The full repertoire of human microRNAs (miRNAs) that could distinguish common (benign) nevi from cutaneous (malignant) melanomas remains to be established. In an effort to gain further insight into ...the role of miRNAs in melanoma, we applied Illumina next-generation sequencing (NGS) platform to carry out an in-depth analysis of miRNA transcriptome in biopsies of nevi, thick primary (>4.0 mm) and metastatic melanomas with matched normal skin in parallel to melanocytes and melanoma cell lines (both primary and metastatic) (n=28). From this data representing 698 known miRNAs, we defined a set of top-40 list, which properly classified normal from cancer; also confirming 23 (58%) previously discovered miRNAs while introducing an additional 17 (42%) known and top-15 putative novel candidate miRNAs deregulated during melanoma progression. Surprisingly, the miRNA signature distinguishing specimens of melanoma from nevus was significantly different than that of melanoma cell lines from melanocytes. Among the top list, miR-203, miR-204-5p, miR-205-5p, miR-211-5p, miR-23b-3p, miR-26a-5p and miR-26b-5p were decreased in melanomas vs. nevi. In a validation cohort (n=101), we verified the NGS results by qRT-PCR and showed that receiver-operating characteristic curves for miR-211-5p expression accurately discriminated invasive melanoma (AUC=0.933), melanoma in situ (AUC=0.933) and dysplastic (atypical) nevi (AUC=0.951) from common nevi. Target prediction analysis of co-transcribed miRNAs showed a cooperative regulation of key elements in the MAPK signaling pathway. Furthermore, we found extensive sequence variations (isomiRs) and other non-coding small RNAs revealing a complex melanoma transcriptome. Deep-sequencing small RNAs directly from clinically defined specimens provides a robust strategy to improve melanoma diagnostics.
miRNAs are key regulatory small non-coding RNAs involved in critical steps of melanoma tumorigenesis; however, the relationship between sequence specific variations at the 5' or 3' termini (isomiR) ...of a miRNA and cancer phenotype remains unclear. Deep-sequencing and qRT-PCR showed reduced expression of miR-144/451a cluster and most abundant isomiR (miR451a.1) in dysplastic nevi, in-situ and invasive melanomas compared to common nevi and normal skin (n = 101). miRNA in situ hybridization reproducibly confirmed lost miR-451a.1 in melanoma compared to nevus cells or adjacent keratinocytes. Significantly higher expression of miR-451a.1 was associated with amelanotic phenotype in melanomas (n = 47). In contrast, miR-451a was associated with melanotic phenotype, absent pagetoid scatter of intraepidermal melanocytes, superficial spreading histological subtype and tumor inflammation. Sequencing miRNAs from cultured melanocytes with cytoplasmic melanin gradient (light, medium to dark) showed absent miR-451a while revealing other melanin-associated miRNAs, e.g. miR-30b, miR-100 and miR-590 in darkly and let-7a, let-7i and let-7f in lightly to moderately pigmented cultured melanocytes. Ectopic expression of miR-144/451a in melanoma cell lines resulted in markedly higher levels of mature miR-451a.1 than miR451a or miR-144; and significantly retarded cell migration and inhibited invasion in a glucose-sensitive manner. Surprisingly, these effects were not mediated by calcium binding protein 39 (CAB39), a proven miR451a gene target. miR-144/miR-451a cluster is a novel miRNA locus with tumor suppressive activity in melanoma.
The mucin-type glycoprotein podoplanin is specifically expressed by lymphatic but not blood vascular endothelial cells in culture and in tumor-associated lymphangiogenesis, and podoplanin deficiency ...results in congenital lymphedema and impaired lymphatic vascular patterning. However, research into the biological importance of podoplanin has been hampered by the lack of a generally available antibody against the human protein, and its expression in normal tissues and in human malignancies has remained unclear. We generated a human podoplanin-Fc fusion protein and found that the commercially available mouse monoclonal antibody D2-40 specifically recognized human podoplanin, as assessed by enzyme-linked immunosorbent assay and Western blot analyses. We found that, in addition to lymphatic endothelium, podoplanin was also expressed by peritoneal mesothelial cells, osteocytes, glandular myoepithelial cells, ependymal cells, and by stromal reticular cells and follicular dendritic cells of lymphoid organs. These findings were confirmed in normal mouse tissues with anti-podoplanin antibody 8.1.1. Podoplanin was also strongly expressed by granulosa cells in normal ovarian follicles, and by ovarian dysgerminomas and granulosa cell tumors. Although podoplanin was primarily absent from normal human epidermis, its expression was strongly induced in 22 of 28 squamous cell carcinomas studied. These findings suggest a potential role of podoplanin in tumor progression, and they also identify the first commercially available antibody for the specific staining of a defined lymphatic marker in archival human tissue sections, thereby enabling more widespread studies of tumor lymphangiogenesis in human cancers.
Papillomaviruses (PVs) induce papillomas, premalignant lesions, and carcinomas in a wide variety of species. PVs are classified first based on their host and tissue tropism and then their genomic ...diversities. A laboratory mouse papillomavirus, MmuPV1 (formerly MusPV), was horizontally transmitted within an inbred colony of NMRI-Foxn1(nu)/Foxn1nu (nude; T cell deficient) mice of an unknown period of time. A ground-up, filtered papilloma inoculum was not capable of infecting C57BL/6J wild-type mice; however, immunocompetent, alopecic, S/RV/Cri-ba/ba (bare) mice developed small papillomas at injection sites that regressed. NMRI-Foxn1(nu) and B6.Cg-Foxn1(nu), but not NU/J-Foxn1(nu), mice were susceptible to MmuPV1 infection. B6 congenic strains, but not other congenic strains carrying the same allelic mutations, lacking B- and T-cells, but not B-cells alone, were susceptible to infection, indicating that mouse strain and T-cell deficiency are critical to tumor formation. Lesions initially observed were exophytic papillomas around the muzzle, exophytic papillomas on the tail, and condylomas of the vaginal lining which could be induced by separate scarification or simultaneous scarification of MmuPV1 at all four sites. On the dorsal skin, locally invasive, poorly differentiated tumors developed with features similar to human trichoblastomas. Transcriptome analysis revealed significant differences between the normal skin in these anatomic sites and in papillomas versus trichoblastomas. The primarily dysregulated genes involved molecular pathways associated with cancer, cellular development, cellular growth and proliferation, cell morphology, and connective tissue development and function. Although trichoepitheliomas are benign, aggressive tumors, few of the genes commonly associated with basal cell carcinoma or squamous cells carcinoma were highly dysregulated.
MicroRNAs (miRNAs) are small non-coding RNAs (18-24 nucleotides) that have recently been shown to regulate gene expression during cancer progression. Dicer, a central enzyme in the multi-component ...miRNA biogenesis pathway, is involved in cutting precursor miRNAs to functionally mature forms. Emerging evidence shows that Dicer expression is deregulated in some human malignancies and it correlates with tumor progression, yet this role has not yet been investigated in skin cancers.
Using an anti-human monoclonal antibody against Dicer and immunohistochemistry, we compared the expression of Dicer protein among 404 clinically annotated controls and skin tumors consisting of melanocytic nevi (n = 71), a variety of melanomas (n = 223), carcinomas (n = 73) and sarcomas (n = 12). Results showed a cell-specific up-regulated Dicer in 81% of cutaneous, 80% of acrolentiginous and 96% of metastatic melanoma specimens compared to carcinoma or sarcoma specimens (P<0.0001). The expression of Dicer was significantly higher in melanomas compared to benign melanocytic nevi (P<0.0001). In patients with cutaneous melanomas, Dicer up-regulation was found to be significantly associated with an increased tumor mitotic index (P = 0.04), Breslow's depth of invasion (P = 0.03), nodal metastasis (P = 0.04) and a higher American Joint Committee on Caner (AJCC) clinical stage (P = 0.009). Using western blot analysis, we confirmed the cell-specific up-regulation of Dicer protein in vitro. A pooled-analysis on mRNA profiling in cutaneous tumors showed up-regulation of Dicer at the RNA level in cutaneous melanoma, also showing deregulation of other enzymes that participate in the biogenesis and maturation of canonical miRNAs.
Increased Dicer expression may be a clinically useful biomarker for patients with cutaneous melanoma. Understanding deregulation of Dicer and its influence on miRNA maturation is needed to predict the susceptibility of melanoma patients to miRNA-based therapy in the future.
A comprehensive repertoire of human microRNAs (miRNAs) that could be involved in early melanoma invasion into the dermis remains unknown. To this end, we sequenced small RNAs (18–30 nucleotides) ...isolated from an annotated series of invasive melanomas (average invasive depth, 2.0 mm), common melanocytic nevi, and matched normal skin (n=28). Our previously established bioinformatics pipeline identified 765 distinct mature known miRNAs and defined a set of top 40 list that clearly segregated melanomas into thin (0.75 mm) and thick (2.7 mm) groups. Among the top, miR-21-5p, let-7b-5p, let-7a-5p, miR-424-5p, miR-423-5p, miR-21-3p, miR-199b-5p, miR-182-5p, and miR-205-5p were differentially expressed between thin and thick melanomas. In a validation cohort (n=167), measured expression of miR-21-5p and miR-424-5p, not previously reported in melanoma, were significantly increased in invasive compared with in situ melanomas (P<0.0001). Increased miR-21-5p levels were significantly associated with invasive depth (P=0.038), tumor mitotic index (P=0.038), lymphovascular invasion (P=0.0036), and AJCC stage (P=0.038). In contrast, let-7b levels were significantly decreased in invasive and in situ melanomas compared with common and dysplastic nevi (P<0.0001). Decreased let-7b levels were significantly associated with invasive depth (P=0.011), Clark's level (P=0.013), ulceration (P=0.0043), and AJCC stage (P=0.011). These results define a distinct set of miRNAs associated with invasive and aggressive melanoma phenotype.
Sustained lymph stagnation engenders a pathological response that is complex and not well characterized. Tissue inflammation in lymphedema may reflect either an active or passive consequence of ...impaired immune traffic.
We studied an experimental model of acute post-surgical lymphedema in the tails of female hairless, immunocompetent SKH-1 mice. We performed in vivo imaging of impaired immune traffic in experimental, murine acquired lymphatic insufficiency. We demonstrated impaired mobilization of immunocompetent cells from the lymphedematous region. These findings correlated with histopathological alterations and large-scale transcriptional profiling results. We found intense inflammatory changes in the dermis and the subdermis. The molecular pattern in the RNA extracted from the whole tissue was dominated by the upregulation of genes related to acute inflammation, immune response, complement activation, wound healing, fibrosis, and oxidative stress response.
We have characterized a mouse model of acute, acquired lymphedema using in vivo functional imaging and histopathological correlation. The model closely simulates the volume response, histopathology, and lymphoscintigraphic characteristics of human acquired lymphedema, and the response is accompanied by an increase in the number and size of microlymphatic structures in the lymphedematous cutaneous tissues. Molecular characterization through clustering of genes with known functions provides insights into processes and signaling pathways that compose the acute tissue response to lymph stagnation. Further study of genes identified through this effort will continue to elucidate the molecular mechanisms and lead to potential therapeutic strategies for lymphatic vascular insufficiency.
Some melanocytic tumors can be histologically ambiguous causing diagnostic difficulty, which could lead to overtreatment of benign lesions with an unwarranted psychological distress or undertreatment ...of malignant cancers. Previously, we demonstrated that significantly decreased miR-211 expression in melanomas compared with melanocytic nevi could accurately discriminate malignant from benign tumors. Herein we show microRNA in situ hybridization for fluorescent detection of miR-211, suitable for paraffin-embedded tissues in 109 primary melanocytic tumors. miR-211 expression was significantly lower in melanomas vs nevi (P<0.0001), and receiver operating characteristic curve (area under the curve=0.862) accurately discriminated melanomas from nevi with 90% sensitivity and 86.2% specificity. Qualitatively, all dysplastic nevi expressed miR-211 at high (86%) and low (14%) levels, independent of the degree of nuclear atypia. All 35 melanocytic tumors with Spitz morphology expressed miR-211 independent of morphological classification, where clinical follow-up of these patients showed no recurrence at the site or metastasis in mean and median of 3 (ranging 2-5) years. Moreover, a decision tree learning analysis selected age and miR-211 miRNA in situ hybridization as the predictive variables for benign or malignant outcome in 88 patients correctly classified 92% (81 out of 88) of cases as proven by receiver operating characteristic curve (area under the curve=0.9029). These results support miR-211 as a leading miRNA candidate for melanoma diagnosis and miRNA in situ hybridization as a uniquely uncomplicated ancillary test.
Histologic differentiation of melanoma in situ (MIS) from solar keratosis on chronically sun‐damaged skin is challenging. The first‐line immunostain is usually MART‐1/Melan‐A, which can exaggerate ...the epidermal melanocytes, causing a diagnostic pitfall for MIS. By comparing MART‐1 and SOX10 immunostaining, we scored the percentage of epidermal melanocytes per 2‐mm diameter fields in pigmented actinic keratosis (n = 16), lichenoid keratosis (n = 7), junctional melanocytic nevus (n = 6), keratosis with atypical melanocytic proliferation (n = 17) and MIS (n = 10). These cases represented an older population (68 years median age) and the head and neck (50%) was the most common anatomic site. MART‐1 score was significantly higher than SOX10 (P value <.05) in solar keratoses, but showed no difference in detecting melanocytic proliferations, demonstrating their equal detection rate of melanocytes. The sensitivity of both MART‐1 and SOX10 was 100%, while their specificities were 17% and 96%, respectively. These results show that SOX10 is more specific than MART‐1 in distinguishing epidermal melanocytes on sun‐damaged skin by avoiding overdiagnosis of melanoma.
Discerning lymphatics from blood vessels using lineage-specific markers has advanced our understanding of tumor lymphangiogenesis. Many studies have demonstrated that the newly formed lymphatics are ...the result of a dynamic and complex interplay between lymphatic endothelium, tumor cells, secreted growth factors, and inhibitors in the tumor microenvironment. Results provide evidence that lymphatics play an active role in cancer metastasis rather than once thought to be passively invaded by infiltrating tumor cells. The latest discovery of Bracher et al. (2012) further supports a dynamic role for lymphatics-mediated melanoma metastasis to sentinel lymph node prompted by tumor-derived epidermal growth factor.
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