Progression Of Melanoma Cells Research Articles

Control of Melanoma Invasiveness by Anticollagenolytic Agents: A Reappraisal of an Old Concept

Collagen, the major constituent of human dermis, represents the main barrier against progression of melanoma cells. Several matrix metalloproteinases (MMPs), i.e. collagenase-1 (MMP-1), gelatinase A (MMP-2) and membrane-type 1-MMP (MMP-14), favor melanoma cell invasion through their capacity of degrading collagen and thus, are considered as main targets. Potent inhibitors, as hydroxamate-derived pseudopeptides were first proposed as pharmacological agents to control melanoma invasiveness. These molecules have major drawbacks linked to i) toxicity and ii) absence of specificity, in keeping with the high Zn chelating property of hydroxamates, that might hinder the contribution of the occupancy of other subsites in enzyme inhibition. To date, research focuses on the design of compounds which display a lower affinity for Zn in enzyme active site. For instance, hydroxamate can be replaced by phosphinic acid or hydrazide which further allows synthesis of both right- and left- hand side inhibitors and therefore occupancy of non-primed enzyme subsites. Novel types of selective MMP inhibitors also include non-competitive and mechanism-based inhibitors. Finally, collagenolysis may be controlled by modulating enzyme-substrate interaction through the identification of substances that bind to MMP exosites. Such compounds could be of value by impeding collagenases to associate to plasma-membrane of invading cancer cells.

Direct role of nucleotide metabolism in C-MYC-dependent proliferation of melanoma cells

To identify C-MYC targets rate-limiting for proliferation of malignant melanoma, we stably inhibited C-MYC in several human metastatic melanoma lines via lentivirus-based shRNAs approximately to the levels detected in normal melanocytes. C-MYC depletion did not significantly affect levels of E2F1 protein reported to regulate expression of many S-phase specific genes, but resulted in the repression of several genes encoding enzymes rate-limiting for dNTP metabolism. These included thymidylate synthase (TS), inosine monophosphate dehydrogenase 2 (IMPDH2) and phosphoribosyl pyrophosphate synthetase 2 (PRPS2). C-MYC depletion also resulted in reduction in the amounts of deoxyribonucleoside triphosphates (dNTPs) and inhibition of proliferation. shRNA-mediated suppression of TS, IMPDH2 or PRPS2 resulted in the decrease of dNTP pools and retardation of the cell cycle progression of melanoma cells in a manner similar to that of C-MYC-depletion in those cells. Reciprocally, concurrent overexpression of cDNAs for TS, IMPDH2 and PRPS2 delayed proliferative arrest caused by inhibition of C-MYC in melanoma cells. Overexpression of C-MYC in normal melanocytes enhanced expression of the above enzymes and increased individual dNTP pools. Analysis of in vivo C-MYC interactions with TS, IMPDH2 and PRPS2 genes confirmed that they are direct C-MYC targets. Moreover, all three proteins express at higher levels in cells from several metastatic melanoma lines compared to normal melanocytes. Our data establish a novel functional link between C-MYC and dNTP metabolism and identify its role in proliferation of tumor cells.

Development of IFN-γ resistance is associated with attenuation of SOCS genes induction and constitutive expression of SOCS 3 in melanoma cells

The resistance to interferons (IFNs) limits their anticancer therapeutic efficacy. Here we studied the evolution of an IFN-resistant state in vitro using melanoma cell lines. We found that the cells became less sensitive to antiproliferative effect of IFN-γ after prolonged cultivation enabling us to isolate sensitive and resistant subclones of the parental line. We investigated transcription of signal transducer and activator of transcription (STAT) 1–6 and suppressor of cytokine signalling (SOCS) 1–3 genes, and phosphorylation of STAT 1 protein. The resistant subline (termed WM 1158R) differed from the sensitive subline (WM 1158S) by a constitutive expression of SOCS 3, lack or weak SOCS 1–3 activation following IFN-γ, and short duration of cytokine activatory signal. Similar correlations were observed in additional melanoma lines differing in IFN sensitivities. At the protein level, IFN-γ induced strong and prolonged STAT 1 activation at serine 727 (S727) in WM 1158R while in WM 1158S cells phosphorylation of this amino acid was much less pronounced. On the other hand, phosphorylation of tyrosine 701 (Y701) was stimulated regardless of the sensitivity phenotype. In conclusion, constitutive expression of SOCS 3 is correlated with attenuation of its induction following IFN treatment. These results suggest that progression of melanoma cells from IFN sensitivity to IFN insensitivity associates with changes in SOCS expression.

Angiotropism of Human Melanoma: Studies Involving In Transit and Other Cutaneous Metastases and the Chicken Chorioallantoic Membrane

Melanoma cell migration along the outside of vessels has been termed "extravascular migratory metastasis" (EVMM), as distinct from intravascular dissemination. Previous studies in both human and experimental melanoma models have shown angiotropism of melanoma cells, suggesting EVMM. Our objectives are to study the mechanism of dissemination of human melanoma cells in the chick chorioallantoic membrane (CAM) and to compare the histopathology in the CAM with that of patients with in transit and other cutaneous melanoma metastases. Human and murine melanoma cells were inoculated onto the CAM and observed over a 10-day period for tumor dissemination. Both human melanoma specimens from 26 patients and melanoma cells growing on the CAM showed the presence of tumor cell angiotropism at the invasive front of the tumor and at some distance from the tumor mass. In addition, a clear progression of melanoma cells spreading on the CAM was observed along the abluminal surface of vessels, where they occupied a perivascular location. By day 10 after injection, small micrometastases had developed along vessels, in a pattern similar to that in transit and other cutaneous melanoma metastases. In addition, the results suggested that the number of micrometastases directly correlated with increasing tumor volume. Taken together, these data suggest that the CAM is a relevant model for studying tumor cell dissemination, and that EVMM may be a mechanism by which some melanoma cells spread to nearby and even distant sites.

Expression of SOCS-1, Suppressor of Cytokine Signalling-1, in Human Melanoma

Cytokine resistance is a well-established feature of melanoma cell progression and represents also a major obstacle in immunotherapy of patients with metastatic melanoma. To check whether suppressors of cytokine signalling (SOCS) play a role in cytokine resistance and tumor progression of melanoma, we investigated the expression and regulation of SOCS-1, an established negative regulator of interleukin-6 (IL-6) and interferon (IFN) signalling. In vitro SOCS-1 transcripts were detectable by RT-PCR in 8 out of 8 human melanoma cell lines derived from different tumor stages. Normal human melanocytes also expressed SOCS-1 mRNA in the presence or absence of artificial growth factors. Both IL-6 and alpha-IFN induced rapid and transient SOCS-1 mRNA expression in WM35 and WM9 melanoma cells. At the protein level, SOCS-1 was undetectable in normal human melanocytes whereas uniformly expressed in all tested melanoma cell lines. The aberrant SOCS-1 protein expression in melanoma cells was recapitalized in situ as shown by immunohistochemical analysis. SOCS-1 immunoreactivity was closely related to tumor invasion (Clark level), tumor thickness according to Breslow, and stage of the disease. In contrast, melanocytes in normal skin or melanocytic nevi lacked SOCS-1 protein expression. Our findings show that melanoma cells express a member of the SOCS family, SOCS-1, in vitro and in situ. SOCS-1 is a progression marker of human melanoma and may downregulate biological responses by endogenous and/or therapeutically administered cytokines.

Identification of AP-2-regulated genes by macroarray profiling of gene expression in human A375P melanoma.

Transcription factor AP-2 is a negative regulator of metastasis. Its expression is down regulated with progression of melanoma cells to metastasis. In this study, we performed macroarray profiling of gene expression of human A375P melanoma cells and their derivatives with overexpression of AP-2 and dominant-negative AP-2. Such comprehensive analysis lead to an identification of genes such as MMP-2, E-cadherin, melanoma adhesion molecule, early growth response 1, fibroblast growth factor 3, ubiquitin C, histone deacetylase 3 and integrin alpha 5,7, beta 3,5 as regulated by AP-2. Whereas some of these are known as AP-2-regulated genes, the others are not so far. Thus the study reports for the first time identification of new genes regulated by AP-2 that may be involved in metastasis of melanoma.

P53 alone or in combination with antisense cyclin D1 induces apoptosis and reduces tumor size in human melanoma.

Melanoma incidence is growing at a faster rate than any other human malignancy. Wild-type (wt) p53 is important in both G(1) and G(2) cell cycle arrest, and cyclin D1 (CD1) is necessary for G(1)-->S progression in melanoma cells. We reported that an adenoviral vector containing wt p53 significantly reduced [(3)H]thymidine uptake in melanoma cells containing mutant but not wt p53. Subsequently we showed that CD1 decreased melanoma proliferation and increased apoptosis. We now extend these findings by evaluating the effect on preformed melanomas of (1) intratumoral therapy with wt p53 alone, (2) wt p53 in combination with antisense (AS) CD1, both short (< or =14 days) and longer term, and (3) doubling the dose or repeat doses of wt p53 or AS CD1. Two melanoma cells lines that metastasize in SCID mice (451 and 1205) were used, one containing a p53 mutation (451) and the other a normal p53 gene sequence (1205). Compared to injection with a control adenoviral vector containing beta-galactosidase (LacZ), intratumoral injection of wt p53 slowed the growth of tumors formed from 451 cells. Using 5 x 10(8) plaque forming units as our standard intratumoral dose, neither doubling the dose of LacZ, p53 or AS CD1, nor repeat doses of the vectors, was as effective as combined therapy with wt p53+AS CD1, which resulted in the shrinkage of all tumors treated and 4/7 (57%) tumors vanished. No tumors treated with wt p53 or AS CD1 alone vanished. Wt p53+AS CD1 treatment resulted in significantly more cells undergoing apoptosis compared to either therapy alone. In summary, combining the separately effective treatment vectors p53 and AS CD1 led to an enhanced growth-suppressive and apoptotic effect, supporting a role for combination gene therapy to treat human malignant melanoma.

Gene regulation in melanoma progression by the AP-2 transcription factor.

The molecular changes associated with the transition of melanoma cells from radial growth phase (RGP) to vertical growth phase [(VGP), metastatic phenotype] are not very well defined. We previously demonstrated that expression of the cell-surface adhesion molecule MCAM/MUC18 correlates directly with the metastatic potential of human melanoma cells. In addition, the progression of human melanoma towards the metastatic phenotype is associated with loss of expression of the tyrosine-kinase receptor c-KIT. In this review, I will summarize our recent studies demonstrating that the expression of both genes is regulated by the AP-2 transcription factor. Moreover, we have observed a loss of AP-2 expression in metastatic melanoma cells. Re-expression of AP-2 in the highly metastatic A375SM cells decreased their tumorigenicity and inhibited their metastatic potential in nude mice. MCAM/MUC18 mRNA and protein expression was significantly down-regulated while c-KIT expression was up-regulated in the AP-2-transfected cells. To further investigate the role of AP-2 in the progression of human melanoma, we attempted to inactivate AP-2 in primary cutaneous melanoma by using a dominant-negative AP-2, or the AP-2B gene. Expression of AP-2B in SB-2 cells augmented their tumorigenicity in nude mice, and upregulated MMP-2 expression and activity. As AP-2 also regulates other genes that are involved in the progression of human melanoma such as E-cadherin, p21/WAF-1, HER2/neu, Bcl-2, FAS/APO-1, IGF-R-1, VEGF and the thrombin receptor (PAR-1), we therefore propose that loss of AP-2 is a crucial event in the development of malignant melanoma. In addition, the transition of melanoma cells from RGP to VGP is also associated with over-expression of the transcription factors CREB and ATF-1. The notion that the balance between AP-2 and CREB/ATF-1 expression determines the progression of melanoma cells towards the metastatic phenotype will be discussed.