Introduction
The complement system may play a dual role in the pathogenesis of cancer. On the one hand, it may contribute to the clearance of potentially tumourigenic cells harbouring pathogen- or danger-associated molecular patterns (PAMPs or DAMPs). On the other hand, complement activity may favour tumour development. For example, it has been suggested (based on an animal model) that myeloid-derived suppressor cells (MDSC), recruited in a C5a-dependent way, stimulate production of reactive oxygen species and reactive nitrogen species (ROS and RNS) and then contribute to the inhibition of a CD8+ T cell-mediated anti-tumour response [
1].
Mannose-Binding Lectin (MBL) may interact directly with neoplastic cells (e.g. by MBL-dependent cell-mediated cytotoxicity [
2]), or inhibit metalloproteases that degrade extracellular matrix, or protect against chemotherapy-related infections or infections with cancerogenic agents (reviewed by Swierzko et al. [
3]). Moreover, MBL may interact with antigen-presenting cells (often present in the tumour microenvironment), influence their activity/proliferation and thereby contribute to the outcome of the anti-tumour immune response [
4,
5].
The gene responsible for MBL synthesis (
MBL2) is localized to chromosome 10 (10q11.2–q21). Single nucleotide polymorphisms (SNPs) in its first exon are responsible for altered MBL serum concentration and impaired function. The dominant alleles
D,
B and
C (collectively designated
O), corresponding to mutations in codons 52, 54 and 57, respectively, are associated with lower MBL levels compared with the
A (wild-type or normal) allele. Polymorphisms in the promoter region (
H/
L and
Y/
X at positions −550 and −221, respectively) also influence the serum protein concentration. Homozygotes or compound heterozygotes for variant alleles (
O/
O) as well as
LXA/
O heterozygotes are considered to be MBL deficient (reviewed in [
3]). Although MBL is predominantly synthesized in the liver, some
MBL2 gene expression (at the mRNA level) has been found in bone marrow, fetal lung, small intestine and testis [
6]. We ourselves have reported the presence of MBL protein and specific mRNA in both normal and malignant ovaries. Moreover, MBL protein was detected in ascites from women with ovarian cancer [
7]. The transcription of the
MBL2 gene is known to be up-regulated by IL-6, dexamethasone, heat shock, thyroid hormones and growth hormone while down-regulated by IL-1 [
8,
9].
The ability of MBL to activate the complement cascade results from its cooperation with MBL-associated serine proteases (MASPs), encoded by the
MASP1/
3 and
MASP2 genes, localized to chromosomes 3 (3q27–q28) and 1 (1p36.2–3), respectively [
10]. MASP-2 cleaves complement factors C4 and C2 with high efficiency. Among Caucasians, one SNP (+
359 A>
G;
D120G) leads to diminished MASP-2 activity in heterozygotes and total MASP-2 deficiency in homozygotes. This mutation affects the structure of the CUB1 domain and abolishes interaction with the pattern recognition molecules of the lectin pathway of complement activation [
11].
MASP-1 was demonstrated to associate with MASP-2 in the same complex with MBL and to activate MASP-2 directly. Moreover, it is able to activate factor C2, producing the majority (60 %) of C2a molecules, necessary for the C3 convertase. Thus, it is considered to be crucial for lectin pathway activation [
12‐
14]. MASP-1 could possibly also be involved in the coagulation cascade with its substrates being fibrinogen, factor XIII and thrombin-activatable fibrinolysis inhibitor (TAFI). Moreover, its thrombin-like activity enables cleaving of protease-activated receptor-4, a mediator of inflammation and platelet activation (reviewed by Yongqing et al. [
15] and Matsushita et al. [
16]). Recently, Dobo et al. [
17] found evidence that another MASP-1 substrate is high-molecular-weight kininogen. This activity (like that of kallikrein) enables release of bradykinin, a highly pro-inflammatory mediator of the contact system. Although these authors noted that MASP-2 also cleaved kininogen, no bradykinin was released. To date, no case of MASP-1 deficiency has been described, but curiously, fish and birds do well without possessing MASP-1 [
18,
19].
The liver is the main source of both MASP-1 and MASP-2, but low expression at the mRNA level has also been found in colon, heart, lung, kidney, placenta, brain (for MASP-1), and in testis and small intestine (for both MASP-1 and MASP-2) [
20]. In contrast to the
MBL2 gene, transcription of
MASP1/
3 and
MASP2 was shown to be stimulated by IL-1β and abolished by IL-6. Moreover,
MASP1/
3 expression is down-regulated by IFN-γ [
21].
We previously reported an association between low MBL-conferring haplotypes and ovarian cancer, but surprisingly no corresponding relationship with serum MBL concentration or activity [
7]. Also,
MBL2 gene expression was detected in all ovarian tissues examined, but significant
MASP2 expression was confined to malignant ovarian tissue only. We later found elevated serum Ficolin-2 (L-ficolin) and Ficolin-3 (H-ficolin) in ovarian cancer patients, while the local expression of the corresponding genes was decreased [
22]. In the present case-controlled, retrospective study, we have revisited the relationship between MBL and ovarian cancer with an entirely separate series of patients and controls in order to confirm or refute the apparent paradox (association with genetic MBL deficiency, but not serum MBL deficiency) reported earlier, and we have supplemented our investigations to include MASP-2 concentrations,
MASP2 genotyping and MBL–MASP-1 complex activities. Additionally, gene expression data have been markedly extended.
Discussion
Various disorders (endometriosis, pelvic inflammatory disease, etc.) are postulated to increase the risk for epithelial ovarian cancer (reviewed by Maccio and Madeddu [
32]). MBL, by participating in the clearance of microorganisms, might help to limit inflammation in the reproductive system. As mentioned, it may also interact directly with certain cancer cells or inhibit extracellular matrix-degrading enzymes. The results reported here broadly confirm our previous finding (from another cohort) [
7] that MBL deficiency-associated genotypes are over-represented in ovarian cancer. In contrast to the earlier study, controls (previously defined as “healthy women with no history of cancer”) were recruited on the basis of histopathological examination enabling us to distinguish between patients with benign ovarian tumours and those without any ovarian pathology. The
MBL2 relationship was stronger in comparison with the latter group. Recently, Nevadunsky et al. [
33] have also postulated that the
MBL2 gene
B variant may be a risk factor for ovarian cancer. Several reports demonstrated the significance of
MBL2 gene SNPs in gastric [
34,
35], hepatic [
36] or colon cancers [
37], glioma [
38] and acute lymphoblastic leukaemia [
39]. In contrast, Ytting et al. [
40] found no such association with colorectal cancer.
We also confirmed the absence of association of serum MBL activities (both binding to mannan and complement-activating ability) and ovarian cancer. Indeed, OC patients with normal (wild-type) genotypes had higher levels of serum MBL than their controls. This is likely to be a nonspecific response to an inflammatory stimulus and accounts for the correlation with C-reactive protein. Earlier, elevated MBL concentrations in patients with papillary thyroid carcinoma (compared with subjects with thyroid adenoma and healthy controls) were reported. However,
MBL2 polymorphisms were not analysed in that study [
41].
The reason for an association with genetically defined MBL deficiency but not serum protein deficiency is not obvious. One possibility is the existence of a cancer susceptibility gene in linkage disequilibrium with variant
MBL2 alleles, which can account for a number of inconsistent findings in the context of MBL and various malignant and non-malignant diseases (discussed by Kilpatrick [
26]). Alternatively, this putative linked gene may act as a disease modifier, accounting for the prediction of longer survival by MBL deficiency-associated (
LXA/
O,
O/
O) genotypes. Conversely, the association between very high serum MBL and poor prognostic indicators (grade 3 tumours and FIGO stage III-IV) is consistent with this view as all the very high serum MBL patients had wild-type genotypes.
Neither MASP-2 serum concentration nor the +
359 A>
G polymorphism of its gene was associated with ovarian cancer. The former correlated with Ficolin-3 levels (reported previously by Szala et al. [
22]), within both OC and C groups (not shown). Earlier, Ytting et al. [
42,
43] reported high MASP-2 serum level to be a biomarker predicting recurrence and poor survival of patients with colorectal cancer. That was not associated with
MASP2 polymorphism [
40]. Moreover, elevated MASP-2 levels in children with tumours of the central nervous system [
44] and in patients with papillary thyroid carcinoma [
41] have been reported.
An important novel aspect of this study was investigation of local
MBL2 and
MASP2 expression, at the mRNA level. The
MASP2 gene relative expression was significantly higher in malignant ovaries compared with normal organs or those affected by benign tumours. For the
MBL2 gene, the differences were less evident, but a clear difference between OC and BT patients was observed. Interestingly, earlier, we found opposite relationships for Ficolin-2 and Ficolin-3 genes [
22]. Consequently, relative expression levels of both
MBL2 and
MASP2 inversely correlated with that of
FCN2 [
22] in both OC and C groups (not shown). Recently, expression of the
MBL2 gene in papillary thyroid carcinoma tissue specimens was shown to be higher than in those from adenoma or normal thyroid glands [
45].
Since many statistical tests have been performed to compare frequencies of MBL2 and MASP2 genotypes/alleles (MBL2 O/O, XA/O + O/O genotypes, O alleles; MASP2 A/G heterozygosity and G alleles), protein concentrations/activities (MBL serum levels, MBL–MASP-1 and MBL–MASP-2 activities together with frequencies of extreme values) as well as relative gene expression levels (MBL2 and MASP2), there is a real possibility that some apparently “significant” findings may be due to chance (type 1 error). However, that consideration does not apply to the main conclusions and the statistics underpinning them, because the central findings are confirmations of apparent relationships previously obtained. However, the other relationships indicated here for the first time cannot be considered established, but serve to formulate hypotheses, which may be subsequently confirmed or refuted (ideally by independent researchers).
To summarize, our previous [
7,
22] and current data demonstrate certain abnormalities in expression (mRNA, protein levels) of factors specific for the lectin pathway of complement in ovarian cancer. This possibly indicates involvement in pathogenesis, although it is possible that at least some of these disturbances are effects of carcinogenesis. Genetic polymorphisms might influence both risk of disease (
MBL2) and prognosis (probably
MBL2 and
MASP2).