Conceived and designed the experiments: SSD AF. Performed the experiments: ZM EF. Analyzed the data: SSD ZM HS EF. Contributed reagents/materials/analysis tools: SSD. Wrote the paper: SSD. Confirmed histological diagnoses and build TMA: DC.
The authors have declared that no competing interests exist.
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 (
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.
Small (18–24 nucleotides) non-coding RNAs, including microRNAs (miRNAs), regulate gene expression in many biological processes
Dicer, a member of the RNase III family of double-stranded RNases, is a central enzyme in a multi-component miRNA biogenesis pathway where the Drosha/DGCR8 complex and Dicer act sequentially to crop long primary and precursor miRNAs into functionally mature miRNAs
Although changes in the expression level of Dicer, and possibly other miRNA processing enzymes, are of clinical significance in some cancers, these alterations or their clinical consequences in melanoma or other skin cancers remain unknown.
We demonstrate herein that Dicer protein is specifically up-regulated in melanoma compared to other skin cancers such as carcinomas and sarcomas. This up-regulation is further specific to melanoma subtype and is significantly associated with clinical stage in patients with cutaneous melanoma. Additional analyses of Dicer levels in tissue culture cells support a general up-regulation of Dicer in melanoma and suggest an autonomous up-regulation in the absence of supporting cells.
We used seven different tissue microarray (TMA) slides, prepared from formalin-fixed paraffin embedded (FFPE) specimens, representing a wide variety of human skin tumors, both benign and malignant, from 404 different patients arrayed onto slides at 80 to 100 cores per slide (in duplicates or triplicates). This set also included complete tumor FFPE sections. The TMA slides included normal tissue (skin and other organs), melanocytic nevi (compound, intradermal and blue), primary melanomas (acrolentiginous, cutaneous, desmoplastic, mucosal, ocular), metastatic melanomas, squamous cell carcinoma, adenocarcinoma (eccrine, sebaceous and metastatic), basal cell carcinoma, sarcomas (dermatofibrosarcoma protuberans and fibrosarcomas), neurofibromas, and benign and malignant schwannomas. We purchased TMA slides ME1001, ME801, SK803 and BC21011 from US Biomax, Inc. (Rockville, MD) and CS38-01-001 from Cybrdi (Rockville, MD). The squamous or melanocytic differentiation of tumors was confirmed by immunohistochemical staining for cytokeratins or HMB-45, respectively, by the manufacturers. DC-1 and DC-2 TMAs were built at Stanford University Pathology Department (DC). For most TMAs, the information on age, sex and anatomic sites were available. For primary melanomas arrayed on DC-1 and DC-2 TMAs, the information on age, tumor thickness (Breslow's depth, Clark's level of invasion and histological type were available. Complete tumor sections were also examined that included cutaneous melanomas (n = 19), metastatic melanomas to lymph node (n = 5) and common nevi (n = 9). At least two pathologists/dermatopathologists (SSD and/or DC) confirmed all diagnoses. The institutional review boards of the Stanford University Medical Center and the University of Connecticut Health Center approved this protocol.
Out of 95 patients diagnosed with primary cutaneous melanoma, we had complete clinical follow-up information for 19 patients (mean = 26.6 month, range 7 to 64 months). The majority of cases were obtained from the Stanford University Pathology archive from 1997-2006. Clinical information included gender, age, anatomic site of the primary tumor, relapse-free survival, American Joint Committee on Cancer (AJCC) pathological (tumor, node, metastasis) stage
Immunostaining for all cases (TMAs and complete sections) was performed on 4-mm-thick FFPE sections mounted on charged slides and incubated at 60°C overnight. All slides were incubated with the anti-human Dicer monoclonal antibody through Clonegene (1∶100, clone mab 13D6, Hartford, CT), generously provided by Dr. Henry Furneaux. We performed antigen retrieval using DakoCytomation Target Retrieval Solution (High pH, Catalogue No. S-3308) and biocare digital decloaking chamber (Biocare Medical, Concord, CA) at 100°C for 10 minutes, followed by treatment of 3% H2O2 to block the endogenous peroxidase activity. The slides were incubated at room temperature for 1 hour with anti-Dicer antibodies at 1∶100 dilution. Hematoxylin was used for counterstaining. Following the manufacturer's instructions, we developed the immunohistochemical stain using EnVision™+ kit (DAKO, Carpinteria, CA).
The expression of Dicer was examined in normal skin and other organs; Dicer immunoreactivity was seen in the cytoplasm without nuclear staining. A semi-quantitative, four-point ordinal immunoreactivity score was established: “0” reflected the lack of Dicer immunoreactivity and was the most common pattern in normal skin, carcinomas and sarcomas. Weakly positive (“1”) staining was observed in epidermal keratinocytes and melanocytic nevi. Moderately positive (“2”) staining was assigned to modest granular staining. Strongly positive (“3”) staining consisted of diffuse and homogenous staining. Basal levels of Dicer were detected in epidermal keratinocytes. Only staining of tumor cells was scored in comparison to adjacent keratinocytes (internal positive control). Melanophages, specialized macrophages containing dark, brown coarse melanin pigment were not scored. Two investigators scored the stained slides independently. Scores for multiple cores from one case were averaged, and final Dicer scores were categorized into a three-level grouping of Negative, Low (>0 and ≤1.5) or High (>1.6) for analyses that included all 404 patients. For the exploratory analyses (n = 19), a dichotomous breakdown of Negative or Positive (>0) Dicer expression was used. These analyses included the following clinical and histopathological variables: AJCC Stage (I, II, III, IV); Distant Metastases, Non-Sentinel Node Metastases, Organ Metastases, Evidence of Regression, and Ulceration, which were treated as dichotomous variables; Melanoma Histology (Superficial Spreading, Nodular, Acrolentiginous or Lentigo Maligna); Vital status (alive, dead); and, Mitotic Index (mm2), Clark's Level and Breslow's depth of invasion, i.e. Tumor Thickness (mm) were treated as continuous variables. Pearson Chi-Square tests were employed to assess categorical levels of Dicer status. Kruskal-Wallis (k = 3) or Mann-Whitney (k = 2) non-parametric tests were used for analyses when Dicer expression was treated as a continuous value as well as for analyses of Mitotic Index, Clarks Level and Breslow's depth of invasion. SPSS version 18.0 was used, and tests were two-sided in all analyses.
Using NextBio (nextbio.com), we mined and pooled publically available gene expression profiling data interrogating the mRNA levels of the genes encoding all of the known enzymes involved in the canonical miRNA biogenesis and maturation pathway (
The detailed summary of cell lines is shown (
Cultured cells were lysed in NP40 Cell Lysis Buffer (Invitrogen, Carlsbad, CA) and spun at 16,000×g to extract soluble proteins. Twenty microgram of total protein was resolved on a 4-20% Tris-Glycine gradient gel (BioRad) and blotted onto a nitrocellulose membrane. The membrane was blocked with 5% powdered nonfat milk in TBST buffer for 1 hour, then incubated with 1∶500 dilution of anti-Dicer or 1∶50,000 dilution of anti-SDHA antibody (Abcam) or 1∶1000 dilution of α-tubulin antibody overnight at 4°C. The membrane was then washed three times with TBST buffer and incubated with anti mouse HRP conjugated secondary antibody, washed thrice with TBST, developed with the ECL Western Blotting Substrate (Pierce, Rockford, IL) and imaged and analyzed on a Kodak Image Station 4000 MM Pro (Carestream Health, Rochester, NY). Relative band intensity for Dicer was normalized against SDHA or α-tubulin as a loading control and quantified according to pixel intensity using Adobe Photoshop.
For measuring mRNA levels, total RNA was extracted from cultured cells with TRIzol and the reverse transcription of purified RNA was performed using oligo(dT) priming and superscript II reverse transcription according to the manufacturer's instructions (Invitrogen). The quantification of Dicer and GAPDH transcripts by Real-time quantitative PCR amplification of a cDNA template corresponding to 15 ng total RNA was performed using TaqMan Universal PCR Master Mix and TaqMan gene expressions assay probes (Applied Biosystems, Foster City, CA, USA). As previously described
We initially sought to determine if Dicer is expressed in any of the major categories of human cutaneous malignancies, namely melanoma, carcinoma or sarcoma. To this end, we tested a large clinical sample set of formalin-fixed paraffin-embedded (FFPE) benign and malignant tumors (n = 404) including melanocytic nevi (benign melanocytic hyperplasia), a variety of melanoma subtypes (cutaneous, acrolentiginous, mucosal, ocular and desmoplastic), a variety of carcinomas (squamous, basal cell and eccrine) and sarcomas (
A) Dicer immunoreactivity was variable across different types of skin cancer by immunohistochemistry using tissue microarrays. B) Normal epidermal keratinocytes exhibited low, basal level of Dicer expression. C–E) The cytoplasm of basal cell carcinoma, squamous cell carcinoma and sarcoma cells were negative for Dicer, respectively. In contrast, melanoma cells strongly and diffusely expressed Dicer in both primary cutaneous (F) and metastatic (G) melanoma. Original magnification: A, 20X; B–C, 100X and D–G, 200X.
A) Intradermal melanocytic nevus cells weakly expressed Dicer in a small group of cells in the superficial dermis (arrowheads) whereas melanoma cells diffusely expressed Dicer at higher levels (B). However, two independent individuals with cutaneous melanoma (CM), both excised from the thigh, expressed Dicer at different levels (B, left and right cores). C–D) Both tissue cores are shown at a higher magnification (CM, thigh-1 and CM, thigh-2). The immunoreactivity for Dicer in melanoma cells was granular and cytoplasmic. E) Cancer cells strongly expressed Dicer in an acrolentiginous melanoma while they were negative for Dicer in a mucosal melanoma (F). Original magnification: A–B, 100X; C, E–F, 200X and D, 400X.
Dicer Immunoreactivity | |||||||||||
Negative | Low (≤1.5) | High (>1.6) | Mean ± SD | ||||||||
Total | n | % | n | % | n | % | |||||
|
Normal Tissue | 12 | 11 | 91.7% | 1 | 8.3% | 0 | - | 0.04±0.14 | ||
Melanocytic Nevus | 71 | 13 | 18.3% | 48 | 67.6% | 10 | 14.1% | 0.83±0.60 | |||
Cutaneous Melanoma | 95 | 18 | 18.9% | 33 | 34.7% | 44 | 46.4% | 1.40±0.96 | |||
Acrolentiginous Melanoma | 40 | 4 | 10.0% | 14 | 35.0% | 22 | 55.0% | 1.61±0.92 | |||
Mucosal Melanoma | 24 | 8 | 33.3% | 13 | 54.2% | 3 | 12.5% | 0.79±0.65 | |||
Ocular Melanoma | 4 | 0 | - | 2 | 50.0% | 2 | 50.0% | 1.50±0.58 | |||
Desmoplastic Melanoma | 8 | 5 | 62.5% | 2 | 25.0% | 1 | 12.5% | 0.44±0.73 | |||
Metastatic Melanoma | 52 | 2 | 3.8% | 21 | 40.4% | 29 | 55.8% | 1.71±0.83 | |||
Carcinoma | 73 | 45 | 61.6% | 24 | 32.9% | 4 | 5.5% | 0.39±0.59 | |||
Sarcoma | 12 | 11 | 91.7% | 1 | 8.3% | 0 | - | 0.08±0.29 | |||
Neural Tumors | 13 | 9 | 69.2% | 4 | 30.8% | 0 | - |
|
0.23±0.39 | < |
Pearson Chi-Square test for proportions.
Kruskal-Wallis (k = 3 or more) non-parametric test for continuous values.
Overall, when compared among all examined cutaneous malignancies, Dicer up-regulation was tumor-type specific by immunostaining, as Dicer was highly expressed by melanomas (metastatic and cutaneous) compared to carcinomas or sarcomas (
A) Primary cutaneous (n = 95) and metastatic (n = 52) melanomas had the highest levels of Dicer immunoreactivity vs. carcinomas (n = 73) and sarcomas (n = 12). B) Cutaneous (n = 95) and acrolentiginous (n = 40) melanomas had the highest levels of Dicer immunoreactivity vs. melanocytic nevi (n = 71), mucosal (n = 24) and desmoplastic (n = 8) melanomas. Dicer immunoreactivity is shown as mean (boxed) ±2 standard error (SE). The statistical significance was measured for all independent samples comparing to each other (Kruskal-Wallis Test,
DICER1 | ||
Fold change | ||
|
NS | NS |
|
NS | NS |
|
NS | NS |
|
0.0108 | +2.39 |
|
0.0055 | +2.54 |
|
0.0006 | −2.17 |
|
0.0074 | −1.49 |
|
NS | NS |
|
NS | NS |
|
NS | NS |
|
NS | NS |
|
0.0323 | +2.21 |
|
0.0391 | −3.25 |
|
0.0244 | +2.95 |
|
0.0242 | +1.85 |
|
NS | NS |
|
0.0082 | +2.36 |
|
0.0045 | +2.42 |
|
1.70E-06 | +2.04 |
|
NS | NS |
To stay consistent with the direction of disease progression, the sign (+ or −) was changed appropriately. Fold changes with a (−) sign are lower and those with a (+) sign are higher than the disease group to which it was compared. For example, PM showed 2.36-fold increase in Dicer mRNA levels when compared to NS. The abbreviations of disease groups are as follows: DNL-dysplastic nevus low-grade atypia; DNH-dysplastic nevus high-grade atypia; CN-common nevus; C I RGP-Clark's level I radial growth phase; C II RGP-Clark's level II radial growth phase; C III VGP-Clark's level III vertical growth phase; C IV VGP-Clark's level IV vertical growth phase; C V VGP-Clark's level V vertical growth phase; LNMM-lymph node metastatic melanoma; DMM-dermal metastatic melanoma; MIS-melanoma in situ; PM-primary melanoma invasive; MM-metastatic melanoma; NS-normal skin; BCC-basal cell carcinoma; SCC-squamous cell carcinoma. NS-not significant.
To characterize Dicer expression pattern and distribution, we immunostained melanocytic nevi, cutaneous and metastatic melanomas in complete sections (n = 33). In 30% of cutaneous melanomas, the intratumoral expression of Dicer varied where immunoreactivity was focally high compared no expression in other areas within the same lesion (
A–B) The mean values for melanoma mitotic index (per mm2) and Breslow's depth of invasion significantly correlated with Dicer expression (
Dicer Immunoreactivity | |||
Negative n = 5 | Positive n = 14 | ||
Mean ± SD | Mean ± SD | ||
|
0.40±0.55 | 3.07±4.16 |
|
|
1.07±0.43 | 2.25±1.31 |
|
|
3.20±0.84 | 3.79±0.43 | 0.08 |
Mann-Whitney (k = 2) non-parametric test for continuous values.
Dicer Immunoreactivity | ||||||||
Negative | Positive | Mean ± SD | ||||||
n | % | n | % | |||||
|
Superficial spreading | 5 | 29.4% | 12 | 70.6% | 1.35±0.99 | ||
Nodular | 0 | - | 2 | 100% | 0.99 | 2.00±1.41 | 0.43 |
|
|
Absent | 2 | 28.6% | 5 | 71.4% | 1.43±1.13 | ||
Present | 3 | 25.0% | 9 | 75.0% | 0.99 | 1.41±0.99 | 0.96 |
|
|
Absent | 4 | 25.0% | 12 | 66.7% | 1.50±1.03 | ||
Present | 1 | 50.0% | 1 | 50.0% | 0.49 | 0.50±0.71 | 0.18 |
|
|
Absent | 5 | 38.5% | 8 | 61.5% | 1.23±1.09 | ||
Present | 0 | - | 6 | 100% | 0.13 | 1.83±0.75 | 0.28 |
|
|
I | 5 | 71.4% | 2 | 28.6% | 0.71±1.25 | ||
II | 0 | - | 4 | 100% | 1.75±0.50 | |||
III | 0 | - | 2 | 100% | 1.50±0.71 | |||
IV | 0 | - | 6 | 100% |
|
2.00±0.63 | 0.17 |
|
|
Negative | 5 | 38.5 | 8 | 61.5% | 1.23±1.09 | ||
Positive | 0 | - | 6 | 100% | 0.12 | 1.83±0.75 | 0.28 |
|
|
Negative | 5 | 45.5% | 6 | 54.5% | 1.00±1.10 | ||
Positive | 0 | - | 5 | 100% | 0.12 | 2.20±0.45 |
|
|
|
Absent | 5 | 33.3% | 10 | 66.7% | 1.33±1.11 | ||
Present | 0 | - | 4 | 100% | 0.53 | 1.75±0.50 | 0.52 |
|
|
M0 | 5 | 38.5% | 8 | 61.5% | 1.15±1.07 | ||
M1 | 0 | - | 6 | 100% | 0.13 | 2.00±1.41 | 0.10 |
|
|
Alive | 0 | - | 4 | 100% | 1.75±0.50 | ||
Dead | 5 | 33.3% | 10 | 66.7% | 0.53 | 1.33±1.11 | 0.52 |
Pearson Chi-Square test for Stage and Tumor Thickness (
Mann-Whitney (k = 2) non-parametric test for continuous values.
Kruskal-Wallis (k = 3 or more) non-parametric test for continuous values.
Three cases with unknown nodal status.
Our findings in clinical melanoma specimens raised the question of whether Dicer up-regulation might be intrinsic to the tumor cells. We compared Dicer protein levels between primary melanocytes, primary (n = 3) and metastatic (n = 3) melanoma cell lines. Western blot analysis combined with measured relative band intensity, normalized against succinate dehydrogenase (SDHA), showed >2 to 4-fold higher Dicer levels in melanoma cell lines (WM278, WM1552C and A375P) when compared to melanocyte-L or other melanoma cell lines (WM35 and A375M) (
A) Western blot analysis of Dicer shows a 219-kDa band. Relative band intensity was compared to succinate dehydrogenase (SDHA, 68 kDa) as a loading control. B) Western blot quantification showed >2 to 4-fold change in Dicer levels in melanoma cell lines (WM278, WM1552C and A375P) when compared to melanocyte-L or other melanoma cell lines (WM35 and A375M). C) Dicer mRNA expression did not correlate with mature let-7a expression
Given the differential levels of Dicer expression in melanoma cell lines and the prior finding of let-7a miRNA exerting a negative feedback loop on Dicer expression in lung and pancreatic cell lines
Since the expression of Dicer is significantly altered from common to dysplastic nevi to melanoma in situ to invasive and to metastatic melanoma, we interrogated the same combined dataset, which included 20 different disease groups and 25, 135 genes, for the mRNA levels of all the known enzymes involved in canonical miRNA biogenesis by performing a pooled analysis mining published whole genome oligo-microarray dataset
Combined Dicer immunoreactivity, presented herein (denoted by asterisk ‘*’), and mRNA transcriptional profiling
In this study, we examined the expression and the clinical relevance of Dicer in cutaneous melanoma. We showed that a large portion of cutaneous melanomas exhibited up-regulation of Dicer significantly associated with aggressive cancer features. We demonstrated definitive evidence that Dicer up-regulation is specific to the malignant proliferation of melanocytes (melanoma) and not keratinocytes (carcinoma) or fibroblasts (sarcoma) in 404 human skin tumors. Given that the “melanoma disease group” is a heterogeneous cancer, to have a complete representation, we compared Dicer expression among the various subtypes of melanomas occurring in glabrous (subungual, palm and sole) skin, non-glabrous skin, eye, mucosal sites (e.g. oral, urothelial and anal mucosa) and metastatic sites (variety of organs) to melanocytic nevi. These immunostaining results clearly showed that Dicer up-regulation was specific to cutaneous, acrolentiginous and metastatic melanomas. To complement the immunostaining, we carried out a pooled analysis using two recent large studies that profiled gene expression pattern in cutaneous tumors. This analysis corroborated our immunostaining data and indicated that at least a component of Dicer up-regulation in melanoma is due to differences in mRNA accumulation.
Although our results provide strong evidence that up-regulated Dicer was tumor-cell specific and reflective of mRNA levels, these results provided little mechanistic explanation. Given the involvement of Dicer, Dicer products, and associated components of the RNAi machinery in diverse cellular processes
First, the presence of cell-specific transcription factors might be expected to partially explain the difference in Dicer expression between melanocytes vs. keratinocytes or fibroblasts. Recently, it has been shown that Dicer is a direct transcriptional target of microphthalmia-associated transcription factor (MITF); tissue-restricted master transcriptional regulator of melanocytes; and that targeted KO of Dicer is lethal to melanocytes
We found that the expression of Dicer was variable among cutaneous melanomas (n = 95) where, the great majority (81%) of cases expressed it while 19% of cases demonstrated an absence of immunoreactivity. Postulating that this difference could be clinically relevant, we examined correlations with other clinical features, observing a statistically significant association between Dicer expression and melanoma mitotic index and Breslow's depth of invasion, both indicative of a more aggressive cancer (these are two of the three most important AJCC staging parameters) currently used to determine prognosis for melanoma patients
Overall, our results show definitive up-regulation of Dicer in cutaneous melanoma, compared to other skin cancer types, which correlated with a more aggressive behavior. When confirmed by independent studies in larger cohorts, increased Dicer expression may serve as a clinically useful prognostic biomarker for cutaneous melanoma patients. Beyond this, a combined understanding of deregulated Dicer and its influence on the expression pattern of mature miRNAs may lead to indications of directions in which small RNA modulations may contribute therapeutically in melanoma treatment. During the revision of this manuscript, we noted an abstract for a small pilot study
Expression of Dicer in primary cutaneous and metastatic melanomas by immunohistochemistry using complete tumor sections. A) Cancer cells focally expressed Dicer at high levels in the left margin (arrowhead) compared to the cancer cells in the center (asterisks) that were negative for Dicer in the same cutaneous melanoma (CM). B) In another CM, cancer cells expressed Dicer along the dermal-epidermal junction and follicular epithelium (
(TIF)
Dicer mRNA expression did not correlate with the expression of any mature miRNA members in the let-7 family
(TIF)
Pooled analysis performed on enzymes in miRNA biogenesis pathway in publically available gene expression profiling studies (clinical sample size = 139, disease groups = 20).
(DOCX)
Summary of cell lines, source and type.
(DOCX)
Dicer expression in relation to cutaneous tumors, sex (n = 328) and age (n = 335).
(DOCX)
Dicer expression in relation to melanoma type and anatomic site (n = 133).
(DOCX)
We acknowledge Dr. Stanley N. Cohen for the gift of melanoma cell lines (A2058, A375P, C32 and A375SM).