Published in Volume
115, Issue 1
(January 3, 2005)J Clin Invest.
Copyright © 2005, American Society for Clinical Investigation
A new mechanism of BRAF activation in human thyroid papillary carcinomas
1Dipartimento di Biologia e Patologia Cellulare e Molecolare, Istituto di Endocrinologia ed Oncologia Sperimentale del CNR, Facolt¤ di Medicina e Chirurgia di Napoli, Universit¤ degli Studi di Napoli “Federico II,” Naples, Italy.
2NOGEC (Naples Oncogenomic Center)–CEINGE, Biotecnologia Avanzate, Naples, Italy.
Address correspondence to: Alfredo Fusco, Dipartimento di Biologia e Patologia Cellulare e Molecolare c/o Istituto di Endocrinologia ed Oncologia Sperimentale del CNR, Facoltà di Medicina e Chirurgia di Napoli, Università degli Studi di Napoli “Federico II,” via Pansini, 5, 80131 Naples, Italy. Phone: 0039-0813722857; Fax: 0039-0817463749; E-mail: firstname.lastname@example.org.
Published January 3, 2005
In this issue of the JCI, Ciampi et al. report the identification of a novel oncogene in patients affected by radiation-associated thyroid papillary carcinomas. This oncogene derives from a paracentric inversion of the long arm of chromosome 7, which results in an in-frame fusion of the N-terminus of the A-kinase anchor protein 9 (AKAP9) gene with the C-terminal catalytic domain (exons 9–18) of the serine-threonine kinase BRAF. The resulting AKAP9-BRAF fusion protein shows constitutive kinase activity, and it is able to transmit mitogenic signals to the MAPK pathways and to promote malignant transformation of NIH3T3 cells.
The Chernobyl nuclear power plant accident in 1986 caused severe radioiodide contamination of several areas in Belarus, Ukraine, and western Russia, leading to high radioactive exposure of the thyroid gland among the general population, including children. Beginning in 1992, a sharp increase in the incidence of childhood thyroid tumors, predominantly papillary thyroid carcinomas (PTCs), was reported. The molecular analysis of these tumors has provided a unique opportunity to study the mechanisms of radiation-dependent carcinogenesis in humans (1).
In this issue of the JCI, Ciampi et al. (2) report the identification of a novel oncogene in PTCs that developed in irradiated patients after a short latency period. This oncogene derives from the in-frame fusion of the first 8 exons of the A-kinase anchor protein 9 (AKAP9) gene with the C-terminal–encoding region (exons 9–18) of the proto-oncogene BRAF. AKAP9-BRAF fusion results from a paracentric inversion [inv (7)(q21–22q34)] of the long arm of chromosome 7 (Figure 1). BRAF is a serine-threonine kinase involved in the transmission of signals from membrane receptors and RAS small GTPases to MAPK (Figure 2). This pathway transduces mitogenic signals in response to the activation of tyrosine kinase receptors. The AKAP9-BRAF fusion event results in the loss of 2 BRAF regulatory domains, CR1 and CR2 (Figure 2), which exert autoinhibitory effects on the kinase activity of BRAF; CR1 includes the RAS-GTP binding domain (3). Accordingly, the AKAP9-BRAF recombination and the loss of CR1 results in a RAS-independent gain-of-function of BRAF that is able to induce transformation of NIH3T3 cells that become tumorigenic after injection into athymic mice.
Molecular mechanism of the chromosomal rearrangement generating the transforming AKAP9-BRAF oncogene in PTCs.
The MAPK pathway. Once activated, tyrosine kinase (TK) receptors activate the monomeric G protein RAS (pathway I), which in turn binds the serine-threonine kinase BRAF by inducing a conformational change that allows its activation (pathway I) and hence activation of the MAPK pathway. The activation of the MAPK pathway results in DNA synthesis in response to the external mitogenic stimulus (pathway I). When the RET/PTC (pathway II) or the BRAF (pathway II) oncogenes are generated through chromosomal rearrangements, activation of the MAPK pathway becomes constitutive, and cells become able to proliferate indefinitely, to grow in an anchorage-independent manner, and to induce tumors after injection into athymic mice (pathway II). MEK, MAPK/ERK kinase; L, ligand.
Oncogene activation in human PTCs
The AKAP9-BRAF fusion event reported by Ciampi and coworkers (2) represents an additional example of oncogene formation due to chromosomal rearrangements in human PTCs. Other examples include RET/PTC and TRK-T oncogenes. RET/PTC, present in about 40% of human PTCs, are chimeric genes generated by the fusion of the C-terminal catalytic domain of the RET receptor tyrosine kinase with the N-terminal region encoded by heterologous genes. In the most prevalent variants, RET/PTC1 (4) and RET/PTC3 (5), the fusion occurs between RET and the H4 (also named D10S170) or RFG (also named Ele1/ARA70/Ncoa4) genes. RET, H4, and RFG map to the long arm of chromosome 10, and paracentric chromosomal inversions account for RET/PTC1 and RET/PTC3 generation (6). Similarly, TRK-T oncogenes, present in about 10% of human PTCs, are generated by structural rearrangements of the NTRK1 (also named TRKA) gene, coding for the high-affinity nerve growth factor (NGF) receptor. The TRK-T1 and TRK-T2 oncogenes are both generated by NTRK1 fusion with the TPR gene. NTRK1 and TPR genes are both located on chromosome 1; therefore, as is this case for AKAP9-BRAF fusion, the structural rearrangement is mediated by an intrachromosomal event (7).
Chromosomal rearrangements are typical of PTCs
In PTCs, the prevalence of gene rearrangements, particularly chromosomal inversions, is quite high. This is at odds with the low frequency of gene rearrangements in most epithelial tumors, where point mutations are prevalent (8). Interestingly, this tendency is not restricted to PTCs, since thyroid tumors of the follicular histotype, follicular thyroid carcinomas (FTCs), and follicular adenomas (FAs), are also characterized by chromosomal translocations. A [t(2;3)(q13;p25)] translocation involving the PAX8 and PPARγ1 genes is a characteristic of a significant number of FTCs (9), whereas 2 chromosomal regions, 19q13 and 2p21, are frequently rearranged in FAs.
The study by Ciampi and coworkers (2) provides further evidence supporting the concept that chromosomal inversions represent the most typical molecular lesion in tumors occurring in Belarus and the surrounding region after the Chernobyl accident. In contrast, very rare point mutations have been detected in these tumors (10). The peculiar susceptibility of thyroid follicular cells to chromosomal rearrangements is remarkable. Previously, Yuri Nikiforov and coworkers (11) proposed an intriguing mechanism to explain how ionizing radiation can cause RET/PTC rearrangements and why this occurs preferentially in thyrocytes. They showed that while RET and H4 loci are about 30 Mb apart in the linear map of chromosome 10, they frequently juxtapose within the nuclei of thyroid cells but not in other cell types; this contiguity provides the structural basis for radiation-induced illegitimate nonhomologous recombination of the 2 genes (11). Another explanation was proposed to explain the peculiar susceptibility of thyroid follicular cells to chromosomal rearrangements. In vitro exposure of thyroid cells to ionizing radiation did not induce apoptosis but significantly increased DNA end-joining enzymatic activity (12). Thus, thyrocytes may respond to DNA damage with chromosomal alterations rather than cell death (6).
BRAF activation is a common feature of both sporadic and post-Chernobyl PTCs
BRAF mutations have previously been implicated in the carcinogenesis of PTCs. Indeed, several groups have reported that about 50% of adult PTCs harbor a specific point mutation (V600E, formerly designated V599E) in BRAF. In human cancer, BRAF mutations only appear more frequently in melanomas (13). V600 maps to the activation segment of the BRAF kinase. Structural analysis has revealed that the V600E mutation constitutively activates BRAF by destabilizing the inactive form of the kinase, thereby shifting the equilibrium toward its active conformation (3). However, prior to this report by Ciampi et al. (2), BRAF activation was considered to be a feature of sporadic, non–radiation-associated PTCs; a negligible frequency of BRAF mutations having been detected in radiation-induced childhood PTCs (14–16). In this context, this JCI study represents a major breakthrough in our knowledge of molecular events involved in PTC initiation. Indeed, it demonstrates that BRAF activation is a common feature of both sporadic and post-Chernobyl thyroid carcinomas and that it is the molecular mechanism underlying BRAF activation that differentiates the 2 types of tumors, with the higher prevalence of point mutations occurring in sporadic PTCs and intrachromosomal inversion responsible for a larger percentage of radiation-induced PTCs.
The identification of the AKAP9-BRAF oncogene in post-Chernobyl PTCs represents an important step forward in thyroid cancer research. Certainly, it will foster new lines of research aimed at addressing still unanswered questions. Is the level of transforming ability exerted by the AKAP9-BRAF oncogene different from that exerted by BRAF point mutants? Indeed, biological differences in the potency of these 2 oncogenic versions of the BRAF protein are expected, since childhood and adult tumors differ in some of their clinical characteristics (i.e., childhood PTCs are often characterized by distant metastases, which are rather rare in the adult).
Among the most intriguing issues to be addressed is the role played by the likely inactivation of the AKAP9 gene following the paracentric inversion [inv (7)(q21–22q34)] that generates the AKAP9-BRAF fusion protein. AKAP9 is capable of binding the type II regulatory subunit of the cAMP-dependent PKA; intriguingly, the type I regulatory subunit — RIα — of PKA is fused to RET in the RET/PTC2 oncogene (17). Thus, the possibility that functional alterations in the PKA pathway are involved in the pathogenesis of PTCs carrying the AKAP9-BRAF or RET/PTC2 oncogenes deserves further investigation. It is also worth mentioning that loss-of-function mutations in the RIα regulatory subunit are responsible for the development of the Carney complex, a multiple endocrine neoplasia syndrome (18).
The report by Ciampi et al. (2) presents what we believe to be the first example of an intracellular effector in the MAPK pathway activated by recombination occurring in vivo and supports the concept that one specific signaling pathway that leads to MAPK activation plays a major role in the generation of PTCs. At this stage, this pathway is the most attractive target for novel pharmacological intervention in human PTCs.
See the related article beginning on page 94.
Nonstandard abbreviations used: AKAP9, A-kinase anchor protein 9; FA, follicular adenoma; FTC, follicular thyroid carcinoma; PTC, papillary thyroid carcinoma.
Conflict of interest: The authors have declared that no conflict of interest exists.
Williams, D. Cancer after nuclear fallout: lessons from the Chernobyl accident. Nat. Rev. Cancer. 2002. 2:543-549.
Ciampi, R, et al. Oncogenic AKAP9-BRAF fusion is a novel mechanism of MAPK pathway activation in thyroid cancer. J. Clin. Invest. 2005. 115:94-101. doi:10.1172/JCI200523237.
Dibb, NJ, Dilworth, SM, Mol, CD. Switching on kinases: oncogenic activation of BRAF and the PDGFR family. Nat. Rev. Cancer. 2004. 4:718-727.
Grieco, M, et al. PTC is a novel rearranged form of the ret proto-oncogene and is frequently detected in vivo in human thyroid papillary carcinomas. Cell. 1990. 60:557-563.
Santoro, M, et al. Molecular characterization of RET/PTC3; a novel rearranged version of the RETproto-oncogene in a human thyroid papillary carcinoma. Oncogene. 1994. 9:509-516.
Pierotti, MA. Chromosomal rearrangements in thyroid carcinomas: a recombination or death dilemma. Cancer Lett. 2001. 166:1-7.
Alberti, L, Carniti, C, Miranda, C, Roccato, E, Pierotti, MA. RET and NTRK1 proto-oncogenes in human diseases. J. Cell. Physiol. 2003. 195:168-186.
Futreal, PA, et al. A census of human cancer genes. Nat. Rev. Cancer. 2004. 4:177-183.
Kroll, TG, et al. PAX8-PPARgamma1 fusion oncogene in human thyroid carcinoma. Science. 2000. 289:1357-1360.
Fagin, JA. Challenging dogma in thyroid cancer molecular genetics — role of RET/PTC and BRAF in tumor initiation. J. Clin. Endocrinol. Metab. 2004. 89:4264-4266.
Nikiforova, MN, et al. Proximity of chromosomal loci that participate in radiation-induced rearrangements in human cells. Science. 2000. 290:138-141.
Yang, T, et al. p53 induced by ionizing radiation mediates DNA end-jointing activity, but not apoptosis of thyroid cells. Oncogene. 1997. 14:1511-1519.
Kimura, ET, et al. High prevalence of BRAF mutations in thyroid cancer: genetic evidence for constitutive activation of the RET/PTC-RAS-BRAF signaling pathway in papillary thyroid carcinoma. Cancer Res. 2003. 63:1454-1457.
Lima, J, et al. BRAF mutations are not a major event in post-Chernobyl childhood thyroid carcinomas. J. Clin. Endocrinol. Metab. 2004. 89:4267-4271.
Kumagai, A, et al. Low frequency of BRAFT1796A mutations in childhood thyroid carcinomas. J. Clin. Endocrinol. Metab. 2004. 89:4280-4284.
Nikiforova, MN, et al. Low prevalence of BRAF mutations in radiation-induced thyroid tumors in contrast to sporadic papillary carcinomas. Cancer Lett. 2004. 209:1-6.
Bongarzone, I, et al. Molecular characterization of a thyroid tumor-specific transforming sequence formed by the fusion of ret tyrosine kinase and the regulatory subunit RI alpha of cyclic AMP-dependent protein kinase A. Mol. Cell. Biol. 1993. 13:358-366.
Kirschner, LS, et al. Mutations of the gene encoding the protein kinase A type I-alpha regulatory subunit in patients with the Carney complex. Nat. Genet. 2000. 26:89-92.