TNF-alpha and metalloproteases as key players in melanoma cells aggressiveness

Melanoma incidence and mortality are steeply increased in the last century [1‐3]. Melanoma is the most aggressive skin cancer and the cutaneous form (Cutaneous Melanoma, CM) is the most common one. Recent data on novel pathways involved in melanoma development opened new opportunities to identify novel therapeutic targets [4, 5], nevertheless additional key players underlying melanoma onset and progression need to be identified. Indeed, further elucidation of the molecular mechanisms underlying melanoma malignancy is expected to improve prognostic assessment and therapeutic options. The role of altered RAS/BRAF/MEK/ERK pathway in melanoma pathogenesis and progression is well known [6]. Mutated BRAF^V600E represents a major target in the current therapeutic strategies, despite the fact that more than half of melanomas do not harbor this mutation. Melanoma highly aggressive behavior depends on migration, invasion, proliferation of metastatic cells and on their ability to promote angiogenesis [7]. Invasive CM cells metastasize changing cytoskeletal organization and modifying the interaction with the extracellular matrix (ECM) and the surrounding stromal cells. During the vertical growth phase, primary tumor cells invade the dermis [8], via a cross-talk with the neighboring microenvironment [9, 10]. Proteolysis in the pericellular and stromal compartments, in fact, exerts a key role in the invasion process and it is well know that several protease, such as matrix metalloproteases (MMPs), are mediators of melanoma development [11]. On the other hand, MMP-9 activation was associated with cancer growth and dissemination [12], its role in cutaneous melanoma was reported and its activation, mediated by NF-κB, was associated with the BRAFV600E mutation status [13]. Another recent study reports the correlation of MMP-9 hypermethylation with its overexpression in melanoma [14] indicating novel molecular mechanisms underlying the MMPs activity and their modulatory role in melanoma aggressiveness.

Different molecules play a role in cancer progression, including chemokines and their receptors, as well as cytokines and growth factors [15]. Melanoma cells often express variable levels of cytokines and cytokine receptors at different stages of disease progression. Interleukin (IL-)1β, IL-6 and IL-8, for example, are known to be important drivers of cell proliferation and melanoma progression [16, 17]. Nevi and thin primary melanomas (less than < 1 mm of thickness) express low levels of IL-8, tumor necrosis factor-alpha (TNF-α), transforming growth factor-beta (TGF-β) and c-kit [18]. On the contrary, primary melanomas at more advanced stage (> 1 mm of thickness) show up-regulation of IL-1α, IL-1β, IL-8, TNF-α, TGF-β and granulocyte-macrophage colony stimulating factor (GM-CSF). TGF-β is considered a marker of melanoma metastatic spreading [18]. Moreover, a link between high levels of TNF-α and increased risk of tumor formation and development has been described in vivo [19]. An additional study in a murine model shows that more aggressive tumors express lower levels of TNF-α and other inflammatory cytokines, as determined by qRT-PCR analyses [20], confirming previously reported controversial role of TNF-α [21]. This suggests that melanoma development and progression is a complex process based on a well-organized interplay of intrinsic proliferation ability combined to the immune and angiogenic response, as coordinated action of several cell types. Cytokines controlling inflammation and immune cells can influence host immune response and melanoma cells can activate and/or reshape the surrounding environment to secrete factors mediating metastatic progression. Furthermore, melanoma cells can secrete inhibitory modulators and thereby arrest recognition and maturation of effector immune cells [22] or other signals affecting cancer cells microenvironments [23]. Therefore, several different mechanisms control CM ability to rapidly grow, invade and disseminate metastases in other tissues and organs. This highlights the importance of microenvironment and immune response to CM, as strongly influenced by intrinsic characters of primary melanoma cells.

Understanding such multifaceted functional interactions, involving different cell types and molecules, requires to integrate information gathered by different analytical approaches [24‐26]. The aim of the present study was to investigate intrinsic factors affecting human melanoma cell aggressiveness, investigating at different levels human melanoma cells that hold highly different malignancy grade. The study led to the novel identification of molecular targets and functional pathways likely responsible of the melanoma aggressive phenotype.

One of the most critical issues regarding cutaneous melanoma is related to its aggressiveness, which was also related either to mutational and immunological state or anatomical site [4, 50], or to the intrinsic behavior of melanoma cells. To assess the latter point, the proliferation, migration and invasion abilities of ten human melanoma cell lines were studied. To summarize aggressiveness rate of such cancer cells in one unique parameter, the MAG Score was calculated as a single number able to recapitulate proliferation, migration and invasion ability of each melanoma cell. According to these values, it was possible to classify these human melanoma cell lines as high and low aggressive cells. A375 and SK-MEL-28 cells, two of the most studied human melanoma cells, were chosen as model of different aggressiveness and malignancy, also confirmed by analyzing in depth their melanosphere forming capabilities.

It should be noted that the in vitro aggressive phenotype quantified according to MAGS perfectly matches the in vivo aggressiveness of the two cell lines [51]. This suggests that MAGS may have important clinical applications when patient derived organoids cultures are available. In these cases, the quantitative approach of MAGS may evaluate the organoids aggressiveness for prognostic purposes and to monitor the efficacy of new drugs under development, or drugs combinations, also within a precision medicine framework. In order to identify novel intrinsic molecular pathways responsible for melanoma aggressiveness, the two selected cell lines were analyzed at transcriptomic, proteomic and secretome analysis level. Ingenuity Pathway Analysis from these integrated multiomic investigations highlighted the prominent role of inflammatory response, as well as metalloproteases and secretion of inflammatory cytokines as potentially involved in determining human melanoma cells aggressive phenotype. In the current study, melanoma cells were investigated in vitro without any contact with immune-competent cells, suggesting that intrinsic pathways are likely to be involved in determining their aggressive phenotype. IPA analyses of transcriptomic expression profiles indicated TNF, the MMP-2 and IL-6 pathways as the most significantly upstream regulators, strongly suggesting them as possible key modulators of the melanoma cell aggressiveness. Several pathways resulted particularly dragged into cell aggressive phenotype such as Aryl Hydrocarbon Receptor Signaling; OX40 Signaling Pathway, Antigen Presentation Pathway, Estrogen-mediated S-phase Entry, Cell Cycle, G1/S Checkpoint Regulation, Vitamin D and seleno-proteins, known as potentially important in tumor development and progression. To confirm the results indicating the important role of TNF, MMP and IL-6, the proteomic profile by LC-MS/MS analysis of both cell lines extracts was achieved by applying a multi-denaturation protocol recently developed to increase analytical sensitivity of complex mixtures of proteins [45]. Functional annotation analyses of the collected data revealed a strong implication in aggressiveness traits of post-transcriptional modifications, molecular transport and protein traffic networks and cytokines signaling pathways. Proliferation rates, calculated under serum deprivation conditions and in cells seeded at three different densities, strongly suggested that melanoma cell aggressiveness is related to cell density, highlighting the possibility that a cell-cell interaction crosstalk and/or the secretion of autocrine signals may play a role in melanoma aggressiveness and progression. In the present study, cytokines secretion, evaluated by Luminex technology, showed different levels of pro-inflammatory cytokines like IL-1β, IL-8 and TNF both in melanoma cell lysates and in supernatants. TNF-α showed a higher (more than 20-fold increase) secretion by the most aggressive cell lines. It is noteworthy that TNF has been found involved in the enhancement of tumor invasion partially by upregulating matrix metalloproteases in human skin [52], therefore the low transcript levels of TIMP coupled to the increased enzymatic activity of the MMP2 in more aggressive cells, reported in this study, may be a direct consequence of TNF action, as predicted by IPA. It is important to note that both transcriptional (Illumina - Affymetrix) and bioinformatic (IPA) analyses supported the increased activity of metalloproteases in A375 cells and that such data were perfectly confirmed by the MMP2 enzymatic activity measured. These results were further reinforced by a complementary approach based on analyses of transcriptomic data from biopsies of melanoma patients vs benign nevi, with a consensus within the transcriptomic, proteomic, cytokinomic and zymography data reported in Figs. 3, 4 and 5, in Tables 2, 3 and 4 and in Additional file 1. The hypothesis that TNF-α may be an intrinsic crucial player in melanoma growth and aggressiveness was further tested by inhibiting the TNF secretion through a chimeric monoclonal antibody (INFLIXIMAB-IFX). Upon IFX treatment, the proliferation rate was significantly reduced in 3 out of 4 human melanoma cell lines; the highly aggressive A375 cell line exhibited the lower sensitivity to this drug. The MAG score, based on proliferation, migration and invasion abilities, showed a marked reduction that was very striking for the very aggressive melanoma cell line A375. Recent studies based on mRNA and protein expression show that several MMPs, namely MMP-9, MMP-12 MMP-2, MMP-14, and MMP-19, play a role in melanoma aggressiveness and consequently may represent useful prognostic biomarkers [53‐55]. The role of TNF-dependent pathways in melanoma cells growth and malignant phenotype proposed in the current study confirms previous data carried out in similar cellular models [56] as well as the controversial role of TNF in cutaneous melanoma [21]. However, our study suggests, for the first time, a cooperation between MMP-2 enzymatic activity, measured by means of zymography approach, and TNF secretion to define melanoma cells aggressive phenotype, as summarized in Fig. 7. The controversial role of TNF, reported to both inhibit and promote cancer growth, has been explained by the ability of tumor cells to attract TNF-secreting cells through MHC class II molecules expression [21]. Our study investigates expression, secretion and function of molecular signals produced by melanoma cells. A multiomic approach combined with different cellular functions such as proliferation, migration and invasion, lead to develop a new quantitative score called MAGS. In fact, in the present study, for the first time melanoma aggressiveness was assayed by simultaneous multiomic and multifunctional points of view, including enzymatic activities quantification.

Unexpectedly, MMPs mRNA levels were found to be similar in both melanoma cell lines, while bioinformatics analyses indicated that MMPs-related pathways are significantly involved in the phenotypic features of A375 cells (very aggressive) and SK-MEL-28 cells (less aggressive cell type). Such apparently contradictory finding was explained by the functional analysis, which confirmed that, despite similar expression profiles, MMPs enzymatic activity was strongly and significantly different, likely due to the observed different TIMPs expression. Therefore, our findings indicate that the MMPs pathway considered from a functional- rather than just the expression-point of view, may explain, at least in part, the higher A375 cells aggressiveness.

In our in vitro studies, TIMP3 mainly accounts for the observed reduction of TIMPs expression in A375 compared to SK-MEL-28 cells (Fig. 4), strongly matching the in patients validation reported in Table 4, and according to previous studies [57].

Further, we found expression of several cyto- and chemokines to be strongly different in the two cell types, e.g. IP-10 more than 32 times down-modulated, RANTES more than 22 times up-regulated and Eotaxin more than 500 time up-regulated in A375 compared to SK-MEL-28 cells (see Table 2). This signature and the corresponding specific molecular-balance may represent the scenario underlying, at least in part, the melanoma aggressiveness. As an example, a significant increase of eotaxin was reported in humor aqueous samples from uveal melanoma patients [58], compared to non-melanoma samples, but its involvement has never been reported in aggressive cutaneous melanoma models. Thus, the combined analysis of transcriptomic, proteomic, secretomic and functional data may represent a powerful and novel way to further investigate cancer aggressiveness molecular signatures, as shown in Fig. 7, reporting a simplified model where other important players for melanoma microenvironment and immune response are not taken into account (e.g. lymphocytes and dendritic cells and other molecular signals highly significantly modulated in our study). A crucial concept emerging from the present study is the need to approach complex issues by different and simultaneous functional points of view. The controversial role of TNF [21], as well as TIMPs versatility [58], may be better understood in simplified cellular models carefully characterized in terms of aggressiveness by applying a functional-quantitative approach such as the MAGS reported in this study. It is noteworthy that one of the side effects of long-term therapies with anti-TNF drugs is an increase of cancer risk, including melanoma, or demyelination [27]. The role played by growth factors and cytokines in regulating melanoma cells behaviors was investigated in the past indicating the presence of a complex network with autocrine and paracrine effects [59]. Interestingly, when further investigated at immuno-histochemical level on fresh specimens from melanocytic nevi and primary cutaneous and metastatic melanomas, the expression of some inflammatory mediators and their receptors was found increased with tumor progression [19]. The present study confirmed these findings by more quantitative approaches, indicating that melanoma cell itself secretes large amount of TNF-α, IL-6 and other cytokines, triggering a cascade of effects like, for instance, the increase of MMP2 enzymatic activity, possibly related to the aggressive phenotype of the cell.

The reported findings indicate i) a novel functional scoring method potentially useful for prognostic purposes and to better characterize cancer cells from patients-derived organoids, ii) new mechanisms underlying melanoma cells aggressiveness and novel molecular targets.