P53 In Melanoma Research Articles

Abstract 4785: S-nitrosylation of p53 in melanoma alters p53-DNA binding and downstream gene expression

Abstract Aberrant expression of inducible nitric oxide synthase (iNOS) and chronic nitrosative stress is correlated with growth, metastasis, resistance to therapy, and poor patient survival of metastatic melanoma. One physiological influence of nitric oxide (NO) is exerted directly through the post-translational modification of many proteins, including the unique S-nitrosylation (SNO). To date, the role of SNO in melanoma development has not been fully characterized. Herein, we focus on the mechanism of how nitrosative stress regulates the tumor suppressor p53 in melanoma cells. We first examined the effects of different levels of NO donors, S-nitrosoglutathione (GSNO) and diethylenetriamine (DETA) NONOate, on the growth of melanoma cells by the MTT assay. Low levels of nitrosative stress (NO donors ≤ 20 µM) did not suppress the growth of human melanoma A375 and SB2 cells, but high levels of nitrosative stress (NO donors ≥ 50 µM) inhibited melanoma cell growth. Our biotin switch assays (BSA) showed that the treatment of NO donors significantly increased total S-nitrosylated proteins in A375 and SB2 cells. Markedly, p53 is confirmed to be one of the S-nitrosylated proteins derived from BSA. Our mass spectrometry analysis of SNO proteins in melanoma cells confirms that p53 is S-nitrosylated in A375 cells under nitrosative stress. Further proteomic characterization identifies Cys242, Cys275, and Cys277 as the SNO sites of p53. We conducted a molecular dynamics (MD) simulation of p53-DNA binding under SNO modification. The structural analysis shows that the SNO of Cys277 reduces the hydrogen bonds between p53 and the targeted DNA, interfering with the recognition and binding of p53 to DNA. Moreover, the chelating ability of Cys242 for Zn2+ is weakened after SNO, indicating the SNO of p53 reduces p53 binding to Zn2+ and affects its active form. The electrophoretic mobility shift assay also confirmed that the treatment of 100 µM of DETA NONOate, significantly decreased the nuclear p53 of A375 and SB2 cells binding to the consensus p53-binding DNA. The induction of nitrosative stress by NO donors also significantly increased mouse double minute 2 homolog (MDM2), p21, and p53 expression but decreased p53 upregulated modulator of apoptosis (PUMA) expression.This study confirms that the crucial tumor suppressor, p53, is S-nitrosylated in melanoma cells under nitrosative stress. Notably, for the first time, the SNO sites of p53 and the effects of SNO on p53 functions were characterized. Our results describe an important mechanism of how nitrosative stress modifies crucial cysteines of p53, alters p53 DNA-binding activities, and regulates downstream gene expression through SNO. Our study provides much-needed insights into identifying novel NO-driven SNO proteins in melanoma as new biomarkers and targets for drug development. Citation Format: Jordan Winfield, Mariana Grigoruta, Yiliang Li, Fancui Meng, Yong Qin, Elizabeth Grimm, Leyuan Chen, Kevin Rosenblatt, Li Li. S-nitrosylation of p53 in melanoma alters p53-DNA binding and downstream gene expression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4785.

P53 Promotes Cytokine Expression in Melanoma to Regulate Drug Resistance and Migration.

The transcription factor p53 is frequently lost during tumor development in solid tumors; however, most melanomas retain a wild type p53 protein. The presence of wild type p53 in melanoma has fueled speculation that p53 may play a neutral or pro-tumorigenic role in this disease. Here we show that p53 is functional in human melanoma cell lines, and that loss of p53 results in a general reduction in basal NF-kB regulated cytokine production. The reduced cytokine expression triggered by p53 loss is broad and includes key inflammatory chemokines, such as CXCL1, CXCL8, and the IL6 class cytokine LIF, resulting in a reduced ability to induce chemotactic-dependent migration of tumor cells and immune cells and increased sensitivity to BRAF inhibition. Taken together, this result indicates that wild type p53 regulates cytokine expression and induces cytokine-dependent phenotype on melanoma.

Metastatic Melanoma Patient-Derived Xenografts Respond to MDM2 Inhibition as a Single Agent or in Combination with BRAF/MEK Inhibition.

Over 60% of patients with melanoma respond to immune checkpoint inhibitor (ICI) therapy, but many subsequently progress on these therapies. Second-line targeted therapy is based on BRAF mutation status, but no available agents are available for NRAS, NF1, CDKN2A, PTEN, and TP53 mutations. Over 70% of melanoma tumors have activation of the MAPK pathway due to BRAF or NRAS mutations, while loss or mutation of CDKN2A occurs in approximately 40% of melanomas, resulting in unregulated MDM2-mediated ubiquitination and degradation of p53. Here, we investigated the therapeutic efficacy of over-riding MDM2-mediated degradation of p53 in melanoma with an MDM2 inhibitor that interrupts MDM2 ubiquitination of p53, treating tumor-bearing mice with the MDM2 inhibitor alone or combined with MAPK-targeted therapy. To characterize the ability of the MDM2 antagonist, KRT-232, to inhibit tumor growth, we established patient-derived xenografts (PDX) from 15 patients with melanoma. Mice were treated with KRT-232 or a combination with BRAF and/or MEK inhibitors. Tumor growth, gene mutation status, as well as protein and protein-phosphoprotein changes, were analyzed. One-hundred percent of the 15 PDX tumors exhibited significant growth inhibition either in response to KRT-232 alone or in combination with BRAF and/or MEK inhibitors. Only BRAFV600WT tumors responded to KRT-232 treatment alone while BRAFV600E/M PDXs exhibited a synergistic response to the combination of KRT-232 and BRAF/MEK inhibitors. KRT-232 is an effective therapy for the treatment of either BRAFWT or PAN WT (BRAFWT, NRASWT) TP53WT melanomas. In combination with BRAF and/or MEK inhibitors, KRT-232 may be an effective treatment strategy for BRAFV600-mutant tumors.

AXL receptor signalling suppresses p53 in melanoma through stabilization of the MDMX-MDM2 complex.

Deregulation of the tyrosine kinase signalling is often associated with tumour progression and drug resistance, but its underlying mechanisms are only partly understood. In this study, we investigated the effects of the receptor tyrosine kinase AXL on the stability of the MDMX-MDM2 heterocomplex and the activity of p53 in melanoma cells. Our data demonstrated that AXL overexpression or activation through growth arrest-specific 6 (Gas6) ligand stimulation increases MDMX and MDM2 protein levels and decreases p53 activity. Upon activation, AXL stabilizes MDMX through a post-translational modification that involves phosphorylation of MDMX on the phosphosite Ser314, leading to increased affinity between MDMX and MDM2 and favouring MDMX nuclear translocation. Ser314 phosphorylation can also protect MDMX from MDM2-mediated degradation, leading to stabilization of the MDMX-MDM2 complex. We identified CDK4/6 and p38 MAPK as the two kinases mediating AXL-induced modulation of the MDMX-MDM2 complex, and demonstrated that suppression of AXL, either through siRNA silencing or pharmacological inhibition, increases expression levels of p53 target genes P21, MDM2, and PUMA, improves p53 pathway response to chemotherapy, and sensitizes cells to both Cisplatin and Vemurafenib. Our findings offer an insight into a novel signalling axis linking AXL to p53 and provide a potentially druggable pathway to restore p53 function in melanoma.

Targeting p53 in melanoma

Melanoma is the sixth most common cancer in the United States with the numbers of annual cases increasing faster than any other tumor type. Approximately 50% of melanomas harbor oncogenic BRAF mutations, 90% of which are BRAFV600E. Recently, US Food and Drug Administration (FDA) approval of the selective BRAFV600E inhibitor, Zelboraf (vemurafenib), has inspired rapid development of new targeted therapeutic strategies for melanoma. Subsequently, the first FDA approved combination of molecularly targeted cancer therapies has emerged with GlaxoSmithKline’s Tafinlar (dabrafenib) and Mekinist (trametinib) that inhibit BRAFV600E and the primary downstream target, MEK (mitogen-activated protein kinase kinase), respectively. As BRAF and MEK function within the same oncogenic RAS pathway, there is much anticipation about forthcoming clinical trials investigating molecularly targeted combination therapies that attack two or more different pathways.

High-level expression of wild-type p53 in melanoma cells is frequently associated with inactivity in p53 reporter gene assays.

BackgroundInactivation of the p53 pathway that controls cell cycle progression, apoptosis and senescence, has been proposed to occur in virtually all human tumors and p53 is the protein most frequently mutated in human cancer. However, the mutational status of p53 in melanoma is still controversial; to clarify this notion we analysed the largest series of melanoma samples reported to date.Methodology/Principal FindingsImmunohistochemical analysis of more than 180 melanoma specimens demonstrated that high levels of p53 are expressed in the vast majority of cases. Subsequent sequencing of the p53 exons 5–8, however, revealed only in one case the presence of a mutation. Nevertheless, by means of two different p53 reporter constructs we demonstrate transcriptional inactivity of wild type p53 in 6 out of 10 melanoma cell lines; the 4 other p53 wild type melanoma cell lines exhibit p53 reporter gene activity, which can be blocked by shRNA knock down of p53.Conclusions/SignificanceIn melanomas expressing high levels of wild type p53 this tumor suppressor is frequently inactivated at transcriptional level.

P53 in human melanoma fails to regulate target genes associated with apoptosis and the cell cycle and may contribute to proliferation

BackgroundMetastatic melanoma represents a major clinical problem. Its incidence continues to rise in western countries and there are currently no curative treatments. While mutation of the P53 tumour suppressor gene is a common feature of many types of cancer, mutational inactivation of P53 in melanoma is uncommon; however, its function often appears abnormal.MethodsIn this study whole genome bead arrays were used to examine the transcript expression of P53 target genes in extracts from 82 melanoma metastases and 6 melanoma cell lines, to provide a global assessment of aberrant P53 function. The expression of these genes was also examined in extracts derived from diploid human melanocytes and fibroblasts.ResultsThe results indicated that P53 target transcripts involved in apoptosis were under-expressed in melanoma metastases and melanoma cell lines, while those involved in the cell cycle were over-expressed in melanoma cell lines. There was little difference in the transcript expression of P53 target genes between cell lines with null/mutant P53 compared to those with wild-type P53, suggesting that altered expression in melanoma was not related to P53 status. Similarly, down-regulation of P53 by short-hairpin RNA (shRNA) had limited effect on P53 target gene expression in melanoma cells, whereas there were a large number of P53 target genes whose mRNA expression was significantly altered by P53 inhibition in melanocytes. Analysis of whole genome gene expression profiles indicated that the ability of P53 to regulate genes involved in the cell cycle was significantly reduced in melanoma cells. Moreover, inhibition of P53 in melanocytes induced changes in gene expression profiles that were characteristic of melanoma cells and resulted in increased proliferation. Conversely, knockdown of P53 in melanoma cells resulted in decreased proliferation.ConclusionsThese results indicate that P53 target genes involved in apoptosis and cell cycle regulation are aberrantly expressed in melanoma and that this aberrant functional activity of P53 may contribute to the proliferation of melanoma.

Abstract 587: B-Raf status predicts response to pharmacological activation of p53 in melanoma

Abstract The formulation of highly-potent and specific inhibitors of the MAPK pathway has yielded several promising candidates and these are currently under advanced clinical development; however, early data suggests that inhibition of the MAPK pathway will not be sufficient to elicit sustained tumor regression in malignant melanoma. Thus, other pathways likely need to be therapeutically exploited to achieve sustained responses in melanoma patients. Because the tumor suppressor protein p53 is functional, yet inactive, in nearly 100% of melanomas, we sought to identify novel pharmacological activators of p53 in melanoma. To accomplish this goal, we employed a high-throughput screening strategy that identified hycanthone, a compound previously characterized for anti-schistosomal activity, as a specific activator of p53 function. In vitro characterization of hycanthone demonstrated that it activates the p53-transcriptional response leading to cell cycle arrest and subsequent apoptosis in a panel of melanoma cell lines harboring wild-type p53; these effects are largely absent in isogenic cells expressing p53 shRNA or alternative lines expressing mutant p53. Screening of a substantial panel of cell lines revealed that activation of p53 initiated anti-melanoma effects exclusively in cells that expressed mutant Braf, indicating that activated MAPK signaling may serve as a correlate to predict response to pharmacological activators of p53 in melanoma. However, additional experimentation has demonstrated that, instead, it is the presence of mutations in Braf, rather than the resultant MAPK activity, that predicts response to p53 activation. Studies investigating the mechanistic details governing this biological phenomenon are ongoing. Collectively, our data indicate that reactivation of p53 in melanoma, like multiple other tumors, can elicit an anti-tumor phenotype; however, this response is negatively modulated by the expression of mutant Braf, which would suggest that patients should be prospectively genotyped to identify potential responders to p53 reactivators. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 587. doi:10.1158/1538-7445.AM2011-587

Abstract 2328: 15N-backbone and side-chain dynamics of the mutant calcium-binding protein S100B D63N

Abstract Similar to calmodulin and other EF-hand-containing proteins, S100 proteins have no inherent enzymatic activity and perform their biological functions via calcium-dependent protein-protein interactions. Elevated protein levels of S100B have been linked to various types of cancers, including malignant melanomas, glioblastomas, and anaplastic astrocytomas. In the case of malignant melanomas, S100B contributes to cell proliferation by binding to p53 and inhibiting its tumor suppressing properties. However, one property of S100 proteins, including S100B, is that it only binds to calcium tightly when its target protein is bound, a feature that drug design studies for restoring p53 in melanoma must mimic. Therefore, the purpose of this study is to determine the mechanism for how target protein binding to S100B causes an increase in calcium-binding affinity. Previous studies suggest that the increase in calcium affinity in the presence of target is not the result of a structural modification. As an alternative explanation, the mechanism behind this event may involve a reduction in the ligand dynamics coordinating calcium in the canonical EF-hand when target peptide is present. To test this hypothesis with S100B, an asparagine mutant was made in order to have an 15N-probe in the tight-binding EF-hand of S100B for NMR side-chain dynamics with and without target peptide. 15N backbone dynamic experiments (T1, T2, and hetNOE) were collected for the S100B D63N mutant (+/- TRTK-12) at two magnetic fields to determine the effects of peptide binding at multiple time scales. Relaxation dispersion experiments were also collected for backbone and side-chain amides of S100B in the presence and absence of TRTK-12. For the backbone dynamic experiments, motion was detected in the hinge region and C-terminus of helix-4 at multiple timescales in the absence of TRTK-12 (ns-sec timescale). A similar profile was observed for those backbone residues undergoing motion in the chemical exchange time scale seen with the relaxation dispersion data. However, when a molecular target was introduced, the majority of these backbone amides that make up the hydrophobic pocket displayed a general decrease in dynamics. Side chain dynamics in the EF-hand was appreciated in the absence of TRTK-12 peptide, however when the target was present, no chemical exchange was seen with the 15N-D63N probe in the EF-hand. These findings imply that In the presence of target peptide, S100 proteins undergo a decrease in protein dynamics that translates from the site of target binding to the EF-hand. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2328. doi:10.1158/1538-7445.AM2011-2328

PRIMA-1Met/APR-246 induces wild-type p53-dependent suppression of malignant melanoma tumor growth in 3D culture and in vivo

Disseminating malignant melanoma is a lethal disease highly resistant to radio- and chemotherapy. Therefore, the development of new treatment strategies is strongly needed. Tumor suppressor p53-mediated apoptosis is essential for the response to radio- and chemotherapy. Although p53 is not frequently mutated in melanoma, it is inactivated by integrin αv-mediated signaling, as we previously demonstrated 1, which may account, at least partially, for increased apoptosis resistance of malignant melanoma. In this study we addressed the question whether functional restoration of p53 by APR-246 (PRIMA-1Met), which can reactivate mutant p53 and induce massive apoptosis in cancer cells, is able to restore the function of inactive p53 in melanoma. Using a three-dimensional collagen gel (3D-collagen) to culture melanoma cells carrying wild-type p53, we found that APR-246 treatment resulted in activation of p53, leading to increased expression of p53 pro-apoptotic targets Apaf1 and PUMA and activation of caspase- 9 and -3. Moreover, APR-246 triggered melanoma cell apoptosis that was mediated by p53 and caspase 9. Importantly, APR-246 treatment also suppressed human melanoma xenograft tumors in vivo in a p53-dependent manner. Thus, wild-type p53 reactivation may provide a novel approach for malignant melanoma treatment, with APR-246 as a candidate drug for such a development.

Ubiquitin proteasomal pathway mediated degradation of p53 in melanoma

Ubiquitin proteasomal pathway (UPP) is the principle mechanism for protein catabolism and affects cellular processes critical for survival and proliferation. Levels of tumor suppressor protein p53 are very low in cells due to its rapid turnover by UPP-mediated degradation. While p53 is mutated in human cancers, most human melanomas maintain wild-type conformation. In this study, to investigate the effects of UPP inhibitor in vitro and in vivo, we used a genetically-engineered mouse model (GEMM) that has the same genetic alterations as those of human melanomas . Melanoma cells were established from mouse tumors and named 8B20 cells. Treatment of 8B20 cells with the UPP inhibitors, MG132 and clasto-lactacystin-β-lactone, led to an increase in levels of p53 while treatment with non-proteasomal inhibitors did not alter p53 levels. UPP inhibitors induced formation of heavy molecular weight ubiquitinated proteins, a hallmark of UPP inhibition, and p53-specific poly-ubiquitinated products in 8B20 cells. To further decipher the mechanism of p53 stabilization, we investigated half-life of p53 in cells treated with cycloheximide to block de novo protein synthesis. Treatment of 8B20 cells with MG132 led to an increase in the half-life of p53. Further analysis revealed that p53 stabilization was not mediated by phosphorylation of Ser-15 and Ser-20 residues. In vivo studies showed that MG132 induced p53 overexpression and reduced tumor growth, suggesting an important role of p53 stabilization in controlling melanoma. Taken together, our studies provide a proof of principle for using a GEMM to address the mechanisms of action and efficacy of melanoma treatment.

Tanning, p53 and PCMR

Tanning, p53 and PCMR

Potent p53‐independent tumor suppressor activity of ARF in melanoma‐genesis

It is generally accepted that human cancer is a resultof genetic mutations in molecular pathways controllingprocesses that normally harness the capacity for prolif-eration and survival. An emerging picture has begun toevolve from our understanding of these particular path-ways as they become disrupted in various cancers, in afashion which shows significant lineage-dependence.Cutaneous malignant melanoma, the most commonform of melanoma in man, appears to invariably harbormutations leading to activation of the canonical MAPK-pathway wherein the oncogene BRAF is found activatedin approximately 70% of all melanomas (Davies et al.,2002) as well as benign nevi (Pollock et al., 2003) andcorrespondingly NRAS in another 25% (Demunteret al., 2001) leaving only a small fraction unaccountedfor. In addition, disruption of the pRB-pathway throughloss of pRB, mutation or amplification of CDK4, amplifi-cation of CCND1, missense mutation or deletion of theINK4A-locus is similarly common. However, contrastingmany other human malignancies, melanomas exhibit astrikingly low frequency of canonical p53-mutations,amounting to approximately 20–25% of all cases(Ragnarsson-Olding et al., 2002). This is particularlynotable because of the discordance between the p53’swild-type status and the near-uniform relative refractori-ness of melanomas to numerous triggers of apoptosis,including cytotoxic chemotherapeutic agents. Addition-ally, disruption of the TP53-pathway through amplifica-tion of the HDM2 oncogene occasionally occurs, but itsupstream inhibitor ARF, the alternative product of theINK4A-locus (Kamijo et al., 1997), is a well-known mela-noma tumor-suppressor whose loss together withCDKN2A⁄p16 is relatively common during melanoma-genesis. It is broadly accepted that ARF functions toinhibit MDM2⁄HDM2, the canonical negative regulatorof p53, in mouse and man while there are additionalARF-interacting proteins that may execute importantfunctions various settings of tumorigenesis (Sherr,2006). The common occurrence of wild-type p53together with ARF (and CDKN2A⁄p16) deficiency in mel-anoma has appropriately raised the prospect of a thera-peutic opportunity based upon reactivating p53-function,to which end multiple lead drugs are in various stagesof development.In a recently published study, Merlino and colleagues(Ha et al., 2007) have utilized a powerful genetic modelof murine melanoma to assess the functional roles ofp53 and ARF in murine melanoma-genesis. Their dataclearly demonstrate p53-independent tumor suppressiveactivity of ARF- an observation which carries importantimplications for therapeutic targeting⁄activation of p53in melanoma patients.In this study, Ha et al. employ an established mousemodel that develops UV-induced cutaneous melanomaas well as rhabdomyosarcoma (RMS) via transgenicexpression of hepatocyte growth factor⁄scatter factor(Noonan et al., 2001), the ligand for the cMET receptortyrosine kinase. Merlino et al. previously showed thatmelanoma latency following UV irradiation was signifi-cantly reduced when the transgenic mice were crossedinto an INK4A⁄ARF-null background, suggesting thatloss of both the TP53- (through ARF) and pRB- (throughCDKN2A⁄p16) pathways likely contribute to develop-ment of UV-induced cutaneous melanoma. In this study,the authors have overlaid a more refined approach, ofseparating the functions of the two products from theINK4A-locus. They observe that ARF-nullizygosity exhib-its nearly the same frequency and timing of RMS devel-opment as genetic ablation of both products. Similarly,p53-nullizygousity exhibited similar frequency and timingas ARF-alone for induction of RMS, thus consistent withthe predicted role of the ARF-(MDM2)-p53-pathway fortumor suppression. Importantly, however, UV-inducedmelanoma incidence in these transgenic animalsshowed dependence on both CDKN2A and ARF statuswhereas p53-nullizygousity had no overt effect, suggest-ing that ARF tumor suppressor function is mechanisti-cally distinct from p53-function. The importance of thismelanoma-specific behavior is underscored by the dif-ferences seen in RMS tumors within the same geneticmodel. Interestingly, in a UV-independent RAS-driventransgenic model, CDKN2A, ARF and p53 all displayedimportant contributions as suppressors of melanomaincidence (Bardeesy et al., 2001; Sharpless and Chin,

An Organometallic Protein Kinase Inhibitor Pharmacologically Activates p53 and Induces Apoptosis in Human Melanoma Cells

Unlike other tumors, melanomas harbor wild-type (WT) p53 but exhibit impaired p53-dependent apoptosis. The mechanisms for the impaired p53 activation are poorly understood but may be linked to the high expression of the p53 suppressor Mdm2, which is found in >50% of melanoma lesions. Here, we describe an organometallic glycogen synthase kinase 3beta (GSK3beta) inhibitor (DW1/2) as a potent activator of p53 and inducer of cell death in otherwise highly chemoresistant melanoma cells. Using RNA interference and pharmacologic approaches, we show that p53 is required for the cytotoxic effects of this organometallic inhibitor. The DW1/2 compound was barely able to induce cell death in melanoma cells with p53 mutations, further confirming the requirement for p53-WT in the cytotoxic effects of the GSK3beta inhibition. Mechanistic analysis of the p53-dependent cell death indicated an apoptotic mechanism involving depolarization of mitochondrial membrane potential, caspase cleavage, and elevated NOXA expression. The effect of p53 was not simply due to passive up-regulation of protein expression as adenoviral-mediated overexpression of p53 was not able to induce cell death. Treatment of melanoma cells with DW1/2 was instead found to decrease levels of Mdm2 and Mdm4. The importance of Mdm2 down-regulation in DW1/2-induced apoptosis was confirmed by treating the p53-WT cells with the p53/Mdm2 antagonist Nutlin-3. Taken together, our data provide a new strategy for the pharmacologic activation of p53 in melanoma, which may be a viable approach for overcoming apoptotic resistance in melanoma and offer new hope for rational melanoma therapy.

A Merging Duo in Melanoma Formation

A Merging Duo in Melanoma Formation

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 T:A 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. 1999 Cancer Research Campaign

Mutation of p53 in melanoma

Mutation of p53 in melanoma