Role of BRAF in Thyroid Oncogenesis - Clinical Cancer Researchproduce thyroid hormone, and parafollicular cells (or C-cells), which are much less prevalent and produce calcito-nin,

Molecular Pathways

Role of BRAF in Thyroid Oncogenesis

Lisa M. Caronia1, John E. Phay2, and Manisha H. Shah3

AbstractBRAF, a cytoplasmic serine–threonine protein kinase, plays a critical role in cell signaling as an activator

within the mitogen-activated protein kinase (MAPK) pathway. The most common BRAF mutation is the

V600E transversion, which causes constitutive kinase activity. This mutation has been found in amultitude

of human cancers, including both papillary thyroid cancer (PTC) and papillary-derived anaplastic thyroid

cancer (ATC), in which it initiates follicular cell transformation. With such a high frequency of BRAF

mutations in PTC (44%) and PTC-derived ATC (24%), research in BRAFV600E detection for diagnostic

purposes has shown high sensitivity and specificity for tumor cell presence. BRAFV600E in PTC has also

provided valuable prognostic information, as its presence has been correlated with more aggressive and

iodine-resistant phenotypes. Such findings have initiated research in targeting oncogenic BRAF in cancer

therapeutics. Althoughmultiple phase II clinical trials in patients with iodine-refractorymetastatic PTChave

shown significant efficacy for sorafenib, a first-generation BRAF inhibitor, the mechanism by which it

mediates its effect remains unclear because of multiple additional kinase targets of sorafenib. Additionally,

preclinical and clinical studies investigating combination therapy with agents such as selective (PLX 4032)

and potent (BAY 73-4506 and ARQ 736) small-molecule BRAF inhibitors and MAP/extracellular signal-

regulated kinase (ERK) kinase inhibitors (AZD6244) hold great promise in the treatment of BRAFV600E

cancers andmay eventually play a powerful role in changing the clinical course of PTC and ATC.Clin Cancer

Res; 17(24); 7511–7. �2011 AACR.

Background

RAF, a cytoplasmic serine–threonine protein kinase, isa member of the RAS–RAF–MEK–ERK cell-signaling path-way [also known as the MAP kinase (MAPK) pathway],and it plays an essential role in mediating cellular differ-entiation, proliferation, senescence, and survival inresponse to extracellular cues. Physiologic activation ofthis pathway typically occurs through a variety of plasmamembrane receptors that activate Ras, a membrane-bound small G protein. Activated Ras recruits Raf to theplasma membrane for activation. Subsequently, Rafphosphorylates and activates MAP–ERK kinase (MEK),which phosphorylates and activates extracellular signal-regulated kinase (ERK). Phosphorylated Erk has morethan 150 downstream targets, both nuclear and cytosolic(1). After nuclear translocation, Erk can directly phos-

phorylate multiple transcription factors, including c-Myc,c-Jun, Ets, and c-Fos (2). These transcription factors, inturn, have been shown to regulate cell cycle, growth, andsurvival (Fig. 1). Erk also phosphorylates many cytosolicproteins, including cell-cycle proteins such as retinoblasto-ma, apoptotic proteins such as Bad, MCL-1, and caspase 9,and cytoskeletal proteins such as paxillin, calnexin, andvinexin. The overall effects can be very divergent, and clearlyare dependent on specific cell types.

The regulation of this pathway is complex becausemultiple isoforms of every pathway protein exist, eachencoded by different genes and having both overlappingand distinct functions. There are 3 RAF isoforms: ARAF,BRAF, and CRAF (also known as Raf-1). The BRAF gene,which is found on chromosome 7, is the strongest MAPKpathway activator (3, 4) and the most frequently mutatedhuman oncogene in the kinase superfamily (5). Addi-tional pathway complexity arises from its lack of linearity,as BRAF can form a heterodimer with CRAF, resulting indownstream MEK–ERK signaling (6, 7), which can alsooccur even when one of the heterodimers is inactive.Additionally, kinase suppressor of Ras (KSR), which func-tions primarily as a scaffold, colocalizing Raf, Mek, andErk, is able to trigger BRAF activation through side-to-sideheterodimerization (8, 9). Thus, the intricacy of theMAPK pathway and the regulation of BRAF within itcreate a variety of opportunities whereby a mutationcould result in aberrant BRAF signaling.

Authors' Affiliations: 1OhioStateUniversity College ofMedicine, Divisionsof 2Surgical Oncology and 3Medical Oncology, James Cancer Center &Solove Research Institute, Columbus, Ohio

Note: L.M. Caronia and J.E. Phay contributed equally to this article.

Corresponding Author:Manisha H. Shah, A438 Starling-Loving Hall, 320West 10th Ave., Columbus, OH 43210. Phone: 614-293-8629; Fax: 614-293-3112; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-11-1155

�2011 American Association for Cancer Research.

ClinicalCancer

Research

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Published OnlineFirst September 7, 2011; DOI: 10.1158/1078-0432.CCR-11-1155

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Oncogenic Mutations in BRAF

The first activating mutations in BRAF were described in2002 and clustered in the kinase domain (10). The mostcommon BRAF mutation is the T1799A transversionresulting in a glutamic acid for valine (V600E) adjacentto an activating phosphorylation site at Ser599. TheCatalogue of Somatic Mutations in Cancer (COSMIC)database currently reports on its website that the V600Emutation represents more than 95% of all BRAF muta-tions of the 78,000 unique samples reported (11). In itswild-type conformation, residues G597 to V601 form ahydrophobic interaction with residues G465 to V472 inthe ATP-binding side (P-loop), keeping it inactivated. TheBRAFV600E mutation disrupts the hydrophobic interac-tion, enabling the BRAF kinase to fold into a catalyticallyactive formation, resulting in an almost 500-fold increasein kinase activity (12). Presently, more than 40 BRAFmutations have been reported, with a majority located inthe kinase domain and P-loop, resulting in a directincrease in MEK phosphorylation. Paradoxically, severalmutations have reduced in vitro kinase activity towardMEK, but they possess enough activity to transphosphor-ylate and activate CRAF though differences in heterodi-merization (6, 12). The BRAFD594V variant is termed"kinase-dead" as the BRAF is catalytically inactive, yet ithas been found in multiple cancers. This kinase-deadBRAF or wild-type BRAF that has been chemically inhib-

ited has been shown to bind to CRAF and potentiateoncogenic Ras mutations, thereby further stimulatingthe MAPK-signaling cascade and resulting in increasedtumor growth (13). This aberrant growth highlights aparticular danger in targeting BRAF activity in a tumorwith a RAS mutation. Finally, a rare genetic alteration inthe BRAF gene has also been identified, in which the longarm of chromosome 7 becomes paracentrically inverted,leading to recombinant AKAP9-BRAF oncogene forma-tion (14). This rearrangement results in loss of the BRAFautoinhibitory domains and, thus, constitutive kinaseactivation.

Thyroid Cancer and BRAF Mutations

The thyroid gland is composed of 2 hormone-producingcell types: follicular cells, which incorporate iodine toproduce thyroid hormone, and parafollicular cells (or C-cells), which are much less prevalent and produce calcito-nin, a hormone that regulates calcium. There are 4 types ofthyroid cancer. Papillary thyroid cancer (PTC) is the mostprevalent, accounting for more than 80% all of thyroidcancer cases, and arises from follicular cells. Follicularthyroid cancer (FTC) is also derived from follicular cellsand, similar to PTC, is treated primarily with surgery andradioactive iodine ablation. Medullary thyroid cancer(MTC) is derived from the parafollicular cell and is treatedby surgery. Finally, anaplastic thyroid cancer (ATC), which

© 2011 American Association for Cancer Research

ERK

MEK

MutantBRAF

RAS

CRAFBRAFBRAF

BRAF

BRAF

Receptortyrosine kinase

Cell cycleRb

ApoptosisBad

MCL-1Caspase 9

CytoskeletalPaxillin

CalnexinVinexin

c-Myc

Etsc-Jun

Nucleus

c-Fos

KSR

Figure 1. The MAPK-signalingpathway is typically initiatedthrough activation of a receptortyrosine kinase, which activatesRAS, which, in turn, facilitateshomo- or heterodimerization ofwild-type BRAF. Activated BRAFphosphorylates MEK (which isbound to KSR), whichphosphorylates ERK, resulting inmultiple cellular effects such asproliferation and survival. MutantBRAF can dimerize and activateMEK without Ras activation.

Caronia et al.

Clin Cancer Res; 17(24) December 15, 2011 Clinical Cancer Research7512




probably arises from PTC or FTC, is the most aggressivethyroid cancer, with an average survival of less than 6months. Very few effective therapies are available exceptsurgery and radioactive iodine for all thyroid cancers.BRAF mutations have been discovered in a variety of

human cancers, includingmalignant melanoma, colorectalcancer, ovarian cancer, lung cancer, and thyroid cancer(4, 10, 15, 16). BRAF mutations were initially reported inthyroid cancer in 2003 with a frequency ranging from 26%to 44% (17, 18). Mutations have only been reported in 2types of thyroid cancer, namely PTC and ATC (19). BRAFmutations have not been identified in FTC, MTC, benignthyroid adenomas, or hyperplasia (20). From 29 studiesreporting on BRAFmutations inmore than 2,000 examinedthyroid cancers, the average frequency of mutations in PTCis 44% and in ATC is 24% (20). Besides BRAF mutations, 2other well-described mutations activate the MAPK-signal-ing pathway in PTC: RET/PTC rearrangements and activat-ing Ras mutations. RET/PTC rearrangements are a somaticchromosomal fusion in which the 30 terminal activationsequence of RET (a receptor tyrosine kinase that activatesRas) is placed under the expressional control of anothergene, leading to ligand-independent activation of RETand, subsequently, Ras. BRAF, RET/PTC, and Ras muta-tions are generally mutually exclusive in PTC and occur inapproximately 70% to 80% of cases, indicating theimportance of the MAPK-signaling pathway in this par-ticular tumor (21).BRAFV600E has been shown to initiate thyroid follic-

ular cell transformation both in culture and in trans-genic mice (22). In transgenic mice, conditional endog-enous expression of BRAFV600E in melanocytes and lungalveolar epithelial cells generally results in initialincreased proliferation, frequently followed by senes-cence, as opposed to endogenous expression in thyro-cytes, where fully penetrant PTC is seen by 5 weeks (23).The knock-in of BRAFV600E in mice thyrocytes, under thecontrol of the thyroid peroxidase promoter, results inclassic appearing PTC with frequent local invasion, andits short latency seems dependent on the presence ofthyrotropin receptor signaling. BRAFV600E has beenfound in microcarcinomas, further supporting the ideathat this mutation may be an inciting factor in theoncogenic transformation.The prevalence of BRAF mutations within PTC has a

subtype-specific pattern, such that BRAFmutations aremostcommon in tall-cell PTC, slightly less common in conven-tional PTC, and rarely found in follicular-variant PTC (24,25). This preferential cell-specific distributionmay partiallyexplain the wide range in BRAF mutation prevalencereported inPTC, asmany reportsmaynot stratify by subtypeduringdata analysis (20). There also seems tobe a reciprocalage association between BRAF mutation and RET/PTCrearrangements in PTC, such that increasing age is a pre-disposing factor to sporadic BRAF mutations (20, 26).Conversely, RET/PTC rearrangements are more prevalentin childhoodPTC, aswell as in all age groupswith radiation-induced PTC.

Clinical–Translational Advances

BRAF mutation analysis for diagnosisWith such a high prevalence of BRAF mutations in PTC

and PTC-derived ATC, a great deal of interest has beenexpressed in the use of BRAFV600E detection for diagnosticpurposes. Fine-needle aspiration (FNA) with cytologicanalysis is widely used as the initial step for evaluatingthyroid nodules. BRAFV600E detection in FNA specimenshas been evaluated in multiple studies (27, 28). Using acolorimetric assay to detect the BRAFV600E mutation froma FNA sample, Xing and colleagues showed 100% sensitiv-ity and specificity at a prevalence similar to that of theBRAFV600E PTC patient population (44%), with no false-positive findings; however, negative BRAFV600E resultscould not discriminate between the presence or absence ofPTC, especially in specimens with unclear cytology (27).Because 20% of FNAs yield ambiguous or indeterminateresults, surgical intervention is often the next step, despitethe fact that approximately 80%of this population does nothave thyroid cancer upon further histologic analysis (29).Most indeterminate FNAs aredue to the inability of cytologyto distinguish a benign follicular adenoma from a follicularcarcinoma. Because BRAF mutations are not found infollicular carcinomas, mutational analysis is generally nothelpful for this group of thyroid nodules (28). In a recentstudy of 110 indeterminate FNAs with definitive surgicalpathology, 3 had BRAF mutations (29 cancers; ref. 30). Areview of several studies of BRAFmutations in FNA samplestotaled 7 BRAF mutations in 100 samples (28).

Detection of BRAF mutations in serum DNA is also ofinterest, as cancerous cells can sometimes break off a tumorand circulate peripherally in the blood. In previous lines ofresearch, detection sensitivity was insufficient; however,with the use of a mutant allele-specific PCR amplificationtechnique, plasma DNA detection of BRAF mutations inpatients with colon cancer was completed with 100%sensitivity (31). Another study was able to detect 1 hetero-zygous BRAFmutation in a population ofmore than 20,000cells using real-time PCR, and when this technique wasapplied to blood samples from PTC patients, a BRAF muta-tion was detected in 20% of patients (32). The benefit ofthese detection methods is the potential to eliminate theneed to perform FNA in patients with a positive BRAFmutation; however, additional research investigating spec-ificity and sensitivity will need to be completed before thesemethods can be put into practice clinically.

BRAF mutation analysis for prognosisMore than 30 studies have examined the correlation of

BRAFV600E mutation with a variety of clinicopathologicPTCcharacteristics (33, 34).Most of these studies, includinga meta-analysis, have shown a correlation with at least 1poor prognostic factor, such as extrathyroidal extension,lymph node metastases, advanced stage, greater predilec-tion to develop iodine-131 resistance, and recurrent disease(35–38). Conversely, several studies have not shown anycorrelation, including 2 that analyzed a relatively large

BRAF and Thyroid Cancer

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number of patients (631 and 260; refs. 39–41). Only 1study has shown a correlation with distant metastases(18, 42, 43). Papillary thyroid microcarcinomas are tumorsless than 1 cm in diameter and are generally indolent, buttheyhave alsobeen found tohaveBRAFV600Emutations thatare associated with extrathyroidal extension and lymphnode metastasis (44, 45). An increased frequency of BRAFmutation, up to 85%, has been reported in recurrent PTCtumors (42, 43). Additionally, in the setting of advancedPTC, BRAF mutations were noted to be at an increasedfrequency (62%) of recurrent and/or metastatic tumorsfrom iodine-refractory PTC patients (46). In this study,frequency of BRAF mutation varied based on the type ofPTC: 100% (12 of 12) of tall-cell variant of PTC, 85% (6 of7) of well-differentiated PTC, and 47% (15 of 32) of poorlydifferentiated PTC had BRAF mutations. Among patientswith metastases frommultiple sites, 8 of 8 patients showedbetween-sample concordance for BRAF mutations. Despitethese conflicting studies, a BRAF mutation is likely anindependent marker for a more aggressive PTC, and itspresence may aid in the management of PTC throughoutits clinical course.

Targeting BRAF in thyroid cancer for treatmentWith the findings that BRAF mutations in PTC, ATC, and

other human tumors tend to have more aggressive pheno-types and often become resistant to traditional therapies,developing a therapeutic agent that can selectively targetoncogenic BRAF kinase may be of great clinical utility.Multikinase inhibitors, which act on multiple componentsof the MAPK pathway, have shown great promise in thetreatment of malignancies harboring a BRAF mutation.Sorafenib (BAY 43-9006) is one such therapeutic agent thattargets BRAF, CRAF, VEGF receptors 1 to 3, platelet-derivedgrowth factor (PDGF) receptor, and RET kinases to inhibittumor proliferation and angiogenesis (47, 48). Using RNAinterference (siRNA) to knock down BRAF in human ATCcell lines, preclinical studies showed the importance ofBRAF for intracellular MAPK signaling and proliferation,as tumor growth was significantly inhibited (49, 50). Thesefindings suggested that BRAF could be an effective target forthyroid cancer treatment. The effect of sorafenibwas similarto that of siRNA BRAF knockdown, inhibiting BRAFV600E-mediated intracellular signaling in vitro in xenograft modelsand in thyroid carcinoma cells, yet having minimal effectson normal thyrocytes (49).

Our group reported results of a National Cancer Insti-tute–sponsored investigator-initiated phase II clinical trialin patients with iodine-refractory metastatic PTC (51, 52).This study was the first phase II trial of a multikinaseinhibitor showing significant clinical and biologic activityof sorafenib in patients with iodine-refractory metastaticPTC. Based on the Response Evaluation Criteria in SolidTumors (RECIST), sorafenib showed a 15% (6 of 41patients) partial response rate in patients with metastaticPTC. A median progression-free survival in this single-armstudy was 15 months (51). Significant and sustaineddecreases in the serum tumor marker thyroglobulin were

also observed. Fourteen (64%) of 22 patient tumor sampleshad a BRAFV600E mutation, whereas 3 (14%) had aBRAFK601E mutation. Of 9 PTC patients who had tumorsamples from multiple sites, 8 showed concordant BRAFmutation status. Because of the high frequency (78%) ofBRAF mutations in the study population, statistical com-parison of objective response with BRAF mutation statuswas not possible. Paired tumor biopsies were collected in asubset of patients and showed a significant reduction inphosphorylated VEGFR and phosphorylated ERK and anincrease in VEGF expression after 8 weeks of sorafenibcompared with baseline. Tumor perfusion was alsodecreased when assessed with serial dynamic contrast-enhanced MRI (DCE-MRI). In 10 of 14 assessable PTCpatients, the 8- or 16-week on therapy DCE-MRI scansrevealed a median decrease of 46% (range, 27%–92%) inexchange rate (Kep, exchange rate constant) in the indexlesions compared with baseline. Of note, no objectiveresponse occurred in any of the 4 patients who did notshow a change in Kep. Results of correlative studies in thistrial reveal significant antiangiogenic activity of sorafenib inaddition to inhibiting the BRAF pathway. Another single-institution phase II clinical trial of sorafenib in metastatic,iodine-refractory thyroid carcinoma (N ¼ 30) yielded sim-ilar results, with a 23% partial response rate and 18-monthmedian progression-free survival (53). Differences inpatient population may underlie the variation in outcomesobserved between the 2 trials. Sorafenib failed to restoreiodine avidity in patients with iodine-refractory PTC whentested in a phase II clinical trial (54). Taken together,these phase II clinical trials of sorafenib show its efficacyin the treatment of iodine-refractory metastatic PTC. Aphase II study of sorafenib in ATC patients and an inter-national multicenter phase III trial of sorafenib versusplacebo in patients with iodine-refractory thyroid cancer(NCT00984282) are ongoing.

Despite its effectiveness in the treatment of thyroidcancer, sorafenib has a range of side effects that must beconsidered prior to the initiation of therapy. The mostcommon adverse events reported include diarrhea, hyper-tension, fatigue, and hand–foot syndrome (51, 53). Oth-er serious, yet rare events include bowel perforation,thromboembolism, and bleeding. Additionally, keratoa-canthomas of the skin have been reported in a minorityof patients, and they seem to be related to class effects ofBRAF inhibitors. As keratoacanthoma is a low-gradesquamous cell carcinoma variant that has the potentialto become invasive or metastatic, the papules are gener-ally treated surgically.

The mechanism by which sorafenib mediates its ther-apeutic effect in PTC remains unclear, although it is likelyto be related to multiple target inhibition, includingBRAF, RET, VEGF, and PDGF. It seems that angiogenesisis a major mechanism that is targeted, because severalmultikinase inhibitors (such as sunitinib, axitinib, mote-sanib, and pazopanib) that are not shown to inhibitBRAF, but target VEGF and PDGF, are also effective inpatients with PTC.

Caronia et al.





Future directionsWith the discovery of BRAF-targeted therapy producing

exciting but modest clinical benefit, improving efficacy ofsuch therapy in thyroid cancer is the obvious next step. Tothis end, attempts are being made to design combinationtherapies that target pathways responsible for resistance toBRAF as well as to improve specificity and potency of BRAFinhibitors. A phase I trial examined the effect of bothsorafenib and tipifarnib, a farnesyltransferase inhibitor, in50 patients with a variety of advancedmalignancies, includ-ing ATC and PTC (55). All 4 patients with PTC showedsignificant regression lasting over 18 months, despite thesepatients having disease progression prior to study entry.Another study investigated the effectiveness of sorafeniband AZD6244, a MEK kinase inhibitor, in the treatment ofhuman gastric cancer–derived xenografts (56). In vitro andin vivo, this combination therapy showed a decrease in bothtumor growth and angiogenesis and an increase in apopto-sis, responses that were an amplification of those seen withsorafenib alone.Several new drugs that are either potent BRAF inhibitors

(i.e., BAY 73-4506 and ARQ 736) or selectively targetBRAFV600E mutation (i.e., PLX4032 and GSK2118436) arebeing tested in various phases of clinical trials. PLX4032showed apreferential inhibition of cell proliferation,migra-tion, and invasion of BRAFV600E human ATC cell lines (57).Furthermore, PLX4032 decreased tumor growth and aggres-siveness in an animal model using human ATC cell lines.Although phase II clinical trials are still under developmentusing this drug, data are available in a few patients withthyroid cancers who were treated on a phase I clinical trial.In this trial, 2 of 3 patients with PTC had stable diseaselasting 11 to 13months, whereas 1 patient had either partialor complete response lasting 8 months (56). Of note, 32patients withmelanomawith the BRAFV600E mutation wereenrolled in this phase I study. Partial or complete response

was noted in 81% of patients, with an estimated medianprogression-free survival of more than 7 months. Recentstudies have unveiled pathways of acquired resistance toBRAF-targeted therapy in melanoma (58, 59).

Overexpression of MAP kinase kinase kinase 8 (MAP3K8or COT), CRAF, or PDGF-b, as well as mutations in NRAS,result in secondary resistance to BRAF inhibitors. Interest-ingly, secondary mutations in BRAFV600E are not found inthis setting.

Conclusions

BRAF mutations were first identified in malignant mel-anoma by Davies and colleagues in 2002 while screeninggenes encoding the components of theMAPKpathway (10).In the past 9 years, remarkable progress has been made inthe BRAF field, including the identification of oncogenicBRAF in a wide range of human malignancies includingPTC, development of diagnostic techniques, and prognosticcriteria for thyroid cancers based on BRAF mutations, andcompletion of phase II clinical trials in thyroid cancer fortherapeutics targeting aberrant BRAF signaling. Thisresearch has already begun to change the clinical course ofiodine-refractory PTC, for which the previous therapeuticoptions were limited to supportive care. In the future,further exploration of the mechanisms of primary andsecondary resistance for BRAF-targeted therapies in thyroidcancer and preclinical studies identifying types of effectivecombination therapies and optimum dose and sequence ofcombination therapies will be critical.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Received May 2, 2011; revised August 9, 2011; accepted August 9, 2011;published OnlineFirst September 7, 2011.

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2011;17:7511-7517. Published OnlineFirst September 7, 2011.Clin Cancer Res Lisa M. Caronia, John E. Phay and Manisha H. Shah Role of BRAF in Thyroid Oncogenesis

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