Background
Accumulation of biological evidence has led to the establishment of inflammation as a hallmark of cancer [
1]. It has been postulated that a state of low-grade inflammation can increase mutation rates and augment the proliferation of mutated cells by supplying trophic signals [
2]. In addition to potential direct cell growth promotion effects, activated inflammatory cells can stimulate reactive oxygen species and the accumulation of reactive nitrogen intermediates in neighbouring cells [
1]. These processes may damage DNA and its protein products, directly or indirectly, thus having tumour promoting effects [
3,
4].
Observational evidence has shown that diseases characterised by a chronic inflammatory state are associated with an increased risk of several cancers, including lung, prostate and colorectal cancer, while use of nonsteroidal anti-inflammatory drugs such as aspirin, may have a chemopreventive role in several cancers, including colorectal, lung, breast, prostate, endometrial and ovarian [
5‐
8]. Specific circulating inflammatory markers have been linked to cancer development in prospective cohort studies. For example, higher concentrations of circulating C-reactive protein (CRP), a highly sensitive but non-specific marker of elevated inflammatory response, were associated with a higher risk of several cancers, including breast, lung, prostate, ovarian and colorectal cancer [
9]. Higher pre-diagnostic concentrations of interleukin 1 alpha (IL-1a), IL-8 and tumour necrosis factor alpha (TNF-A) have been associated with higher risk of ovarian cancer, whereas concentrations of serum amyloid A, soluble tumour necrosis factor receptor-2 (sTNF-RII) and monokine induced by gamma interferon (MIG) have been positively associated with lung cancer risk [
10,
11]. If such associations are causal, preventing or intervening on inflammation pathways could be a strategy to reduce cancer risk.
While the reduction of chemokine levels for inhibiting cancer progression has been much discussed in the context of cancer therapy, observational studies linking specific circulating inflammatory cytokine concentrations to cancer risk are sparse, have relatively small sample sizes, and the results from which may be impacted by unmeasured confounding, reverse causation and other biases [
12,
13]. An approach to overcome the potential limitations of observational epidemiology and strengthen the evidence for a potential causal role of chronic inflammation on cancer risk is Mendelian randomisation (MR). In MR, germline genetic variants are used as instrumental variables to proxy lifetime exposure for an exposure of interest, in this case circulating cytokines, chemokines, growth factors and interferons (hereafter cytokines). In the present study, MR was used to capture usual cytokine concentration experience over the life course, rather than expression variations such as those resulting from epigenetic alterations. We used genetic variants robustly associated with circulating cytokines to estimate the association of genetically proxied inflammatory cytokine concentrations on risk of breast, endometrial, lung, ovarian and prostate cancer. We used outcome-data from large well-established consortia that were either publicly available or for which we had access to base on active research proposals.
Discussion
We used MR analyses to investigate potential causal links between genetically proxied circulating concentrations of several inflammatory-related cytokines and risk of breast, endometrial, lung, ovarian and prostate cancer. We found an inverse association between genetically proxied concentrations of MIF and breast cancer, a positive association of MIG and an inverse association of IL-1ra with endometrial cancer, a positive association of CTACK and an inverse association of IL-18 with lung cancer and an inverse association of RANTES with epithelial ovarian cancer. These findings were similar among sensitivity analyses and were supported in colocalization analyses. We also found a positive association of GROa with breast cancer, an inverse association of sICAM with endometrial cancer, positive associations of MCSF and MIP1a with epithelial ovarian cancer and a positive association of MIP1a and an inverse association of VEGF with prostate cancer. These findings were similar in sensitivity analyses but were not supported by colocalization analyses.
Previous MR analyses have investigated associations of cytokines with cancer, but in general used trans SNPs in the construction of instrumental variables. One MR study that investigated the effect of 27 cytokines and growth factors on the risk of prostate cancer, drawing IV estimates from a previously published GWAS on 8293 Finnish individuals and the same population that we included in our analysis for prostate cancer, found that higher genetically proxied circulating concentrations of C-C motif chemokine ligand 2 (MCP-1/
CCL2) were associated with a higher risk of prostate cancer [
42]. Another MR study using the same source of instrumental variables investigated 24 cytokines in relation to breast cancer risk and demonstrated positive associations for MCP-1, MIP1b and IL-13 [
43]. Such associations were not replicated in our analysis, most likely due to a different instrument definition. Contrary to the previous MR studies, we used a cis definition to instrument the inflammatory biomarkers, selecting variants in close proximity to the encoding gene region, thus reducing the likelihood of horizontal pleiotropy [
44]. Furthermore, previous MR studies used publicly available GWAS estimates for cytokines that were adjusted for BMI and these estimates may suffer from collider bias (a variable that is a common effect of two other variables) [
45,
46], when cytokine concentrations affect directly BMI levels that has been observed in the literature for some cytokines [
15].
We found an inverse association between genetically proxied MIF concentrations and breast cancer risk. MIF is a pro-inflammatory cytokine, aberrantly expressed in many solid tumours, including breast, and it has been shown to promote tumour progression and metastasis [
47]. Additionally, due to its functional properties, it has been characterised as a promising target for anti-cancer treatment development [
48]. Studies in various breast cancer cell lines and human breast cancer tissue have indicated a potential role in breast cancer invasion and immunomodulation, though its functional role is not fully understood [
49]. Overexpression of MIF has also correlated with worse survival in triple-negative breast cancer compared to other hormonal status [
50]. Despite findings from several experimental studies suggesting a positive association, our study showed an inverse association for genetically proxied circulating MIF concentrations in relation to breast cancer and a similar observation was made in another study using the MR approach [
43]. A reason for this discrepancy could be a potential pleiotropic effect that MIF might have depending on its cellular localization and tumour stage and type [
47]. It has been speculated that intracellular MIF in the breast cells has a protective function, whereas extracellular MIF, whether it is tumour-associated macrophage (TAM)-derived or produced by carcinoma cells upon stroma/tumour interactions, is pathogenic [
47].
In our study, we also found an inverse association between genetically proxied circulating IL-1ra concentrations and endometrial cancer risk. Few prospective studies have evaluated IL-1ra in relation to endometrial cancer [
51‐
53]. In a nested case-control study in the European Prospective Investigation into Cancer (EPIC), elevated concentrations of IL-1ra were associated with higher endometrial cancer risk [
52]. It should be noted though that a large proportion of the measurements of IL-1ra (52%) was below the assay limit of detection. Another EPIC study, in the context of a factor analysis, provided evidence for a positive association between IL-1ra and endometrial cancer only in post-menopausal women, while adjustment for BMI markedly attenuated risk estimates [
51]. A case-control study nested within the PLCO cohort reported null associations between IL-1ra and endometrial cancer risk [
53]. Even though observational studies have shown that IL-1ra might act as a pro-inflammatory agent, mechanistic plausibility for a protective role of IL-1ra in cancer was demonstrated in experimental studies [
54‐
56]. Little epidemiological and experimental evidence is currently available to support the observed positive association for MIG in relation to endometrial cancer, although associations with cancer outcomes other than endometrial have been reported [
57].
A positive association was found between genetically proxied CTACK and lung cancer in never smokers and a nominally significant association in small-cell lung cancer. Findings from experimental studies have shown that CTACK is highly expressed in tumour cells with metastatic potential [
58]. Additionally, a recent biomarker analysis using an antibody array demonstrated that 17 cytokines, among them CTACK, were differentially expressed in serum samples of non-small-cell lung cancer patients compared to healthy controls [
59]. On the other hand, a nested case-control study within the PLCO, including 526 cases and 592 controls, was inconclusive on the association between pre-diagnostic plasma CTACK concentrations and lung cancer (Q4 versus Q1: OR = 0.93, 0.64 to 1.35) [
57]. Our analysis also showed an inverse association between genetically proxied IL-18 concentrations and overall lung cancer and lung adenocarcinoma. Our observations are in line with findings from experimental research that has demonstrated an antitumour activity of IL-18 on lung cancer [
60,
61]. It has been shown that IL-18 exhibits a variety of biological activities with implications in tumour initiation and development. IL-18 can activate T-helper cells, which produce cytokines that interact with activated natural killer cells and mediate the antitumour activity of IL-18. Furthermore, IL-18 has anti-angiogenic and pro-lymphangiogenesis properties, which contribute to its antitumour activity.
We also found inverse associations for RANTES in relation to invasive epithelial ovarian cancer and serous ovarian cancer. Such an inverse association is not supported by the limited to date evidence that largely comes from experimental studies and suggest that RANTES is positively associated with cancer stem-like cells differentiation and tumour angiogenesis, tumour immune tolerance and invasion, and chemoresistance [
62‐
64]. On the other hand, a study that used publicly available microarray data-sets, deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) demonstrated that the association between RANTES and overall survival (OS) among ovarian cancer patients was dependent on the TP53 mutation status and higher expression of RANTES was associated with better OS only on TP53 mutant serous ovarian cancer [
65].
Among the strengths of our analyses are the wide range of inflammatory cytokines that we covered, and the large sample size that was used in most of the analyses that we performed. Another strength is the approach that we used for instrument selection by using variants in close proximity to the encoding gene region, minimising the likelihood of horizontal pleiotropy [
44]. Since cis-acting regulatory variants in the vicinity of genes influence mRNA and protein expression, and the majority of drug targets are proteins, an MR analysis using cis defined instruments is likely to have translational relevance [
66]. In support of this notion, a number of studies during the last decade have demonstrated that variants in genes encoding a drug target mimic the effect of modifying the same target by use of pharmacological agents [
67]. In addition, lifestyle changes such as changes in dietary habits, weight reduction and smoking cessation have been associated with changes in plasma concentrations of inflammatory biomarkers [
68‐
70]. Considering that such changes can have a significant impact on the incidence of cancer when applied at the population scale, with minimal adverse effects, future studies that will aid in delineating such mediating effects are warranted. Our study’s primary limitation is the use of a single instrument or few instruments in some of the analyses, which may have affected power to reject the null hypothesis for some associations. The null findings for some associations are not necessarily indicative of the cytokines having no effect, since there were several cytokines with weak instruments. In addition, there may be non-linear effects, time-dependent effects or inflammation-environment interactions that are not captured by the current analysis. Moreover, potential synergistic effects between the studied cytokines and network cytokine approaches were not considered. Furthermore, since the number of independent SNPs required for the MR sensitivity analyses (i.e. weighted median, ConMix, MR Egger and PRESSO) to work properly is quite large, which is not the case in most of the analyses that we performed due to the cis-instrument definition approach, results of these analyses should be interpreted with caution. Another important point is that, although using the cis-eQTL definition (by retaining variants that are additionally associated with tissue specific expression), the cytokine expression component throughout the body (including in target organs) is partially captured, as measured cytokine concentrations in circulation may not relate to tissue expression. In addition, different parameters of gene expression, namely tissue specific and exposure specific expression are not accounted for in MR analyses. Even though we used a wide panel of inflammatory cytokines, genetic instruments were not available for several additional cytokines that may be implicated in cancer, such as IL-13, IFN-gamma and CXCL13. Future larger single- and multi-trait GWASs of cytokine concentrations, and MR studies with individual-level data could address some of the latter issues.
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