Polyclonality of BRAF mutations in acquired melanocytic nevi.
ABSTRACT: Melanocytic nevi are thought to be senescent clones of melanocytes that have acquired an oncogenic BRAF mutation. BRAF mutation is considered to be a crucial step in the initiation of melanocyte transformation. However, using immunomagnetic separation or laser-capture microdissection, we examined BRAF mutations in sets of approximately 50 single cells isolated from acquired melanocytic nevi from 13 patients and found a substantial number of nevus cells that contained wild-type BRAF mixed with nevus cells that contained BRAF(V600E). Furthermore, we simultaneously amplified BRAF exon 15 and a neighboring single nucleotide polymorphism (SNP), rs7801086, from nevus cell samples obtained from four patients who were heterozygous for this SNP. Subcloning and sequencing of the polymerase chain reaction products showed that both SNP alleles harbored the BRAF(V600E) mutation, indicating that the same BRAF(V600E) mutation originated from different cells. The polyclonality of BRAF mutations in acquired melanocytic nevi suggests that mutation of BRAF may not be an initial event in melanocyte transformation.
Project description:BRAF(V600E) mutations are frequent in melanomas originating from intermittently sun-exposed skin and also in common acquired melanocytic nevi, suggesting that BRAF mutation is an early event in melanocytic neoplasia. All neoplastic melanocytes within such a nevus would be expected to carry the BRAF mutation, and thus we evaluated the frequency of cells with BRAF(V600E) mutations within acquired nevi by droplet digital polymerase chain reaction. In BRAF-mutant nevi the number of BRAF mutant alleles equaled the number of wild-type (WT) alleles in the neoplastic cell population, consistent with a fully clonal heterozygous BRAF mutation. The allelic ratio of BRAF(V600E) to BRAF(WT) in the eight VE1-positive nevi, adjusted for degree of stromal contamination, ranged from 0.84 to 1.12 with an average ratio of 1.01. This was confirmed by immunohistochemistry with an antibody specific for BRAF(V600E), which uniformly labeled the neoplastic cells without any evidence of heterogeneity. We found BRAF(V600E) mutations in the melanocytic nevi to be fully clonal, strongly suggesting that BRAF-activating mutations typically are early initiating events in melanocytic neoplasia.
Project description:According to the prevailing multistep model of melanoma development, oncogenic BRAF or NRAS mutations are crucial initial events in melanoma development. It is not known whether melanocytic nevi that are found in association with a melanoma are more likely to carry BRAF or NRAS mutations than uninvolved nevi. By laser microdissection we were able to selectively dissect and genotype cells either from the nevus or from the melanoma part of 46 melanomas that developed in association with a nevus. In 25 cases we also genotyped a control nevus of the same patients. Available tissue was also immunostained using the BRAF(V600E)-mutation specific antibody VE1. The BRAF(V600E) mutation was found in 63.0% of melanomas, 65.2% of associated nevi and 50.0% of control nevi. No significant differences in the distribution of BRAF or NRAS mutations could be found between melanoma and associated nevi or between melanoma associated nevi and control nevi. In concordant cases immunohistochemistry showed a higher expression (intensity of immunohistochemistry) of the mutated BRAF(V600E)-protein in melanomas compared to their associated nevi. In this series the presence of a BRAF- or NRAS mutation in a nevus was not associated with the risk of malignant transformation. Our findings do not support the current traditional model of stepwise tumor progression.
Project description:Braf(V600E) induces benign, growth-arrested melanocytic nevus development, but also drives melanoma formation. Cdkn2a loss in Braf(V600E) melanocytes in mice results in rare progression to melanoma, but only after stable growth arrest as nevi. Immediate progression to melanoma is prevented by upregulation of miR-99/100, which downregulates mTOR and IGF1R signaling. mTORC1 activation through Stk11 (Lkb1) loss abrogates growth arrest of Braf(V600E) melanocytic nevi, but is insufficient for complete progression to melanoma. Cdkn2a loss is associated with mTORC2 and Akt activation in human and murine melanocytic neoplasms. Simultaneous Cdkn2a and Lkb1 inactivation in Braf(V600E) melanocytes results in activation of both mTORC1 and mTORC2/Akt, inducing rapid melanoma formation in mice. In this model, activation of both mTORC1/2 is required for Braf-induced melanomagenesis.
Project description:Deletion of the entire CDKN2B-CDKN2A gene cluster is among the most common genetic events in cancer. The tumor-promoting effects are generally attributed to loss of CDKN2A-encoded p16 and p14ARF tumor suppressors. The degree to which the associated CDKN2B-encoded p15 loss contributes to human tumorigenesis is unclear. Here, we show that CDKN2B is highly upregulated in benign melanocytic nevi, contributes to maintaining nevus melanocytes in a growth-arrested premalignant state, and is commonly lost in melanoma. Using primary melanocytes isolated directly from freshly excised human nevi naturally expressing the common BRAF(V600E)-activating mutation, nevi progressing to melanoma, and normal melanocytes engineered to inducibly express BRAF(V600E), we show that BRAF activation results in reversible, TGF?-dependent, p15 induction that halts proliferation. Furthermore, we engineer human skin grafts containing nevus-derived melanocytes to establish a new, architecturally faithful, in vivo melanoma model, and demonstrate that p15 loss promotes the transition from benign nevus to melanoma.Although BRAF(V600E) mutations cause melanocytes to initially proliferate into benign moles, mechanisms responsible for their eventual growth arrest are unknown. Using melanocytes from human moles, we show that BRAF activation leads to a CDKN2B induction that is critical for restraining BRAF oncogenic effects, and when lost, contributes to melanoma.
Project description:BACKGROUND: Oncogenic BRAF mutation had been considered to be a founder event in the formation of melanocytic tumours; however, we recently argued against this notion by showing marked polyclonality of BRAF mutations in acquired melanocytic nevi (Lin et al, J Natl Cancer Inst., 2009; 101:1423-7). Here, we tested whether similar heterogeneity of BRAF mutations exists in primary melanomas. METHODS: We isolated and sequenced single melanoma cells from five primary melanoma tissues using antibodies against human high-molecular-weight melanoma-associated antigen. We also examined 10 primary melanomas by the sensitive Mutector assay detecting the BRAF(V600E) mutation, as well as by cloning and sequencing of separated alleles. Furthermore, we estimated the frequency of BRAF mutant alleles in paired samples of primary tumour and recurrence or metastasis in three patients. RESULTS: Single-cell mutation analyses revealed that four of five primary melanomas contained both BRAF-wild-type and BRAF-mutant tumour cells. Tumour heterogeneity in terms of BRAF mutations was also shown in 8 of 10 primary melanomas. Selection of BRAF mutant alleles during progression was demonstrated in all the three patients. CONCLUSION: Acquisition of a BRAF mutation is not a founder event, but may be one of the multiple clonal events in melanoma development, which is selected for during the progression.
Project description:Human melanocytic nevi (moles) are benign lesions harboring activated oncogenes, including BRAF. Although this oncogene initially acts mitogenically, eventually, oncogene-induced senescence (OIS) ensues. Nevi can infrequently progress to melanomas, but the mechanistic relationship with OIS is unclear. We show here that PTEN depletion abrogates BRAF(V600E)-induced senescence in human fibroblasts and melanocytes. Correspondingly, in established murine BRAF(V600E)-driven nevi, acute shRNA-mediated depletion of PTEN prompted tumor progression. Furthermore, genetic analysis of laser-guided microdissected human contiguous nevus-melanoma specimens recurrently revealed identical mutations in BRAF or NRAS in adjacent benign and malignant melanocytes. The PI3K pathway was often activated through either decreased PTEN or increased AKT3 expression in melanomas relative to their adjacent nevi. Pharmacologic PI3K inhibition in melanoma cells suppressed proliferation and induced the senescence-associated tumor suppressor p15(INK4B). This treatment also eliminated subpopulations resistant to targeted BRAF(V600E) inhibition. Our findings suggest that a significant proportion of melanomas arise from nevi. Furthermore, these results demonstrate that PI3K pathway activation serves as a rate-limiting event in this setting, acting at least in part by abrogating OIS. The reactivation of senescence features and elimination of cells refractory to BRAF(V600E) inhibition by PI3K inhibition warrants further investigation into the therapeutic potential of simultaneously targeting these pathways in melanoma.
Project description:Melanocytic nevi frequently harbor oncogenic BRAF mutations, but only a minority progress to melanoma. In human melanocytes, persistent BRAF(V600E) expression triggers oncogene-induced senescence, which implies that bypass of oncogene-induced senescence is necessary for malignant transformation of melanocytes. We show that a subpopulation of primary human melanocytes with persistent expression of BRAF(V600E) do not enter oncogene-induced senescence, but instead survive despite heightened MAPK activity. Disruption of the p53 pathway using short-hairpin RNA initiated rapid growth of these V600E(+) melanocytes in vitro. The resultant V600E(+)/p53(sh) melanocytes grew anchorage-independently in soft agar, formed pigmented lesions reminiscent of in situ melanoma in artificial skin reconstructs, and were weakly tumorigenic in vivo. Array comparative genomic hybridization analysis demonstrated that the transformed melanocytes acquired a substantial deletion in chromosome 13, which encodes the Rb1 tumor suppressor gene. Gene expression profiling study of nevi and melanomas showed that p53 target genes were differentially expressed in melanomas compared with nevi, suggesting a dysfunctional p53 pathway in melanoma in vivo. In summary, these data demonstrate that a subpopulation of melanocytes possesses the ability to survive BRAF(V600E)-induced senescence, and suggest that p53 inactivation may promote malignant transformation of these cells.
Project description:Most melanomas are driven by BRAF(V600E)-activating mutations, while nevi harboring the same mutations have growth arrest. Although decreased p16 expression has been associated with melanoma formation, in recent work, p15 represented a primary effector of oncogene-induced senescence in nevomelanocytes that was diminished in melanomas. This study determined whether decreased p15 levels represent a general biomarker for the transition from nevus to melanoma. We performed p15 and p16 IHC analyses on a random series of nevi and melanomas. Staining was evaluated and graded for percentage and intensity to determine the H score. For real-time quantitative RT-PCR analysis of p15, RNA was extracted from FFPE sections from 14 nevus and melanoma samples via macrodissection. A two-sided t-test was used to evaluate between-group differences in mean H scores and q?Ct values. p15 Expression was significantly increased in melanocytic nevi compared with melanomas (mean H scores, 254.8 versus 132.3; P < 0.001). On p15 staining, the H score differential was greater than that with p16 staining [122.5 (P < 0.001) and 64.8 (P = 0.055), respectively]. Real-time quantitative RT-PCR analysis revealed a lower mean q?Ct value in melanomas, consistent with lower p15 expression (P = 0.018). Together, these data support the hypothesis that decreased p15 expression is a robust biomarker for distinguishing nevus from melanoma.
Project description:Conjunctival melanoma (CM) is a rare but lethal form of cancer. Similar to cutaneous melanoma, CM frequently carries activating mutations in BRAF and NRAS. We studied whether CM as well as conjunctival benign and premalignant melanocytic lesions express targets in the mitogen-activated protein kinase (MAPK) and AKT pathways, and whether specific inhibitors can suppress CM growth in vitro.131 conjunctival lesions obtained from 129 patients were collected. The presence of BRAF V600E mutation and expression of phosphorylated (p)-ERK and p-AKT were assessed by immunohistochemistry. We studied cell proliferation, phosphorylation, cell cycling and apoptosis in three CM cell lines using two BRAF inhibitors (Vemurafenib and Dabrafenib), a MEK inhibitor (MEK162) and an AKT inhibitor (MK2206).The BRAF V600E mutation was present in 19% of nevi and 26% of melanomas, but not in primary acquired melanosis (PAM). Nuclear and cytoplasmic p-ERK and p-AKT were expressed in all conjunctival lesions. Both BRAF inhibitors suppressed growth of both BRAF mutant CM cell lines, but only one induced cell death. MEK162 and MK2206 inhibited proliferation of CM cells in a dose-dependent manner, and the combination of these two drugs led to synergistic growth inhibition and cell death in all CM cell lines.ERK and AKT are constitutively activated in conjunctival nevi, PAM and melanoma. While BRAF inhibitors prohibited cell growth, they were not always cytotoxic. Combining MEK and AKT inhibitors led to more growth inhibition and cell death in CM cells. The combination may benefit patients suffering from metastatic conjunctival melanoma.
Project description:We conducted small RNA sequencing on low passage human melanocytes derived from melanocytic nevi and normal skin. Overall design: 8 normal skin derived melanocyte cultures and 6 nevus derived melanocyte cultures were sequenced.