Melanomas originate from cutaneous or extracutaneous melanocytes. In case of cutaneous melanoma, 25–35% of the tumors are derived from pre-existing nevi, although the origin of the remaining tumors is unknown. The transformation of nevi to melanoma can be reconstructed by a well-described progression model (1). By various oncogenic or epigenetic events, nevi can be stimulated to develop into the radial growth phase (RGP). At this stage, the pre-malignant cells expand in horizontal direction with restriction to the epidermis. RGP melanomas are <0.75 mm thick. When excised at this early stage, patients have an excellent prognosis, as the RGP tumors are generally not able to metastasize, and angiogenesis does not occur. During the next step, the transition to the vertical growth phase (VGP), melanomas gain the ability to cross the basal layer of the epidermis and to invade the deeper dermal layers of the skin. VGP tumors, which are >0.75 mm in size, have the potential to metastasize and to induce angiogenic events. The “angiogenic switch”, describing the gain of the ability to induce numerous pro-angiogenic factors, is a characteristic feature of the transition from RGP to VGP melanomas (2), and the pro-angiogenic features are even more enhanced in melanoma metastases. Pro-angiogenic factors can already be secreted by RGP melanoma cells in vitro under appropriate conditions, such as the presence of fibroblasts (3). However, because of their epidermal location, they are not able to attract blood vessels. Overall, the process of tumor angiogenesis contributes to the initial events of melanoma development, but it is also important for the maintenance, progression, and further spreading of already metastasized melanomas.

The diffusion limit of oxygen between cell and blood vessel is ~100–200 μm (4). Consequently, in absence of nutrient supply by the blood, the tumor size is limited to 0.2–2 mm in diameter (5). Interestingly, observations from animal models of melanoma show that tumor angiogenesis can already occur when melanomas are clearly below this size limit. In order to enable the detection of pro-tumorigenic events at this small-size scale, transgenic fish models with blood vessel-specific expression of fluorescent proteins are a valuable tool. In a xenotransplant model performed in zebrafishes which express eGFP under the flk1 promoter (with flk1 encoding Vegfr-2), it was shown that 15–30 injected melanoma cells already elicit an angiogenic response (6). For their experiments, the authors used the strongly metastatic murine B16-F10 melanoma cells. In our own laboratory, we investigated tumor angiogenesis in a genetic medaka fish melanoma model where the expression of a fish-specific oncogenic epidermal growth factor receptor (EGFR) leads to strong pro-tumorigenic signaling which is enhanced by autocrine loops (7, 8) and results in the malignant transformation of pigment cells and tumor development (9, 10). This fish EGFR is termed Xiphophorus melanoma receptor kinase (Xmrk) and was identified in naturally occurring melanoma of Xiphophorus fishes, the first described animal model for melanoma (11, 12). To visualize blood vessels, we used eGFP expression driven by the fli-1 promoter, with Fli1 being a transcription factor strongly expressed in myeloid and endothelial cells. Strikingly, we observed that single melanoma cells had already the capacity to induce neoangiogenesis (13). These data are corroborated by earlier observations in a murine angiogenesis model of other tumor types based on dorsal skin-fold window imaging. Using fluorescent HCT116 human colon carcinoma cells and 4T1 murine mammary carcinoma, Cao and colleagues observed that incipient tumor angiogenesis can occur independently from hypoxia (14). In line with the results from these animal models, an association of angiogenesis with small cutaneous melanomas <1 mm in diameter was already described >20 years ago (15). To better comprehend the early steps of melanomagenesis, the following sections aim at increasing the understanding of angiogenic processes which can occur before a critical tumor mass is reached.

In general, tumor angiogenesis is regarded as a result from intratumoral hypoxia, which leads to the activation of hypoxia-inducible factor-1 (HIF1), one of the best-studied angiogenesis-related transcription factors. HIF1 is composed of the subunits HIF1α and HIF1β. HIF1β [also called aryl hydrocarbon receptor nuclear translocator (ARNT)] is constitutively expressed, whereas the abundance of HIF1α is strictly regulated and thereby determines the activity of the dimeric transcription factor. Under normoxic conditions, human HIF1α is hydroxylated on Pro402 and Pro564 by prolyl-4 hydroxylases (PDHs), thus leading to the recognition by the ubiquitin ligase component von Hippel Lindau protein (pVHL) and proteasomal degradation (16). PDHs exhibit an absolute requirement for oxygen, Fe²⁺ and α-ketoglutarate, the latter being a co-substrate for the enzymes. When pO₂ is low, prolyl hydroxylation of HIF1α is suppressed, and the protein is stabilized (17). HIF1 is a potent inducer of pro-angiogenic factors including vascular endothelial growth factor (VEGF), angiopoietin-2, matrix metalloprotease 14 (MMP14), and angiogenin (18–20). Notably, next to angiogenesis, HIF1 regulates a large number of genes involved in invasion, differentiation, and metabolism as well as protection from apoptosis in melanoma. These processes are not in the scope of this review and are summarized elsewhere (21, 22).

Although HIF1α is already expressed in nevi, most likely as a result from the mild hypoxic conditions, which occur in skin (23), HIF1 expression and activity is increased in melanoma and correlates with decreased differentiation and VEGF expression (22, 24, 25). In a genetic mouse melanoma model driven by melanocyte-specific Braf^V600E induction and Pten depletion, HIF1α is required for angiogenesis and metastasis without affecting primary tumor formation (26).

In addition to the well-described hypoxia-dependent regulation mechanism, there are several hypoxia-independent processes, which can lead to the stabilization of HIF1α. These seem to play an important role in melanoma, where HIF1α is constitutively expressed – a fact, which is not necessarily due to the prevalence of hypoxic conditions. As an example, protein expression of HIF1α is strong in melanoma cells predominantly expressing HIF1α-785, a splice isoform lacking a crucial part of the oxygen-dependent degradation domain (27). Under normoxic conditions, HIF1α can be stabilized by various growth factors, cytokines and oncogenes, as shown for BRAF^V600E in melanoma (28). Next to BRAF/MEK/ERK signaling, the PI3K pathway is involved in the increase of HIF1α protein levels, e.g., via the PI3K downstream component 70 kDa ribosomal S6 kinase 1 (p70S6K1) (29–31). The PI3K pathway is frequently activated in melanomas because of deletions or inactivating mutations in the phosphatase PTEN or activation mechanisms, which encompass upstream components. In addition, endothelin-1 (ET-1)-dependent activation of the PI3K pathway could be directly linked to HIF1α stability under normoxic conditions in melanoma cells. Here, RNA and protein levels of the prolyl hydroxylase PHD2 were reduced in response to ET-1-dependent PI3K activation, and this was accompanied by increases in HIF1α (32). HIF1α accumulation can also take place in presence of enhanced reactive oxygen species (ROS) and NF-κB in melanoma (33, 34), both being commonly deregulated in this cancer type (35, 36).

The melanocytic master regulator and melanoma oncogene microphthalmia-associated transcription factor (MITF) is another important melanoma player. It was first described as a lineage-determining factor in melanocytes, which regulates the expression of genes involved in pigment production (37, 38). Nowadays, many more target genes of MITF are identified [reviewed in Ref. (39)], including HIF1A, which is regulated by MITF in a hypoxia-independent manner (40, 41). Notably, MITF cannot generally be considered as oncogenic, as its effects seem to depend on the expression level. While the entire depletion of MITF in melanoma cells can lead to cell cycle arrest and senescence, low MITF activity is associated with stem cell-like properties and invasiveness (42, 43). High MITF activity, however, promotes differentiation and exit from the cell cycle (44, 45). Different MITF levels lead to the targeting of different gene sets, which explains the observed concentration dependent effects. Next to the MITF expression level, protein modifications exhibit another measure to alter target gene expression. It could be demonstrated that the inheritance of mutant MITF^E318K predisposes to familial melanoma. This mutant exhibits defective SUMOylation, which results in an altered transcription of target gene subtypes in comparison to wildtype MITF. Among others, MITF^E318K shows enhanced binding to the HIF1α promoter and results in enhanced transcriptional activity of HIF1α (46).

However, HIF1α is not necessarily required for tumor angiogenesis. Early studies performed with RAS-transformed MEFs derived from Hif1a-/- mice showed that oncogenic HRAS is able to induce full tumor vascularization in the xenotransplants (47). Although HRAS plays a negligible role in melanoma, NRAS, which is activated in 15–20% of cutaneous melanoma, has strong functional overlap with the other RAS isoforms. These data are corroborated by the observation that mutant NRAS cooperates with the scaffold protein GAB2, which is often co-expressed with oncogenic NRAS, to increase angiogenesis and VEGF transcription in hypoxic and normoxic conditions in melanoma (48). By stimulating the expression of several matrix metalloproteases and urokinase plasminogen activator, RAS isoforms can furthermore contribute to the release and maturation of VEGF (49).

Next to harboring the equipment for protein synthesis and maturation, the endoplasmatic reticulum (ER) is a crucial stress sensor and a regulator of cellular homeostasis. Nutrient starvation as well as nutrient excess are received as ER stress and are translated into different cellular responses which protect the cellular or tissue integrity. Owing to their high proliferation rate and altered metabolism, the demand for protein production in the ER is very high in tumor cells, thus generating conditions, which permit ER stress (79). The unfolded protein response (UPR) is a mechanism activated in the ER by the accumulation of mis- or unfolded protein, which can occur in fast growing tumors or under conditions of hypoxia, low pH, or low nutrient supply. The UPR generally serves the cellular integrity by enabling ER homeostasis under ER stress and is composed of three pathways, which stimulate protein folding, protein degradation, and the transient inhibition of protein synthesis (80). UPR activity also plays a role during tumor development, and the angiogenic switch was reported to be induced by the PERK/ATF4 pathway in a HIF1α independent manner (81). UPR activation is able to enhance the transcription of pro-angiogenic factors including VEGFA, FGF2, angiogenin, and angiopoietin-2 and to downregulate the expression of several angiogenesis inhibitors (81, 82). In melanoma, activation of the UPR component GRP78 is correlated with metastases and shorter survival (83).

Notably, the UPR can also be considered as a part of the ROS network: On the one hand, ROS can trigger the UPR and on the other hand, all three UPR pathways are implicated in the generation of ROS (79).

Observations from neoangiogenic processes of normal healthy tissues also provide valuable information, which can be relevant for tumor biology. In adipose tissue, hypoxia-independent angiogenesis during cold acclimation is regulated by mitochondrial proteins (84). Adipose tissue is prone to constant changes of size and volume and is accompanied by corresponding adaptations of tissue vascularization. When the organism is exposed to cold temperature, expression of the mitochondrial biogenesis regulator PGC-1α is induced. PGC-1α is able to activate VEGFA expression at the transcriptional level in an HIF1α-independent manner (85). Consequently, several pro-angiogenic factors including VEGFA are produced hypoxia independently by cold acclimation, leading to VEGFR2-dependent angiogenesis of the adipose tissue (84). A high expression of PGC-1α is found in ~10% of melanomas and is associated with poor overall survival (86). The link between PGC-1α, VEGF, and angiogenesis has not been investigated in melanoma to date. It would be interesting to analyze whether the survival disadvantage of patients with highly PGC-1α expressing melanoma is related to an increased tumor angiogenesis. Of note, BRAF inhibitors, which are routinely used in the clinic for the treatment of patients with BRAF^V600E positive melanomas, have been reported to suppress PGC-1α expression (87). Whether PGC-1α is re-expressed in recurrent melanoma and contributes to the fast progression after BRAF inhibitor resistance development is currently unknown. Table 1 summarizes the mentioned hypoxia-independent pro-angiogenic mechanisms in melanoma.

Pro-angiogenic events caused by hypoxia-independent cues allow incipient tumors to recruit blood vessels even before a critical tumor mass is reached. This provides the first entry point into the vasculature and might enable small tumor cell colonies, which produce the appropriate signals, to metastasize. Genomic information from several melanomas from different stages in the same patients lead to the conclusion that the first step of metastasis can occur very early during tumor formation (88, 89). The underlying reasons are most likely found in the incipient tumor-initiated angiogenesis of blood and lymph vessels. Although the colonization at distant sites is also determined by additional factors, the knowledge of the first possible metastatic events is invaluable to understand the timing of melanoma development.

SM reviewed the literature and wrote the article.

The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.