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Cancer Therapy: Preclinical |
Authors' Affiliations: 1 Istituto di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, c/o Dipartimento di Biologia e Patologia Cellulare e Molecolare; 2 Cattedra di Oncologia Medica; 3 Dipartimento di Scienze Biomorfologiche e Funzionali, Università di Napoli Federico II, Naples, Italy; and 4 Bayer HealthCare Pharmaceuticals, West Haven, Connecticut
Requests for reprints: Massimo Santoro, Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università di Napoli Federico II, via S. Pansini 5, 80131 Naples, Italy. Phone: 39-081-7463056; Fax: 39-081-7463037; E-mail: masantor{at}unina.it.
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Abstract |
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44% of papillary thyroid carcinomas and 24% of anaplastic thyroid carcinomas. In papillary thyroid carcinomas, this mutation is associated with an unfavorable clinicopathologic outcome. Our aim was to exploit BRAF as a potential therapeutic target for thyroid carcinoma. Experimental Design: We used RNA interference to evaluate the effect of BRAF knockdown in the human anaplastic thyroid carcinoma cell lines FRO and ARO carrying the BRAF V600E (V600EBRAF) mutation. We also exploited the effect of BAY 43-9006 [N-(3-trifluoromethyl-4-chlorophenyl)-N'-(4-(2-methylcarbamoyl pyridin-4-yl)oxyphenyl)urea], a multikinase inhibitor able to inhibit RAF family kinases in a panel of six V600EBRAF-positive thyroid carcinoma cell lines and in nude mice bearing ARO cell xenografts. Statistical tests were two sided.
Results: Knockdown of BRAF by small inhibitory duplex RNA, but not control small inhibitory duplex RNA, inhibited the mitogen-activated protein kinase signaling cascade and the growth of ARO and FRO cells (P < 0.0001). These effects were mimicked by thyroid carcinoma cell treatment with BAY 43-9006 (IC50 = 0.5-1 µmol/L; P < 0.0001), whereas the compound had negligible effects in normal thyrocytes. ARO cell tumor xenografts were significantly (P < 0.0001) smaller in nude mice treated with BAY 43-9006 than in control mice. This inhibition was associated with suppression of phospho–mitogen-activated protein kinase levels.
Conclusions: BRAF provides signals crucial for proliferation of thyroid carcinoma cells spontaneously harboring the V600EBRAF mutation and, therefore, BRAF suppression might have therapeutic potential in V600EBRAF-positive thyroid cancer.
2% to 5% of all thyroid cancers (3). Despite its rarity, more than half of the deaths attributed to thyroid cancer result from anaplastic thyroid carcinoma (3–5). More than 25% of anaplastic thyroid carcinoma patients have coincidentally detected well-differentiated carcinoma, suggesting that, at least in some cases, anaplastic thyroid carcinoma derives from a preexisting well-differentiated carcinoma (3–5). Rapid growth and dissemination characterize the clinical course of anaplastic thyroid carcinoma. Virtually, all anaplastic thyroid carcinoma patients die from their disease in 2 to 7 months; death is attributable to upper airway obstruction and suffocation in half of the patients (3–5). Anaplastic thyroid carcinomas do not concentrate radioiodine and do not respond to conventional chemotherapy (3–5).
BRAF belongs to the RAF family of serine/threonine kinases. RAF proteins are components of the RAF-MEK [mitogen activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase]-ERK pathway, a highly conserved signaling module in eukaryotes. They are activated through binding to RAS in its GTP-bound state. Once activated, RAF kinases phosphorylate MEK, which in turn phosphorylates and activates ERK (6). Activation of BRAF has emerged as the most prevalent oncogenic mutation in thyroid carcinoma (7–14). Overall, this genetic alteration is found in 44% of papillary thyroid carcinoma and 24% of anaplastic thyroid carcinoma (reviewed in ref. 15). In the case of anaplastic thyroid carcinoma, BRAF mutations are restricted to those cases that arose in association with papillary thyroid carcinoma (12, 14). A transversion from thymine to adenine (T1799A), leading to a Glu for Val substitution at residue 600 (V600E), accounts for >90% of BRAF mutations in thyroid carcinomas. Other more rare mutations have been described (reviewed in ref. 15). The V600E mutation enhances BRAF activity by disrupting the autoinhibited state of the kinase (16). Another interesting mechanism for BRAF activation has been described in radiation-induced papillary thyroid carcinoma, where a paracentric inversion of chromosome 7q resulted in the in-frame fusion between the AKAP9 gene and BRAF (17).
Consistent with a pivotal role in thyroid cancer initiation, V600EBRAF has been found in microcarcinomas (15), and it was shown to induce transformed features in thyroid follicular cells in culture (18, 19) and thyroid carcinoma formation in transgenic mice (20). Many evidences suggest that V600EBRAF plays a role in thyroid cancer progression as well: (a) Adoptive expression of V600EBRAF induces genomic instability in cultured thyrocytes (19); (b) thyroid tumors in V600EBRAF-transgenics undergo dedifferentiation and metastasis formation (20); and (c) papillary thyroid carcinoma with the V600EBRAF mutation often presents with extrathyroidal invasion, lymph node metastasis, and advanced tumor stage (14). Importantly, the V600EBRAF mutation was frequently associated to loss of I-131 avidity and papillary thyroid carcinoma recurrence (14).
In this framework, BRAF could be an appealing therapeutic target for thyroid carcinomas, especially for aggressive papillary thyroid carcinoma subtypes and anaplastic thyroid carcinoma. Here, we show that suppression of BRAF expression exerts cytostatic activity in V600EBRAF-positive thyroid carcinoma cell lines. Moreover, we show that BAY 43-9006 [N-(3-trifluoromethyl-4-chlorophenyl)-N'-(4-(2-methylcarbamoyl pyridin-4-yl)oxyphenyl)urea], a multikinase ATP-competitive inhibitor able to obstruct RAF kinases (21–24), reduces tumor growth in an anaplastic thyroid carcinoma xenograft model.
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Materials and Methods |
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Cell cultures. We used six cancer cell lines in this study: (a) the anaplastic thyroid carcinoma cell lines ARO (25), FB1 (26), KAT4 (27), and FRO (28); (b) the 8505C cell line (29), established from an anaplastic thyroid carcinoma containing areas of papillary thyroid carcinoma; (c) the NPA cell line (28) established from a poorly differentiated thyroid carcinoma. The ARO (11), KAT4 (12), and FB1 (12) cells harbor a heterozygous BRAF V600E mutation, whereas 8505C (12), NPA, and FRO (13) express only the mutated BRAF allele. Cells were grown in DMEM supplemented with 10% fetal bovine serum (Life Technologies, Paisley, PA), 2 mmol/L L-glutamine, and 100 units/mL penicillin-streptomycin (Life Technologies). The P5 primary culture of normal human thyroid follicular cells was kindly donated by Francesco Curcio (Dipartimento Di Patologia e Medicina Sperimentale e Clinica, Udine, Italy) and was grown as described elsewhere (30). For cell proliferation assays, 5 x 104 cells were plated in 35 mm dishes in 2.5% serum. The day after plating, BAY 43-9006 or vehicle was added. Cells were counted in triplicate every day. For flow cytometry analysis, 5 x 105 cells were plated in 100 mm dishes in 2.5% serum, and the next day they were treated with different concentrations of BAY 43-9006 or vehicle. After harvesting, cells were fixed in cold 70% ethyl alcohol in PBS. Propidium iodide (25 µg/mL) was added in the dark and samples were analyzed with a FACSscan flow cytometer (Becton Dickinson, San Jose, CA) interfaced with a Hewlett-Packard computer (Palo Alto, CA).
RNA silencing. The small inhibitor duplex RNAs targeting human BRAF we used in this study (31) were chemically synthesized by PROLIGO (Boulder, CO). Sense strands for small inhibitory duplex RNA (siRNA) targeting were as follows: BRAF, 5'-AGAAUUGGAUCUGGAUCAUTT-3'; lamin A/C, 5'-CUGGACUUCCAGAAGAACATT-3'. As a control, we used a nonspecific siRNA duplex containing the same nucleotides but in irregular sequence (scrambled). For siRNA transfection, cells were grown under standard conditions. The day before transfection, 1 x 105 cells were plated in 35 mm dishes in DMEM supplemented with 10% fetal bovine serum and without antibiotics. Transfection was done using 360 pmol siRNA and 18 µL OligofectAMINE reagent (Invitrogen, Groningen, the Netherlands) following the instructions of the manufacturer. Cells were kept in 2.5 serum and counted 48 and 72 hours after transfection.
Protein studies. Immunoblotting experiments were done according to standard procedures. Briefly, cells were harvested in lysis buffer [50 mmol/L HEPES (pH 7.5), 150 mmol/L NaCl, 10% glycerol, 1% Triton X-100, 1 mmol/L EGTA, 1.5 mmol/L MgCl2, 10 mmol/L NaF, 10 mmol/L sodium PPi, 1 mmol/L Na3VO4, 10 µg aprotinin/mL, and 10 µg leupeptin/mL] and clarified by centrifugation at 10,000 x g. For protein extraction, samples of mouse xenografts were snap frozen and immediately homogenized in lysis buffer by using the Mixer Mill MM300 (Qiagen, Crawley, West Sussex, United Kingdom). Protein concentration was estimated with a modified Bradford assay (Bio-Rad, Munich, Germany). Antigens were revealed by an enhanced chemiluminescence detection kit (ECL, Amersham Pharmacia Biotech, Little Chalfont, United Kingdom). Signal intensity was evaluated with the Phosphorimager (Typhoon 8600, Amersham Pharmacia Biotech) interfaced with the ImageQuant software. Anti-phospho-p44/42 MAP kinase (MAPK), specific for MAPK (ERK1/2) phosphorylated at Thr202/Tyr204, anti-p44/42 MAPK, anti-phospho-p90RSK (90 kDa ribosomal S6 kinase), specific for p90RSK phosphorylated at Thr359/Ser363, anti-p90RSK, anti-phospho-MEK1/2 (MAPK1 and MAPK2), specific for MEK1 and MEK2 phosphorylated at Ser217/Ser221, and anti-MEK1/2 were purchased from Cell Signaling (Beverly, MA). Anti-BRAF antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-
-tubulin was from Sigma Chemical. Secondary antibodies coupled to horseradish peroxidase were from Santa Cruz Biotechnology. For the BRAF kinase assay, cells were cultured for 12 hours in serum-deprived medium. Thereafter, cells were treated with BAY 43-9006 for 1 hour; BRAF kinase was immunoprecipitated with the anti-BRAF antibody and resuspended in a kinase buffer containing 25 mmol/L sodium PPi, 10 µCi [32P]ATP, and 1 µg recombinant glutathione S-transferase–MEK (Upstate Biotechnology, Inc., Lake Placid, NY). After 30-minute incubation at 4°C, reactions were stopped by adding 2x Laemmli buffer. Proteins were then subjected to 12% SDS gel electrophoresis. The radioactive signal was analyzed using a Phosphorimager (Molecular Dynamics, Piscataway, NJ).
Tumorigenicity in nude mice. Animals were housed in barrier facilities at the Dipartimento di Biologia e Patologia Cellulare e Molecolare (University of Naples "Federico II," Naples, Italy). They were exposed to a 12-hour light-dark cycle and received food and water ad libitum. All manipulations were conducted in accordance with Italian regulations for experimentation on animals. No mouse showed signs of wasting or other signs of toxicity. ARO cells (1 x 106/mouse) were inoculated s.c. into the right flank of 4-week-old male BALB/c nu/nu mice (The Jackson Laboratory, Bar Harbor, ME). Tumors (100 mm3) were treated with BAY 43-9006 (30 or 60 mg/kg) or vehicle alone by oral gavage for 5 consecutive days per week for 3 weeks. Tumor diameters were measured with calipers. Tumor volumes (V) were calculated by the formula: V = A x B2 / 2 (A = axial diameter; B = rotational diameter). Another group of animals (surrogate) was treated with vehicle or 60 mg/kg of BAY 43-9006 (five animals per group) for 5 days starting when the tumors reached 300 mm3. Tumors were excised 3 hours after the last dose and divided in two parts. Half of the tissue was snap-frozen in liquid nitrogen and used for protein extraction. The other half of the tissue was fixed overnight in neutral buffered formalin and processed by routine methods. Paraffin-embedded blocks were sliced into 5 µm sections and stained by H&E for histologic examination or processed for immunohistochemistry. Briefly, sections were deparaffinized, alcohol-rehydrated, subjected to heat-induced antigen retrieval, and incubated overnight with anti-Ki67/MIB-1 (1:50, 3,3'-diaminobenzidine, DAKO, Carpinteria, CA) or anti-CD31 antibodies [platelet/endothelial cell adhesion molecule 1 (M-20) goat polyclonal; Santa Cruz Biotechnology; ref. 32]. Finally, the slides were incubated with biotinylated anti-IgG and with premixed avidin-biotin complex (Vectostain ABC kits, Vector Laboratories, Burlingame, CA). The immune reaction was revealed with 0.06 mmol/L diaminobenzidine (DAKO) and 2 mmol/L hydrogen peroxide. As a negative control, tissue slides were incubated with preimmune serum. Apoptotic cell death rate was assessed in tissue slides by in situ labeling of DNA strand breaks as previously described (33). Briefly, dewaxed tissue sections were digested with Proteinase K (Boehringher Mannheim, Mannheim, Germany) and processed with the in situ cell death detection kit (Roche Diagnostics, Mannheim, Germany) used according to the instructions of the manufacturer.
Statistical analysis. Two-tailed unpaired Student's t test (normal distributions and equal variances) were used for statistical analysis. Differences were significant when P < 0.05. Statistical analysis was done using the Graph Pad InStat software program (version 3.06.3, San Diego, CA).
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5-fold) and MEK1/2 (3-fold) phosphorylation levels 48 hours after transfection (Fig. 1A and C).
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Inhibition of oncogenic BRAF signaling in thyroid carcinoma cell lines by BAY 43-9006. Of the small-molecule RAF kinase inhibitors in clinical development, BAY 43-9006 is the furthest along (35). BAY 43-9006 is a multikinase inhibitor able to target not only RAF kinases but also receptor tyrosine kinases, including vascular endothelial growth factor receptor-2 (KDR) and platelet-derived growth factor receptor B. Thus, its anticancer activity is currently thought to be the result of the dual inhibition of RAF signaling and KDR-mediated and platelet-derived growth factor receptor B–mediated tumor angiogenesis (23, 35).
Because BRAF expression was found to be essential for thyroid carcinoma cell growth, chemical BRAF blockade by BAY 43-9006 could exert cytostatic effects; thus, BAY 43-9006 could be exploited as a therapeutic tool for BRAF mutation–positive thyroid carcinoma models. To investigate this possibility, we studied the effects of BAY 43-9006 on the anaplastic thyroid carcinoma cell lines ARO, KAT4, and FB1, which carry the V600EBRAF mutation at the heterozygous level, and 8505C, FRO, and NPA, which carry only the mutated allele. After 12 hours of cultivation in low serum (2.5%), cells were treated for 6 hours with different concentrations of BAY 43-9006 or vehicle (NT) and the activity of MEK1/2, p44/p42 MAPK, and p90RSK (a p44/p42 MAPK substrate) was monitored by immunoblot with phosphospecific antibodies. Antibodies that recognize the same proteins also when nonphosphorylated were used for normalization. Immunoblots were examined with the Phosphorimager. Representative experiments are reported in Fig. 2. Consistent with the expression of an oncogenic BRAF, the MAPK cascade was constitutively active (even in low serum) in all the thyroid carcinomas tested. Treatment with BAY 43-9006 reduced the phosphorylation of MEK1/2, p44/p42 MAPK, and p90RSK with a IC50 of 1 µmol/L for ARO, KAT4, and NPA cells and of 500 nmol/L for FB1, 8505C, and FRO cells. After treatment with 5 µmol/L BAY 43-9006, only residual phosphorylation levels of MEK1/2, p44/p42 MAPK, and p90RSK were detected in the carcinoma cell lines (Fig. 2).
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1 µmol/L; at 5 µmol/L, BAY 43-9006 virtually arrested thyroid carcinoma cell growth (P < 0.0001; Fig. 4). Examination of the ARO cell cycle profile after BAY 43-9006 treatment (in 2.5% serum) by flow cytometry showed a marked G1 arrest upon treatment with 2.5 µmol/L BAY 43-9006. There were a few cells in the sub-G1 fraction, which indicates that BAY 43-9006 treatment results mainly in a cytostatic effect in these cells (not shown).
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10 days, when tumors had reached 100 mm3, animals (seven for each group) were randomized and treated orally 5 d/wk with BAY 43-9006 (30 or 60 mg/kg) or with vehicle. Tumor growth was monitored with calipers. The experiment was done twice and a representative experiment is shown in Fig. 5A. After 22 days of treatment, mice treated with BAY 43-9006 at either 30 or 60 mg/kg had significantly smaller tumors than control mice (P < 0.0001). No significant improvement of the therapeutic effect was noted at 60 mg/kg with respect to 30 mg/kg, indicating that maximal therapeutic efficacy was already achieved at 30 mg/kg.
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300 mm3 were treated daily with 60 mg/kg of BAY 43-9006 or with vehicle for 5 days. Three hours after the final dose, tumors were excised. Half the tissue was used for protein extraction and immunoblot analysis, and the other half was used for histologic examination. There were large areas of necrosis in tumors from treated animals at the H&E staining (Fig. 5B). Moreover, Ki67/MIB-1 immunolocalization was reduced and terminal deoxynucleotidyl transferase–mediated nick end labeling reactivity increased in treated tumors (Fig. 5B). These in vivo cell death effects were in contrast with the lack of apoptotic effects of the drug (see above) as well as of BRAF RNAi (data not shown) in cultured cells. However, by directly targeting vascular endothelial growth factor receptors in tumor endothelium (23) and by targeting RAF-regulated vascular endothelial growth factor receptor secretion (36), BAY 43-9006 might prevent the development of tumor neovascularization and therefore cause tumor cell death. Accordingly, blood vessel counting by immunoperoxidase staining with anti-CD31 revealed that BAY 43-9006 treatment reduced the number of vessels (from 20 ± 4 to 10 ± 3 per microscopic field), demonstrating that BAY 43-9006 exerted antiangiogenic effects in ARO cell xenografts. To ensure that BRAF kinase inhibition in vivo participated to the therapeutic effect as well, proteins were extracted from ARO tumors treated with BAY 43-9006. As shown in Fig. 5C, tumor growth inhibition was associated with a remarkable reduction of p44/42 MAPK, MEK1/2, and, at a lower extent, RSK in vivo phosphorylation levels. |
Discussion |
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At a preclinical level, recent insights have shown that chemically modified siRNAs can silence endogenous genes after i.v. injection in mice and, therefore, be exploited for treatment of disease (37). Moreover, injection of lentiviral vectors that produce RNAi-mediated silencing of specific genes proved efficacious in animal models of disease (38). Nevertheless, in clinical setting, molecular targeting of specific protein kinases, like ABL and KIT, with small-molecule inhibitors has already proved efficacious (39). Various BRAF inhibitors have been reported and, among them, the orally available by-aryl urea BAY 43-9006 has reached the clinical testing stage (35). BAY 43-9006 inhibits RAF kinases and the tyrosine kinases vascular endothelial growth factor receptor receptors 2/3, Flt-3, platelet-derived growth factor receptor B, FGFR1, and KIT (23). It inhibits the V600E BRAF mutant albeit with a slightly lesser potency than the wild-type kinase (23). BAY 43-9006 is undergoing advanced clinical trials (35). It is being tested in a phase II study of patients with locally advanced, metastatic, or recurrent thyroid cancer (www.cancer.gov/clinicaltrials). Thus far, phase III is achieving promising results on renal cell carcinoma, where probably BAY 43-9006 is effective for its activity on angiogenic kinases (35). Here, we show that BAY 43-9006 targets signal transduction along the MAPK cascade and tumor cell proliferation in V600EBRAF-positive thyroid carcinoma cell lines. Although in vitro, the compound mainly exerted cytostatic effects, it also caused tumor cell death in nude mice xenografts very likely for the concomitant angiogenesis inhibition. Tumor cells are often selected to bypass the effects of antineoplastic agents and the simultaneous assault on both neoplastic and endothelial cells may circumvent the development of resistance. This might be an advantage of drugs like BAY 43-9006 that are able to pinpoint more than one target simultaneously (40). However, BAY 43-9006 treatment did not cause a complete regression of ARO cell tumors. Similar observations have been reported upon mutant BRAF targeting in melanomas (41).
In conclusion, these findings provide the proof-of-concept that BRAF is a therapeutic target in thyroid cancer analogous to ABL and KIT in other tumors. Thus, BAY 43-9006, and perhaps other small molecules with a similar specificity profile, holds promise for molecular therapy of thyroid cancer. In a clinical setting, it will be mandatory to know the BRAF mutational status of treated patients and to show that the compound has sufficient activity to inhibit the BRAF kinase at the concentration achieved in patient tissues. Based on the preclinical data, one possibility could be to combine the drug with other synergistic therapeutics that may facilitate tumor regression.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 10/31/05; accepted 12/22/05.
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