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The Long Noncoding RNA SPRIGHTLY Regulates Cell Proliferation in Primary Human Melanocytes

Open ArchivePublished:January 29, 2016DOI:https://doi.org/10.1016/j.jid.2016.01.018
      The long noncoding RNA SPRIGHTLY (formerly SPRY4-IT1), which lies within the intronic region of the SPRY4 gene, is up-regulated in human melanoma cells compared to melanocytes. SPRIGHTLY regulates a number of cancer hallmarks, including proliferation, motility, and apoptosis. To better understand its oncogenic role, SPRIGHTLY was stably transfected into human melanocytes, which resulted in increased cellular proliferation, colony formation, invasion, and development of a multinucleated dendritic-like phenotype. RNA sequencing and mass spectrometric analysis of SPRIGHTLY-expressing cells revealed changes in the expression of genes involved in cell proliferation, apoptosis, chromosome organization, regulation of DNA damage responses, and cell cycle. The proliferation marker Ki67, minichromosome maintenance genes 2-5, antiapoptotic gene X-linked inhibitor of apoptosis, and baculoviral IAP repeat-containing 7 were all up-regulated in SPRIGHTLY-expressing melanocytes, whereas the proapoptotic tumor suppressor gene DPPIV/CD26 was down-regulated, followed by an increase in extracellular signal-regulated kinase 1/2 phosphorylation, suggesting an increase in mitogen-activated protein kinase activity. Because down-regulation of DPPIV is known to be associated with malignant transformation in melanocytes, SPRIGHTLY-mediated DPPIV down-regulation may play an important role in melanoma pathobiology. Together, these findings provide important insights into how SPRIGHTLY regulates cell proliferation and anchorage-independent colony formation in primary human melanocytes.

      Abbreviations:

      CDK (cyclin-dependent kinase), DPPIV (dipeptidyl peptidase-IV), ERK (extracellular signal-regulated kinase), lncRNA (long noncoding RNA), MAPK (mitogen-activated protein kinase), MCM (minichromosome maintenance), siRNA (small interfering RNA)

      Introduction

      Melanoma is a skin cancer that arises from pigment-producing cells called melanocytes and is the leading cause of skin cancer-related death in the United States. Because melanoma is intrinsically resistant to many existing therapies, there is a pressing need to better understand the gene regulatory pathways that contribute to melanomagenesis.
      A class of regulatory RNAs greater than 200 nucleotides in length known as long noncoding RNAs (lncRNAs) has recently gained attention as oncogenes or tumor suppressor genes (
      • Amaral P.P.
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      Noncoding RNA in development.
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      • Taft R.J.
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      ). The lncRNAs were originally dismissed as nonfunctional transcriptional noise (
      • Clark M.B.
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      The reality of pervasive transcription.
      ) because although some lncRNAs are translated into short polypeptides, the majority of lncRNAs are rarely or never translated (
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      Long noncoding RNAs are rarely translated in two human cell lines.
      ,
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      Pinstripe: a suite of programs for integrating transcriptomic and proteomic datasets identifies novel proteins and improves differentiation of protein-coding and non-coding genes.
      ). However, lncRNAs exhibit exquisite spatial and temporal context-dependent expression in different cell types, commensurate with their presumed regulatory role (
      • Khaitan D.
      • Dinger M.E.
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      • Crawford J.
      • Smith M.A.
      • Mattick J.S.
      • et al.
      The melanoma-upregulated long noncoding RNA SPRY4-IT1 modulates apoptosis and invasion.
      ,
      • Mercer T.R.
      • Dinger M.E.
      • Sunkin S.M.
      • Mehler M.F.
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      Specific expression of long noncoding RNAs in the mouse brain.
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      MEN epsilon/beta nuclear-retained non-coding RNAs are up-regulated upon muscle differentiation and are essential components of paraspeckles.
      ). At the molecular level, lncRNAs influence target gene expression at specific genomic loci either by directly interacting with chromatin regulatory proteins and/or by modulating the activity of their interacting partners (
      • Dinger M.E.
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      Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression.
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      ,
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      ). The lncRNAs can function as decoys for bound proteins and can alter protein structure and function (
      • Rinn J.L.
      • Chang H.Y.
      Genome regulation by long noncoding RNAs.
      ). They play important physiological roles in normal cellular development and differentiation (
      • Dinger M.E.
      • Amaral P.P.
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      • Pang K.C.
      • Bruce S.J.
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      • et al.
      Long noncoding RNAs in mouse embryonic stem cell pluripotency and differentiation.
      ), but changes in lncRNA expression are also associated with several diseases, including cancer, heart disease, Alzheimer disease, psoriasis, and spinocerebellar ataxia type 8 (
      • Esteller M.
      Non-coding RNAs in human disease.
      ). For example, in cancer, increased HOTAIR expression is associated with poor prognosis pancreatic cancer (
      • Kim K.
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      HOTAIR is a negative prognostic factor and exhibits pro-oncogenic activity in pancreatic cancer.
      ), and increased expression of PCGEM1 and PCA3/DD3 is associated with the development of prostate cancer (
      • Ifere G.O.
      • Ananaba G.A.
      Prostate cancer gene expression marker 1 (PCGEM1): a patented prostate-specific non-coding gene and regulator of prostate cancer progression.
      ).
      We previously identified a number of lncRNAs that are differentially expressed in melanoma cell lines relative to melanocytes and keratinocytes (
      • Khaitan D.
      • Dinger M.E.
      • Mazar J.
      • Crawford J.
      • Smith M.A.
      • Mattick J.S.
      • et al.
      The melanoma-upregulated long noncoding RNA SPRY4-IT1 modulates apoptosis and invasion.
      ,
      • Mazar J.
      • Sinha S.
      • Dinger M.E.
      • Mattick J.S.
      • Perera R.J.
      Protein-coding and non-coding gene expression analysis in differentiating human keratinocytes using a three-dimensional epidermal equivalent.
      ). One of these, SPRIGHTLY (GenBank Accession ID AK024556), was highly expressed and localized predominantly in the cytoplasm in melanoma cells but expressed at low levels in primary human melanocytes (
      • Khaitan D.
      • Dinger M.E.
      • Mazar J.
      • Crawford J.
      • Smith M.A.
      • Mattick J.S.
      • et al.
      The melanoma-upregulated long noncoding RNA SPRY4-IT1 modulates apoptosis and invasion.
      ). SPRIGHTLY is derived from the intronic region of the SPRY4 gene, and its predicted secondary structure contains several long hairpins (
      • Khaitan D.
      • Dinger M.E.
      • Mazar J.
      • Crawford J.
      • Smith M.A.
      • Mattick J.S.
      • et al.
      The melanoma-upregulated long noncoding RNA SPRY4-IT1 modulates apoptosis and invasion.
      ). Loss of function of SPRIGHTLY in melanoma cells prevented cell growth and differentiation and induced apoptosis (
      • Khaitan D.
      • Dinger M.E.
      • Mazar J.
      • Crawford J.
      • Smith M.A.
      • Mattick J.S.
      • et al.
      The melanoma-upregulated long noncoding RNA SPRY4-IT1 modulates apoptosis and invasion.
      ).
      In this study, we sought to examine how SPRIGHTLY contributes to melanocyte dedifferentiation and melanomagenesis by characterizing its molecular function. We hypothesized that the lncRNA SPRIGHTLY and its target genes dedifferentiate melanocytes and contribute to the development of human melanomas. To test the hypothesis, we ectopically expressed SPRIGHTLY in normal human melanocytes and knocked it down in melanoma cells.
      SPRIGHTLY ectopically expressed in human melanocytes increased cellular proliferation, invasion, and colony formation, and induced a multinucleated dendritic-like phenotype. RNA sequencing and mass spectrometric analysis revealed changes in subsets of genes and proteins involved in cell proliferation, apoptosis, chromosome organization, regulation of DNA damage response, and cell cycle progression. Accordingly, the cell proliferation marker Ki67, minichromosome maintenance (MCM) genes (MCM2-5), and antiapoptotic genes X-linked inhibitor of apoptosis and baculoviral IAP repeat-containing 7 were all up-regulated in SPRIGHTLY-expressing melanocytes. In contrast, expression of the proapoptotic tumor suppressor gene DPPIV was down-regulated. Loss-of-function experiments in the melanoma cell line A375 confirmed the opposite effects. SPRIGHTLY contributes to the regulation of proliferation and apoptosis pathway genes in melanocytes and melanomas.

      Results

      Ectopic expression of SPRIGHTLY in normal human melanocytes results in multinuclear and multidendrite cells

      SPRIGHTLY is expressed at significantly lower levels in human melanocytes than melanoma cells (
      • Khaitan D.
      • Dinger M.E.
      • Mazar J.
      • Crawford J.
      • Smith M.A.
      • Mattick J.S.
      • et al.
      The melanoma-upregulated long noncoding RNA SPRY4-IT1 modulates apoptosis and invasion.
      ). To establish the molecular and cellular functions of SPRIGHTLY in melanocytes, normal human melanocytes were engineered to ectopically express the SPRIGHTLY transcript using a lentiviral vector. The same vector without SPRIGHTLY was used as a control. Ribonucleic acid-fluorescence in situ hybridization (see Supplementary Figure S1a online) and RNA sequencing (see Supplementary Figure S1b) confirmed ectopic expression of SPRIGHTLY in engineered cells. Interestingly, morphological examination of melanocytes that ectopically express SPRIGHTLY (SPRIGHTLY-EE) after 1 month of transfection revealed approximately 25% altered dendritic-like cell morphology with multiple enlarged nuclei compared to vector-only controls (Figure 1a). Next, we stained SPRIGHTLY-EE cells by immuno-histochemistry with α-MELAN-A antibody to observe probable pigmentation changes. Results reveal that SPRIGHTLY did not interfere with the melanocyte pigmentation (see Supplementary Figure S2 online). It has previously been reported that oncogene-induced senescence results in multinucleated giant cells (
      • Leikam C.
      • Hufnagel A.
      • Schartl M.
      • Meierjohann S.
      Oncogene activation in melanocytes links reactive oxygen to multinucleated phenotype and senescence.
      ). Oncogene-induced senescence is thought to be a natural antitumorigenic effect that occurs in response to extreme growth stimulatory signals from activated oncogenes.
      Figure 1
      Figure 1Morphological and gene ontology changes in melanocytes that ectopically express SPRIGHTLY. (a) Melanocytes ectopically expressing SPRIGHTLY (melanocyte SPRIGHTLY-EE) show a multinucleated and multidendritic-like phenotype. (b) Gene ontology and KEGG pathway enrichments determined from genes significantly differentially expressed between SPRIGHTLY-EE melanocytes compared to vector-only control melanocytes by RNA sequencing. P < 0.005. Bar = 10 μm.

      SPRIGHTLY regulates chromatin and cell cycle pathway genes in normal human melanocytes

      To identify the molecular mechanisms by which ectopic expression of SPRIGHTLY modifies cell phenotype, SPRIGHTLY-EE and vector-only cells were subjected to RNA sequencing, mass spectrometry, quantitative PCR, and protoarray-based analyses. Comparison of the transcriptomic profiles revealed 740 significantly (P < 0.05) differentially expressed genes (see Supplementary Dataset S1 online). There was gene ontology enrichment for biological processes involved in DNA packaging, chromosome organization, DNA repair, cell cycle, and maintenance or establishment of chromatin architecture (mainly due to up-regulation of core histone genes) in SPRIGHTLY-EE cells (Figure 1b and Supplementary Dataset S2 online). Pathway analysis demonstrated significant (P < 0.005) enrichment for cell cycle, apoptosis, and DNA replication pathways, which was in keeping with the proliferative and multinuclear phenotype of SPRIGHTLY-expressing melanocytes. Notably, enrichment for chromatin-related gene expression is consistent with the multinucleated character of SPRIGHTLY-overexpressing cells and suggests that SPRIGHTLY may have a role in inducing DNA replication and nuclear division.
      Further enrichment analysis of RNA sequencing data for biological processes and cellular and molecular function revealed the presence of distinct gene clusters. These functional cluster categories are depicted in Figure 2 a–c. There was significant enrichment for genes in the chromosome organization, assembly, segregation, and condensation, DNA packaging, nucleosome positioning and assembly, DNA replication, and maintenance of chromatin architecture biological process groups (Figure 2a). Genes in the cellular function catenin complex and chromosome groups were overrepresented (Figure 2b), and genes in the molecular function structural constituent of myelin sheath and insulin-binding groups were also enriched (Figure 2c).
      Figure 2
      Figure 2Gene ontology (GO) enrichment. Statistically significant and differentially expressed genes were used for GO enrichment (SPRIGHTLY-EE vs melanocyte vector-only [VO]). GoGraphViz (Principe Felipe Research Center, Valencia, Spain) visualizations show (a) biological process, (b) cellular component, and (c) molecular function GO terms. Adjusted P < 0.005 for GO terms were considered significant. The GO enrichments and visualizations were performed using Babelomics 4.3 (Principe Felipe Research Center) (
      • Medina I.
      • Carbonell J.
      • Pulido L.
      • Madeira S.C.
      • Goetz S.
      • Conesa A.
      • et al.
      Babelomics: an integrative platform for the analysis of transcriptomics, proteomics and genomic data with advanced functional profiling.
      ).

      Cell proliferation genes are perturbed when SPRIGHTLY is ectopically expressed in melanocytes and knocked down in melanoma cells

      Because cell cycle and proliferative pathway genes appear to be perturbed in gene ontology analyses, RNA samples were subjected to cell cycle and proliferation quantitative PCR array analysis (Qiagen, Germany). Notably, MCM (MCM2, MCM3, MCM4, and MCM5), antigen-MKI67, cyclin-dependent kinase 1 (CDK1), and cell-division cycle protein 20 (CDC20) genes were significantly up-regulated in SPRIGHTLY-EE cells, suggesting that SPRIGHTLY plays an important role in cell cycle progression and proliferation (Figure 3). Therefore, we performed cell proliferation, invasion and colony formation assays in SPRIGHTLY-EE cells and found that SPRIGHTLY-expressing melanocytes are more proliferative, invasive, and form anchorage-independent colonies (see Supplementary Figure S3a–c online), further supporting the potential role of SPRIGHTLY in melanomagenesis. We additionally assayed SPRIGHTLY-EE cells for changes in cell senescence (β-galactosidase assay) but could detect no differences with control cells (data not shown).
      Figure 3
      Figure 3Differentially expressed proliferation, cell cycle, and apoptosis-related genes in SPRIGHTLY-EE cells compared to melanocyte vector-only. Relative expression of CDK1, CDC20, MCM2, MCM3, MCM4, MCM5, Ki-67, and TNFRSF25 in SPRIGHTLY-EE versus melanocyte vector-only.
      Because we observed SPRIGHTLY-EE cells showing increased cell proliferation, invasion, and colony forming ability, we hypothesize that these cells may be capable of forming tumors in SCID mice. To examine the oncogenic potential of SPRIGHTLY in vivo using mouse models, we implanted SPRIGHTLY-EE cells into SCID mice. As a control, we injected vector-only parental melanocytes and included groups of five mice for each experiment. The results are depicted in Supplementary Figure S4 online. Three months after cell implantation, we observed that SPRIGHTLY-EE cells, but not vector-only parental cells, initiated subcutaneous cell migration.
      MCM2 is the most widely studied MCM family member, and expression of MCM2 has been directly linked to cancer cell proliferation and invasion (
      • Guzinska-Ustymowicz K.
      • Stepien E.
      • Kemona A.
      MCM-2, Ki-67 and PCNA protein expressions in pT3G2 colorectal cancer indicated lymph node involvement.
      ,
      • Lian M.
      • Fang J.
      • Han D.
      • Ma H.
      • Feng L.
      • Wang R.
      • et al.
      Microarray gene expression analysis of tumorigenesis and regional lymph node metastasis in laryngeal squamous cell carcinoma.
      ,
      • Liu M.
      • Li J.S.
      • Tian D.P.
      • Huang B.
      • Rosqvist S.
      • Su M.
      MCM2 expression levels predict diagnosis and prognosis in gastric cardiac cancer.
      ,
      • Wojnar A.
      • Pula B.
      • Piotrowska A.
      • Jethon A.
      • Kujawa K.
      • Kobierzycki C.
      • et al.
      Correlation of intensity of MT-I/II expression with Ki-67 and MCM-2 proteins in invasive ductal breast carcinoma.
      ). MCM2 is up-regulated in primary cutaneous melanomas and cutaneous melanoma metastases compared to benign nevi (
      • Boyd A.S.
      • Shakhtour B.
      • Shyr Y.
      Minichromosome maintenance protein expression in benign nevi, dysplastic nevi, melanoma, and cutaneous melanoma metastases.
      ,
      • de Andrade B.A.
      • Leon J.E.
      • Carlos R.
      • Delgado-Azanero W.
      • Mosqueda-Taylor A.
      • de Almeida O.P.
      Expression of minichromosome maintenance 2, Ki-67, and geminin in oral nevi and melanoma.
      ). Because ectopic expression of SPRIGHTLY in melanocytes increased MCM2, we decided to investigate whether MCM2 knockdown by small interfering RNA (siRNA) in the stage IV melanoma cell line A375 has opposite effects. Quantitative PCR confirmed excellent MCM2 knockdown efficiency (∼90%; Figure 4a).
      • Boyd A.S.
      • Shakhtour B.
      • Shyr Y.
      Minichromosome maintenance protein expression in benign nevi, dysplastic nevi, melanoma, and cutaneous melanoma metastases.
      reported that MCM2 protein expression differs significantly in melanocytic neoplasms and can help distinguish benign tumors from their malignant counterparts. Therefore, we used an anti-MCM2 antibody to measure MCM2 protein expression in A375 parental and MCM2 siRNA knockdown to further confirm siRNA knockdown efficiency (Figure 4b).
      Figure 4
      Figure 4MCM2 knockdown on cell viability, growth, and invasiveness. A375 cells transfected with an MCM2-specific small interfering RNA (siRNA) and compared to scramble control siRNA. (a) Quantitative reverse transcriptase-PCR demonstrates the effect of MCM2-specific knockdown relative to scrambled control. (b) Immunohistochemistry staining of A375 cells using MCM2-specific antibodies. MCM2 staining shown in yellow (fluorescein isothiocyanate) and nuclei in blue (4',6-diamidino-2-phenylindole). Bar = 200 μm. (c) Knockdown of MCM2 affects A375 cell viability (CellTiter 96 AQueous). (d) Knockdown of MCM2 affects A375 cell number. (e) Knockdown of MCM2 suppresses A375 invasion. (f) Staining of A375 cells (crystal violet) 48 hours after transfection with MCM2 siRNA reveals a decrease in cell invasion. All results are expressed as mean ± standard deviation of three experiments. Bar = 200 μm. Data for (a) and (e) were normalized to the scramble-transfected control.
      Because MCM2 is a known cell proliferation gene/marker, we next measured cell proliferation in MCM2 knockdown A375 cells by direct cell counting. MCM2 knockdown reduced A375 cell numbers by ∼30% over a 48-hour period compared to scramble siRNA control cells (Figure 4c). This was corroborated in cell viability assays (CellTiter 96 AQueous, Promega, WI), which also revealed ∼30% decrease in viability in MCM2 knockdown cells over the same period of time (Figure 4d). Cell invasion was reduced by ∼50% in MCM2 knockdown A375 cells compared to controls (Figure 4e and f), an effect replicated by knockdown of the SPRIGHTLY transcript (
      • Khaitan D.
      • Dinger M.E.
      • Mazar J.
      • Crawford J.
      • Smith M.A.
      • Mattick J.S.
      • et al.
      The melanoma-upregulated long noncoding RNA SPRY4-IT1 modulates apoptosis and invasion.
      ). Next, we examined whether MCM2 knockdown had any effect on the activity of effectors of apoptosis but did not find any changes either in caspase 3 or 7 activity. Of note, SPRIGHTLY expression did not change when MCM2 was knocked down in A375 cells. However, SPRIGHTLY knockdown in A375 melanoma cells did result in down-regulation of both MCM2 and Ki67, suggesting that SPRIGHTLY does regulate cell proliferation and likely is located upstream of MCM2 and Ki67 gene regulatory pathways.
      Next, to further confirm cell proliferation, Ki67 immunostaining was performed in normal human melanocytes, SPRIGHTLY-EE cells, and A375 cells (Figure 5a). Ki67 was poorly expressed in melanocytes, showed increased expression in SPRIGHTLY-EE cells, and was highly expressed in A375 melanoma cells, supporting the notion that SPRIGHTLY contributes to increased cell proliferation in SPRIGHTLY-EE cells. This observation was further confirmed by knockdown of SPRIGHTLY in A375 cells, followed by measurement of Ki67 expression using RNA sequencing. As expected, Ki-67 expression was profoundly down-regulated in SPRIGHTLY knockdown A375 cells (see Supplementary Figure S5 online). Moreover RNA sequencing data were further analyzed for proliferation pathway genes using Ingenuity pathway analysis software (Qiagen), and several proliferative pathway genes were perturbed by SPRIGHTLY knockdowns (see Supplementary Figure S6 online). Together, these data indicate that SPRIGHTLY is important for cell proliferation in melanocytes and melanoma cells.
      Figure 5
      Figure 5Ki-67 and dipeptidyl peptidase-IV (DPPIV) expression in melanocytes ectopically expressing SPRIGHTLY. (a) Immunofluorescence staining of Ki-67 in SPRIGHTLY-EE, vector-only melanocytes, and A375 cells. Ki-67 nuclear staining is highest in A375 cells. Ki-67 staining is elevated in SPRIGHTLY-EE cells compared to vector-only controls. Bar = 80 μm. (b) Immunofluorescence staining of DPPIV in SPRIGHTLY-EE cells, vector-only melanocytes, and A375 cells. Control melanocytes display abundant cell-surface DPPIV, A375 cells express little or no DPPIV, and SPRIGHTLY-EE cells show intermediate expression levels, that is, an inverse pattern to Ki-67. Bar = 20 μm. (c) DPPIV protein levels (western blot) in SPRIGHTLY-EE cells relative to melanocytes alone and melanocytes transfected with empty vector. (d) Ectopic expression of SPRIGHTLY down-regulates DPPIV mRNA levels in melanocytes. (e) Small interfering RNA-mediated knockdown of SPRIGHTLY up-regulates DPPIV mRNA levels in A375 melanoma cells. Ab, antibody; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

      Antiapoptotic X-linked inhibitor of apoptosis and baculoviral IAP repeat-containing 7 are up-regulated and proapoptotic dipeptidyl peptidase-IV is down-regulated in SPRIGHTLY-EE cells

      Further analysis of protein array data revealed that a group of antiapoptotic proteins, including baculoviral IAP repeat-containing 7 and the X-linked inhibitor of apoptosis proteins, was induced in SPRIGHTLY-EE cells compared to controls. Conversely, proapoptotic proteins such as TRAIL-R2 (DR5) and dipeptidyl peptidase-IV (DPPIV) were clearly decreased (see Supplementary Figure S7 online). These results suggest that SPRIGHTLY expression is associated with changes in antiapoptotic, proproliferative, and DNA-packaging genes, although whether these are indirect or direct effects remains to be determined.
      Of the identified gene products modulated by SPRIGHTLY, DPPIV is known to be directly associated with the molecular etiology of human melanomas (
      • Nielsen P.S.
      • Riber-Hansen R.
      • Steiniche T.
      Immunohistochemical double stains against Ki67/MART1 and HMB45/MITF: promising diagnostic tools in melanocytic lesions.
      ,
      • Pethiyagoda C.L.
      • Welch D.R.
      • Fleming T.P.
      Dipeptidyl peptidase IV (DPPIV) inhibits cellular invasion of melanoma cells.
      ). DPPIV is a known proapoptotic tumor suppressor that is usually highly expressed in normal melanocytes but shows reduced expression in SPRIGHTLY-EE cells and has undetectable expression in A375 cells (Figure 5b). Therefore, we analyzed protein expression of DPPIV in SPRIGHTLY-EE and vector-only cells by western blotting, which confirmed that DPPIV protein expression is significantly lower in SPRIGHTLY-EE cells compared to controls (Figure 5c). Because it has been previously reported that DPPIV expression is known to inhibit mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase 1/2 (ERK1/2) activation (
      • Wesley U.V.
      • McGroarty M.
      • Homoyouni A.
      Dipeptidyl peptidase inhibits malignant phenotype of prostate cancer cells by blocking basic fibroblast growth factor signaling pathway.
      ), we decided to check ERK1/2 phosphorylation by western blot analysis. The results are depicted in the Supplementary Figure S8 online. An examination of SPRIGHTLY-EE cells revealed an increase in phosphorylation of ERK1/2 compared to that seen in melanocyte vector-only control cells, suggesting that the loss of DPPIV may allow for an increase in activation of MAPK. Given that DPPIV expression can be silenced by promoter hypermethylation in melanomas (
      • McGuinness C.
      • Wesley U.V.
      Dipeptidyl peptidase IV (DPPIV), a candidate tumor suppressor gene in melanomas is silenced by promoter methylation.
      ), DPPIV promoter CpG islands were examined for possible promoter hypermethylation. However, no aberrant CpG island methylation was identified (see Supplementary Figure S9 online), indicating that DPPIV down-regulation may occur via an alternate (i.e., other than epigenetic) mechanism.
      To further examine the relationship between SPRIGHTLY and DPPIV, SPRIGHTLY and DPPIV mRNA levels were measured by quantitative PCR under two opposing conditions (increased and decreased levels of SPRIGHTLY). Our results revealed a reciprocal relationship between DPPIV and SPRIGHTLY expression levels: decreased DPPIV mRNA levels were associated with increased SPRIGHTLY, whereas increased DPPIV mRNA levels were associated with decreased SPRIGHTLY expression (Figure 5d and e). Because DPPIV is a known proapoptotic tumor suppressor gene that is highly expressed in normal human melanocytes but its expression is lost in malignant melanoma, our results imply that a higher SPRIGHTLY:DPPIV ratio most likely contributes to the malignant phenotype. Thus, our study provides evidence that SPRIGHTLY may contribute to malignant transformation of melanocytes by regulating a number of cancer hallmarks.

      Discussion

      The lncRNAs are involved in numerous aspects of human physiology and pathology. Until recently, these transcripts had been dismissed as nonfunctional noise and were regarded as junk. Although our understanding of the function of lncRNAs has increased rapidly, large knowledge gaps remain about how lncRNAs are regulated and how they regulate other genes and proteins.
      In this study, we provide evidence that stable lentiviral expression of the lncRNA SPRIGHTLY in normal human melanocytes contributes to the regulation of cell proliferation, apoptosis, chromosome organization, regulation of DNA damage response, and cell cycle in melanocytes. Previously, we reported that SPRIGHTLY, which is transcribed from the first intron of the Sprouty 4 gene (SPRY4), is expressed at low levels in normal human melanocytes but is highly up-regulated in human melanoma cells and patient samples (
      • Khaitan D.
      • Dinger M.E.
      • Mazar J.
      • Crawford J.
      • Smith M.A.
      • Mattick J.S.
      • et al.
      The melanoma-upregulated long noncoding RNA SPRY4-IT1 modulates apoptosis and invasion.
      ). Importantly, siRNA-mediated knockdown of SPRIGHTLY in melanoma cells decreased cell proliferation and invasion and increased apoptosis. To investigate this further, we affinity purified SPRIGHTLY from melanoma cells and used mass spectrometry to identify protein interactions. Of these, the protein phosphatidate phosphatase (lipin 2) was found to be a major binding partner involved in lipid metabolism (
      • Mazar J.
      • Zhao W.
      • Khalil A.M.
      • Lee B.
      • Shelley J.
      • Govindarajan S.S.
      • et al.
      The functional characterization of long noncoding RNA SPRY4-IT1 in human melanoma cells.
      ). These findings support a role for SPRIGHTLY in multiple regulatory pathways in melanomas and suggest that SPRIGHTLY and its target genes may be important in melanocyte dedifferentiation and their transformation into melanomas.
      Here we report that melanocytes engineered to stably express SPRIGHTLY display increased cell proliferation and a multinucleated dendritic-like morphology. The melanocyte dendrite is a specialized structure that is formed in response to hormones (e.g., α–melanocyte-stimulating hormone) and UV light and functions to transport melanosomes to neighboring keratinocytes.
      • Scott G.A.
      • Cassidy L.
      Rac1 mediates dendrite formation in response to melanocyte stimulating hormone and ultraviolet light in a murine melanoma model.
      reported that transfection of an expression vector encoding constitutively active rac1 protein induces the formation of branching actin-based structures in B16F1 murine melanoma cells and melanoma cell lines. However, we did not observe an increase in rac1 expression in SPRIGHTLY-EE cells compared to parental controls. Therefore, we believe that SPRIGHTLY-induced multidendrite formation is rac1 independent.
      RNA sequencing and mass spectrometric analysis of SPRIGHTLY-EE cells revealed changes in subsets of genes involved in cell proliferation, apoptosis, chromosome organization, regulation of DNA damage response, and cell cycle. Interestingly, the cell proliferation marker MKI67 and MCM2-5 genes as well as the antiapoptotic genes X-linked inhibitor of apoptosis and baculoviral IAP repeat-containing 7 were all up-regulated in SPRIGHTLY-expressing melanocytes. In contrast, expression of the proapoptotic and tumor suppressor gene DPPIV was down-regulated.
      The protein encoded by the DPPIV gene is a transmembrane glycoprotein with serine exopeptidase activity. It is constitutively expressed on the surface of numerous cell types (including melanocytes) and plays an important role in immune regulation, signal transduction, and apoptosis (
      • Pro B.
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      CD26/dipeptidyl peptidase IV and its role in cancer.
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      • et al.
      The dipeptidyl peptidase IV family in cancer and cell biology.
      ). In melanocytes, down-regulation of DPPIV/CD26 is associated with malignant transformation (
      • Morrison M.E.
      • Vijayasaradhi S.
      • Engelstein D.
      • Albino A.P.
      • Houghton A.N.
      A marker for neoplastic progression of human melanocytes is a cell surface ectopeptidase.
      ,
      • Van den Oord J.J.
      Expression of CD26/dipeptidyl-peptidase IV in benign and malignant pigment-cell lesions of the skin.
      ,
      • Wesley U.V.
      • Albino A.P.
      • Tiwari S.
      • Houghton A.N.
      A role for dipeptidyl peptidase IV in suppressing the malignant phenotype of melanocytic cells.
      ). Conversely, inducible transduction of DPPIV into melanoma cells reverses the malignant phenotype to that of normal melanocytes (
      • Wesley U.V.
      • Albino A.P.
      • Tiwari S.
      • Houghton A.N.
      A role for dipeptidyl peptidase IV in suppressing the malignant phenotype of melanocytic cells.
      ), suggesting that DPPIV/CD26 down-regulation represents an important event in the pathogenesis of melanoma. Supporting this, DPPIV/CD26 has successfully been used as a diagnostic biomarker to discriminate malignant melanomas from deep penetrating nevi (
      • Roesch A.
      • Wittschier S.
      • Becker B.
      • Landthaler M.
      • Vogt T.
      Loss of dipeptidyl peptidase IV immunostaining discriminates malignant melanomas from deep penetrating nevi.
      ).
      • Morrison M.E.
      • Vijayasaradhi S.
      • Engelstein D.
      • Albino A.P.
      • Houghton A.N.
      A marker for neoplastic progression of human melanocytes is a cell surface ectopeptidase.
      showed that DPPIV/CD26 loss occurs late during malignant transformation and is associated with the emergence of growth factor independence and the development of specific chromosomal abnormalities. In addition, melanoma cells transfected with DPPIV have a longer lag period before entering the logarithmic growth phase, with growth inhibited when cells reach confluence. DPPIV reduces melanoma tumor growth proportional to expression (
      • Wesley U.V.
      • Albino A.P.
      • Tiwari S.
      • Houghton A.N.
      A role for dipeptidyl peptidase IV in suppressing the malignant phenotype of melanocytic cells.
      ), and DPPIV has been shown to promote apoptosis and inhibit cell migration and angiogenesis (
      • Arscott W.T.
      • LaBauve A.E.
      • May V.
      • Wesley U.V.
      Suppression of neuroblastoma growth by dipeptidyl peptidase IV: relevance of chemokine regulation and caspase activation.
      ). DPPIV expression increases the percentage of cells in G0–G1 phase, indicating that DPPIV/CD26 may promote cell cycle arrest (
      • Wesley U.V.
      • Tiwari S.
      • Houghton A.N.
      Role for dipeptidyl peptidase IV in tumor suppression of human non small cell lung carcinoma cells.
      ). DPPIV/CD26 expression decreases the ability of melanoma cells to grow in soft agar, indicating that DPPIV/CD26 is important for the inhibition of anchorage-independent growth, and DPPIV/CD26 inhibits the invasion of malignant melanoma cell lines (
      • Pethiyagoda C.L.
      • Welch D.R.
      • Fleming T.P.
      Dipeptidyl peptidase IV (DPPIV) inhibits cellular invasion of melanoma cells.
      ). Interestingly, ectopic expression of DPPIV/CD26 has been shown to induce marked phenotypic changes, with cells expressing DPPIV/CD26 exhibiting a more organized growth pattern and sheetlike appearance compared to parental melanoma cells and cells transfected with control vector (
      • Pro B.
      • Dang N.H.
      CD26/dipeptidyl peptidase IV and its role in cancer.
      ).
      In our study, ectopic expression of SPRIGHTLY was associated with down-regulation of DPPIV/CD26 in transfected melanocytes but not in parental melanocytes or control cells. This was validated using multiple assays including protein arrays, western blotting, and immunofluorescent staining. Conversely, knockdown of SPRIGHTLY in melanoma cells up-regulated DPPIV/CD26. Consistent with these findings, SPRIGHTLY-transfected melanocytes showed an increase in growth rate, invasiveness, and anchorage-independent colony formation (see Supplementary Figure S3). Because overexpression of SPRIGHTLY promoted anchorage-independent growth of melanocytes, we conducted in vivo tumor formation studies (utilizing SCID mice). As depicted in Supplementary Figure S4, 3 months after cell implantation we observed that SPRIGHTLY-EE cells, but not vector-only parental cells, produced subcutaneous cell migration. However, we believe that had we been able to maintain the mice for a longer period (>3 months), then even greater transformational phenotypes might have become evident. Likewise, combining SPRIGHTLY with mutations in genes such as PTEN, CDKN2A, BRAF, NRAS, GNQ11, and TP53 may have revealed additional oncogenic potential.
      Although the exact mechanism by which SPRIGHTLY regulates DPPIV/CD26 remains to be determined, DPPIV/CD26 has been reported to be regulated at the transcriptional level by promoter methylation, interferons, and retinoic acid (
      • Bauvois B.
      • Djavaheri-Mergny M.
      • Rouillard D.
      • Dumont J.
      • Wietzerbin J.
      Regulation of CD26/DPPIV gene expression by interferons and retinoic acid in tumor B cells.
      ) and at the protein level by posttranslational events (
      • Pereira D.A.
      • Gomes L.
      • El-Cheikh M.C.
      • Borojevic R.
      Dipeptidyl peptidase IV (CD26) activity in the hematopoietic system: differences between the membrane-anchored and the released enzyme activity.
      ,
      • Swenson L.I.
      Progress in tumor marker research.
      ). Here, DPPIV promoter CpG islands were examined for possible promoter hypermethylation; however, no aberrant CpG island methylation was identified. Because SPRIGHTLY is predominantly present in the cytoplasm in melanoma cells (
      • Khaitan D.
      • Dinger M.E.
      • Mazar J.
      • Crawford J.
      • Smith M.A.
      • Mattick J.S.
      • et al.
      The melanoma-upregulated long noncoding RNA SPRY4-IT1 modulates apoptosis and invasion.
      ) and localized to the polysome fraction (the site of protein synthesis), it is possible that SPRIGHTLY regulates DPPIV posttranscriptionally or translationally by an unknown mechanism.
      Interestingly, we found that the cellular proliferation marker Ki-67, CDK1, and MCM protein family genes MCM2-5 were significantly up-regulated in SPRIGHTLY-EE cells. MCM proteins are central players in both the initiation and the elongation of eukaryotic DNA replication (
      • Forsburg S.L.
      Eukaryotic MCM proteins: beyond replication initiation.
      ) and are regulated by CDKs (
      • Nguyen V.Q.
      • Co C.
      • Li J.J.
      Cyclin-dependent kinases prevent DNA re-replication through multiple mechanisms.
      ), including CDK1 (synonym: CDC2), which was up-regulated in melanocytes that ectopically expressed SPRIGHTLY. Activation of CDK1/CDC2 is dependent on MAPK (
      • Guadagno T.M.
      • Ferrell Jr., J.E.
      Requirement for MAPK activation for normal mitotic progression in Xenopus egg extracts.
      ,
      • Palmer A.
      • Gavin A.C.
      • Nebreda A.R.
      A link between MAP kinase and p34(cdc2)/cyclin B during oocyte maturation: p90(rsk) phosphorylates and inactivates the p34(cdc2) inhibitory kinase Myt1.
      ). Of note, DPPIV expression is known to inhibit MAPK-ERK1/2 activation (
      • Wesley U.V.
      • McGroarty M.
      • Homoyouni A.
      Dipeptidyl peptidase inhibits malignant phenotype of prostate cancer cells by blocking basic fibroblast growth factor signaling pathway.
      ). It is not surprising that in our study, the loss of DPPIV may have led to increased phosphorylation of ERK1/2, suggesting increased activation of MAPK. Therefore, it is possible that down-regulation of DPPIV/CD26 may be responsible for up-regulation of MCMs and CDK1/CDC2 in melanocytes ectopically expressing SPRIGHTLY via reduction of the inhibitory activity on MAPK. Interestingly, because SPRIGHTLY forced expression led to increased phosphorylation of ERK1/2 and increased cell proliferation and anchorage-independent colony formation, it will be important to investigate the activation and mutation status of other melanoma-related MAPK pathway genes (e.g., BRAF, NRAS). This continues as ongoing research in our laboratory.
      The down-regulation of DPPIV by SPRIGHTLY has significant implications with respect to the biological behavior and metastatic potential of melanoma. As a serine protease and membrane glycoprotein, DPPIV inactivates glucagon-like peptide 1, an incretin and gut-derived peptide important for postprandial glucose homeostasis. However, DPPIV has many other substrates, including CXCL12/SDF1, the unique CXCR4 ligand.
      • Wesley U.V.
      • Albino A.P.
      • Tiwari S.
      • Houghton A.N.
      A role for dipeptidyl peptidase IV in suppressing the malignant phenotype of melanocytic cells.
      first highlighted that DPPIV expression suppresses the malignant phenotype of melanocytes, and others have recently demonstrated that CXCL12/CXCR4 signaling antagonists suppress CXCR4+ pulmonary metastases in preclinical models of melanoma (
      • O'Boyle G.
      • Swidenbank I.
      • Marshall H.
      • Barker C.E.
      • Armstrong J.
      • White S.A.
      • et al.
      Inhibition of CXCR4-CXCL12 chemotaxis in melanoma by AMD11070.
      ). Because stromal CXCL12 production drives the recruitment and migration of melanoma, it is tempting to speculate that SPRIGHTLY participates in malignant transformation by down-regulating melanocyte DPPIV activity and dissipating the metastatic milieu created by the stroma, thereby promoting metastasis.
      Collectively, our study demonstrates that ectopic expression of SPRIGHTLY in melanocytes results in down-regulation of DPPIV/CD26 and up-regulation of cell proliferation genes, consequently altering the melanocytic phenotype toward malignant melanoma. The findings provide direct evidence for the melanomagenic role of SPRIGHTLY and how it regulates cell proliferation in melanocytes and melanomas. We also postulate that SPRIGHTLY and its interacting molecular partners may be involved in the development and malignant transformation of congenital melanocytic nevi. Further clinicopathological studies to determine the association between SPRIGHTLY and its target genes in malignant transformation of melanocytic nevi and clinical outcomes are warranted. Finally, elevated expression of SPRIGHTLY in melanoma cells compared to melanocytes, its accumulation in the cytoplasm, and its effects on cell dynamics suggest that this lncRNA and its target genes may play an important role in primary human melanocyte development and the molecular etiology of human melanomas.

      Materials and Methods

      Further details are available in the Supplementary Materials online.

      Cell lines

      Human melanocytes (HEM-l, catalog no. 2200; ScienCell, Carlsbad, CA) were grown in Melanocyte Media media containing Melanocyte Growth Supplement, 0.5% fetal bovine serum, penicillin, and streptomycin. A375 melanoma cells (ATCC CRL-1619, VA) were grown in Complete Tu Medium containing a 4:1 mixture of MCDB-153 medium with 1.5 g/L sodium bicarbonate and Leibovitz L-15 medium with 2 mM L-glutamine, 2% fetal bovine serum, and 1.68 mM CaCl2. The cells were grown in a humidified tissue culture incubator at 37 °C and 5% CO2 atmosphere.

      Lentiviral vector constructs

      A third-generation replication-defective HIV-1–based lentiviral vector was obtained from the Sanford-Burnham (Orlando, FL) virus core facility. This vector system includes the 5′ and 3′ lentiviral long terminal repeats and all necessary elements for effective transduction. Green fluorescent protein served as a transduction marker, and woodchuck hepatitis virus posttranscriptional regulatory element, which is believed to promote RNA processing and nuclear export, was used to boost expression of the gene of interest in target cells by facilitating the production of mature mRNA from transcripts initiated by the vector's internal promoter (pCMV). To construct the SPRIGHTLY expression lentiviral vector, DNA encoding full-length SPRIGHTLY was cloned into the lenti-transfer vector MCS region NheI site and then packaged into viral particles in 293T cells by cotransfection with packaging-defective helper plasmids using FuGENE 6 Transfection Reagent (Roche, Mannheim, Germany). Empty vector was also packaged in 293T cells as vector control. Virus-containing supernatants were collected 48 hours later, and the lentiviral titers were determined by high-content screening.

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      We thank the Sanford-Burnham Analytical Genomics core facility for deep sequencing, Bioinformatics core for data analysis support, Histology and Microscope facility for immunohistochemistry studies, and Ms. Debbie McFadden (SBMRI) for formatting the manuscript. This work was supported by National Institutes of Health Grants CA165184, NCI 5P30CA030199, and FL Biomed BHC 5BC08 to RJP and 2R01CA125255, Bankhead Coley 4BB17, and NSF CBET-1403535 to MK. MED is supported by a Career Development Award and an Australia Fellowship from the National Health and Medical Research Council of Australia, respectively.

      Supplementary Material

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