SUMMARY : This review summarizes current knowledge of
oncogenes and tumour-suppressor genes in development and progression
of malignant melanoma.
Molekularni aspekty maligniho melanomu. Uloha protoonkogenu a onkosupresorovych genu. Kremen J., Stribrna J., Pohlreich P., Hradecky P.
Sborn. lék., Vol. 95 (1994) N° 4, p. 321-331.
SOUHRN : Nase referatové sdeleni shrnuje soucasné
poznatky o mozné roli onkogenu a onkosupresorovych genu
pri vzniku a rozvoji maligniho melanomu.
Mailing address : Doc. MUDr. J. Kremen, Csc., 2nd Dept.
of Medical Chemistry and Biochemistry, 1st Medical Faculty, Charles
Univeristy, U nemocnice 5, 128 53 Prague 2 (Czech Republic)
Universita Karlova, vydavatelstvi Karolinum, Praha 1994
In human pathology, malignant melanoma has been traditionally
regarded as one of the most malignant neoplasms. It has recently
attracted a considerable degree of interest for several reasons
1. The incidence of melanoma and mortality from this disease has been rising. Younger age-groups of population - those in reproductive and the professionally most active period of their lives - are particularly affected. It is estimated that in the year 2,000 approximately 1 of 90 Caucasians will be affected. These facts are most probably related to whole body suntanning, particularly during childhood and adolescence, and to the destruction of a protecting ozone layer in the Earth's atmosphere.
2. Although changes in cutaneous pigmentation are easily accessible, the diagnosis is often established at later stages of the disease. Because of relatively high resistance of melanoma to current means of oncological therapy, the mortality from melanoma is high.
3. Appropriate experimental models are available - in particular
due to advances in techniques of culturing normal melanocytes
and melanoblastoma cells.
Oncogenesis is a multi-step process in which a wide array of factors are involved. Viewed from the perspective of molecular biology, malignant transformation is a result of accumulation of several primary and secondary changes in the genome. The primary change is a defect in the genome that was inherited from one or both parents. A defect that develops during the prenatal or postnatal period of life as a result of spontaneous or induced mutation, is termed a secondary change. Such a defect can be induced by the effect of environment or by infection with oncogenic DNA viruses or retroviruses. Genetic alterations can lead to significant changes in the regulation of cell growth. These changes can give rise to a clone of transformed cells which acts as a primary lesion in melanoma development. In transformed cells, the genetic alterations are found in genes that participate on the control of the cell growth and differentiation - i.e. in protooncogenes and tumour suppressor genes (oncosuppressors).
As in other types of neoplasm, the etiologic factors leading
to the development of malignant melanoma have not been fully
determined yet. The degree of skip pigmentation and concentration
of melanocytes in pigment nevi play a significant role. Statistics
shows that in whites the incidence of melanoma is ten times higher
than in blacks>. Mutagenic effect of ultraviolet (UV) light
present in sun radiation is another etiologic factor in melanoma
development. This factor can contribute to the genesis of melanoma
in association with a hereditary defect of DNA repair mechanism
(Xeroderma pigmentosum) or with a defect of tumour suppressor
gene (familial melanoma).
The UV-B part (290-320 nm) of sun radiation participates on the induction of skin malignancies by several mechanisms. These include inhibition of endogenous anti-oxydant system and suppression of immune processes. They can also result in the induction of mutations that lead to activation of some growth factors and protooncogenes or to inactivation of oncosuppressor genes. Activation of the N-ras protooncogene by replacement of AT with TA or CG at the third position of codone 61 was detected in a melanoma cell line from a patient with Xeroderma pigmentosum.
This mutation occurred at a dipyrimidine site and was probably a result of pyrimidine dimers formation induced by UV radiation (24). Mediators of subsequent inflammatory reaction (especially leucotriene C4) are likely to contribute to stimulation of melanocyte proliferation. The effect of chronic inflammation on melanocytes in vitro leads to the loss of the contact inhibition of cell growth, changes in the cell shape (to spheroidal cells) and to constitutive expression of fos protooncogene (30). These factors can be involved especially in the early phase of melanoma genesis.
Among other physical factors in melanoma development, traumatization of pigment nevi should be mentioned. It leads to repeated proliferation of cells with high frequency of mutations.
Chemical factors of the environment have not been proved to be involved in melanoma development.
In general, effect of physical and chemical cancerogenes is potentiated
by the dose and exposure time.
Familial melanoma accounts for 5 to 10% of all melanoma cases. In recent years attention has been concentrated on the localization of a genetic locus associated with familial melanoma. Linkage analyses and cytogenetic studies have been used to find the regions in the genome that are associated with melanoma development. These regions are located on chromosomes 1, 6, 9 and 10. A region associated with familial melanoma has been localized to chromosome 1 (17,50). Another group has found deletions in the long arm of chromosome 6 in melanoma cell lines (51). This locus is probably implicated in later stages of tumour progression as deletions have been found in melanoma metastases. Translocations between chromosome 1 (p22 or q12-21) and chromosome 6 (6q11-13) have been found (52, 4). The region 6q11-13 probably contains a tumour suppressor gene. The inactivation of this gene can be associated with competence of the tumour to metastasis (58). Moreover, prtooncogenes pim-1, myb, ros and mas-1 are located on chromosome 6. Except for overexpression of myb, activation of these protooncogenes have not been found in melanoma (20).
Recent findings show that a melanoma susceptibility locus is probably located on the short arm of chromosome 9 (9p21-23). In a number of melanoma cell lines, deletions or rearrangements have been found in the region flanked by the sequence of alpha-interferon gene (IFNA locus) and the marker D953 (14). Support for localization of a melanoma predisposition gene to this region has also been brought in a study of Petty et al. (38). In one patient with multiple melanomas and dysplastic nevi, a germline rearrangement of chromosome 9 was detected. Molecular analysis using the same markers (IFNA, D953) and the D9S126 marker lying between the two, confirmed a loss of genetic material in the region 9p21. Using cytogenetic studies several kindreds with familial melanoma were analysed and deletions and rearrangements in the short arm of chromosome 9 were found (9). It is assumed that a melanoma oncosuppressor gene is located in that region. Its position has recently been established using physical mapping of 84 melanoma cell lines. It has been localized to a region less than 40 kb of length proximally from the IFNA gene cluster (56). An oncosuppressor gene has been located to this region and the gene has been designated the multiple tumour suppressor 1 (MTS-1). MTS-1 deletions have been found in melanoma cell lines and in many other cancer cell lines (23). The MTS-1 codes for previously identified protein inhibitor (p16) of the cycline-dependent kinase 4 (xdk-4). The cdk-4 drives the cells into the S phase of cell cycle (43). Parmiter et al. (35) described multiple alterations of the long arm of chromosome 10 (q23 and q25) in early stages of melanoma development. These findings suggest that 10q region contains a gene or a group of genes implicated in early phases of melanoma development.
A Homozygous deletion in neurofibromatosis 1 gene (NF-1 gene)
has recently been reported (49). The gene is located on chromosome
17 (17q11.2) (3) and encodes protein product called neurofibromin
containing a domain homologous to GTPase activating protein (GAP).
GAP is known to down-regulate the function of ras protein
(transduction of signal) by stimulating its intrinsic GTPase activity.
NF-1 gene is a possible candidate for an oncosuppressor gene
as the loss of its function is associated with development of
neurofibromatosis and malignant tumours originating in the cells
of neuroectodermal origin (41).
It is known that abnormalities in the expression of some protooncogenes
and oncosuppressor genes participate in the genesis and progression
of human tumours. About one hundred protooncogenes are known
at the moment (25). They can be divided into 5 or 6 classes according
to the function of their products :
1. Growth factors
2. Growth factor receptors with protein tyrosine kinase activity
3. G-proteins (GDP/GDP binding proteins) functioning as plasma membrane signal
4. Intracellular transducers of growth signals :
a - Protein tyrosine kinases
b- Protein Ser/Thr kinases, some of them are likely to be involved in the regulation
of cell cycle.
5. Nuclear proteins - involved in activation of DNA replication and transcription.
Products of oncosuppressor genes (such as RB-1 and p53) can be classified as
6. Others protooncogenes - genes involved in the control of entering into apoptosis
(e.g. bcl-2) belong to this group.
Some groups of the protooncogenes form so called gene families on the basis of their structural similarities.
Expression of protooncogenes can be induced during physiological processes, such as tissue regeneration. Activation of protooncogenes can be a result of point mutations, chromosome rearrangements, gene amplification and insertion of viral oncogenes or their regulatory sequences (e. g. LTR and trans-regulating sequences of retroviruses).
Expression of protooncogenes in normal cells is a highly regulated process. On the other hand, their expression in transformed cells is usually constitutive and of a high degree.
Oncosuppressor genes (antioncogenes) belong to a group of so called recessive genes. They act as negative regulators of cell growth. Loss of their regulatory function requires defects in both copies of the gene present in a cell.
Alterations of various oncogenes and tumour suppressor genes
have been found in human malignant melanoma. They are implicated
in the development and progression of the malignancy. Table 1
showes the chromosomal location of cellular protooncogenes and
tumour suppressor genes and functions of their protein products.
Protooncogenes of the ras family (H-ras, K-ras and N-ras) code for proteins of molecular weight of 21,000 (p21). These protein product are related to membrane-bound G-proteins that are activated by extracellular signals and that play an important role in regulation of second messenger activity, i.e. they function as signal transducers. In many mammalian and human neoplasms mutations in codones 12, 13 and 61 have been found. These mutations result in constitutive activation of the ras-proteins (7).
Biological function of the products of ras genes has not been fully determined. It is assumed that they may play a specific role in regulation of cytokine expression in human malignancies (H-ras-cytokines of fibroblasts; N-ras - IL-1, IL-6 and TNF) (12).
In melanoma cells, mutations in all three ras-oncogenes have been detected (1,42,40,33). They were not found in normal or dysplastic nevi (2). Activation of these oncogenes was detected in 10 to 25% of melanoma cell lines (2,32). Albino found mutations in codone 61 of N-ras and H-ras genes in 24% of melanoma cell lines (2). Mutations in K-ras were detected in codone 12 in 20% of melanomas under study (44). Close correlation has been found between mutations in N-ras and exposure to UV radiation (53). Activation of ras genes in primary melanomas gives evidence that they are implicated in the development of this malignancy.
Activation of another transforming oncogen, c-mel, has
been reported in a melanoma cell line. This gene, located on
chromosome 19, is weakly related to ras oncogenes (34,46).
This protoncogene belongs to a group of nuclear protein that are involved in the regulation of cell growth, most probably as transcription factors. They act by binding to specific DNA sequences and by interaction with other nuclear proteins. The C-myb gene is located on chromosome 6 (6q22-23).
Overexpression of the myb gene has been reported in several
melanoma cell lines (20). In one melanoma cell line, rearrangement
of c-myb in connection with alteration in 6q22 region has
been found. More detailed analysis revealed a deletion in the
3' end of c-myb locus with concomitant translocation of
a portion of chromosome 12 to chromosome 6(27).
Like ras protooncogene family, the myc family is
represented by several genes (c-myc, N-myc, L-myc). Their
products are nuclear proteins functioning as transcription modulators.
Table 1 - Oncogenes and suppressor genes involved in the development and progression of melanoma tumors
|Gene symbol||subcellular location of protein||function||chromosomal
|H-Ras||plasma membrane||GTP/GDP binding protein||11pter-p15.5||7,2|
|K-Ras-1||plasma membrane||GTP/GDP binding protein||6p12-p21||7,44|
|N-Ras||plasma membrane||GTP/GDP binding protein||1p13||7,53,2|
|Myb||nuclear protein||transcription factor||6q22-23||20,27|
|c-Myc||nuclear protein||DNA binding protein||8q24-qter||36,37|
|N-Myc||nuclear protein||DNA binding protein||2p23-24||5|
|Fos||nuclear protein||transcription factor (AP-1)||14q21-31||60|
|Jun||nuclear protein||transcription factor (AP-1)||1p32-34||60,62|
|Ski||nuclear protein||DNA binding protein||1q22-24||15|
|Kit||plasma membrane||mast cell growth factor receptor (MGFR)||4q12||26,61|
|Met||plasma membrane||hepatocyte growth factor (HGFR)||7q31||31|
|Ret||plasma membrane||insuline like growth factor receptor (IGFR)||10q11.2||22|
|Src||membrane associated||nonreceptor protein-tyrosine kinase||20q12-13||28|
|Yes||membrane associated or cytosol||nonreceptor protein-tyrosine kinase||18q21.3||28|
|p53||nuclear protein||transcription factor||17p13||21,47,55,57|
|MTS-1||nuclear protein||cell cycle inhibitor||9p21||23,56|
Overexpression of the c-myc protooncogene caused by its amplification is frequently found in melanoma cells. We have found amplification of the N-myc gene in melanomas of two patients. Number of extra copies of the gene correlated with a degree of tumour progression (5).
In melanoma, correlation has recently been found between c-myc
overproduction and down-regulation of expression of HLA-I genes.
Deficiency in HLA-I antigens could lead to decreased competence
of the organism to recognize transformed cells (36,37).
These protooncogenes are classified as nuclear proteins and function as transcription factors. Changes in the expression of these genes can participate in transformation of melanocytes. Expression of fos and jun genes in normal melanocytes cultured in vitro requires the presence of growth factors in the medium, whereas in melanoblastoma cells these genes are expressed independently from those factors (60).
Ski protooncogene is one of the genes coding for nuclear
DNA-binding proteins. The chromosome locus of the gene is found
on 1q22-24. The level of its expression in normal melanocytes
is considerably lower than in melanoma cells. Alteration in the
long arm of chromosome 1, a frequent finding in malignant melanomas,
have not been proved to alter the ski protooncogene expression
This gene belongs to a class of protooncogenes that code for transmembrane receptors with protein tyrosine kinase autophosphorylation activity. It is located on the long arm of chromosome 4 (4q12) and encodes a receptor protein for the mast cell growth factor (MGF), also known as the kit-ligand or "stem cell factor". It has been shown that normal function of kit and its ligand is essential for proliferation and distribution of murine melanocyte precursors during certain stages of embryonic development. Mutations (rearrangements or deletions) in the c-kit locus or in the MGF locus in mice lead to the emergence of "piebalt" phenotype. Normal function of kit-receptor is apparently required for the development of human melanocytes as well, as mutations in kit gene have been found in piebaldism, autosomal disorder of pigment distribution in the skin (16,45).
The role of kit in melanoma has not been precisely
defined yet. Expression of this gene in murine and human melanoma
cells have been shown to be markedly decreased compared to expression
in normal melanocytes (26). This reduction is probably caused
by inhibition of transcription as no point mutations or rearrangements
in the gene have been found. Such suppression could be caused
by alteration in expression of other genes - e.g. those
for IL-3, GM-CSF and erythropoietin (61).
Protooncogene c-met is located on the segment q31 of chromosome
7 and encodes the receptor for hepatocyte growth factor (HGF).
The factor is known to mediate mitogenic and invasive response
of certain cell types.
Comparative analyses of benign and malignant melanocytic lesions
have shown a significant increase of met gene expression
in metastatic lesions. The gene is expressed at a large stage
of melanoma progression and can be essential for tumour invasivity
Ret protooncogene codes for a receptor of protein tyrosine kinase type. It is frequently expressed in neoplasms of neuroectodermal origin such as familial thyroid medullar carcinoma and multiple endocrine neoplasia type 2A and 2B (MEN-2A and MEN-2B). In MEN-2A, the carriers of mutation are predisposed to medullar thyroid carcinoma, pheochromocytoma and benign parathyroid hyperplasia. MEN-2B is characterized by early development of neoplasia. Ganglioneuromas of lips, tongue and colon and skeletal and ocular abnormalities develop in addition to the features of MEN-2A. C-ret gene has been localized to chromosome 10 (10q11.2). Oncogene activation is caused by the gene rearrangement. Deletion in a part of this protooncogene leading to inactivation of its product is associated with Hirschprung disease (54).
In mice transgenic for ret gene, hyperpigmentation caused
by aberrant melanogenesis and development of melanoma have been
described (22). Cells of the lines cultured from these tumours
have been shown to be highly invasive when transplanted to nude
mice, with subsequent metastases formation. Activation of ret
protooncogene in human melanoma has not been described (48).
Src protooncogene is located in the chromosomal bands 20q12-13 and belongs to genes encoding non-receptor protein tyrosine kinases. Its product is predominantly anchored to the inner surface of cell membrane. Activation of this protooncogene has not been described in melanoma.
C-yes, mapped to chromosome 18 (18q21.3), also belongs to src family of non-receptor protein tyrosine kinases. In most melanoma cell lines kinase activity of c-yes product reaches levels 5 to 10 times higher those that in normal melanocytes. In melanoma cells in which an elevated activity of this kinase has been detected, a tyrosine-phosphorylated protein of Mr 39,000 has been found. This protein does not occur in normal melanocytes. Increased expression of c-yes protein kinase may have influence on signal transduction processes in melanocytes and may play a role in their malignant transformation (28).
cDNA sequence of a new oncogene nck has recently been
established. The nck gene is highly related to src.
Its product is phosphorylated on tyrosine, serine and threonine
residues and may function as an adaptor for physical and functional
coordination of signal transduction proteins (10). The role of
this gene in melanoma etiology has not been fully determined yet.
Mutations in the p53 gene (TP53) have been found in a variety of human malignancies. The gene lies on chromosome 17 (17p13) and encodes nuclear phosphoprotein p53. It seems that p53 protein can influence passage through the G1 phase of the cell cycle. This protein apparently suppresses cell division by stimulating the synthesis of a protein inhibitor p21 that blocks the activity of cyclin-dependent kinase 2 (Cdk-2) and other types of cyclin-dependent kinases. Therefore, p53 is classified as an oncosuppressor (29,59). Various mutations of the gene inactivate the p53 tumor suppressor function.
Acquired mutations of p53 gene have been found in epithelial, mesenchymal, hematopoietic and central nervous system neoplasms (21).
Alteration of p53 and overproduction of its abnormal product
have also been found in cutaneous malignant melanoma. Some authors
report high frequency of such mutations (up to 80% in primary
and metastatic melanomas (47). However, in other studies, much
lower frequency of p53 mutations have been reported-ranging
from 1 of 9 to 4 of 13 melanoma cell lines under study (55,57).
In some cases, C to T transition has been detected. Such mutations
occur in other types of skin neoplasms (basal and squamous cell
carcinomas) and are likely to be induced by UV radiation.
Various growth factors and cytokines are involved in interactions between normal melanocytes and surrounding epidermal tissue. An important role in the regulation of melanocyte growth is played by interactions between keratinocytes and melanocytes. Keratinocytes induce the dendritic morphology of melanocytes. They also regulate the proliferation of melanocytes so that a constant ratio of keratinocytes to melanocytes is maintained during the entire exponential phase of growth. Keratinocytes control the expression of adhesion receptors on the surface of melanocytes and nevus cells. The function of these receptors is to attach the cells to the basement membrane of epidermis. It seems that growth of melanoma cells is independent from the control mechanisms exerted by keratinocytes. Constitutive synthesis of growth factors and cytokines lead to autocrine stimulation of their growth.
Comparative studies of normal melanocytes and melanoma cells have shown that the development and progression of melanoma depends on growth stimulation by endogenous basic fibroblast growth factor (bFGF). Expression of bFGF and its receptor (bFGF-R) in melanoma cells generates an autocrine loop that forms a basis for cell proliferation. The growth of melanoma cellos is stimulated by endogenous secretion of bFGF and is not dependent on exogenous bFGF production. bFGF may accomplish a key role in melanoma development as the proliferation of melanoma cells in vitro can be inhibited by antisense oligodeoxynucleotides or by anti-bFGF antibodies introduced into the cytoplasm of melanoma cellos (18).
In nevus cells, the influence of exogenous bFGF on cell proliferation has also been found to be reduced. In nevus cells of the junctional region between the epidermis and the dermis, expression of bFGF mRNA has been detected. This suggests that the expression of this factor can be involved in migration of nevus cells through the basement membrane (19).
Melanoma-derived bFGF can also be essential for tumour proliferation and dissemination. This growth factor can stimulate angiogenesis and stroma formation through its mitogenic effect on endothelial cells, fibroblasts and keratinocytes of the surrounding tissue. These effects are potentiated by its involvement in the activation of proteolytic enzymes (tissue and urokinase type of plasminogene activator and collagenases). Proteolytic activity of these enzymes increases the competence of the tumour cells to invade into the surrounding tissue as well as blood and lymphatic vessels (39, 8). Expression of FGFR-1 (a member of FGF-receptor family) can be detected in normal melanocytes and melanoma cells and may play a role in genesis of melanoma. It has been shown that inhibition of FGFR-1 mRNA function by antisense oligodeoxynucleotides blocks proliferation of normal melanocytes and melanoma cells and leads to dendrites formation. The oligodeoxynucleotides are targeted against the region of the initiation of translation or against the donor-acceptor site of the primary transcript of the FGFR-1 gene (6).
Increased expression of other members of the FGF family (K-fg/hst,
int-2 and KGF) has also been detected in melanomas (13,11).
In recent years, etiology of malignant melanoma has been a subject to intensive research. Molecular, biochemical, biological and immunological aspects of melanoma development have been studied.
To date, over 100 genes have been known to be involved in the
development and progression of malignant melanoma. None of these
genes seems to be responsible for the entire process of melanocytes
transformation. Deletions in tumour suppressor genes located
on chromosomes 1 and 9 are of a particular significance for the
genesis of this malignancy.
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