RESUMEN
Extracellular vesicles secreted from uveal melanoma (UM) cells are involved in the establishment of the premetastatic niche and display transforming potential for the formation of metastases, preferentially in the liver. In this study, we cultivated human primary UM cells and uveal melanoma-associated fibroblasts in vitro to be transduced by infection with a retrovirus containing the suicide gene-fused yeast cytosine deaminase::uracil phospho-ribosyl transferase (yCD::UPRT). A homogenous population of yCD::UPRT-UM cells with the integrated provirus expressed the gene, and we found it to continuously secrete small extracellular vesicles (sEVs) possessing mRNA of the suicide gene. The yCD::UPRT-UM-sEVs were internalized by tumor cells to the intracellular conversion of the prodrug 5-fluorocytosine (5-FC) to the cytotoxic drug 5-fluorouracil (5-FU). The host range of the yCD::UPRT-UM-sEVs was not limited to UMs only. The yCD::UPRT-UM-sEVs inhibited the growth of the human cutaneous melanoma cell line A375 and uveal melanoma cell line MP38, as well as other primary UMs, to various extents in vitro. The yCD::UPRT-UM-sEVs hold the therapeutic and prophylactic potential to become a therapeutic drug for UM. However, the use of yCD::UPRT-UM-sEVs must first be tested in animal preclinical studies.
Asunto(s)
Vesículas Extracelulares , Melanoma , Neoplasias Cutáneas , Neoplasias de la Úvea , Animales , Humanos , Melanoma/genética , Melanoma/terapia , Neoplasias de la Úvea/genética , Neoplasias de la Úvea/terapia , Flucitosina/farmacología , FluorouraciloRESUMEN
Uveal melanoma (UM) is an ocular tumor with a dismal prognosis. Despite the availability of precise molecular and cytogenetic techniques, clinicopathologic features with limited accuracy are widely used to predict metastatic potential. In 51 UM tissues, we assessed a correlation between the expression of nine proteins evaluated by immunohistochemistry (IHC) (Melan-A, S100, HMB45, Cyclin D1, Ki-67, p53, KIT, BCL2, and AIFM1) and the presence of UM-specific chromosomal rearrangements measured by multiplex ligation-dependent probe amplification (MLPA), to find IHC markers with increased prognostic information. Furthermore, mRNA expression and DNA methylation values were extracted from the whole-genome data, achieved by analyzing 22 fresh frozen UM tissues. KIT positivity was associated with monosomy 3, increasing the risk of poor prognosis more than 17-fold (95% CI 1.53-198.69, p = 0.021). A strong negative correlation was identified between mRNA expression and DNA methylation values for 12 of 20 analyzed positions, five located in regulatory regions of the KIT gene (r = -0.658, p = 0.001; r = -0.662, p = 0.001; r = -0.816; p < 0.001; r = -0.689, p = 0.001; r = -0.809, p < 0.001, respectively). DNA methylation ß values were also inversely associated with KIT protein expression (p = 0.001; p = 0.001; p = 0.015; p = 0.025; p = 0.002). Our findings, showing epigenetic deregulation of KIT expression, may contribute to understanding the past failure to therapeutically target KIT in UM.
Asunto(s)
Biomarcadores de Tumor/genética , Metilación de ADN , Regulación Neoplásica de la Expresión Génica , Melanoma/genética , Proteínas Proto-Oncogénicas c-kit/genética , Neoplasias de la Úvea/genética , Adulto , Anciano , Anciano de 80 o más Años , Terapia Combinada , Femenino , Estudios de Seguimiento , Humanos , Masculino , Melanoma/patología , Melanoma/terapia , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa Multiplex , Pronóstico , Neoplasias de la Úvea/patología , Neoplasias de la Úvea/terapiaRESUMEN
Despite outstanding advances in diagnosis and the treatment of primary uveal melanoma (UM), nearly 50% of UM patients develop metastases via hematogenous dissemination, driven by the epithelial-mesenchymal transition (EMT). Despite the failure in UM to date, a liquid biopsy may offer a feasible non-invasive approach for monitoring metastatic disease progression and addressing protracted dormancy. To detect circulating tumor cells (CTCs) in UM patients, we evaluated the mRNA expression of EMT-associated transcription factors in CD45-depleted blood fraction, using qRT-PCR. ddPCR was employed to assess UM-specific GNA11, GNAQ, PLCß4, and CYSLTR2 mutations in plasma DNA. Moreover, microarray analysis was performed on total RNA isolated from tumor tissues to estimate the prognostic value of EMT-associated gene expression. In total, 42 primary UM and 11 metastatic patients were enrolled. All CD45-depleted samples were negative for CTC when compared to the peripheral blood fraction of 60 healthy controls. Tumor-specific mutations were detected in the plasma of 21.4% patients, merely, in 9.4% of primary UM, while 54.5% in metastatic patients. Unsupervised hierarchical clustering of differentially expressed EMT genes showed significant differences between monosomy 3 and disomy 3 tumors. Newly identified genes can serve as non-invasive prognostic biomarkers that can support therapeutic decisions.
Asunto(s)
Biomarcadores de Tumor/genética , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Melanoma/genética , Células Neoplásicas Circulantes/patología , Neoplasias de la Úvea/genética , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Deleción Cromosómica , Cromosomas Humanos Par 3/genética , ADN de Neoplasias/análisis , ADN de Neoplasias/genética , Femenino , Estudios de Seguimiento , Humanos , Biopsia Líquida , Masculino , Melanoma/secundario , Melanoma/terapia , Persona de Mediana Edad , Células Neoplásicas Circulantes/metabolismo , Pronóstico , Neoplasias de la Úvea/secundario , Neoplasias de la Úvea/terapiaRESUMEN
Metastatic disease in a cancer patient still remains a therapeutic challenge. Metastatic process involves many steps, during which malignant cells succeed to activate cellular pathways promoting survival in hostile environment, engraftment and growth at the distant site from the primary tumor. Melanoma is known for its high propensity to produce metastases even at the early stages of the disease. Here we summarize the most important molecular mechanisms which were associated with the melanoma metastasis. Then, we specifically focus on the signaling pathway mediated by hepatocyte growth factor (HGF) and its receptor c-Met, which play an important role during physiological processes and were been associated with tumorigenesis. We also focus on the effect of the small molecule inhibitors of the tyrosine kinase domain of the c-Met receptor and its effects on properties of melanoma cell. We summarize recent studies, which involved inhibition of the HGF/c-Met signaling in order to decrease melanoma growth and metastatic capacity.
Asunto(s)
Inhibidores Enzimáticos/uso terapéutico , Factor de Crecimiento de Hepatocito/metabolismo , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Metástasis de la Neoplasia/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-met/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Línea Celular Tumoral , Factor de Crecimiento de Hepatocito/genética , Humanos , Melanoma/genética , Metástasis de la Neoplasia/genética , Proteínas Proto-Oncogénicas c-met/genética , Transducción de Señal/genéticaRESUMEN
BACKGROUND: Small molecule inhibitor of tyrosine kinase activity, compound SU11274, was reported to have antitumorigenic and antimetastatic effect in melanoma. In this study, we evaluated, whether similar effect could be achieved also in other melanoma cells including highly tumorigenic and hypermetastatic variant. METHODS: The effect of SU11274 was evaluated in adherent and non-adherent melanosphere cultures of human melanoma cells M14, M4Beu, A375 and EGFP-A375/Rel3. Tumorigenicity of SU11274-treated cells was tested by limiting dilution assay in xenograft model in vivo. RESULTS: Here we show that SU11274 enriched for melanoma-initiating cells in vivo. SU11274 substantially decreased number of cells in adherent and spheroid cultures, but increased their tumorigenic potential as determined by higher frequency of tumor-initiating cells in vivo. SU11274 treatment was not associated with any significant alteration in the expression of stem cell markers, but the inhibitor stimulated higher level of pluripotent markers. SU11274-treated melanoma cells exhibited higher ATP content and lactate release indicative of increased glycolysis. Our data suggest that the SU11274 altered bioenergetic state of the cells. Indeed, pharmacological intervention with a glycolytic inhibitor dichloroacetate significantly reduced SU11274-promoted increase in melanoma-initiating cells and decreased their tumorigenicity. CONCLUSIONS: Our data suggest critical role of glycolysis regulation in melanoma-initiating cells. Moreover, these data unravel substantial plasticity of melanoma cells and their adoptive mechanisms, which result in ambivalent response to therapeutic targeting.
Asunto(s)
Metabolismo Energético/efectos de los fármacos , Indoles/farmacología , Melanoma/metabolismo , Melanoma/patología , Células Madre Neoplásicas/patología , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Sulfonamidas/farmacología , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Humanos , Melanoma/tratamiento farmacológico , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Fosforilación/efectos de los fármacos , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Cells of the tumor microenvironment are recognized as important determinants of the tumor biology. The adjacent non-malignant cells can regulate drug responses of the cancer cells by secreted paracrine factors and direct interactions with tumor cells. RESULTS: Human mesenchymal stromal cells (MSC) actively contribute to tumor microenvironment. Here we focused on their response to chemotherapy as during the treatment these cells become affected. We have shown that the secretory phenotype and behavior of mesenchymal stromal cells influenced by cisplatin differs from the naïve MSC. MSC were more resistant to the concentrations of cisplatin, which was cytotoxic for tumor cells. They did not undergo apoptosis, but a part of MSC population underwent senescence. However, MSC pretreatment with cisplatin led to changes in phosphorylation profiles of many kinases and also increased secretion of IL-6 and IL-8 cytokines. These changes in cytokine and phosphorylation profile of MSC led to increased chemoresistance and stemness of breast cancer cells. CONCLUSION: Taken together here we suggest that the exposure of the chemoresistant cells in the tumor microenvironment leads to substantial alterations and might lead to promotion of acquired microenvironment-mediated chemoresistance and stemness.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Cisplatino/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Microambiente Tumoral/efectos de los fármacos , Apoptosis/efectos de los fármacos , Mama/efectos de los fármacos , Mama/inmunología , Neoplasias de la Mama/inmunología , Línea Celular Tumoral , Senescencia Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Femenino , Humanos , Interleucina-6/inmunología , Interleucina-8/inmunología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/inmunologíaRESUMEN
Decitabine (DAC), a DNA methyltransferase (DNMT) inhibitor, is tested in combination with conventional anticancer drugs as a treatment option for various solid tumors. Although epigenome modulation provides a promising avenue in treating resistant cancer types, more studies are required to evaluate its safety and ability to normalize the aberrant transcriptional profiles. As deoxycytidine kinase (DCK)-mediated phosphorylation is a rate-limiting step in DAC metabolic activation, we hypothesized that its intracellular overexpression could potentiate DAC's effect on cell methylome and thus increase its therapeutic efficacy. Therefore, two breast cancer cell lines, JIMT-1 and T-47D, differing in their molecular characteristics, were transfected with a DCK expression vector and exposed to low-dose DAC (approximately IC20). Although transfection resulted in a significant DCK expression increase, further enhanced by DAC exposure, no transfection-induced changes were found at the global DNA methylation level or in cell viability. In parallel, an integrative approach was applied to decipher DAC-induced, methylation-mediated, transcriptomic reprogramming. Besides large-scale hypomethylation, accompanied by up-regulation of gene expression across the entire genome, DAC also induced hypermethylation and down-regulation of numerous genes in both cell lines. Interestingly, TET1 and TET2 expression halved in JIMT-1 cells after DAC exposure, while DNMTs' changes were not significant. The protein digestion and absorption pathway, containing numerous collagen and solute carrier genes, ranking second among membrane transport proteins, was the top enriched pathway in both cell lines when hypomethylated and up-regulated genes were considered. Moreover, the calcium signaling pathway, playing a significant role in drug resistance, was among the top enriched in JIMT-1 cells. Although low-dose DAC demonstrated its ability to normalize the expression of tumor suppressors, several oncogenes were also up-regulated, a finding, that supports previously raised concerns regarding its broad reprogramming potential. Importantly, our research provides evidence about the involvement of active demethylation in DAC-mediated transcriptional reprogramming.
RESUMEN
RNF43 is an E3 ubiquitin ligase and known negative regulator of WNT/ß-catenin signaling. We demonstrate that RNF43 is also a regulator of noncanonical WNT5A-induced signaling in human cells. Analysis of the RNF43 interactome using BioID and immunoprecipitation showed that RNF43 can interact with the core receptor complex components dedicated to the noncanonical Wnt pathway such as ROR1, ROR2, VANGL1, and VANGL2. RNF43 triggers VANGL2 ubiquitination and proteasomal degradation and clathrin-dependent internalization of ROR1 receptor and inhibits ROR2 activation. These activities of RNF43 are physiologically relevant and block pro-metastatic WNT5A signaling in melanoma. RNF43 inhibits responses to WNT5A, which results in the suppression of invasive properties of melanoma cells. Furthermore, RNF43 prevented WNT5A-assisted development of resistance to BRAF V600E and MEK inhibitors. Next, RNF43 acted as melanoma suppressor and improved response to targeted therapies in vivo. In line with these findings, RNF43 expression decreases during melanoma progression and RNF43-low patients have a worse prognosis. We conclude that RNF43 is a newly discovered negative regulator of WNT5A-mediated biological responses that desensitizes cells to WNT5A.
Asunto(s)
Melanoma , Transducción de Señal , Ubiquitina-Proteína Ligasas/genética , Proteína Wnt-5a/genética , Animales , Masculino , Melanoma/genética , Melanoma/patología , Melanoma/prevención & control , Ratones , Ratones Endogámicos NOD , Invasividad Neoplásica/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteína Wnt-5a/metabolismoRESUMEN
Mesenchymal stromal cells (MSC) exert either tumor-stimulatory or tumor-inhibitory effect. The outcome of the tumor-MSC interaction is dictated by the tumor-specific activating signals. We analyzed the alterations in MSC phenotype in response to stimulation by tumor-secreted paracrine factors. Paracrine factors from human melanoma A375 and glioblastoma 8MGBA cells were used for prolonged culture of MSC to produce derived cells designated DIFF(A)-MSC or DIFF(G)-MSC, respectively. Derived cells were analyzed for the specific surface markers, the expression pattern of MSC markers and fibroblast-specific proteins. Changes in the cell phenotype were evaluated using scratch wound assay and tube formation in vitro; and xenotransplant growth in vivo. Our data show induced expression of vascular endothelial growth factor 2, CD146, fibroblast-specific protein, vimentin and endosialin in DIFF(A)-MSC cells. This indicates their differentiation towards the cells with features of tumor-associated fibroblasts upon stimulation with melanoma-secreted cytokines. Paracrine stimulation in DIFF(G)-MSC led to up-regulation of the genes involved in the MSC differentiation. MSC-specific surface marker characteristics were preserved in derived DIFF(A)-MSC and DIFF(G)-MSC cells. However, we observed increased proportion of CD146 and GD2 (neural ganglioside) positive cells and decreased expression of marker NG2 in the MSC exposed to tumor-conditioned medium. Melanoma-CM increased MSC migration, glioblastoma-CM compromised angiogenic capacity of MSC in vitro and the protumorigenic effect in vivo. Our data directly compare the pleiotropic effects mediated by the malignant cells on the MSC. Secreted paracrine factors from melanoma or glioblastoma differently changed molecular traits in MSC, which explains the dual role of MSC in tumor growth.