Melanoma in the liver: Oxidative stress and the mechanisms of metastatic cell survival

Abstract

Metastatic melanoma is a fatal disease with a rapid systemic dissemination. The most frequent target sites are the liver, bone, and brain. Melanoma metastases represent a heterogeneous cell population, which associates with genomic instability and resistance to therapy. Interaction of melanoma cells with the hepatic sinusoidal endothelium initiates a signaling cascade involving cytokines, growth factors, bioactive lipids, and reactive oxygen and nitrogen species produced by the cancer cell, the endothelium, and also by different immune cells. Endothelial cell-derived NO and H₂O₂ and the action of immune cells cause the death of most melanoma cells that reach the hepatic microvascularization. Surviving melanoma cells attached to the endothelium of pre-capillary arterioles or sinusoids may follow two mechanisms of extravasation: a) migration through vessel fenestrae or b) intravascular proliferation followed by vessel rupture and microinflammation. Invading melanoma cells first form micrometastases within the normal lobular hepatic architecture via a mechanism regulated by cross-talk with the stroma and multiple microenvironment-related molecular signals. In this review special emphasis is placed on neuroendocrine (systemic) mechanisms as potential promoters of liver metastatic growth. Growing metastatic cells undergo functional and metabolic changes that increase their capacity to withstand oxidative/nitrosative stress, which favors their survival. This adaptive process also involves upregulation of Bcl-2-related antideath mechanisms, which seems to lead to the generation of more resistant cell subclones.

Introduction

Malignant melanoma (MM) is derived from uncontrolled proliferation of melanocytes. In the last 50 years its incidence has risen faster than almost any other cancer, an increase directly related to sun exposure for aesthetic and leisure reasons [1,2]. About 81 % of cases are located in developed countries [3].

Although most melanomas originate in the skin, they can also appear on other surfaces of the body (such as the mucosa of the mouth, rectum or vagina, or choroid layer inside our eyes). It represents approximately 1.5 % of tumors in both sexes. In Europe it is more frequent among women, unlike in the rest of the world. The highest incidence is recorded in countries with strong solar irradiation and with a non-native white population (i.e. Australia, New Zealand, South Africa and USA). Cases are recorded at virtually any age, although most are diagnosed between 40 and 70 years of age [2]. According to recent reports released by the World Health Organization, MM causes 50,000–55,000 deaths annually, and approx. 75 % of all deaths associated with skin cancer (www.who.int).

Melanoma is most likely due to a multistep process of genetic mutations that alter the cell cycle and render the melanocytes more susceptible to carcinogens (mainly UV radiation) [4]. It is also known that UV radiation causes reactive oxygen species (ROS)-mediated oxidative stress [5]. The biological behavior of primary skin melanoma involves, at least, two phases: one in which the growth of the tumor is radial and, therefore, cannot produce metastasis; and another in which the growth is vertical. This last phase will imply that the melanoma increases in thickness and invades the deeper layers of the skin and the tissue under it, and will have the capacity to produce lymphatic or blood metastases (stages III and IV, TNM classification) [6].

Breslow [7] first described the depth of tumor invasion correlating with patient outcomes: melanomas <0.76 mm thickness were low-risk lesions, seldom metastasized, and prognostically favorable, whereas melanomas >4 mm had a high risk of recurrence or metastasis, and bad prognosis. The American Joint Committee on Cancer considers Breslow thickness as the most important prognostic factor of survival in localized melanoma. The 10-year survival rates for each T-stage are approx. 92 % for T1 (<1.0 mm thickness), 80 % for T2 (1.0–2.0 mm), 63 % for T3 (2.0–4.0 mm), and 50 % for T4 melanomas (>4.0 mm) [8,9].

Besides Breslow’s staging the primary melanoma mitotic rate is also considered a prognostic value in localized melanoma survival. The mitotic rate reflects the tumor's proliferation in the vertical growth phase. The 10-year survival rate for melanomas with 0 mitosis/mm² is of approx. 93 % compared to 48 % for melanomas with >20/mm². Thicker or ulcerated tumors are associated with higher mitotic rates [9].

Steven Paget’s classical “seed and soil” hypothesis introduced the concept that a receptive microenvironment is required for the development of metastasis [10]. The liver is a frequent site of metastasis for several cancers including, melanoma, lung, colorectal, breast, esophagus, stomach, pancreas, and neuroendocrine tumors [11]. However, molecular determinants of this organotropism are poorly understood. Recently, Lee et al. using mouse models of pancreatic ductal adenocarcinoma have shown that almost all hepatocytes showed STAT3 activation in mice with cancer, compared to less than 2% of hepatocytes in mice without tumors. Knockout of STAT3 only in mouse hepatocytes blocked the increased susceptibility of the liver to metastatic seeding [12]. These authors also showed that a specific cytokine, IL6 (released into the blood circulation by non-malignant cells), promotes STAT3 signaling in hepatocytes leading to an increased production of serum amyloid A1 and A2 (SAA), which acts as an alarm to attract inflammatory cells and initiate a fibrotic reaction that together establish the "soil." Therefore, hepatocytes appear to coordinate myeloid cell accumulation and fibrosis within the liver and, in doing so, increase the susceptibility of the liver to metastatic seeding and outgrowth. In parallel studies, it has been observed that overexpression of SAA by hepatocytes also occurs in patients with pancreatic and colorectal cancers that have metastasized to the liver, and that many patients with locally advanced and metastatic disease show increases in circulating SAA [12].

To make this signaling mechanism even more interesting, previous findings demonstrated that IL6 (but mainly release by murine metastatic B16-F10 melanoma cells), also induces glutathione (GSH) efflux from hepatocytes [13]. This mechanism is also STAT3-dependent [13]. GSH, the most prevalent non-protein thiol in mammalian cells and a main physiological antioxidant, acts as a metastatic growth promoter [[14], [15], [16]].