Growth factor independence and growth regulatory pathways in human melanoma development

Summary This review concentrates on growth autonomy of tumor cells in relation to tumor progression. Human malignant melanoma serves as an example for progressive growth factor independence at subsequent stages of tumor progression. Mechanisms by which malignant cells acquire growth factor independence are discussed. In melanoma, deregulation of growth regulatory pathways has been described on four levels: 1) aberrant production of autocrine growth factors that substitute for exogenous growth factors (basic fibroblast growth factor [bFGF]); 2) alterations in the response to negative autocrine growth factors (interleukin [IL]-6 and transforming growth factor [TGF]-); 3) overexpression of epidermal growth factor receptors (EGF-R); and 4) alterations of cellular protooncogenes involved in signal transduction (RAS, MYB) and growth suppression (p53). In addition to bFGF and IL-6, multiple other growth factor genes are activated in malignant melanoma cells but not normal melanocytes. These include both chains of platelet-derived growth factor (PDGF), TGF-, IL-1, IL-8, and tumor necrosis factor (TNF)-. Of these, PDGF-B has been investigated in more detail. Melanoma-derived PDGF clearly does not act in a direct autocrine mode, but has important paracrine effects on normal tissue constituents, notably fibroblasts and endothelial cells, that are essential for tumor developmentin vivo. It is speculated that other melanoma-derived growth factors with as yet undefined functions similarly exert such paracrine or indirect autocrine effects that cannot be sufficiently addressed in studies on cultured cells.     

Insulin-like growth factor expression in breast cancer epithelium and stroma

Summary The insulin-like growth factors (IGFs) are mitogens for many cancer cell types. In breast cancer cells, IGF-I and IGF-II have both been shown to stimulate cell proliferation. However, IGF-I mRNA has not been found in human breast cancer cell lines, making it unlikely that IGF-I is commonly expressed as an autocrine growth factor for breast cancer cells. Nevertheless, IGF-I mRNA can be detected in breast cancer tissue samples, and in situ hybridization studies have shown that the message originates from the stromal cells adjacent to normal lobules. IGF-II, on the other hand, has been detected in some breast cancer cell lines. In the estrogen receptor positive cell line T47-D, IGF-II mRNA was induced by estradiol. Furthermore, transfection of an IGF-II expression vector into a previously estrogen-dependent cell line resulted in hormone independent growth. Thus, IGF-II can be expressed as an autocrine growth factor in some breast cancers and its expression may, in part, result in hormone independence. Finally, stromal cells obtained from breast tissues showed that IGF-I was commonly expressed in fibroblasts derived from non-malignant biopsy specimens, while IGF-II mRNA was detected in fibroblasts adjacent to malignant tissue. These studies suggest that IGF-II expression may be important in both autocrine and paracrine regulation of breast cancer cell growth.     

The role of growth factors in breast cancer

A series of teleconferences has been organized under the auspices of Bristol-Myers to address several major current questions in oncology. A panel of recognized experts with a moderator has been assembled to discuss each question, and we are reporting a number of these discussions in Breast Cancer Research and Treatment. This is reprinted from Oncology Viewpoints, courtesy of Bristol-Myers Oncology Division, Evansville IN 47721, USA.     

Human melanoma: Development and progression

Clinical and histopathological evidence suggests that melanoma develops in a sequence of steps, progressing from benign proliferative lesions, to primary melanomas that do not show evidence for metastasis, to invasive primary lesions, and to metastases. This review focuses on the experimental studies examining the phenotypic characteristics of cultured primary melanoma cells as they relate to cells from non-malignant nevi and metastases. Genetic, biologic, and immunologic criteria have been established to distinguish melanocytes from different steps of tumor development. These include non-random chromosomal abnormalities, expression of melanocyte-and melanoma-specific antigens, requirements for exogenous growth factors, production of endogenous growth factors, and expression of receptors for growth factors. The transformation of melanocytes and nevus cells with viral oncogenes has facilitated studies on the malignant phenotype. Variants have been developed through successive selections from primary melanoma cell populations that have one or several characteristics of metastatic cells. The study of melanocytes isolated from various stages of tumor development and the generation of cell variants with specific properties should enable a long-term search for the molecular mechanisms of melanoma development and progression.     

Combined risk factors for melanoma in a Mediterranean population.

A case-control study of non-familial melanoma including 183 incident cases and 179 controls was conducted in North-Eastern Italy to identify important risk factors and determine how combination of these affects risk in a Mediterranean population. Presence of dysplastic nevi (OR = 4.2, 95% CI = 2.4-7.4), low propensity to tan (OR = 2.4, 95% CI = 1.1-5.0), light eye (OR = 2.4, 95% CI = 1.1-5.2), and light skin colour (OR = 4.1, 95% CI = 1.4-12.1) were significantly associated with melanoma risk after adjustment for age, gender and pigmentation characteristics. A chart which identifies melanoma risk associated with combinations of these factors is presented; it can be used to identify subjects who would most benefit from preventive measures in Mediterranean populations. According to the combination of these factors, a relative risk range from 1 to 98.5 was found. Light skin colour, high number of sunburns with blistering, and low propensity to tan were significantly associated with melanoma thickness, possibly indicating that individuals with these characteristics underestimate their risk and seek attention when their lesion is already advanced.     

Polyamines and the synthesis of estradiol-regulated growth factors in rat mammary cancer in culture

We have recently provided evidence to suggest that the polyamine pathway plays an essential role in the expression of the growth-promoting effect of estradiol (E₂) regulated growth factors in the N-nitrosomethylurea (NMU) induced rat mammary tumor culturedin vitro in the soft agar clonogenic assay. To further explore the interaction between the polyamine pathway and autocrine control of tumor growth by E₂, we tested whether, in our system, polyamines play a role in the synthesis of E₂-regulated growth factors. Conditioned medium (CM) obtained from tumors treated with E₂ and the polyamine biosynthesis inhibitor -difluoromethyl-ornithine (DFMO) (1 mM) no longer exhibited the colony-stimulating effect which was consistently observed with E₂-CM. Such growth promoting activity was restored in a dose-dependent fashion with CM obtained from tumors treated with E₂, DFMO, and increasing concentrations of spermidine (from 1 to 100µM). Conditioned medium obtained from tumors treated with DFMO with and without spermidine in the absence of E₂ had no discernible effects on colony formation. The colony stimulating effect of the CM employed could not be accounted for by the contaminating presence in the media of E₂, DFMO, or polyamines. These results indicate that, in our system, the polyamine pathway plays an important role in the synthesis of E₂-regulated growth factors.     

Prognostic factors for survival after isolated limb perfusion for malignant melanoma

AIMS: Risk factors were determined for mortality within 1 year after isolated limb perfusion (ILP). METHODS: All of 439 patients who underwent ILP for melanoma of the extremities were studied. Ninety percent of the patients had MD Anderson stage IIB or III disease at the time of ILP. ILP was performed with melphalan with or without TNFalpha under mild hyperthermic (38-40 degrees C) or normothermic (37-38 degrees C) conditions in 80% of the cases. RESULTS: Sixty-nine patients died within this period, 64 of metastatic melanoma. The indication for ILP was an unresectable primary (n=3), a local recurrence (n=24) or adjuvant to excision of primary lesions (n=17) in patients with stage IIIB regional lymph node metastases. These patients or patients with stage IIIAB melanoma with satellites and/or in-transit metastases with regional lymph node metastases had a relative risk of 4.6 (95% CI 2.0-6.6) and 3.6 (95% CI 2.1-10) of dying within 1 year from ILP, respectively (p<0.001). In patients with stage IV disease (distant metastases), the relative risk was 22 (95% CI 3.8-127, p=0.001). CONCLUSION: Patients with advanced limb melanoma have an increased risk of death within 1 year after ILP when regional lymph node or distant metastases are present.     

Regulation of insulin-like growth factors by antiestrogen

Insulin-like growth factors are potent mitogens for breast cancer cell proliferation. This effect is modulated by the cirulatory and extracellular IGFBPs as well as by the affinity of ligand binding receptors on the target cells. Antiestrogens have been shown to reduce both circulatory and microenvironmental IGF levels and thus suppress the IGF-I-induced growth of both ER-positive and ER-negative breast cancer cells. However, the effects of antiestrogens in down regulation of type I IGF receptor and in altering the autophosphorylation tyrosine kinase activity of EGF receptors are mainly observed in ER-positive cells. Furthermore, alteration of IGFBP by antiestrogens such as a marked increase of IGFBP-I production have been shown to inhibit the proliferative effect of IGF-I on ER-positive, but stimulate this effect, on ER-negative cells. Such differential effects from IGF receptor and IGFBP may explain the clinical outcome that tumor regression from antiestrogens is mainly observed in ER-positive type. This assumption based on IGF regulation alone is certainly an oversimplistic view amid the complexity of autocrine, paracrine, and endocrine functions.     

An analysis of risk factors for cutaneous melanoma by anatomical site (Australia)

Objective: Emerging evidence suggests that melanomas arising on the head and neck that are not lentigo maligna melanomas have different associations with phenotypic and environmental risk factors than those on the trunk and other sites. We sought to test this hypothesis in a population-based study in Queensland, Australia.Methods: Risk factor data were collected from 2360 participants with incident cutaneous melanoma diagnosed 1982–1990, including 167 participants with lentigo maligna melanoma. For each risk factor, polytomous logistic regression analysis, using the trunk as a reference category, was used to estimate the odds ratio and 95% confidence interval for cutaneous melanomas by anatomical site.Results: Participants with melanomas of the head and neck were significantly older than those with melanomas of the trunk (males 52.7 versus 49.7 years; females 47.8 versus 40.5 years). Compared with patients with truncal melanomas, those of the head and neck were less likely to have many nevi (OR 0.41, 95% CI 0.13–1.31), although this did not reach statistical significance. Among females, melanomas of the lower limb were negatively associated with a past history of non-melanoma skin cancer (OR 0.41, 95% CI 0.23–0.74).Conclusions: We have observed heterogeneity for melanoma risk by anatomical site, lending weight to the hypothesis that cutaneous melanomas may develop through multiple causal pathways.     

Prognostic factors in cutaneous desmoplastic melanoma

BACKGROUND:

Desmoplastic melanoma (DM) is a rare subtype of melanoma that is characterized by malignant spindle cells separated by prominent, fibrocollagenous stroma. Primary melanomas either may be entirely desmoplastic or almost entirely desmoplastic (pure DM [pDM]) or may exhibit a desmoplastic component admixed with a nondesmoplastic component (combined DM [cDM]).

METHODS:

Patients who were diagnosed between 1993 and 2007 at a single institution with clinically localized, primary cutaneous melanoma (PCM) that contained a desmoplastic component and who underwent sentinel lymph node (SLN) biopsy were identified. Clinical and pathologic features of the primary tumors were correlated with DM type, SLN status, and patient outcome.

RESULTS:

Two hundred fifty‐two patients (167 men, 85 women) were identified (median age, 61 years). The median tumor thickness was 2.0 mm. One hundred twenty‐three patients (48.8%) had pDM, and 129 patients (51.2%) had cDM. Overall, 17 patients (6.7%) had positive SLN status, including 12 patients with cDM and 5 patients with pDM. Because of the low SLN‐positive rate, a statistically significant difference in SLN status between patients with cDM (8.5%) and patients with pDM (4.9%; P = .25) could not be demonstrated. Older patient age, being a man, positive SLN status, and increasing tumor thickness were associated significantly with poorer disease‐free survival (P < .05), although only the latter 2 variables were independently predictive. In addition, cDM type (P = .017) was associated significantly and independently with a shorter time to recurrence.

CONCLUSIONS:

In this largest study to date of patients with DM who underwent SLN biopsy, the SLN‐positive rate in patients with DM was lower than that in patients with conventional melanoma. The results indicated that DM type is associated significantly and independently with the time to recurrence and should be evaluated routinely in all patients with PCM. Cancer 2010. © 2010 American Cancer Society.     

The effects of type beta transforming growth factor on proliferation and epidermal growth factor receptor expression in a human glioblastoma cell line

Summary Type beta transforming growth factor (B-TGF) is a potent growth inhibitor to many human tumor cell lines. Very little is known about the mechanism for this growth inhibitory action of BTGF We here report the effect of B-TGF on proliferation and epidermal growth factor receptor (R-EGF) expression in a human glioblastoma cell line named T-MG1.B-TGF inhibite the soft agar growth of T-MG1 cells. Maximum inhibition was 70%, achieved with 0.5 units BTGF. BTGF had no effect on monolayer growth of T MGl cells.T-MG1 cells contained abundant R-EGF, which could be divided into two subpopulations, one high affinity and one low affinity population of R-EGF. Treatment with B-TGF caused an initial decrease (0-6 h) in EGF-binding, followed by an increase in EGF-binding which reached maximum after 24 h exposure to B-TGE. Since addition of EGF to agar cultures gave no additional increase in inhibition by B-TGF and EGF alone had no inhibitory effect, we believe that binding of EGF to its receptor is not part of the pathway mediating the inhibitory effect of B-TGF.All neoplastic cells have lost some measure of growth control and the cellular elements involved are growth factors, growth factor receptors and oncogenes. T-MG1 cells contain abundant R-EGF and this may partly explain their malignant nature (malignant nature is here defined as ability to proliferate in agarose). Type alpha transforming growth factors, which in some cancer cells act as uncontrolled autocrine growth factors, were not found in protein extracts from T-MG1 cells. BTGF, which is normally found in all cells, could not be detected in protein extracts from T-MG1 cells. The lack of B-TGF as an autocrine growth inhibitor may be of importance for maintaining the malignant nature of T-MG1 cells.     

Malignant melanoma metastasis to brain: role of degradative enzymes and responses to paracrine growth factors

Mouse and human melanoma cells metastatic to the brain express degradative enzyme activities that are used for invasion of brain basement membrane and parenchyma. Compared to poorly metastatic or lung- or ovary-metastatic murine melanoma lines, the brain-metastatic sublines secreted higher levels of a variety of degradative enzymes. Brain-metastatic murine and human melanoma cells also degraded subendothelial basement membrane and reconstituted basement membrane at rates higher than other metastatic melanoma cells. In some cases these degradative activities in mouse and human melanoma cells can be induced by paracrine factors known to be present in the brain parenchyma, such as nerve growth factor (NGF). NGF stimulates the expression of degradative enzymes, such as the endo--glucuronidase heparanase, that are important in basement membrane penetration but this factor does not stimulate melanoma cell growth. The growth of brain-metastasizing melanoma cells appears to be stimulated by other paracrine growth factors, such as paracrine transferrin. Melanoma cells metastatic to brain express higher numbers of transferrin receptors and respond and proliferate at lower concentrations of transferrin than do melanoma cells metastatic to other sites or poorly metastatic melanoma cells. The results suggest that degradation and invasion of brain basement membrane and responses to paracrine neurotrophins and paracrine transferrins are important properties in brain metastasis of murine and human malignant melanoma cells.     

Secreted growth factors from estrogen receptor-negative human breast cancer do not support growth of estrogen receptor-positive breast cancer in the nude mouse model

Estrogen receptor (ER)-negative MDA-231 human breast cancer cells have been shown to secrete high concentrations of several growth factors including transforming growth factor-alpha and insulin-like growth factor I, which could have important autocrine or paracrine growth regulatory functions and, additionally, could explain the rapid autonomous growth of these cells. In contrast, the hormone-responsive, ER-positive MCF-7 cells secrete low levels of these factors constitutively. Since estrogen treatment increases secretion of these growth factors in MCF-7 cells, it has been postulated that these growth factors mediate estrogen's growth effects through an autocrine mechanism. To test this hypothesis we reasoned that growth factors supplied by MDA-231 cells should support growth of MCF-7 cells in an estrogen-depleted environment. Inoculation of castrated female athymic nude mice with MDA-231 cells resulted in rapid tumor growth. However, MDA-231 tumors did not support growth of MCF-7 cells inoculated on the opposite flank by an endocrine mechanism; MCF-7 tumors required estrogen supplementation for growth. To determine if MDA-231 cells could support MCF-7 growth by a paracrine mechanism, various mixtures of the two cell lines were coinoculated at the same site in castrated or in estrogen-supplemented mice. ER was not detectable in tumors derived from a mixed inoculum, indicating the absence of MCF-7 cell growth. Furthermore, DNA flow cytometry of these tumors revealed only a single G₁ peak representative of MDA-231 cells in estrogen-deprived mice. On the other hand, two distinct G₁ peaks representing both MDA-231 and MCF-7 cells were detected in tumors grown in estrogen-supplemented mice. These data demonstrate that growth factors from estrogen-independent MDA-231 cells are not capable of replacing estrogen for growth stimulation of MCF-7 cells. Either estrogen-stimulated growth of MCF-7 cells requires other secreted factors not supplied by MDA-231 cells, or it involves a different mechanism.     

Primary malignant melanoma of the lip

Melanoma of the lip is a very rare clinical entity. As such, no large studies are available to help in diagnosis and treatment. We report our results on 11 patients with lip melanoma. Patients with lip melanoma should be approached in a similar fashion to patients with other cutaneous melanomas.     

Prognostic factors in metastatic malignant melanoma treated with combined radiation therapy and hyperthermia

From May 1981 to September 1991, 38 patients with metastatic malignant melanoma were treated with combined radiation therapy and hyperthermia to a total of 97 hyperthermia treatment fields. Prior treatments to these sites included surgery (31 patients, 76 fields), chemotherapy (18 patients, 54 fields), immunotherapy (14 patients, 42 fields) and radiation therapy (7 patients, 13 hyperthermia fields). Hyperthermia was given to fields located in the head and neck region, trunk and extremities in 30, 45 and 22 cases, respectively. Nodular-diffuse tumours were present in 86 fields while 11 fields were treated for microscopic residual tumour deposits. Concurrent radiation therapy was given in 180–400 cGy per fraction, 2–5 times per week for a mean total dose of 4098 cGy per field. Hyperthermia treatments were delivered using either microwave or ultrasound devices (286 and 48 treatments, respectively) with a mean (range) of 3. 4 (1–14) hyperthermia treatments per field for a mean (range) of 43 (10–70) min per field. Patients (n = 34; 84 fields) were available for follow-up for a mean (range) of 14–6 (0–4-82.5) months. At 3 weeks post-treatment, 34 fields had complete, ongoing, or partial responses; 39 fields had no response; and there were no recurrences in the 11 fields treated for microscopic residual disease. Local control was maintained in 31% (26/84) fields with a mean follow-up of 14.6 months. At 36 months, five patients remained alive with complete control of their treated local disease. Statistical analyses revealed that patients with soft tissue metastases only, who were older at the time of hyperthermia, had a longer time between initial diagnosis and hyperthermia treatment, received a higher dose of radiation, had no previous chemotherapy, and had small tumour volumes, had a higher initial response. Multivariate analyses revealed that the three-covariate model including time interval between initial diagnosis and hyperthermia treatment, previous chemotherapy, and metastases to soft tissue only, best predicted response. The results of the investigation support the continued study of combined radiation therapy and hyperthermia treatments for selected patients with metastatic melanoma, and indicate that long-term survival can occasionally be obtained with this approach.     

Familial aggregation of melanoma risks in a large population-based sample of melanoma cases

ObjectiveMelanoma has been shown in numerous studies to be associated with sun exposure, and with host phenotypic factors of genetic origin. In this study we use information from a large series of incident cases of melanoma from an international population-based study to examine the patterns of incidence of melanoma in the first-degree relatives of these cases. Methods: A total of 2508 incident cases of melanoma provided information on basic demographic data and pigmentary characteristics, in addition to detailed information on family history of melanoma. These data were used to examine the incidence rates ratios of melanoma in the relatives of cases in relation to population rates, and also with respect to phenotypic characteristics of the probands that have been shown to be associated with melanoma: mole counts, hair color, eye color, and skin sensitivity to the sun. Results: The incidence rates reflect the underlying patterns of incidence in the source populations, with generally higher rates in the Australian sample, low rates in Italy, and intermediate rates in the USA and Canada. Also, rates are higher in men than in women, except at very young ages. Phenotypic characteristics of the probands were only weakly associated with the observed rates in the relatives although there is a strong inverse association with age at diagnosis. Cumulative risk of melanoma rises to 6.9 (6.1) at age 80 in male (female) first-degree relatives of cases, and to 10.8 (9.5) in relatives of cases diagnosed before age 50. Conclusions: Relatives of cases diagnosed with melanoma are at considerable lifetime risk of the disease, especially if the case is diagnosed at a young age.     

Suppression of invasive behavior of melanoma cells by stable expression of anti-sense perlecan cDNA

Background: Heparan sulfate proteoglycans are one of the major componentsof extracellular matrix and are secreted at different levels by severalnormal and tumoral cells. Perlecan, the basement membrane proteoglycan, hasstructural domains involved in cell/matrix interactions and growth factorstorage. Metastatic melanoma cells show an increase in perlecan expressionas compared to low metastatic ones. We examined whether reduction ofperlecan expression could down-modulate the malignant phenotype in melanomaclones.Materials and methods: We transfected B16-F10 murine malignant melanomacells with a perlecan antisense cDNA construct and tested the in vitrobehavior of the selected clones.Results: The expression of antisense mRNA corresponded to a reduction ofperlecan synthesis. The clones with reduced perlecan synthesis showed adown-regulation of proliferation and invasion.Conclusions: These results further indicate the importance of perlecan asa regulator of growth factor activity affecting the biological properties ofmetastatic cells, and suggest the potential use of antisense perlecan DNA inanti-melanoma gene therapy approaches.     

Gamma Knife radiosurgery for intracranial metastatic melanoma: an analysis of survival and prognostic factors

Objective of this study was to evaluate retrospectively the effectiveness of Gamma Knife radiosurgery for intracranial metastatic melanoma and to identify prognostic factors related to survival. Twenty-six patients with intracranial metastases (72 lesions) from melanoma underwent Gamma Knife radiosurgery. In 14 patients (54%) whole-brain radiotherapy (WBRT) was performed as part of the initial treatment, and in 12 patients (38%) immunotherapy and/or chemotherapy was given after Gamma Knife radiosurgery. The median tumor volume for Gamma Knife radiosurgery treated lesions was 1.72 cm³. The median prescribed radiation dose was 18 Gy (range 8–22 Gy) typically prescribed to the isodose at the tumor margin. Univariate and multivariate analyses were used to determine significant prognostic factors affecting survival. Overall median survival was 6 months after Gamma Knife radiosurgery, and 1-year survival was 25%. The median survival from the onset of brain metastases was 9 months and from the original diagnosis of melanoma was 50 months (range 4–160 months). There were no major acute or late GKS complications. In univariate testing, the Karnofsky score equal to or higher than 90% (P < 0.01, log-rank test), supratentorial localization (P < 0.001, log-rank test), intracranial tumor volume less than 1 cm³ (P < 0.02, log-rank test), and absence of neurological signs or symptoms before Gamma Knife radiosurgery (P < 0.003, log-rank test) were significant favorable factors for survival. In multivariate regression analyses, the most important predictors associated with increased survival were a KPS 90 (P < 0.023), female sex (P < 0.004), supratentorial localization (P < 0.01), and absence of neurological symptoms (P < 0.008). Radiosurgery is a noninvasive, safe, and effective treatment option for patients with single or multiple intracranial metastases from melanoma. Female sex, Karnofsky score 90, supratentorial localization and lack of symptoms before the Gamma Knife radiosurgery were good independent predictors of survival.