The Nuclear Factor Kappa-B Signaling Pathwayas a Therapeutic Target Against Thyroid Cancers

Xinying Li,1,2 Asim B. Abdel-Mageed,1 Debasis Mondal,1 and Emad Kandil1

Background: The nuclear factor kappa-B (NF-jB) proteins, a family of transcription factors found virtually in allcells, are known to play crucial roles in the growth of a number of human malignancies. The ability of NF-jB totarget a large number of genes that regulate cell proliferation, differentiation, survival, and apoptosis, providesclues toward its deregulation during the process of tumorigenesis, metastatic progression, and therapeuticresistance of tumors.Summary: In addition to the signaling pathways known to be involved in thyroid tumorigenesis, such as themitogen-activated protein kinase and janus kinase cascades, studies implicate the NF-jB pathway in the develop-ment of both less aggressive thyroid cancers, papillary and follicular adenocarcinomas, and progression to ag-gressive thyroid cancers, such as anaplastic adenocarcinomas. A constitutively activated NF-jB pathway also closelylinks Hashimoto’s thyroiditis with increased incidence of thyroid cancers. The NF-jB pathway is becoming one ofthe major targets for drug development, and a number of compounds have been developed to inhibit this pathway atdifferent levels in cancer cells. Some of these targets have shown promising outcomes in both in vitro and in vivoinvestigations and a handful of them have shown efficacy in the clinical setting.Conclusions: This review discusses the recent findings that demonstrate that the inhibition of NF-jB, alone or withother signaling pathway inhibitors may be of significant therapeutic benefits against aggressive thyroid cancers.

Introduction

According to the 2011 SEER analysis (1), there will be anestimated 56,460 new cases of thyroid cancer in 2012,

and 1,780 deaths will occur due to this disease in the UnitedStates. Currently, there are over 490,000 patients living withdifferent stages of thyroid cancer in the United States. Themajority of thyroid cancers, such as the papillary and follicularcancers (PTC & FTC), generally have good prognosis afterappropriate treatment; however, once they progress to themore aggressive anaplastic (ATC) type, patients manifest sig-nificantly poorer prognosis, and tumor resistance to both ra-dioactive iodine therapy and conventional chemo- andradiotherapy. In addition, some variants of PTC, which arefeathered with columnar cells, tall cells, and diffusely scleros-ing solid tumors, as well as a variety of poorly differentiatedFTCs defined as ‘‘follicular thyroid epithelium,’’ retain suffi-cient differentiation to produce scattered small follicularstructures, but generally lack the usual morphologic charac-teristics of PTC and FTC, and are also highly aggressive (2).In patients with such advanced and progressive thyroid can-cers, routine chemotherapeutic strategies have provided lowresponses and are also refractory to radiation therapy (3).

Therefore, many novel approaches are being developed fortargeted therapy of such aggressive cancers, in which nuclearfactor kappa-B (NF-jB) emerges as one of the most promisingmolecular targets. The NF-jB signaling pathway is now wellknown to play a major role in the initiation and progressionof thyroid carcinoma (4–6). Therefore, blockade of NF-jB sig-naling may represent a novel therapeutic strategy for thyroidcarcinoma, especially the advanced disease. The followingsections first describe the transcription regulatory effects of NF-jB signaling, followed by the inhibitory outcomes based ondifferent levels of the signal pathway inhibitors in thyroidcarcinoma, and finally, the suggested strategies to treat thyroidcarcinoma with the available NF-jB inhibitors.

NF-jB and its activation pathway

The NF-jB protein was first discovered as a DNA-bindingprotein that interacts with an 11-base pair sequence in theimmunoglobulin kappa light-chain enhancer in B cells. Sinceits first discovery in 1986, the NF-jB cascade has been wellrecognized as a transcription factor, which can regulate theexpression of a large number of genes that are critical forapoptosis, tumorigenesis, inflammation, and various

1Department of Surgery and Tulane Cancer Center, Tulane University School of Medicine, New Orleans, Louisiana.2Department of General Surgery, Xianya Hospital, Central South University, Changsha, China.

THYROIDVolume 23, Number 2, 2013ª Mary Ann Liebert, Inc.DOI: 10.1089/thy.2012.0237

209

autoimmune diseases (7). In mammals, the NF-jB family oftranscription factors is composed of five distinct members:RelA (p65), RelB and c-Rel, NF-jB1 (p50 and its precursorprotein p105), and NF-jB2 (p52 and its precursor proteinp100). All these NF-jB members share a highly conserved 300amino acid dimerization domain known as the Rel HomologyDomain, which is required for binding DNA and enhances avariety of target genes. RelA (p65), RelB, and c-Rel containanother common carboxy-terminal structure called transacti-vation domain, which exerts a positive role in transcriptioncomplex formation by binding to numerous other proteins,such as transcriptional coregulators. Several different struc-tural combinations of NF-jB dimers exist in the cytoplasm, inwhich p65/p50 is the most common heterodimer. Thesesubunits can also combine as homodimers (p50/p50, p52/p52) and regulate target genes positively or negatively bybinding the inhibitor of NF-jB (IjB) family member Bcl-3(8).

The NF-jB signaling pathway, which can be activated byboth extracellular and intracellular stimuli, includes, the cell

surface receptors (tumor necrosis factor receptor [TNFR],interleukin-1 receptor [IL-1R], Toll-like receptor 4 [TLR4],nucleotide oligomerization domain–like receptor [NLR],lymphotoxin beta receptor [LTbR], B-cell activating factorreceptor [BAFF-R], receptor activator of NF-jB [RANK], etc.),the receptor signal adaptor protein (tumor necrosis factor[TNF], TNF receptor–associated factors [TRAFs], receptor-interacting proteins [RIPs]), IjB kinase (IKK) complex, IjBproteins, and dimers of NF-jB subunits. An IKK complexconsists of three subunits, including IKKa, IKKb, and IKKc(also named NEMO). IKKb can induce phosphorylation ofIjBa at serine 32 and 36 and causes the subsequent degrada-tion of IjBa. NEMO, as a regulatory subunit of the IKKcomplex, plays an integral role in the activation of NF-jB bymodulating degradation of IjBa. Following a wide variety ofexternal stimuli, the IKK complex is activated, which results inphosphorylation and ubiquitination of the IjB protein. AfterIjB protein degradation by the proteasome, bound NF-jBdimers are released and translocate to the nucleus. After fur-ther post-transcriptional modification in the nucleus, NF-jB isable to transcribe a selective subset of target genes controllingdifferent aspects of cell physiology.

Thus, the activation of the NF-jB pathway can be triggeredby various stimuli in a canonical (classical) and noncanonical(alternative) manner. The canonical pathway introduced bymost physiological stimuli involves cell membrane receptors,such as TNFR, IL-1R, and TLR4, which are able to recruit theadaptors TRADD, TRAF2, and RIP in the cytoplasm and se-quentially phosphorylate the IKK complex. Activation of theIKK complex leads to phosphorylation and ubiquitination ofIjBa and releases the heterodimer p65/p50 from the cyto-plasm leading to its nuclear translocation, which sequentiallyactivates target genes. The activation of the noncanonicalpathway triggered by CD40, RANK, LTbR, and BAFF de-pends on phosphorylation of IKKa, which is not necessary inthe canonical pathway. Activation of IKKa sequentially pro-cesses p100 into p52, which dimerizes with RelBand andforms the NF-jB subunit to trigger transcription of targetgenes in the nucleus (9). The complete NF-jB pathway acti-vation pathway is illustrated in Figure 1.

Signaling pathways cross-talk with NF-jB and regulateits function

Multiple parallel signal transduction pathways are also in-volved in the activation of NF-jB. As a major regulator of cellsurvival, NF-jB can act synergistically with other importantoncogenic signaling cascades to ultimately regulate tumori-genesis. The cross-talk between NF-jBand and these parallelpathways are well documented in a recent review article (10).In this article, we have emphasized the role of several of theseimportant signaling pathways, including the c-Jun-NH2-terminal kinase ( JNK) pathway, PI3K (phosphatidylinositol 3-kinase) pathway, and P53 pathway, which are known to playan essential role in the pathogenesis of thyroid cancer.

The JNK pathway (a signaling pathway that is known topromote apoptosis) and NF-jB have a common regulatorynode via the TRAFs. External stimulus, such as TNFa, cantarget TRAF2, which can then activate both JNK and NF-jBsignal pathways. The balance between JNK and NF-jB ac-tivities is considered crucial to determine the cell fate in re-sponse to external stimuli (11,12). Similarly, with IL-1R or

FIG. 1. The NF-jB signaling cascade. In cells, NF-jB acti-vation is triggered by extracellular stimuli, which transducethe signal via membrane receptors to sequentially phos-phorylate the IKK complex. The IKK then phosphorylatesIjBa, which is released from the NF-jB complex, ubiquiti-nated and degradated by the proteasomes. The released NF-jB heterodimers are able to translocate from the cytoplasmto the nucleus, where they bind to cognate DNA sequencesand activate numerous target genes. In cancer cells, theseNF-jB–induced genes are crucial in regulating cell prolif-eration, metastasis, survival, and therapeutic resistance.TRAF, tumor necrosis factor receptor–associated factor; RIP,receptor-interacting protein; NEMO, NF-jB essential mod-ulator; P, proteasome; IKK, IjB kinase; IjB, inhibitor ofNF-jB; NF-jB, nuclear factor kappa-B; Ub, ubiquitination.Color images available online at www.liebertpub.com/thy

210 LI ET AL.

TLR4 signaling, TRAF6 can induce TAK1 to trigger the acti-vation of both of these two signaling pathways (13,14). Al-ternatively, NF-jB-induced gene products may also affectJNK activity. For instance, the ferritin heavy chain or man-ganese-superoxide dismutase blunts sustained JNK activity.Proteins inhibiting JNK signal activation, such as GADD45b,A20, and XIAP, can also be produced due to the activation ofthe NF-jB signal pathway (11). These signaling pathways andtheir cross-talk with NF-jB are depicted in Figure 2.

The tumor suppressor PTEN, which acts as a negativeregulator of the PI3K/Akt pathway, is downregulated byNF-jB to prevent apoptosis (15). In addition to the down-regulation of PTEN by NF-jB, RIP1 activates PI3K/Aktthrough a mechanism involving NF-jB-mediated inhibitionof the mTOR-S6K–IRS1 negative feedback loop (16,17). TNF-related activation-induced cytokine, a TNF family member,activates the PI3K/Akt pathway through a signaling complexinvolving TRAF6 (18), which is also involved in the canonicalactivation of NF-jB pathway. It was revealed (19,20) that thePI3K/Akt pathway can switch second messenger signalingfrom the occupied IL-1 receptor to NF-jB, and specific PI3Kinhibitors can strongly inhibit both PI3K activation andNF-jB-dependent gene expression. Interestingly, both over-expression of the p110 catalytic subunit of PI3K and the PI3K-activated protein kinase Akt can induce p65/RelA-mediatedtransactivation.

The tumor suppressor P53 is known to be an importantregulator of cellular apoptosis. Under certain stimuli, such asTNFa, various tumor cell lines elicit different responses,which depend predominantly on activation of the P53 and

NF-jB pathways. Indeed, P53 is known to be a competitiveinhibitor of NF-jBp65 that can function through the seques-tration of the CREB-binding protein (CBP/P300), a cofactorrequired for activation of both the P53 and the NF-jB path-ways (21). Huang et al. (22) showed that phosphorylation ofCBP at serine 1382 and serine 1386 increases IKKa-inducedHAT activity of CBP, which then enhances its binding capa-bility with NF-jBp65 and reduces its binding to P53.

The aforementioned studies argued that NF-jB activationacts in concert and as part of a complex network of othersignal transduction pathways, under both physiologic con-ditions and especially under diseased states, such as cancer.Therefore, systemic blockade of NF-jB may intervene withthese other pathways and cause unexpected adverse effects.Thus, understanding the cross-talk with other pathways willbe helpful to elucidate how to selectively inhibit NF-jB incancer cells without influencing its normal physiologic func-tions in normal tissues.

The crucial role of NF-jB pathway in thyroid cancers

Over the past two decades, the diverse roles of NF-jB havebeen well documented in multiple carcinomas, includingleukemia, lymphoma, head and neck squamous carcinoma,ovarian, prostate, colon, and breast cancers (23). These diverseeffects have been linked to the activation of cell proliferation,anti-apoptosis, tumor progression, and invasion, as well asresistance to chemotherapy and radiotherapy (24,25). In mostof the above cancers, although NF-jB has been primarilyconsidered as a tumor-promoting factor, studies suggest that

FIG. 2. Cross-talk betweenNF-jB and parallel signalingpathways. Several secondmessenger signaling pathwayscan cross-talk with the NF-jBactivation cascade. Thesemultiple nodes of regulationof the NF-jB pathway are alsosuggested to be involved in thegrowth of aggressive thyroidcancers. Green lines show theNF-jB pathway, blue linesshow the JNK pathway, andthe yellow lines shows thePI3K pathway. The pink linesdemonstrate how NF-jB acti-vation can alter the functioningof these parallel signalingpathways in tumor cells. IL-1R,interleukin-1 receptor; TLR4,Toll-like receptor 4; TRANCE,TNF-related activation-induced cytokine; JNK, c-Jun-NH2-terminal kinase; GADD,growth arrest and DNA dam-age; XIAP, X-linked inhibitorof apoptosis protein; PI3K,phosphatidylinositol 3-kinase;PTEN, phosphatase and tensinhomolog; mTOR, mammaliantarget of rapamycin. Colorimages available online atwww.liebertpub.com/thy

NF-jB AS A THERAPEUTIC TARGET FOR THYROID CANCER 211

the induction of NF-jB signaling may actually have disparateeffects in different thyroid cancers.

An earlier study had reported constitutive activation of NF-jB in a panel of thyroid carcinoma cell lines (4). With theexception of two lines (NPA and ARO) that were found not tobe of thyroid origin, thyroid cancer cell lines included in thisstudy were found to have significantly increased p65 mRNAand protein expression compared to normal thyroid cells,further complicating the role of NF-jB in thyroid cancers (26).Interestingly, by immunohistochemical staining using ananti-p65 antibody, tissue specimens from papillary, follicular,and anaplastic thyroid cancers were shown to have consti-tutively activated NF-jB (5,27), clearly implicating the im-portance of this family of transcription factors in differentstages of thyroid cancers. A number of recent molecularstudies also suggest the reason for this activated NF-kB levels.

Thyroid carcinoma comprises a group of different typesof tumor cells with distinctive clinical and pathological char-acteristics that occur due to different genetic mutations in-volving specific oncogenes. The RET/PTC rearrangements, aswell as BRAF and RAS mutations, are often seen in aggressivecancers. These genetic alterations potently activate the MAPKpathway, which can in turn cause NF-jB activation andoncogene-mediated progression and aggressive behavior ofpapillary thyroid carcinomas (28–31). A common mutationfound in the BRAF gene (i.e., BRAFV600E) can also activateNF-jB, which occurs via the direct activation of BRAF, butis independent of ERK signaling (32). Neely et al. (33) recentlyreported that the RET/PTC-3 gene can also activate the ca-nonical NF-jB pathway by stabilizing the NF-jB-inducingkinase (NIK) in both papillary thyroid carcinomas and inthyroid epithelial cells found in Hashimoto’s thyroiditis. Lossof function of tumor suppressors, including PTEN down-regulation, P53 mutations, and b-CATENIN, also contributesto the progression of thyroid cancer (34–37). Inactivation ofPTEN in thyroid cancer can oppose the activation of PI3K/Akt pathway, thus, in turn, increasing NF-jB activity andaccelerating tumor progression (15,38). PI3K/Akt can alsodownregulate the FOXO3a activity, which is a central medi-ator of cell cycle and apoptosis (39). In contrast with othercarcinomas, mutations in P53 and its family members P63 andP73 are not common in early stages of thyroid cancer, but aremainly manifested in the poorly differentiated and aggressivephenotype of thyroid cancers (40–42). Indeed, the down-regulation of cyclin B1 by NF-jB inhibition in the 8505C-ATCcell line showed P53-induced cell cycle arrest through an in-crease in the level of p21WAF1 (43). Kroll et al. (44) were the firstto report that the PAX8-PPARc fusion, which results from achromosomal translocation of t (2; 3) (q13; p25) in humanthyroid follicular tumors, lacks the ability of wild-type PPARcto inhibit NF-jB activation and results in the activation ofcyclin D1, which causes repression of critical genes involvedin apoptosis (45).

The relationship between inflammation and cancer wasfirst noted by Rudolf Virchow in 1863 (46). Subsequent studiesprovided the connection between gastritis associated withchronic Helicobacter pylori infection and the increased occur-rence of gastric cancers. Further studies have also linked co-lonitis with colon cancer, cholangitis with cholangiocellularcarcinoma, and hepatitis with hepatocellular carcinoma (47).Extensive studies have proposed further molecular mecha-nisms, which show that the NF-jB pathway is a major link

between inflammation and neoplastic transformation, as wellas cancer progression. The inflammation process chronicallyactivates NF-jB. This stimulates the expression of cytokines,chemokines, growth factors, and protease cascades, whichfavors initiation and progression of tumors, and provides aniche for malignant transformation. Arif et al. (48) observedthat Hashimoto’s thyroiditis, a chronic inflammatory condi-tion, shares many common morphological features and im-munohistochemical staining patterns as observed in thyroidcancers. Asioli et al. (49) also found that Hashimoto’s thy-roiditis shows atypical nuclear features, including prominentnuclear grooves, enlarged overlapping nuclei, and nuclearclearing, which are often evident in thyroid cancers. The co-existence of Hashimoto’s thyroiditis in PTC cases has beenassociated in some studies with unfavorable clinical outcomescompared with those without Hashimoto’s thyroiditis (48–52), further strengthening the link between chronic inflam-mation and progression to thyroid carcinomas.

The oncogenes found to be relevant in different signalingpathways in thyroid cancers, such as RAS, RET, and BRAF,can recruit and activate inflammatory cells and reduce anti-cancer immune surveillance. With the loss of PTEN functionin thyroid cancers, downstream PI3K/Akt pathway activa-tion is observed in both Hashimoto’s thyroiditis and thyroidcancer (34,51). As previously discussed, the NF-jB pathwaycan be activated by PI3K/Akt, which can then promote an-giogenic and metastatic gene expression (53). The RET/PTC3(RP3) fusion gene product is an oncogenic protein that is fre-quently expressed in papillary thyroid carcinoma accompaniedby Hashimoto’s thyroiditis. The RP3 genetic background is alsoknown to induce the secretion of proinflammatory mediators,including granulocyte-macrophage colony–stimulating factorand macrophage chemotactic protein 1, and suppresses innateimmunity against cancers, which results in the onset and pro-gression of thyroid cancer (54,55). Indeed, it has been shownthat the RP3 oncogene can activate the canonical NF-jB path-way by stabilizing NIK (33).

NF-jB inhibitory strategies as targets for thyroidcancer therapy

Due to the important role played by NF-jB, inhibitors ofthis pathway may be promising targets against thyroidcancers (Fig. 3). The sustained activation of NF-jB has alsobeen associated with tumor recurrence following both che-motherapy and radiotherapy. Therefore, adjunct treatmentwith NF-jB inhibitors may be of great benefit to reversetherapeutic resistance (56–58). Numerous NF-jB inhibitorsare currently being tested in different laboratories and sev-eral of them are in clinical trials. These compounds can in-hibit the NF-jB activation pathway at different levels, suchas at the activation of IKK complex, stabilization of the IjBa,activation of NF-jB inhibitory proteins, activation of theproteasome cascade, and lastly, the targeting of the tran-scriptionally active NF-jB heterodimers, such as the RelAsubunit. In addition, NF-jB inhibitory strategies may befeasible by targeting signal pathways from upstream effec-tors, such as TRAFs and NIK, as well as the inhibitionof downstream factors, for example, direct blockade ofNF-jB binding to DNA. Furthermore, parallel inhibition ofNF-jB-independent pathways and its cross-talk with thesepathways may provide more efficient and long-lasting

212 LI ET AL.

therapeutic outcomes for tumors, especially due to themultiple mechanisms via which NF-jB and its regulatorysignaling events might ultimately affect cancer initiation,progression, and recurrence/resistance (32,43).

Notably, it is well established that TRAFs transduce signalsthat emanate from most members of the TNFR superfamilyand the IL-1 receptor/Toll-like receptor super family; theseare able to activate both canonical and noncanonical NF-jBpathway (59,60). A recent study has shown that dominant-negative TRAF2 mutants or downregulation of TRAF2achieved by siRNA can significantly inhibit NF-jB activa-tion in response to TNF-a (61). Similarly, in human 293cells, IL-1-induced NF-jB activation could be blocked byusing a dominant-negative mutant of TRAF6 (62). It shouldalso be noted that, in addition to suppressing the NF-jBpathway, inhibition of TRAFs resulted in the inactivation ofthe JNK pathway, as well. The NIK protein that is induced byseveral NF-jB activators, such as TRAF-2, TRAF-6, CIAP1,and CIAP2, was found to activate NF-jB through the non-canonical pathway. This activation of NF-jB was inhibited inNIK-deficient murine embryonic fibroblasts, despite an in-crease in NIK induced by the RET/PTC (RP3) gene product(33). Also, a kinase-dead mutant of IKKa, an RIP mutantlacking a kinase domain, or direct suppression of IKKa bysiRNA technology, were able to significantly block the PDCasp8/10-mediated NF-jB activation (63). Since the activation ofNF-jB requires the activity of the IKK complex (IKKa, IKKb,and IKKc), repression of IKKs has been recognized as anotherstrategy to inhibit NF-jB activation by increasing the accu-

mulation of IjBa. Many pharmacologically available anti-in-flammatory drugs, such as NSAIDS, manifest their inhibitoryeffects on NF-jB activation via this pathway. These includeaspirin, ibuprofen, mitricoxide, prostaglandin, sanguinarine,4-hydroxy-2-nonenal, curcumin, and ursolic acid, whichfunction by suppressing the phosphorylation of IjBa viathe IKK complex (64–71). Of note, several highly selectiveinhibitors of IKKb, such as PS1145 and BAY 11-7082, whichsuppresses proinflammatory stimuli-induced IjBa phos-phorylation, are under investigation for the treatment of dif-ferent carcinomas (72). Furthermore, since the proteasome isnecessary to degrade the IjB inhibitory subunit after itsphosphorylation and ubiquitination in the cytoplasm (73),proteasome inhibitors are potent drugs to block NF-jB acti-vation by preventing 26S proteasome from degrading IjBa.Therefore, several proteasome inhibitors, such as MG132,lactacystin, epoxomicin, MLN519, and bortezomib, are beingevaluated for their therapeutic effects against diverse humancancers. Indeed, bortezomib has been clinically approved forthe therapy of several different types of solid tumors, and itsuse in combination therapies has been proven to enhancesensitivity to both chemotherapy and radiotherapy (72,74–79).

The most direct strategy for blocking NF-jB activation is toblock its binding to its cognate nucleotide sequences in theenhancer regions of numerous genes. Oligonucleotide decoys(OD), short synthetic fragments of DNA or RNA, can com-petitively inhibit NF-jB binding to these complementary re-gions of specific DNA, and have been very successful in thisrespect. The first reported OD was a double-stranded

FIG. 3. NF-jB inhibitors astargets for thyroid cancertherapy. The content in thestars show numerous strate-gies being used to inhibit theactivation of NF-jB at differ-ent levels, such as the IKKcomplex, the IjBa, inhibitoryproteins, the proteasome, andthe RelA subunit of the tran-scriptionally active hetero-dimers. Color imagesavailable online at www.liebertpub.com/thy

NF-jB AS A THERAPEUTIC TARGET FOR THYROID CANCER 213

oligonucleotide, which was able to compete with the endog-enous DNA for NF-jB binding (80). Direct injection of the NF-jB OD into colon carcinomas implanted in mice was shown toinhibit cachexia (81). Some sesquiterpene lactones have alsobeen reported to inhibit NF-jB binding by interacting withCys-38 in the DNA-binding loop of RelA and through ananalogous Cys residue in the DNA-binding loops of p50 andc-Rel (82,83). These sesquiterpene lactone compounds havebeen shown to possess potent anticancer activities, as well.Small polypeptides that are capable of penetrating the cellmembrane and block nuclear translocation of NF-jB is an-other approach to inhibit its activation. SN50 is a 41 residuesynthetic peptide. Its potent effects in blocking the nucleartranslocation of NF-jB have shown significant promise as ananticancer agent. However, SN50 also blocks nuclear trans-location of other transcription factors (84), thus obscuring theexact contribution of NF-jB blockade to its anticancer ef-fects, and to its potential for manifesting side effects. Severalother reports have shown that novel compounds, such as o,o¢-bismyristoyl thiamine disulfide, and dehydroxymethylepoxy-quinomicin (85,86) have also been reported to be specific in-hibitors of NF-jB nuclear translocation. These are currentlybeing tested as anticancer agents. Although not well inves-tigated, alternate mechanisms are available to suppressNF-jB binding to its cognate DNA element, such as the useof specific methyl transferases, to methylate these sequencesand by inhibition of p65 acetylation using specific acetyltransferases.

As illustrated above, NF-jB inhibitors can suppress its ac-tivation by different mechanisms. They are being extensivelystudied in diverse human cancers. However, whether theseinhibitors can result in therapeutic benefits in the clinic is stillcontroversial. Long-term suppression of NF-jB activation viathese inhibitors will probably induce a homeostatic switch andprevent its efficacy in therapy-resistant tumors. In addition,prophylactic therapy using these inhibitors should also takeinto account the adverse effects of these agents and how theymay result in imbalance of physiological functions in normalcells and tissues. Therefore, it is currently envisioned that thestrategies toward safe and efficacious use of these NF-jBpathway inhibitors should be tailored to the patient’s geneticbackground and according to the subtype of tumors that arebeing targeted. Using a panel of thyroid cancer lines harboringdifferent activating mutations, including the HRAS G13Rmutation (C643), BRAFV600E mutations (BCPAR, SW1736), andRET/PTC1 rearrangement (TPC1), Bauerle et al. (43) monitoredthe antiproliferative effects of a selective genetic inhibitor ofNF-jB, a mutant IjBa. Interestingly, the responses to this NF-jBinhibitor were quite different in different cell lines, and ontheir inhibitory effects on proliferation, apoptosis, and inva-sion capabilities. These disparate results were believed due todifferent genetic backgrounds in thyroid cell lines, which re-presented the potential of combinational strategies to treatthyroid cancer. Most of the activating mutations in thyroidcancer, such as in the RAS, RET, and BRAF genes, are in-volved in the MAPK signal pathway, which is crucial for tu-mor initiation (87–89). Studies have shown that NF-jBactivation is involved in the development of resistance to ei-ther MAPK or BRAF inhibitors, but the mechanism remainsunclear (90–94). Interestingly, mutant BRAF has been shownto activate NF-jB in melanoma cells by enhancing the ubi-quitination and degradation of IjB (95). Liu and Xing (96) had

explored the efficacy of dually targeting the NF-jB andMAPK pathways in thyroid cancer. A recent study showedthat bortezomib induces apoptosis and growth suppression inhuman medulloblastoma cells, associated with inhibition ofAKT and NF-jB signaling, and synergizes with an ERK in-hibitor (97). Studies in thyroid cancer cells suggested thatBRAF activates NF-jB and this pathway is MEK-independent(32). Therefore, cotargeting of both MAPK and NF-jB path-ways should be effective in suppressing growth of thyroidtumors harboring BRAFV600E mutations, while abrogatingresistance due to inhibition of the MEK/ERK pathway.Therefore, it has been postulated that in future studies NF-jBinhibitors should be used in combination with drugs targetingalready known abnormalities in parallel signaling pathways,and as an adjunct to cytotoxic chemotherapy or immuno-therapy. Multikinase inhibitors, such as Lenvatinib, motesa-nib, sorafenib, sunitinib, and vandetanib, that can target RET,VEGFR, and other kinases, are studied in clinical trials (3).

Drugs studied as NF-jB inhibitors in thyroid cancer

The underlying mechanisms linked to NF-jB activationand the different inhibitory strategies illustrated above pro-vide the rationale for new therapeutic approaches to treataggressive thyroid cancers. Some of these inhibitors, such asthe proteosomal inhibitor bortezomib, have been clinicallyapproved for multiple myeloma and mantle cell lymphoma.Many other NF-jB inhibitors are also in phase I/II clinicaltrials for different types of cancers (98,99). However, theseNF-jB inhibitors have not been tested for the treatment ofthyroid cancers, and no ongoing clinical trials against thyroidcancer are under way even with approved NF-jB inhibitors.Several recent studies using drugs that are able to inhibit theNF-jB pathway in thyroid cancer are mostly in the experi-mental phase, and data generated have only shown in vitroeffects, and few studies showed in vivo effects (43,100–102).However, it should also be noted that the in vivo targeting ofcancer cells by therapeutic drugs is a complex process thatinvolves proper documentation of pharmacokinetic andpharmacodynamic efficacies of these agents. Furthermore, theefficacy of NF-jB inhibitors may only be achieved throughsimultaneous targeting of multiple pathways. At present,several drugs that have been tested for their antitumor effectsin poorly differentiated and anaplastic thyroid cancers in-clude, PS-1145, Bay-11-7082, IKK inhibitor VII, CDDO-Me,and bortezomib, a small-molecule triptolide. Bauerle et al. (43)had used three NF-jB inhibitors, that is, Bay-11-7082, IKKinhibitor VII, and CDDO-Me in a panel of advanced thyroidcancer lines, only one of which showed a significant decreasein cell growth, which was shown to occur via the inhibition ofthe S-G2/M transition in these cells. Two out of the five thy-roid cell lines, SW1736 and TPC1, were sensitive to inhibitionof NF-jB by a dominant-negative IjBa (mIjBa), which is re-sistant to IKK-induced phosphorylation and proteasomaldegradation. Studies also showed that triptolide, a smallmolecule from a Chinese herb, can inhibit the NF-jB activityvia blocking the association of p65 with CBP/p300 in the earlystage and decreasing the protein level of p65 in the late stagein a human ATC cell line (101,103).

Out of all the NF-jB inhibitors tested so far, only bortezo-mib was shown to induce significant apoptosis in anaplasticthyroid cancer cell lines. However, this may be due to the

214 LI ET AL.

effects of bortezomib in targeting numerous other moleculesin the cell, such as p21, p27, and BCL-2 family members, aswell as several parallel signaling pathways, such as JNK andP53 (100,104). Two recent reports suggested that bortezomibmay be a promising agent to treat thyroid cancers (100,102).Therefore, in light of the findings that the NF-jB pathwaymay play an important role in aggressive thyroid cancers andespecially in certain therapy-resistant subtypes which harbormultiple mutations, there needs to be a concerted and sys-temic effort toward preclinical and ultimately clinical studiesto investigate the efficacy of NF-jB inhibitors.

Conclusions

Current investigations clearly suggest that the inhibition ofthe NF-jB pathway may be a promising strategy for thetreatment of advanced thyroid cancers. Despite bortezomib’sapproval for multiple myeloma and mantle cell lymphoma,there are no inhibitors of NF-jB clinically approved fortreatment of thyroid cancer. The NF-jB inhibitors should basetheir antitumor efficacies by targeting this specific pathway inspecific types of thyroid tumors, while avoiding the risk ofunexpected side effects. One should also consider that globaland prolonged inhibition of NF-jB could manifest detri-mental effects to the immune system of the patient. Thus, toavoid long-term immunosuppression and achieve efficacy ofNF-jB inhibition, the dosage and schedule for administrationof these inhibitors should be carefully considered. Furtherinvestigation is needed to develop effective and clinicallysafe NF-jB inhibitors against aggressive and therapy-resistant thyroid cancers.

Acknowledgments

The authors wish to acknowledge funding from the Na-tional Natural Science Foundation of China (no. 30600601),China, and funds from the Tulane Cancer Center (TCC) andLouisiana Cancer Research Consortium (LCRC).

Disclosure Statement

No competing financial interests exist.

Reference

1. Howlader N NA, Krapcho M, Neyman N, Aminou R, Al-tekruse SF, Kosary CL, Ruhl J, Tatalovich Z, Cho H, Mar-iotto A, Eisner MP, Lewis DR, Chen HS, Feuer EJ, CroninKA (eds). SEER Cancer Statistics Review, 1975–2009 (Vin-tage 2009 Populations). National Cancer Institute, Bethes-da, MD. Available online at http://seer.cancer.gov/csr/1975_2009_pops09 (based on November 2011 SEER datasubmission, posted to the SEER web site April 2012).

2. Patel KN, Shaha AR 2006 Poorly differentiated and ana-plastic thyroid cancer. Cancer Control 13:119–128.

3. Schlumberger M, Sherman SI 2012 Approach to the patientwith advanced differentiated thyroid cancer. Eur J En-docrinol 166:5–11.

4. Visconti R, Cerutti J, Battista S, Fedele M, Trapasso F, ZekiK, Miano MP, de Nigris F, Casalino L, Curcio F, Santoro M,Fusco A 1997 Expression of the neoplastic phenotype byhuman thyroid carcinoma cell lines requires NFkappaB p65protein expression. Oncogene 15:1987–1994.

5. Pacifico F, Mauro C, Barone C, Crescenzi E, Mellone S,Monaco M, Chiappetta G, Terrazzano G, Liguoro D, Vito P,

Consiglio E, Formisano S, Leonardi A 2004 Oncogenic andanti-apoptotic activity of NF-kappa B in human thyroidcarcinomas. J Biol Chem 279:54610–54619.

6. Palona I, Namba H, Mitsutake N, Starenki D, Podtcheko A,Sedliarou I, Ohtsuru A, Saenko V, Nagayama Y, UmezawaK, Yamashita S 2006 BRAFV600E promotes invasiveness ofthyroid cancer cells through nuclear factor kappaB activa-tion. Endocrinology 147:5699–5707.

7. Sen R, Baltimore D 1986 Inducibility of kappa immuno-globulin enhancer-binding protein Nf-kappa B by a post-translational mechanism. Cell 47:921–928.

8. Thornburg NJ, Pathmanathan R, Raab-Traub N 2003 Acti-vation of nuclear factor-kappaB p50 homodimer/Bcl-3complexes in nasopharyngeal carcinoma. Cancer Res63:8293–8301.

9. Gupta SC, Sundaram C, Reuter S, Aggarwal BB 2010 In-hibiting NF-kappaB activation by small molecules as atherapeutic strategy. Biochim Biophys Acta 1799:775–787.

10. Oeckinghaus A, Hayden MS, Ghosh S 2011 Crosstalk inNF-kappaB signaling pathways. Nat Immunol 12:695–708.

11. Papa S, Zazzeroni F, Pham CG, Bubici C, Franzoso G 2004Linking JNK signaling to NF-kappaB: a key to survival. JCell Sci 117:5197–5208.

12. Zhang Y, Chen F 2004 Reactive oxygen species (ROS),troublemakers between nuclear factor-kappaB (NF-kappaB)and c-Jun NH(2)-terminal kinase ( JNK). Cancer Res64:1902–1905.

13. Chung JY, Park YC, Ye H, Wu H 2002 All TRAFs are notcreated equal: common and distinct molecular mechanismsof TRAF-mediated signal transduction. J Cell Sci 115:679–688.

14. Wajant H, Henkler F, Scheurich P 2001 The TNF-receptor-associated factor family: scaffold molecules for cytokinereceptors, kinases and their regulators. Cell Signal 13:389–400.

15. Vasudevan KM, Gurumurthy S, Rangnekar VM 2004Suppression of PTEN expression by NF-kappa B preventsapoptosis. Mol Cell Biol 24:1007–1021.

16. Park S, Zhao D, Hatanpaa KJ, Mickey BE, Saha D, Booth-man DA, Story MD, Wong ET, Burma S, Georgescu MM,Rangnekar VM, Chauncey SS, Habib AA 2009 RIP1 activatesPI3K-Akt via a dual mechanism involving NF-kappaB-mediated inhibition of the mTOR-S6K-IRS1 negative feed-back loop and down-regulation of PTEN. Cancer Res 69:

4107–4111.17. Dan HC, Cooper MJ, Cogswell PC, Duncan JA, Ting JP,

Baldwin AS 2008 Akt-dependent regulation of NF-{kappa}B is controlled by mTOR and Raptor in associationwith IKK. Genes Dev 22:1490–1500.

18. Wong BR, Besser D, Kim N, Arron JR, Vologodskaia M,Hanafusa H, Choi Y 1999 TRANCE, a TNF family member,activates Akt/PKB through a signaling complex involvingTRAF6 and c-Src. Mol Cell 4:1041–1049.

19. Sizemore N, Leung S, Stark GR 1999 Activation of phos-phatidylinositol 3-kinase in response to interleukin-1 leadsto phosphorylation and activation of the NF-kappaB p65/RelA subunit. Mol Cell Biol 19:4798–4805.

20. Reddy SA, Huang JH, Liao WS 1997 Phosphatidylinositol3-kinase in interleukin 1 signaling. Physical interaction withthe interleukin 1 receptor and requirement in NFkappaBand AP-1 activation. J Biol Chem 272:29167–29173.

21. Ravi R, Mookerjee B, van Hensbergen Y, Bedi GC, Gior-dano A, El-Deiry WS, Fuchs EJ, Bedi A 1998 p53-mediatedrepression of nuclear factor-kappaB RelA via the tran-scriptional integrator p300. Cancer Res 58:4531–4536.

NF-jB AS A THERAPEUTIC TARGET FOR THYROID CANCER 215

22. Huang WC, Ju TK, Hung MC, Chen CC 2007 Phosphor-ylation of CBP by IKKalpha promotes cell growth byswitching the binding preference of CBP from p53 to NF-kappaB. Mol Cell 26:75–87.

23. Escarcega RO, Fuentes-Alexandro S, Garcia-Carrasco M,Gatica A, Zamora A 2007 The transcription factor nuclearfactor-kappa B and cancer. Clin Oncol (R Coll Radiol)19:154–161.

24. Luo JL, Kamata H, Karin M 2005 IKK/NF-kappaB signal-ing: balancing life and death—a new approach to cancertherapy. J Clin Invest 115:2625–2632.

25. Malara N, Foca D, Casadonte F, Sesto MF, Macrina L, San-toro L, Scaramuzzino M, Terracciano R, Savino R 2008 Si-multaneous inhibition of the constitutively activated nuclearfactor kappaB and of the interleukin-6 pathways is necessaryand sufficient to completely overcome apoptosis resistanceof human U266 myeloma cells. Cell Cycle 7:3235–3245.

26. Schweppe RE, Klopper JP, Korch C, Pugazhenthi U, Ben-ezra M, Knauf JA, Fagin JA, Marlow LA, Copland JA,Smallridge RC, Haugen BR 2008 Deoxyribonucleic acidprofiling analysis of 40 human thyroid cancer cell linesreveals cross-contamination resulting in cell line redun-dancy and misidentification. J Clin Endocrinol Metab93:4331–4341.

27. Mitsiades CS, Kotoula V, Poulaki V, Sozopoulos E, Negri J,Charalambous E, Fanourakis G, Voutsinas G, Tseleni-Balafouta S, Mitsiades N 2006 Epidermal growth factorreceptor as a therapeutic target in human thyroid carcino-ma: mutational and functional analysis. J Clin EndocrinolMetab 91:3662–3666.

28. Kimura ET, Nikiforova MN, Zhu Z, Knauf JA, NikiforovYE, Fagin JA 2003 High prevalence of BRAF mutations inthyroid cancer: genetic evidence for constitutive activationof the RET/PTC-RAS-BRAF signaling pathway in papillarythyroid carcinoma. Cancer Res 63:1454–1457.

29. Mitsutake N, Miyagishi M, Mitsutake S, Akeno N, Mesa C,Jr., Knauf JA, Zhang L, Taira K, Fagin JA 2006 BRAF me-diates RET/PTC-induced mitogen-activated protein kinaseactivation in thyroid cells: functional support for require-ment of the RET/PTC-RAS-BRAF pathway in papillarythyroid carcinogenesis. Endocrinology 147:1014–1019.

30. Tuyt LM, Dokter WH, Birkenkamp K, Koopmans SB,Lummen C, Kruijer W, Vellenga E 1999 Extracellular-regulated kinase 1/2, Jun N-terminal kinase, and c-Jun areinvolved in NF-kappa B-dependent IL-6 expression inhuman monocytes. J Immunol 162:4893–4902.

31. Xing M 2007 BRAF mutation in papillary thyroid cancer:pathogenic role, molecular bases, and clinical implications.Endocr Rev 28:742–762.

32. Bommarito A, Richiusa P, Carissimi E, Pizzolanti G, Ro-dolico V, Zito G, Criscimanna A, Di Blasi F, Pitrone M,Zerilli M, Amato MC, Spinelli G, Carina V, Modica G,Latteri MA, Galluzzo A, Giordano C 2011 BRAFV600Emutation, TIMP-1 upregulation, and NF-kappaB activation:closing the loop on the papillary thyroid cancer trilogy.Endocr Relat Cancer 18:669–685.

33. Neely RJ, Brose MS, Gray CM, McCorkell KA, LeibowitzJM, Ma C, Rothstein JL, May MJ 2011 The RET/PTC3 on-cogene activates classical NF-kappaB by stabilizing NIK.Oncogene 30:87–96.

34. Nagy R, Ganapathi S, Comeras I, Peterson C, Orloff M,Porter K, Eng C, Ringel MD, Kloos RT 2011 Frequency ofgermline PTEN mutations in differentiated thyroid cancer.Thyroid 21:505–510.

35. Gujral TS, van Veelen W, Richardson DS, Myers SM, MeensJA, Acton DS, Dunach M, Elliott BE, Hoppener JW, Mulli-gan LM 2008 A novel RET kinase-beta-catenin signalingpathway contributes to tumorigenesis in thyroid carci-noma. Cancer Res 68:1338–1346.

36. Kroll TG 2004 Molecular events in follicular thyroid tu-mors. Cancer Treat Res 122:85–105.

37. Yang D, Zhang H, Hu X, Xin S, Duan Z 2012 Abnormalityof p16/p38 MAPK/p53/Wip1 pathway in papillary thy-roid cancer. Gland Surg 1:33–38.

38. Edwards CQ, Griffen LM, Kushner JP 1991 Coincidentalhemochromatosis and viral hepatitis. Am J Med Sci 301:

50–54.39. Karger S, Weidinger C, Krause K, Sheu SY, Aigner T,

Gimm O, Schmid KW, Dralle H, Fuhrer D 2009 FOXO3a: anovel player in thyroid carcinogenesis? Endocr Relat Can-cer 16:189–199.

40. Tan A, Etit D, Bayol U, Altinel D, Tan S 2011 Comparisonof proliferating cell nuclear antigen, thyroid transcriptionfactor-1, Ki-67, p63, p53 and high-molecular weight cyto-keratin expressions in papillary thyroid carcinoma, follic-ular carcinoma, and follicular adenoma. Ann Diagn Pathol15:108–116.

41. Puppin C, Passon N, Frasca F, Vigneri R, Tomay F, To-maciello S, Damante G 2011 In thyroid cancer cell linesexpression of periostin gene is controlled by p73 and is notrelated to epigenetic marks of active transcription. CellOncol (Dordr) 34:131–140.

42. Malaguarnera R, Vella V, Vigneri R, Frasca F 2007 p53family proteins in thyroid cancer. Endocr Relat Cancer14:43–60.

43. Bauerle KT, Schweppe RE, Haugen BR 2010 Inhibition ofnuclear factor-kappa B differentially affects thyroid cancercell growth, apoptosis, and invasion. Mol Cancer 9:117.

44. Kroll TG, Sarraf P, Pecciarini L, Chen CJ, Mueller E, Spie-gelman BM, Fletcher JA 2000 PAX8-PPARgamma1 fusiononcogene in human thyroid carcinoma [corrected]. Science289:1357–1360.

45. Kato Y, Ying H, Zhao L, Furuya F, Araki O, WillinghamMC, Cheng SY 2006 PPARgamma insufficiency promotesfollicular thyroid carcinogenesis via activation of the nuclearfactor-kappaB signaling pathway. Oncogene 25:2736–2747.

46. Balkwill F, Mantovani A 2001 Inflammation and cancerback to virchow? Lancet 357: 539–545.

47. Ben-Neriah Y, Karin M 2011 Inflammation meets cancer,with NF-kappaB as the matchmaker. Nat Immunol 12:715–723.

48. Arif S, Blanes A, Diaz-Cano SJ 2002 Hashimoto’s thyroiditisshares features with early papillary thyroid carcinoma.Histopathology 41:357–362.

49. Asioli S, Erickson LA, Lloyd RV 2009 Solid cell nests inHashimoto’s thyroiditis sharing features with papillarythyroid microcarcinoma. Endocr Pathol 20:197–203.

50. Ahn D, Heo SJ, Park JH, Kim JH, Sohn JH, Park JY, ParkSK, Park J 2011 Clinical relationship between Hashimoto’sthyroiditis and papillary thyroid cancer. Acta Oncol50:1228–1234.

51. Larson SD, Jackson LN, Riall TS, Uchida T, Thomas RP, QiuS, Evers BM 2007 Increased incidence of well-differentiatedthyroid cancer associated with Hashimoto thyroiditis and therole of the PI3k/Akt pathway. J Am Coll Surg 204:764–773;discussion 773–765.

52. Loh KC, Greenspan FS, Dong F, Miller TR, Yeo PP 1999Influence of lymphocytic thyroiditis on the prognostic

216 LI ET AL.

outcome of patients with papillary thyroid carcinoma. JClin Endocrinol Metab 84:458–463.

53. Agarwal A, Das K, Lerner N, Sathe S, Cicek M, Casey G,Sizemore N 2005 The AKT/I kappa B kinase pathwaypromotes angiogenic/metastatic gene expression in colo-rectal cancer by activating nuclear factor-kappa B and beta-catenin. Oncogene 24:1021–1031.

54. Pufnock JS, Rothstein JL 2009 Oncoprotein signaling me-diates tumor-specific inflammation and enhances tumorprogression. J Immunol 182:5498–5506.

55. Russell JP, Engiles JB, Rothstein JL 2004 Proinflammatorymediators and genetic background in oncogene mediatedtumor progression. J Immunol 172:4059–4067.

56. Xiang Y, Remily-Wood ER, Oliveira V, Yarde D, He L,Cheng JQ, Mathews L, Boucher K, Cubitt C, Perez L,Gauthier TJ, Eschrich SA, Shain KH, Dalton WS, Hazle-hurst L, Koomen JM 2011 Monitoring a nuclear factor-kappaB signature of drug resistance in multiple myeloma.Mol Cell Proteomics 10:M110 005520.

57. Liu J, Qu X, Xu L, Zhang Y, Qu J, Hou K, Liu Y 2010Phosphoinositide 3-kinase/Akt and nuclear factor kappaBpathways are involved in tumor necrosis factor-relatedapoptosis-inducing ligand resistance in human gastriccancer cells. Mol Med Report 3:491–496.

58. Kim DS, Park SS, Nam BH, Kim IH, Kim SY 2006 Reversalof drug resistance in breast cancer cells by transglutami-nase 2 inhibition and nuclear factor-kappaB inactivation.Cancer Res 66:10936–10943.

59. Hayden MS, Ghosh S 2008 Shared principles in NF-kappaBsignaling. Cell 132:344–362.

60. Bonizzi G, Karin M 2004 The two NF-kappaB activationpathways and their role in innate and adaptive immunity.Trends Immunol 25:280–288.

61. Garrison JB, Samuel T, Reed JC 2009 TRAF2-binding BIR1domain of c-IAP2/MALT1 fusion protein is essential foractivation of NF-kappaB. Oncogene 28:1584–1593.

62. Cao Z, Xiong J, Takeuchi M, Kurama T, Goeddel DV 1996TRAF6 is a signal transducer for interleukin-1. Nature383:443–446.

63. Shikama Y, Yamada M, Miyashita T 2003 Caspase-8 andcaspase-10 activate NF-kappaB through RIP, NIK andIKKalpha kinases. Eur J Immunol 33:1998–2006.

64. Kopp E, Ghosh S 1994 Inhibition of NF-kappa B by sodiumsalicylate and aspirin. Science 265:956–959.

65. Rossi A, Elia G, Santoro MG 1997 Inhibition of nuclearfactor kappa B by prostaglandin A1: an effect associatedwith heat shock transcription factor activation. Proc NatlAcad Sci USA 94:746–750.

66. Spiecker M, Liao JK 1999 Assessing induction of I kappa Bby nitric oxide. Methods Enzymol 300:374–388.

67. May MJ, D’Acquisto F, Madge LA, Glockner J, Pober JS,Ghosh S 2000 Selective inhibition of NF-kappaB activationby a peptide that blocks the interaction of NEMO with theIkappaB kinase complex. Science 289:1550–1554.

68. Weerasinghe P, Hallock S, Liepins A 2001 Bax, Bcl-2, andNF-kappaB expression in sanguinarine induced bimodalcell death. Exp Mol Pathol 71:89–98.

69. Shishodia S, Majumdar S, Banerjee S, Aggarwal BB 2003Ursolic acid inhibits nuclear factor-kappaB activationinduced by carcinogenic agents through suppression ofIkappaBalpha kinase and p65 phosphorylation: correla-tion with down-regulation of cyclooxygenase 2, matrixmetalloproteinase 9, and cyclin D1. Cancer Res 63:4375–4383.

70. Deeb D, Jiang H, Gao X, Hafner MS, Wong H, Divine G,Chapman RA, Dulchavsky SA, Gautam SC 2004 Curcuminsensitizes prostate cancer cells to tumor necrosis factor-related apoptosis-inducing ligand/Apo2L by inhibitingnuclear factor-kappaB through suppression of IkappaBal-pha phosphorylation. Mol Cancer Ther 3:803–812.

71. Dauletbaev N, Lam J, Eklove D, Iskandar M, Lands LC2010 Ibuprofen modulates NF-kB activity but not IL-8production in cystic fibrosis respiratory epithelial cells.Respiration 79:234–242.

72. Gasparian AV, Guryanova OA, Chebotaev DV, ShishkinAA, Yemelyanov AY, Budunova IV 2009 Targeting tran-scription factor NFkappaB: comparative analysis of pro-teasome and IKK inhibitors. Cell Cycle 8:1559–1566.

73. Palombella VJ, Rando OJ, Goldberg AL, Maniatis T 1994The ubiquitin-proteasome pathway is required for proces-sing the NF-kappa B1 precursor protein and the activationof NF-kappa B. Cell 78:773–785.

74. Roccaro AM, Vacca A, Ribatti D 2006 Bortezomib in thetreatment of cancer. Recent Pat Anticancer Drug Discov1:397–403.

75. Williams AJ, Dave JR, Tortella FC 2006 Neuroprotectionwith the proteasome inhibitor MLN519 in focal ischemicbrain injury: relation to nuclear factor kappaB (NF-kappaB), inflammatory gene expression, and leukocyteinfiltration. Neurochem Int 49:106–112.

76. Sunwoo JB, Chen Z, Dong G, Yeh N, Crowl Bancroft C,Sausville E, Adams J, Elliott P, Van Waes C 2001 Novelproteasome inhibitor PS-341 inhibits activation of nuclearfactor-kappa B, cell survival, tumor growth, and angiogenesisin squamous cell carcinoma. Clin Cancer Res 7:1419–1428.

77. Daroczi B, Kari G, Ren Q, Dicker AP, Rodeck U 2009 Nu-clear factor kappaB inhibitors alleviate and the proteasomeinhibitor PS-341 exacerbates radiation toxicity in zebrafishembryos. Mol Cancer Ther 8:2625–2634.

78. Van Waes C, Chang AA, Lebowitz PF, Druzgal CH, ChenZ, Elsayed YA, Sunwoo JB, Rudy SF, Morris JC, Mitchell JB,Camphausen K, Gius D, Adams J, Sausville EA, Conley BA2005 Inhibition of nuclear factor-kappaB and target genesduring combined therapy with proteasome inhibitor bor-tezomib and reirradiation in patients with recurrent head-and-neck squamous cell carcinoma. Int J Radiat Oncol BiolPhys 63:1400–1412.

79. Cusack JC, Jr., Liu R, Houston M, Abendroth K, Elliott PJ,Adams J, Baldwin AS, Jr. 2001 Enhanced chemosensitivityto CPT-11 with proteasome inhibitor PS-341: implicationsfor systemic nuclear factor-kappaB inhibition. Cancer Res61:3535–3540.

80. Bielinska A, Baier L, Hailat N, Strahler JR, Nabel GJ, HanashS 1991 Expression of a novel nuclear protein in activated andin tat-I expressing T cells. J Immunol 146:1031–1036.

81. Kawamura I, Morishita R, Tomita N, Lacey E, Aketa M,Tsujimoto S, Manda T, Tomoi M, Kida I, Higaki J, KanedaY, Shimomura K, Ogihara T 1999 Intratumoral injection ofoligonucleotides to the NF kappa B binding site inhibitscachexia in a mouse tumor model. Gene Ther 6:91–97.

82. Zhang S, Won YK, Ong CN, Shen HM 2005 Anti-cancerpotential of sesquiterpene lactones: bioactivity and molecularmechanisms. Curr Med Chem Anticancer Agents 5:239–249.

83. Garcia-Pineres AJ, Lindenmeyer MT, Merfort I 2004 Role ofcysteine residues of p65/NF-kappaB on the inhibition bythe sesquiterpene lactone parthenolide and N-ethyl mal-eimide, and on its transactivating potential. Life Sci 75:841–856.

NF-jB AS A THERAPEUTIC TARGET FOR THYROID CANCER 217

84. Torgerson TR, Colosia AD, Donahue JP, Lin YZ, Hawiger J1998 Regulation of NF-kappa B, AP-1, NFAT, and STAT1nuclear import in T lymphocytes by noninvasive deliveryof peptide carrying the nuclear localization sequence of NF-kappa B p50. J Immunol 161:6084–6092.

85. Umezawa K 2006 Inhibition of tumor growth by NF-kappaB inhibitors. Cancer Sci 97:990–995.

86. Shoji S, Furuishi K, Ogata A, Yamataka K, Tachibana K,Mukai R, Uda A, Harano K, Matsushita S, Misumi S 1998An allosteric drug, o,o¢-bismyristoyl thiamine disulfide,suppresses HIV-1 replication through prevention of nucleartranslocation of both HIV-1 Tat and NF-kappa B. BiochemBiophys Res Commun 249:745–753.

87. Tang KT, Lee CH 2010 BRAF mutation in papillary thyroidcarcinoma: pathogenic role and clinical implications. J ChinMed Assoc 73:113–128.

88. O’Neill CJ, Bullock M, Chou A, Sidhu SB, Delbridge LW,Robinson BG, Gill AJ, Learoyd DL, Clifton-Bligh R, SywakMS 2010 BRAF(V600E) mutation is associated with an in-creased risk of nodal recurrence requiring reoperativesurgery in patients with papillary thyroid cancer. Surgery148:1139–1145; discussion 1145–1136.

89. Salvatore G, De Falco V, Salerno P, Nappi TC, Pepe S,Troncone G, Carlomagno F, Melillo RM, Wilhelm SM,Santoro M 2006 BRAF is a therapeutic target in aggressivethyroid carcinoma. Clin Cancer Res 12:1623–1629.

90. Ahmed KM, Dong S, Fan M, Li JJ 2006 Nuclear factor-kappaB p65 inhibits mitogen-activated protein kinase sig-naling pathway in radioresistant breast cancer cells. MolCancer Res 4:945–955.

91. Kim BY, Kim KA, Kwon O, Kim SO, Kim MS, Kim BS, OhWK, Kim GD, Jung M, Ahn JS 2005 NF-kappaB inhibitionradiosensitizes Ki-Ras-transformed cells to ionizing radia-tion. Carcinogenesis 26:1395–1403.

92. Yeh PY, Yeh KH, Chuang SE, Song YC, Cheng AL 2004Suppression of MEK/ERK signaling pathway enhancescisplatin-induced NF-kappaB activation by protein phos-phatase 4-mediated NF-kappaB p65 Thr dephosphoryla-tion. J Biol Chem 279:26143–26148.

93. Yu C, Rahmani M, Conrad D, Subler M, Dent P, Grant S2003 The proteasome inhibitor bortezomib interacts syn-ergistically with histone deacetylase inhibitors to induceapoptosis in Bcr/Abl + cells sensitive and resistant toSTI571. Blood 102:3765–3774.

94. Yeh PY, Chuang SE, Yeh KH, Song YC, Ea CK, Cheng AL2002 Increase of the resistance of human cervical carcinomacells to cisplatin by inhibition of the MEK to ERK signal-ing pathway partly via enhancement of anticancer drug-induced NF kappa B activation. Biochem Pharmacol 63:

1423–1430.95. Liu J, Suresh Kumar KG, Yu D, Molton SA, McMahon M,

Herlyn M, Thomas-Tikhonenko A, Fuchs SY 2007 Onco-genic BRAF regulates beta-Trcp expression and NF-

kappaB activity in human melanoma cells. Oncogene26:1954–1958.

96. Liu D, Xing M 2008 Potent inhibition of thyroid cancer cellsby the MEK inhibitor PD0325901 and its potentiation bysuppression of the PI3K and NF-kappaB pathways. Thyroid18:853–864.

97. Yang F, Jove V, Chang S, Hedvat M, Liu L, Buettner R, TianY, Scuto A, Wen W, Yip ML, Van Meter T, Yen Y, Jove R2012 Bortezomib induces apoptosis and growth suppres-sion in human medulloblastoma cells, associated with in-hibition of AKT and NF-kB signaling, and synergizes withan ERK inhibitor. Cancer Biol Ther 13:349–357.

98. Kane RC, Dagher R, Farrell A, Ko CW, Sridhara R, JusticeR, Pazdur R 2007 Bortezomib for the treatment of mantlecell lymphoma. Clin Cancer Res 13:5291–5294.

99. Richardson PG, Hideshima T, Anderson KC 2003 Borte-zomib (PS-341): a novel, first-in-class proteasome inhibitorfor the treatment of multiple myeloma and other cancers.Cancer Control 10:361–369.

100. Wunderlich A, Arndt T, Fischer M, Roth S, Ramaswamy A,Greene BH, Brendel C, Hinterseher U, Bartsch DK, Hoff-mann S 2012 Targeting the proteasome as a promisingtherapeutic strategy in thyroid cancer. J Surg Oncol105:357–364.

101. Zhu W, Ou Y, Li Y, Xiao R, Shu M, Zhou Y, Xie J, He S, QiuP, Yan G 2009 A small-molecule triptolide suppresses an-giogenesis and invasion of human anaplastic thyroid carci-noma cells via down-regulation of the nuclear factor-kappaB pathway. Mol Pharmacol 75:812–819.

102. Stenner F, Liewen H, Zweifel M, Weber A, Tchinda J, BodeB, Samaras P, Bauer S, Knuth A, Renner C 2008 Targetedtherapeutic approach for an anaplastic thyroid cancerin vitro and in vivo. Cancer Sci 99:1847–1852.

103. Zhu W, He S, Li Y, Qiu P, Shu M, Ou Y, Zhou Y, Leng T,Xie J, Zheng X, Xu D, Su X, Yan G 2010 Anti-angiogenicactivity of triptolide in anaplastic thyroid carcinoma ismediated by targeting vascular endothelial and tumor cells.Vascul Pharmacol 52:46–54.

104. Mitsiades CS, McMillin D, Kotoula V, Poulaki V, McMullanC, Negri J, Fanourakis G, Tseleni-Balafouta S, Ain KB, Mit-siades N 2006 Antitumor effects of the proteasome inhibitorbortezomib in medullary and anaplastic thyroid carcinomacells in vitro. J Clin Endocrinol Metab 91:4013–4021.

Address correspondence to:Emad Kandil, MD, FACS

Tulane Cancer CenterTulane University School of Medicine

1430 Tulane Ave. SL-22New Orleans, LA 70112-2699

E-mail: ekandil@tulane.edu

218 LI ET AL.