Oncogene (2011) 30, 631–641 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc REVIEW Emerging roles for the non-canonical IKKs in

RR Shen1 and WC Hahn1,2

1Department of Medical Oncology, Dana Farber Cancer Institute, Boston, MA, USA and 2Broad Institute of Harvard and MIT, Cambridge, MA, USA

The IjB (IKK)-related TBK1 and IKKe to form the mature p50 and p52 . This have essential roles as regulators of innate immunity by processing is necessary for p50 and p52 to function as modulating interferon and NF-jB signaling. Recent work transcription factors (Dolcet et al., 2005). Distinct has also implicated these non-canonical IKKs in malignant NF-kB complexes are formed from combinations of transformation. IKKe is amplified in B30% of breast homo- and heterodimers of these family members and transforms cells through the activation of (Bonizzi and Karin, 2004). NF-kB complexes are NF-jB. TBK1 participates in RalB-mediated inflamma- retained in the cytoplasm by a family of NF-kB-binding tory responses and cell survival, and is essential for the proteins known as the inhibitors of NF-kB(IkBs). survival of non-small cell lung cancers driven by oncogenic A variety of inflammatory stimulants initiate the KRAS. The delineation of target substrates and down- induction of NF-kB and trigger activation of the IkB stream activities for TBK1 and IKKe has begun to define Kinase (IKK) complex, which is composed of the their role(s) in promoting tumorigenesis. In this review, we catalytic kinases IKKa and IKKb, and the regulatory will highlight the mechanisms by which IKKe and TBK1 NF-kB essential modifier (NEMO, or IKKg) (Perkins, orchestrate pathways involved in inflammation and cancer. 2007). One important function of the IKK complex is to Oncogene (2011) 30, 631–641; doi:10.1038/onc.2010.493; mark IkB for and ubiquitination, published online 1 November 2010 which in turn, leads to proteasomal degradation. This activity facilitates the release and accumulation NF-kB Keywords: NF-kB; cancer; IKK dimers in the nucleus where a transcriptional program involving many target related to immune response are activated. Although the classical NF-kB pathway primarily involves the activation of RelA and p50 and is Introduction strictly facilitated by the IKK complex, an alternative pathway is active in B cells where NF-kB inducing The NF-kB pathway is a pivotal regulator of several kinase (NIK) promotes the activation of RelB and p52/ important physiological functions including the inflam- p100 complexes (Ghosh and Hayden, 2008). Genetic matory immune response, proliferation, cell survival experiments have demonstrated that IKKb is the and cell invasion. These activities are well-described principal IKK for the canonical NF-kB pathway, hallmarks of cancer, and NF-kB activation has been whereas IKKa has a more dominant role in the non- observed in a wide range of tumors (Dolcet et al., 2005; canonical pathway (Bonizzi and Karin, 2004; Paspar- Baud and Karin, 2009; Prasad et al., 2010), leading some akis et al., 2006; Perkins, 2007). In addition to the to suggest that NF-kB serves as a bridge between conventional IKKs, a related pair of non-canonical inflammation and cancer (Baldwin, 2001; Karin, 2006). kinases, IKKe (IKKi, encoded by IKBKE) and TBK1 Activation of NF-kB in cancer arises either from (NAK), have been identified as important mediators of extrinsic signals in the tumor microenvironment or both inflammatory and oncogenic signaling. from intrinsic dysregulation of the pathway within the tumor. In either case, several different components of the NF-kB signaling cascade may contribute to this IKKe and TBK1 function in inflammation activity in particular cancers. In mammals five NF-kB members exist including IKKe was simultaneously identified in a subtractive RelA (p65), RelB, c-Rel, p50/p105 (NF-kB1) and p52/ hybridization screen as a LPS-inducible and as a p100 (NF-kB2). Although RelA, RelB and cRel are PMA-inducible with a kinase domain that is synthesized as mature forms, p105 and p100 are 27% identical to IKKa and IKKb (Shimada et al., 1999; synthesized as longer precursor proteins that are cleaved Peters et al., 2000). Concurrently, TBK1, which exhibits 49% identity and 65% similarity to IKKe was identified Correspondence: Dr WC Hahn, Department of Medical Oncology, as an interaction partner with the scaffolding molecule, Dana Farber Cancer Institute, 44 Binney Street, Dana 1538, Boston, TRAF-associated NF-kB activator (TANK) (Pomerantz MA 02115, USA. E-mail: [email protected] and Baltimore, 1999). Both IKKe and TBK1 are Received 30 July 2010; revised 15 September 2010; accepted 18 comprised of an N-terminal kinase domain, an ubiquitin- September 2010; published online 1 November 2010 like domain, a C-terminal LZ and a HLH motif Roles for the non-canonical IKKs in cancer RR Shen and WC Hahn 632 455 483 738 743 double stranded RNA and DNA initiate signaling IKKα 1 Kinase domainLZ HLH NBD 745 through intracellular RNA and DNA sensors such as 15 301 599 638 RIG-I, MDA-5 and DAI (Andrejeva et al., 2004; 311 388 603 642 Yoneyama et al., 2004; Kawai et al., 2005; Meylan IKKβ 1 Kinase domainULD LZ HLH NBD 756 et al., 2005; Seth et al., 2005; Takaoka et al., 2007). IFNb also activates a toll-like receptor-independent 15 300 458 486 737 742 350 383 W445S 578 619 pathway by stimulating IKKe phosphorylation of STAT1 to facilitate binding with ISGF3, a complex ε IKK 1Kinase domain ULD LZ HLH 716 that serves as the transcriptional machinery important 9 300G417D 500 527 for activating a subset of interferon response genes 305 383 591 632 (Tenoever et al., 2007). Moreover, engagement of TBK1 1Kinase domain ULD LZ HLH 730 both toll-like receptor-dependent and -independent 9 300 499 527 P675L pathways recruits additional scaffolding molecules Figure 1 Structural comparison of the classical and non-canonical including FADD, TRADD, MAVS, NAP1, HSP90 IKKs. The major domains of each IKK kinase are depicted with and SINTBAD which are necessary for IKKe amino-acid numbers that correspond to the human proteins. The and TBK1-mediated interferon activation (Rothe kinase domain of IKKe exhibits 27% and 24% identity to IKKa et al., 1996; Balachandran et al., 2004; Hacker et al., and IKKb, respectively, and TBK1 shares 49% identity and 65% 2006; Oganesyan et al., 2006; Yang et al., 2006; Gatot similarity to IKKe. Somatic mutations of IKKe and TBK1 recently identified in lung adenocarcinomas are marked in red. ULD, et al., 2007; Guo and Cheng, 2007; Ryzhakov and Ubiquitin-like domain; LZ, leucine zipper; HLH, helix-loop-helix; Randow, 2007; Michallet et al., 2008). Thus, IKKe and NBD, NEMO-binding domain (May et al., 2004; Hiscott et al., TBK1 form several protein complexes, the composition 2006; Perkins, 2007; Kan et al., 2010). of which is dependent on the type of cellular stimuli. Ultimately, these signaling complexes share a role in activating interferon responses for achieving an antiviral state. (Figure 1). Despite their similarity in structure, TBK1 The ability of IKKe and TBK1 to activate the and IKKe exhibit differential expression patterns. interferon response is also dependent on post transla- TBK1, like IKKa and IKKb, is ubiquitously expressed tional modifications by -independent Lys-63 (Shimada et al., 1999). In contrast, IKKe expression is linked ubiquitin chains (Figure 2). Both IKKe and restricted to particular tissue compartments, with high- TBK1 have an ubiquitin like domain, and Lys-63 est levels detected in lymphoid tissues, peripheral blood ubiquitination of both kinases are promoted by TRAF3, lymphocytes and the pancreas (Shimada et al., 1999). which itself is Lys-63 ubiquitinated. Disruption of this Various epithelial-derived cell lines also exhibit IKKe activity by TAXBP1 and the deubiquitinase A20 ablates expression (Shimada et al., 1999; Gravel and Servant, the interferon response (Ikeda et al., 2007; Parvatiyar 2005; Honda et al., 2005; Bibeau-Poirier et al., 2006). et al., 2010). Both TBK1 and IKKe also mediate Mitogenic stimulation with LPS and TNFa can also ubiquitination of TANK by an unknown E3 induce IKKe and TBK1 expression in a NF-kB (Gatot et al., 2007). Although TANK further serves as a dependent manner (Shimada et al., 1999; Kravchenko phosphorylation target of TBK1 and IKKe, TANK et al., 2003; Hemmi et al., 2004). With these partially ubiquitination seems to occur independently of this overlapping characteristics, IKKe and TBK1 are func- kinase activity. The mechanism by which TANK tionally more similar to each other than the canonical ubiquitination contributes to the activation of IKKe IKKs (Clement et al., 2008). and TBK1 is unknown. Lys-63 ubiquitination also has a role in the negative regulation of IKKe and TBK1 induced responses. For example, during- by RNA The non-canonical IKKs coordinate the interferon viruses, Lys-63 ubiquitination of MAVS is essential for response the recruitment of IKKe and leads to the inhibition of IKKe and TBK1 are critical inducers of interferon antiviral and NF-kB induced inflammatory genes (Paz signaling in response to viral infection (Fitzgerald et al., et al., 2009). Ultimately, these modifications will likely 2003; Sharma et al., 2003). Following activation of toll- dictate a dynamic system of regulating IKKe and like receptors by viral components, IKKe and TBK1 TBK1-mediated function in both inflammation and assemble with TRAF3 and TANK to phosphorylate cancer. interferon regulatory factors (IRFs) 3, 5 and 7 at multiple serine and threonine residues (Pomerantz and Baltimore, 1999; McWhirter et al., 2004; Mori et al., The non-canonical IKKs function as NF-kB effectors 2004; Caillaud et al., 2005; Cheng et al., 2006). This Although TBK1 and IKKe are not a part of the classical activity allows for heterodimerization and nuclear IKKa/b/g signaling complex, these kinases were origin- translocation of the IRFs and induction of pro- ally characterized as activators of NF-kB and target inflammatory and antiviral genes, including IFN-a/b multiple NF-kB members and effectors. Both IKK- (Lin et al., 1998; Sato et al., 2000). Toll-like receptor- related kinases phosphorylate IkBa at, one of the two- independent mechanisms also activate IKKe and TBK1 serine residues typically targeted on IkBa. Although to induce the interferon response. In this scenario, viral IKKe phosphorylates Ser36, TBK1 phosphorylates Ser32

Oncogene Roles for the non-canonical IKKs in cancer RR Shen and WC Hahn 633 Non-Cell Autonomous Cell Autonomous

TLR

TRAF3 TRAF6 RAS ?

p γ CYLD IKK p RalGEF IKKα IKKβ TRAF3

TANK

TBK1 IKKε IKKε p p p p TBK1 p κ α p TANK cREL p65 I B Sec5 RalB IRF3 Exocyst Complex p p IRF5 IRF7 IκBα degradation BCL-xL

Type I IFN Survival/inflammatory NF-κB NF-κB genes genes IRF IRF

p phosphorylation

K63-linked polyubiquitination

K48-linked polyubiquitination

Figure 2 Cell autonomous and non-cell autonomous roles of IKKe and TBK1 in inflammation and cancer. (a) Engagement of toll- like receptors initiates Lys-63 linked ubiquitination of TRAF3 and recruitment of IKKe and TBK1 through the adaptor molecule TANK. This recruitment promotes Lys-63 linked ubiquitination and activation of IKKe/TBK1. TANK is modified by both ubiquitination and phosphorylation through IKKe/TBK1, although it is unclear how these activities are related. The TANK/IKKe/ TBK1 complex can then target interferon response factors (IRF3, IRF5 and IRF7) for phosphorylation, and promote translocation to the nucleus where IFN target genes are activated. This cascade primarily defines the non-cell autonomous functions of the IKK-related kinases. In parallel to the interferon pathways, IKKe and TBK1 also phosphorylate many NF-kB effectors such as IkBa, p65 and cREL in response to cellular stimuli. This activity facilitates NF-kB nuclear translocation as a homo or hetero dimer and induces numerous inflammatory and survival-related target genes. (b) The IKKe/TBK1 oncogenic pathways function in a cell autonomous manner and are predominantly driven by aberrant levels of IKKe/TBK1. In this context, oncogenic RAS activates RalGEF, which in turn recruits the exocyst complex consisting of TBK1, RalB and Sec5, among other secretory proteins. TBK1 mediates phosphorylation of multiple effectors including Sec5, IKKb and IkBa that ultimately lead to the activation of both BCL-xL and NF-kB. Similarly, aberrant levels of IKKe in cancer also promote NF-kB activation through the phosphorylation of CYLD. CYLD negatively regulates NF-kB signaling by deubiquitinating Lys-63 linked ubiquitin chains on TRAF6 and IKKg. It is hypothesized that IKKe-mediated phosphorylation of CYLD leads to either its degradation or sequestration, thereby inactivating its negative role on the NF-kB signaling cascade.

(Pomerantz and Baltimore, 1999; Shimada et al., 1999; has a similar role in IKKe/TBK1-mediated NF-kB Tojima et al., 2000). IKKe overexpression reduces IkBa activation as it does in interferon activation and levels suggesting that phosphorylation at a single residue functions as both a substrate and critical adaptor may result in increased IkBa turnover. However, it molecule for these kinases. Furthermore, in most cells, remains possible that the IKK-related kinases phos- of TBK1 and IKKe is readily phorylate a canonical IKK that in turn leads to IkBa detected. However, it has also been suggested that other turnover (Eddy et al., 2005; Boehm et al., 2007). The kinases may phosphorylate the IKK-related kinases canonical NF-kB, RelA/p65, is another substrate for (Gatot et al., 2007; Ikeda et al., 2007). The significance IKKe and TBK1, and phosphorylation of RelA at of these phosphorylation events remains unclear. serine 536 by these kinases occurs at a basal level Although closely related, TBK1 and IKKe also independently of extracellular stimuli (Fujita et al., phosphorylate distinct substrates, and may activate 2003; Buss et al., 2004). cRel is also phosphorylated by NF-kB through different mechanisms (Table 1). For both IKK-related kinases. This activity is sufficient to example, TBK1 is the only of the two IKK-related promote nuclear translocation, but does not modulate kinases to phosphorylate and activate IKKb, suggesting downstream NF-kB activity (Harris et al., 2006). TANK that TBK1 may function preferentially on canonical

Oncogene Roles for the non-canonical IKKs in cancer RR Shen and WC Hahn 634 Table 1 Differential substrates of the non-canonical IKKs Substrates Targeted region Phosphorylation site Suggested function References

IKKe specific substrates RelA/p65 TAD Ser 468 NF-kB activation Mattioli et al. (2006) p100/p52 — Not phosphorylated Transactivation by p65 Wietek et al. (2006) STAT1 C-Terminal Ser 708 ISGF3 stability Tenoever et al. (2007) CYLD Central Serine 418 NF-kB activation Hutti et al. (2009) ERa N-Terminal, AF-1 Serine 167 ER Transactivation Guo et al. (2010)

TBK1 specific substrates Sec5 Ral binding domain Unknown Interferon induction Chien et al. (2006) IKKb Activation loop Ser 177/181 IKK activation Tojima et al. (2000) DDX3X DEAD and helicase domains Ser 181/183/240/269/429/ Interferon induction Soulat et al. (2008) 442/456/520, Thr 438 IR C-Terminal Ser 994 Insulin resistance Munoz et al. (2009)

Abbreviations: ER, estrogen receptor; IKK, IkB Kinase; IR, insulin Receptor.

NF-kB signaling (Tojima et al., 2000). On the other These models demonstrate that although the non- hand, in stimulated T cells, IKKe targets a second canonical IKKs are sufficient, but not essential for residue on p65/RelA, serine 468. Although phosphor- NF-kB activation, IKKe and TBK1 have a key role in ylation at this site occurs in addition to the serine 536 the induction of interferon signaling. residue that is phosphorylated by both IKK-related kinases, it is not clear whether this event occurs in other cellular contexts (Mattioli et al., 2006). IKKe also associates with p100/p52 in a ternary complex Extrinsic versus intrinsic NF-kB activation in cancer with p65 following TNF induction, and this interac- tion facilitates transactivation of p52-dependent genes In addition to a role in facilitating inflammatory (Wietek et al., 2006). Moreover, recent experiments responses to foreign agents, the IKK-related kinases indicate that IKKe can associate with the chromatin were recently recognized as NF-kB effectors that of promoters of specific NF-kB target genes (Moreno contribute to tumorigenesis, and thus represent a link et al., 2010). between NF-kB-mediated inflammation and cancer. Activation of NF-kB by the IKK-related kinases The contribution of NF-kB to inflammation-asso- occurs in response to specific stimuli. PMA-induced NF- ciated cancer has been studied extensively, and inhibi- kB activation is dependent on IKKe (Peters et al., 2000). tion of the canonical pathway in mouse models of Similarly, induction of NF-kB by the T-cell receptor inflammation-associated cancer demonstrates that involves TBK1 (Tojima et al., 2000). In contrast, neither NF-kB is often essential for cancer initiation and IKKe or TBK1 are required for TNF- or IL-1-induced progression (Greten et al., 2004; Pikarsky et al., 2004; NF-kB activation (Pomerantz and Baltimore, 1999; Lawrence et al., 2005; Karin, 2006). For example, Peters et al., 2000). The role of the IKK-related kinases deletion of IKKb in either epithelial or myeloid cells in NF-kB activation is not well understood and seems to of a mouse model of colitis-associated cancer dramati- be highly dependent on specific cellular stimuli. This cally decreases tumor incidence by B80% (Greten et al., area has been extensively reviewed elsewhere (Hacker 2004). Anti-TNFa treatment and introduction of the et al., 2006; Chau et al., 2008; Clement et al., 2008). NF-kB super repressor in a model of inflammatory Ultimately, the multiple modes of IKKe and TBK1- hepatocellular carcinoma also induces and mediated NF-kB activation highlight both independent inhibition of tumor progression (Pikarsky et al., 2004). and synergistic relationships that are likely dictated by These observations underscore how malignant cells are cellular and signal-induced contexts. critically dependent on the inflammatory microenviron- In consonance with these biochemical observations, ment for their survival. However, it is difficult to assess genetically engineered mice lacking either Tbk1 or Ikbke whether there are additional cell autonomous contribu- exhibit distinct phenotypes. Tbk1 deficient animals are tions of NF-kB activation in inflammation-associated embryonic lethal and die at E14.5 due to extensive fetal cancer models, and it is likely that both cell autonomous liver degeneration, a phenotype that is also observed in and non-cell autonomous functions are involved. IKKg, IKKb and RelA deficient animals (Bonnard Although much focus of NF-kB activation has et al., 2000; Hemmi et al., 2004). By contrast, Ikbke revolved around inflammation-associated cancer, this deficient animals are viable, but are impaired in pathway has a direct cell autonomous role in transfor- initiating a productive IFN-b response (Bonnard et al., mation and tumorigenesis. Activation of NF-kBin 2000; Hemmi et al., 2004). Interestingly, NF-kB activa- tumor cells is frequently observed in both solid tumors tion in either Tbk1 or Ikbke single and double knockout and hematological malignancies (Dolcet et al., 2005; models is predominantly normal, apart from minimal Basseres and Baldwin, 2006; Baud and Karin, 2009). defects in the induction of select NF-kB target genes. Until recently, it was assumed that this activation is the

Oncogene Roles for the non-canonical IKKs in cancer RR Shen and WC Hahn 635 result of immune stimuli originating from the micro- catalytic subunit alpha transgenic mouse environment. However, somatic mutations of compo- model of mammary adenocarcinoma exhibit higher nents in the NF-kB pathway were recently described, levels of IKKe. Likewise, overexpression of casein and provide an alternative means of NF-kB activation kinase 2 in MCF10F cells also increased IKKe expres- in tumors. For example, mutations in NF-kB family sion. These results suggested that casein kinase 2 might members NFKB1 (encoding p105/p50) and NFKB2 have an important role in regulating IKKe. In addition, (encoding p100/p52) as well as genes of which protein expression of the kinase-inactive form of IKKe in breast products regulate the IKK complex such as CIAP1, cancer cells reduced levels of two NF-kB target genes, CIAP2, CYLD, NIK and TRAF3 are associated with the D1 and RelB, as well as anchorage-independent pathogenesis of multiple myeloma (Annunziata et al., growth and invasion in matrigel (Eddy et al., 2005). Adli 2007; Keats et al., 2007; Baud and Karin, 2009). The et al. also found elevated IKKe expression in prostate application of targeted sequencing analyses in other and breast lines. In this work, IKKe was lymphoid malignancies including diffuse large found to activate NF-kB at a basal level by phosphor- and marginal zone has led to the ylating serine 536, and inhibition of this activity identification of mutations in additional regulators of significantly suppressed cancer cell proliferation (Adli the NF-kB pathway including A20, CARD11, TRAF2, and Baldwin, 2006). Together, these studies provided TRAF5, TAK1 and RANK (Compagno et al., 2009; the first evidence of IKKe involvement in breast cancer. Novak et al., 2009). Furthermore, a more recent More recently, IKBKE was identified as a breast genomic analysis of somatic copy-number alterations oncogene through the intersection of three complemen- across a large subset of 3131 human cancer tissues and tary genomic approaches. First, Boehm et al. developed cell lines found amplifications of major NF-kB regula- an experimental model of cell transformation that was tors including TRAF6, IKBKB, IKBKG, IRAK1 and dependent on both MEK and AKT signaling. By RIPK1 (Beroukhim et al., 2010). Importantly, all of screening kinases that could replace AKT and induce these genes were significantly amplified in epithelial tumorigenesis, we found that IKKe and CSNK1E were cancers, suggesting that alterations of NF-kB effectors able to transform such cells (Boehm et al., 2007). In are far more frequent than previously appreciated. parallel, loss-of-function screens to identify kinases that In addition to direct mutations in the NF-kB path- were essential for the proliferation and survival of breast way, NF-kB activation is observed in tumors harboring cancer cell lines revealed that both IKKe and CSNK1E mutations in numerous oncogenes including HRAS, were required for the survival of breast cancer cell lines. ERB2, PI3K and Bcr-Abl, the viral oncoprotein TAX, Of these two genes, IKBKE is amplified in 8 of 49 Vav-Pim2, BRAF, MUC1 and BMI-1 (Finco et al., (16.3%) breast cancer cell lines and in over 30% of 1997; Reuther et al., 1998; Pianetti et al., 2001; Xiao primary breast tumors (Boehm et al., 2007). FISH et al., 2001; Ikenoue et al., 2003; Biswas et al., 2004; analysis further confirmed increased copy number of Gustin et al., 2004; Hammerman et al., 2004; Basseres IKBKE by up to 10 additional copies is the consequence and Baldwin, 2006; Li et al., 2010) Although activation of these amplification events. Taken together, these of the NF-kB pathway has been documented in each of studies suggested that IKBKE was a breast cancer these contexts, the mechanism by which these oncogenes oncogene. activate NF-kB signaling, and the role of such signaling IKKe activates both interferon and NF-kB signaling, in tumorigenesis remains incompletely understood. and overexpression of IKKe in mammary epithelial cells However, H-RASV12-induced transformation depends induced both interferon regulated genes and several on NF-kB activity (Mayo et al., 1997), and recent NF-kB target genes including MMP9, BCL2, CIAP1 work in genetically-engineered mouse models demon- and CIAP2. In agreement with these observations, strated that NF-kB activity was required for the primary breast carcinomas in which IKKe expression development of lung adenocarcinomas driven by onco- was elevated showed nuclear localization of the NF-kB genic KRAS in the setting of p53 loss (Meylan et al., factor cREL. Many cancer cells with increased levels of 2009; Basseres et al., 2010). Other recent studies suggest IKKe also exhibit increased levels of serine 536 that the non-canonical IKKs, IKKe and TBK1 may phosphorylated p65. Strikingly, expression of the have key roles in the cell autonomous activation of NF- NF-kB super repressor in IKKe-transformed cells kB in cancer. blocked IKKe-induced cell transformation. In contrast, suppression of IRF3 and IRF7 by small hairpin RNA failed to alter the transformation phenotype, indicating that the role of IKKe in mediating inflammatory IKKe in cancer interferon responses is unrelated to its function in cancer (Boehm et al., 2007). A potential role for IKKe in breast cancer was first Recent observations demonstrate that IKKe also shown by Sonenshein and her colleagues who demon- functions to protect cells from DNA damage-induced strated that IKKe was constitutively expressed in MDA- death, and the underlying mechanism for this response is MB-231 and Hs578T breast cancer cells lines, four of six mediated by IKKe sumoylation (Renner et al., 2010). breast carcinomas patient, and carcinogen-induced Following genotoxic stress, IKKe translocates to the mouse mammary tumors (Eddy et al., 2005). In this nucleus and induces phosphorylation of the pro- study, tumors and mammary glands derived from a myelocytic leukemia tumor suppressor PML. This

Oncogene Roles for the non-canonical IKKs in cancer RR Shen and WC Hahn 636 activity results in the sequestration of IKKe in PML Sec15, Exo70 and Exo84. TBK1 is able to participate in nuclear bodies. Multiple nuclear targets including the exocyst complex through its direct interaction with p65 are then phosphorylated following critical sumoyla- Sec5, and further facilitates transformation through the tion of IKKe by the SUMO ligase TOPORS. phosphorylation of Sec5 (Camonis and White, 2005; Ultimately, these events facilitate an NF-kB-mediated Chien et al., 2006). This cascade is not only a critical antiapoptotic response to DNA damage (Renner et al., function in transformation, but is also essential for 2010). These observations identify a novel function mediating the host defense to viral infection. Impor- for IKKe in promoting cell survival and suggest that tantly, suppression of either TBK1 or Sec5 was shown to IKKe functions in parallel to the p53 pro-apoptotic induce apoptosis in Ras-transformed cells, and expres- pathway. sion of oncogenic alleles of KRAS induced cell death in Although IKBKE copy number gain does not TBK1-deficient murine embryonic fibroblasts (Chien correlate with estrogen receptor (ER) or HER2/Neu et al., 2006). status, Guo et al. demonstrated that IKKe phosphor- In recent work, TBK1 was identified as a gene of ylates estrogen receptor alpha (ERa). In ER-positive which expression was selectively required in KRAS- cells, this activity is necessary for activation of Cyclin dependent cancer cell lines (Barbie et al., 2009). D1. However, IKKe is also sufficient to activate Cyclin Suppression of TBK1 affects the survival of KRAS- D1 in ER-negative cells (Boehm et al., 2007; Guo et al., dependent cells, while having little to no effect in cells 2010). Furthermore, although overexpression of IKKe where KRAS is not essential. Transcriptional profiling promotes tamoxifen resistance, suppression of IKKe using gene set enrichment analysis in KRAS-trans- sensitizes cells to tamoxifen-induced death (Guo et al., formed cells revealed the enrichment of several NF-kB 2010). These findings highlight potential crosstalk activation signatures. Expression of the NF-kB super between NF-kB dependent and independent pathways repressor also selectively induced apoptosis in KRAS- in IKKe transformed breast cells. dependent lung cell lines. In these cells, TBK1 promoted In addition to breast cancer, IKKe also seems to have degradation of IkBa and induced expression of the a role in ovarian cancer. A recent study showed that survival factor BCL-xL, which in part sustains the IKKe is overexpressed in 63 of 95 ovarian carcinomas, survival of KRAS-driven tumors (Barbie et al., 2009). and elevated IKKe levels were associated with poor In addition, a recent study identified another novel prognosis (Guo et al., 2009). Notably, 76.5% of breast function for TBK1 as an inducer of angiogenesis. cell lines and 47.6% of breast carcinomas exhibit Korherr et al. screened a genome-wide complementry increased expression of IKKe in the absence of 1q32 DNA library in HEK293 cells for genes that regulate copy number changes. Furthermore, recent deep vascularization. Supernatants isolated from growth sequencing of a set of human cancers including, breast, factor-producing HEK293 cells were evaluated for the ovarian, lung and prostate tumors identified two IKKe ability to induce proliferation of HUVEC endothelial mutations, G417D and W445S, in lung adenocarcino- cells. TBK1 was found to function as a trigger in mas (Kan et al., 2010). The function of these mutations, stimulating a secretion program consisting of endothe- which do not map to any previously described domains lial growth factors RANTES and IL-8, as well as other of IKKe, remains unclear (Figure 1). Taken together, pro-angiogenic factors such as CXCL10, CXCL11 and these observations suggest that IKKe expression may be IFN-b. Moreover, several cancer cell lines with upregu- dysregulated through multiple mechanisms in various lated TBK1 exhibit a similar autocrine makeup cancer types. (Korherr et al., 2006).

TBK1 in cancer New and old substrates of IKKS and TBK1 in cancer

TBK1 is highly expressed in lung, breast and colon The initial characterization of IKKe as a breast cancer cancers and a TBK1 mutation, P675L, was recently oncogene indicated that its intrinsic kinase activity was identified in lung adenocarcinoma (Korherr et al., 2006; essential to activate NF-kB and transform cells, but the Barbie et al., 2009; Kan et al., 2010) (Figure 1). RalB is a substrates and effectors responsible for these phenotypes key factor that mediates TBK1 activation in cancer remained undefined. Both TBK1 and IKKe seem to (Bodemann and White, 2008). The Ras-like RalB have many substrates that modulate NF-kB (Figure 2) proteins function downstream of RAL-GEF (Ras-like- in response to extracellular stimuli, namely p65, cREL guanine nucleotide exchange factor) to initiate a variety and IkBa. However, engagement of these targets by the of regulatory processes associated with oncogenesis non-canonical IKKs is different than what has been (Bodemann and White, 2008). The activation of RalB observed for the other IKKs that lead to NF-kB by Ral-GEF is necessary for RAS-induced transforma- activation. For example, it is uncertain how phosphor- tion (Rangarajan et al., 2004; Yoneyama et al., 2004; ylation of one of the two required sites on IkBa is Lim et al., 2006). Downstream of Ral-GEF, RalB- sufficient to activate NF-kB. Similarly, although IKKe mediated activation of TBK1 promotes TBK1 assembly and TBK1 can phosphorylate cREL, this modification with the exocyst complex, which contains several core fails to promote significant NF-kB activation (Harris secretory proteins, namely Sec3, Sec5, Sec6, Sec8, Sec10, et al., 2006).

Oncogene Roles for the non-canonical IKKs in cancer RR Shen and WC Hahn 637 To identify IKKe and TBK1 substrates, Hutti et al. substrate of IKKe discussed above that typically developed and used an unbiased screening functions in mediating the interferon response, but method coupled with bioinformatic analyses to identify may have an additional role in cancer progression a potential substrate recognition motif. In this approach, (Tenoever et al., 2007). STAT proteins, including a collection of 198 biotinylated peptide libraries was STAT1, STAT3 and STAT5, are critical for the employed to perform simultaneous kinase assays with regulation of several genes that function in antiviral recombinant IKKe or TBK1 and radiolabeled ATP. immunity and also function as oncogenic transcription Each library contains mixtures of degenerate factors in various malignancies (Bowman et al., 2000). with two fixed residues, a serine at the central position STAT1 expression is constitutively active in many breast and another natural amino acid at one additional cancer cell lines, but also has a paradoxical role in position neighboring the serine. The relative IKKe promoting apoptosis in tumor cells (Kim and Lee, 2007; or TBK1 preference for each amino acid at each Yarilina et al., 2008). In addition to the STAT proteins, position was determined after capture of the biotiny- IRF3, IRF5 and IRF7 also behave as tumor suppressors lated peptides on a streptavidin-coated membrane. The and facilitate anti-tumorigenic effects in various cancers. resulting IKKe/TBK1 substrate recognition motif was However, as suppression of these factors in IKKe- used to identify potential substrates through the transformed cells fails to decrease tumorigenic potential, proteomic search engine Scansite (Hutti et al., 2009). they likely have a minimal role in the IKKe oncogenic The IKKe and TBK1 phosphorylation motif consists pathway. of a central serine that is surrounded by a hydrophobic residue at the þ 1 position relative to the phosphoryla- tion site, an aromatic residue at the À2 position and bulky hydrophobic residues at the þ 3 position (x-Y/F/ Conclusions P-x-pS-L/I-x-Y/W/F-x). Parallel analysis of IKKe and TBK1 using the scanning peptide library screen, IKKe and TBK1 seem to behave as integrators of independently identified the same target motif for each signals induced not only by pro-inflammatory stimuli, kinase (Hutti et al., 2009). In vitro kinase assays but also by oncogenes and tumor suppressors (Figure 2). demonstrate that IKKe efficiently targets peptides with Through the phosphorylation and modulation of several the identified IKKe recognition motif. In contrast, the NF-kB effectors and IRFs, the IKK-related kinases observed activity of IKKe toward a peptide representing primarily function as inflammatory regulators. More the IkBa target sequence was much less robust, recently, IKKe was identified as a breast cancer suggesting that IkBa is unlikely to be a preferred oncogene that requires NF-kB activation to mediate substrate of IKKe (Hutti et al., 2009). transformation, and TBK1 was demonstrated to be Bioinformatic analysis of the IKKe recognition motif essential for the survival of KRAS-dependent non-small identified many potential IKKe substrates. Interestingly, cell lung cancers. The discovery of IKKe and TBK1 as the top scoring candidates consisted of many oncogenic kinases, which are intricately associated with NF-kB regulators including the known IKKe/TBK1 Ras-mediated transformation, suggests that these non- substrate TANK. Additional putative substrates seem to canonical IKK regulators are subverted in cancer cells. be involved in ubiquitination. Although further func- Although recent studies have defined CYLD as an tional validation of these substrates is necessary, these important IKKe substrate that contributes to transfor- findings suggest that IKKe and TBK1 function in a mation, it is not known whether other targets are network of pathways that converge to activate NF-kB involved in the IKKe oncogenic pathway. Likewise, it (Hutti et al., 2009). will be important to define the critical substrates for The proteomic/bioinformatic approach taken by TBK1 in KRAS-dependent lung cancers. It is clear for Hutti et al. further identified the familial tumor IKKe that kinase activity contributes to its transforming suppressor CYLD as an IKKe substrate that was activity. essential for transformation. CYLD is a deubiquitinase The finding that both TBK1 and IKKe preferentially that attenuates NF-kB signaling by targeting various phosphorylate the same consensus motif, and for the effectors in the pathway (Brummelkamp et al., 2003; most part share the same substrates, raises the question Kovalenko et al., 2003; Trompouki et al., 2003). IKKe as to how these kinases function differently in cell phosphorylates CYLD at serine 418 and disruption of transformation (Hutti et al., 2009). One possibility is this activity substantially hinders both IKKe-induced that IKKe and TBK1 may be differentially regulated. NF-kB activation and tumorigenicity. Although CYLD Negative-feedback mechanisms involving CYLD and expression is necessary for IKKe-mediated transforma- A20 have been shown to control both TBK1 and IKKe tion, it is likely that other substrates also facilitate IKKe activity (Zhang et al., 2008). However, other negative function in cancer (Hutti et al., 2009). regulators such as the phosphatase SHP2 and the NOD- Several other cancer-related substrates have also been like protein NLRC5 primarily affect TBK1 and not described for IKKe/TBK1. As previously discussed, IKKe activity (An et al., 2006; Lin et al., 2006; Cui et al., IKKe phosphorylates ERa through a direct interaction. 2010). In addition, the participation of IKKe and TBK1 This activity occurs on serine 167 and promotes ERa in different protein complexes that are modified by Lys- transactivation and induction of target genes such 63 linked ubiquitin chains proposes that the regulation, as Cyclin D1 (Guo et al., 2010). STAT1 is another stability and activation of complex formation could

Oncogene Roles for the non-canonical IKKs in cancer RR Shen and WC Hahn 638 contribute to differential oncogenic signaling. Another Although there is some concern that prolonged NF-kB explanation for the differential roles of the non-canonical inhibition will lead to unacceptable side effects in IKKs in cancer is the growing list of substrates that are inflammatory disease, such inhibitors could be used distinct for IKKe and TBK1 (Table 1). The subtle but transiently to treat cancer patients. Surprisingly, IKKb disparate regulation and activity of the non-canonical inhibitors increase the production of IL-1b and other IKKs would likely influence divergent activities of TBK1 inflammatory cytokines that are normally suppressed by and IKKe in cancer. NF-kB, and treatment with these agents results in an Current work suggests that IKKe and TBK1 partici- adverse exacerbation of inflammation (Greten et al., pate in both cell autonomous and cell non-autonomous 2007). The non-canonical IKKs may thus serve as more functions in inflammation and cancer (Figure 2). The attractive candidates for therapeutic development in non-canonical IKKs may augment inflammatory signal- cancer. Although further work is necessary to decipher ing in addition to its intrinsic role of promoting cell whether these non-canonical IKKs are therapeutic survival. Integration of these pathways may explain the targets in certain malignancies, the finding that these high levels of NF-kB activation observed in aggressive non-canonical IKKs have important roles in the inflammatory breast cancer (Van Laere et al., 2006; maintenance of epithelial tumors suggests that targeting Lerebours et al., 2008). The recent finding that IKKe the NF–kB pathway may be an attractive strategy in a regulates fat metabolism, adds another potential me- wide range of epithelial cancers. chanism by which IKKe may contribute to transforma- tion (Chiang et al., 2009). Both IKK-related kinases increase the production of IL-6 and TNF, two Conflict of interest inflammatory cytokines associated with insulin resis- tance and the induction of oncogenic STAT3 signaling The authors declare no conflict of interest. (Chiang et al., 2009; Peant et al., 2009; Park et al., 2010). These findings begin to suggest crosstalk between pathways involving inflammation, insulin resistance Acknowledgements and cellular transformation. Several drug discovery efforts have focused on the We thank the members of the Hahn laboratory for advice. development of NF-kB inhibitors for various malig- This work was supported in part by the US NIH (R01 nancies (Karin et al., 2004; Lee and Hung, 2008). CA130988).

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