Ординатура / Офтальмология / Английские материалы / Ocular Oncology_Albert, Polans_2003

independent apoptosis. Bc1-2 is upregulated in more than 70% of uveal melanomas [70,72,93]. Bc1-2 expression does not have an effect on prognosis, although an inverse relation was found between Bc1-2 expression and myc expression in uveal melanomas.

We have recently identified the apoptosis linked protein ALG-2 in uveal melanomas cells. ALG-2 is a calcium-binding protein that is involved in apoptosis in T-cell hybridomas [94]. Its expression is necessary for response to a number of apoptotic signals. Levels of this protein are decreased in certain more malignant uveal melanoma cell lines as compared to normal uveal melanocytes.

3.Angiogenesis

Another important acquired property of tumor cells is their ability to induce angiogenesis when the size of the primary tumor exceeds 1–2 mm. This allows the primary tumor to continue growing while simultaneously allowing tumor cells to intravasate and metastasis to occur. The leakiness of the new vessels is thought to aid in this process. Tumor angiogenesis is especially important for uveal melanomas, since the eye lacks lymphatics and metastasis occurs almost exclusively via the hematogenous route. Vascular endothelial growth factor (VEGF) is a well-described angiogenic factor; it is induced by hypoxia and is upregulated in many tumors. Reports of VEGF expression in uveal melanomas have been inconsistent. While some studies have found no expression [95,96], others have found diffuse or inconsistent expression of VEGF within the tumor [60,97,98]. More recently, VEGF expression was reported in the majority of uveal melanomas analyzed, although levels were variable [99]. VEGF levels correlated with the presence of necrosis but not with angiogenesis or metastasis. In a study of uveal melanoma cell lines, high levels of VEGF expression were found in all lines [100]. In the same study, expression of angiopoietin 2 was found in all cell lines, and of interleukin 8 in some cell lines. These factors may increase the angiogenic properties of these melanomas.

We have recently identified two additional angiogenic factors, tissue factor and Cyr61, which are upregulated in uveal melanomas and certain uveal melanoma cell lines [60]. Tissue factor correlated with blood vessel density in paraffin sections from uveal melanomas.

Recently, a process called vasculogenic mimicry has been described for certain aggressive uveal melanomas in which tumor cells form patterned tubular networks that mimic endothelial-formed vasculogenic networks [101]. It is not clear whether these networks have a role in perfusion of the tumor, though they are associated with aggressive growth. Comparison by chip array analysis of aggressive tubular network–forming uveal melanoma cells to poorly aggressive ones that lack this ability identified various protein kinases such as epithelial cell kinase (Eck/EphA2). This kinase was exclusively expressed and phosphorylated in the aggressive cells [102]. When this protein was transiently knocked out, these tumor cells lost the ability to form tubular networks. Eck/Eph2A has been previously implicated in angiogenesis, growth and proliferation as well as induction of vascularization of melanoma cells [103,104]. Several other genes associated with the endothelial/ vascular phenotype, such TIE-1, an endothelial receptor kinase, urokinase-type plasminogen activator (uPA), and keratin 8 intermediate filament were also identified by this same technique [101]. Other genes that were upregulated and

could be involved in the microvascular channel formation include connective tissue growth factor (CTGF), fibrillin, collagens VI and I, and fibronectin.

4.Invasion and Metastasis

An important property of many tumors, and of uveal melanomas in particular, is the ability to escape the primary tumor mass and then to invade neighboring tissues and metastasize to more distant sites. One of the natural barriers against tumor spread is the extracellular matrix (ECM). Many tumor cells excrete proteolytic enzymes, such as matrix metalloproteinases (MMPs) and plasminogen activators, to degrade basal membranes and ECM components. At least half of the uveal melanomas express MMP2 and or MMP9 [105–108]. In a different study, all epithelioid tumors and only 30% of the spindle cell tumors were found to be positive for MMP2 [107]. MMP9 is mainly expressed in the more malignant epithelioid uveal melanomas [109]. Tumors positive for MMP2 and/or 9 are associated with a significantly higher metastatic incidence and lower survival rate. In addition, 65% of uveal melanomas express MMP3, which can activate pro-MMP9. Microarray gene chip analysis revealed increased expression of MMP1, 2, 9, and 14 in aggressive, compared to poorly aggressive, uveal melanoma cells [110]. It has been suggested that another function of MMPs is in vasculogenic mimicry, a process whereby tumor cells form ECM-rich patterned tubular networks [101]. Both MMPs and laminin 5, which is also overexpressed, may be involved in this remodeling of the ECM [110].

Tissue inhibitors of MMPs (TIMP) are natural inhibitors of MMPs. Levels of TIMP1 and 2 tend to be lower in patients with uveal melanomas that had developed metastatic disease [109]. TIMP1 may also have growth factor-like effects, as has been described in colon cancer cells [111]. This might explain why upregulation of this protein may lead to increased tumorigenicity in some tumors. In addition, TIMP2 also induces activation of proMMP2 [112], thus leading to increased tumorigenicity at intermediate levels. MMPs work synergistically with plasminogen activators. Urokinase-type activator (uPA) and tissue plasminogen activator (tPA) have been implicated in metastasis. The presence of uPA on primary uveal tumors correlates with metastatic disease and poor prognosis [113]. uPA and inhibitors of plasminogen activity (PAI-1, PAI-2) were detected on all of a series of 10 uveal melanomas [106]. tPA activity from primary cultures of uveal melanoma correlates with scleral invasion in the tumor lesion. Furthermore, tPA activity is present in the invasive front of uveal melanoma [113]. The importance of the plasminogen activator system in metastasis was also apparent from gene transfer experiments of PAI-1 into uveal melanoma cells, which inhibits metastasis of uveal melanoma [114].

For tumor cells to colonize new sites, adaptations in their cell surface receptors occur in order to adhere to their new microenvironment. These interactions are mediated by integrins, which consist of an alpha and a beta subunits. Sixteen alpha and eight beta subunits can form 20 different integrins. The combination of these subunits displayed on the cell surface determines the adhesive properties of the cell to the matrix in a certain environment. Some integrins induce expression of MMP’s and are thus involved in the invasive process.

Integrin expression is heterogeneous in both cutaneous and uveal melanoma cell lines [115]. Although no link between invasiveness or cell type and integrin expression has been observed in one study [116]; others have reported a correlation

between spindle cell morphology and the lack of a6b1 integrin expression in a group of 10 uveal melanomas [108]. In addition, all uveal melanomas were positive for a1b1, whereas ocular melanocytes were negative. Both a1 and a6 integrins bind laminin, which is a constituent of basement membranes and may thus be important for invasive potential. avb3, an important integrin for the invasive potential of cutaneous melanoma, does not appear to be important to uveal melanoma: some studies do not find it on these tumors whereas others find it on melanoma cells and melanocytes [108,115,116]. A potential caveat in some of these studies is that integrin expression can be altered as a result of culturing cells [106].

Expression of the a4 subunit gene was found to be downregulated in a uveal melanoma cell line and its metastatic derivatives as compared to uveal melanocytes [117]. Study of the promoter of this gene showed that transcriptional activity and transcription factor binding was inversely correlated with metastatic potential of the lines.

Another class of proteins involved in cell adhesion and implicated in invasion and metastasis are the cell adhesion molecules (CAM), which mediate calciumdependent adhesion. The role of the intercellular adhesion molecule 1 (ICAM-1) in uveal melanoma seems unclear [118–120]. Neural cell adhesion molecule (NCAM) expression was correlated to metastatic potential of uveal melanomas [121]. NCAM isoforms that lack the HNK-1 epitope may play a role in organ specific metastatic behavior of uveal melanomas. This epitope may serve as a ligand for cell adhesion.

Organ-specific metastasis is not only a result of organ-specific homing mediated by cell surface receptors on the tumor cells but also paracrine stimulation of tumor cells by organ-derived growth factors. It has been shown that expression of the epidermal growth factor receptor (EGFR) in uveal melanoma cell lines correlates with the capacity of tumor cells to localize in the liver [122]. These tumor cells may be stimulated to proliferate by transforming growth factor alpha (TGF-a) and hepatocyte growth factor (HGF), similar to colon carcinoma cells, which also preferentially metastasize to the liver [123]. EGFR receptor also protects uveal melanoma cells against lysis mediated by TNF-a. There is some controversy regarding a correlation of EGFR expression and metastatic disease. Hurks et al. [124] have shown that such a correlate exists in patients with uveal melanoma. However, Scholes et al. [125] find no such correlation and, in addition, find EGFR immunoreactivity restricted to macrophages.

Another receptor involved in dissemination of uveal melanoma cells to the liver is the proto-oncogene c-met, the receptor for HGF/scatter factor (SF) [126]. Expression of c-met correlates with the appearance of cells coexpressing vimentin and keratin intermediate filaments (interconverted phenotype) [127] and invasive ability. HGF/SF is expressed both in the primary uveal tumor and in metastatic foci in the liver. An important source of HGF in the primary tumor may be the tumorinfiltrating lymphocytes. Two-thirds of surgically removed uveal melanomas contain moderate to high numbers of these cells [128]. A downstream target of c-met is ezrin, which acts as a linker between the plasma membrane and the actin cytoskeleton [129,130]. Ezrin is involved in cell motility, cell adhesion, and intracellular signaling [131,132]. It is expressed in a number of different tumors and tumor cell lines. More than 60% of uveal melanomas express ezrin [133]. The presence of ezrin in these melanomas is associated with higher mortality, increased microvascular density, and the presence of infiltrating macrophages.

Another growth factor that is mainly produced in the liver is insulin-like growth factor 1 (IGF-1), which binds to the IGF-1 receptor (IGF-1R). Activation of the IGF-1R leads to activation of the mitogenic cascade. IGF-1R is upregulated in many different tumor types and is important in tumorigenesis and cell transformation. It also protects cells from apoptosis [134]. In uveal melanomas, high IGF-1R expression is associated with death due to metastatic disease [135]. In addition, decreasing the IGF-1R levels of uveal melanoma cell lines in vitro decreased cell viability.

We have recently found that the tyrosine kinase receptor Axl is upregulated in uveal melanoma [136]. We show that Axl can mediate mitogenesis and survival of cultured uveal melanoma cells through its ligand Gas6. Therefore this receptor may enable uveal melanoma cells to remain dormant in the liver as micrometastases till outgrowth occurs years later.

The gene nm23 is a metastasis suppressor that is downregulated in many human cancers. The mechanism by which this suppression occurs is unclear. In a mouse model of uveal melanoma, the level of nm23 expression and the development of liver metastases were inversely correlated, demonstrating the importance of nm23 in limiting metastasis in uveal melanoma [137].

Table 1 Suppression Subtractive Hybridization of Mel290 vs. Uveal Melanocytes

technologies to scan for genomewide changes in gene expression will identify many more gene products that are relevant for the growth of these uveal tumors. Many of these genes will be part of different signal transduction pathways. Identification of these key pathways holds prospects for treatment even in the absence of one distinct disease causing gene mutation. We have used SSH [138] to compare the epithelioid uveal melanoma cell line Me1290 to cultured normal uveal melanocytes. This cell line is especially interesting because of the relatively few genomic changes it has undergone and the stability of the changes in tissue culture [60]. We obtained a list of genes that were upregulated in the tumor cell line and may be relevant to the disease (Table 1). Many of these genes play a role in other types of cancers. The described functions of these genes span the different categories of alterations in cell physiology necessary for malignant growth—namely, cell cycle regulation/proliferation, apoptosis, angiogenesis and invasion, and metastasis [139]. We have similarly compared an epithelioid (OCM-3) to a spindle-type (OCM-1) uveal melanoma cell line using SSH to begin to study the molecular basis for the difference between these two types of uveal melanoma cells (Table 2). Although these are one-way comparisons screening only for genes upregulated in Me1290 and OCM-3 cells respectively, it does show the wealth of data that can be obtained by these sorts of studies. The use of increasingly sophisticated gene expression analysis software will allow the further sorting of these data for example by signal transduction pathways

[140].Similarly, chip arrays have been used to do cluster analysis to compare tumor cell lines that differ in their ability to form tubular networks (vasculogenic mimicry)

[141].The growing complement of human expressed sequences represented on the chips and their relative ease of use make them a powerful tool for further identification of genes that are important for the growth and development of uveal melanoma. However, ultimately, these descriptive data will have to lead to biological experiments that identify the roles of these gene products and their pathways in this specific cancer. Development of suitable animal models will then allow for the design of therapeutic interventions.

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