Tetracycline-inducible transgenic models of oncogenes such as MYC, RAS, and EGFR in different cell types helped establish the concept of oncogene addiction in vivo by showing that deinduction of the initiating oncoprotein causes tumor regression (16
). Our objective in this study was to demonstrate that conditional BRAF expression in thyroid cells of adult mice was sufficient to induce tumorigenesis and that the cancer cells required the continued presence of the oncoprotein for tumor maintenance. In addition, we wanted this information to serve as the gold standard to evaluate the therapeutic effects of selective inhibitors of BRAF signaling, particularly focusing on their effects on iodine transport and retention, since treatment with RAI is a key therapeutic modality in this disease.
One of the striking aspects of the tumor phenotype was the very short latency required for transformation, which occurred nonfocally throughout the entire thyroid gland. Mice expressing endogenous levels of BrafV600E
as of E14.5 develop histological features of PTC beginning at approximately 3 weeks postnatally (32
), whereas adult-onset activation of a latent knockin Braf
allele generates PTCs after approximately 6 months (33
). The more rapid onset seen in the current study may be due to overexpression of the oncogene. Braf-induced thyroid tumor initiation requires TSH action (32
), but once established, further growth and viability are largely independent of TSH. This is also likely the case after dox induction of oncogenic BRAF, because the TSH receptor is rapidly and profoundly downregulated by BRAF in this model, thereby rendering the thyroid cells unresponsive to the ligand. Oncogenic BRAF also impairs responses to TSH at steps distal to the TSH receptor within 48 hours, including the ability of its intracellular signaling effector cAMP to induce Nis
). In contrast, conditional activation of an endogenous mutant Braf
allele in melanocytes primarily induces benign nevi, which express markers of senescence and have a low mitotic rate. Malignant melanomas do ultimately arise, but only after a prolonged latency, suggesting a requirement for cooperating genetic events (21
). This is not likely the case for the malignant phenotype observed in our model, because it manifests ubiquitously throughout the entire thyroid gland after a short latency. However, we did not observe nodal or distant metastases here or in mice with thyroid-specific expression of a latent Braf allele (32
), suggesting that other oncogenic hits are needed for metastatic spread.
-induced PTCs closely recapitulate the histology of BRAF-positive advanced human thyroid cancer, with classic PTC architecture, presence of tall cell features, and evidence of extrathyroidal extension, consistent with their proclivity for invasion (3
). Upon dox withdrawal, thyrocyte apoptosis occurs in an organized pattern, beginning within the center of the tumor micronodules and radiating outward, leaving behind thyroid cells in the periphery of the nodule that appear to secrete Tg into the apical lumen. The restoration of gland architecture after oncogenic withdrawal is remarkable, but not unprecedented. Similar observations have been reported in MYC- and K-RAS–induced murine breast acinar 3D primary cultures following deinduction of oncoprotein expression. This was associated with clearance by caspase-3–mediated apoptosis of cells within the center of the disorganized mammospheres, leaving behind repolarized acinar structures (34
Approximately 70% of human PTCs are associated with mutually exclusive mutations of RET
) or BRAF
). The oncoproteins encoded by these genes share the common property of constitutively activating MAPK signaling, which has been taken as evidence supporting a critical role of this pathway in the pathogenesis of the disease. The expression of many of the genes required for thyroid hormone biosynthesis (e.g., NIS, TG, TPO) is particularly sensitive to the activity of the MAPK pathway. This has been demonstrated in PCCL3 cells, a rat thyroid cell line that retains most of the differentiated properties of normal thyrocytes, in which conditional activation of RET/PTC1 or RET/PTC3 downregulates expression of Nis, Tg, and Tpo (37
). This effect was abolished upon deletion of RET-Y1062, which is required to couple to Shc-Sos-Ras. The inhibitory effect was recapitulated by conditional expression of oncogenic RAS or of constitutively active MEK and restored by treatment with MEK inhibitors (39
). Accordingly, conditional expression of oncogenic BRAF has a similar effect on these cells (14
The mechanism or mechanisms by which constitutive MAPK pathway activation inhibits NIS expression have recently been clarified. BRAF activation has been proposed to induce a TGF-β autocrine loop (40
), which downregulates Pax8 and evokes a Smad3-dependent inhibition of Pax8 binding to the Nis promoter (41
). Interestingly, short-term pretreatment of PCCL3 cells prior to induction of BRAFV600E
with MEK inhibitors did not restore Nis activity, whereas treatment with a TGF-β–R1 inhibitor was more effective. These results contrast with our data in mice, in which PD0325901 treatment for 6 days resulted in a clear reactivation of iodine uptake. It is possible that a longer treatment time is required to switch off both the direct and indirect consequences of MEK overactivity on Nis expression.
In this study, we used 2 compounds working at different levels of the MAPK signaling pathway to attempt to reverse the biological consequences of oncogenic BRAF activation. The exquisite reversibility of the entire phenotype by switching off the oncoprotein set the scene for establishing the comparative efficacy of drugs targeting BRAF itself or its downstream effector MEK. Neither PLX4720 nor PD0325901 resulted in regression of the papillary thyroid cancers after 2 weeks of therapy. Recent data show that in order to achieve full therapeutic benefit with RAF inhibitors, it is not sufficient merely to inhibit the MAPK pathway, but that it needs to be almost completely suppressed (42
). Indeed, as opposed to the dramatic reversibility of the phenotype after dox withdrawal, treatment of thyroid cancers with 25 mg/kg/d PD0325901 failed to decrease mitotic activity or to induce tumor regression. This is likely explained by the fact that inhibition of MAPK signaling was transient with this dosing schedule. Although p-ERK inhibition was not as profoundly inhibited when mice were treated with 12.5 mg/kg of the compound every 12 hours, the effect was more sustained, which was associated with improved responses, i.e., decreased proliferation and partial reduction of TSH levels. The fact that oncogenic BRAF was overexpressed in this model may also have contributed to the attenuated responses to small molecule MAPK pathway inhibitors as compared with dox withdrawal. Notably, the 2 drugs had a robust effect on 124
I-iodide incorporation, yet the recovery of Nis
mRNA expression was modest when compared with switching off BRAF expression. This could be due in part to the continued presence of stroma in drug-treated animals, resulting in dilution of the Nis
mRNA signal. Indeed, evaluation of NIS by IHC showed clear restoration of the symporter, which was appropriately targeted to the plasma membrane. Besides restoring iodine uptake, the striking increase in 124
I-iodide incorporation after drug treatment could also be due to greater iodine organification, since these compounds increased Tg and Tpo levels.
Withdrawal of dox was associated with a marked response to RAI treatment, as determined by the induction of DNA damage and cell death. Treatment with PLX4720 elicited similar, albeit weaker responses. This is likely due to lower uptake as well as a shorter half-life of the isotope in drug-treated versus BRAF deinduced animals. The retention of inorganic iodide in thyroid cells required the activity of Tpo for its incorporation into tyrosine residues and subsequently into iodoproteins. Overall, the kinase inhibitors had a more attenuated effect on restoration of Nis expression levels, as well as those of Tpo and Tg, compared with those in dox-withdrawn mice. This likely explains not only the weaker uptake, but also the shorter 124I-iodide residence time after PLX4720. Indeed, after dox withdrawal, there was a 2.9-fold higher uptake as well as 1.6-fold longer biological half-life, which together predict a 4.7-fold higher radiation dose compared with that of drug-treated animals.
A clinical trial of the RAF inhibitor PLX4032 in patients with metastatic melanomas elicited spectacular clinical regressions of the disease in the large majority of cases, which were only observed in patients harboring BRAF
). Interestingly, 3 patients with BRAF
-positive PTC included in the dose-escalation phase of the study also showed tumor control, including 1 patient with a partial response. The RAF inhibitor PLX4720 had a profile of activity similar to that of PLX4032 and showed the most promising effects on RAI incorporation in this mouse model. Together, these data show that the BRAF-induced inhibition of iodine incorporation is a reversible process and that optimal inhibition of MAPK signaling represents a promising strategy to achieve this.
mutations are overrepresented in RAI-refractory thyroid cancers (6
), and these have lower expression of TG, TPO, and NIS compared with PTCs harboring either RAS or RET/PTC (43
). Accordingly, they are more refractory to 131
I-iodide therapy (4
). Hence, patients with BRAF
(+) PTCs would be ideally suited for therapeutic trials with MAPK pathway inhibitors to restore RAI treatment efficacy, in particular with relatively selective BRAF inhibitors. However, it should be noted that tumors with RET/PTC
rearrangements or RAS
mutations also have comparatively lower RAI uptake than normal thyrocytes. As these oncoproteins inhibit NIS, TG, and TPO expression in a MEK-ERK–dependent manner (39
), it is likely that MAPK pathway inhibitors may also have a beneficial effect on iodine transport and organification in thyroid cancers harboring these defects.