Prognostic Value of IGF2 mRNA-Binding Protein 3 (IGF2BP3) Intratumoral Expression in Melanoma Patients at the Time of Diagnosis: Comparative Analysis of RT-qPCR Versus Immunohistochemistry.

Screening for prognostic biomarkers is crucial for clinical melanoma management. Insulin-like growth factor-II mRNA-binding protein 3 (IGF2BP3) has emerged as a potential melanoma diagnostic and prognostic biomarker. It is commonly tested by immunohistochemistry (IHC). Our study retrospectively examines IGF2BP3 mRNA and protein expression in primary melanomas, their correlation with clinicopathologic factors, clinical outcome, and selected miRNAs expression, and their efficiency in predicting melanoma progression and survival. RT-qPCR and IHC on IGF2BP3 expression were performed in 61 cryopreserved and 63 formalin-fixed paraffin-embedded primary melanomas, respectively, and correlated to clinicopathologic factors, distant metastasis-free survival (DMFS), and melanoma -specific survival (MSS). The correlation between RT-qPCR and IHC was significant but moderate. IGF2BP3 mRNA showed a stronger association with clinicopathologic factors (Breslow thickness, ulceration, mitosis rate, growth phase, development of metastasis, and melanoma-specific survival) than its protein counterpart. Interestingly, higher IGF2BP3 mRNA expression was detected in primary melanomas that further metastasized to distant sites and was an independent prognostic factor for the risk of unfavorable DMFS and MSS. RT-qPCR outperformed IHC in sensitivity and in predicting worse clinical outcomes. Therefore, RT-qPCR may successfully be implemented for routine IGF2BP3 assessing for the selection of melanoma patients with a higher risk of developing distant metastasis and dying of melanoma.

Cutaneous Lymphadenoma Is a Distinct Trichoblastoma-like Lymphoepithelial Tumor With Diffuse Androgen Receptor Immunoreactivity, Notch1 Ligand in Reed-Sternberg-like Cells, and Common EGFR Somatic Mutations.

The term "cutaneous lymphadenoma" was coined in this journal for an unusual lymphoepithelial cutaneous adnexal neoplasm, possibly with immature pilosebaceous differentiation. Some authors further proposed that cutaneous lymphadenoma was an adamantinoid trichoblastoma. However, although a hair follicle differentiation is widely accepted, the fact that this is a lymphoepithelial tumor is not appropriately explained by the trichoblastoma hypothesis. Our goal was to further clarify the phenotypic and genotypic features of cutaneous lymphadenoma in a series of 11 cases. Histologically, a lobular architecture surrounded by a dense fibrous stroma was present in all cases. The lobules were composed of epithelial cells admixtured with small lymphocytes and isolated or clustered large Reed-Sternberg-like (RS-L) cells. The epithelial cells were diffusely positive for the hair follicle stem cell markers CK15, PHLDA1, and for androgen receptor. No immunostaining for markers of sebaceous differentiation was found. Intraepithelial lymphocytes were predominantly CD3+, CD4+, FoxP3+ T cells. RS-L cells showed both strong Jagged-1 and Notch1 cytoplasmic immunostaining. Androgen-regulated NKX3.1 nuclear immunostaining was present in a subset of large intralobular cells in all cases. Double immunostaining showed coexpression of NKX3.1 and CD30 in a subset of RS-L cells. No immunostaining for lymphocytic or epithelial markers was present in RS-L cells. EGFR, PIK3CA, and FGFR3 somatic mutations were found by next-generation sequencing in 56% of the cases. We consider that cutaneous lymphadenoma is a distinct benign lymphoepithelial tumor with androgen receptor and hair follicle bulge stem cell marker expression, RS-L cell-derived Notch1 ligand, and common EGFR gene mutations.

Transcriptomic identification of miR-205 target genes potentially involved in metastasis and survival of cutaneous malignant melanoma.

Cutaneous melanoma is an aggressive neoplasm and is responsible for the majority of skin cancer deaths. Several miRNAs are involved in melanoma tumor progression. One of them is miR-205, the loss of which contributes to the development of melanoma metastasis. We evaluated whole-genome mRNA expression profiling associated with different miR-205 expression levels in melanoma cells. Differential expression analysis identified 243 differentially expressed transcripts including inositol polyphosphate 5'-phosphatase-like protein-1 (INPPL1) and BTB/POZ Domain-Containing Protein 3 (BTBD3). INPPL1 and BTBD3 were downregulated when melanoma cells expressed miR-205, indicating that these genes are potential miR-205 targets. Additionally, the target prediction algorithm TargetScan revealed that INPPL1 and BTBD3 genes had predicted target sites of miR-205 in their 3'UTRs and functional analysis demonstrated that these genes were directly linked to miR-205. Interestingly, our clinical data showed that INPPL1 was significantly associated with lymph node metastasis-free survival (LNMFS), distant metastasis-free survival (DMFS) and melanoma specific survival (MSS). This study supports INPPL1 as a miR-205 target gene and, therefore, that the involvement of miR-205 in the metastatic dissemination of malignant melanoma is, at least in part, via INPPL1.

Downregulation of intratumoral expression of miR-205, miR-200c and miR-125b in primary human cutaneous melanomas predicts shorter survival.

While only 15-25 percent of melanoma patients develop distant metastasis and die, this disease is still responsible for the majority of skin cancer-related deaths. The availability of adjuvant therapies makes the selection of high-risk patients essential. We evaluated the intratumoral expression of ten miRNAs in primary melanomas in relation to its ability to predict melanoma survival. To this end, we correlated miRNA expression in 132 cryopreserved primary and metastatic tumors with clinicopathological factors and clinical outcome. We found sequential downregulation of intratumoral expression of miR-125b, miR-182, miR-200c and miR-205 over the full spectrum of melanoma progression. Moreover, downregulation of these miRNAs occurred in primary melanomas that further disseminated to distant sites. Furthermore, miR-125b, miR-200c and miR-205 correlated as independent factors with shorter survival. Our in vitro findings demonstrate that loss of miR-205 potentiates the invasive ability of melanoma cells. We conclude that the downregulation of miR-205 in primary melanomas is an intrinsic property that might contribute to distant metastasis. In particular, the interaction of melanoma cells with the extracellular matrix is one of the key mechanisms by which miR-205 influences melanoma metastasis. In conclusion, miR-125b, miR-200c and miR-205 are useful prognostic biomarkers at the time of diagnosis to select high-risk patients.

Null mutants of Candida albicans for cell-wall-related genes form fragile biofilms that display an almost identical extracellular matrix proteome.

By two-dimensional gel electrophoresis (2-DE) and mass spectrometry, we have characterized the polypeptide species present in extracts obtained by 60% ethanol treatment of whole mature (48 h) biofilms formed by a reference strain (CAI4-URA3) and four Candida albicans null mutants for cell-wall-related genes (ALG5, CSA1, MNN9 and PGA10) Null mutants form fragile biofilms that appeared partially split and weakly attached to the substratum contrary to those produced by the reference strain. An almost identical, electrophoretic profile consisting of about 276 spots was visualized in all extracts examined. Proteomic analysis led to the identification of 131 polypeptides, corresponding to 86 different protein species, being the rest isoforms-83 displayed negative hydropathic indexes and 82 lack signal peptide. The majority of proteins appeared at pI between 4 and 6, and molecular mass between 10 and 94 kDa. The proteins identified belonged to the following Gene Ontology categories: 21.9% unknown molecular function, 16.2% oxidoreductase activity, 13.3% hydrolase activity and 41.8% distributed between other different GO categories. Strong defects in biofilm formation appreciated in the cell-wall mutant strains could be attributed to defects in aggregation due to abnormal cell wall formation rather than to differences in the biofilm extracellular matrix composition.

Deregulation of glyceraldehyde-3-phosphate dehydrogenase expression during tumor progression of human cutaneous melanoma.

BACKGROUND/AIM: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a highly abundant housekeeping gene. GAPDH overexpression has been reported in diverse types of human cancers including cutaneous melanoma. Our goal was to quantify GAPDH mRNA and protein expression in the whole spectrum of primary and metastatic melanomas in the search for a specific role for this ubiquitous molecule during tumor progression. MATERIALS AND METHODS: Intratumoral GAPDH mRNA expression was quantified by real-time PCR in 71 cases, including 29 primary melanomas and 42 metastatic cases. Relative expression levels in thin (≤1 mm) and thick (>1 mm) primary tumors and 'in-transit', lymph node and distant metastases were compared. Similarly, protein expression was investigated by means of immunohistochemistry. Specific exons of GAPDH were analyzed by DNA sequencing. RESULTS: GAPDH mRNA expression was significantly up-regulated in thick melanomas when compared to primary thin melanomas. Similar differences were also encountered between metastatic melanomas when compared to lymph-node metastatic melanomas. Interestingly, GAPDH protein immunoexpression was higher in thick melanomas and distant metastases than in thin tumors and lymph node metastases, respectively. However, no specific point-mutations in GAPDH-specific exons were found in any patient. CONCLUSION: Deregulation of GAPDH during melanoma progression was demonstrated in our series by mRNA and protein expression studies.

CCL27-CCR10 and CXCL12-CXCR4 chemokine ligand-receptor mRNA expression ratio: new predictive factors of tumor progression in cutaneous malignant melanoma.

CXCR4, CCR7 and CCR10 chemokine receptors are known to be involved in melanoma metastasis. Our goal was to compare the relative intratumoral mRNA expression of these receptors with that of their corresponding chemokine ligands, CXCL12, CCL19, CCL21, and CCL27 across the full spectrum of human melanoma progression: thin and thick primary melanomas, as well as "in transit", lymph node, and distant metastases. Expression was quantified by real-time RT-PCR in 103 melanoma samples: 51 primary tumors and 52 metastases. Particular emphasis was focused on chemokine ligand-receptor expression ratios. Immunohistochemistry was performed to identify the cell types expressing these molecules. CXCL12-CXCR4 and CCL27-CCR10 ratios were higher in thin than in thick primary melanomas, and all four chemokine-receptor ratios were higher in primary tumors than in melanoma metastases. CCL27-CCR10 and CXCL12-CXCR4 expression ratios in primary tumors were inversely associated with the development of distant metastases, and improved the predictive value of tumor thickness for distant metastasis, which is important since chemokine ligand-receptor ratios are not affected by the endogenous gene employed for normalizing mRNA expression. Both receptor and ligand immunolabeling were detected in neoplastic cells suggesting autocrine mechanisms. Our results support the concept that low CCL27/CCR10 and CXCL12/CXCR4 intratumoral mRNA ratios are associated with melanoma progression, and in combination with Breslow thickness, are the best predictive factors for the development of distant metastases in primary cutaneous melanoma.

Some biological features of Candida albicans mutants for genes coding fungal proteins containing the CFEM domain.

Several biological features of Candida albicans genes (PGA10, RBT5 and CSA1) coding for putative polypeptide species belonging to a subset of fungal proteins containing an eight-cysteine domain referred as common in several fungal extracellular membrane (CFEM) are described. The deletion of these genes resulted in a cascade of pleiotropic effects. Thus, mutant strains exhibited higher cell surface hydrophobicity levels and an increased ability to bind to inert or biological substrates. Confocal scanning laser microscopy using concanavalin A-Alexafluor 488 (which binds to mannose and glucose residues) and FUN-1 (a cytoplasmic fluorescent probe for cell viability) dyes showed that mutant strains formed thinner and more fragile biofilms. These apparently contained lower quantities of extracellular matrix material and less metabolically active cells than their parental strain counterpart, although the relative percentage of mycelial forms was similar in all cases. The cell surface of C. albicans strains harbouring deletions for genes coding CFEM-domain proteins appeared to be severely altered according to atomic force microscopy observations. Assessment of the relative gene expression within individual C. albicans cells revealed that CFEM-coding genes were upregulated in mycelium, although these genes were shown not to affect virulence in animal models. Overall, this study has demonstrated that CFEM domain protein-encoding genes are pleiotropic, influencing cell surface characteristics and biofilm formation.

Candida albicans ABG1 gene is involved in endocytosis.

The human fungal pathogen Candida albicans undergoes reversible morphogenetic transitions between yeast, hyphal and pseudohyphal forms. The fungal vacuole actively participates in differentiation processes and plays a key role supporting hyphal growth. The ABG1 gene of C. albicans encodes an essential protein located in the vacuolar membranes of both yeast and hyphae. Using fluorescence microscopy of a green fluorescent protein-tagged version of Abg1p, a fraction of the protein was detected in hyphal tips, not associated with vacuolar membranes. Live cell imaging of emerging germ tubes showed that Abg1p migrated to the polarized growth site and colocalized with endocytic vesicles. Phenotypic analysis of a methionine-regulated conditional mutant confirmed that Abg1p is involved in endocytosis.

Biofilm formation by Candida albicans mutants for genes coding fungal proteins exhibiting the eight-cysteine-containing CFEM domain.

Several features and functions of a Candida albicans gene, PGA10 (also designated as RBT51), coding for a putative polypeptide species belonging to a subset of fungal proteins containing an eight-cysteine domain referred as CFEM (Common in several Fungal Extracellular Membrane proteins), are described. The ORF of the gene (ORF19.5674) encoded a protein of 250 amino acids, with a predicted molecular mass of 25.17 kDa. The product of the PGA10 gene also exhibited some features reminiscent of a class II-type hydrophobin. Deletion of PGA10 resulted in a cascade of pleiotropic effects, mostly affecting cell-surface-related properties. Thus, the null pga10Delta mutant displayed an increased sensitivity to cell-wall-perturbing agents and formed fragile biofilms that appeared partially split and weakly attached to the substratum. The biofilm-forming ability of several C. albicans mutants with single, double and triple deletions of genes encoding other protein species also containing the CFEM domain (RBT5 and WAP1/CSA1) was determined. These mutants also exhibited an abnormal ability to form biofilms. Overall, the evidence presented here suggests that fungal proteins containing the CFEM domain (Pga10p/Rbt51p, Rbt5p and Wap1p/Csa1p) may play a key role in the formation, development and/or maintenance of the biofilm structure in C. albicans.

Rapid PCR-based test for identifying Candida albicans by using primers derived from the pH-regulated KER1 gene.

A PCR-based method in combination with a simple, reliable and inexpensive DNA extraction procedure for rapid detection of Candida albicans clinical isolates is described here. The extraction protocol is based on a combination of chemical (NaOH and detergents) and physical (boiling) treatments, thus avoiding many of the problems inherent in the currently available DNA extraction protocols (basically the use of expensive and/or toxic chemical reagents), and may be useful for daily clinical routine. The PCR-based system described here uses a single pair of primers (SC1F and SC1R) deduced from the C. albicans-specific KER1 gene sequence. These primers amplify a 670-bp fragment of the KER1 gene. All the clinical C. albicans isolates generated the expected 670-bp amplicon. Other non-albicans Candida species, including the azole-resistant C. krusei and C. glabrata, and the very closely related C. dubliniensis, failed to amplify any DNA fragment. The PCR results reported here suggest that amplification with SC1F and SC1R primers is species specific and, consequently, may be useful for specifically identifying C. albicans strains.

ABG1, a novel and essential Candida albicans gene encoding a vacuolar protein involved in cytokinesis and hyphal branching.

Immunoscreening of a Candida albicans expression library resulted in the isolation of a novel gene encoding a 32.9-kDa polypeptide (288 amino acids), with 27.7% homology to the product of Saccharomyces cerevisiae YGR106c, a putative vacuolar protein. Heterozygous mutants in this gene displayed an altered budding growth pattern, characterized by the formation of chains of buds, decreasingly in size towards the apex, without separation of the daughter buds. Consequently, this gene was designated ABG1. A conditional mutant for ABG1 with the remaining allele under the control of the MET3 promoter did not grow in the presence of methionine and cysteine, demonstrating that ABG1 was essential for viability. Western analysis revealed the presence of a major 32.9-kDa band, mainly in a particulate fraction (P40) enriched in vacuoles, and tagging with green fluorescent protein confirmed that Abg1p localized to the vacuole. Vacuole inheritance has been linked to the regulation of branching frequency in C. albicans. Under repressing conditions, the conditional mutant had an increased frequency of branching under hyphal inducing conditions and an altered sensitivity to substances that interfered with cell wall assembly. Repression of ABG1 in the conditional mutant strain caused disturbance of normal size and number of vacuoles both in yeast and mycelial cells and also in the asymmetric vacuole inheritance associated with the characteristic pattern of germ tubes and branching in C. albicans. These observations indicate that ABG1 plays a key role in vacuole biogenesis, cytokinesis, and hyphal branching.

The Candida albicans pH-regulated KER1 gene encodes a lysine/glutamic-acid-rich plasma-membrane protein that is involved in cell aggregation.

Immunoscreening of a Candida albicans cDNA library with a polyclonal germ-tube-specific antibody (pAb anti-gt) resulted in the isolation of a gene encoding a lysine/glutamic-acid-rich protein, which was consequently designated KER1. The nucleotide and deduced amino acid sequences of this gene displayed no significant homology with any other known sequence. KER1 encodes a 134 kDa lysine (14.5%)/glutamic acid (16.7%) protein (Ker1p) that contains two potential transmembrane segments. KER1 was expressed in a pH-conditional manner, with maximal expression at alkaline pH and lower expression at pH 4.0, and was regulated by RIM101. A Deltaker1/Deltaker1 null mutant grew normally but was hyperflocculant under germ-tube-inducing conditions, yet this behaviour was also observed in stationary-phase cells grown under other incubation conditions. Western blotting analysis of different subcellular fractions, using as a probe a monospecific polyclonal antibody raised against a highly antigenic domain of Ker1p (pAb anti-Ker1p), revealed the presence of a 134 kDa band in the purified plasma-membrane fraction from the wild-type strain that was absent in the homologous preparation from Deltaker1/Deltaker1 mutant. The pattern of cell-wall protein and mannoprotein species released by digestion with beta-glucanases, reactive towards pAbs anti-gt and anti-Ker1p, as well as against concanavalin A, was also different in the Deltaker1/Deltaker1 mutant. Mutant strains also displayed an increased cell-surface hydrophobicity and sensitivity to Congo red and Calcofluor white. Overall, these findings indicate that the mutant strain was affected in cell-wall composition and/or structure. The fact that the ker1 mutant had attenuated virulence in systemic mouse infections suggests that this surface protein is also important in host-fungus interactions.

Antibody response to Candida albicans cell wall antigens.

The cell wall of Candida albicans is not only the structure where many essential biological functions reside but is also a significant source of candidal antigens. The major cell wall components that elicit a response from the host immune system are proteins and glycoproteins, the latter being predominantly mannoproteins. Both carbohydrate and protein moieties are able to trigger immune responses. Proteins and glycoproteins exposed at the most external layers of the wall structure are involved in several types of interactions of fungal cells with the exocellular environment. Thus, coating of fungal cells with host antibodies has the potential to profoundly influence the host-parasite interaction by affecting antibody-mediated functions such as opsonin-enhanced phagocytosis and blocking the binding activity of fungal adhesins to host ligands. In this review we examine various members of the protein and glycoprotein fraction of the C. albicans cell wall that elicit an antibody response in vivo. Some of the studies demonstrate that certain cell wall antigens and anti-cell wall antibodies may be the basis for developing specific and sensitive serologic tests for the diagnosis of candidiasis, particularly the disseminated form. In addition, recent studies have focused on the potential of antibodies against the cell wall protein determinants in protecting the host against infection. Hence, a better understanding of the humoral response triggered by the cell wall antigens of C. albicans may provide the basis for the development of (i) effective procedures for the serodiagnosis of disseminated candidiasis, and (ii) novel prophylactic (vaccination) and therapeutic strategies to control this type of infections.

Different adhesins for type IV collagen on Candida albicans: identification of a lectin-like adhesin recognizing the 7S(IV) domain.

Adherence of the opportunistic pathogen Candida albicans to basement membrane (BM) proteins is considered a crucial step in the development of candidiasis. In this study the interactions of C. albicans yeast cells with the three main domains of type IV collagen, a major BM glycoprotein, were analysed. C. albicans adhered to the three immobilized domains by different mechanisms. Adhesion to the N-terminal cross-linking domain (7S) required the presence of divalent cations, whereas interaction with the central collagenous domain (CC) was cation-independent. Recognition of the C-terminal non-collagenous domain (NC1) was partially cation-dependent. Binding inhibition assays with the corresponding domains in soluble form showed that these interactions were specific. Both Ca(2+) and Mg(2+) promoted adhesion to the 7S domain and the interaction was completely abolished by EDTA. Treatment of the 7S domain, or its subunits, with N-glycosidase F reduced yeast binding by approximately 70%. Moreover, several sugars known to be part of the N-linked oligosaccharide chains of collagen IV inhibited adhesion to immobilized 7S; N-acetylglucosamine, L-fucose and methylmannoside caused a similar inhibition whereas N-acetyllactosamine was a more effective inhibitor. In contrast, glucose, galactose, lactose or heparan sulfate did not affect yeast binding. Combinations of the inhibitory sugars at suboptimal inhibition concentrations did not reduce C. albicans adhesion more than the individual sugars, pointing to a single lectin as responsible for the interaction. These results taken together show that C. albicans utilizes several adhesins for interacting with type IV collagen, and that at least one of them is a lectin which recognizes the 7S(IV) oligosaccharide residues as its receptor.