1. Introduction
Uveal melanoma (UM) is a malignancy that originates from melanocytes in the eye, and 50% of the patients will ultimately develop metastases [
1,
2]. Immune cell infiltration, secretion of inflammatory cytokines and an increase in HLA expression are frequently observed in UM, leading to an inflamed tumor environment, inside the immune-privileged eye [
3]. This inflammatory phenotype is linked to an increased risk for metastasis [
4,
5,
6,
7], and has been associated with loss of chromosome 3 [
8,
9]. It was recently noticed that an extra copy of chromosome 8q in the tumor is associated with an influx of macrophages, while loss of chromosome 3 (Monosomy 3, M3) correlates with an increased influx of T cells [
10]. M3 occurs in almost 50% of patients and is associated with a bad prognosis [
11,
12,
13]. Almost all cases with M3 also show gain of chromosome 8q, but this may even occur in tumors with two chromosomes 3 (Disomy 3, D3); it is considered a bad prognostic sign [
14,
15,
16]. Another alteration is a gain of 6p, which occurs in 25–40% of cases, and is a sign of good prognosis [
17,
18].
Besides chromosomal changes, important genetic driver mutations have been identified: these include activating mutations in
GNAQ and
GNA11 [
19,
20], which are thought to lead to the transformation of melanocytic cells by upregulation of YAP1 [
21]. These mutations are already seen in choroidal nevi, where YAP upregulation is also present [
22]. Loss of expression of a key tumor suppressor gene,
BAP1, encoding a deubiquitinating protein and located on chromosome 3, together with the loss of the other chromosome 3, is strongly correlated with the development of metastases [
23]. Major signalling pathways such as the Rb, PI3K/Akt, MAPK and NFkB pathways are frequently disregulated in prognostically bad UM [
24,
25,
26]. This disregulation will lead to inhibition of apoptosis and stimulation of cell proliferation, while the cells will become more invasive and tend to migrate to other organs.
Inflammation is considered one of the hallmarks of cancer [
27], but its regulation is not always clear. Although in UM a relation is seen between M3/loss of BAP1 expression and the presence of an inflammatory phenotype, little is known about the pathways which regulate this inflammation. One of the major regulators of inflammation in cancer is the NFkB-signalling pathway [
28]. Members of the NFkB transcription family include RELA (p65), RELB, c-REL, NFkB1 (p105/p50), and NFkB2 (p100/p52), which are regulated by proteins such as IKK and NEMO (IkBkG). The canonical pathway, in which NFkB1 is the main player, is involved in many different aspects of the immune system while the non-canonical pathway, in which NFkB2 is involved, regulates specific functions of the adaptive immune system.
During tumorigenesis, the NFkB pathway is often upregulated and leads to uncontrolled proliferation, apoptosis resistance, angiogenesis, inhibition of senescence, invasion, metastasis and resistance to different treatments [
28]. P53 and PTEN proteins can function as negative regulators of NFkB signalling and mutations in these genes can affect the pathway’s activity. Other oncogenic mutations, such as amplifications and point mutations in
RELA and other
NFkB signalling genes, have been identified in several lymphoid malignancies, and give rise to inflammation [
29]. Stromal M2 macrophages produce various cytokines and play a major role in NFkB upregulation. In cutaneous melanoma, the NFkB pathway is known as a molecular switch which is turned on during tumor progression [
30,
31].
Both the canonical and non-canonical NFkB pathway have been shown to be active in primary and metastatic UM and are related to progression of the disease [
32,
33,
34], while inhibition of NFkB has been shown to reduce cell proliferation. As far as we know, the relation between NFkB signalling, HLA Class I expression and genetic tumor progression in UM has not been reported. As we observed in our previous studies that genetic aberrations in UM are related to progression of the inflammatory environment, we set out to investigate whether NFkB plays a role in the expression of HLA Class I and whether the expression of NFkB itself is related to the tumor’s chromosome 3/BAP1 status.
3. Discussion
We hypothesized that the inflammatory phenotype with an increased HLA Class I expression in UM is linked to the activation of the NFkB pathway. Therefore, we explored the expression of some of the most important components of the NFkB signalling pathway and investigated their association with HLA-A and HLA-B expression and with different types of infiltrating leukocytes. In accordance with previous studies, we noticed an association between upregulation of most of the factors involved in the NFkB pathway with inflammatory factors [
33,
34]. We found that NFkB1, NFkB2 and RELB expression were positively correlated with the expression levels of HLA-A and HLA-B, their regulators and their antigen-loading machinery, suggesting that these NFkB pathway molecules may be positive regulators of HLA-A and HLA-B transcription. It has been suggested that NFkB is responsible for the secretion of several proinflammatory cytokines and chemokines and thereby guarantees the attraction, activation, survival and differentiation of immune cells [
39] which might lead to the increase of HLA Class I expression in the tumor cells. Singh et al. [
34], in a recent study, similarly observed that infiltration increases during progression of UM and that the canonical NFkB pathway is more active in high-risk inflamed UM. They did not report on the chromosome or BAP1 status of the tumors they studied.
We observed that M3 tumors showed higher expression levels of NFkB1, NFkB2, and RELB. Similarly, BAP1-negative tumors showed higher levels of NFkB1 and NFkB2. We previously demonstrated that M3/loss of BAP1 is related to increased leukocytic infiltration [
8,
9], and now show an association between BAP1 loss and upregulation of the NFkB pathway. As the link between the expression of the NFkB molecules and T cell infiltrate may also be caused by the expression of these molecules in the immune infiltrate itself, we repeated the analyses on the same set but now without one-third of the tumors, which based on the expression of CD8, were highly infiltrated with T cells. We again looked for associations between HLA and NFkB, and the genetic constitution. In this group, we similarly observed that the tumors with M3/loss of BAP1 had an increased NFkB and HLA Class I expression, indicating that the infiltrating leukocytes may enhance expression, but that the genetic basis determines expression in the first place.
We also explored the presence of negative regulators of inflammation in UM: SPP1 and PPAR-γ. The SPP1/osteopontin protein is involved in different aspects of tumor biology such as cell survival and proliferation, invasion and metastasis, and is involved in neural crest development. Kadkol et al. [
40] reported that the level of SPP1 expression was increased in the serum of patients with UM metastasis, and that tumors with looping high-risk vasculogenic mimicry patterns had increased expression of osteopontin. Another study [
41] also looked at protein expression using a monoclonal antibody and did not observe a relation between expression and survival. In contrast to Kadkol and Simoes, we looked at mRNA and observed that decreased levels of SPP1 mRNA were associated with an unfavorable prognosis in UM. Onken et al. have similarly reported a decreased expression of SPP1 mRNA in high-risk UM [
42]. It is of interest to further investigate the mechanism of SPP1 downregulation during UM progression to determine its function.
The upregulation of the NFkB pathway upon loss of one copy of chromosome 3 could possibly be explained by the fact that the
PPAR-γ gene is located on chromosome 3. The PPAR-γ protein is a negative regulator of the NFkB pathway, so it has been hypothesized that loss of chromosome 3 leads to insufficient inactivation of NFkB by PPAR-γ [
43]. In our study, SPP1 and PPAR-γ expression were negatively correlated to T lymphocyte and macrophage markers. As expected, we found a positive correlation between the expression of these two factors with BAP1 expression. We previously observed [
35] that a high expression of HLA-A, HLA-B, B2M, TAP1, IRF1 and IRF8 was related to an increase in the presence of T cells. van Essen and colleagues compared the expression levels of these genes in primary tumors with their corresponding xenograft in immunodeficient mice, in which, of course, no human leukocytes are present. They showed that the expression of HLA-A, HLA-B, B2M, TAP1, TAP2, IRF1, IRF8, CIITA and NLRC5 were all down-regulated in xenografts compared to the primary tumors, suggesting that the increased expression depended on the presence of infiltrate. Cytokines potentially involved in the upregulation of these genes were interferon-gamma and CCL2. In the current study, we observed a correlation between the expression of the HLA and HLA-associated genes, the presence of infiltrating immune cells, and the NFkB pathway, but we also see this correlation in tumors without inflammation. From this, we conclude that the genetic basis of the tumor, i.e., Monosomy 3 with BAP1 loss, determines the primary upregulation of the NFkB pathway, which leads to an increase in HLA Class I expression in the tumor cells and production of cytokines and chemokines, which then attracts immune cells, further upregulating expression of HLA Class I. These data indicate that absence of the deubiquitinating function of BAP1 allows the NFkB pathway to be active, potentially by affecting the regulators of NFkB directly (
Figure 4).
These findings imply that BAP1 (UCHL2) might have a similar function in UM as observed for UCHL1 in Human papilloma virus (HPV)-infected keratinocytes. UCHL1 and UCHL2 are both ubiquitin hydrolase enzymes which belong to the ubiquitin hydrolase family [
44]. HPV-infected keratinocytes produce increased levels of the ubiquitin protease UCHL1, which reduces tumor necrosis factor receptor-associated factor 3 (TRAF3) K63 poly-ubiquitination, resulting in the suppression of the production of interferon and other pro-inflammatory cytokines [
45]. Higher UCHL1 expression also resulted in increased degradation of an essential NFkB modulator, IkBkG. In this manner, canonical NFkB signalling was reduced, thereby suppressing a potential anti-viral immune response. HPV-infected keratinocytes with a high UCHL1 expression showed greatly decreased HLA-A and -B levels [
46]. The virus uses UCHL1 to escape from the immune system, while in UM, the presence of an inflammatory infiltrate helps to produce blood vessels [
9].
Taken together, our data suggest that the main regulator of the NFkB pathway in uveal melanoma is loss of chromosome 3 and BAP1: loss of chromosome 3/loss of BAP1 expression correlates with upregulation of the NFkB pathway and affects the activity of the NFkB pathway in UM tumors, which leads to upregulation of HLA Class I expression and attraction of infiltrating cells to the tumor environment which is a well known factor in the development of metastasis in this disease.