P53 prevents progression of nevi to melanoma predominantly through cell cycle regulation.
ABSTRACT: 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.
Project description:The inactivation of the p53 tumor suppressor pathway, which often occurs through mutations in TP53 (encoding tumor protein 53) is a common step in human cancer. However, in melanoma-a highly chemotherapy-resistant disease-TP53 mutations are rare, raising the possibility that this cancer uses alternative ways to overcome p53-mediated tumor suppression. Here we show that Mdm4 p53 binding protein homolog (MDM4), a negative regulator of p53, is upregulated in a substantial proportion (?65%) of stage I-IV human melanomas and that melanocyte-specific Mdm4 overexpression enhanced tumorigenesis in a mouse model of melanoma induced by the oncogene Nras. MDM4 promotes the survival of human metastatic melanoma by antagonizing p53 proapoptotic function. Notably, inhibition of the MDM4-p53 interaction restored p53 function in melanoma cells, resulting in increased sensitivity to cytotoxic chemotherapy and to inhibitors of the BRAF (V600E) oncogene. Our results identify MDM4 as a key determinant of impaired p53 function in human melanoma and designate MDM4 as a promising target for antimelanoma combination therapy.
Project description:The identification of the essential role of cyclin-dependent kinases (CDKs) in the control of cell division has prompted the development of small-molecule CDK inhibitors as anticancer drugs. For many of these compounds, the precise mechanism of action in individual tumor types remains unclear as they simultaneously target different classes of CDKs - enzymes controlling the cell cycle progression as well as CDKs involved in the regulation of transcription. CDK inhibitors are also capable of activating p53 tumor suppressor in tumor cells retaining wild-type p53 gene by modulating MDM2 levels and activity. In the current study, we link, for the first time, CDK activity to the overexpression of the MDM4 (MDMX) oncogene in cancer cells. Small-molecule drugs targeting the CDK9 kinase, dinaciclib, flavopiridol, roscovitine, AT-7519, SNS-032, and DRB, diminished MDM4 levels and activated p53 in A375 melanoma and MCF7 breast carcinoma cells with only a limited effect on MDM2. These results suggest that MDM4, rather than MDM2, could be the primary transcriptional target of pharmacological CDK inhibitors in the p53 pathway. CDK9 inhibitor atuveciclib downregulated MDM4 and enhanced p53 activity induced by nutlin-3a, an inhibitor of p53-MDM2 interaction, and synergized with nutlin-3a in killing A375 melanoma cells. Furthermore, we found that human pluripotent stem cell lines express significant levels of MDM4, which are also maintained by CDK9 activity. In summary, we show that CDK9 activity is essential for the maintenance of high levels of MDM4 in human cells, and drugs targeting CDK9 might restore p53 tumor suppressor function in malignancies overexpressing MDM4.
Project description:The purpose of the present study was to investigate the prognostic significance of murine double minute 4 (MDM4) in chronic lymphocytic leukemia (CLL) and to characterize the role of MDM4 in the p53 pathway. Full-length MDM4 (FL-MDM4), a splicing variant of MDM4 (S-MDM4) and murine double minute 2 (MDM2) mRNA expressions were detected by quantitative PCR in 140 Chinese patients with CLL, and primary CLL cells were treated in vitro with either fludarabine or Nutlin-3 to explore the interaction between p53 status and MDM4 or MDM2 expression. A marked increase of FL-MDM4 and S-MDM4 expressions were observed in the CLL patients with p53 aberrations (deletion and/or mutation) (P = 0.024, P < 0.001). A high level of S-MDM4 mRNA expression was associated with short treatment free survival (TFS) (P = 0.004). FL-MDM4 expression was significantly decreased after fludarabine treatment (P = 0.001) but increased after Nutlin-3 treatment (P = 0.008) of primary CLL cells without p53 aberrations. Both S-MDM4 and MDM2 expressions were significantly increased after fludarabine treatment of CLL cells without p53 aberrations (P = 0.013 and P = 0.030). MDM2 overexpression also occurred in CLL cells with p53 wild type after Nutlin-3 treatment (P = 0.018). FL-MDM4 and S-MDM4 overexpression are indicators of p53 aberrations in CLL patients, suggesting that those patients have a poor prognosis. FL-MDM4 inhibitory effects on p53 can be removed by MDM2-p53 and saved by Nutlin-3.
Project description:Loss of p16(INK4a)-RB and ARF-p53 tumor suppressor pathways, as well as activation of RAS-RAF signaling, is seen in a majority of human melanomas. Although heterozygous germline mutations of p16(INK4a) are associated with familial melanoma, most melanomas result from somatic genetic events: often p16(INK4a) loss and N-RAS or B-RAF mutational activation, with a minority possessing alternative genetic alterations such as activating mutations in K-RAS and/or p53 inactivation. To generate a murine model of melanoma featuring some of these somatic genetic events, we engineered a novel conditional p16(INK4a)-null allele and combined this allele with a melanocyte-specific, inducible CRE recombinase strain, a conditional p53-null allele and a loxP-stop-loxP activatable oncogenic K-Ras allele. We found potent synergy between melanocyte-specific activation of K-Ras and loss of p16(INK4a) and/or p53 in melanomagenesis. Mice harboring melanocyte-specific activated K-Ras and loss of p16(INK4a) and/or p53 developed invasive, unpigmented and nonmetastatic melanomas with short latency and high penetrance. In addition, the capacity of these somatic genetic events to rapidly induce melanomas in adult mice suggests that melanocytes remain susceptible to transformation throughout adulthood.
Project description:Although p53 is found mutated in almost 50% of all cancers, p53 mutations in leukaemia are relatively rare. Acute myeloid leukaemia (AML) cells employ other strategies to inactivate their wild type p53 (WTp53), like the overexpression of the p53 negative regulators Mdm2 and Mdm4. As such, AMLs are excellent candidates for therapeutics involving the reactivation of their WTp53 to restrict and destroy cancer cells, and the Mdm2 antagonist nutlin-3 is one such promising agent. Using AML cell lines with WTp53, we identified stable and high levels of p53 in the OCI/AML-2 cell lines. We demonstrate that this nutlin-3 sensitive cell line overexpressed Mdm4 to sequester, stabilise and inhibit p53 in the cytoplasm. We also show that elevated Mdm4 competed with Mdm2-p53 interaction and therefore extended p53 half-life while preventing p53 transcriptional activity. Our results provide biochemical evidence on the dynamics of the p53-Mdm2-Mdm4 interactions in affecting p53 levels and activity, and unlike previously reported findings derived from genetically manipulated systems, AML cells with naturally high levels of Mdm4 remain sensitive to nutlin treatment.Endogenously high levels of Mdm4 inhibit and sequester p53 in AML. High levels of Mdm4 do not block function of Mdm2 inhibitors in AML.
Project description:Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors are an established treatment in estrogen receptor-positive breast cancer and are currently in clinical development in melanoma, a tumor that exhibits high rates of CDK4 activation. We analyzed melanoma cells with acquired resistance to the CDK4/6 inhibitor palbociclib and demonstrate that the activity of PRMT5, a protein arginine methyltransferase and indirect target of CDK4, is essential for CDK4/6 inhibitor sensitivity. By indirectly suppressing PRMT5 activity, palbociclib alters the pre-mRNA splicing of MDM4, a negative regulator of p53, leading to decreased MDM4 protein expression and subsequent p53 activation. In turn, p53 induces p21, leading to inhibition of CDK2, the main kinase substituting for CDK4/6 and a key driver of resistance to palbociclib. Loss of the ability of palbociclib to regulate the PRMT5-MDM4 axis leads to resistance. Importantly, combining palbociclib with the PRMT5 inhibitor GSK3326595 enhances the efficacy of palbociclib in treating naive and resistant models and also delays the emergence of resistance. Our studies have uncovered a mechanism of action of CDK4/6 inhibitors in regulating the MDM4 oncogene and the tumor suppressor, p53. Furthermore, we have established that palbociclib inhibition of the PRMT5-MDM4 axis is essential for robust melanoma cell sensitivity and provide preclinical evidence that coinhibition of CDK4/6 and PRMT5 is an effective and well-tolerated therapeutic strategy. Overall, our data provide a strong rationale for further investigation of novel combinations of CDK4/6 and PRMT5 inhibitors, not only in melanoma but other tumor types, including breast, pancreatic, and esophageal carcinoma.
Project description:BACKGROUND:The causative link between UV exposure and melanoma development is well known, however the mechanistic relationship remains incompletely characterised. UVA and UVB components of sunlight are implicated in melanomagenesis; however the majority of studies have focused on the effects of UVB and UVC light. Interestingly, melanoma tumour sequencing has revealed an overrepresentation of mutations signature of unrepaired UV-induced DNA damage. Repair of UVA-induced DNA damage is thought to occur primarily through the Nucleotide Excision Repair (NER) pathway, which recognises and repairs damage either coupled to transcription (Transcription Coupled Repair; TCR), or through global genome scanning (Global Genome Repair; GGR). Current literature suggests NER is deficient in melanoma, however the cause of this remains unknown; and whether reduced NER activity in response to UVA may be involved in melanoma development remains uncharacterised. In this study we aimed to determine if melanoma cells exhibit reduced levels of NER activity in response to UVA. METHODS:Melanocyte and melanoma cell lines were UVA-irradiated, and DNA damage levels assessed by immunodetection of Cyclobutane Pyrimidine Dimer (CPD) and (6-4) Photoproduct [(6-4)PP] lesions. Expression of NER pathway components and p53 following UVA treatment was quantified by qPCR and western blot. RESULTS:UVA did not induce detectable induction of (6-4)PP lesions, consistent with previous studies. Repair of CPDs induced by UVA was initiated at 4 h and complete within 48 h in normal melanocytes, whereas repair initiation was delayed to 24 h and >40 % of lesions remained in melanoma cell lines at 48 h. This was coupled with a delayed and reduced induction of GGR component XPC in melanoma cells, independent of p53. CONCLUSION:These findings support that NER activity is reduced in melanoma cells due to deficient GGR. Further investigation into the role of NER in UVA-induced melanomagenesis is warranted and may have implications for melanoma treatment.
Project description:BRAF mutations play a well-established role in melanomagenesis; however, without additional genetic alterations, tumor development is restricted by oncogene-induced senescence (OIS). Here, we show that mutations in the NF1 tumor suppressor gene cooperate with BRAF mutations in melanomagenesis by preventing OIS. In a genetically engineered mouse model, Nf1 mutations suppress Braf-induced senescence, promote melanocyte hyperproliferation, and enhance melanoma development. Nf1 mutations function by deregulating both phosphoinositide 3-kinase and extracellular signal-regulated kinase pathways. As such, Nf1/Braf-mutant tumors are resistant to BRAF inhibitors but are sensitive to combined inhibition of mitogen-activated protein/extracellular signal-regulated kinase kinase and mTOR. Importantly, NF1 is mutated or suppressed in human melanomas that harbor concurrent BRAF mutations, NF1 ablation decreases the sensitivity of melanoma cell lines to BRAF inhibitors, and NF1 is lost in tumors from patients following treatment with these agents. Collectively, these studies provide mechanistic insight into how NF1 cooperates with BRAF mutations in melanoma and show that NF1/neurofibromin inactivation may have an impact on responses to targeted therapies.
Project description:MDM4 is a promising target for cancer therapy, as it is undetectable in most normal adult tissues but often upregulated in cancer cells to dampen p53 tumor-suppressor function. The mechanisms that underlie MDM4 upregulation in cancer cells are largely unknown. Here, we have shown that this key oncogenic event mainly depends on a specific alternative splicing switch. We determined that while a nonsense-mediated, decay-targeted isoform of MDM4 (MDM4-S) is produced in normal adult tissues as a result of exon 6 skipping, enhanced exon 6 inclusion leads to expression of full-length MDM4 in a large number of human cancers. Although this alternative splicing event is likely regulated by multiple splicing factors, we identified the SRSF3 oncoprotein as a key enhancer of exon 6 inclusion. In multiple human melanoma cell lines and in melanoma patient-derived xenograft (PDX) mouse models, antisense oligonucleotide-mediated (ASO-mediated) skipping of exon 6 decreased MDM4 abundance, inhibited melanoma growth, and enhanced sensitivity to MAPK-targeting therapeutics. Additionally, ASO-based MDM4 targeting reduced diffuse large B cell lymphoma PDX growth. As full-length MDM4 is enhanced in multiple human tumors, our data indicate that this strategy is applicable to a wide range of tumor types. We conclude that enhanced MDM4 exon 6 inclusion is a common oncogenic event and has potential as a clinically compatible therapeutic target.
Project description:Deregulation of signaling pathways that control differentiation, expansion and migration of neural crest-derived melanoblasts during normal development contributes also to melanoma progression and metastasis. Although several epithelial-to-mesenchymal (EMT) transcription factors, such as zinc finger E-box binding protein 1 (ZEB1) and ZEB2, have been implicated in neural crest cell biology, little is known about their role in melanocyte homeostasis and melanoma. Here we show that mice lacking Zeb2 in the melanocyte lineage exhibit a melanoblast migration defect and, unexpectedly, a severe melanocyte differentiation defect. Loss of Zeb2 in the melanocyte lineage results in a downregulation of the Microphthalmia-associated transcription factor (Mitf) and melanocyte differentiation markers concomitant with an upregulation of Zeb1. We identify a transcriptional signaling network in which the EMT transcription factor ZEB2 regulates MITF levels to control melanocyte differentiation. Moreover, our data are also relevant for human melanomagenesis as loss of ZEB2 expression is associated with reduced patient survival.