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Functional Characterization of the Novel T599I-VKSRdel BRAF Mutation in a Follicular Variant Papillary Thyroid Carcinoma

Istituto di Endocrinologia ed Oncologia Sperimentale del CNR (V.D.F., A.T., M.S.), Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università di Napoli Federico II, 80131 Naples, Italy; Department of Surgery (R.G., C.U., C.L., F.B.), University of Pisa, 56127 Pisa, Italy; and Dipartimento di Medicina Interna (E.P.), Università degli Studi di Perugia, 06123 Perugia, Italy

Address all correspondence and requests for reprints to: Massimo Santoro, Dipartimento di Biologia e Patologia Cellulare e Molecolare, "L. Califano", Universita’ Federico II di Napoli, via S. Pansini 5, 80131 Naples, Italy. E-mail: masantor{at}unina.it.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
Context: Mutations in BRAF are rare in the follicular variant of papillary thyroid carcinoma (FV-PTC).

Objective: We identified and functionally characterized a novel T599I-VKSR(600–603)del BRAF mutation in a FV-PTC patient. We analyzed in vitro the effects of this novel mutation in comparison with other thyroid cancer-associated mutations.

Design: Expression vectors for the BRAF mutants were generated and their in vitro kinase activity, signaling along the MAPK pathway, and capability of stimulating transcription from an AP1-responsive reporter evaluated.

Results: BRAF kinase and signaling were increased to a similar extent by the T599I-VKSR (600–603)del, V600E, and K601E mutations. Instead, the G474R, a mutation previously found in a FV-PTC, knocked down the BRAF kinase and its intracellular signaling. Some cancer-associated low-activity BRAF mutants stimulate the MAPK cascade via CRAF; however, the G474R protein lacked also this property.

Conclusion: The T599I-VKSR(600–603)del is a novel gain-of-function mutation that targets BRAF in FV-PTC. Moreover, G474R is the first example of a mutation knocking down enzymatic BRAF activity in a FV-PTC. These findings underscore the importance of functional studies to characterize the role of BRAF mutations associated with thyroid cancer.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
Point mutations in BRAF are the most common genetic event in papillary thyroid carcinoma (PTC) and can initiate thyroid carcinogenesis in vivo (1, 2, 3, 4). V600E is the most frequent mutation in thyroid cancer. Less frequent are the K601E (5), V599ins (6), G474R (7), and G469R (8) point mutations as well as complex genetic alterations such as the AKAP9-BRAF rearrangement (9), the V600E-K601del (10, 11), and V600D-FLAGT601–605ins (12).

There are three main PTC subtypes: the tall cell variant (TCV)-PTC, conventional variant (CV)-PTC, and follicular variant (FV)-PTC. BRAF is mutated in about 77% of TCV-PTC cases, in 60% of CV-PTC cases, and in only 12% of FV-PTC cases (3). Moreover, there is an association between PTC subtypes and particular BRAF mutations. Although V600E accounts for most BRAF-mutated cases of CV-PTC and TCV-PTC, mutants K601E (7% of patients) and G474R (in only one patient) are associated with FV-PTC (3, 5, 7, 13).

Under basal conditions, hydrophobic interactions between the activation loop (A-loop), in the C-lobe, and the glycine-rich phosphate-binding loop (P-loop), in the N-lobe, of the BRAF kinase stabilize its inactive conformation. Oncogenic mutations mostly cluster at the A- and P-loops and destabilize the inactive conformation, thereby promoting constitutive activity of the enzyme (14). Functionally, cancer-associated BRAF mutants can be divided into three categories: high activity (exemplified by V600E in the A-loop), low activity (exemplified by G466E/V in the P-loop and G596R in the A-loop), and rare impaired activity (exemplified by D594V in the A-loop) (14, 15). These mutants differ in the extent and mechanism of MAPK kinase (MEK), the BRAF downstream kinase, activation. Low-activity mutants, which have impaired MEK kinase activity, can activate MEK indirectly by binding and allosterically activating CRAF. High-activity BRAF mutants signal directly to MEK (although they can also activate MEK through CRAF). No effect has yet been ascribed to impaired-activity BRAF mutants (14, 15).

	Patients and Methods

Top Abstract Introduction Patients and Methods Results Discussion References

 
Thyroid sample

The patient (a 23-yr-old woman) had a 2-cm thyroid nodule and was treated at the Institute of Endocrinology of the University of Pisa with Ethics Committee approval (16). Histological diagnosis, encapsulated FV-PTC stage pT1NxMx with a neoplastic blood embolus, was made in a blinded fashion by two pathologists (C.U. and F.B.) according to the World Health Organization guidelines (17). She belonged to a series of 500 PTC patients treated at the same institution (16). Among these samples, 230 were micro-PTC, 82 were CV-PTC, 114 were FV-PTC, 40 were TCV-PTC, and 34 belonged to other variants; 219 and three of the PTC samples of this series had the V600E or the K601E mutation, respectively. The BRAF mutant cases were 90 micro-PTC, 56 CV-PTC, 32 TCV-PTC, 21 FV-PTC, and 15 other subtypes. The K601E mutation was found in one FV-PTC and in two micro-PTC with a FV-PTC pattern (16).

Detection of BRAF mutation

DNA was isolated by using QIAGEN spin columns (QIAGEN GmbH, Hilden, Germany) and processed for PCR amplification. Single-strand conformation polymorphism and sequencing were carried out as described elsewhere (16).

Expression vectors and cell transfections

BRAF and CRAF expression vectors were kindly donated by C. J. Marshall. The T599I-VKSRdel mutant was generated by PCR. The thyroid cancer-associated (V600E, G474R, and K601E), the melanoma-associated (G596R), and the kinase-dead K483M (BRAF K-) BRAF constructs were obtained by site-directed mutagenesis (Stratagene, La Jolla, CA). The constructs were myc-tagged and cloned in the pEF vector. The mutations were confirmed by DNA sequencing. HEK293 cells were grown in DMEM supplemented with 10% fetal calf serum (Invitrogen, Carlsbad, CA). PC Cl 3 (hereafter referred to as PC), a differentiated rat thyroid follicular cell line, was cultured in Coon’s modified Ham F12 medium supplemented with 5% calf serum and a mixture of six hormones (6H), including TSH as described (18). Transfections were carried out with the Lipofectamine reagent according to the manufacturer’s instructions (GIBCO, Paisley, PA). In HEK293, green fluorescent protein transfection was used to measure transfection efficacy (about 30%).

Protein studies

Immunoblotting experiments were performed according to standard procedures. Signal intensity was evaluated with the Phosphorimager (Typhoon 8600; Amersham Pharmacia Biotech, Little Chalfont, UK) interfaced with the ImageQuant software. Anti-phospho-p44/42 MAPK (9102), anti-p44/42 MAPK (9101), anti-phospho-p90RSK (90-kDa ribosomal S6 kinase) (9344), anti-p90RSK (9347), anti-phospho-MEK1/2 (MAPK 1 and 2) (9121), and anti-MEK1/2 (9122) were from Cell Signaling (Beverly, MA). Anti-BRAF (sc-9002) was from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti--tubulin was from Sigma Chemical Co. (St. Louis, MO). Secondary antibodies coupled to horseradish peroxidase were from Santa Cruz Biotechnology. For the BRAF kinase assay, HEK293 cells were transiently transfected with the indicated constructs, cultured for 18 h in serum-deprived medium, and harvested 48 h after transfection. BRAF kinase was immunoprecipitated with anti-myc and resuspended in a kinase buffer containing 100 mM MgCl₂, 20 µM ATP, and 0.5 µg recombinant glutathione S-transferase-MEK. After 15 min incubation at 30 C, reactions were stopped by adding 2x Laemmli buffer. Proteins were then subjected to 10% SDS-PAGE and immunoblotted with anti-phospho-MEK1/2 antibody. Immunoblots with anti-MEK antibodies served as loading controls.

Luciferase assays

HEK293 cells were transiently transfected with BRAF vectors and the AP1-Luc reporter containing six AP1 binding sites upstream from the Firefly luciferase cDNA (Stratagene, Garden Grove, CA). Twenty-four hours after transfection, cells were serum starved and harvested 48 h after transfection. Ten nanograms of pRL-null (a plasmid expressing the enzyme Renilla luciferase from Renilla reniformis) were used for normalization. Firefly and Renilla luciferase activities were assayed using the Dual-Luciferase reporter system (Promega Corp., Madison, WI) in protein extracts normalized for equal level expression of the BRAF constructs and expressed as fold increase with respect to empty vector-transfected cells. Average results of three independent assays ± SD are indicated.

Statistical analysis

The two-tailed unpaired Student’s t test (normal distributions and equal variances) was used for statistical analysis (GraphPad InStat software program, version 3.06.3; GraphPad, San Diego, CA). Differences were significant when P < 0.001.

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
Identification of the T599I-VKSRdel mutation in BRAF

We identified a novel BRAF mutation in a FV-PTC (Fig. 1A). The mutation was a heterozygous 14-bp deletion with a 2-bp insertion. This resulted in a complex amino acid change consisting in replacement of threonine 599 with isoleucine and deletion of valine 600, lysine 601, serine 602, and arginine 603 [T599I-VKSR (600–603)del, abbreviated to T599I-VKSRdel] (Fig. 1, B and C).

View larger version (74K): [in this window] [in a new window]   FIG. 1. A, Representative hematoxylin- and eosin-stained sections of the FV-PTC at x1.6 (left) and x40 (right) magnification; B, sequence analysis (reverse strand) of the BRAF exon 15 region containing the T599I-VKSR (600–603)del mutation (K represents T/G overlap; W represents T/A overlap); C, partial BRAF exon 15 sequence of the mutated (mut) sample aligned with the wild-type (wt) sequence; mutated residues are in bold, and deleted residues are marked with an asterisk.

After being phosphorylated by RAF kinases at serine 217 and 221, MEK1/2 phosphorylates threonine 202 and tyrosine 204 of p44 and p42 MAPK (ERK1 and 2). In turn, p90RSK is a p44/p42 MAPK substrate. We transiently expressed the BRAF T599I-VKSRdel mutant and other BRAF alleles previously identified in thyroid carcinoma (V600E, K601E, and G474R) in HEK293 and evaluated their effect on the MAPK cascade using immunoblotting with phosphospecific antibodies. Forty-eight hours after transfection, V600E, K601E, and T599I-VKSRdel expression resulted in higher phosphorylation levels of MEK1/2 (2.8-, 2.4-, and 4-fold, respectively), p44/42 MAPK (3-, 2.5-, and 3-fold, respectively), and RSK (3-, 2.7-, and 1.8-fold, respectively) compared with wild-type BRAF (P < 0.001) (Fig. 2B). Differently, the activity of G474R was impaired compared with wild-type BRAF (2.5-fold reduction of both phospho-MEK and phospho-MAPK) and virtually indistinguishable from that of kinase-dead BRAF (Fig. 2B). Accordingly, in an in vitro immunocomplex phosphorylation assay, V600E, K601E, and T599I-VKSRdel exerted strong kinase activity, whereas G474R exerted no activity (Fig. 2C).

View larger version (41K): [in this window] [in a new window]   FIG. 2. A, Schematic representation of the BRAF protein structure and the various mutants used in this study. CR1, -2, and -3 represent conserved regions; CR3 is a kinase domain; A represents an activation loop. B, HEK293 cells expressing the indicated BRAF mutants were kept in serum-deprived medium (18 h) and harvested. Total cell lysates (50 µg) were immunoblotted with the indicated phosphospecific antibodies. Total amounts of MEK, MAPK, and RSK are shown for normalization. The results were quantified at the Phosphorimager. Data are representative of at least three different experiments. C, Protein lysates (500 µg) were immunoprecipitated with anti-BRAF and subjected to a kinase assay with recombinant glutathione S-transferase-MEK. Data are representative of at least three different experiments. D, HEK293 cells were transiently transfected with BRAF mutant-expressing vectors and the AP1-Luc reporter. Average results of three independent Luciferase assays ± SD are expressed as fold increase compared with empty vector-transfected (–) cells. Statistical significance was assessed with the Student’s t test. E, HEK293 cells expressing the indicated BRAF mutants, and when indicated CRAF, were kept in serum-deprived medium and harvested. Total cell lysates (50 µg) were immunoblotted with the indicated antibodies. Data are representative of at least three different experiments.

Although unable to signal to MEK directly, low-activity BRAF mutants (exemplified by G466E/V and G596R) can induce MEK phosphorylation in trans by allosterically activating CRAF. We tested whether G474R retained the ability to cooperate with CRAF. G474R was coexpressed in HEK293 cells in combination with a suboptimal CRAF dose, which alone is unable to stimulate MAPK. The G596R mutant, previously reported to activate MEK through CRAF (14, 15), was used as control. Overexpressed wild-type BRAF exerted modest levels of MEK stimulation that was not further potentiated by CRAF (Fig. 2E). G596R, although inactive alone, triggered MEK and MAPK phosphorylation in the presence of CRAF. G474R was not able to active the MEK/ERK pathway also when coexpressed with CRAF (Fig. 2E).

BRAF V600E drives TSH-independent proliferation of thyroid PC cells (18). Thus, equal numbers (25,000) of marker-selected PC cells transfected with V600E, T599I-VKSRdel, or G474R mutants were plated in triplicate in the absence of TSH and counted 7 d later. Empty vector-transfected cells did not proliferate; BRAF(V600E) cells were able to proliferate (cell number increased by 4.5-fold) in the absence of TSH. The BRAF(T599I-VKSRdel) also triggered TSH-independent proliferation (cell numbers increased by 3.5-fold). Although to a lesser extent (2.5-fold), also G474R was able to mediate a modest TSH-independent proliferation.

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

 
Here we show that the newly identified T599I-VKSRdel mutant activates the kinase function and signaling ability of BRAF. Interestingly, while this manuscript was under revision, a second patient with the T599I-VKSRdel mutation was found in the frame of a routine analysis for BRAF mutations at the University of Pisa; also in this case (a 46-yr-old women), the tumor sample was a small (T1) encapsulated FV-PTC. T599I-VKSRdel targets the A-loop of BRAF, which is also targeted by most activating mutations (V600E, K601E, V599ins, V600E-K601del, and V600D-FLAGT601–605ins). It is likely that all these mutations disturb the interaction +between the A- and P-loops that normally keep BRAF under check, thereby activating its catalytic function (14). In contrast, the other FV-PTC-associated mutant (G474R), targeting the glycine-rich loop, impaired BRAF kinase. G474R has been identified only in one FV-PTC (7), and to the best of our knowledge, it has not been described in other tumor types. It remains unclear whether G474R is only a passenger mutation or whether it signals in a kinase-independent manner through pathway(s) alternative to the MAPK one. The observation that in vitro G474R is able to drive, albeit at low levels, TSH-independent thyroid cell proliferation suggests that it may contribute to thyroid tumorigenesis in a kinase-independent manner. In vivo experiments may help to address this issue.

In conclusion, the identification of T599I-VKSRdel confirms that FV-PTC often harbor BRAF mutations other than the classic V600E one, information to be taken into account to optimize the search of BRAF mutations in thyroid cancer patients.

	Acknowledgments

 
We thank Jean Ann Gilder for text editing and Ciotola Presentation for the artwork.

	Footnotes

 
This study was supported by the Associazione Italiana per la Ricerca sul Cancro and the Naples Oncogenomic Center and by grants from Italian Ministero della Salute and Ministero dell’Università e della Ricerca.

Disclosure Statement: The authors have declared no conflict of interest.

First Published Online August 12, 2008

Abbreviations: A-loop, Activation loop; CV, conventional variant; FV, follicular variant; MEK, MAPK kinase; P-loop, phosphate-binding loop; PTC, papillary thyroid carcinoma; TCV, tall cell variant.

Received April 23, 2008.

Accepted August 1, 2008.

	References

Top Abstract Introduction Patients and Methods Results Discussion References

 

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor View responses Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via Google Scholar Google Scholar Articles by De Falco, V. Articles by Basolo, F. Search for Related Content PubMed PubMed Citation Articles by De Falco, V. Articles by Basolo, F. Related Collections Thyroid Endocrine Oncology