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Oncogene (2010) 29, 5006–5018 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 www.nature.com/onc REVIEW The unexpected role of b receptor signaling in : from lymphoid tissue formation to and prostate development

MJ Wolf1, GM Seleznik1, N Zeller1,3 and M Heikenwalder1,2

1Department of Pathology, Institute of Neuropathology, University Hospital Zurich, Zurich, Switzerland and 2Institute of Virology, Technische Universita¨tMu¨nchen/Helmholtz Zentrum Mu¨nchen, Munich, Germany

The lymphotoxin (LT) a, b and their receptor genesis. Consequently, the inflammatory microenviron- (LTbR) belong to the (TNF) super- ment was added as the seventh hallmark of cancer family, whose founder—TNFa—was initially discovered (Hanahan and Weinberg, 2000; Colotta et al., 2009). due to its tumor necrotizing activity. LTbR signaling This was ultimately the result of more than 100 years of serves pleiotropic functions including the control of research—indeed—the first observation that tumors lymphoid organ development, support of efficient immune often arise at sites of inflammation was initially reported responses against pathogens due to maintenance of intact in the nineteenth century by Virchow (Balkwill and lymphoid structures, induction of tertiary lymphoid organs, Mantovani, 2001). Today, understanding the underlying liver regeneration or control of lipid homeostasis. Signal- mechanisms of why immune cells can be pro- or anti- ing through LTbR comprises the noncanonical/canonical carcinogenic in different types of tumors and which nuclear factor-jB (NF-jB) pathways thus inducing cellular and molecular inflammatory mediators (for , or adhesion molecule expression, cell example, , , or proliferation and cell survival. Blocking LTbR signaling or cytokines) drive or potentially prevent carcinogenesis Fcc-receptor mediated immunoablation of LT-expressing is subject of intense research (Supplementary material). cells was demonstrated to be beneficial in various infectious Here, we focus on the cytokines lymphotoxin (LT) a or noninfectious inflammatory or autoimmune disorders. and b and their involvement in inflammation-induced Only recently, LTbR signaling was shown to initiate carcinogenesis. inflammation-induced carcinogenesis, to influence primary tumorigenesis and to control reemergence of carcinoma in various cancer models through distinct mechanisms. Indeed, LTbR signaling inhibition has already been used LT, a molecule with a long history as efficient anti-inflammatory, anti-cancer therapy in some experimental models. Here, we review the pleiotropic In the 1960s and early 1970s, two molecules were functions attributed to LT, the effects of its deregulation described based on their ability to promote cell death and extensively discuss the recent literature on LT’s in target cells (for example, L cells; fibroblasts) (Ruddle link to carcinogenesis. and Waksman, 1967; Granger and Williams, 1968). Oncogene (2010) 29, 5006–5018; doi:10.1038/onc.2010.260; In 1967, a ‘cytotoxic factor’ was described causing published online 5 July 2010 toxicity on fibroblasts in presence of specific (Ruddle and Waksman, 1967, 1968). Further, in 1968, Keywords: lymphotoxin b receptor signaling; inflamma- a cytotoxic molecule produced by lymphocytes was tion-induced carcinogenesis; NF-kB signaling; lympho- reported and named LT (Granger and Williams, 1968; toxin a; lymphotoxin b Granger et al., 1969). In 1975, a substance was depicted causing efficient necrosis in various tumor types in vivo throughout a cytotoxic activity and was thus denomi- nated ‘tumor necrosis factor’ (TNF) (Carswell et al., Inflammation-induced carcinogenesis 1975). These molecules subsequently caught attention of several research groups using these cytotoxic, tumor- In the last decade, several studies have corroborated the necrotizing capabilities for their therapeutic potential in tight link between chronic inflammation and carcino- the treatment of cancer. In 1984, human LT was the first cytokine to be purified from a b-lymphoblastoid cell line (Aggarwal et al., 1984) and thereafter its amino- Correspondence: Dr M Heikenwalder, Department of Pathology, Institute of Neuropathology, University Hospital Zurich, Schmelz- acid sequence and structure were determined (Aggarwal bergstrasse 12, CH-8091 Zurich, Switzerland. et al., 1985a; Nedwin et al., 1985a). Neutralization of E-mail: [email protected] or LT activity by LT-specific antibodies led to the isolation [email protected] of a second cytotoxic factor from a human myeloid cell 3Current address: Department of Neuropathology, University of Freiburg, D-79106 Freiburg, Germany. line, already known as TNFa (Aggarwal et al., 1985b). Received 1 March 2010; revised 21 May 2010; accepted 27 May 2010; Amino-acid sequence determination revealed close published online 5 July 2010 relationship of TNF to LT, resulting in the renaming Unexpected role of LTbR signaling in carcinogenesis MJ Wolf et al 5007 of LT into TNFb. It became evident that these two cellular responses, development and maintenance cytokines are prototypic members of a super- of lymphoid organs (Figure 1). family, later defined as the TNF superfamily (TNFSF) Activation of the canonical NF-kB pathway mainly (Hehlgans and Pfeffer, 2005). Interestingly, murine and occurs in response to inflammatory cytokines such as human TNFa and b are close to each other within TNF, and IL-1, engagement of the T-cell receptor and in the major histocompatibility complex region (mouse, response to bacterial infection (for example, lipopoly- 17 (Nedospasov et al., 1986a, b); man, saccharide). Interestingly, LTbR activation (through short arm of (Nedwin et al., 1985b; LIGHT or LTab heterotrimers) can also activate the Muller et al., 1987)). Later a third —homologous canonical NF-kB (Dejardin et al., 2002; Muller and to TNFb—was identified within the major histocompa- Siebenlist, 2003; Haybaeck et al., 2009). tibility complex and named LTb (TNFSF3; In addition to the canonical pathway a parallel (Hiserodt et al., 1977; Ware and Granger, 1979; signaling cascade exists called noncanonical pathway Browning et al., 1993; Pokholok et al., 1995), whereas of NF-kB activation. Stimulation of a subset of TNFR subsequently TNFb was termed LTa or TNFSF1B. At family members, such as LTbR and CD40, can activate present, 19 different ligands belong to the TNFSF, the noncanonical NF-kB pathway (Coope et al., 2002; including, for example, TRAIL, RANKL, BAFF, CD40 Pomerantz and Baltimore, 2002). These stimuli appear (Aggarwal, 2003), mediating their cellular response to converge upon NF-kB-inducing (Xiao et al., through 29 receptors, which contain an extracellular 2001), which is required for IKKa phosphorylation cysteine-rich domain (Aggarwal, 2003) and in some (Ling et al., 1998). In this pathway, IKKa is thought to cases (for example, TNFR1, TNFR2, TRAIL R1, exist as a homodimer, independent of NEMO/IKKg TRAIL R2, Fas) a . and IKKb (Senftleben et al., 2001). Activated IKKa phosphorylates p100, thereby inducing its - dependent processing to p52. The p52/RelB dimer LT, LTbR and their cellular origin then translocates into the nucleus to induce gene expres- sion through NF-kB activation by the LTa1b2,LTbR, Under physiological conditions, LT is expressed by NF-kB-inducing kinase, IKKa pathway. This pathway T-, B-, natural killer (NK)- and lymphoid tissue-inducer is crucial for development and maintenance of cells (Aggarwal et al., 2003). Although LTa mRNA architecture, follicular maturation, lymph expression is inducible in B-cells, LTb mRNA is node, Peyer’s patch and NALT organogenesis as well as constitutively produced (Browning et al., 1993; Worm regulation of B-cell survival (Weih and Caamano, 2003). and Geha, 1994). LTb is exclusively anchored in Studies with mutant mice showed a phenotypic overlap the membrane as a type II transmembrane , between LT or LTbR deficiencies and inactivated NF- binding LTa to form membrane-anchored heterotrimers kB signaling (Bonizzi and Karin, 2004; Bonizzi et al., (LTa1b2 and LTa2b1; Browning et al., 1993, 1995). 2004). Some of these findings are currently challenged LTab heterotrimers can also be cleaved from the cell by recently published results in vitro (Britanova et al., surface by matrix metalloproteases eliciting responses 2008). Thus, explanations for the noncanonical pathway on distal cells (Young et al., 2010). In contrast to LTb, specificity await further experiments. À/À À/À À/À LTa can be secreted as a soluble LTa3 homotrimer. LTa ,LTb and LTbR mice display severe LTa and TNF homotrimers bind and signal through the developmental defects in and Peyer’s TNF receptors TNFR1 or TNFR2. LTa1b2 triggers patch neogenesis, a disorganized lymphoid micro- LTbR, whereas LTa2b1 was reported to bind TNFR1 architecture and lack of mature follicular dendritic cells and TNFR2, but this is a diminutive LT form expressed (Pfeffer et al., 1993; De Togni et al., 1994; Koni et al., by T-cells (o2%) with an undefined biological role 1997; Futterer et al., 1998; Ngo et al., 1999). In addition, (Ware, 2005). LIGHT (TNFSF14) is an alternative LTaÀ/À and LTbRÀ/À mice show inflammatory disorders ligand for the LTbR but also interacts with herpesvirus in nonlymphoid organs (for example, lung, liver, kidney; entry mediator (Schneider et al., 2004). LTbR is mainly (Fu et al., 1997, 2000; Fu and Chaplin, 1999; Chin et al., expressed on parenchymal and stromal cells—but 2003). Whether these inflammatory disorders are due apparently also on myeloid cells (Norris and Ware, to decreased expression of the autoimmune regulator, 2007). Its signaling might enable the communication a key mediator of central tolerance for peripherally between lymphocytes and stromal cells, thereby influen- restricted antigens, in LTaÀ/À,LTbÀ/À and LTbRÀ/À mice cing various biological processes (Gommerman and (Chin et al., 2003) is challenged by various publications Browning, 2003; Ware, 2005; Lo et al., 2007; Tumanov (Boehm et al., 2003; Rossi et al., 2007). et al., 2009).

LT and inflammation LTbR activation and NF-jB signaling A major role of LT in inflammation was first implied by LTa,LTb and LTbR, as other TNFSF members its ability to activate endothelial cells in vitro to express activate canonical/noncanonical nuclear factor-kB adhesion molecules (for example, V-CAM, I-CAM) (NF-kB) signaling, a major regulator of innate and (Pober, 1987; Pober et al., 1987; Cavender et al., 1989), adaptive immune responses, cell survival or , to regulate expression of homeostatic chemokines

Oncogene Unexpected role of LTbR signaling in carcinogenesis MJ Wolf et al 5008 Canonical pathway Noncanonical pathway  LT 3 TNF   LT 1 2

TNFR1 LTR TRAF2 TRAF2 TRAF6 TRAF5 TAB1 membrane- cytoplasm TAK1   TRAF3 NIK bound LT 1 2 TAB2/3 P P

P

  IKK IKK IKK phosphory- lation  NEMO IKK P P P

P P P proteasomal P degradation IB p100 RelB p50 RelA p50 RelA p52 RelB induction of

p50 RelA p52 RelB

target genes target genes nucleus

e.g. , cancer e.g. lymphoid neogenesis, cancer

Figure 1 TNFR and LTbR signaling pathways lead to the activation of NF-kB through different pathways. (Left) The canonical pathway of NF-kB activation is induced by a large number of agonists, representatively, TNF and LTa3 are shown. Stimulation of the TNF receptor (TNFR) leads to the activation of the TAK1 complex through TRAF . This leads to the activation of the IKK complex by phosphorylation of NEMO/IKKg and IKKb, which results in the phosphorylation of IkBa, its subsequent ubiquitylation and proteasomal degradation. Finally, the heterodimeric p50-RelA complex translocates to the nucleus, where it binds to specific kB-sequences and induces the expression of target genes, including pro-inflammatory cytokines or genes involved in cell survival, and pro-carcinogenesis. (Right) The noncanonical pathway becomes activated by membrane-bound LTa1b2 heterotrimers, which results in the activation of NF-kB-inducing kinase (NIK). This leads to the phosphorylation and activation of the homodimeric IKKa complex. As a consequence, the precursor p100 becomes phosphorylated and proteasomally processed to the p52 subunit establishing now the RelB-p52 heterodimeric complex that translocates to the nucleus and induces the expression of target genes. Besides, LTbR was shown to activate the canonical NF-kB signaling pathway.

(for example, CXCL13, CCL19, CCL21) (Ansel et al., promoter II (RIP) induces chemokine expression and 2000; McCarthy et al., 2006) and to induce a variety of follicular lymphocytic infiltrations with mature follicular pro- and anti-inflammatory chemokines (for example, dendritic cell networks, resulting in chronic insulitis and CCL5, CCL2, CXCL10) (Cuff et al., 1998, 1999). glomerulonephritis (Picarella et al., 1992; Kratz et al., The role of LT in the context of lymphoid neogenesis, 1996; Drayton et al., 2003). Inflamed sites in RIPLTa development of autoimmune and inflammatory or RIPLTab mice are vascularized with endothelia disorders was further investigated in vivo (Picarella showing morphologic characteristics of high endothelial et al., 1992; Kratz et al., 1996; Luther et al., 2000; venules (Picarella et al., 1992; Cuff et al., 1999; Drayton Drayton et al., 2003, 2006; Gommerman and Browning, et al., 2003). Further, it has been reported that LTbR 2003; Martin et al., 2004; Heikenwalder et al., 2005, signaling is a requirement for the homeostatic control of 2008; Haybaeck et al., 2009). These studies revealed high endothelial venule differentiation and function that ectopic LT expression suffices to generate (Browning et al., 2005). Overexpression of LTab under lymphoid-like structures (Picarella et al., 1992; Kratz the liver-specific albumin promoter (AlbLTab) led et al., 1996; Gommerman and Browning, 2003; to chronic hepatitis, organized portal and lobular Heikenwalder et al., 2005; Haybaeck et al., 2009), also lymphocytic infiltrates, follicular dendritic cells and termed tertiary lymphoid organs or tissues. LTa or germinal centers leading to mild, chronic liver damage LTab expression under the control of the rat insulin (Heikenwalder et al., 2005; Haybaeck et al., 2009).

Oncogene Unexpected role of LTbR signaling in carcinogenesis MJ Wolf et al 5009 Development of tertiary lymphoid organs is observed in encephalomyelitis and collagen-induced arthritis, reflect- hi multiple human infectious diseases, such as viral-induced ing a correlation between LTa TH1 and TH17 cells and chronic hepatitis (for example, hepatitis B virus (HBV), progression in the respective models (Chiang hepatitisCvirus(HCV))(Llovetet al., 2003), Helicobacter et al., 2009). pylori-driven gastritis (Genta et al., 1993; Kobayashi et al., 2004) or chronic lyme disease (Steere et al., 1988; Ghosh et al., 2005). Moreover, tertiary lymphoid organs also LT and cancer frequently appear in autoimmune diseases such as Sjo¨ gren’s syndrome (Hjelmervik et al.,2005),chronic As if the biological functions of LT were not multi- obstructive pulmonary disease, (RA) faceted enough, various studies have also suggested the (Young et al., 1984; Takemura et al., 2001), Graves’ involvement of LTbR signaling in cancer development. disease (Armengol et al., 2003; Aust et al.,2004)or Of these reports, some have focused more on the multiple sclerosis (Prineas 1979; Serafini et al., 2004). In integrity of LT at the genetic/genomic level and most of these diseases, elevated expression of LT or its the association of polymorphisms in the human LTA target genes has been detected, supporting LT’s role in gene with cancer, for example, as being predictive for lymphoid neogenesis (Drayton et al.,2006).Suppression the outcome of various cancer types. In the case of of LTbR signaling by injection of LTbR-Ig was diffuse, large B-cell lymphoma, a polymorphism in the efficacious in the treatment of various inflammatory or LTA gene, was described to be linked with faster rate of models (Gommerman and Brown- disease progression (Chae et al., 2010). It remains elusive ing, 2003; Ware, 2005; Drayton et al., 2006; Browning, how instructive these genetic studies will be, because 2008; Haybaeck et al., 2009). most LTA polymorphisms and their pathological conse- quences are not described in detail. Other studies have focused on the activation of Depletion of LThi expressing cells and curing autoimmune LTbR signaling pathways in various cancer types. diseases Bergsagel and colleagues reported a constitutive acti- vation of NF-kB signaling in as a Pleiotropic functions are attributed to LT in both health result of mutations in the LTbR gene (Keats et al., and disease (Tumanov et al., 2007)—which in some 2007), emphasizing a critical role for the NF-kB instances can go beyond the well-documented role of pathway in its pathogenesis. Furthermore, constitutive LT in the development and maintenance of lymphoid NF-kB activity was detected in diffuse large B-cell organs. This includes an important role of LT signaling lymphoma, indicating a role for this signaling pathway in the acceptance of donor stem cells, controlling as a primary pathogenic event in lymphomagenesis cuprizone-induced demyelination, the progression of (Compagno et al., 2009). experimental autoimmune encephalomyelitis and the In addition, LTbR-mediated signaling—most likely control of lipid metabolism or liver regeneration through the noncanonical NF-kB pathway—is also (Gommerman et al., 2003; Lo et al., 2007; Plant et al., involved in tumor . Activation of LTbR 2007; Markey et al., 2009; Ruddell et al., 2009; on fibrosarcoma cells by activated host lymphocytes is Tumanov et al., 2009). Studies with different mouse capable of initiating a novel, pro-angiogenic path- models revealed a role of LTbR signaling in athero- way leading to organized tumor tissue development. sclerosis development (Schreyer et al., 2002; Grabner Conversely, in this model, inhibition of LTbR signaling et al., 2009) and an involvement of LTbR signaling was prevented tumor angiogenesis and neovascularization, discovered in the pathogenesis of RA (Fava et al., 2003). resulting in tumor growth arrest (Hehlgans et al., 2002). Blocking LTbR signaling using a LTbR-Ig could prevent the onset of collagen-induced arthritis or reduce its severity, depending on the time Hepatic LTbR signaling and HCC point of LTbR-Ig treatment (Fava et al., 2003). On the basis of these results, clinical trials with patients Chronic inflammation, NF-kB activation and signaling suffering from RA have been initiated by Biogen Idec by members of the TNFSF have been shown to (Boston, MA, USA) to investigate the beneficial role of contribute to hepatocellular carcinoma (HCC) progres- LTbR-Ig (Baminercept). Baminercept has been studied sion. This is substantiated by analysis of multidrug in both healthy volunteers and in several phase 2 trials in resistance gene 2 knockout (mdr2À/À) mice, which RA with patients who have incomplete responses to spontaneously develop cholestatic hepatitis, elevated either disease-modifying anti-rheumatic drugs or TNF hepatocyte proliferation and subsequently HCC inhibitor therapy. The RA studies did not meet their end (Pikarsky et al., 2004). points for American College of (ACR) In a model of chemically-induced carcinogenesis, the score improvement and other indications are now being anticipated pro-tumorigenic role of canonical NF-kB pursued (Biogen Idec, unpublished data). signaling in hepatocytes was confounded by the finding hi Recently, targeted depletion of LTa TH1 and TH17 that mice lacking IKKb only in hepatocytes exhibited cells with a against LTa could a profound increase in hepatocarcinogenesis after inhibit autoimmune disease in mouse models for diethlynitrosamine (DEN) application (Maeda et al., delayed-type hypersensitivity, experimental autoimmune 2005; Maeda and Omata, 2008). This was associated

Oncogene Unexpected role of LTbR signaling in carcinogenesis MJ Wolf et al 5010 with augmented hepatocyte death, indicating that To determine whether sustained hepatic LTbR compensatory proliferation might be a crucial step in signaling is causally linked to hepatitis and liver cancer HCC development. However, the absence of IKKb in development, we analyzed mice expressing LTa and b both hepatocytes and bone marrow-derived Kupffer in a liver-specific manner (AlbLTab mice) (Haybaeck cells decreased compensatory proliferation and carcino- et al., 2009). From 6 months onward transgenic mice genesis. This suggests that canonical NF-kB activation developed hepatitis characterized by infiltrating B-, in Kupffer cells is essential for the promotion of T-cells, macrophages, hepatocyte and oval cell proli- compensatory hepatocyte proliferation resulting in feration. This caused mild liver damage documented by DEN-induced HCC. Using a transplant system which elevated serum transaminase (ALT/AST) levels and allows initiated mouse hepatocytes to form HCC, He enhanced production of cytokines/chemokines such as and colleagues studied late events of tumor progression IL6, IL1b, IFNg and CCL2. Chronic hepatitis incidence and malignant conversion in the DEN model (He et al., reached 100% in 9-month-old mice, with infiltrates 2010). Deletion of IKKb long after tumor initiation increasing in size over time (Figure 2). In approximately accelerated HCC development in a cell-autonomous one-third of the mice at an age X12 months chronic manner (He et al., 2010). Furthermore, the absence of inflammation caused the development of HCC. Tumors IKKb in hepatocytes was associated with STAT3 are characterized by the presence of Ki67 þ hepatocytes, activation. Hepatocyte-specific ablation of STAT3 expression of gp73 (GOLPH2), a-fetoprotein and gluta- blocked HCC development, suggesting an interface mine synthetase (Figure 2, Haybaeck et al., 2009). between NF-kB and STAT3 signaling in the context of Furthermore, array comparative genomic hybridization DEN-mediated HCC development. analysis revealed that HCC displayed chromosomal Other members of the IKK complex were also shown aberrations. to be crucially involved in the protection or promo- But is LT itself an oncogene causing HCC develop- tion of HCC. Ablation of NEMO/IKKg in hepatocytes ment and is hepatic activation of LTbRsufficientto results in the development of steatohepatitis and HCC cause inflammation-induced carcinogenesis? To address (Luedde et al., 2007). In this model, spontaneous HCC these questions, we crossed AlbLTab to various knock- development is preceded by steatohepatitis, increased out mice. Intercrossings with TNFR1À/À mice should hepatocyte apoptosis and compensatory liver regenera- answer whether the absence of TNFR1 signaling could tion. These findings suggest that NF-kB has an active prevent HCC formation. Moreover, the role of lympho- role in protecting the liver from cancer and emphasizes cytes and NF-kB signaling in the development the role of NEMO/IKKg as a tumor suppressor of inflammation-induced HCC formation was assessed (Luedde et al., 2007). Of note, it was described that by intercrossing AlbLTab mice to RAG1À/À mice, which the MAP3-kinase TGF-b-activated kinase 1 (TAK1) lack mature B- and T-cells, and to IKKbDhep mice, devoid suppresses a NEMO-dependent but NF-kB-independent of canonical NF-kB signaling in hepatocytes. AlbLTab/ pathway to liver cancer (Bettermann et al., 2010). RAG1À/À or AlbLTab/IKKbDhep mice did not display Recently, a causal involvement of LTbR signaling in chronic hepatitis or HCC, suggesting that lymphocytes chronic hepatitis and HCC was proposed. Patients and NF-kB signaling are tumor promoting. Further, this suffering from HBV- or HCV-induced chronic hepatitis suggested that LT itself is not an oncogene but rather that or HCC displayed increased hepatic expression of LTa, its sustained hepatic expression creates a pro-carcinogenic LTb and LTbR (Haybaeck et al., 2009). But which cell environment. Intercrossing to TNFR1À/À mice did not type is responsible for the elevated expression of LT in alter hepatitis or HCC incidence, suggesting that chronic chronic HCV-infected patients? Under physiological hepatitis and HCC formation in AlbLTab mice does not conditions, LT is predominantly produced by lympho- depend on pathways involving TNFR1. cytes. However, infection of Huh-7.5 cells with fully The major LT responsive liver cell type was identified infectious, cell culture-derived HCV also leads to by using IKKbDhep mice. I.v. administration of TNF elevated levels of LTa,LTb, LIGHT and LTbR to IKKbDhep mice did not cause p65 translocation transcripts 48 and 72 h after infection. Therefore, LT in hepatocytes but upregulated NF-kB target genes, expression in HCV-infected could stem from presumably through TNF-activated non-parenchymal lymphocytes and hepatocytes. Cells were collected cells (NPC). In contrast, treatment with an agonistic from curative resections of HCV-induced hepatitis and LTbR antibody (3C8) in IKKbDhep mice neither induced HCC specimens and sorted according to their CD45 nuclear p65 translocation in hepatocytes and NPC nor surface expression, resulting in CD45-enriched (T-, B- upregulation of selected NF-kB target genes. Therefore, and NK-cells; , macrophages/Kupffer cells; hepatocytes but not NPC are likely to be the major dendritic and NK-cells) or CD45-depleted (hepatocytes, cells integrating LT signaling in the liver. In addition, oval cells, bile duct epithelial and endothelial cells) upon 3C8 treatment, IKKaAA/AA livers upregulated fractions. CD45 þ cells showed elevated expression of selected NF-kB target genes, similar to C57BL/6 mice. LTa and LTb, both in HCV-induced hepatitis and Thus, IKKa deficiency in both hepatocytes and NPC HCC. However, also the CD45À cell fraction showed an does not block NF-kB target gene expression upon 3C8 elevation in LTa,LTb and LTbR mRNA expression in treatment, suggesting the involvement of the canonical liver tissues from HCV-induced hepatitis and HCC, NF-kB pathway in LTbR-induced hepatic signaling. which was corroborated by immunohistochemical Treatment of 9-month-old AlbLTab mice with analysis for LTb (Haybaeck et al., 2009). LTbR-Ig reduced the incidence of chronic hepatitis

Oncogene Unexpected role of LTbR signaling in carcinogenesis MJ Wolf et al 5011 a wild-type AlbLTαβ b wild-type AlbLTαβ AlbLTαβ B220 CD3 c H/E Collagen IV Ki67 wild-type F4/80 αβ Ki67 AlbLT

Figure 2 Development of chronic inflammation and HCC in mice expressing LTa and LTb under the control of the albumin promoter. (a) Immunohistochemical analysis of representative 9-month-old C57BL/6 and AlbLTab livers. B220 þ stained B-cells, CD3 þ T-cells, F4/80 þ macrophages, Kupffer cells and A6 þ oval cells (scale bar: 150 mm). Ki67 þ proliferating hepatocytes (arrowheads) and inflammatory cells are indicated (scale bar: 50 mm). (b) Macroscopy of C57BL/6 (left panel) and AlbLTab livers at the age of 12 (middle panel) and 18 months (right panel). White arrows indicate tumor nodules. White arrowhead indicates a liver lobe completely affected by HCC. Scale bar size is indicated. (c) Histological analysis of livers derived from C57BL/6 and AlbLTab mice at the age of 12 months. Dashed line depicts the HCC border. Collagen IV staining highlights the broadening of the liver cell cords and loss of collagen IV networks indicative of HCC in AlbLTab mice (scale bar: 200 mm). High numbers of Ki67 þ proliferating hepatocytes (arrowheads) are only found in AlbLTab HCC (right column; scale bar: 100 mm). Adapted from Haybaeck et al. (2009). and chronic hepatitis-driven HCC formation compared and enhances tumor chemosensitivity. Using a syngeneic to untreated mice, suggesting that suppression of LTbR colon carcinoma tumor xenograft model (CT26), it was signaling reduces chronic hepatitis incidence and subse- shown that treatment of tumor-bearing mice with a quent transition towards HCC (Figure 3a). This shows murine agonistic LTbR antibody (AC6H) caused in- that LT signaling is critically involved in hepatitis and creased infiltration and tumor necrosis. subsequent HCC development. Blocking LTbR signal- However, extensive studies revealed that there are human ing might become beneficial in liver diseases with cell lines resistant to CBE11 treatment, indicating that sustained hepatic LTbR signaling. A clinical trial using treatment success may depend on the cancer cell type. baminercept as an anti-viral and anti-cancer drug is Moreover, CBE11 treatment decreased tumor size and/or currently being initiated (Villanueva et al., 2009) improved long-term animal survival in two out of six and potential side effects of long-term blockade of LTbR orthotopic xenografts derived from independent surgical signaling in (for example, effects on lymphoid colorectal carcinoma samples. Inhibition of tumor growth tissue microarchitecture; NK-cell compartment) have by CBE11 could possibly result from direct activation of already been and will continue to be investigated. LTbR on tumor endothelial cells proposing two potential effects: (1) Involvement of LTbR signaling in interacting with mechanisms that control cell survival and tumor- Agonistic LTbR activation and suppression of solid tumor igenesis (Rayet and Gelinas, 1999; Wang et al., 2002). growth (2) LTbR-induced tumor inhibition may involve modula- tion of cell-cycle progression, thereby contributing to LTbR was shown to be expressed in several solid tumors, the tumor chemosensitivity (Lukashev et al., 2006). for example, colorectal, larynx/pharynx and lung tumors, Further, LTbR activation may interfere with tumor as well as in (Lukashev et al.,2006).Recently, progression through induction of host-mediated Lukashev et al. (2006) have proposed a mechanism by immunological responses, for example by the upregula- which agonistic activation of the LTbR using a mono- tion of pro-inflammatory and homeostatic cytokines clonal antibody (mAb; CBE11) suppresses tumor growth attracting various anti-tumor immune cells.

Oncogene Unexpected role of LTbR signaling in carcinogenesis MJ Wolf et al 5012 reduces/prevents primary tumorigenesis, Hepatic LT LT

causes HCC (1)   R-signaling inhibition (LT Lack of LT - re-emergence, (1-5) signaling causes enhanced tumor metastasis (7) B-cell derived LT causes CRCaP (2)

LTR-activaton Genomic LTR causes anti-tumor amplification 

immune-responses causes NPC (3) R-Ig/siRNA) (6)

Pro-angiogenesis, pro-tumor growth Negative selection of anti-tumor T-cells by activated LTR/MIP-2 signaling - FSC (5) by LTR signaling - causes CaP (4)

Figure 3 (a) Scheme of chronic inflammation-induced liver carcinogenesis in mice expressing LTa and LTb under the control of the albumin promoter. Transgenic hepatocytes (brown) express LTa, b and induce chemokine production (for example, CCL2, CCL7, CXCL1, CXCL10) in the presence of IKKb and intrahepatic lymphocytes. Chemoattraction and activation of myeloid cells and lymphocytes expressing particular chemokine receptors (for example, CXCR3, CXCR2, CCR2, CCR1) cause hepatitis: CXCL10 attracts CXCR3 þ T- and NK-cells, CXCL1 CXCR2 þ T- and B-cells and , CCL2 CCR2 þ macrophages and CCL7 attracts CCR1 þ monocytes. Activated, infiltrating immune cells secrete cytotoxic cytokines (for example, IL6, IL1b, TNFa, IFNg,LTab) that cause tissue destruction, hepatocyte proliferation, cell death and tissue remodeling. In such an environment, hepatocytes are susceptible to chromosomal aberrations leading to HCC. Tissue destruction and remodeling supports the infiltration of activated inflammatory cells (for example, myeloid cells) leading to a feed-forward loop towards chronic aggressive hepatitis. Asterisks indicate that genetic depletion of those components (IKKb; T- and B-cells) blocks chronic hepatitis development and HCC. Blocking LTbR signaling with LTbR-Ig in 9-month-old AlbLTab mice reduces chronic hepatitis incidence and prevents HCC. þ , indicates the fortification of a described process; B, indicates the suppression of a described process. The RelA is schematically depicted as a green circle, inducing transcription of NF-kB target genes (for example, chemokines) (arrow). B, T, B- and T-cells; MØ, macrophages; N, neutrophils; NK, NK-cells. Adapted from Haybaeck et al. (2009). (b) Scheme summarizing in vivo studies showing the pro- and anti-tumorigenic effects of LTbR activation or inhibition as well as consequences of LTa deficiency on anti-tumor surveillance (colored sectors). Experiments describing the pro- or anti-carcinogenic effects (depicted with þ or À) of activated LTbR signaling in vivo (Hehlgans et al., 2002 (5); Lukashev et al., 2006 (6); Haybaeck et al., 2009 (1); Or et al., 2009 (3); Zhou et al., 2009 (4); Ammirante et al., 2010 (2)). (Gray sector) The gray sector indicates that also lack of LTa signaling can influence anti-tumor surveillance causing significant enhancement of experimental pulmonary metastases upon injection of B16F10 (Ito et al., 1999 (7)). (Right column) Depicts various studies indicating that block of LTbR signaling might work as an anti-tumor strategy. CaP, prostate cancer; CRCaP, castration resistant prostate cancer; FSC, fibrosarcoma; HCC, hepatocellular carcinoma; NPC, nasopharyngeal carcinoma.

Oncogene Unexpected role of LTbR signaling in carcinogenesis MJ Wolf et al 5013 LT and anti-tumor T-cells current therapy. Interventions that prevent LT production or ablate LT signaling could delay regressing tumors in In contrast, genetic or conditional blockade of LTbR patients with androgen ablation therapy. signaling was recently shown to lead to an effective anti- tumor T-cell response (Zhou et al., 2009). The researchers investigated a nonantigen-based cancer immune preven- Involvement of LTbR signaling in head and neck cancer tion strategy using the transgenic adenocarcinoma pros- tate mouse model (TRAMP) that expresses the SV40 In a study on nasopharyngeal carcinoma (NPC), a T- controlled by rat probasin regulatory elements. distinct type of head and neck cancer, LTbR was shown TRAMP mice spontaneously develop prostate cancer to be frequently overexpressed in primary NPC tumors, with 100% penetrance and metastases. LTa deficiency in concomitant with an amplification of chromosome TRAMP mice prevented efficient negative selection of 12p13.3 (Or et al.,2009).In vitro studies in nasopharyn- tumor-reactive T-cells, reduced primary prostate cancer geal epithelial cells revealed increased NF-kBactivityand incidence by approximately 50%, significantly reduced the cell proliferation due to LTbR overexpression. Addition size of prostate and almost completely ablated of LTa1b2 led to increased proliferation of naso- metastases. Similarly, treatment with LTbR-Ig interrupted pharyngeal epithelial cells and siRNA knockdown of clonal T-cell deletion, reduced the size of the primary LTbR expression inhibited growth of the NPC tumors cancer and completely prevented further metastasis, with 12p13.3 amplification (Or et al.,2009),further showing the value of nonantigen-based immune preven- strengthening a tumor-promoting role of LTbRsignaling tion for individuals with genetic predisposition to cancer in this type of carcinoma. (Zhou et al., 2009). Involvement of LTa signaling in anti-tumor surveillance

B-cell-derived LT controls reemergence of castration- Interestingly, LTa signaling may also be involved in the resistant prostate cancer maturation and recruitment of NK-cells and may have an important role in anti-tumor surveillance. Ito et al. A study by Ammirante et al. (2010) deals with (1999) have shown that LTa deficiency leads to defects progression of prostate cancer (CaP), mainly focusing in NK maturation and recruitment and that formation on the mechanisms underlying the recurrence of of experimental pulmonary metastases upon injection of castration-resistant (CR) metastatic carcinomas. In B16F10 melanoma cells was enhanced in LTaÀ/À mice addition to the previously described TRAMP model, (Ito et al., 1999). Therefore, LTa signaling may be subcutaneous allografts of mouse androgen-dependent involved in NK-cell maturation, recruitment and anti- AD-CaP cell line myc-CaP, were used to facilitate better tumor surveillance. mechanistic analysis of CR-CaP development. Of note, LT’s involvement is very unlikely to be To determine whether inflammation-responsive IKKb universal to all cancer types or signaling pathways participates in development of CR-CaP, Ammirante and contributing to or enabling carcinogenesis: In an colleagues deleted IKKb in prostate epithelial cells of illuminating study, Kuprash et al. (2008) showed that TRAMP mice. This neither affected primary tumor neither lack of TNF nor LTa reduced tumor burden growth nor CR-CaP emergence after castration on a -deficient background, suggesting that at least (Ammirante et al., 2010). In contrast, specific deletion in the context of p53-deficiency LTa signaling does not of IKKb in bone marrow-derived cells delayed devel- aggravate carcinogenesis. opment of CR-CaP emergence, although lacking effects on primary tumor development. These results, suggested an involvement of immune cells promoting tumor Summary emergence after castration. Indeed, after androgen ablation the regressing tumors were infiltrated with Recent reports convincingly show an involvement of LTbR T- and B-cells, NK-cells and myeloid cell types. signaling in inflammatory processes and carcinogenesis Furthermore, inflammatory cytokines, for example, (Table 1)—however, they may involve distinct mechan- IL-6, IL-12, TNFa and LT, were upregulated in the isms in the various in vivo paradigms tested so far. What regressing myc-CaP allografts. Further, STAT3, an anti- could be the reason(s) why increased LTbR signaling apoptotic and pro-tumorigenic transcription factor, was generates in some instances a pro-carcinogenic (Hay- activated during CR-CaP emergence. Notably, LT baeck et al., 2009) and in others an anti-carcinogenic ablation on B-cells, but not on T-cells delayed CR- environment (Lukashev et al., 2006)? CaP growth. Accordingly, in addition to mediators Data by Lukashev et al. (2006) indicate that agonistic released by dying CaP cells, critical participants in LTbR antibody treatment induces lymphocyte tumor emergence are tumor-infiltrating B-cells, produ- infiltration and tumor necrosis in a syngeneic colon cing LT that signals through LTbR on CaP cells to tumor model. One proposed mechanism might be the induce IKKa nuclear translocation and STAT3 activa- induction of chemokine expression, functioning as tion. This enhances androgen-independent growth of chemoattractant for T-, NK- and dendritic cells, driving CaP cells. Therefore, the inflammatory response elicited by tumor destruction. Whether these results are applicable the dying primary tumor contributes to the failure of to other tumor types needs to be investigated.

Oncogene Unexpected role of LTbR signaling in carcinogenesis MJ Wolf et al 5014 Table 1 Summary of experiments leading to disease regression in rodent models due to conditional interference with LTbR signaling Target Disease Model References

LT signaling in cancer LTab expressing Prostate cancer (primary tumor, TRAMP (SV40-Tag); AD-CaP cell (Ammirante et al., 2010) B-cells reemergence of CRCaP) line, myc-CaP LTbR signaling Colon carcinoma Syngeneic colon carcinoma tumor (Lukashev et al., 2006) xenograft model Fibrosarcoma LTbR-Ig expression on tumor cells (Hehlgans et al., 2002) Hepatocellular carcinoma AlbLTab mice (Haybaeck et al., 2009) Nasopharyngeal carcinoma Transplantation of tumors with (Or et al., 2009) amplification 12p13.3 Prostate cancer (primary tumor, TRAMP (SV40-Tag) (Zhou et al., 2009) metastasis)

LT signaling in infectious diseases Prion disease Scrapie infection (Montrasio et al., 2000) Viral LCMV infection (Puglielli et al., 1999)

LT signaling in autoimmune disease Arthritis Classical CIA (Fava et al., 2003) Adjuvant arthritis (Fava et al., 2003) Hepatitis Con A hepatitis (An et al., 2006; Anand et al., 2006) AlbLTab mice (Haybaeck et al., 2009) Inflammatory bowel disease Inflammatory CD45RBhi (Mackay et al., 1998) CD3eTg26 (Mackay et al., 1998) DSS (Stopfer et al., 2004) TNBS (An et al., 2005) Th2-like TNBS (Dohi et al., 2001) Multiple sclerosis PLP_SJL EAE (Columba-Cabezas et al., 2006; Gommerman et al., 2003) MBP-rat acute EAE (Gommerman et al., 2003) Cuprizone (Plant et al., 2007) Sjo¨ gren’s syndrome Male NOD (Gatumu et al., 2009) Systemic erythematosis Adeno-IFN BWFI Biogen Idec (Jeffrey Browning et al., unpublished data) Pristane SNFI Biogen Idec (Jeffrey Browning et al., unpublished data) GVHD skin (Wu et al., 2004) GVHD chronic (Tamada et al., 2000, 2002) Type I diabetes Female NOD (Ettinger et al., 2001; Lee et al., 2006; Wu et al., 2001) R16 immunization (Shao et al., 2003)

LTa or LTab Arthritis CIA (Chiang et al., 2009) expressing T-cells Delayed type hypersensitivity KLH/CFA (Chiang et al., 2009) Multiple sclerosis EAE (MBP-TCR tg mice) (Chiang et al., 2009)

Abbreviations: AdCaP, androgen-dependent prostate cancer; CIA, collagen-induced arthritis; CRCaP, castration-resistant prostate cancer; DSS, dextran sulfate sodium; EAE, experimental autoimmune encephalomyelitis; GVHD, graft vs host disease; KLH/CFA, Keyhole Limpet Hemocyanin/complete Freud’s adjuvant; mycCaP, c-myc transgenic mouse with prostate cancer; TNBS, 2,4,6-trinitrobenzenesulfonic acid.

LTbR-Ig treatment or genetic depletion of LTa signaling and cancer: (1) at the site of carcinogenesis in TRAMP mice interrupted clonal T-cell deletion, and (2) at the site where the host’s adaptive immune reduced the size of the primary CaP and prevented response is triggered against cancer (Figure 3b). Exciting further metastasis (Zhou et al., 2009), suggesting a times are ahead, verifying the utility of LTbR antago- role of LT in controlling anti-tumor T-cell selection and nists or agonists in the different clinical settings primary cancer development. Moreover, depletion and hopefully revealing the detailed mechanisms of LT expression on B-cells—and not on T-cells— of LTbR signaling in the context of inflammation and delayed CR-CaP recurrence (Ammirante et al., 2010), cancer. pointing toward a therapeutic application of LTbR signaling inhibition even as a tool to fight cancer re-emergence. Conflict of interest In summary, there is a compelling case for potential roles of LTbR signaling in carcinogenesis. However, Dr Mathias Heikenwa¨ lder is in the process of designing more is to be learned about the link between LT a clinical trial using LTBR-Ig (baminercept) in hepatitis

Oncogene Unexpected role of LTbR signaling in carcinogenesis MJ Wolf et al 5015 C virus-infected patients, which would be partially was supported by grants of the Oncosuisse foundation (OCS funded by Biogen Idec. The planned trial is not yet 02113-08-2007), the Novartis Stiftung fu¨ r Biologisch-Medizi- approved. Dr Nicolas Zeller, Monika Wolf and Gitta nische Forschung (Nr. 09C62), the ‘Stiftung zur Schweizer- Seleznik declare no conflict of interest. ischen Krebsbeka¨ mpfung’, the Helmholtz-foundation, the research foundation at the Medical Faculty Zurich, the Acknowledgements Julius-Mu¨ ller foundation and the ‘Kurt und Senta Hermann Stiftung’. MJW was supported by a grant of the Roche We thank Dr Tracy O’Connor, Dr Barbara Stecher, Jay Research Foundation. MH is a fellow of the Professor Dr Max Tracy and Lukas Frick for critically reading this paper. MH Cloe¨ tta foundation.

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