdehyde gelatin (ADA-GEL) and a mixture of alginate, hyaluronic acid, and gelatin (Alg/HA/Gel), on the melanoma cell line Mel Im and vice versa in terms of stiffness, shrinkage, cellular behavior and colony formation over 15 days. Rheological stiffness measurements revealed two soft gels with similar storage moduli. The cells did not have a significant impact on the overall stiffness, whereas ADA-GEL (2.5/2.5%) was significantly stiffer than Alg/HA/Gel (0.5/0.1/3%). Regarding the shrinkage of printed constructs, cells had a significant influence, especially in ADA-GEL, which has covalent bonds between the oxidized alginate and gelatin. Multi-photon microscopy exhibited proliferation, cell spreading and migration in ADA-GEL with cell-cell and cell-matrix interaction, dissimilarly to Alg/HA/Gel, in which cells formed spherical, encapsulated colonies. Scanning electron microscopy and histology showed degradation and multi-layered growth on ADA-GEL and fewer examples of escaped cells on Alg/HA/Gel. Both gels serve as proliferation bioink for melanoma with more necrosis in deeper Alg/HA/Gel colonies and differences in spreading and matrix interaction. These findings show the importance of proper characterization of the bioinks for different applications.
Asunto(s)
Alginatos , Bioimpresión , Proliferación Celular , Gelatina , Melanoma , Impresión Tridimensional , Alginatos/química , Melanoma/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Gelatina/química , Bioimpresión/métodos , Humanos , Tinta , Ácido Hialurónico/química , Reología , Andamios del Tejido/química , Ingeniería de Tejidos/métodosRESUMEN
Malignant melanoma, the most aggressive form of skin cancer, is often incurable once metastatic dissemination of cancer cells to distant organs has occurred. We investigated the role of Transcription Factor Activating Enhancer-Binding Protein 2ε (AP2ε) in the progression of metastatic melanoma. Here, we observed that AP2ε is a potent activator of metastasis and newly revealed AP2ε to be an important player in melanoma plasticity. High levels of AP2ε lead to worsened prognosis of melanoma patients. Using a transgenic melanoma mouse model with a specific loss of AP2ε expression, we confirmed the impact of AP2ε to modulate the dynamic switch from a migratory to a proliferative phenotype. AP2ε deficient melanoma cells show a severely reduced migratory potential in vitro and reduced metastatic behavior in vivo. Consistently, we revealed increased activity of AP2ε in quiescent and migratory cells compared to heterogeneously proliferating cells in bioprinted 3D models. In conclusion, these findings disclose a yet-unknown role of AP2ε in maintaining plasticity and migration in malignant melanoma cells.
Asunto(s)
Movimiento Celular , Progresión de la Enfermedad , Melanoma , Factor de Transcripción AP-2 , Animales , Humanos , Ratones , Línea Celular Tumoral , Proliferación Celular , Melanoma/patología , Melanoma/metabolismo , Melanoma/genética , Ratones Transgénicos , Metástasis de la Neoplasia , Fenotipo , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/genética , Factor de Transcripción AP-2/metabolismo , Factor de Transcripción AP-2/genéticaRESUMEN
Targeted therapies with chemotherapeutic agents and immunotherapy with checkpoint inhibitors are among the systemic therapies recommended in the guidelines for clinicians to treat melanoma. Although there have been constant improvements in the treatment of melanoma, resistance to the established therapies continues to occur. Therefore, the purpose of this study was to explore the function of garcinol with regards to specific cancer properties such as proliferation and apoptosis. Garcinol, a natural compound isolated from the plant also known as mangosteen (Garcinia mangostana), is a newly discovered option for cancer treatment. Numerous pharmaceutical substances are derived from plants. For example, the derivates of camptothecin, extracted from the bark of the Chinese tree of happiness (Camptotheca acuminate), or paclitaxel, extracted from the bark of the Western yew tree (Taxus brevifolia), are used as anti-cancer drugs. Here, we show that garcinol reduced proliferation and induced apoptosis in melanoma cell lines. In addition, we found that those cells that are positive for the expression of the cell-cell adhesion molecule T-cadherin (CDH13) respond more sensitively to treatment with garcinol. After knock-down experiments with an siRNA pool against T-cadherin, the sensitivity to garcinol decreased and proliferation and anti-apoptotic behavior of the cells was restored. We conclude that patients who are T-cadherin-positive could especially benefit from a therapy with garcinol.
RESUMEN
Although 2D cancer models have been the standard for drug development, they don't resemble in vivo properties adequately. 3D models can potentially overcome this. Bioprinting is a promising technique for more refined models to investigate central processes in tumor development such as proliferation, dormancy or metastasis. We aimed to analyze bioinks, which could mimic these different tumor stages in a cast vascularized arteriovenous loop melanoma model in vivo. It has the advantage to be a closed system with a defined microenvironment, supplied only with one vessel-ideal for metastasis research. Tested bioinks showed significant differences in composition, printability, stiffness and microscopic pore structure, which led to different tumor stages (Matrigel and Alg/HA/Gel for progression, Cellink Bioink for dormancy) and resulted in different primary tumor growth (Matrigel significantly higher than Cellink Bioink). Light-sheet fluorescence microscopy revealed differences in vascularization and hemorrhages with no additional vessels found in Cellink Bioink. Histologically, typical human melanoma with different stages was demonstrated. HMB-45-positive tumors in progression inks were infiltrated by macrophages (CD163), highly proliferative (Ki67) and metastatic (MITF/BRN2, ATX, MMP3). Stainings of lymph nodes revealed metastases even without significant primary tumor growth in Cellink Bioink. This model can be used to study tumor pathology and metastasis of different tumor stages and therapies.
RESUMEN
BACKGROUND: Melanoma is a highly heterogeneous cancer, in which frequent changes in activation of signaling pathways lead to a high adaptability to ever changing tumor microenvironments. The elucidation of cancer specific signaling pathways is of great importance, as demonstrated by the inhibitor of the common BrafV600E mutation PLX4032 in melanoma treatment. We therefore investigated signaling pathways that were influenced by neurotrophin NRN1, which has been shown to be upregulated in melanoma. METHODS: Using a cell culture model system with an NRN1 overexpression, we investigated the influence of NRN1 on melanoma cells' functionality and signaling. We employed real time cell analysis and spheroid formation assays, while for investigation of molecular mechanisms we used a kinase phosphorylation kit as well as promotor activity analysis followed by mRNA and protein analysis. RESULTS: We revealed that NRN1 interacts directly with the cleaved intracellular domain (NICD) of Notch1 and Notch3, causing a potential retention of NICD in the cytoplasm and thereby reducing the expression of its direct downstream target Hes1. This leads to decreased sequestration of JAK and STAT3 in a Hes1-driven phosphorylation complex. Consequently, our data shows less phosphorylation of STAT3 while presenting an accumulation of total protein levels of STAT3 in association with NRN1 overexpression. The potential of the STAT3 signaling pathway to act in both a tumor suppressive and oncogenic manner led us to investigate specific downstream targets - namely Vegf A, Mdr1, cMet - which were found to be upregulated under oncogenic levels of NRN1. CONCLUSIONS: In summary, we were able to show that NRN1 links oncogenic signaling events between Notch and STAT3 in melanoma. We also suggest that in future research more attention should be payed to cellular regulation of signaling molecules outside of the classically known phosphorylation events.
Asunto(s)
Melanoma , Neuropéptidos , Factor de Transcripción STAT3 , Transducción de Señal , Humanos , Carcinogénesis/genética , Carcinogénesis/metabolismo , Línea Celular Tumoral , Melanoma/metabolismo , Melanoma/genética , Melanoma/patología , Fosforilación , Unión Proteica , Receptor Notch1/metabolismo , Receptor Notch1/genética , Receptor Notch3/metabolismo , Receptor Notch3/genética , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT3/genéticaRESUMEN
Malignant melanoma remains the most lethal form of skin cancer, exhibiting poor prognosis after forming distant metastasis. Owing to their potential tumor-suppressive properties by regulating oncogenes and tumor suppressor genes, microRNAs are important player in melanoma development and progression. We defined the loss of miR-101-3p expression in melanoma cells compared with melanocytes and melanoblast-related cells as an early event in tumor development and aimed to understand the tumor suppressive role of miR-101-3p and its regulation of important cellular processes. Reexpression of miR-101-3p resulted in inhibition of proliferation, increase in DNA damage, and induction of apoptosis. We further determined the nuclear structure protein Lamin B1, which influences nuclear processes and heterochromatin structure, ATRX, CASP3, and PARP as an important direct target of miR-101-3p. RNA sequencing and differential gene expression analysis after miR-101-3p reexpression supported our findings and the importance of loss of mir-101-3p for melanoma progression. The validated functional effects are related to genomic instability, as recent studies suggest miRNAs plays a key role in mediating this cellular process. Therefore, we concluded that miR-101-3p reexpression increases the genomic instability, leading to irreversible DNA damage, which leads to apoptosis induction. Our findings suggest that the loss of miR-101-3p in melanoma serves as an early event in melanoma progression by influencing the genomic integrity to maintain the increased bioenergetic demand.
Asunto(s)
Melanoma , MicroARNs , Neoplasias Cutáneas , Humanos , Melanoma/genética , MicroARNs/metabolismo , Neoplasias Cutáneas/genética , Apoptosis/genética , Genómica , Inestabilidad Genómica , Línea Celular Tumoral , Proliferación Celular/genética , Regulación Neoplásica de la Expresión GénicaRESUMEN
Oncogene-induced senescence (OIS) is an important process that suppresses tumor development, but the molecular mechanisms of OIS are still under investigation. It is known that BRAFV600E-mutated melanocytes can overcome OIS and develop melanoma, but the underlying mechanism is largely unknown. Using an established OIS model of primary melanocytes transduced with BRAFV600E, YAP activity was shown to be induced in OIS as well as in melanoma cells compared to that in normal epidermal melanocytes. This led to the assumption that YAP activation itself is not a factor involved in the disruption of OIS. However, its role and interaction partners potentially change. As Wnt molecules are known to be important in melanoma progression, these molecules were the focus of subsequent studies. Interestingly, activation of Wnt signaling using AMBMP resulted in a disruption of OIS in BRAFV600E-transduced melanocytes. Furthermore, depletion of Wnt6, Wnt10b or ß-catenin expression in melanoma cells resulted in the induction of senescence. Given that melanoma cells do not exhibit canonical Wnt/ß-catenin activity, alternative ß-catenin signaling pathways may disrupt OIS. Here, we discovered that ß-catenin is an interaction partner of YAP on DNA in melanoma cells. Furthermore, the ß-catenin-YAP interaction changed the gene expression pattern from senescence-stabilizing genes to tumor-supportive genes. This switch is caused by transcriptional coactivation via the LEF1/TEAD interaction. The target genes with binding sites for LEF1 and TEAD are involved in rRNA processing and are associated with poor prognosis in melanoma patients. This study revealed that an alternative YAP-Wnt signaling axis is an essential molecular mechanism leading to OIS disruption in melanocytes.
Asunto(s)
Melanoma , Humanos , Melanoma/patología , beta Catenina/metabolismo , Vía de Señalización Wnt/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Senescencia Celular/genética , OncogenesRESUMEN
In this work, we present a promising diagnostic tool for melanoma diagnosis. With the proposed terahertz biosensor, it was possible to selectively and sensitively detect the early growth response protein 2, a transcription factor with an increased activity in melanoma cells, from a complex sample of cellular proteins. Fundamentally, the sensor belongs to the frequency selective surface type metamaterials and consists of a two-dimensional array of asymmetrically, doubly split ring resonator unit cells. The single elements are slits in a metallic layer and are complemented by an undercut etch. This allows a selective functionalization of the active area of the sensor and increases the sensitivity towards the target analyte. Hereby, specific detection of a defined transcription factor is feasible.
Asunto(s)
Melanoma , Factores de Transcripción , Humanos , Melanoma/diagnóstico , Regulación de la Expresión Génica , Agitación PsicomotoraRESUMEN
Obesity is characterized by the expansion of the adipose tissue, usually accompanied by inflammation, with a prominent role of macrophages infiltrating the visceral adipose tissue (VAT). This chronic inflammation is a major driver of obesity-associated comorbidities. Four-and-a-half LIM-domain protein 2 (FHL2) is a multifunctional adaptor protein that is involved in the regulation of various biological functions and the maintenance of the homeostasis of different tissues. In this study, we aimed to gain new insights into the expression and functional role of FHL2 in VAT in diet-induced obesity. We found enhanced FHL2 expression in the VAT of mice with Western-type diet (WTD)-induced obesity and obese humans and identified macrophages as the cellular source of enhanced FHL2 expression in VAT. In mice with FHL2 deficiency (FHL2KO), WTD feeding resulted in reduced body weight gain paralleled by enhanced energy expenditure and uncoupling protein 1 (UCP1) expression, indicative of activated thermogenesis. In human VAT, FHL2 was inversely correlated with UCP1 expression. Furthermore, macrophage infiltration and the expression of the chemokine MCP-1, a known promotor of macrophage accumulation, was significantly reduced in WTD-fed FHL2KO mice compared with wild-type (wt) littermates. While FHL2 depletion did not affect the differentiation or lipid metabolism of adipocytes in vitro, FHL2 depletion in macrophages resulted in reduced expressions of MCP-1 and the neuropeptide Y (NPY). Furthermore, WTD-fed FHL2KO mice showed reduced NPY expression in VAT compared with wt littermates, and NPY expression was enhanced in VAT resident macrophages of obese individuals. Stimulation with recombinant NPY induced not only UCP1 expression and lipid accumulation but also MCP-1 expression in adipocytes. Collectively, these findings indicate that FHL2 is a positive regulator of NPY and MCP-1 expression in macrophages and herewith closely linked to the mechanism of obesity-associated lipid accumulation and inflammation in VAT. Thus, FHL2 appears as a potential novel target to interfere with the macrophage-adipocyte crosstalk in VAT for treating obesity and related metabolic disorders.
Asunto(s)
Grasa Intraabdominal , Neuropéptido Y , Animales , Humanos , Ratones , Tejido Adiposo/metabolismo , Dieta , Dieta Alta en Grasa , Inflamación/metabolismo , Grasa Intraabdominal/metabolismo , Proteínas con Homeodominio LIM/metabolismo , Lípidos , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Neuropéptido Y/metabolismo , Obesidad/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Activated hepatic stellate cells (HSCs) play a key role in hepatic fibrosis and, thus, build the "soil" for hepatocarcinogenesis. Furthermore, HSCs are known to promote the progression of hepatocellular carcinoma (HCC), but the molecular mechanisms are only incompletely understood. Recently, we newly described the expression of bone morphogenetic protein 13 (BMP13) by HSCs in fibrotic liver tissue. In addition, BMP13 has mostly been studied in the context of cartilage and bone repair, but not in liver disease or cancer. Thus, we aimed to analyze the expression and function of BMP13 in HCC. Expression analyses revealed high BMP13-expression in activated human HSCs, but not in human HCC-cell-lines. Furthermore, analysis of human HCC tissues showed a significant correlation between BMP13 and α-smooth muscle actin (α-SMA), and immunofluorescence staining confirmed the co-localization of BMP13 and α-SMA, indicating activated HSCs as the cellular source of BMP13 in HCC. Stimulation of HCC cells with recombinant BMP13 increased the expression of the inhibitors of differentiation 1 (ID1) and 2 (ID2), which are known targets of BMP-signaling and cell-cycle promotors. In line with this, BMP13-stimulation caused an induced SMAD 1/5/9 and extracellular signal-regulated kinase (ERK) phosphorylation, as well as reduced expression of cyclin-dependent kinase inhibitors 1A (CDKN1A) and 2A (CDKN2A). Furthermore, stimulation with recombinant BMP13 led to increased proliferation and colony size formation of HCC cells in clonogenicity assays. The protumorigenic effects of BMP13 on HCC cells were almost completely abrogated by the small molecule dorsomorphin 1 (DMH1), which selectively blocks the intracellular kinase domain of ALK2 and ALK3, indicating that BMP13 acts via these BMP type I receptors on HCC cells. In summary, this study newly identifies stroma-derived BMP13 as a potential new tumor promotor in HCC and indicates this secreted growth-factor as a possible novel therapeutic target in HCC.
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Línea Celular Tumoral , Células Estrelladas Hepáticas/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Proliferación CelularRESUMEN
The bulge of hair follicles harbors Nestin+ (neural crest like) stem cells, which exhibit the potential to generate various cell types including melanocytes. In this study, we aimed to determine the role of Sox9, an important regulator during neural crest development, in melanocytic differentiation of those adult Nestin+ cells. Immunohistochemical analysis after conditional Sox9 deletion in Nestin+ cells of adult mice revealed that Sox9 is crucial for melanocytic differentiation of these cells and that Sox9 acts as a fate determinant between melanocytic and glial fate. A deeper understanding of factors that regulate fate decision, proliferation and differentiation of these stem cells provides new aspects to melanoma research as melanoma cells share many similarities with neural crest cells. In summary, we here show the important role of Sox9 in melanocytic versus glial fate decision of Nestin+ stem cells in the skin of adult mice.
RESUMEN
Originally considered to act as a transcriptional co-factor, Pirin has recently been reported to play a role in tumorigenesis and the malignant progression of many tumors. Here, we have analyzed the diagnostic and prognostic value of Pirin expression in the early stages of melanoma, and its role in the biology of melanocytic cells. Pirin expression was analyzed in a total of 314 melanoma biopsies, correlating this feature with the patient's clinical course. Moreover, PIR downregulated primary melanocytes were analyzed by RNA sequencing, and the data obtained were validated in human melanoma cell lines overexpressing PIR by functional assays. The immunohistochemistry multivariate analysis revealed that early melanomas with stronger Pirin expression were more than twice as likely to develop metastases during the follow-up. Transcriptome analysis of PIR downregulated melanocytes showed a dampening of genes involved in the G1/S transition, cell proliferation, and cell migration. In addition, an in silico approach predicted that JARID1B as a potential transcriptional regulator that lies between PIR and its downstream modulated genes, which was corroborated by co-transfection experiments and functional analysis. Together, the data obtained indicated that Pirin could be a useful marker for the metastatic progression of melanoma and that it participates in the proliferation of melanoma cells by regulating the slow-cycling JARID1B gene.
Asunto(s)
Melanoma , Humanos , Pronóstico , Melanocitos , Biopsia , Factores de Transcripción , Proliferación Celular , Proteínas Nucleares , Proteínas Represoras , Histona Demetilasas con Dominio de JumonjiRESUMEN
Cold atmospheric plasma (CAP) describes a partially ionized gas carrying large amounts of reactive oxygen (ROS) and nitrogen species (RNS). Numerous studies reported strong antitumor activity of CAP, thus rendering it a promising approach for tumor therapy. Although several cellular mechanisms of its cytotoxicity were identified in recent years, the exact molecular effects and contributing signaling pathways are yet to be discovered. We discovered a strong activation of unfolded protein response (UPR) after CAP treatment with increased C/EBP homologous protein (CHOP) expression, which was mainly caused by protein misfolding and calcium loss in the endoplasmic reticulum. In addition, both ceramide level and ceramide metabolism were reduced after CAP treatment, which was then linked to the UPR activation. Pharmacological inhibition of ceramide metabolism resulted in sensitization of melanoma cells for CAP both in vitro and ex vivo. This study identified a novel mechanism of CAP-induced apoptosis in melanoma cells and thereby contributes to its potential application in tumor therapy.
RESUMEN
Melanoma inhibitory activity/cartilage-derived retinoicacid-sensitive protein (MIA/CD-RAP) is a protein expressed and secreted by chondrocytes and cartilaginous tissues. MIA/CD-RAP-deficient mice develop milder osteoarthritis than wildtype mice. In this study, we investigated MIA/CD-RAP downstream targets to explain this reduced disease development. As a possible mediator, we could detect matrix metalloproteinase 13 (MMP13), and the influence of MIA/CD-RAP on MMP13 regulation was analyzed in vitro using SW1353 chondrosarcoma cells and primary chondrocytes. The femoral head cartilage of WT and MIA/CD-RAP -/- mice were cultured ex vivo to further investigate MMP13 activity. Finally, osteoarthritis was surgically induced via DMM in C57BL/6 mice, and the animals were treated with an MIA/CD-RAP inhibitory peptide by subcutaneously implanted pellets. MMP13 was regulated by MIA/CD-RAP in SW1353 cells, and MIA/CD-RAP -/- murine chondrocytes showed less expression of MMP13. Further, IL-1ß-treated MIA/CD-RAP -/- chondrocytes displayed less MMP13 expression and activity. Additionally, MIA/CD-RAP-deficient ex vivo cultured cartilage explants showed less MMP13 activity as well as reduced cartilage degradation. The mice treated with the MIA/CD-RAP inhibitory peptide showed less osteoarthritis development. Our findings revealed MIA/CD-RAP as a new regulator of MMP13 and highlighted its role as a potential new target for osteoarthritis therapy.
Asunto(s)
Cartílago , Osteoartritis , Animales , Ratones , Cartílago/metabolismo , Condrocitos , Metaloproteinasa 13 de la Matriz/metabolismo , Ratones Endogámicos C57BL , Osteoartritis/tratamiento farmacológico , Osteoartritis/metabolismoRESUMEN
Cutaneous malignant melanoma is a highly proliferative and aggressive skin cancer with a steadily increasing incidence and a low long-term survival rate after metastatic progression. The protein MAGOH and its highly identical homologue MAGOHB are core components of the exon junction complex (EJC), which regulates splicing, stability and translation of mRNAs. The EJC, and especially MAGOH, has been shown to be involved in the development and progression of several cancers. In melanoma, the expression and function of both homologues remain essentially unexplored. This study identifies high MAGOH and MAGOHB protein expression in cutaneous melanoma cell lines and patient derived tissue samples. An siRNA-mediated knockdown of MAGOH significantly inhibits melanoma cell proliferation. The loss of MAGOH does not affect cell cycle progression, but induces apoptosis, an effect that is enhanced by a simultaneous knockdown of MAGOH and MAGOHB. MAGOH and MAGOHB do not influence the expression of the pro-apoptotic protein Bcl-XS or exon skipping. However, the knockdown of MAGOH and MAGOHB strongly decreases nonsense-mediated decay (NMD) activity, leading to an upregulation of the pro-apoptotic protein GADD45A. In conclusion, simultaneous inhibition of MAGOH and MAGOHB expression substantially affects cell survival, indicating both MAGOH homologues as promising new targets for the treatment of melanoma.
Asunto(s)
Melanoma , Neoplasias Cutáneas , Humanos , Melanoma/genética , Proteínas Nucleares/metabolismo , Neoplasias Cutáneas/genética , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis , Proteínas de Ciclo Celular/genéticaRESUMEN
Hepatic metastasis is the critical factor determining tumor-associated mortality in different types of cancer. This is particularly true for uveal melanoma (UM), which almost exclusively metastasizes to the liver. Hepatic stellate cells (HSCs) are the precursors of tumor-associated fibroblasts and support the growth of metastases. However, the underlying mechanisms are widely unknown. Fibroblast growth factor (FGF) signaling is dysregulated in many types of cancer. The aim of this study was to analyze the pro-tumorigenic effects of HSCs on UM cells and the role of FGFs in this crosstalk. Conditioned medium (CM) from activated human HSCs significantly induced proliferation together with enhanced ERK and JNK activation in UM cells. An in silico database analysis revealed that there are almost no mutations of FGF receptors (FGFR) in UM. However, a high FGFR expression was found to be associated with poor survival for UM patients. In vitro, the pro-tumorigenic effects of HSC-CM on UM cells were abrogated by a pharmacological inhibitor (BGJ398) of FGFR1/2/3. The expression analysis revealed that the majority of paracrine FGFs are expressed by HSCs, but not by UM cells, including FGF9. Furthermore, the immunofluorescence analysis indicated HSCs as a cellular source of FGF9 in hepatic metastases of UM patients. Treatment with recombinant FGF9 significantly enhanced the proliferation of UM cells, and this effect was efficiently blocked by the FGFR1/2/3 inhibitor BGJ398. Our study indicates that FGF9 released by HSCs promotes the tumorigenicity of UM cells, and thus suggests FGF9 as a promising therapeutic target in hepatic metastasis.
Asunto(s)
Neoplasias Hepáticas , Neoplasias de la Úvea , Proliferación Celular , Medios de Cultivo Condicionados/metabolismo , Medios de Cultivo Condicionados/farmacología , Factores de Crecimiento de Fibroblastos/metabolismo , Células Estrelladas Hepáticas/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Melanoma , Compuestos de Fenilurea , Pirimidinas , Neoplasias de la Úvea/metabolismoRESUMEN
AMPHIREGULIN (AREG) is a multifaceted molecule, which acts not only as an extracellular ligand for EGF receptor (EGFR), but also as an intracellular signaling molecule. It remains elusive, however, whether AREG has a tumor suppressive or oncogenic role in melanoma. Here, we found that several melanoma cell lines express AREG, but the expression does not correlate with that of EGFR. Recombinant AREG and the neutralizing antibody experiments showed that intracellular AREG plays an important role in melanoma, implying a divergent function of AREG in addition to the role as a ligand for EGFR. Further investigation of this mechanism revealed that particularly nuclear-localized AREG regulates IGF-1R, P21 (Cip1/Waf1), TP53 and JARID1B protein accumulation in the nucleus. Furthermore, manipulation of nuclear AREG levels has influence on heterochromatin condensation (HP1beta, SETDB1) and trimethylation of histones H3K9 and H3K4. As these molecules correspond to previously identified markers for slow-cycling drug resistant cells, we speculate that nuclear AREG predisposes cells to resistance to therapy. According to the hypothesis, we detected the accumulation of AREG in the nucleus of SK-Mel-28-VR, which was cultured under Vemurafenib (VR) selection pressure, and this correlates with JARID1B expression. Here, knockdown of AREG makes the previously resistant cells more sensitive to VR treatment, resulting in inhibited proliferation. Taken together, we suggest that nuclear AREG affects a slow-cycling phenotype and increases resistance to VR, raising a possibility that AREG might be a potential therapeutic target for resistance in melanoma.
Asunto(s)
Histonas , Melanoma , Humanos , Anfirregulina/genética , Ligandos , Vemurafenib , Histonas/genética , Heterocromatina , Receptores ErbB/genética , Melanoma/tratamiento farmacológico , Melanoma/genética , Fenotipo , Resistencia a Medicamentos , Anticuerpos NeutralizantesRESUMEN
In malignant melanoma, a highly aggressive form of skin cancer, many microRNAs are aberrantly expressed contributing to tumorigenesis and progression. Further, deregulation of microRNA processing enzymes, like the miRNA-binding protein Argonaute 2, significantly impacts microRNA function. This study characterizes a novel splice variant of Argonaut 2, AGO2-ex1/3. AGO2-ex1/3 is substantially expressed in different melanoma cell lines and patient-derived tissue samples. It is a mature mRNA, which is translated into an N-terminally truncated Argonaute 2 protein form. Molecular dynamics simulations show that the PAZ, MID, and PIWI domain largely retain their structure in AGO2-ex1/3 and that the truncation of the N-terminus leads to an increased interdomain flexibility. Expression of AGO2-ex1/3 provides a survival advantage for melanoma cells while the knockdown causes significantly reduced proliferation and increases apoptosis. RNA-sequencing revealed that in cells lacking AGO2-ex1/3 expression many miRNA target genes are deregulated, implicating a considerable role of AGO2-ex1/3 for miRNA function. This study inaugurates insights into an important role of a so far unknown splice variant of Argonaute 2 for the miRNA pathway as well as the mechanisms which drive growth and survival of melanoma cells. This knowledge provides the basis for potential new promising therapeutic targets focusing on small RNA-mediated gene regulation in melanoma.
Asunto(s)
Melanoma , MicroARNs , Neoplasias Cutáneas , Apoptosis/genética , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Humanos , Melanoma/genética , MicroARNs/genética , MicroARNs/metabolismo , Interferencia de ARN , Neoplasias Cutáneas/genéticaRESUMEN
The skin of adult mammals protects from radiation, physical and chemical insults. While melanocytes and melanocyte-producing stem cells contribute to proper skin function in healthy organisms, dysfunction of these cells can lead to the generation of malignant melanoma-the deadliest type of skin cancer. Addressing cells of the melanocyte lineage in vivo represents a prerequisite for the understanding of melanoma on cellular level and the development of preventive and treatment strategies. Here, the inducible Cre-loxP-system has emerged as a promising tool to specifically target, monitor, and modulate cells in adult mice. Re-analysis of existing sequencing data sets of melanocytic cells revealed that genes with a known function in neural cells, including neural stem cells (Aldh1L1 and Nestin), are also expressed in melanocytic cells. Therefore, in this study, we explored whether the promoter activity of Nestin and Aldh1L1 can serve to target cells of the melanocyte lineage using the inducible CreERT2 -loxP-system. Using an immunohistochemical approach and different time points of analysis, we were able to map the melanocytic fate of recombined stem cells in the adult hair follicle of Nestin-CreERT2 and Aldh1L1-CreERT2 transgenic mice. Thus, we here present two new mouse models and propose their use to study and putatively modulate adult melanocytic cells in vivo.
Asunto(s)
Melanoma , Neoplasias Cutáneas , Animales , Ratones , Integrasas/genética , Melanocitos/patología , Melanoma/patología , Ratones Transgénicos , Nestina/genética , Neoplasias Cutáneas/genéticaRESUMEN
Due to the occurrence of THz-excited vibrational modes in biomacromolecules, the THz frequency range has been identified as particularly suitable for developing and applying new bioanalytical methods. We present a scalable THz metamaterial-based biosensor being utilized for the multifrequency investigation of single- and double-stranded DNA (ssDNA and dsDNA) samples. It is demonstrated that the metamaterial resonance frequency shift by the DNA's presence depends on frequency. Our experiments with the scalable THz biosensors demonstrate a major change in the degree of the power function for dsDNA by 1.53 ± 0.06 and, in comparison, 0.34 ± 0.11 for ssDNA as a function of metamaterial resonance frequency. Thus, there is a significant advantage for dsDNA detection that can be used for increased sensitivity of biomolecular detection at higher frequencies. This work represents a first step for application-specific biosensors with potential advantages in sensitivity, specificity, and robustness.