ABSTRACT
Diagnostic imaging techniques provide a new aspect of the ante-mortem and post-mortem diagnostics in fish medicine. Ultrasonography, computed tomography (CT) and magnetic resonance imaging (MRI) can provide more information about the internal organs and pathognomic lesions. The authors used diagnostic imaging techniques to evaluate and describe the neoplastic malformation in a 3-year-old female rainbow trout (Oncorhynchus mykiss). The fish was examined with Siemens Somatom Definition AS + CT scanner and Siemens Biograph mMR scanner. The animal was lethargic and showed anorectic signs and muscular dystrophy. During the post-mortem investigation, histopathology and immunohistochemistry were also performed allowing us to identify the neoplasms. The results showed a large soft tissue mass in the first mid-intestine segment, which proved to be an adenocarcinoma. This subsequently led to digestion problems and absorption disorders. Immunohistochemically, neoplastic cells of carcinoma revealed E-cadherin and pancytokeratin positivity. This is the first study to report the use of MRI and CT for studying gastrointestinal adenocarcinoma in rainbow trout.
Subject(s)
Adenocarcinoma , Fish Diseases , Magnetic Resonance Imaging , Oncorhynchus mykiss , Animals , Adenocarcinoma/veterinary , Adenocarcinoma/diagnostic imaging , Adenocarcinoma/pathology , Female , Fish Diseases/diagnostic imaging , Fish Diseases/pathology , Magnetic Resonance Imaging/veterinary , Tomography, X-Ray Computed/veterinary , Ultrasonography/veterinaryABSTRACT
The aim of this study was to apply a state-of-the-art quantitative lipidomic profiling platform to uncover lipid alterations predictive of melanoma progression. Our study included 151 melanoma patients; of these, 83 were without metastasis and 68 with metastases. Plasma samples were analyzed using a targeted Lipidyzer™ platform, covering 13 lipid classes and over 1100 lipid species. Following quality control filters, 802 lipid species were included in the subsequent analyses. Total plasma lipid contents were significantly reduced in patients with metastasis. Specifically, levels of two out of the thirteen lipid classes (free fatty acids (FFAs) and lactosylceramides (LCERs)) were significantly decreased in patients with metastasis. Three lipids (CE(12:0), FFA(24:1), and TAG47:2-FA16:1) were identified as more effective predictors of melanoma metastasis than the well-known markers LDH and S100B. Furthermore, the predictive value substantially improved upon combining the lipid markers. We observed an increase in the cumulative levels of five lysophosphatidylcholines (LPC(16:0); LPC(18:0); LPC(18:1); LPC(18:2); LPC(20:4)), each individually associated with an elevated risk of lymph node metastasis but not cutaneous or distant metastasis. Additionally, seventeen lipid molecules were linked to patient survival, four of which (CE(12:0), CE(14:0), CE(15:0), SM(14:0)) overlapped with the lipid panel predicting metastasis. This study represents the first comprehensive investigation of the plasma lipidome of melanoma patients to date. Our findings suggest that plasma lipid profiles may serve as important biomarkers for predicting clinical outcomes of melanoma patients, including the presence of metastasis, and may also serve as indicators of patient survival.
Subject(s)
Lipidomics , Lipids , Melanoma , Humans , Melanoma/blood , Melanoma/pathology , Male , Female , Middle Aged , Lipids/blood , Lipidomics/methods , Aged , Biomarkers, Tumor/blood , Adult , Neoplasm Metastasis , Lymphatic Metastasis , Skin Neoplasms/blood , Skin Neoplasms/pathologyABSTRACT
Recently, liquid biopsy, as a promising approach was introduced for the analysis of different tumor-derived circulating markers including tumor DNA and cell free DNA (ct/cfDNA). Identification of mutations in cfDNA may allow the early detection of tumors, as well as predicting and monitoring treatment responses in a minimally invasive way. In the present study, we used commercially available gene panels to verify the mutation overlap between liquid biopsy and abnormalities detected in colorectal tumor tissue. The two panels (Archer®VariantPlex®Solid Tumor and LIQUIDPlexTM ctDNA) overlap in 23 genes, which enables a comprehensive view of tumor-plasma mutational status by next generation sequencing. We successfully analyzed 16 plasma and 16 tumor samples. We found that 87% of tumor tissues contained 44 mutations in 12 genes and 43.8% of cfDNA harbored 13 mutations in 5 genes. To verify whether the mutation pattern of the tumor DNA could be consistently detected in plasma cfDNA, we compared the alterations between cfDNA and matched tissue DNA in nine patients. Six of the 9 tumor tissues harbored mutations in TP53, KRAS or MET genes, those were not detectable by the ctDNA kit, even eventhough the exons of these genes overlap in both panels. Comparing the mutational patterns of the matched samples, we found that only one cfDNA had the same mutations (KRAS, SMAD4 and TP53) in the paired tissue. The results of the comparison between tumor tissue DNA and matched plasma cfDNA underline the importance of studying the paired solid tumor and plasma samples together.
Subject(s)
Cell-Free Nucleic Acids , Circulating Tumor DNA , Colorectal Neoplasms , Humans , Circulating Tumor DNA/genetics , Proto-Oncogene Proteins p21(ras)/genetics , Mutation , Liquid Biopsy , Colorectal Neoplasms/genetics , High-Throughput Nucleotide Sequencing/methods , Biomarkers, Tumor/geneticsABSTRACT
Cytokines and chemokines (chemotactic cytokines) are soluble extracellular proteins that bind to specific receptors and play an integral role in the cell-to-cell signaling network. In addition, they can promote the homing of cancer cells into different organs. We investigated the potential relationship between human hepatic sinusoidal endothelial cells (HHSECs) and several melanoma cell lines for the expression of chemokine and cytokine ligands and receptor expression during the invasion of melanoma cells. In order to identify differences in gene expression related to invasion, we selected invasive and non-invasive subpopulations of cells after co-culturing with HHSECs and identified the gene expression patterns of 88 chemokine/cytokine receptors in all cell lines. Cell lines with stable invasiveness and cell lines with increased invasiveness displayed distinct profiles of receptor genes. Cell lines with increased invasive capacity after culturing with conditioned medium showed a set of receptor genes (CXCR1, IL1RL1, IL1RN, IL3RA, IL8RA, IL11RA, IL15RA, IL17RC, and IL17RD) with significantly different expressions. It is very important to emphasize that we detected significantly higher IL11RA gene expression in primary melanoma tissues with liver metastasis as well, compared to those without metastasis. In addition, we assessed protein expression in endothelial cells before and after co-culturing them with melanoma cell lines by applying chemokine and cytokine proteome arrays. This analysis revealed 15 differentially expressed proteins (including CD31, VCAM-1, ANGPT2, CXCL8, and CCL20) in the hepatic endothelial cells after co-culture with melanoma cells. Our results clearly indicate the interaction between liver endothelial and melanoma cells. Furthermore, we assume that overexpression of the IL11RA gene may play a key role in organ-specific metastasis of primary melanoma cells to the liver.
Subject(s)
Liver Neoplasms , Melanoma , Humans , Cytokines/genetics , Receptors, Chemokine , Endothelial Cells/metabolism , Melanoma/metabolism , Chemokines/genetics , Liver Neoplasms/genetics , Gene ExpressionABSTRACT
Cytokine and chemokine receptors can promote tumor progression, invasion, and metastasis development by inducing different intracellular signaling pathways. The aim of this study was to determine the cytokine and chemokine receptor gene expression patterns in human melanoma cell lines. We found a large set of cytokine and chemokine receptor genes that were significantly differentially expressed between melanoma cell lines that originated from different subtypes of primary melanomas as well as cell lines that originated from melanoma metastases. The relative expressions of two receptor genes (CCR2 and TNFRSF11B) were positively correlated with the invasive potential of the cell lines, whereas a negative correlation was observed for the TNFRSF14 gene expression. We also found a small set of receptor genes that exhibited a significantly decreased expression in association with a BRAFV600E mutation. Based on our results, we assume that the analyzed cytokine and chemokine receptor collection may provide potential to distinguish the different subtypes of melanomas, helping us to understand the biological behavior of BRAFV600E-mutated melanoma cells.
Subject(s)
Melanoma , Proto-Oncogene Proteins B-raf , Cell Line, Tumor , Cytokines/genetics , Humans , Melanoma/metabolism , Mutation , Proto-Oncogene Proteins B-raf/genetics , Receptors, Chemokine/genetics , Skin Neoplasms , Melanoma, Cutaneous MalignantABSTRACT
Following a case of mandibular fracture in a giraffe (Giraffa camelopardalis) at the Budapest Zoo, a survey was conducted across European zoological institutions with the support of the European Association of Zoos and Aquariums Giraffe Ex situ Programme (EAZA Giraffe EEP) to help identify the incidence, potential causes, and management of such injuries. Two hundred sixteen zoos keeping giraffes were invited to participate in the survey. Eighty-six responses were received, 14 of which (16.3%) reported jaw fractures with various causes in their giraffes. Of these responses, hay racks, where the muzzle of the giraffe could fit between the grid of feeding contraptions, were associated with mandibular fractures in seven cases (50.0%). Most giraffes were managed surgically (10/14; 71.4%), two were managed medically (14.3%), one was euthanized because of the severity of the injury (7.1%), and one was only diagnosed with a mandibular fracture postmortem. One giraffe died owing to postanesthetic complications; all other individuals had a full recovery. Most mandible fractures occurred in relatively young giraffes (between 5 wk and 8 yr with an average of 3.4 yr). Based on these findings, facilities with giraffe should evaluate their hay feeders for mandible entrapment risk, especially if they house young individuals.
Subject(s)
Giraffes , Mandibular Fractures , Animals , Giraffes/physiology , Mandibular Fractures/veterinaryABSTRACT
Tumor cell invasion is one of the key processes during cancer progression, leading to life-threatening metastatic lesions in melanoma. As methylation of cancer-related genes plays a fundamental role during tumorigenesis and may lead to cellular plasticity which promotes invasion, our aim was to identify novel epigenetic markers on selected invasive melanoma cells. Using Illumina BeadChip assays and Affymetrix Human Gene 1.0 microarrays, we explored the DNA methylation landscape of selected invasive melanoma cells and examined the impact of DNA methylation on gene expression patterns. Our data revealed predominantly hypermethylated genes in the invasive cells affecting the neural crest differentiation pathway and regulation of the actin cytoskeleton. Integrative analysis of the methylation and gene expression profiles resulted in a cohort of hypermethylated genes (IL12RB2, LYPD6B, CHL1, SLC9A3, BAALC, FAM213A, SORCS1, GPR158, FBN1 and ADORA2B) with decreased expression. On the other hand, hypermethylation in the gene body of the EGFR and RBP4 genes was positively correlated with overexpression of the genes. We identified several methylation changes that can have role during melanoma progression, including hypermethylation of the promoter regions of the ARHGAP22 and NAV2 genes that are commonly altered in locally invasive primary melanomas as well as during metastasis. Interestingly, the down-regulation of the methylcytosine dioxygenase TET2 gene, which regulates DNA methylation, was associated with hypermethylated promoter region of the gene. This can probably lead to the observed global hypermethylation pattern of invasive cells and might be one of the key changes during the development of malignant melanoma cells.
Subject(s)
DNA Methylation , Melanoma/genetics , Melanoma/secondary , Skin Neoplasms/genetics , Skin Neoplasms/pathology , Actin Cytoskeleton/genetics , Actin Cytoskeleton/metabolism , Cell Differentiation/genetics , Cell Line, Tumor , DNA Helicases/genetics , DNA-Binding Proteins/genetics , Dioxygenases , Epigenesis, Genetic , GTPase-Activating Proteins/genetics , Gene Expression/genetics , Gene Expression Profiling , Humans , Neoplasm Invasiveness/genetics , Neoplasm Metastasis/genetics , Phenotype , Promoter Regions, Genetic , Proto-Oncogene Proteins/geneticsABSTRACT
It was shown that osteopontin (OPN), a glycophosphoprotein, plays divergent roles in cancer progression. In addition to multiple intra- and extracellular functions, it facilitates migration of tumour cells, has crucial role in cell adhesion and is associated with increased metastasis formation. In previous studies, we performed global gene expression profiling on a series of primary melanoma samples and found that OPN was significantly overexpressed in ulcerated melanomas. The major purpose of this study was to define OPN expression in primary melanomas with differing biological behaviours. OPN mRNA expression was analysed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in primary melanoma tissues. Immunohistochemistry was performed using a tissue microarray. Cox regression tests were used for survival analysis. Greater than 50 % of the tissues exhibited high protein expression that was significantly associated with tumour thickness and metastasis. OPN mRNA expression was significantly increased in thicker melanomas and lesions with an ulcerated surface. Increased expression was primarily detected in advanced-stage tumours. A multivariate Cox regression analysis revealed that high OPN expression, tumour thickness and metastasis were significantly associated with reduced relapse-free survival. In summary, high OPN mRNA and protein expression were associated with a less favourable clinical outcome of primary melanoma patients. We determined that OPN is a significant predictive factor for the survival of primary melanoma patients. Based on our and others data, the high expression of OPN may have a crucial stimulatory role in tumour progression and metastasis formation, which, thus, have been proposed as potential targets for cancer diagnosis and therapy.
Subject(s)
Biomarkers, Tumor/genetics , Gene Expression Regulation, Neoplastic , Melanoma/genetics , Osteopontin/genetics , Adult , Biomarkers, Tumor/metabolism , Disease Progression , Female , Follow-Up Studies , Gene Expression Profiling , Humans , Immunoenzyme Techniques , Male , Melanoma/metabolism , Melanoma/mortality , Melanoma/pathology , Middle Aged , Neoplasm Metastasis , Neoplasm Staging , Osteopontin/metabolism , Prognosis , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Survival Rate , Young AdultABSTRACT
Osteopontin (OPN) is a secreted integrin-binding protein that plays a role in inflammation, cellular viability, cell adhesion and migration, cancer development, and diabetes through different mechanisms. The splice variants of OPN can play essential roles in cancer development, progression, and metastasis formation; however, limited data are available about the role of OPN isoforms in human malignant melanoma. Our goal was to define the gene expression patterns of five OPN variants (OPN4, OPN5, OPNa, OPNb, and OPNc), integrin, and CD44 receptor genes in primary and metastatic melanoma-originated cell lines (n = 19), and to explore the association of the expression patterns with clinicopathological parameters. We evaluated the invasive property of the cell lines and investigated the potential association between the invasion and gene expression of OPN isoforms. We found a significant rise in the expression of OPNc in the invasive cell lines compared to the noninvasive cells and detected significantly higher expression of the OPN splice variants in melanoma cell lines originating from more advanced stages tumors than cell lines originating from early-stage melanomas. The correlation analysis revealed that all five OPN variants positively correlated with ITGB3 and ITGA9, whereas OPN5 positively correlated with ITGB1, ITGAV, ITGA6, and CD44. OPN can activate extracellular signal-regulated kinase signaling through binding to α9ß1 integrin, promoting melanoma tumor cell migration. It is possible that such associations between OPN splice variants and integrin receptors may play a role in melanoma progression. In conclusion, our findings suggest that high expression of OPNc correlates with the invasive behavior of melanoma cells.
Subject(s)
Melanoma , Humans , Osteopontin/genetics , Osteopontin/metabolism , Cell Line, Tumor , Protein Isoforms/genetics , Integrins , Cell MovementABSTRACT
We describe clinical cases caused by Microsporum gypseum in two subadult male California sea lions (Zalophus californianus). Dermatomycosis is uncommonly reported in pinnipeds, including this species. In these cases, skin lesions were multifocal to coalescing, involved all flippers, and were most pronounced on the ventral surfaces of flippers. They were well-demarcated, depigmented, and covered with crusts. The definitive diagnosis was obtained through microscopic examination and fungal culture of skin scrapings. Oral terbinafine and topical enilconazole were used as treatments for 65 days, and complete recovery was subsequently achieved. California sea lion, dermatomycosis, Microsporum gypseum, terbinafine, enilconazole
Subject(s)
Dermatomycoses/veterinary , Sea Lions , Animals , Antifungal Agents/administration & dosage , Antifungal Agents/therapeutic use , Dermatomycoses/drug therapy , Extremities/pathology , Imidazoles/administration & dosage , Imidazoles/therapeutic use , Male , Naphthalenes/administration & dosage , Naphthalenes/therapeutic use , Skin/pathology , TerbinafineABSTRACT
The article deals with the primary aspects of how to maintain healthy integument in zoo mammals and in particular why husbandry-related health problems can occur in general in a zoologic setting. While working with these species we are often faced with diagnostic challenges, which may include a general approach (often requiring anesthesia or medical training), lack of "normal" references, and difficulties, especially if the cutaneous signs are not a primary ailment, but a manifestation of a generalized disease (eg, immune-suppression). The different etiologies of skin problems are discussed with clinical examples.
Subject(s)
Animals, Zoo , Mammals , AnimalsABSTRACT
The early detection of melanoma relapse can improve patient survival; thus, there is a great need for easily accessible biomarkers that facilitate the diagnosis of metastatic disease. We investigated the diagnostic effect of blood biomarkers such as lactate dehydrogenase (LDH), S100B, and osteopontin in the detection of metastases. Clinical data and peripheral blood samples of 206 melanoma patients were collected (no metastasis, N = 120; metastasis, N = 86). The discriminative power of blood biomarkers, patient demographics, and clinicopathological parameters of primary melanomas were evaluated using univariate and multivariate logistic regression models and receiver operating characteristic (ROC) analysis to determine the area under the curve (AUC). Plasma osteopontin levels showed a significant and independent effect on the probability of metastasis, similar to serum S100B levels. In addition, the location of the primary tumor on the lower extremities and the American Joint Committee on Cancer (AJCC) categories pT2b-3a, pT3b-4a, and pT4b were associated with the diagnosis of metastasis. Importantly, the combination of the three blood biomarkers and primary tumor localization and AJCC pT category yielded excellent discrimination (AUC: training set: 0.803; validation set: 0.822). In conclusion, plasma osteopontin can be classified as a melanoma biomarker; moreover, by combining clinicopathological prognostic variables, the diagnostic effect of blood biomarkers in the detection of metastatic melanoma can be improved.
ABSTRACT
It is well demonstrated that CCND1 amplification is a frequent event in the acral subtype of cutaneous malignant melanoma; however, its role in the other subtypes of the disease is still controversial. The objectives of this study were to evaluate genetic and expression alterations of CCND1 with a focus on primary cutaneous melanomas, to define BRAF and NRAS mutation status, and correlate the data with clinical-pathological parameters. CCND1 amplification was associated with ulceration and the localization of the metastasis. After correction for the mutation state of BRAF and NRAS genes, CCND1 amplification in samples without such mutations was associated with ulceration and sun exposure. The cyclin D1 (CCND1) mRNA level decreased in lesions with multiple metastases and was correlated with both the mRNA levels and mutation state of BRAF and NRAS genes. Primary melanomas with BRAF(V600) or NRAS(Q61 ) mutations exhibited lower CCND1 mRNA level. CCND1 protein expression was associated with Breslow thickness, metastasis formation, and shorter survival time. These observations suggest that CCND1 alterations are linked to melanoma progression and are modified by BRAF and NRAS mutations. Our data show that CCND1 amplification could have a prognostic relevance in cutaneous melanoma and highlight that altered CCND1 gene expression may influence the metastatic progression, survival, and the localization of metastases.
Subject(s)
Cyclin D1/genetics , Genes, ras/genetics , Melanoma/genetics , Mutation/genetics , Proto-Oncogene Proteins B-raf/genetics , Skin Neoplasms/genetics , Adult , Comparative Genomic Hybridization , Disease Progression , Female , Follow-Up Studies , Humans , Immunoenzyme Techniques , In Situ Hybridization, Fluorescence , Lymphatic Metastasis , Male , Melanoma/mortality , Melanoma/secondary , Middle Aged , Neoplasm Staging , Prognosis , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Skin Neoplasms/mortality , Skin Neoplasms/pathology , Survival Rate , Young AdultABSTRACT
Tumour cell invasion is the first step in metastasis, during which cells are able to infiltrate surrounding tissues. We aimed to investigate genetic and epigenetic differences associated with the invasiveness in melanoma. To determine the invasiveness of cells, we used Matrigel invasion chamber. Genetic analyses were performed by array CGH, DNA methylation was assessed by Illumina array, gene expression changes were determined by Affymetrix array. Our results showed significantly higher copy numbers of GDNF, GPAA1, PLEC and SHARPIN genes in invasive cells compared to non-invasive ones. We observed that the invasive cells were characterized by a hypermethylated pattern. Most of the hypermethylated genes were associated with decreased expression, however, increased gene expression was observed for EGFR and RBP4 genes with hypermethylation extending into the gene body. Hypermethylation of the ARHGAP22 and NAV2 genes characterized invasive cells and melanoma metastasis samples. Our results point to the hypermethylation pattern of invasive cells, which may be related to the invasive property.
Subject(s)
Glial Cell Line-Derived Neurotrophic Factor , Melanoma , DNA Methylation , Epigenesis, Genetic , ErbB Receptors , Gene Expression Regulation, Neoplastic , Genomics , Glial Cell Line-Derived Neurotrophic Factor/genetics , Humans , Melanoma/genetics , Melanoma/pathology , Retinol-Binding Proteins, Plasma/genetics , Skin Neoplasms , Melanoma, Cutaneous MalignantABSTRACT
Osteopontin (OPN) is a multifunctional glycoprotein that physiologically interacts with different types of integrins. It is considered to be a possible prognostic biomarker in certain tumor types; however, various splicing isoforms exist, which have not been investigated in melanoma. We aimed to define the relative expression pattern of five OPN isoforms and clarify the prognostic significance of the splice variants in melanoma. We also aimed to investigate the expression pattern of eight integrins in the same tumors. Gene expression analyses revealed that the relative expression of OPNa, OPNb, and OPNc is significantly higher in metastatic tumors compared to primary lesions (p < 0.01), whereas the expression of OPN4 and OPN5 was low in both. The more aggressive nodular melanomas had higher expression levels compared to the superficial spreading subtype (p ≤ 0.05). The relative expression of the eight tested integrins was low, with only the expression of ITGB3 being detectable in nodular melanoma (Medianlog2 = 1.274). A positive correlation was found between Breslow thickness and the expression of OPNc variant, whereby thicker tumors (>4 mm) had significantly higher expression (p ≤ 0.05). The Breslow thickness was negatively correlated with the expression of OPN4, and similarly with ITGA2. OPNc also exhibited significant positive correlation with the presence of metastasis. Our data show that high expression of OPNa, OPNb, and especially OPNc and low expression of OPN4 and ITGA2 are associated with an advanced stage of tumor progression and poor prognosis in melanoma.
Subject(s)
Integrins , Melanoma , Osteopontin , Skin Neoplasms , Gene Expression , Humans , Integrins/genetics , Integrins/metabolism , Melanoma/genetics , Melanoma/metabolism , Melanoma/pathology , Osteopontin/genetics , Osteopontin/metabolism , Prognosis , Protein Isoforms/genetics , Protein Isoforms/metabolism , Skin Neoplasms/genetics , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , Melanoma, Cutaneous MalignantABSTRACT
Target-specific inhibition of the BRAFV600E mutant protein has been a major breakthrough in the treatment of metastatic cutaneous melanoma. However, the success of therapies is significantly overshadowed by the development of resistance. Understanding the molecular mechanisms associated with acquired resistance is an important step to increase the effectiveness of melanoma treatment. Our aim was to elucidate the molecular differences underlying the development of drug resistance using a mutant BRAF protein inhibitor (vemurafenib analogue: PLX4720) in BRAFV600E mutant melanoma cell lines. We developed four BRAF inhibitor-resistant cell lines and examined the effect of BRAF inhibitor "withdrawal" on cell division. ArrayCGH was used to define genetic, and Affymetrix HumanGene 1.0 microarray to monitor gene expression alterations between the sensitive and resistant cell lines. Protein expression was determined using Proteome Profiler Human XL Oncology Array. We found that withdrawal of the inhibitor reduces cell proliferation in the resistant cells. The invasive potential of the resistant cells increased. Using genomic and proteomic methods we described new molecular alterations associated with acquired resistance.
Subject(s)
Melanoma , Skin Neoplasms , Cell Line, Tumor , Drug Resistance, Neoplasm/genetics , Humans , Melanoma/drug therapy , Melanoma/genetics , Mutation , Protein Kinase Inhibitors/pharmacology , Proteomics , Proto-Oncogene Proteins B-raf/genetics , Skin Neoplasms/drug therapy , Skin Neoplasms/geneticsABSTRACT
Combination treatment using BRAF/MEK inhibitors is a promising therapy for patients with advanced BRAFV600E/K mutant melanoma. However, acquired resistance largely limits the clinical efficacy of this drug combination. Identifying resistance mechanisms is essential to reach long-term, durable responses. During this study, we developed six melanoma cell lines with acquired resistance for BRAFi/MEKi treatment and defined the molecular alterations associated with drug resistance. We observed that the invasion of three resistant cell lines increased significantly compared to the sensitive cells. RNA-sequencing analysis revealed differentially expressed genes that were functionally linked to a variety of biological functions including epithelial-mesenchymal transition, the ROS pathway, and KRAS-signalling. Using proteome profiler array, several differentially expressed proteins were detected, which clustered into a unique pattern. Galectin showed increased expression in four resistant cell lines, being the highest in the WM1617E+BRes cells. We also observed that the resistant cells behaved differently after the withdrawal of the inhibitors, five were not drug addicted at all and did not exhibit significantly increased lethality; however, the viability of one resistant cell line (WM1617E+BRes) decreased significantly. We have selected three resistant cell lines to investigate the protein expression changes after drug withdrawal. The expression patterns of CapG, Enolase 2, and osteopontin were similar in the resistant cells after ten days of "drug holiday", but the Snail protein was only expressed in the WM1617E+BRes cells, which showed a drug-dependent phenotype, and this might be associated with drug addiction. Our results highlight that melanoma cells use several types of resistance mechanisms involving the altered expression of different proteins to bypass drug treatment.
ABSTRACT
HA15 is a new anti-melanoma drug that triggers endoplasmic reticulum (ER) stress and causes deleterious effects on melanoma cell viability due to autophagy and apoptosis, regardless of driver mutations or drug resistance. In this study, we investigated the effect of HA15 on the viability/proliferation of BRAFV600E-mutant melanoma cells using different culture conditions. In contrast to the published data, we did not detect significant melanoma cell death under normal culture conditions using HA15 treatment. Indeed, only cells that were cultured under long-term starvation conditions were sensitive to the drug. Quantitative measurements of ER stress and autophagy markers showed that the compound HA15 does not trigger stress alone but synergistically enhances ER stress under starvation conditions. Importantly, we observed that the viability of normal melanocytes decreased significantly with treatment, even at low HA15 concentrations. Finally yet importantly, we were able to generate HA15-resistant cell lines, which failed by Cerezo et al. In summary, HA15 only influences the viability of cells that are starved for several hours before and during treatment. However, this in vitro setting is far from the in vivo conditions. In addition, our data clearly show that melanoma cells can acquire HA15 resistance. Further studies are needed to prove that HA15 is an effective anti-cancer agent.
ABSTRACT
Osteopontin (OPN) is a multifunctional phosphoprotein that is expressed in different types of cancers, including melanoma. OPN overexpression is associated with tumor progression and metastasis formation; however, the role of OPN in cell invasion and metastasis formation is not completely understood. In this study we aimed to define OPN expression in melanoma tissues and cell lines and investigate the effect of OPN expression on cell proliferation and invasion after inhibiting OPN expression with small interfering RNA (siRNA). OPN gene expression was determined by qRT-PCR, while protein expression was examined using a Proteome Profiler Oncology Array. siRNA-mediated OPN knockdown led to decreased OPN expression in melanoma cell lines, which was associated with decreased cell proliferation and invasion. Proteome profile analysis revealed significantly different protein expression between the original and transfected cell lines. The altered expression of the differently expressed proteins was validated at the mRNA level. Furthermore, OPN-specific siRNA was able to reduce OPN expression and inhibit the invasiveness of melanoma cells. Our results revealed for the first time that silencing the OPN gene influences proliferation and invasion of melanoma cells by effecting EGFR, tenascin C, survivin, galectin-3 and enolase 2 expression. To predict protein-protein interactions along with putative pathways we used STRING analysis for the differentially expressed proteins. These proteins formed multiple clusters, including extracellular matrix organization, regulation of angiogenesis, cell death and cell migration, PI3K-Akt, MAPK and focal adhesion signaling pathways. Taken together these data suggest that OPN might be an ideal target for drug development and therapies.
Subject(s)
Cell Proliferation/genetics , Melanoma/genetics , Osteopontin/genetics , Cell Line, Tumor , Cell Movement/genetics , Gene Expression , Gene Expression Regulation, Neoplastic , Gene Silencing , Humans , Melanoma/metabolism , Melanoma/pathology , Osteopontin/metabolism , Protein Interaction Maps , RNA, Small Interfering/genetics , Signal Transduction/geneticsABSTRACT
In vitro cell cultures are frequently used to define the molecular background of drug resistance. The majority of currently available data have been obtained from 2D in vitro cultures, however, 3D cell culture systems (spheroids) are more likely to behave similarly to in vivo conditions. Our major aim was to compare the gene expression signature of 2D and 3D cultured BRAFV600E mutant melanoma cell lines. We successfully developed BRAF-drug resistant cell lines from paired primary/metastatic melanoma cell lines in both 2D and 3D in vitro cultures. Using Affymetrix Human Gene 1.0 ST arrays, we determined the gene expression pattern of all cell lines. Our analysis revealed 1049 genes (562 upregulated and 487 downregulated) that were differentially expressed between drug-sensitive cells grown under different cell cultures. Pathway analysis showed that the differently expressed genes were mainly associated with the cell cycle, p53, and other cancer-related pathways. The number of upregulated genes (72 genes) was remarkably fewer when comparing the resistant adherent cells to cells that grow in 3D, and were associated with cell adhesion molecules and IGF1R signalling. Only 1% of the upregulated and 5.6% of the downregulated genes were commonly altered between the sensitive and the resistant spheroids. Interestingly, we found several genes (BNIP3, RING1 and ABHD4) with inverse expression signature between sensitive and resistant spheroids, which are involved in anoikis resistance and cell cycle regulation. In summary, our study highlights gene expression alterations that might help to understand the development of acquired resistance in melanoma cells in tumour tissue.