Repigmentation of Human Vitiligo Skin by NBUVB Is Controlled by Transcription of GLI1 and Activation of the ß-Catenin Pathway in the Hair Follicle Bulge Stem Cells.

Vitiligo repigmentation is a complex process in which the melanocyte-depleted interfollicular epidermis is repopulated by melanocyte precursors from hair follicle bulge that proliferate, migrate, and differentiate into mature melanocytes on their way to the epidermis. The strongest stimulus for vitiligo repigmentation is narrow-band UVB (NBUVB), but how the hair follicle melanocyte precursors are activated by UV light has not been extensively studied. To better understand this process, we developed an application that combined laser capture microdissection and subsequent whole transcriptome RNA sequencing of hair follicle bulge melanocyte precursors and compared their gene signatures to that of regenerated mature epidermal melanocytes from NBUVB-treated vitiligo skin. Using this strategy, we found up-regulation of TNC, GJB6, and THBS1 in the hair follicle bulge melanocytes and of TYR in the epidermal melanocytes of the NBUVB-treated vitiligo skin. We validated these results by quantitative real-time-PCR using NBUVB-treated vitiligo skin and untreated normal skin. We also identified that GLI1, a candidate stem cell-associated gene, is significantly up-regulated in the melanocytes captured from NBUVB-treated vitiligo bulge compared with untreated vitiligo bulge. These signals are potential key players in the activation of bulge melanocyte precursors during vitiligo repigmentation.

Whole-exome sequencing identifies recurrent SF3B1 R625 mutation and comutation of NF1 and KIT in mucosal melanoma.

Mucosal melanomas are a rare subtype of melanoma, arising in mucosal tissues, which have a very poor prognosis due to the lack of effective targeted therapies. This study aimed to better understand the molecular landscape of these cancers and find potential new therapeutic targets. Whole-exome sequencing was performed on mucosal melanomas from 19 patients and 135 sun-exposed cutaneous melanomas, with matched peripheral blood samples when available. Mutational profiles were compared between mucosal subgroups and sun-exposed cutaneous melanomas. Comparisons of molecular profiles identified 161 genes enriched in mucosal melanoma (P<0.05). KIT and NF1 were frequently comutated (32%) in the mucosal subgroup, with a significantly higher incidence than that in cutaneous melanoma (4%). Recurrent SF3B1 R625H/S/C mutations were identified and validated in 7 of 19 (37%) mucosal melanoma patients. Mutations in the spliceosome pathway were found to be enriched in mucosal melanomas when compared with cutaneous melanomas. Alternative splicing in four genes were observed in SF3B1-mutant samples compared with the wild-type samples. This study identified potential new therapeutic targets for mucosal melanoma, including comutation of NF1 and KIT, and recurrent R625 mutations in SF3B1. This is the first report of SF3B1 R625 mutations in vulvovaginal mucosal melanoma, with the largest whole-exome sequencing project of mucosal melanomas to date. The results here also indicated that the mutations in SF3B1 lead to alternative splicing in multiple genes. These findings expand our knowledge of this rare disease.

Use of a MCL-1 inhibitor alone to de-bulk melanoma and in combination to kill melanoma initiating cells.

MCL-1 (BCL-2 family anti-apoptotic protein) is responsible for melanoma's resistance to therapy. Cancer initiating cells also contribute to resistance and relapse from treatments. Here we examined the effects of the MCL-1 inhibitor SC-2001 in killing non melanoma-initiating-cells (bulk of melanoma), and melanoma-initiating-cells (MICs). By itself, SC-2001 significantly kills melanoma cells under monolayer conditions in vitro and in a conventional mouse xenograft model. However, even at high doses (10µM), SC-2001 does not effectively eliminate MICs. In contrast, the combination of SC-2001 with ABT-737 (a BCL-2/BCL-XL/BCL-W inhibitor) significantly decreases ALDH+ cells, disrupts primary spheres, and inhibits the self-renewability of MICs. These results were observed in multiple melanomas, including short term cultures of relapsed tumors from current treatments, independent of the mutation status of BRAF or NRAS. Using a low-cell-number mouse xenograft model, we examined the effects of these treatments on the tumor initiating ability of MIC-enriched cultures. The combination therapy reduces tumor formation significantly compared to either drug alone. Mechanistic studies using shRNA and the CRISPR-Cas9 technology demonstrated that the upregulation of pro-apoptotic proteins NOXA and BIM contribute to the combination-induced cell death. These results indicate that the MCL-1 inhibitor SC-2001 combined with ABT-737 is a promising treatment strategy for targeting melanoma.

Combining a GSI and BCL-2 inhibitor to overcome melanoma's resistance to current treatments.

Major limitations of current melanoma treatments are for instances of relapse and the lack of therapeutic options for BRAF wild-type patients who do not respond to immunotherapy. Many studies therefore focus on killing resistant subpopulations, such as Melanoma Initiating Cells (MICs) to prevent relapse. Here we examined whether combining a GSI (γ-Secretase Inhibitor) with ABT-737 (a small molecule BCL-2/BCL-XL/BCL-W inhibitor) can kill both the non-MICs (bulk of melanoma) and MICs. To address the limitations of melanoma therapies, we included multiple tumor samples of patients relapsed from current treatments, with a diverse genetic background (with or without the common BRAF, NRAS or NF1 mutations) in these studies. Excitingly, the combination treatment reduced cell viability and induced apoptosis of the non-MICs; disrupted primary spheres, decreased the ALDH+ cells, and inhibited the self-renewability of the MICs in multiple melanoma cell lines and relapsed patient samples. Using a low-cell-number mouse xenograft model, we demonstrated that the combination significantly reduced the tumor initiating ability of MIC-enriched cultures from relapsed patient samples. Mechanistic studies also indicate that cell death is NOXA-dependent. In summary, this combination may be a promising strategy to address treatment relapse and for triple wild-type patients who do not respond to immunotherapy.

Isolating RNA from precursor and mature melanocytes from human vitiligo and normal skin using laser capture microdissection.

To characterize the gene expression profile of regenerated melanocytes in the narrow band UVB (NBUVB)-treated vitiligo epidermis and their precursors in the hair follicle, we present here a strategy of RNA isolation from in situ melanocytes using human frozen skin. We developed a rapid immunostaining protocol using the NKI-beteb antibody, which labels differentiated and precursor melanocytes, followed by fluorescent laser capture microdissection. This technique enabled the direct isolation, from melanocyte and adjacent keratinocyte populations, of satisfactory quality RNA that was successfully amplified and analysed by qRT-PCR. The melanocyte-specific gene transcripts TYR, DCT, TYRP1 and PMEL were significantly upregulated in our NBUVB-treated melanocyte samples as compared with the keratinocyte samples, while keratinocyte-specific genes (KRT5 and KRT14) were expressed significantly higher in the keratinocyte samples as compared with the melanocyte samples. Furthermore, in both NBUVB-treated vitiligo skin and normal skin, when bulge melanocytes were compared with epidermal melanocytes, we found significantly lower expression of melanocyte-specific genes and significantly higher expression of three melanocytic stem cell genes (SOX9, WIF1 and SFRP1), while ALCAM and ALDH1A1 transcripts did not show significant variation. We found significantly higher expression of melanocyte-specific genes in the epidermis of NBUVB-treated vitiligo, as compared to the normal skin. When comparing bulge melanocyte samples from untreated vitiligo, NBUVB-treated vitiligo and normal skin, we did not find significant differences in the expression of melanocyte-specific genes or melanocytic stem cell genes. These techniques offer valuable opportunities to study melanocytes and their precursors in vitiligo and other pigmentation disorders.

IMPACT: a whole-exome sequencing analysis pipeline for integrating molecular profiles with actionable therapeutics in clinical samples.

OBJECTIVE: Currently, there is a disconnect between finding a patient's relevant molecular profile and predicting actionable therapeutics. Here we develop and implement the Integrating Molecular Profiles with Actionable Therapeutics (IMPACT) analysis pipeline, linking variants detected from whole-exome sequencing (WES) to actionable therapeutics. METHODS AND MATERIALS: The IMPACT pipeline contains 4 analytical modules: detecting somatic variants, calling copy number alterations, predicting drugs against deleterious variants, and analyzing tumor heterogeneity. We tested the IMPACT pipeline on whole-exome sequencing data in The Cancer Genome Atlas (TCGA) lung adenocarcinoma samples with known EGFR mutations. We also used IMPACT to analyze melanoma patient tumor samples before treatment, after BRAF-inhibitor treatment, and after BRAF- and MEK-inhibitor treatment. RESULTS: IMPACT Food and Drug Administration (FDA) correctly identified known EGFR mutations in the TCGA lung adenocarcinoma samples. IMPACT linked these EGFR mutations to the appropriate FDA-approved EGFR inhibitors. For the melanoma patient samples, we identified NRAS p.Q61K as an acquired resistance mutation to BRAF-inhibitor treatment. We also identified CDKN2A deletion as a novel acquired resistance mutation to BRAFi/MEKi inhibition. The IMPACT analysis pipeline predicts these somatic variants to actionable therapeutics. We observed the clonal dynamic in the tumor samples after various treatments. We showed that IMPACT not only helped in successful prioritization of clinically relevant variants but also linked these variations to possible targeted therapies. CONCLUSION: IMPACT provides a new bioinformatics strategy to delineate candidate somatic variants and actionable therapies. This approach can be applied to other patient tumor samples to discover effective drug targets for personalized medicine.IMPACT is publicly available at http://tanlab.ucdenver.edu/IMPACT.

Narrow Band Ultraviolet B Treatment for Human Vitiligo Is Associated with Proliferation, Migration, and Differentiation of Melanocyte Precursors.

In vitiligo, the autoimmune destruction of epidermal melanocytes produces white spots that can be repigmented by melanocyte precursors from the hair follicles, following stimulation with UV light. We examined by immunofluorescence the distribution of melanocyte markers (C-KIT, DCT, PAX3, and TYR) coupled with markers of proliferation (KI-67) and migration (MCAM) in precursors and mature melanocytes from the hair follicle and the epidermis of untreated and narrow band UVB (NBUVB)-treated human vitiligo skin. NBUVB was associated with a significant increase in the number of melanocytes in the infundibulum and with restoration of the normal melanocyte population in the epidermis, which was lacking in the untreated vitiligo. We identified several precursor populations (melanocyte stem cells, melanoblasts, and other immature phenotypes), and progressively differentiating melanocytes, some with putative migratory and/or proliferative abilities. The primary melanocyte germ was present in the untreated and treated hair follicle bulge, whereas a possible secondary melanocyte germ composed of C-KIT+ melanocytes was found in the infundibulum and interfollicular epidermis of UV-treated vitiligo. This is an exceptional model for studying the mobilization of melanocyte stem cells in human skin. Improved understanding of this process is essential for designing better treatments for vitiligo, ultimately based on melanocyte stem cell activation and mobilization.

Combining a BCL2 inhibitor with the retinoid derivative fenretinide targets melanoma cells including melanoma initiating cells.

Investigations from multiple laboratories support the existence of melanoma initiating cells (MICs) that potentially contribute to melanoma's drug resistance. ABT-737, a small molecule BCL-2/BCL-XL/BCL-W inhibitor, is promising in cancer treatments, but not very effective against melanoma, with the antiapoptotic protein MCL-1 as the main contributor to resistance. The synthetic retinoid fenretinide N-(4-hydroxyphenyl)retinamide (4-HPR) has shown promise for treating breast cancers. Here, we tested whether the combination of ABT-737 with 4-HPR is effective in killing both the bulk of melanoma cells and MICs. The combination synergistically decreased cell viability and caused cell death in multiple melanoma cells lines (carrying either BRAF or NRAS mutations) but not in normal melanocytes. The combination increased the NOXA expression and caspase-dependent MCL-1 degradation. Knocking down NOXA protected cells from combination-induced apoptosis, implicating the role of NOXA in the drug synergy. The combination treatment also disrupted primary spheres (a functional assay for MICs) and decreased the percentage of aldehyde dehydrogenase (high) cells (a marker of MICs) in melanoma cell lines. Moreover, the combination inhibited the self-renewal capacity of MICs, measured by secondary sphere-forming assays. In vivo, the combination inhibited tumor growth. Thus, this combination is a promising treatment strategy for melanoma, regardless of mutation status of BRAF or NRAS.

Long term storage of dry versus frozen RNA for next generation molecular studies.

The standard method for the storage and preservation of RNA has been at ultra-low temperatures. However, reliance on liquid nitrogen and freezers for storage of RNA has multiple downsides. Recently new techniques have been developed for storing RNA at room temperature utilizing desiccation and are reported to be an effective alternative for preserving RNA integrity. In this study we compared frozen RNA samples stored for up to one year to those which had been desiccated using RNAstable (Biomatrica, Inc., San Diego, CA) and stored at room temperature. RNA samples were placed in aliquots and stored after desiccation or frozen (at -80°C), and were analyzed for RNA Integrity Number (RIN), and by qPCR, and RNA sequencing. Our study shows that RNAstable is able to preserve desiccated RNA samples at room temperature for up to one year, and that RNA preserved by desiccation is comparable to cryopreserved RNA for downstream analyses including real-time-PCR and RNA sequencing.

Therapeutic monoclonal antibodies in human breast milk: a case study.

Recently, therapeutic monoclonal antibodies have been introduced for the treatment of advanced melanoma and other diseases. It remains unclear whether these drugs can be safely administered to women who are breast feeding because of the potential hazardous side effects for nursing infants. One such therapy for metastatic melanoma is ipilimumab, a human monoclonal antibody that blocks cytotoxic T-lymphocyte-antigen-4, and is the preferred treatment for patients with metastatic melanoma when other molecular therapies are not viable. This study measured ipilimumab levels in the breast milk of a patient undergoing treatment that were enough to raise concerns for a nursing infant exposed to ipilimumab.

Extracellular vesicles secreted from cancer cell lines stimulate secretion of MMP-9, IL-6, TGF-ß1 and EMMPRIN.

Extracellular vesicles (EVs) are key contributors to cancer where they play an integral role in cell-cell communication and transfer pro-oncogenic molecules to recipient cells thereby conferring a cancerous phenotype. Here, we purified EVs using straightforward biochemical approaches from multiple cancer cell lines and subsequently characterized these EVs via multiple biochemical and biophysical methods. In addition, we used fluorescence microscopy to directly show internalization of EVs into the recipient cells within a few minutes upon addition of EVs to recipient cells. We confirmed that the transmembrane protein EMMPRIN, postulated to be a marker of EVs, was indeed secreted from all cell lines studied here. We evaluated the response to EV stimulation in several different types of recipient cells lines and measured the ability of these purified EVs to induce secretion of several factors highly upregulated in human cancers. Our data indicate that purified EVs preferentially stimulate secretion of several proteins implicated in driving cancer in monocytic cells but only harbor limited activity in epithelial cells. Specifically, we show that EVs are potent stimulators of MMP-9, IL-6, TGF-ß1 and induce the secretion of extracellular EMMPRIN, which all play a role in driving immune evasion, invasion and inflammation in the tumor microenvironment. Thus, by using a comprehensive approach that includes biochemical, biological, and spectroscopic methods, we have begun to elucidate the stimulatory roles.

CtBP1 is expressed in melanoma and represses the transcription of p16INK4a and Brca1.

Carboxyl-terminal binding protein 1 (CtBP1) has been shown to suppress the transcription of several tumor suppressors in vitro. Paradoxically, a previous report showed that CtBP1 mRNA was downregulated in melanoma. Using immunostaining, we found that a large percentage of human melanomas were positive for CtBP1 protein. Furthermore, we demonstrated that CtBP1 expression in melanoma cells contributes to cell proliferation and genome instability, two aspects promoting melanoma initiation and progression. Breast cancer susceptibility gene 1 (Brca1), a core protein in DNA-damage repair, was repressed by CtBP1 in melanoma cells. Consistently, Brca1 loss in human malignant melanoma tissues was found to be inversely correlated with CtBP1 expression levels. In addition, the inhibitor of cyclin-dependent protein kinases (CDKs), p16INK4a, whose loss has been related to the pathogenesis of melanoma, was repressed by CtBP1 as well. Our findings suggest an important role of CtBP1 in the transcriptional control of p16INK4a and Brca1, with CtBP1 overexpression potentially contributing to increased proliferation and DNA damage in melanoma.

ALDH1A isozymes are markers of human melanoma stem cells and potential therapeutic targets.

Although the concept of cancer stem cells (CSCs) is well-accepted for many tumors, the existence of such cells in human melanoma has been the subject of debate. In this study, we demonstrate the existence of human melanoma cells that fulfill the criteria for CSCs (self-renewal and differentiation) by serially xenotransplanting cells into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. These cells possess high aldehyde dehydrogenase (ALDH) activity with ALDH1A1 and ALDH1A3 being the predominant ALDH isozymes. ALDH-positive melanoma cells are more tumorigenic than ALDH-negative cells in both NOD/SCID mice and NSG mice. Biological analyses of the ALDH-positive melanoma cells reveal the ALDH isozymes to be key molecules regulating the function of these cells. Silencing ALDH1A by siRNA or shRNA leads to cell cycle arrest, apoptosis, decreased cell viability in vitro, and reduced tumorigenesis in vivo. ALDH-positive melanoma cells are more resistant to chemotherapeutic agents and silencing ALDH1A by siRNA sensitizes melanoma cells to drug-induced cell death. Furthermore, we, for the first time, examined the molecular signatures of ALDH-positive CSCs from patient-derived tumor specimens. The signatures of melanoma CSCs include retinoic acid (RA)-driven target genes with RA response elements and genes associated with stem cell function. These findings implicate that ALDH isozymes are not only biomarkers of CSCs but also attractive therapeutic targets for human melanoma. Further investigation of these isozymes and genes will enhance our understanding of the molecular mechanisms governing CSCs and reveal new molecular targets for therapeutic intervention of cancer.

Side population cells from human melanoma tumors reveal diverse mechanisms for chemoresistance.

Side population (SP) cells are identified as cells capable of excluding the fluorescent Hoechst dye and anticancer drugs, and it represents hematopoietic stem cells and chemoresistant cells from several solid tumors. In this study, we confirmed the presence of SP cells in tumors from melanoma patients. Melanoma SP cells overexpressed ATP-binding-cassette (ABC) transporters, ABCB1 and ABCB5. We generated a direct in vivo xenograft model, and demonstrated that SP cells were resistant to paclitaxel, a substrate of ABCB1, both in vitro and in vivo. However, melanoma SP cells were also resistant to temozolomide, which is not a substrate for ABC transporters, through IL-8 upregulation. In addition, gene profiling studies identified three signaling pathways (NF-κB, α6-ß4-integrin, and IL-1) as differentially upregulated in melanoma SP cells, and there was a significant increase of PCDHB11 and decrease of FUK and TBX2 in these cells. Therefore, we provide evidence that SP is an enriched source of chemoresistant cells in human melanomas, and suggest that the selected genes and signaling pathways of SP cells may be a potential target for effective melanoma therapies. To our knowledge, this is a previously unreported study to isolate SP cells from melanoma patients and to investigate the gene expression profiling of these cells.

The RET G691S polymorphism is a germline variant in desmoplastic malignant melanoma.

The RET protooncogene was originally identified in 1985. It encodes for a receptor tyrosine kinase. The RET receptor is activated by its ligand glial cell-derived neurotrophic factor. A polymorphism, RETp (G691S), in the intracellular juxtamembrane domain of RET, which enhances signaling by glial cell-derived neurotrophic factor has been described and studied previously in pancreatic cancer, medullary thyroid cancer, the multiple endocrine neoplasia 2 syndromes, and recently in cutaneous malignant melanoma. In particular, it has been shown that desmoplastic melanomas, which have neurotrophic features, have a high frequency of this polymorphism. In previous studies, however, it was not clear whether this was a germline or somatic change. Previous studies on pancreatic cancer indicated that both mechanisms may occur. To clarify this further we examined peripheral blood cell DNA from 30 patients with desmoplastic melanomas and 30 patients with nondesmoplastic melanoma for the RETp. In this study, a germline polymorphism was found in 30% of the patients with desmoplastic melanomas and 21% of the patients with nondesmoplastic melanoma. These findings indicate that the RETp may be a genetic risk factor for the development of desmoplastic melanoma.

Recovery of MicroRNA from Stored Human Peripheral Blood Samples.

Recovery of microRNAs (miRs) from tissue samples of many kinds has become an area of increasing interest and importance in recent years. We isolated and then amplified and quantitated miRs from human peripheral blood samples stored in the University of Colorado Denver Skin Cancer Biorepository to determine whether miR recovery was possible and consistent over time in storage. Forty-five blood samples from patients with different stages of malignant melanoma were collected in PAXgene Blood RNA tubes and then stored at -80°C prior to RNA preparation. The samples examined had been stored from 4 weeks to 3 years. Total RNA was prepared, followed by miR isolation, amplification, and quantitation using real-time polymerase chain reaction. A widely expressed miR, miR-221, was used as a standard for comparison across samples and storage time. miR-221 was recovered from all samples, with no differences observed with longer storage time. These studies show that miRs can be recovered and quantified from human blood samples stored for up to 3 years.

p53 prevents progression of nevi to melanoma predominantly through cell cycle regulation.

p53 is the central member of a critical tumor suppressor pathway in virtually all tumor types, where it is silenced mainly by missense mutations. In melanoma, p53 predominantly remains wild type, thus its role has been neglected. To study the effect of p53 on melanocyte function and melanomagenesis, we crossed the 'high-p53'Mdm4+/− mouse to the well-established TP-ras0/+ murine melanoma progression model. After treatment with the carcinogen dimethylbenzanthracene (DMBA), TP-ras0/+ mice on the Mdm4+/− background developed fewer tumors with a delay in the age of onset of melanomas compared to TP-ras0/+ mice. Furthermore, we observed a dramatic decrease in tumor growth, lack of metastasis with increased survival of TP-ras0/+: Mdm4+/− mice. Thus, p53 effectively prevented the conversion of small benign tumors to malignant and metastatic melanoma. p53 activation in cultured primary melanocyte and melanoma cell lines using Nutlin-3, a specific Mdm2 antagonist, supported these findings. Moreover, global gene expression and network analysis of Nutlin-3-treated primary human melanocytes indicated that cell cycle regulation through the p21WAF1/CIP1 signaling network may be the key anti-melanomagenic activity of p53.

MicroRNA-137 targets microphthalmia-associated transcription factor in melanoma cell lines.

Micropthalmia-associated transcription factor (MITF) is the master regulator of melanocyte development, survival, and function. Frequent alteration in the expression of MITF is detected in melanoma, but the mechanism(s) underlying the alteration in expression have not been completely determined. In these studies, we have identified microRNA-137 (miR-137) as a regulator of MITF expression. The genomic locus of miR-137 at chromosome 1p22 places it in a region of the human genome previously determined to harbor an allele for melanoma susceptibility. Here, we show that expression of mature miR-137 in melanoma cell lines down-regulates MITF expression. Further, we have identified a 15-bp variable nucleotide tandem repeat located just 5' to the pre-miR-137 sequence, which alters the processing and function of miR-137 in melanoma cell lines.