IUBMB

Life, 62(4): 283289, April 2010

Critical Review
Tumor Necrosis Factor, Lymphotoxin and Cancer
Marina S. Drutskaya1, Grigory A. Emov1, Andrey A. Kruglov2, Dmitry V. Kuprash1 and Sergei A. Nedospasov1,2
1 2

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia German Rheumatism Research Center, a Leibnitz Institute, Berlin, Germany

Summary Initially TNF has been discovered as an anti-tumor factor, but it is now considered as one of the universal effectors of innate signaling implicating its key role in host defense and inammation. Other physiological functions of TNF are primarily linked to organization of lymphoid tissues. TNF can exert deleterious effects on the organism when its local or systemic concentrations exceed certain levels. This is the main reason for the failure of TNF therapy in oncology. Moreover, in certain experimental models TNF to TNFRp55 signaling axis was found to play a pro-tumorigenic role. On the other hand, anti-TNF therapy proved to be benecial in rheumatic and other autoimmune diseases. Taking into consideration the pivotal function of TNF in the immune system, it is obvious that such therapy cannot be entirely free of adverse effects including suppression of host defense and, possibly, predisposition to lymphomas. Lymphotoxins alpha and beta are the two related cytokines that exist in distinct trimeric forms which can signal through TNFR I and TNFR II, as well LTbetaR receptors, depending on the composition of the trimer. These signals have important functions in the development and homeostasis of the immune system. Importantly, there is a recently uncovered link between the LTalpha/LTbeta to LTbetaR signaling axis and cancer. Here we review the current status of the eld with the focus on one particular issue: are TNF and lymphotoxins intrinsically anti-cancer or pro-tumorigenic. 2010 IUBMB IUBMB Life, 62(4): 283289, 2010 Keywords
cytokines; inammation; disease models; conditional mutagenesis.

later utilizing preparations from killed bacteria, known as Coleys mixed toxins. In 1975 Old and coworkers demonstrated that sera from mice primed with BCG and boosted with LPS mimicked the effect of LPS by inducing hemorrhagic necrosis of subcutaneously transplanted sarcomas. The substance which mediated necrosis was named Tumor Necrosis Factor (2). When TNF gene was cloned and expressed in E. coli a decade later (35) both the in vivo activity and the cytotoxicity against some tumor cell lines were reproduced, including the remarkable anti-tumor effect of a single injection of recombinant TNF into tumor-bearing mice. When further tested in vitro TNF was found to possess cytotoxic or cytostatic properties towards approximately half of human tumor cell lines while normal diploid cells were resistant to cytotoxic effects (68). One of the initial hypothesis implicated TNF as part of innate immune surveillance mechanisms against spontaneously emerging tumors. Experimental data in mice supported the idea to use TNF as an anti-tumor agent in humans. However, discovery of deleterious in vivo effects of TNF (see Good Versus Bad TNF section later) has greatly limited the area of possible therapeutic applications.

BRIEF HISTORY OF TNF DISCOVERY Coley was the rst to implement the concept of treating cancer patients by deliberately infecting them with bacteria (1) and
Received 31 October 2009; accepted 2 January 2010 Address correspondence to: Marina S. Drutskaya, Engelhardt Institute of Molecular Biology, 32 Vavilov Street, Russian Federation, Russian Academy of Sciences, Moscow 119991, Russia. Tel: 17 499 1359964. Fax: 17 499 135-1405. E-mail: marinadru@gmail.com
ISSN 1521-6543 print/ISSN 1521-6551 online DOI: 10.1002/iub.309

BRIEF HISTORY OF LYMPHOTOXIN (LT) DISCOVERY AND CHARACTERIZATION The name lymphotoxin (LT) was introduced by Granger and coworkers (9) to describe cytotoxic activity produced by lymphocytes in vitro (10). LT remained poorly understood until its molecular cloning in 1984 (11) which uncovered a TNF-like molecule with similar activity both in vitro and in vivo (including its anti-tumor effects). It was found that LT is essentially a TNF-like activity produced by lymphocytes, rather than by macrophages or neutrophils. These two cytokines were the rst among 1520 TNF-like family members, most of them (except LTa)type II transmembrane molecules. A complimentary family of TNF receptors has been also discovered and

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characterized. As expected, recombinant TNF and LTa can interact (although with different afnities) with the same TNF receptors I and II. However, the discovery of LTb as a second subunit of heteromeric membrane-bound LT complex (consisting of 1 molecule of LTa and 2 molecules of LTb) (12), characterization of the LTb receptor (13) and unexpected phenotype of LTa-decient mice (see Physiological functions of TNF and lymphotoxin as dened by studies of gene-decient mice section) suggested that physiological functions of TNF and LTa are quite distinct.

GOOD VERSUS BAD TNF In 1985 TNF was independently cloned under the name cachectin, as a mediator of wasting syndrome in animals (14). That study immediately suggested the existence of other TNF functions, some of which Cerami and Beutler dened as the dark side of TNF. Indeed, polyclonal anti-TNF serum used for passive immunization of mice could protect animals from LPS induced lethal toxicityan acute model of sepsis (15). Similarly, baboons are protected from otherwise lethal dose of E. coli injected intravenously by monoclonal antibody against human TNF (16). This ndings were consistent with the fact that TNF is primarily produced by innate immune cells (ex. macrophages) in response to various bacterial components. Around the same time, TNF was detected in the inamed joints (17) as well as in the synovial uid (18, 19) of patients with rheumatoid arthritis (RA). These and subsequent studies helped to uncover the deleterious role of TNF in RA. Mouse models also support the hypothesis of the key role of TNF in RA. Collagen-induced arthritis is ameliorated upon antiTNF treatment (2022). Additionally, transgenic mice overexpressing human TNF (hTNF) developed spontaneous polyarthritis which can be completely blocked upon administration of monoclonal antibodies against hTNF (23). On the other hand, already in the pre-knockout era TNF was linked to the formation of protective granulomas during Mycobacterial infections. Anti-TNF antibodies injected 1 or 2 weeks post infection with BCG dramatically interfere with granuloma formation, moreover, fully developed granulomas rapidly regress after anti-TNF treatment (24). In another study depletion of TNF by polyclonal serum from BALB/c mice results in reduced survival time, while the infusion of TNF increases resistance against tuberculosis (TB) (25). Anti-TNF antibodies exacerbate the scope of the Listeria monocytogenes infection, while administration of recombinant mTNF protects otherwise lethally challenged mice (26). In the aggregate, these studies clearly uncovered benecial role of TNF in host defense, but, at the same time, unveiled possible deleterious effects associated with overproduction of TNF, either systemic or local.

PHYSIOLOGICAL FUNCTIONS OF TNF AND LT AS DEFINED BY STUDIES OF GENE-DEFICIENT MICE Studies in various gene-targeted mice, including mice with conditional gene-deciency, were indispensable in establishing physiological functions of TNF and LT (2735). In particular, the signicance of TNF/LT axis in the development and maintenance of microstructure of secondary lymphoid tissues was ve mice and during the uncovered and dissected, both in na immune response [for review see (36)]. Membrane-bound lymphotoxin (LTa1b2) is critical for the development of lymph nodes and Peyers patches during embryogenesis and is indispensable for the maintenance of the structure of secondary lymphoid organs, such as the spleen and the nasopharyngealassociated lymphoid tissue. Furthermore, mice with structural alterations in the organization of lymphoid organs resulting from impaired LT signaling exhibit severe defects in the antiviral immune responses (37). The involvement of TNF in protection against Mycobacteria tuberculosis was conrmed in mice decient either in TNF or its receptor (TNFR1) (3840). The key feature in host protection against this infection is the formation of granulomas, which is considered to be the pathological hallmark of tuberculosis. Extensive studies of infected TNF-decient mice suggest the protective role of TNF as a key mediator in granuloma formation based on the ndings that granulomas formed in the absence of TNF appear less well organized and contain overwhelming bacillary numbers (24). However, it has been recently argued that TNF may not be directly required for granuloma formation, at least in the innate stages of the infection, but rather preserves the integrity of granulomas by restricting M. tuberculosis growth in the infected cells and preventing necrosis of the macrophages (41). Other benecial in vivo functions of TNF, uncovered in studies with gene-decient mice, include its role in the development of Peyers patches (33) and maintenance of the microstructure of the secondary lymphoid tissues (where its role is more modest than that of LT but, nevertheless, distinct and nonredundant) [for review see (42)]. The signicance of TNF-mediated pathway in protection against TB is becoming more evident in view of the widening use of TNF-neutralizing agents in the treatment against a variety of autoimmune and inammatory conditions (in particular, RA). One of the serious side effects associated with anti-TNF therapy is reactivation of latent TB in patients undergoing such therapy. The signicance of LT-mediated pathway in peripheral lymphoid tissue organogenesis was initially demonstrated in studies of mice with complete inactivation of LTa, LTb or LTb receptor (LTbR) which resulted in profound defects in the development and structural maintenance of the secondary lymphoid organs (43). It has been also established that LT is expressed by various cell types but mostly by lymphocytes, natural killer cells and lymphoid tissue-inducing cells (LTICs). The precise contribution from each cell type was studied using

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conditional gene-targeting approach which allowed to dissect the role of the surface LT complex expressed on B and T cells in the formation and maintenance of the lymph nodes, Peyers patches and the spleen and correlate the observed defect with impaired immune response (44).

ANTI-TUMOR VERSUS PRO-TUMORIGENIC FUNCTIONS OF TNF AND LT AS DEFINED IN GENETICALLY MODIFIED MICE Initial attempts to evaluate anti-tumor activity of TNF in vivo were carried out using Meth A mouse sarcoma (45). Subsequent studies concentrated on local effects of TNF on tumor development, in particular, using tumor cells engineered to secrete TNF or LT (4652) (see Table 1 for summary). It has been shown that tumor rejection is dependent on local TNF release by lymphocytes and macrophages (53). These studies revealed that the constitutive TNF expression at the site of malignancy exerts strong and long-term suppression on tumor growth. Such suppression, however, only takes place in the vicinity of cells expressing TNF without affecting distant sites (unless systemic TNF levels are high). Severe toxicity associated with systemic administration of TNF in cancer patients, led researchers to explore the possibility of using LTa for the induction of anti-tumor response (54). On the other hand, targeted disruption of LTa gene in mice resulted in enhanced tumor growth and metastasis of melanoma cells due to impaired NK cell function (55). It should be noted that some of the early studies did not take into consideration the complex biology of the LT system. At least some LTa molecules form heterotrimers with LTb and signal through non-TNF receptors. Recent application of agonistic monoclonal antibodies which specically triggers LTbR but none of the TNF receptors demonstrated anti-tumor effects on colon carcinoma (56). Taken together, this data appeared to suggest a potential anti-tumorigenic role of the TNF/LT axis. Despite the initially discovered anti-tumor properties of TNF, a wide body of evidence implicates this cytokine in tumorigenesis, rather than in protection against the tumor. For example, mice decient in TNF were unexpectedly protected from skin carcinogenesis in model studies (59, 60). Mice lacking TNFR1 and treated with azoxymethane and dextran sodium sulfate to induce colon cancer exhibit fewer signs of the disease, less damage to the mucosal tissue, decreased number of inltrating macrophages and neutrophils, and attenuated tumor growth (62). Additionally, TNF blockade in WT mice upon induction of colitis-associated colon carcinogenesis results in the reduction of tumor growth, as well as decreased colonic inltration by macrophages and neutrophils. Another study of inammation-associated tumorigenesis using Mdr-2 decient mice which spontaneously develop cholestatic hepatitis followed by hepatocellular carcinoma also underlined the potential of TNF blockade in the suppression of tumor development (61).

LT, another cytokine with a misleading name, and its distinct receptor, LTbR, were also implicated in signaling leading to cancer. For example, angiogenesis and solid tumor growth were promoted upon activation of LTbR on brosarcoma tumor cells by activated lymphocytes expressing LTa1/LTb2 heterotrimer (65). Another recent study implicated LTbR as a potential oncogene in nasopharyngeal carcinoma (63). And most interestingly, LT?LTbR signaling pathway was directly implicated in the progression of chronic hepatitis associated with hepatitis B and C virus infections to hepatocellular carcinoma (66). In a mouse model hepatocyte-restricted chronic overexpression of LT was sufcient to induce liver tumors through LTbR-dependent mechanism (66). In another recent study surface LT complex produced by B cells was implicated in the emergence of androgen-independent prostate cancer cells (68). In general, cancerassociated inammation is always accompanied by the presence of the inammatory cells such as tumor-associated macrophages and regulatory T cells, as well as mediators of inammation like TNF in the tumor microenvironment which promote tissue remodeling, angiogenesis, tumor growth and suppress adaptive immunity. Chronic inammation has been shown to promote cancer development [reviewed in (69)], while acute inammatory response has not been associated with increased cancer risk and may even be benecial in ghting tumor growth (70).

RELEVANCE OF MOUSE STUDIES FOR PATIENTS An effective anti-tumor TNF dose in humans turns out to be much higher than the tolerable dose (71) and, thus, clinical trials with the recombinant human TNF as a monotherapy failed. It appears that mice are more resistant to TNF than humans since TNF exerts only minimal toxic effects in mice at the equivalent doses. It should be pointed out that while systemic use of TNF was abandoned, TNF as a drug in oncology has found a small niche for treatment of regionally advanced melanoma and inoperable limb sarcoma by isolated perfusions in combination with chemotherapy (72, 73). On the other hand, during the last decade long-term antiTNF therapy has become a routine treatment for several autoimmune diseases [reviewed in: (74)]. As already mentioned, due to intrinsic and nonredundant functions of TNF in vivo, such treatment cannot be entirely free of side effects. In support of the mouse studies, the direct evidence that TNF blockade may result in the reactivation of latent TB infection comes from the clinical trials. It is believed that in the developing countries the risk of TB reactivation may be signicantly higher. The eligibility of patients for anti-TNF treatment is currently based on the outcome of the screening for latent TB. Such precautions have already resulted in substantial reduction in the incidence of TB among patients undergoing the treatment (75, 76). Since earlier theories concerning in vivo functions of TNF were linked to immunosurveillance mechanisms, systemic TNF inhibition could have resulted in increased malignancies. How-

Table 1 Anti- and pro-tumorigenic effects of TNF and LT, as studied in mice
Tumor type BALB/c Meth A sarcoma Hemorrhagic tumor necrosis Hemorrhagic tumor necrosis Tumor necrosis Tumor necrosis Tumor regression Reduced tumor growth and reduced invasiveness in vivo Experimental manipulation with TNF/LT signaling Effect/comment Refs. 2

Mouse model

Subcutaneous sarcomas in (BALB/c x C57Bl/6)F1 mice

5 11 57 58

Syngeneic melanoma in C57Bl/6 mice Human xenografts in nude mice Tumor cells subcutaneously grown in nude or syngeneic mice

C57Bl/6

Reduced tumor growth

48 47 49 50 51 52 46 59, 60 61 62 63, 64 65, 63

Mdr-2 decient mice Cholestatic hepatitis ID8 murine ovarian cancer Transplanted brosarcoma Same

Injection of sera from mice primed with BCG and boosted with LPS Single injection of recombinant human or mouse TNF Single injection of recombinant human LTa (TNF-b) B16BL6 melanoma cells Injection of recombinant human or mouse TNF Primary breast and bowel Daily injections of rHuTNF tumors intratumorally L929 brosarcoma in nude micea Syngeneic MCA-205 sarcoma in C57Bl/6 micea Syngeneic J558L plasmacytoma in BABL/c micea UV-induced skin tumor 1591-RE in athymic NCR nude micea Syngeneic spontaneous mammary adenocarcinoma TSA in BALB/c micea Mouse mammary tumor line EMT6 in BALB/ca Various tumor cell linesa Chemically induced skin Genetic or biochemical blockade tumors of TNF or TNFR1 signaling Cholestatic hepatitis Same

BALB/c

C57Bl6 C57Bl6

Failure of carcinoma progression Reduced inammation and tumor growth Reduced tumor growth Reduced tumor growth Same Genetic or biochemical blockade of LTbR signaling LTbR-Ig LTa genetic knockout

Abl-LT transgenic

66 55 67 Genetic knockout of TNF/ TNFR1, LTa or both Anti-LTbR agonistic Ab Tumor necrosis or reduced growth 56

C57Bl6 Spontaneous lymphoma Colon carcinomab

Spontaneous hepatocellular carcinoma B16BL6 melanoma cells

p532/2, p531/2

Delay or absence of tumor growth Enhanced tumor growth and metastasis No effect

BALB/c or athymic nude

Tumor cells either selected for TNF production in vitro or engineered to overexpress the TNF gene. Syngeneic murine colon carcinoma in BALB/c mice or human colon carcinoma xenograft.

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ever, the incidence of solid tumors in patients undergoing antiTNF therapy is similar to sex, age and race-matched healthy individuals. Some studies reported slight increase in the incidence of lymphomas in patients receiving iniximab or etanercept (77). Additionally, mouse studies failed to demonstrate correlation between TNF (and also LT) deciency and increased tumor incidence in lymphoma-prone mouse models (67). Nevertheless, recent FDA ruling concerning increased incidence of cancer in young patients placed on anti-TNF therapy reminds us that this issue is not yet completely resolved. More clinical and animal studies are needed to exclude pro-tumorigenic effects of anti-TNF therapy. Surprisingly, (given the name of this cytokine), there were several recent reports revealing benecial effects of cancer treatment using anti-TNF therapy (64,7880). This is attributed to TNF-mediated expansion of TNFR II expressing Tregs which promote tumor escape from the immune system (78), as well as regulation of IL-17-dependent monocyte recruitment to the tumor site (64). Taken together, these studies in mice suggest that either genetic or pharmacological ablation of TNF provides protection in several inammation-based tumor models (81). Recombinant LT has never been used as a single agent in treating cancer. Although earlier quoted study (56) demonstrated therapeutic effect of a monoclonal antibody which selectively triggers LTbR, most recent data implicating LTbR signaling in cancer suggests that inhibitors of LT?LTbR axis (ex. baminercept) should be considered for future clinical trials.

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CONCLUSIONS Tumor Necrosis Factor, once believed to become a potent agent in the ght against cancer, turned out to be a pleiotropic mediator of many immune processes, most notably, of host protection against intracellular bacteria. Its proposed nonspecic immune surveillance function could not be unequivocally demonstrated in mice. Moreover, in certain settings by promoting inammation TNF may play a pro-tumorigenic role. LT, a closely related cytokine, has distinct functions in vivo due to its action through a distinct receptor, LTbR. As in the case of TNF?TNFRp55, the LT?LTbR signaling pathway may lead to carcinogenesis. Based on the current knowledge the latter mechanism appears to be more important for the development of malignancies and pharmacological inhibition of this pathway warrants future clinical evaluation.

ACKNOWLEDGEMENTS The authors thank M. Heikenwalder for many stimulating discussions. This work was supported by the MCB program from the Russian Academy of Sciences, grant 09-04-12185 from Russian Foundation for Basic Research and SFB633 funded by DFG.

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