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Abstract
Up to 20% of all cancers have been linked to chronic inflammation and persistent infections. However, almost all solid tumours contain immune infiltrates, and tumour-associated inflammatory cells play broad roles in different stages of tumour development and malignant progression. Cytokines are important mediators of the inflammatory effect on tumorigenesis both in inflammation-induced cancer and in the inflammation that follows tumour development. We have shown interleukin (IL)-6 to be an important tumour promoter in early colitis-associated cancer (CAC). IL-6 is mainly produced by tumour-infiltrating myeloid cells under the control of NF-κB. IL-6 promotes proliferation of tumour-initiating cells derived from the intestinal epithelium and protects them from apoptotic elimination. These pro-survival and proliferative effects of IL-6 are mainly mediated by STAT3, whose ablation in intestinal epithelial cells significantly reduces CAC tumorigenesis. More recently, we found a critical role for IL-23 and its downstream cytokines IL-17 and IL-22 in the development of CAC. These findings suggest that such cytokines or the cells that produce them may provide new therapeutic or preventive targets in forms of colorectal cancer that are linked to inflammation.
- Cytokines
- Treatment
- Infections
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Introduction
A link between inflammation and cancer has been suspected for long time,1 but experimental evidence demonstrating the important role of inflammation in tumorigenesis has become available only recently.2 It is estimated that 15–20% of cancers arise when underlying chronic inflammation is present. This inflammation can be caused by infections—for example, Helicobacter pylori in gastric cancer, and hepatitis B and C virus in hepatocellular carcinoma. Chronic inflammation can also be autoimmune as manifested in ulcerative colitis (UC) and other inflammatory bowel diseases that significantly increase the probability of colorectal cancer (CRC).3–6 While it is already known that chronic inflammation induces and promotes cancer and that the use of non-steroidal anti-inflammatory drugs, such as aspirin, decreases the lifelong risk of death from cancer,7 ,8 less is known about specific molecular and cellular mechanisms that tie inflammation and cancer together. Recent studies have revealed a critical role for inflammatory cytokines and the upstream pathways that stimulate their production in different gastrointestinal cancers.6 ,9–12
CRC is the third most common and deadly cancer around the globe.13 ,14 Patients with UC have a significantly higher risk of CRC and about 20% of them develop colitis-associated cancer (CAC) in their lifetime.15 ,16 CAC is a classic inflammation-driven cancer, which can be modelled in mice by giving three cycles of dextran sodium sulphate (DSS) in the drinking water subsequent to a single dose of the pro-carcinogen azoxymethane (AOM).17 ,18
We found that an overall reduction of the DSS-evoked intestinal inflammatory response by inactivation of IKKβ (a protein kinase responsible for NF-κB activation) in myeloid cells leads to decreased tumour size, with concomitant reduction in expression of many proinflammatory cytokines, some of which serve as tumour growth factors.19 Importantly, inactivation of IKKβ/NF-κB signalling in intestinal epithelial cells (IECs) also reduced tumour count, but unlike the myeloid-specific ablation the epithelial-specific ablation led to increased apoptosis in tumours and injured tissue.19 That study suggested that cytokines produced by inflammatory cells in an NF-κB-dependent manner can act on premalignant IECs to activate the NF-κB-dependent pro-survival gene expression programme. One of the cytokines that activates NF-κB in epithelial cells is tumour necrosis factor (TNF), whose role in CAC development has been demonstrated. Mice lacking TNFR1 showed reduced mucosal damage, reduced infiltration of macrophages and neutrophils and attenuated formation of colon tumours.20 However, adoptive transfer experiments showed that TNFR1 signalling is particularly important in the radiosensitive compartment,20 whereas the epithelial cells seem to rely on TNFR2 signalling.21 Overall, the TNF-IKKβ/NF-κB signalling activates myeloid cells to produce proinflammatory cytokines, which in turn serve as growth factors that increase malignant cell proliferation. NF-κB signalling in epithelial cells mainly prevents premalignant cells from undergoing apoptosis, therefore in its absence, we observe fewer and smaller tumours being formed upon AOM treatment combined with chronic colitis.19
IL-6 and STAT3
Inactivation of NF-κB in myeloid cells also led to reduced production of IL-6 during DSS-evoked colitis.22 IL-6 is a multifunctional cytokine that plays important roles in immune responses, cell survival and proliferation.23 IL-6 binds to soluble or membrane-bound IL-6Rα, and cell surface gp130, and activates intracellular signalling mediated by STAT3, Ras and PI3K-AKT.23 IL-6 is also important for T cell survival and differentiation, and therefore has a pivotal role in the pathogenesis of autoimmune disorders.24 One type of T cells that is dependent on IL-6 is Th17 cells.25 ,26 Blocking IL-6 signalling by neutralising IL-6R antibody or with a gp130-Fc fusion protein suppressed development of colitis.27 IL-6 also plays an important role in tissue homoeostasis and regeneration, suggesting that it may directly promote tumorigenesis and malignant cell survival.28 ,29 IL-6 mRNA is upregulated in different human cancers, including breast, lung, prostate, liver and colon cancers,30 and its expression often correlates with tumour mass and poor prognosis. In the presence of CAC, IL-6 may perpetuate chronic inflammation and maintain production of proinflammatory cytokines responsible for growth and survival of malignant cells. Blockade of IL-6 signalling with a chimeric gp130-Fc protein, which prevents IL-6 trans-signalling, reduced CAC tumour burden in mice.31
We sought to examine the effect of complete IL-6 deficiency on CAC development. CAC was induced in wild-type (WT) and Il6−/− mice by injection of a single dose of AOM followed by three cycles of DSS. As expected, colonic IL-6 was upregulated upon DSS treatment and ablation of IL-6 reduced tumour number, size and total tumour load in mice.32 These data indicate that IL-6 is important for both tumour development and growth in CAC. Differences in tumour multiplicity and size may be explained by altered cancer cell apoptosis and/or proliferation. Reduced tumour number in Il6−/− mice suggested that IL-6 may contribute to cancer cell survival and/or proliferation. To test this hypothesis, we examined apoptosis and cell proliferation in WT or Il6−/− mice subjected to acute colitis. DSS-exposed Il6−/− mice exhibited increased IEC apoptosis and downregulation of the anti-apoptotic protein Bcl-XL. We also observed a significant decrease in proliferation of crypt cells of Il6−/− mice subjected to DSS-induced intestinal injury. Therefore, IL-6 promotes both proliferation and survival of IECs during acute colitis. Presumably, IL-6 exerts the same effect on premalignant cells.
To determine the origin of IL-6 in CAC, we performed reciprocal adoptive transfer experiments. Reduction in tumour number, size and load was seen in mice deficient in IL-6 in haematopoietic cells, suggesting the importance of immune cells in overall IL-6 production during CAC tumorigenesis. To further delineate the source of IL-6 in CAC, we purified different myeloid and immune cells from CAC adenomas. Analysis of IL-6 mRNA by q-PCR showed that IL-6 is mainly produced by dendritic cells and macrophages, followed by T cells. In patients with UC and CAC, IL-6 is expressed in colonic epithelial cells and more potently by infiltrating immune cells. Expression of IL-6 correlates with downregulation of SOCS3 and activation of STAT3 in both epithelial/tumour cells and immune cells.33
Adenomas in Il6–/– mice showed a reduced number of PCNA cells and decreased levels of cyclin D expression. We also tested the effect of ectopic IL-6 on CAC tumorigenesis. WT mice subjected to the CAC induction protocol were injected with recombinant IL-6 or ‘hyper-IL-6’, which triggers IL-6 trans-signalling.34 ,35 When given at the late stage after the last DSS cycle, both treatments increased tumour size but not tumour multiplicity. When administered during the early stage of CAC induction, they did increase tumour number. Therefore IL-6 promotes both tumour formation and growth.32
Among downstream signalling pathways activated by IL-6, we observed a marked reduction in active STAT3 in IECs of Il6−/− mice subjected to DSS-induced colitis. To test the role of STAT3 in IECs, we crossed Stat3F/F mice36 with Villin-cre mice37 and generated Stat3ΔIEC mice where Stat3 is selectively ablated in IECs. The phenotype of Stat3ΔIEC mice is similar to that of Il6−/− mice, although Stat3ΔIEC mice developed much more severe colitis when exposed to DSS owing to enhanced IEC apoptosis. Consistent with this, expression of Bcl-XL was reduced in IEC lysates of Stat3ΔIEC mice compared with WT controls. Most importantly, fewer adenomas were found in Stat3ΔIEC mice subjected to CAC induction, an effect that was greater than that of the Il6 knockout. These data indicated that STAT3 is required for transduction of the IL-6 tumour-promoting signal and as a result of this, STAT3 promotes the survival, growth and progression of premalignant cells.
IL-23 and Th17 cytokines
Emerging evidence suggests that Th17 cells potentiate tumour-associated inflammation.38 ,39 IL-23 promotes Th17 cell proliferation and IL-17 production, and is important for inflammatory responses including experimental colitis.40–43 IL-23 is a heterodimeric cytokine composed of a unique p19 subunit and a p40 subunit, which is also present in IL-12.44 ,45 IL-23 is upregulated in multiple human cancers and ablation of the Il23p19 gene resulted in reduced tumorigenesis in a mouse model of inflammation-associated skin cancer.46
We tested the role of IL-23 and some of its downstream Th17 effector cytokines in CAC development. Expression of IL-23 was upregulated in intestinal tissues subjected to DSS-induced colitis. Compared with WT controls, Il23−/− (p19−/−) mice developed fewer and smaller colonic adenomas when subjected to CAC induction. Expression of IL-6, IL-17A and IL-22 in CAC was reduced in the absence of IL-23. Ablation of the Il17r or Il22 genes also resulted in reduced CAC tumorigenesis, suggesting an important role for Th17-derived cytokines in CAC promotion. Although the IL-23 receptor (IL-23R) is not expressed on IECs, IL-23 appears to promote CAC development mainly through activation of IEC-expressed STAT3 to enhance the proliferation and survival of transformed enterocytes. This indirect effect of IL-23 may be due to its effects on tumour infiltrating immune cells, particularly Th17 cells, and upregulation of other cytokines like IL-6, IL-17A and IL-22 that are capable of signalling more directly to malignant cells to promote their proliferation.
Therapeutic implications
Our studies indicate that IL-6 produced by myeloid cells serves as tumour promoter by activating STAT3 in IECs.32 Bollrath and coauthors confirmed the important role of IL-6 and IL-11 signalling to STAT3 also in CAC development.47 Taken together, IL-6, IL-11 and possibly other STAT3-activating cytokines promote CAC development. Another important promoter of CAC development is IL-23. Similar to IL-6, IL-23 is produced by lamina propria myeloid cells and it can lead to STAT3 activation in IECs. However, unlike IL-6, IL-23 does not act directly on IECs. Yet, IL-23 controls STAT3 activation in IECs and their premalignant derivatives by affecting the expression of other cytokines, including IL-6, IL-17A and IL-22.
In addition to activation of STAT3 in IECs, IL-6 regulates the recruitment of myeloid cells and neutrophils to the sites of inflammation48 and can also inactivate T regulatory cells by altering FoxP3 expression,49 and promote Th17 cell induction.25 ,26 In future studies, it will be interesting to determine to what extent Th17 cells, which are critical regulators of intestinal inflammation and emerging players in cancer,38 ,39 mediate the tumorigenic effects of IL-6. Indeed, in the CAC model, we observed a reduction in IL-17-producing T cells in CAC tumours of Il6−/− mice.32 Th17 cells may in turn promote tumour development by secreting inflammatory cytokines like IL-6, IL-17A and IL-22. The relative contribution of various cellular targets for IL-6 and the molecular pathways underlying its pro-tumorigenic action remain to be fully unravelled. In addition to its role in CAC, IL-6 also promotes spontaneous intestinal tumorigenesis, as deletion of the Il6 gene in ApcMin mice also results in reduced tumour load.50 The molecular and cellular mechanisms by which IL-6 promotes the development of sporadic CRC are not yet understood, but may be similar to those discovered for CAC.
There is also emerging evidence for the involvement of Th17 cells in CRC development.38 ,39 Colonisation of ApcMin mice with the pathogenic human colonic bacterium, Bacteroides fragilis (ETBF), resulted in development of colitis and increased tumorigenesis.51 ETBF induces robust Th17 response in colonic tissues and elevated STAT3 activation in enterocytes, thereby promoting tumour development.51 Neutralisation of IL-17 and IL-23R by specific antibodies blocked ETBF-induced colitis and inhibited tumorigenesis in ApcMin mice.51 ,52 These results agree with our finding that ablation of IL-23 or its downstream targets IL-17R and IL-22 results in reduced CAC development. While the mechanism by which IL-17 promotes tumorigenesis remains elusive, raised levels of IL-23 and Th-17 cytokines were found in multiple types of human cancers including CRC.46 ,51 ,52 Importantly, the presence of a ‘Th17 signature’ in stage I and stage II human CRC signals poor prognosis and reduced disease-free survival.53 These findings suggest that patients with early-stage CRC should go through ‘Th17 signature’ evaluation and for those who express high Th17 markers, an adjuvant therapy against IL-23 or IL-17 should be employed in addition to the standard treatment.
Taken together, we and others have shown that inflammatory cytokines play pivotal roles in tumour development and malignant progression of CRC, making them prominent targets for therapeutic intervention (figure 1). Reagents that inhibit TNF, IL-6, IL-23 and IL-17 are already available and most have been found to be safe and effective for treatment of autoimmune disorders, including rheumatoid arthritis and inflammatory bowel disease.6 ,54 These drugs include small-molecule inhibitors and antibodies against TNF (infliximab, adalimumab, certolizumab),55 IL-6R (tocilizumab),56 IL-17 (LY2439821),57 and IL-12/23 (ustekinumab, STA 5326).58 ,59 Testing the efficacy of such anti-cytokine drugs, alone or in conjunction with conventional therapeutic options, is an important challenge that may prove beneficial for patients with different forms of CRC, especially if these patients exhibit elevated expression of IL-23, IL-17 and IL-6 in their tumours. It will be particularly interesting to examine the effects of such anti-cytokine drugs used in the treatment of inflammatory and autoimmune diseases on the incidence of CRC and its progression. Based on our mouse studies,60 we expect that therapeutic antibodies targeting IL-23, IL-17 or IL-6 will reduce the incidence of CRC in patients with UC or Crohn's disease.
Tumour-promoting inflammation in colorectal cancer. Tumour-infiltrating myeloid cells and lymphocytes produce inflammatory cytokines, including tumour necrosis factor (TNF), interleukin (IL)-6, IL-23, IL-17 and IL-22. TNF activates NF-κB in myeloid cells and stimulates tumour-associated inflammation. IL-23 signals to T lymphocytes, innate lymphoid cells, NK cells and possibly other cells to stimulate the production of IL-17 and IL-22. TNF, IL-6, IL-17 and IL-22 activate STAT3 and NF-κB signalling in transformed colonic epithelial cells and promote their survival and proliferation.
References
Footnotes
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Competing interests None.
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Provenance and peer review Commissioned; externally peer reviewed.