Absence of ras gene mutations in UV-induced malignant melanomas correlates with a dermal origin of melanocytes in Monodelphis domestica

The South American opossum, Monodelphis domestica, has been used as a model system to study ultraviolet (UV)-induced genetic alterations that lead to the development of melanoma. Suckling young of Monodelphis exposed to multiple doses of UVB radiation can develop benign or malignant melanomas later as adults. Point mutations predominantly at codon 61 of the N-ras gene have been found in melanomas from sun-exposed body sites in humans. To determine if similar mutations are associated with UV-induced melanoma in Monodelphis, the nucleotide sequence of a Monodelphis N-ras cDNA was determined, and the occurrence of ras mutations in melanomas from UV-irradiated opossums was investigated. Single-strand conformation polymorphism analysis revealed no mutations in either the Monodelphis N-ras or H-ras genes in any of 24 primary malignant melanoma samples analyzed in this study. The disparate association of ras mutations with melanoma in humans and Monodelphis may be explained by differences in nucleotide sequence at codon 61 of the N-ras gene as well as differences in skin architecture between the two species. These results support the contention that a mutationally activated N-ras gene contributes to the vertical growth phase, which is specific to the progression of malignant melanoma in humans.     

Screening of N-ras codon 61 mutations in paired primary and metastatic cutaneous melanomas: mutations occur early and persist throughout tumor progression.

PURPOSE: Mutations in the ras genes often occur during tumorigenesis. In malignant melanoma, the most common ras alterations are N-ras codon 61 mutations. This study was aimed to measure the frequency of such mutations in a large series of paired primary and metastatic melanomas to determine their role in melanoma initiation and progression. EXPERIMENTAL DESIGN: Seventy-four primary melanomas and 88 metastases originating from 54 of the primary tumors were screened for N-ras codon 61 mutations using single-strand conformation polymorphism and nucleotide sequence analyses. RESULTS: Twenty-one of the 74 primary tumors (28%) had activating N-ras codon 61 mutations. From 20 of the mutated primary tumors, a total of 34 metastases were analyzed, and all but one showed the same mutation as the primary tumor from which they originated. The remaining 53 primary tumors and corresponding metastases (n = 54) were wild-type for N-ras codon 61. Analysis of the different growth phases of the mutated primary tumors showed that the mutations were already present in the radial growth phase. Mutations were also detected in tumor-associated nevi. N-ras codon 61 mutations were associated with a higher Clark level of invasion (P = 0.012) and a lower age at diagnosis (P = 0.042) but did not affect survival (P = 0.671). CONCLUSIONS: This study shows that N-ras codon 61 mutations occur early in primary melanomas rather than in the metastatic stage and that once the mutations have occurred, they persist throughout tumor progression. This suggests that activated N-ras may be an attractive target for therapy in the subset of melanoma patients carrying such mutations.     

Mutational analysis of 9 different tumour-associated genes in human malignant mesothelioma cell lines

Seven tumour suppressor genes (Chk1, Chk2, Apaf1, Rb1, p53, p16(INK4a) and p14(ARF)) and two oncogenes (N-ras and BRAF) were screened in nine human malignant melanoma (HMM) cell lines for point mutations or small deletions/insertions by DGGE, TGGE and SCCP analysis. For the first time in human mesothelioma, Chk1 gene mutations were detected in two of the nine investigated HMM cell lines. P53 gene mutations were found in three cell lines and p16(INK4a) mutations in 5. Mutation of the Chk1 gene implies a novel disruption mechanism of the p53 pathway in HMM, without affecting p53 itself. According to our knowledge, this is the first mutation screening of Chk1, Chk2, Apaf1 and Rb1 in human malignant mesothelioma.     

p53 mutations in human cutaneous melanoma correlate with sun exposure but are not always involved in melanomagenesis

In melanoma, the relationship between sun exposure and the origin of mutations in either the N-ras oncogene or the p53 tumour-suppressor gene is not as clear as in other types of skin cancer. We have previously shown that mutations in the N-ras gene occur more frequently in melanomas originating from sun-exposed body sites, indicating that these mutations are UV induced. To investigate whether sun exposure also affects p53 in melanoma, we analysed 81 melanoma specimens for mutations in the p53 gene. The mutation frequency is higher than thus far reported: 17 specimens (21%) harbour one or more p53 mutations. Strikingly, 17 out of 22 mutations in p53 are of the C:G to TA or CC:GG to TT:AA transitional type, strongly suggesting an aetiology involving UV exposure. Interestingly, the p53 mutation frequency in metastases was much lower than in primary tumours. In the case of metastases, a role for sun exposure was indicated by the finding that the mutations are present exclusively in skin metastases and not in internal metastases. Together with a relatively frequent occurrence of silent third-base pair mutations in primary melanomas, this indicates that the p53 mutations, at least in these tumours, have not contributed to melanomagenesis and may have originated after establishment of the primary tumour.     

Metastasizing melanoma formation caused by expression of activated N-RasQ61K on an INK4a-deficient background

In human cutaneous malignant melanoma, a predominance of activated mutations in the N-ras gene has been documented. To obtain a mouse model most closely mimicking the human disease, a transgenic mouse line was generated by targeting expression of dominant-active human N-ras (N-RasQ61K) to the melanocyte lineage by tyrosinase regulatory sequences (Tyr::N-RasQ61K). Transgenic mice show hyperpigmented skin and develop cutaneous metastasizing melanoma. Consistent with the tumor suppressor function of the INK4a locus that encodes p16INK4A and p19(ARF), >90% of Tyr::N-RasQ61K INK4a-/- transgenic mice develop melanoma at 6 months. Primary melanoma tumors are melanotic, multifocal, microinvade the epidermis or epithelium of hair follicles, and disseminate as metastases to lymph nodes, lung, and liver. Primary melanoma can be transplanted s.c. in nude mice, and if injected i.v. into NOD/SCID mice colonize the lung. In addition, primary melanomas and metastases contain cells expressing the stem cell marker nestin suggesting a hierarchical structure of the tumors comprised of primitive nestin-expressing precursors and differentiated cells. In conclusion, a novel mouse model with melanotic and metastasizing melanoma was obtained by recapitulating genetic lesions frequently found in human melanoma.     

A novel N-ras mutation in malignant melanoma is associated with excellent prognosis.

Mutations in the ras gene are key events in the process of carcinogenesis; in particular, point mutations in codon 61 of exon 2 of the N-ras gene occur frequently in malignant melanoma (MM). We searched for point mutations in the N-ras gene in a large series of primary and metastatic MM from 81 different retrospectively selected patients using the very sensitive denaturing gradient gel electrophoresis technique, followed by sequencing. The classical codon 12 and codon 61 mutations were found in 21 and 17% of the cases, respectively. No codon 13 mutation was found. A novel mutation at codon 18 of exon 1, consisting of a substitution of alanine (GCA) by threonine (ACA), was found in 15% of the primary MMs but in none of the metastatic MMs. All of the other cases were free of mutations. Using microdissected cells from distinctive MM growth phases as source of DNA for mutation analysis, this particular N-ras exon 1 mutation at codon 18 was already present in the radial growth phase and preserved throughout the successive growth phases; it was also found in a dysplastic nevi in continuity with a MM, indicating a clonal relationship between both lesions. Our findings also illustrate the clonal relationship between the distinctive growth phases in MM and suggest the codon 18 mutation to occur early in MM development. The MM in patients with this mutation were significantly thinner than those without a codon 18 mutation (P = 0.0257). Statistical analysis, comparing the group of codon 18 patients with the group of patients with the classical mutations and without mutations, revealed a highly significant difference in overall outcome. The cumulative probability of developing metastasis was significantly lower for the group patients with a codon 18 mutation (P = 0.0130). We can thus conclude that this codon 18 mutation identifies a group of patients with better prognosis than patients with melanoma that harbor wild-type sequence or classical activating point mutations in codon 12 or 61. Preliminary nucleotide binding measurements could not detect a difference between wild-type Ras protein and the mutant Ras(A18T) protein. However, for a precise elucidation of the role of the N-Ras(A18T) mutant in melanoma, additional studies aimed to measure the affinity to guanine nucleotide exchange factors and GTPase-activating proteins are needed.     

Activating BRAF and N-Ras mutations in sporadic primary melanomas: an inverse association with allelic loss on chromosome 9

We searched and report mutations in the BRAF and N-ras genes in 22 out of 35 (63 percent) primary sporadic melanomas. In three melanomas, mutations were concomitantly present in both genes. In all, 10 out of 12 mutations in the BRAF gene involved the 'hot spot' codon 600 (In all communications on mutations in the BRAF gene, the nucleotide and codon numbers have been based on the NCBI gene bank nucleotide sequence NM_004333. However, according to NCBI gene bank sequence with accession number NT_007914, there is a discrepancy of one codon (three nucleotides) in exon 1 in the sequence with accession number NM_004333. The sequence analysis of exon 1 of the BRAF gene in our laboratory has shown that the sequence derived from NT_007914 is correct (Kumar et al., 2003). Due to the correctness of the latter, sequence numbering of codons and nucleotides after exon 1 are changed by +1 and +3, respectively.), one tandem CT1789-90TC base change represented a novel mutation and another mutation caused a G466R amino-acid change within the glycine-rich loop in the kinase domain. Mutations in the N-ras gene in 11 melanomas were at codon 61 whereas two melanomas carried mutations in codon 12 including a tandem mutation GG>AA. We observed an inverse association between BRAF/N-ras mutations and the frequency of loss of heterozygosity (LOH) on chromosome 9 at 10 different loci. Melanomas with BRAF/N-ras mutations showed a statistically significant decreased frequency of LOH on chromosome 9 compared with cases without mutations (mean fractional allelic loss (FAL)=0.29+/-0.23 vs 0.72+/-0.33; t-test, P=0.0001). Difference in the FAL value between tumours with and without BRAF/N-ras mutations on 33 loci on five other chromosomes was not statistically significant (mean FAL 0.17+/-0.19 vs 0.25+/-0.22; t-test, P=0.24). Melanoma cases with BRAF/N-ras mutations were also associated with lower age at diagnosis than cases without mutations (mean age 80.38+/-7.24 vs 65.77+/-19.79 years; t-test, P=0.02). Our data suggest that the occurrence of BRAF/N-ras mutations compensate the requirement for the allelic loss at chromosome 9, which is one of the key events in melanoma.     

Overexpression of the N-ras proto-oncogene, not somatic mutational activation, associated with malignant tumors in transgenic mice.

We have produced transgenic mice that carry a foreign gene construct consisting of the N-ras proto-oncogene driven by the mouse mammary tumor virus (MMTV) long terminal repeat. Overexpression of the normal N-ras gene is associated with development of hyperplasias and tumors in a variety of tissues. The tumors are clearly malignant, as evidenced by the presence of metastatic lesions. Extensive analysis of the foreign ras gene in these tumors by use of polymerase chain reaction and sequencing demonstrates in all cases the absence of somatically acquired mutations at those codons normally associated with activation of the ras genes. Thus, these tumors develop from overexpression of the proto-oncogene rather than the presence of the mutated oncogene. These data demonstrate that overexpression of a protooncogene of the ras family can predispose cells in vivo to fully malignant behavior.     

The Y152X MC1R gene mutation: occurrence in ethnically diverse Jewish malignant melanoma patients

MC1R sequence variants are associated with malignant melanoma risk, and most commonly are missense mutations. Few (n=9) truncating mutations have been described in this gene as predisposing to malignant melanoma. In this study, three Jewish individuals were found to harbor an identical truncating MC1R mutation--Y152X: an Ashkenazi patient with two malignant melanomas, a non-Ashkenazi malignant melanoma patient with familial malignant melanoma and her asymptomatic mother. Both malignant melanoma patients carried additional, seemingly pathogenic MC1R variants. Haplotype analysis revealed that all three mutation carriers shared the same haplotype. This sequence variant was previously described in ethnically diverse, non-Jewish individuals and in all likelihood represents an error-prone domain that, in conjunction with other genetic and environmental factors, increases malignant melanoma risk.     

Absence of BRAF gene mutations differentiates spitz nevi from malignant melanoma

BACKGROUND: Distinction of Spitz nevus from malignant melanoma is sometimes difficult on the basis of conventional histology. A high rate of BRAF gene mutations in malignant melanomas (66%) and nevi (82%) has recently been reported. MATERIALS AND METHODS: We screened a series of 20 Spitz nevi for BRAF mutations in exons 11 and 15 by denaturing gradient gel electrophoresis (DGGE). RESULTS: BRAF mutations could not be identified in Spitz nevi. CONCLUSION: Our results show that mutations within the BRAF gene are useful markers for the differential diagnosis between Spitz nevus and malignant melanoma.     

Mutational analysis of the BRAF gene in human congenital and dysplastic melanocytic naevi

Eighteen congenital melanocytic naevi (CMN) from 17 patients and 18 dysplastic melanocytic naevi (DMN) from 18 patients were screened for mutations in the BRAF oncogene (present study) and the N-ras oncogene (in the course of two foregoing studies) by single-strand conformational polymorphism (SSCP)/sequencing analysis. BRAF mutations were demonstrated in both types of lesion. As a whole, 17 of 18 CMN (94.4%) and five of 18 DMN (27.7%) harboured either BRAF or N-ras mutations. As the BRAF oncogene is frequently found to be mutated in human cutaneous melanomas, it may constitute a risk factor for melanoma formation within CMN and DMN.     

Mutational analysis of ras gene family in lung cancer in Thai

H-, K- and N-ras gene mutations were analyzed in lung cancer from Thai patients. Thirteen out of 58 cases (22%) harbored the mutations. Ten cases showed K-ras gene mutations at codon 12, 1 case presented a mutation at codon 13 and another case exhibited a mutation at codon 63. Silent mutations of N-ras gene in codons 57 and 62 were seen in one patient, whilst no H-ras mutation was found in these patients. Bases change in K-ras gene were G right curved arrow T transversion (62%), G right curved arrow A transition (15%) and G right curved arrow C transition (15%), whereas T right curved arrow G transversion and A right curved arrow G transition were detected in N-ras mutant gene.     

N-ras gene mutations in acute myeloid leukemia: accurate detection by solid-phase minisequencing.

Mutations in the N-ras gene are found in one-third of patients with acute myeloid leukemia. The N-ras mutations could serve as markers for residual cells, if a highly sensitive method for detecting the mutations was available. We applied a new method, solid-phase minisequencing, to analyze bone-marrow cells from 16 patients with acute myeloid leukemia for mutations in codon 12, 13 and 61 of the N-ras gene. In the solid-phase minisequencing technique the mutations are identified by a primer extension reaction, in which a single labelled nucleoside triphosphate is incorporated into an immobilized DNA fragment previously amplified by the polymerase chain reaction. We identified N-ras mutations in 5 of the patients (30%). In one patient, we observed 2 mutations that were shown to be located in different alleles. With the solid-phase minisequencing method, we were able to determine the proportion of mutated cells in the samples. We found that in 4 of the samples only a fraction (7-64%) of the blasts carried an N-ras mutation, and in one sample practically all blast cells were mutated. The method was highly sensitive, allowing us to identify N-ras mutations even when the sample consisted of 99.7% normal cells and only 0.3% mutated blasts.     

Distinct spectrum of N-ras mutations in aml and mds patients in yugoslavia

Mutations that activate ras genes were demonstrated to be associated with certain types of malignancies. Multiple point mutations were predominantly found in the N-ras and occasionally in the K-ras genes. The analysis of 4 MDS, 23 AML and 11 CML patients from Yugoslavia revealed the prevalence of the N-ras mutation (83%) over K-ras mutations (17%). Although the frequencies of the N- and K-ras mutations in these patients were similar to the ones reported for patients from USA and Japan, the N-ras mutational spectra considerably differed. The prevailing type of mutation in patients from Yugoslavia was G-to-T transversion at the first position in the codon 12 of the N-ras gene. This study supports a hypothesis that different geographical and environmental factors may cause the accumulation of different type of point mutations in the same target gene.     

Analysis of ras gene mutations in human hepatic malignant tumors by polymerase chain reaction and direct sequencing

The ras gene is one of the oncogenes most commonly detected in human cancers, and it consists of three families (H-ras, K-ras, N-ras). These genes are converted to active oncogenes by point mutations occurring in either codon 12, 13, or 61. We analyzed mutations of these codons in 23 primary hepatic malignant tumors (12 hepatocellular carcinomas, nine cholangiocarcinomas, and two hepatoblastomas) by a method to directly sequence nucleotides, using polymerase chain reaction and a direct sequencing method. Of 23 hepatic malignant tumors, point mutations at K-ras codon 12 or K-ras codon 61 were found in six of nine cholangiocarcinomas. In contrast, there were no point mutations in any of 12 hepatocellular carcinomas or two hepatoblastomas around codon 12, 13, or 61 of the ras genes. These observations suggest that ras gene mutations are not related to pathogenesis of hepatocellular carcinoma, but play an important role in pathogenesis of cholangiocarcinoma.     

Sensitivity of melanoma cell lines to natural killer cells: a role for oncogene-modulated HLA class I expression?

N-ras and c-myc oncogenes were found to be activated in melanoma. High c-myc expression renders melanoma cell lines sensitive to lysis by natural killer (NK) cells. This effect is mediated by locus-specific downmodulation of HLA-B expression by c-myc. Cell lines with a mutation in the N-ras gene were relatively sensitive to NK cells irrespective of HLA class I expression. These findings indicate that NK cells can kill tumor cells with activated myc or ras oncogenes in various ways, thus providing potential mechanisms to eliminate cancer cells with an activation of these oncogenes.     

N-ras mutations in myeloid leukemias

The presence of mutations activating the N-ras gene was investigated by the polymerase chain reaction technique in twenty patients with acute myeloblastic leukemia (AML) at onset and in four patients with Ph1 positive chronic myelogeneous leukemia (CML) either in chronic phase or in blast crisis. Four remission samples and four relapses from the AML cases were also studied. Mutations were found in five out of twenty (25%) untreated AML cases at onset. No mutations were detected in the complete remission samples, two of them with N-ras mutations during the leukemic phase. Two out of the four leukemia relapses were positive for the same N-ras mutation shown at presentation, whereas no new mutations were found in the other two initially negative cases. An N-ras mutation appeared during the blast crisis of one of the four CML, which were all negative during the chronic phase. In conclusion, whereas some data appear to be consistent with a role of the N-ras mutations as initiating events in myeloid leukemias, in other cases N-ras activation seems to represent a factor involved in progression. These data suggest that a partial overlapping between initiation and progression factors could exist in naturally occurring tumors.     

Prevalence of N-ras mutations in children with myelodysplastic syndromes and acute myeloid leukemia.

The ras proto-oncogene family encodes a group of 21 kDa nucleotide-binding proteins. Activating mutations of ras genes are associated with certain types of malignancies, indicating that they are related in some way to the malignant process. We have examined bone marrow cells from nine children with myelodysplastic syndromes (MDS) and 35 with acute myeloid leukemia (AML) for activating point mutations of ras genes by in vitro amplification using polymerase chain reaction (PCR), oligonucleotide hybridization and sequencing of PCR products. We found N-ras mutations in cells from 3 of 9 children (33%) with MDS and only 2 of 35 children with AML (6%; 95% confidence interval is 0.7-19%). All mutations the second nucleotide of codon 12 or the first nucleotide of codon 61 of N-ras. There was no apparent correlation with clinical or laboratory characteristics, including karyotype; however, an association of N-ras activation with the most aggressive type of MDS was noted. Among the patients with MDS, 2 of 6 with monosomy 7 had N-ras mutations; however, three children with monosomy 7 which presented with AML lacked ras mutations. One patient was studied at time of diagnosis of MDS and again after progression to AML. At the preleukemic stage of disease, an N-ras mutation was identified; however, after development of AML this mutation was not present in the leukemic clone. In conclusion, these data show that ras mutations, while not necessary for leukemic transformation, may be important for the initiation of preleukemias evolving into overt AML.     

A novel oncogenic role for the miRNA-506-514 cluster in initiating melanocyte transformation and promoting melanoma growth

Malignant melanoma is the most aggressive form of skin cancer and its incidence has doubled in the last two decades. It represents only 4% of skin cancer cases per year, but causes as many as 74% of skin cancer deaths. Early detection of malignant melanoma is associated with survival rates of up to 90%, but later detection (stage III to stage IV) is associated with survival rates of only 10%. Dysregulation of microRNA (miRNA) expression has been linked to tumor development and progression by functioning either as a tumor suppressor, an oncogene or a metastasis regulator in multiple cancer types. To understand the role of miRNA in the pathogenesis of malignant melanoma and identify biomarkers of metastasis, miRNA expression profiles in skin punches from 33 metastatic melanoma patients and 14 normal healthy donors were compared. We identified a cluster of 14 miRNAs on the X chromosome, termed the miR-506-514 cluster, which was consistently overexpressed in nearly all melanomas tested (30-60 fold, P<0.001), regardless of mutations in N-ras or B-raf. Inhibition of the expression of this cluster as a whole, or one of its sub-clusters (Sub-cluster A) consisting of six mature miRNAs, led to significant inhibition of cell growth, induction of apoptosis, decreased invasiveness and decreased colony formation in soft agar across multiple melanoma cell lines. Sub-cluster A of the miR-506-514 cluster was critical for maintaining the cancer phenotype, but the overexpression of the full cluster was necessary for melanocyte transformation. Our results provide new insights into the functional role of this miRNA cluster in melanoma, and suggest new approaches to treat or diagnose this disease.