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Inflammatory cytokines in cancer: tumour necrosis factor and interleukin 6 take the stage
  1. Sergei I Grivennikov,
  2. Michael Karin
  1. Laboratory of Gene Regulation and Signal Transduction, Departments of Pharmacology and Pathology, School of Medicine, University of California, San Diego, California, USA
  1. Correspondence to Dr Michael Karin, Laboratory of Gene Regulation and Signal Transduction, Departments of Pharmacology and Pathology, School of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA; karinoffice{at}ucsd.edu

Abstract

Up to 20% of all cancers arise in association with chronic inflammation and most, if not all, solid tumours contain inflammatory infiltrates. Immune cells have a broad impact on tumour initiation, growth and progression and many of these effects are mediated by proinflammatory cytokines. Among these cytokines, the pro-tumourogenic function of tumour necrosis factor (TNF) and interleukin 6 (IL-6) is well established. The role of TNF and IL-6 as master regulators of tumour-associated inflammation and tumourigenesis makes them attractive targets for adjuvant treatment in cancer

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Introduction

Cytokines were initially discovered as secreted proteins that control various immune functions. It is now clear that cytokine functions extend to many other aspects of biology, including cancer.1 A long-suspected causative connection between inflammation and cancer has been mechanistically established.2,,4 At least 20% of all cancers arise in association with infection and chronic inflammation and even those cancers that do not develop as a consequence of chronic inflammation, exhibit extensive inflammatory infiltrates with high levels of cytokine expression in the tumour microenvironment. Several such cytokines were found to serve as growth and survival factors that act on premalignant cells,5 stimulate angiogenesis, tumour progression and metastasis, and also maintain tumour-promoting inflammation.4 6,,8

Tumour necrosis factor (TNF) and interleukin 6 (IL-6) are perhaps the best characterised pro-tumourigenic cytokines and they were initially suspected to be involved in cancer owing to their ability to activate the oncogenic transcription factors NF-κB, AP-1 (TNF) and STAT3 (IL-6) in epithelial cells.9,,12 Later, many other cytokines and other stimuli were shown to be involved in pro-tumourigenic activation of NF-κB and STAT3 in cancer cells, but TNF and IL-6 remain the model pro-tumourigenic cytokines in quite a few cancers. TNF and IL-6 and other proinflammatory cytokines can influence all stages of tumour development, including initiation, promotion, progression and metastasis.4 13 14 Aberrant activation of NF-κB and/or STAT3 is found in over 50% of all cancers and renders premalignant and fully transformed cells resistant to apoptosis and speeds up their rate of proliferation, thereby increasing tumour growth.7 13 15 Since activating mutations in the genes encoding these transcription factors are rare, autocrine and paracrine mechanisms that lead to NF-κB and STAT3 activation in cancer must be in play. Identification of these mechanisms is of importance as they may provide new means to specifically intervene with the activation of these critical transcription factors in cancer. In this review, we will concentrate on the roles of TNF and IL-6 in tumour promotion and describe some of the mechanisms through which they act in experimental models of colitis-associated colon cancer (CAC) and hepatocellular carcinoma (HCC).

IL-6 in CAC

Elevated expression of IL-6, which can be detected in patient serum, is linked to increased risk of development of colorectal adenomas.16 In general, expression of IL-6 in serum or tissue samples of patients with cancer or tumour-bearing animals correlates with poor prognosis.11 17,,19 Initial studies in the azoxymethane + dextran sulphate sodium model of CAC showed that conditional ablation of IκB kinase (IKKβ), the protein kinase responsible for NF-κB activation, in enterocytes led to a marked reduction in the development of colonic adenomas, but had little effect on adenoma size.20 Therefore, NF-κB in premalignant intestinal epithelial cells is required for cell survival and tumour initiation, rather than for cell proliferation and tumour growth.20 Deletion of the STAT3 gene in enterocytes has a similar inhibitory effect on adenoma development but unlike IKKβ deletion, the STAT3 deletion also curtails adenoma growth.21 22 Most likely, autocrine and paracrine factors produced during colitis and within the future tumour microenvironment lead to the activation of NF-κB and STAT3 in premalignant cells.

The nature and origin of these factors came to light when IKKβ-dependent NF-κB activation in the mouse CAC model was ablated in myeloid cells.20 Lack of NF-κB activity in these cells, mainly lamina propria macrophages, resulted in a significant reduction in both colonic tumour multiplicity and size, and partially phenocopied NF-κB and STAT3 ablation in epithelial cells.20 Since in immune cells genes, encoding proinflammatory cytokines are the most obvious transcriptional targets for NF-κB, it was suggested that proinflammatory cytokines may serve as a critical link between activated immune cells in the tumour microenvironment and aberrant activation of NF-κB and STAT3 in cancer cells.23 The search for such cytokines has yielded several candidates, including TNF and IL-6. Genetic ablation of IL-6 in mice reduced both the multiplicity and size of colonic adenomas in mice subjected to the azoxymethane +dextran sulphate sodium CAC induction protocol.22 Interestingly, ablation of IL-6 resulted in a larger reduction in tumour load in male mice, than in female mice (SG and MK, unpublished results), in agreement with the observation that male mice develop more colon cancer than female mice,24 and IL-6 synthesis is under the repressive control of oestrogen signalling, which is more active in females than in males.25

Inhibition of IL-6 signalling by administration of a soluble gp130-Fc fusion protein (gp130 in the signal transducing subunit of the IL-6 receptor) into mice bearing established CAC tumours led to a reduction in tumour size, indicating inhibition of tumour growth.22 26 Attenuation of IL-6 signalling during tumour induction resulted in a decrease both in tumour multiplicity and growth.22 27 Conversely, mutational activation of gp130-mediated signalling in non-haematopoietic/epithelial cells of mice subjected to the CAC induction protocol increased tumour multiplicity and growth21 and similar observations were made in a mouse model of gastric cancer.28 29 Excessive proliferation of epithelial cells caused by deregulation of gp130 signalling can be reversed by simultaneous deletion of IL-6 and IL-11, the two major cytokines that activate STAT3 via gp130 in the intestine.21 Moreover, genetic inactivation of the inhibitor of gp130 signalling to STAT3, suppressor of cytokine signalling 3, enhances tumour load in the CAC model.30

IL-6 is also a potent stimulator of colon cancer cell proliferation and growth31 and has been implicated in the growth of other cancer cell lines or primary tumours.1 32 Most of the IL-6 in CAC is produced by haematopoietic-derived cells, especially lamina propria macrophages and dendritic cells, during early states of tumour induction,22 27 and by T cells during later stages of tumour progression.26 This is probably due to the high inflammatory content of CAC tumours and the ongoing injury and death of epithelial cells during tumour development. On the other hand, epithelial cells and cancer cells, as well as tumour-associated fibroblasts are also capable of IL-6 production and can significantly contribute to the total pool of this cytokine, especially in sporadic colorectal cancer (CRC) and other cancers not associated with an underlying inflammatory condition.17 33 34

The receptors for IL-6 (IL-6R and gp130) are expressed by epithelial and immune cells, but shedding of membrane-bound IL-6R was detected during CAC development owing to the action of the ADAM17 metalloprotease within the inflammatory microenvironment.26 35 36 The soluble IL-6R being shed into the tumour microenvironment can activate STAT3 in gp130-expressing cells via ‘trans-signalling’.26 36,,38 The proliferative and survival effects of IL-6 on cancer cells are largely mediated through activation of STAT3 in enterocytes.22 26 27 39 Moreover, STAT3 activation in CAC correlates with IL-6 expression levels and inversely correlates with the amount of suppressor of cytokine signalling 3, whose downregulation or loss relieves its inhibitory effects on IL-6-mediated STAT3 activation in premalignant cells.40 Although IL-6 is important for STAT3 activation in enterocytes, STAT3 ablation in these cells has a more profound effect on mucosal injury and regeneration and on tumour multiplicity and growth than IL-6 depletion.13 22 27 This suggests that other cytokines, such as IL-11, IL-22, epidermal growth factor family members (transforming growth factor α and epidermal growth factor), as well as leptin, can potentially contribute to STAT3 activation in cancer cells in CAC.21 28 41,,43 In addition to STAT3, other signalling modules activated by gp130 have also been implicated in tumourigenesis, including the Ras-MAP kinase (MAPK) and the Phosphatidylinositol 3-kinase-AKT-mammalian target of rapamycin (PI3K-AKT-mTOR) pathways. While it is not clear to what degree the IL-6 effects on cancer cells may be mediated by these pathways, MAPK and mTOR signalling are known to be important for intestinal tumourigenesis.42 44 45

Sporadic CRC in the Apcmin mouse model also seems to be positively regulated by IL-6, as IL-6 ablation affected adenoma development,46 and microbially driven CRC cell invasion is also controlled by IL-6.47 Besides its direct action on cancer cells, IL-6 also affects other cells within the tumour microenvironment. Importantly, IL-6 has a pivotal role in induction and maintenance of colitis, since its inactivation blocks colitis in several animal models and in patients.48,,50 This effect may be mostly attributed to IL-6-mediated differentiation of Th17 cells and IL-6 mediated survival of colitogenic T cells,49 51 52 as well as inactivation of T regulatory (Treg) cells.53 Th17 and other types of activated T cells may stimulate CAC tumour growth, whereas Treg cells suppress intestinal inflammation and tumourigenesis.34 54,,56 In addition, IL-6 controls trafficking and recruitment of inflammatory myeloid cells and neutrophils, which are a further source of proinflammatory and pro-tumourigenic cytokines.36 57,,59 Moreover, an important role of IL-6 in differentiation of myeloid-derived suppressor cells was recently described60 61 and these cells are present in both CAC and CRC and may be important for suppression of intratumoural immune responses and tumour growth.62

TNF in CAC

TNF is produced during the initiation of inflammatory responses and is critical for maintenance of chronic inflammation, since TNF blockers are effective in treatment of a variety of acute and chronic inflammatory conditions.63,,66 Given its established role in chronic inflammation, angiogenesis, tissue remodelling, tumour growth and metastasis, TNF is likely to be an important tumour-promoting cytokine in a variety of cancers.11 67 68 The tumour-promoting properties of TNF are probably linked to its ability to activate both AP-1 and NF-κB signalling pathways that stimulate cell proliferation and survival.11 69 70 Importantly, TNF production is increased in ulcerative colitis71 and this cytokine seems to be causative for the pathogenesis of various forms of inflammatory bowel disease, an underlying condition for CAC development. TNF is often produced by multiple cell types during inflammation, including macrophages and T cells.71 72 However, in CAC, the deletion of IKKβ in myeloid cells does not result in decreased TNF production.20 It is not clear whether the LysM-Cre deleter used in these experiments is non-operational in a critical subset of colonic macrophages,73 74 or whether TNF may be produced by other cell types in CAC, including T cells and epithelial cells. TNF expression is elevated during CAC tumourigenesis and genetic inactivation of the type 1 TNF receptor (TNFR1) reduced CAC induction and growth.75 Moreover, interference with TNF signalling using a soluble decoy receptor (Enbrel) resulted in decreased tumourigenicity and tumour growth.75

In another model of spontaneous inflammatory colon cancer, dendritic cell-specific inactivation of transcription factor T-bet resulted in spontaneous intestinal inflammation and CAC development and this process was largely dependent on TNF.76 Although, as described below, in the Mdr2-deficient model of HCC, TNF was implicated in direct activation of NF-κB in hepatocytes,77 it is not yet clear whether the same scenario is applicable to CAC. Bone marrow transplantation experiments showed that the critical sites of TNFR1 expression are cells of haematopoietic origin rather than epithelial cells,75 suggesting that TNF operates as critical regulator of immune cells in CAC, rather than exerting a direct effect on premalignant epithelial cells. Indeed, TNF inhibition dampens the production of other proinflammatory cytokines, which can signal directly to cancer cells, including IL-6 and IL-1711 22 78 However, other studies suggested an important role for TNFR1 and TNFR2 expressed by cancer cells.67 79,,81 Since TNF is a pleiotropic cytokine, it is possible that it actually targets multiple cell types, and future studies using mice with conditional inactivation of TNFR1 and TNFR2 should resolve this question. While TNF itself and NF-κB activation in epithelial cells are critical in CAC, it is not clear whether the TNF–NF-κB axis is essential in these cells. Other stimuli, such as IL-1, IL-18 and Toll receptor (TLR) ligands, can also contribute to NF-κB activation in cancer cells.82,,85

IL-6, TNF and liver cancer

Another inflammation-linked cancer in which inflammatory cytokines have a significant role is HCC, the most common form of liver cancer. HCC often develops in association with chronic liver inflammation caused by either hepatitis B virus or hepatitis C virus infection. However, other risk factors such as alcohol-induced liver injury and obesity-induced fat accumulation followed by chronic liver inflammation (steatohepatitis), significantly contribute to the development of HCC. Increased circulating levels of IL-6 are associated with most HCC risk factors, including hepatosteatosis, obesity and liver cirrhosis, and are the best predictors of rapid progression from viral hepatitis to HCC in humans.86 Similar to its role in CAC, IL-6 is also a critical tumour promoter in HCC, and IL-6-deficient mice are resistant to HCC induction upon challenge with the pro-carcinogen diethylnitrosamine (DEN).25 The early source of IL-6 in DEN-treated mice seems to be Kupffer cells (resident liver macrophages) and inflammatory monocytes recruited to the liver, as inducible ablation of IKKβ in these cells results in decreased IL-6 production and almost complete inhibition of HCC induction.87 In contrast to CAC, however, DEN-induced HCC development is strongly enhanced by inhibition of NF-κB activation in hepatocytes.87 88 Enhanced HCC development in mice lacking hepatocyte NF-κB (or IKKβ) can be explained by increased DEN-induced hepatocyte death, which results in increased compensatory proliferation of differentiated premalignant hepatocytes, which harbour oncogenic mutations.87 89 DEN-induced hepatocyte death (necrosis) causes the release of IL-1α and probably other soluble mediators, which are sensed by sentinel cells, such as resident Kupffer cells.90 IL-1α induces IL-6 production and this response drives the compensatory proliferation of surviving hepatocytes. These effects of IL-6 are also mediated by STAT3 activation, leading to induction of genes that support cell survival and proliferation.91 Correspondingly, STAT3 ablation in hepatocytes curtails HCC development.91

It is important to note that circulating and intrahepatic levels of IL-6 are much higher in men than in women,19 25 but are increased in postmenopausal women.92 Correspondingly, the incidence of HCC is up to five times higher in men than in women93 and male mice treated with DEN develop much more HCC than female mice.25 Most likely, the gender-biased production of IL-6 accounts for the much higher incidence of HCC in male subjects. At least in mice, inactivation of IL-6 eliminates the gender differences in HCC development, whereas ovariectomy enhances IL-6 production and increases HCC induction in female mice.25 The production of IL-6 by Kupffer cells during tumour initiation is subject to negative regulation by oestrogen receptor α.25 While oestrogen signalling inhibits IL-6 production and decreases HCC development, obesity and hepatosteatosis increase IL-6 production and dramatically enhance HCC tumour development.94 Production of TNF is also increased in obese patients and experimental animals and both TNF and IL-6 contribute to obesity-promoted HCC development in mice.94 Interestingly, TNF and IL-6 are needed for obesity-induced hepatosteatosis and steatohepatitis,94 but the mechanism through which they operate is not clear. By contrast, TNF and TNFR1 are not involved in DEN-induced HCC development in lean mice.25 However, spontaneous HCC development in Mdr2−/- knockout mice, which also exhibit steatohepatitis,95 is TNF and TNFR1 dependent.77 Furthermore, in Mdr2−/- mice the inhibition of hepatocyte NF-κB by expression of a non-degradable form of IκBα expressed from an inducible liver-specific promoter results in attenuation of HCC development.77 Most likely in Mdr2−/- mice the critical NF-κB activator is TNF, as inhibition of TNF signalling also decreased HCC tumourigenicity and prevented NF-κB activation in hepatocytes.77 A TNF-related cytokine, lymphotoxin, also triggers NF-κB activation in hepatocytes and promotes HCC development, when overexpressed in the liver.96 In this model, as well as in the Mdr2−/- model, NF-κB activation in hepatocytes is likely to be required for production of chemokines that recruit immune cells into the liver and thereby maintain an inflammatory microenvironment during HCC development.

In conclusion, recent experimental data provide unequivocal evidence for the involvement of both TNF and IL-6 in various inflammation-associated and sporadic cancers. TNF and IL-6 promote tumour development both through direct effects on premalignant cells and by orchestrating a tumour-promoting microenvironment through effects on many different cell types. Long-term TNF or IL-6 blockade, as learnt from their usage in autoimmune diseases, results only in some of the side effects, such as reactivation of tuberculosis in the case of anti-TNF, but does not result in increase in tumour development.68 Such findings suggest that drugs that inhibit TNF and/or IL-6 production and signalling may prove to be useful in cancers as adjuvants for more conventional chemotherapeutic drugs and radiation. By preventing inflammation-promoted cancer cell growth and survival, anti-TNF and anti-IL-6 drugs may enhance the response to more conventional anticancer treatments, although they may not be highly effective anticancer drugs on their own.

References

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Footnotes

  • Funding This work was supported by Crohn's and Colitis Foundation of America (CCFA #2693) to SG and the National Institutes of Health and the American Association for Cancer Research to MK, who is an American Cancer Society research professor.

  • Competing interests None.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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