Inactivation of the p53 tumour suppressor, either by mutation or by overexpression of its inhibitors Hdm2 and HdmX is the most frequent event in cancer. Reactivation of p53 by targeting Hdm2 and HdmX is therefore a promising strategy for therapy. However, Hdm2 inhibitors do not prevent inhibition of p53 by HdmX, which impedes p53-mediated apoptosis. Here, we show that p53 reactivation by the small molecule RITA leads to efficient HdmX degradation in tumour cell lines of different origin and in xenograft tumours in vivo. Notably, HdmX degradation occurs selectively in cancer cells, but not in non-transformed cells. We identified the inhibition of the wild-type p53-induced phosphatase 1 (Wip1) as the major mechanism important for full engagement of p53 activity accomplished by restoration of the ataxia telangiectasia mutated (ATM) kinase-signalling cascade, which leads to HdmX degradation. In contrast to previously reported transactivation of Wip1 by p53, we observed p53-dependent repression of Wip1 expression, which disrupts the negative feedback loop conferred by Wip1. Our study reveals that the depletion of both HdmX and Wip1 potentiates cell death due to sustained activation of p53. Thus, RITA is an example of a p53-reactivating drug that not only blocks Hdm2, but also inhibits two important negative regulators of p53 – HdmX and Wip1, leading to efficient elimination of tumour cells.
p53; HdmX; RITA; Wip1; cancer
While half of all human
tumors possess p53 mutations, inactivation of wild-type p53 can also occur
through a variety of mechanisms that do not involve p53 gene mutation or
deletion. Our laboratory has been interested in tumor cells possessing
wild-type p53 protein and elevated levels of HdmX and/or Hdm2, two critical
negative regulators of p53 function. In this study we utilized RNAi to
knockdown HdmX or Hdm2 in MCF7 human breast cancer cells, which harbor
wild-type p53 and elevated levels of HdmX and Hdm2 then examined gene
expression changes and effects on cell growth. Cell cycle and growth assays
confirmed that the loss of either HdmX or Hdm2 led to a significant growth
inhibition and G1 cell cycle arrest. Although the removal of overexpressed
HdmX/2 appears limited to an anti-proliferative effect in MCF7 cells, the
loss of HdmX and/or Hdm2 enhanced cytotoxicity in these same cells exposed
to DNA damage. Through the use of Affymetrix GeneChips and subsequent
RT-qPCR validations, we uncovered a subset of anti-proliferative p53 target
genes activated upon HdmX/2 knockdown. Interestingly, a second set of
genes, normally transactivated by E2F1 as cells transverse the G1-S phase
boundary, were found repressed in a p21-dependent manner following HdmX/2
knockdown. Taken together, these results provide novel insights into the
reactivation of p53 in cells overexpressing HdmX and Hdm2.
p53; HdmX; Hdm2; RNAi; gene expression profiling
While half of all human tumors possess p53 mutations, inactivation of wild-type p53 can also occur through a variety of mechanisms that do not involve p53 gene mutation or deletion. Our laboratory has been interested in tumor cells possessing wild-type p53 protein and elevated levels of HdmX and/or Hdm2, two critical negative regulators of p53 function. In this study we utilized RNAi to knockdown HdmX or Hdm2 in MCF7 human breast cancer cells, which harbor wild-type p53 and elevated levels of HdmX and Hdm2 then examined gene expression changes and effects on cell growth. Cell cycle and growth assays confirmed that the loss of either HdmX or Hdm2 led to a significant growth inhibition and G1 cell cycle arrest. Although the removal of overexpressed HdmX/2 appears limited to an anti-proliferative effect in MCF7 cells, the loss of HdmX and/or Hdm2 enhanced cytotoxicity in these same cells exposed to DNA damage. Through the use of Affymetrix GeneChips and subsequent RT-qPCR validations, we uncovered a subset of anti-proliferative p53 target genes activated upon HdmX/2 knockdown. Interestingly, a second set of genes, normally transactivated by E2F1 as cells transverse the G1-S phase boundary, were found repressed in a p21-dependent manner following HdmX/2 knockdown. Taken together, these results provide novel insights into the reactivation of p53 in cells overexpressing HdmX and Hdm2.
p53; HdmX; Hdm2; RNAi; gene expression profiling
Cellular senescence is an irreversible state of terminal growth arrest that requires functional p53. Acting to block tumor formation, induction of senescence has also been demonstrated to contribute to tumor clearance via the immune system following p53 reactivation.1,2 the Hdm2-antagonist, Nutlin-3a, has been shown to reactivate p53 and induce a quiescent state in various cancer cell lines,3,4 similar to the G1 arrest observed upon RNAi targeting of Hdm2 in MCF7 breast cancer.5 In the present study we show that HdmX, a negative regulator of p53, impacts the senescence pathway. Specifically, overexpression of HdmX blocks Ras mediated senescence in primary human fibroblasts. the interaction of HdmX with p53 and the re-localization of HdmX to the nucleus through Hdm2 association appear to be required for this activity. Furthermore, inhibiting HdmX in prostate adenocarcinoma cells expressing wild-type p53, mutant Ras and high levels of HdmX-induced cellular senescence as measured by an increase in irreversible β-galactosidase staining. Together these results suggest that HdmX overexpression may contribute to tumor formation by blocking senescence and that targeting HdmX may represent an attractive anti-cancer therapeutic approach.
HdmX; p53; Ras; senescence; LNCaP
The Hdmx protein restricts p53 activity in vivo and is overexpressed in a significant fraction of human tumors that retain the wild type p53 allele. An understanding of how Hdmx limits p53 activation and blocks apoptosis could therefore lead to development of novel therapeutic agents. We previously showed that Hdmx modulates tumor cell sensitivity to Nutlin-3a, a potent antagonist of the p53/Hdm2 interaction. In this report, we demonstrate that this also applies to MI-219, another Hdm2 antagonist. Thus, the inability to disrupt Hdmx/p53 complexes is a potential barrier to the efficacy of these compounds as single agents. We show that sensitivity to apoptosis in cells with high Hdmx levels is restored by combined treatment with Nutlin and a Bcl-2 family member antagonist to activate Bax. The data are consistent with a model in which Hdmx attenuates p53-dependent activation of the intrinsic apoptotic pathway, and that this occurs upstream of Bax activation. Thus, selectively inhibiting Hdm2 and activating Bax is one effective strategy to induce apoptosis in tumors with high Hdmx levels. Our findings also indicate that preferential induction of apoptosis in tumor versus normal cells occurs using appropriate drug doses.
p53; apoptosis; Hdmx; Mdmx; bcl-2
Cancer cells neutralize p53 by deletion, mutation, proteasomal degradation, or sequestration to achieve a pathologic survival advantage. Targeting the E3 ubiquitin ligase HDM2 can lead to a therapeutic surge in p53 levels. However, the efficacy of HDM2 inhibition can be compromised by overexpression of HDMX, an HDM2 homologue that binds and sequesters p53. Here we report that a stapled p53 helix preferentially targets HDMX, blocks the formation of inhibitory p53-HDMX complexes, induces p53-dependent transcriptional upregulation, and thereby overcomes HDMX-mediated cancer resistance in vitro and in vivo. Importantly, our analysis of p53 interaction dynamics provides a blueprint for reactivating the p53 pathway in cancer by matching HDM2, HDMX, or dual inhibitors to the appropriate cellular context.
Selective inhibition of protein-protein interactions important for cellular processes could lead to the development of new therapies against disease. In the area of cancer, overexpression of the proteins human double minute 2 (HDM2) and its homolog HDMX has been linked to tumor aggressiveness. Both HDM2 and HDMX bind to p53 and prevent cell cycle arrest or apoptosis in damaged cells. Developing a strategy to simultaneously prevent the binding of both HDM2 and HDMX to p53 is an essential feature of inhibitors to restore p53 activity in a number of different cancers. Inhibition of protein-protein interactions with synthetic molecules is an emerging area of research that requires new inhibitors tailored to mimic the types of interfaces between proteins. Our strategy to create inhibitors of protein-protein interactions is to develop a non-natural scaffold that may be used as a starting point to identify important molecular components necessary for inhibition. In this study, we report an N-acylpolyamine (NAPA) scaffold that supports numerous sidechains in a compact atomic arrangement. NAPAs were constructed by a series of reductive aminations between amino acid derivatives followed by acylation at the resulting secondary amine. An optimized NAPA was able to equally inhibit the association of both HDM2 and HDMX with p53. Our results demonstrate some of the challenges associated with targeting multiple protein-protein interactions involved in overlapping cellular processes.
Previous studies have suggested that the mdmX gene is constitutively transcribed, and that MdmX protein activity is instead controlled by cellular localization and DNA damage induced Mdm2-mediated ubiquitination leading to proteasomal degradation. In these studies, we report that the human mdmX (hdmX) mRNA is reproducibly decreased in various human cell lines following treatment with various DNA-damaging agents. Repression of hdmX transcripts is observed in DNAdamaged HCT116 colon cancer cells and in isogenic p53−/− cells, suggesting that this effect is p53-independent. Reduction in the amount of hdmX transcript occurs in both human tumor cell lines and primary human diploid fibroblasts, and results in a significant reduction of HdmX protein. Examination of hdmX promoter activity suggests that damage-induced repression of hdmX mRNA is not significantly impacted by transcription initiation. In contrast, changes in hdmX mRNA splicing appear to partly explain the reduction in full-length hdmX mRNA levels in tumor cell lines with the destabilization of full-length hdmX transcripts, potentially through microRNA miR-34a regulation, also impacting transcript levels. Taken together, this study uncovers previously unrecognized cellular mechanisms by which hdmX mRNA levels are kept low following genotoxic stress.
HdmX; p53; transcription; splicing; micro-RNAs
Upregulation of structurally homologous oncoproteins Hdm2 and Hdmx has been linked to the depletion or inactivation of their common regulation target the tumor suppressor p53 protein leading to the progression of cancer. The restoration of the p53 function, rendered suppressed or dormant by these negative regulators, establishes, therefore, a unique opportunity for a targeted induction of apoptosis in cancers that retain wild-type p53. While several small molecules have been reported to rescue the tumor suppressor by antagonizing the Hdm2–p53 interaction, these agents displayed limited application scope by being ineffective in tumors enriched with active Hdmx. Here, we describe the use of a genetic selection system and encoded library of conformationally preorganized peptides to perform functional profiling of each regulator revealing specific recognition features that guide the antagonism of Hdm2–p53 and Hdmx–p53 interactions. Structure-activity relationship analysis of the most effective leads identified functional and structural elements mediating selective recognition of the two structurally related regulators, while providing convenient starting points for further activity optimization.
Hdm2; Hdmx; Protein-protein interactions; Genetic selection; Cyclic peptides
The p53 tumor suppressor plays a major role in maintaining genomic stability. Its activation and stabilization in response to double strand breaks (DSBs) in DNA are regulated primarily by the ATM protein kinase. ATM mediates several posttranslational modifications on p53 itself, as well as phosphorylation of p53's essential inhibitors, Hdm2 and Hdmx. Recently we showed that ATM- and Hdm2-dependent ubiquitination and subsequent degradation of Hdmx following DSB induction are mediated by phosphorylation of Hdmx on S403, S367, and S342, with S403 being targeted directly by ATM. Here we show that S367 phosphorylation is mediated by the Chk2 protein kinase, a downstream kinase of ATM. This phosphorylation, which is important for subsequent Hdmx ubiquitination and degradation, creates a binding site for 14-3-3 proteins which controls nuclear accumulation of Hdmx following DSBs. Phosphorylation of S342 also contributed to optimal 14-3-3 interaction and nuclear accumulation of Hdmx, but phosphorylation of S403 did not. Our data indicate that binding of a 14-3-3 dimer and subsequent nuclear accumulation are essential steps toward degradation of p53's inhibitor, Hdmx, in response to DNA damage. These results demonstrate a sophisticated control by ATM of a target protein, Hdmx, which itself is one of several ATM targets in the ATM-p53 axis of the DNA damage response.
Glioblastoma multiforme (GBM) is one of the deadliest tumors afflicting humans, and the mechanisms of its onset and progression remain largely undefined. Our attempts to elucidate its molecular pathogenesis through DNA copy-number analysis by genome-wide digital karyotyping and single nucleotide polymorphism arrays identified a dramatic focal amplification on chromosome 1q32 in 4 of 57 GBM tumors. Quantitative real-time PCR measurements revealed that HDMX is the most commonly amplified and overexpressed gene in the 1q32 locus. Further genetic screening of 284 low- and high-grade gliomas revealed that HDMX amplifications occur solely in pediatric and adult GBMs and that they are mutually exclusive of TP53 mutations and MDM2 amplifications. Here, we demonstrate that HDMX regulates p53 to promote GBM growth and attenuates tumor response to chemotherapy. In GBM cells, HDMX overexpression inhibits p53-mediated transcriptional activation of p21, releases cells from G0 to G1 phase, and enhances cellular proliferation. HDMX overexpression does not affect the expression of PUMA and BAX proapoptotic genes. While in GBM cells treated with the chemotherapeutic agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), HDMX appears to stabilize p53 and promote phosphorylation of the DNA double-stranded break repair protein H2AX, up-regulate the DNA repair gene VPX, stimulate DNA repair, and confer resistance to BCNU. In summary, HDMX exhibits bona fide oncogenic properties and offers a promising molecular target for GBM therapeutic intervention.
chemoresistance; GBM; HDMX; oncogene; p53
There is great interest in molecules capable of inhibiting the interactions between p53 and its negative regulators hDM2 and hDMX, as these molecules have validated potential against cancers in which one or both oncoproteins are overexpressed. We reported previously that appropriately substituted β3-peptides inhibit these interactions and, more recently, that minimally cationic β3-peptides are sufficiently cell permeable to upregulate p53-dependent genes in live cells. These observations, coupled with the known stability of β-peptides in a cellular environment, and the recently reported structures of hDM2 and hDMX, motivated us to exploit computational modeling to identify β-peptides with improved potency and/or selectivity. This exercise successfully identified a new β3-peptide, β53-16, that possesses the highly desirable attribute of high affinity for both hDM2 as well as hDMX and identifies the 3,4-dichlorophenyl moiety as a novel determinant of hDMX affinity.
Contradictory roles have been attributed to IL-1 in uveal melanoma progression. Inhibiting IL-1 with IL-1ra in experimental uveal melanoma inhibited tumor growth and modified tumor stroma including periodic acid-Schiff loops. Myeloid suppressor cells decreased.
In contrast to many malignancies showing evidence that interleukin-1 (IL-1) promotes progression through effects on tumor vascularity and myeloid suppressor cell populations, in uveal melanoma there is evidence that IL-1 can inhibit progression.
The effects of the IL-1 receptor antagonist IL-1ra against the aggressive/invasive MUM2B and the nonaggressive/noninvasive OCM1 uveal melanoma models were examined in vitro and in vivo in mouse xenografts. Vascularity and myeloid suppressor cell populations and their regulators were assessed.
In vitro, IL-1, and IL-1ra did not affect the proliferation of the uveal melanoma cells or their production of IL-1, IL-6, transforming growth factor (TGF) β, or VEGF. In vivo, IL-1ra treatment resulted in substantial growth inhibition of MUM2B tumors; less inhibition was observed against OCM1 tumors. Periodic acid-Schiff loops and CD11b+ macrophages within the tumor stroma decreased in vivo; CD31+ blood vessels were not altered. IL-1ra treatment in vivo did not affect tumor-derived IL-1, IL-6, TGF-β, or VEGF. In contrast, host IL-1β, IL-6, and tumor necrosis factor decreased. Host VEGF was not altered. Intratumoral IL-12(p40) and CXCL10, markers of host M1 polarization, increased, and intratumoral arginase and CD206, markers of myeloid-derived suppressor cells (MDSC) and M2 macrophage polarization, decreased. IL-1ra treatment in vivo also reduced splenic CD11b+Gr1+ MDSC.
IL-1 may play a role in promoting uveal melanoma progression. Inhibiting IL-1 with IL-1ra inhibits tumor growth in vivo but not in vitro. Tumor stroma is modified, myeloid suppressor cells are reduced, and M1 macrophage polarization is increased in vivo.
The recent identification of frequent activating mutations in GNAQ or GNA11 in uveal melanoma provides an opportunity to better understand the pathogenesis of this melanoma subtype, and to develop rational therapeutics to target the cellular effects mediated by these mutations. Cell lines from uveal melanoma tumors are an essential tool for these types of analyses. We report the mutation status of relevant melanoma genes, expression levels of proteins of interest and DNA fingerprinting of a panel of uveal melanoma cell lines used in the research community.
This study represents the most comprehensive molecular analysis of uveal melanoma cell lines performed to date. The data confirms the mutually exclusive nature of GNAQ and GNA11 mutations in vitro. The lack of BRAF, NRAS, KIT, PI3K, and AKT mutations reveal GNAQ and GNA11 uveal melanoma cells to be distinct among melanoma types. The data provided is intended as a reference for investigators to select appropriate model systems and assist with authentication of uveal melanoma cell lines.
uveal melanoma; GNAQ; GNA11
Human tumors are believed to harbor a disabled p53 tumor suppressor pathway, either through direct mutation of the p53 gene or through aberrant expression of proteins acting in the p53 pathway, such as p14ARF or Mdm2. A role for Mdmx (or Mdm4) as a key negative regulator of p53 function in vivo has been established. However, a direct contribution of Mdmx to tumor formation remains to be demonstrated. Here we show that retrovirus-mediated Mdmx overexpression allows primary mouse embryonic fibroblast immortalization and leads to neoplastic transformation in combination with HRasV12. Furthermore, the human Mdmx ortholog, Hdmx, was found to be overexpressed in a significant percentage of various human tumors and amplified in 5% of primary breast tumors, all of which retained wild-type p53. Hdmx was also amplified and highly expressed in MCF-7, a breast cancer cell line harboring wild-type p53, and interfering RNA-mediated reduction of Hdmx markedly inhibited the growth potential of these cells in a p53-dependent manner. Together, these results make Hdmx a new putative drug target for cancer therapy.
Pigment epithelium–derived factor (PEDF) is expressed in human uveal melanoma. In a mouse model with ocular melanoma, PEDF suppresses tumor angiogenesis, tumor size, and number of hepatic micrometastases.
Pigment epithelium–derived factor (PEDF) is known to be an angiogenesis suppressor and to have antitumor effects. This study investigates whether constitutive overexpression of PEDF inhibits the growth and hepatic micrometastasis of ocular melanoma.
Real-time RT-PCR was used to detect endogenous PEDF expression in human uveal melanoma cell lines and mouse melanoma cells. A lentiviral vector containing a mouse PEDF expression sequence was constructed and transduced into mouse melanoma cells in vitro. Transgene expression was assessed by Western blot analysis. Angiogenesis and transendothelial migration assays were performed in constitutively stable PEDF-overexpressing cells and transduced lentiviral vector control cells. The size and microvessel density of the ocular tumor and the number of hepatic micrometastasis were compared between the mice inoculated with PEDF-overexpressing tumor cells and those mice with the control cell line.
Four human uveal melanoma and three mouse melanoma cell lines were found to express PEDF mRNA. Endogenous overexpressing PEDF melanoma cells lost the ability to migrate and form tubes in vitro. In the animal experiment, the size of the ocular melanoma and the number of hepatic micrometastasis were decreased and microvessel density was also reduced in mice inoculated with constitutively overexpressing PEDF melanoma cells.
Lentivirus-mediated gene transfer of PEDF decreased the growth of ocular melanoma and its hepatic micrometastasis in a mouse ocular melanoma model. Dual antitumor/antiangiogenic activities of PEDF suggest that PEDF gene therapy may be considered an approach for the treatment of ocular melanoma.
To test the efficacy of resveratrol, a nontoxic plant product, in the treatment of uveal melanoma.
The effect of oral administration and peritumor injection of resveratrol was tested on tumor growth in two animal models of uveal melanoma. The mechanism of resveratrol action on uveal melanoma cells was studied in vitro in a cell-viability assay: with JC-1 dye, to measure mitochondrial membrane potential; by Western blot analysis, to analyze the cellular redistribution of cytochrome c and Smac/diablo; and in a fluorescence assay with specific substrates, to measure activation of different caspases.
Resveratrol treatment inhibited tumor growth in animal models of uveal melanoma. Since oral administration resulted in relatively low bioavailability of resveratrol, the effect of increased local levels was tested by peritumor injection of the drug. This method resulted in tumor cell death and tumor regression. In vitro experiments with multiple uveal melanoma cell lines demonstrate that resveratrol causes a decrease in cell viability, resulting at least in part from an increase in apoptosis through a mitochondrial pathway. An early event in drug action is the direct targeting of mitochondria by resveratrol, which leads to a decrease in mitochondrial membrane potential and the eventual activation of caspase-3.
These data suggest that resveratrol can inhibit tumor growth and can induce apoptosis via the intrinsic mitochondrial pathway and that by further increasing bioavailability of resveratrol the potency of the drug can be increased, leading to tumor regression. The nontoxic nature of the drug at levels needed for therapy make resveratrol an attractive candidate for the treatment of uveal melanoma.
Uveal melanomas possess activation of the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT/mammalian Target of Rapamycin (mTOR) pathways. MAPK activation occurs via somatic mutations in the heterotrimeric G protein subunits GNAQ and GNA11 for over 70% of tumors and less frequently via V600E BRAF mutations. In this report, we describe the impact of dual pathway inhibition upon uveal melanoma cell lines with the MEK inhibitor selumetinib (AZD6244/ARRY-142886) and the ATP-competitive mTOR kinase inhibitor AZD8055. While synergistic reductions in cell viability were observed with AZD8055/selumetinib in both BRAF and GNAQ mutant cell lines, apoptosis was preferentially induced in BRAF mutant cells only. In vitro apoptosis assay results were predictive of in vivo drug efficacy as tumor regressions were observed only in a BRAF mutant xenograft model, but not GNAQ mutant model. We went on to discover that GNAQ promotes relative resistance to AZD8055/selumetinib-induced apoptosis in GNAQ mutant cells. For BRAF mutant cells, both AKT and 4E-BP1 phosphorylation were modulated by the combination; however, decreasing AKT phosphorylation alone was not sufficient and decreasing 4E-BP1 phosphorylation was not required for apoptosis. Instead, cooperative mTOR complex 2 (mTORC2) and MEK inhibition resulting in downregulation of the pro-survival protein MCL-1 was found to be critical for combination-induced apoptosis. These results suggest that the clinical efficacy of combined MEK and mTOR kinase inhibition will be determined by tumor genotype, and that BRAF mutant malignancies will be particularly susceptible to this strategy.
The enzyme indoleamine 2, 3-dioxygenase (IDO) catalyzes degradation of tryptophan, an essential amino acid required for lymphocyte activation and proliferation. Many tumors express IDO which implied that it acts as a mechanism to evade T cell-mediated immune attack, and also to establish an immunosuppressive tumor microenvironment. The purpose of this study was to determine whether primary and metastatic uveal melanoma expressed the IDO gene and whether uveal melanoma cells could deplete tryptophan. In situ expression of IDO in primary uveal melanoma from tumor bearing eyes and metastatic uveal melanoma liver tissues was determined by immunohistostaining with IDO-specific antibody. Reverse transcription PCR was used to assess IDO gene transcription by primary and metastatic uveal melanoma cell lines. IDO protein expression was determined by Western blot of uveal melanoma cell protein lysate. IDO catalytic activity was assessed by measuring the presence of kynurenine, a product generated by tryptophan degradation, in uveal melanoma culture supernatants.
Primary uveal melanoma from tumor-bearing eyes and metastatic uveal melanoma from the liver did not express IDO in situ. IDO was not constitutively expressed in either primary or metastatic uveal melanoma cell lines. However, stimulation of primary and metastatic uveal melanoma cell cultures with interferon-gamma (IFN-γ) universally upregulated both IDO gene and protein expression. Culture supernatants from IFN-γ treated primary and metastatic uveal melanoma cell cultures contained elevated levels of kynurenine. Addition of the IDO inhibitor 1-methyl DL-tryptophan significantly diminished kynurenine levels in IFN-γ treated uveal melanoma cell cultures. The results from this study suggest that IFN-γ inducible IDO upregulation by primary and metastatic uveal melanoma may generate a local immune privileged microenvironment to promote escape from T cell-mediated immune surveillance.
PURPOSE: This pilot study examined osteopontin expression in uveal melanoma and focused on the role of measuring serum osteopontin to detect metastatic uveal melanoma.
METHODS: Osteopontin mRNA was measured in 3 uveal melanoma cell lines of varying invasive potential by real time PCR.Tissue sections of primary and metastatic uveal melanomas were stained for osteopontin. Serum osteopontin levels were measured by ELISA assays in 15 patients with metastatic uveal melanoma and in 37 patients who were disease-free for at least 10 years after treatment of the primary tumor. Paired serum samples drawn from 8 patients before and after development of metastasis were analyzed.
RESULTS: By real time PCR, highly invasive primary and metastatic uveal melanoma cells expressed 6 and 250 fold excess osteopontin mRNA respectively compared with poorly invasive primary uveal melanoma cells. Tissue sections of primary uveal melanomas lacking looping vasculogenic mimicry patterns either did not stain for osteopontin or exhibited weak diffuse staining. In primary melanomas containing looping vasculogenic mimicry patterns, strong osteopontin staining was detected in the tumor periphery where patterns were located. Diffuse strong expression of osteopontin was detected in 8 samples of metastatic uveal melanomas to the liver. Serum osteopontin levels were significantly higher in patients with metastatic uveal melanoma compared with patients who were disease-free for at least 10 years after treatment (p=0.0001) or with age matched controls. Serum osteopontin levels were significantly higher (p=0.008) after metastasis than before the detection of metastasis in 8 patients. When a cut-off value of 10ng/ml was used, the sensitivity and specificity of serum osteopontin in detecting metastatic melanoma was 87.5% and the area under the receiver operator characteristic curve was 96%.
CONCLUSIONS: Osteopontin is expressed diffusely in tissue sections of hepatic metastases from uveal melanoma and increased serum osteopontin levels correlate with metastatic melanoma to the liver with high specificity and sensitivity.
We previously described a series of 314-helical β-peptides that bind the hDM2 protein and inhibit its interaction with a p53-derived peptide in vitro. Here we present a detailed characterization of the interaction of these peptides with hDM2 and report two new β-peptides in which non-natural side chains have been substituted into the hDM2-recognition epitope. These peptides feature both improved affinity and inhibitory potency in fluorescence polarization and ELISA assays. Additionally, one of the new β-peptides also binds the hDM2-related protein, hDMX, which has been identified as another key therapeutic target for activation of the p53 pathway in tumors.
Protein–protein interactions; β-Peptides; Peptidomimetics; Inhibitors; Foldamer; p53; hDM2; Cancer
We assessed for mutations in a large number of oncogenes and tumor suppressor genes in primary uveal melanomas using a high-throughput profiling system.
DNA was extracted and purified from 134 tissue samples from fresh-frozen tissues (n = 87) or formalin-fixed, paraffin-embedded tissues (n = 47) from 124 large uveal melanomas that underwent primary treatment by enucleation. DNA was subjected to whole genome amplification and MALDI-TOF mass spectrometry-based mutation profiling (>1000 mutations tested across 120 oncogenes and tumor suppressor genes) using the OncoMap3 platform. All candidate mutations, as well as commonly occurring mutations in GNAQ and GNA11, were validated using homogeneous mass extension (hME) technology.
Of 123 samples, 97 (79%, representing 89 unique tumors) were amplified successfully, passed all quality control steps, and were assayed with the OncoMap platform. A total of 58 mutation calls was made for 49 different mutations across 26 different genes in 34/98 (35%) samples. Of 91 tumors that underwent hME validation, 83 (91%) harbored mutations in the GNAQ (47%) or GNA11 (44%) genes, while hME validation revealed two tumors with mutations in EGFR. These additional mutations occurred in tumors that also had mutations in GNAQ or GNA11.
The vast majority of primary large uveal melanomas harbor mutually-exclusive mutations in GNAQ or GNA11, but very rarely have the oncogenic mutations that are reported commonly in other cancers. When present, these other mutations were found in conjunction with GNAQ/GNA11 mutations, suggesting that these other mutations likely are not the primary drivers of oncogenesis in uveal melanoma.
A high-throughput platform was used to screen 124 uveal melanomas for mutations in 120 oncogenes and tumor suppressor genes. While mutations in GNAQ or GNA11 were found in 91% of tumors, mutations in oncogenes frequently mutated in other more common cancers were found only rarely.
Pigment epithelium-derived factor (PEDF) is known to be an angiogensis suppressor and have anti-tumor effects. This study investigates whether constitutive overexpression of PEDF inhibits the growth and hepatic micrometastasis of ocular melanoma.
Real time RT-PCR was used to detect endogenous PEDF expression in human uveal melanoma cell lines and mouse melanoma cells. A lentiviral vector containing a mouse PEDF expression sequence was constructed and transduced into mouse melanoma cells in vitro. Transgene expression was assessed by Western blot analysis. Angiogenesis and transendothelial migration assays were performed in constitutively stable PEDF overexpressing cells and transduced lentiviral vector control cells. The size and microvessel density of the ocular tumor, and the number of hepatic micrometastasis were compared between the mice inoculated with PEDF overexpressing tumor cells and those mice with the control cell line.
Four human uveal melanoma and three mouse melanoma cell lines were found to express PEDF mRNA. Endogenous overexpressing PEDF melanoma cells lost the ability to migrate and form tubes in vitro. In the animal experiment, the size of the ocular melanoma and the number of hepatic micrometastasis were decreased, and microvessel density was also reduced in mice inoculated with constitutively overexpressing PEDF melanoma cells.
Lentivirus-mediated gene transfer of PEDF decreased the growth of ocular melanoma and its hepatic micrometastasis in a mouse ocular melanoma model. Dual antitumor/anti-angiogenic activities of PEDF suggest that PEDF gene therapy may be considered as an approach for treatment of ocular melanoma.
Radiotherapy-induced radiation retinopathy can develop in over 40% of eyes treated for uveal melanoma. Triamcinolone acetonide (TA) and anecortave acetate (AA) can be used to treat radiation retinopathy. It is not known whether TA or AA has any effect on potentially still viable uveal melanoma cells in the choroid after radiotherapy. We therefore studied the effect of these drugs on the proliferation of uveal melanoma cell lines in vitro. Furthermore, as these drugs are supposed to counteract vascular leakage, we determined their effect on the expression and production of the proangiogenic vascular endothelial growth factor-A (VEGF-A), the antiangiogenic pigment epithelium-derived factor (PEDF), and thrombospondin-1 (TSP-1) in uveal melanoma cells.
Three uveal melanoma cell lines were treated in vitro with TA or AA. Cell proliferation was measured by counting cells and using the Water-Soluble Tetrazolium Salt-1 (WST-1) assay. VEGF-A and PEDF production was measured by ELISA, and intracellular expression of angiogenic-associated genes including VEGF-A, PEDF, and TSP-1 was determined by real-time quantitative RT–PCR.
We found no effect of TA or AA on tumor cell growth or production of VEGF-A and PEDF in any of the three uveal melanoma cell lines tested. Regarding expression as measured by RT–PCR, TA had an inhibiting effect on TSP-1 in only one cell line, and no effect on VEGF-A or PEDF. AA showed a similar lack of effect.
Since TA and AA do not stimulate uveal melanoma cell growth, it seems to be safe to use these drugs to treat radiation retinopathy after irradiation for uveal melanoma. Additional experiments using more cell lines or primary tumor cell cultures are needed to validate this conclusion. Furthermore, the results of our study suggest that TA does not exert its antileakage effect through downregulation of VEGF-A or upregulation of TSP-1 or PEDF in uveal melanoma cell lines. It is possible that TA and AA influence these pro- and antiangiogenic factors only under hypoxic circumstances. Further investigation is needed.
This study was undertaken to determine whether anti-vascular endothelial growth factor (VEGF) therapy inhibits growth of primary uveal melanoma and spread of its hepatic micrometastasis.
Human uveal melanoma cell lines Mel290, Mel 270, and HUVECs, mouse B16LS9 melanoma cells and mouse vascular endothelial cells were separately cultured or co-cultured and incubated with bevacizumab or IgG1. The level of VEGF protein in the culture media was measured by ELISA. In vitro angiognesis and invasion assays were performed under bevacizumab or IgG1 treatment. Mel290 or B16LS9 cells were inoculated into NU/NU or C57Bl/6 mice eyes which were enucleated after 7 days. The sizes of the intraocular tumors were determined. Time and dosing experiments were performed using 50μg or 250μg of bevacizumab starting at day 1 or 4 after inoculation. Hepatic micrometastases were enumerated. Proliferation, apoptosis and angiogenesis markers were detected in the ocular tumor by immunofluorescence staining.
Bevacizumab significantly reduced the level of VEGF in the culture media from human uveal melanoma cells, mouse melanoma cells, and co-cultured cells. Bevacizumab also inhibited cell tube formation and decreased in vitro invasion of tumor cells. We found in a mouse model that bevacizumab suppressed primary ocular melanoma growth and the formation of hepatic micrometastases in a dose-dependent manner. Furthermore, immunohistochemical staining showed decreased Ki67 and unchanged caspase 3 expression after treatment with bevacizumab.
Treatment with bevacizumab suppressed in vitro growth and in vivo hepatic micrometastasis of ocular melanoma cells. Bevacizumab is a potential therapeutic agent for the treatment of uveal melanoma micrometastases.