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. 2009 Sep 1;94(10):4107–4112. doi: 10.1210/jc.2009-0662

Dual Inhibition of Mitogen-Activated Protein Kinase Kinase and Mammalian Target of Rapamycin in Differentiated and Anaplastic Thyroid Cancer

Ning Jin 1, Tianyun Jiang 1, D Marc Rosen 1, Barry D Nelkin 1, Douglas W Ball 1
PMCID: PMC2758734  PMID: 19723757

Abstract

Context: Differentiated thyroid cancer and anaplastic thyroid cancer tumors frequently have activation of the ras/raf /MAPK kinase (MEK)/ERK and phosphatidylinositol 3-kinase (PI-3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathways.

Objective: The objective of the study was to investigate the efficacy of MEK and mTOR inhibitors in preclinical thyroid cancer treatment models with defined mutation status.

Experimental Design: The MEK inhibitor AZD6244 (ARRY-142886) and mTOR inhibitor rapamycin were tested separately and in combination in 10 differentiated thyroid cancer and anaplastic thyroid cancer cell lines and in a xenograft model for evidence of pathway inhibition, growth inhibition, apoptosis, and long-range adaptation and resistance.

Results: Seven of 10 tested lines had evidence of significant basal activity of the PI-3K/AKT/mTOR pathway, with elevated phosphorylated AKT and phosphorylated p70 S6 kinase. Activation of ras/RAF/MEK/ERK was equally common in this panel. All 10 lines exhibited better than 60% growth inhibition with combined MEK and mTOR inhibition, including lines with BRAF, Ret-PTC, ras, and PTEN mutations. Rapamycin or AZD6244 alone achieved this threshold in six and two lines, respectively. Dual-pathway inhibition in the Ret-PTC mutant cell line TPC1 caused an intense G1 arrest in cell culture and reversible cytostatic inhibition in a xenograft model. We did not observe significant feedback up-regulation of AKT activation in either acute or prolonged exposures.

Conclusion: These preclinical results support the inclusion of thyroid cancer patients in early-phase clinical trials combining ras/RAF/MEK/ERK and PI-3K/AKT/mTOR pathway inhibition.

Combined treatment with a MEK inhibitor (AZD6244/ARRY-142886) plus an mTOR inhibitor (Rapamycin) inhibited growth of thyroid cancer cells in vitro, and in a xenograft model, more potently than either agent alone.

Differentiated thyroid cancer (DTC) and anaplastic thyroid cancer (ATC) exhibit frequent genetic alterations activating the ras/RAF/MAPK kinase (MEK)/ERK pathway (1) including ras mutations (∼50% of follicular carcinomas and adenomas), BRAF mutations (∼45% of papillary and 20% of ATC), and RET rearrangements (∼15% of papillary cancer). Many thyroid cancers also exhibit activation of phosphatidylinositol 3-kinase (PI-3K)/AKT/mammalian target of rapamycin (mTOR) signaling, which can lead to increased growth and apoptosis resistance, via targets of AKT, including mTOR (2). mTOR forms two multiprotein complexes. mTORC1 regulates protein translation and cell growth via mediators including p70S6 kinase (p70S6K) and is rapamycin sensitive. mTORC2, activated by distinct cellular inputs, signals via AKT and is rapamycin resistant (2,3). Diverse mechanisms activate the PI-3K/AKT/mTOR pathway in thyroid cancer, including rearrangement, amplification, and mutation of receptor tyrosine kinases; ras mutations; amplification of the PIK3CA and PIK3CB genes; and PTEN inactivation (4,5). Genetic alterations activating the ras/RAF/MEK/ERK and PI-3K/AKT/mTOR pathways occur in 81% of ATC tumors (4).

AZD6244 (ARRY-142886) is a highly selective MEK1/2 inhibitor in clinical trials in DTC, and other tumor types, with significant preclinical activity in BRAF-mutant thyroid and other tumor cell lines (6,7,8). TPC1 and KAT18 are BRAF wild-type thyroid cancer lines with basal AKT activation and partial resistance to this agent (7). We hypothesized that PI-3K/AKT/mTOR activation could promote AZD6244 resistance. Also, growth inhibition by the MEK inhibitor PD0325901 was augmented by the PI-3K inhibitor LY294002; however, neither agent is in clinical development (9). In the current study, we tested whether the combination of MEK and mTORC1 inhibition causes growth reduction across a panel of 10 thyroid cancer lines with varied mutational backgrounds. Both in culture and xenograft models, these data indicate that targeting both pathways provides highly effective growth inhibition in thyroid cancer.

Materials and Methods

Cell lines

TT2609-C02, B-CPAP, 8505C, and Cal62 were from the German Collection of Microorganisms and Cell Culture, U-Hth7 and U-Hth74 (Nils-Erik Heldin, Uppsala University, Uppsala, Sweden), FTC133 (Matthew Ringel, Ohio State University), KAT18 (Kenneth Ain, University of Kentucky, Lexington, KY), and TPC1(Alan Dackiw, Johns Hopkins University). Cell culture conditions were as previously reported (5,6), or as recommended by the supplier, and are detailed in supplemental online methods. Identity of cell lines not obtained from the German Collection of Microorganisms and Cell Culture was confirmed vs. published data (10), using highly polymorphic markers (PowerPlex, Johns Hopkins Genetic Core Facility).

MEK, mTOR, and PI-3K inhibitor treatments

AZD6244 (AstraZeneca, Cheshire, UK) was prepared as described (7). Rapamycin (Sigma, St. Louis, MO) dissolved in dimethylsulfoxide (DMSO) was diluted to 10 nm in media. LY294002 was from Sigma. Media and inhibitors were changed daily.

Western blotting, growth analyses, and flow cytometry cell cycle and apoptosis analyses

Western blotting, growth analyses, and fluorescence-activated cell sorter cell cycle and apoptosis analyses were performed as described previously (7). Antibodies are detailed in supplemental online methods, published as supplemental data on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org.

Animal studies

Animal studies were approved by the Johns Hopkins Institutional Animal Care and Use Committee, in accordance with National Institutes of Health guidelines. TPC1 cells in Matrigel (5 × 106 cells per 200 μl) were inoculated sc into the right flank of 4- to 6-wk-old female nude mice (Harlan, Indianapolis, IN). After tumors reached about 0.1 cm3 average size, animals were sorted into groups of 13 for equal size distribution among treatment groups. Animals were treated twice a day (BID), 5 d/wk, with 50 mg/kg AZD6244 administered by oral gavage, 4 mg rapamycin ip weekly, both agents combined, or control DMSO by gavage and ip Kaplan-Meier analysis (Prism; GraphPad, La Jolla, CA) defined 4-fold tumor volume increase as tumor progression. For immunohistochemistry methods, see supplemental online methods.

Results

Basal activity of PI-3K/AKT/mTOR and ras/RAF/MEK/ERK pathways

To evaluate basal activity of the PI-3K/AKT/mTOR pathway across a panel of thyroid cancer cell lines, we initially performed immunoblots for active AKT, phosphorylated at Ser473, and for the active form of the mTOR target p70S6K, phosphorylated at Thr389. We confirmed that TPC1 cells (RET-PTC mutation) and KAT18 cells (no known mutations) both exhibited significant levels of AKT and p70S6K phosphorylation, reflecting activation of the pathway (Fig 1, A and B). Among eight other tested lines, five exhibited AKT activation. Concordant p70S6K phosphorylation in all five lines implied that AKT, where active, signaled via mTOR. AKT and p70S6K activation were observed in lines with RET-PTC, ras, PTEN, and BRAF mutation, plus three lines with no characterized mutation. The ras/RAF/MEK/ERK pathway, assessed by pERK immunoblot, also appeared activated in seven of ten lines (Fig. 1B). Low or undetectable phosphorylated ERK levels were seen in C643 and Cal62 (ras mutation) and FTC133 (PTEN mutation).

Figure 1.

Figure 1

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PI-3K/AKT/mTOR and ras/raf/MEK/ERK pathway activity in thyroid cancer cell lines is inhibited by rapamycin and AZD6244. A, Immunoblot of basal pAKT expression, showing significant activity in seven of 10 tested lines. Total AKT and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are also indicated. Known mutations in these cell lines are noted at right. B, Immunoblot of pERK and p-p70S6K with and without acute treatment with 400 nm AZD6244 and 10 nm rapamycin, respectively. Total ERK and p70S6K and GAPDH are also indicated. Treatment duration was 4 h. C, pERK, pAKT, and pS6 expression after chronic exposure of TPC1 and Cal62 cells to AZD6244 and rapamycin. Compared with control, no significant up-regulation was noted for pERK, pAKT, or pS6 in dual-treated cells. Total ERK, total AKT, total S6, and GAPDH are also indicated. Treatment duration was 5 d.

Inhibition of PI-3K/AKT/mTOR and ras/raf/MEK/ ERK signaling pathways

The high frequency of PI-3K and ERK activation suggested that inhibiting PI-3K or downstream targets such as mTOR, alone or with MEK inhibitors, could have activity against thyroid cancer. Rapamycin concentrations of 1 nm or greater inhibited mTORC1 by immunoblot for phosphorylated p70S6K (data not shown). We previously showed that AZD6244 completely inhibits MEK phosphorylation of ERK in thyroid cancer cells at about 100 nm (7). Figure 1B illustrates that AZD6244 (400 nm) or rapamycin (10 nm) acutely inhibited their downstream targets, pERK and p70S6K, respectively.

To screen for possible feedback activation of AKT, we treated TPC1 cells or Cal62 cells 5 d using AZD6244, rapamycin, or both. We observed no significant up-regulation of pAKT in either line after dual treatment (Fig. 1C). mTORC1 inhibition was complete after rapamycin or both drugs, assessed by immunoblot for phosphorylated S6 (the target of activated p70S6K). pERK inhibition was complete after AZD6244 or both drugs in Cal62 cells and mildly attenuated after both drugs in TPC1 cells. Both pERK and pAKT became significantly elevated in Cal62 cells treated singly with rapamycin; both activation markers also persisted in TPC1 cells treated with rapamycin alone. Phosphorylated S6 remained elevated when Cal62 cells were treated singly with AZD6244. Thus, compensatory AKT and ERK activation was less significant in dually treated than singly treated cells.

Growth arrest after dual-pathway inhibition

To determine the growth-inhibitory action of PI-3K and MEK pathway inhibitors in thyroid cancer cells in vitro, we performed dimethylthiazol-diphenyltetrazolium bromide (MTT) assays, using AZD6244, rapamycin, or both together for 5 d. Figure 2A shows that all of the lines except the two BRAF mutant lines (8505C and B-CPAP) were inhibited more than 50% by rapamycin. Rapamycin plus AZD6244 was consistently superior to either agent alone, with all lines inhibited by at least 60%. TPC1, FTC133, and Cal62 cells had about 90% growth inhibition by the combination. Among the 10 tested lines, neither mutation status nor any combination of baseline pERK, pAKT, or p70S6K expression appeared to predict response to the combined treatment. Using flow cytometry, we observed an intense G0/G1 arrest in TPC1 cells treated with both inhibitors (data not shown). S phase declined from 14 to 2%, whereas G1 increased from 63 to 94%. Less extensive G1 arrest was seen with rapamycin or the PI-3K inhibitor LY294002. We observed no significantly increased apoptosis (accumulation of sub-G0/G1 nuclei, cell surface annexin V), indicating a predominantly cytostatic rather than apoptotic process.

Figure 2.

Figure 2

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Combined treatment with rapamycin and AZD6244 inhibits growth in thyroid cancer cell lines and xenograft tumors. A, MTT assay comparing three drug regimens with control in 10 different thyroid cancer lines after 5 d. Data are expressed as the mean OD value normalized to the untreated control in three to six independent experiments, with each measurement performed in triplicate. Error bars indicate sem. Known mutations in each cell line are noted at right. B, Relative volume of TPC1 xenograft tumors after treatment with rapamycin 4 mg/wk ip, AZD6244 50 mg BID 5 d/wk, or a combination of both. Treatment was started when established tumors measured about 0.1 cm3; animals were killed when tumors exceeded 1.0 cm3. Combination treatment was highly effective for the 7-wk treatment course. C, Kaplan-Meier analysis of progression-free status in the four treatment groups. Dual treatment was highly effective vs. control (P < 0.0001) and vs. AZD6244 or rapamycin alone (P < 0.0005). AZD6244 and rapamycin were separately effective compared with control (P < 0.0001 and P = 0.004, respectively).

Xenograft model of dual-pathway inhibition

We next investigated the impact of mTORC1 and MEK inhibition in a thyroid cancer xenograft model. Nude mice bearing sc TPC1 cell xenografts (0.1 cm3) were treated with rapamycin (4 mg/kg weekly), AZD6244 (50 mg/kg BID 5 d/wk), combined treatment, or DMSO control. Treated mice exhibited no weight loss or overt toxicity compared with controls (data not shown). Mice receiving dual treatment had minimal progression over the course of 7 wk; many mice had partial tumor regression (Fig. 2B). Mice treated with AZD6244 or rapamycin alone had smaller average tumor volumes than control but still progressed. The calculated median time to progression was 10 d for control mice, 23 d for rapamycin, and 32 d for AZD6244. Median progression was not reached for the dual treatment group at 50 d (Fig. 2C). No histological evidence of hemorrhage or necrosis was observed in any of the treatment groups.

To test the effect of treatment discontinuation, 11 mice receiving dual treatment were randomized to an additional week of drugs or withdrawal. Withdrawn animals had a 4-fold increase in tumor volumes (P = 0.04), whereas treated animals had no significant increase (data not shown). The rapid relapse after treatment withdrawal appears consistent with a cytostatic rather than cytotoxic response. We analyzed pERK, pAKT, and pS6 immunohistochemistry in xenografts under each of the treatment conditions (supplemental Fig. S1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org). Tumors from dual-treated mice had marked down-regulation of both pERK and pS6. Rapamycin had no effect on pERK, and AZD6244 had no impact on pS6 expression. Expression of pAKT was unchanged in dual-treated vs. control tumors. Thus, AZD6244 and rapamycin were highly effective in targeting MEK and mTORC1. The two drugs induced a reversible cytostatic response in the TPC1 xenograft model.

Discussion

The PI-3K/AKT/mTOR and ras/raf/MEK/ERK pathways are frequently activated in thyroid cancer. Dual inhibition with MEK and mTORC1 inhibitors induced a cytostatic response in vitro and in xenograft tumors, exceeding responses to the MEK inhibitor or mTORC1 inhibitor alone. The growth inhibition appeared to be reversible on discontinuation, with little evidence for apoptosis or necrosis.

Dual inhibition of the PI-3K/AKT/mTOR and ras/raf/MEK/ERK pathways has a strong rationale in thyroid cancer. Growing evidence in several tumor types shows that simultaneous inhibition of the PI-3K/AKT/mTOR and ras/raf/MEK/ERK signaling pathways is effective in vitro and in animal models (11,12,13). mTOR inhibitors have additive or synergistic effects with a variety of targeted and cytotoxic agents in solid tumors (14). Many DTC tumors and most ATC tumors have molecular abnormalities activating both pathways. A mouse model with thyroid-specific PTEN knockout underscores the importance of mTOR in thyroidal PI-3K signaling (15). The mTOR inhibitor everolimus caused more than 70% inhibition of the Ki67-staining fraction, indicating that mTOR is a significant mediator of PI-3K-induced growth in thyroid cells.

Numerous studies demonstrated overlap of key downstream actions of the two pathways in different cancer types, including cell growth, survival, and angiogenesis (16,17,18). Cross talk between the two pathways can result in activation of one pathway if the other is inhibited singly. For example, inhibition of mTOR can lead to activation of both AKT and ERK, by a mechanism that includes relief of inhibitory phosphorylation of the key signaling adaptor insulin receptor substrate IRS-1 (19,20). During this study, we monitored pAKT and ERK to determine whether combined therapy with MEK and mTORC1 inhibitors would result in activation through inhibition of negative feedback. Chronic treatment with rapamycin and AZD6244 elicited little induction of pAKT, although AKT activation remains a potential concern with this treatment paradigm. We observed marked compensatory activation of pERK as well as pAKT in ras mutant Cal62 cells treated singly with rapamycin. These findings, potentially stemming from IRS-1 disinhibition or other feedback change, illustrate the value of concurrent MEK inhibition in this mutational context (19).

These data support thyroid cancer clinical trials using combinations of MEK and mTOR inhibitors. Additional combinations targeting these two pathways, including MEK or selective BRAF inhibitors, plus agents that target AKT, PI-3K, or upstream kinases, also may be promising for thyroid cancer.

Supplementary Material

[Supplemental Data]
jc.2009-0662_index.html (1.2KB, html)

Acknowledgments

We thank Paul D. Smith (AstraZeneca) for generously supplying AZD6244 and for his advice on the use of AZD6244 in this study. Manuel Hidalgo (Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins) and Matthew Ringel (Ohio State University, Columbus, OH) provided valuable discussion.

Footnotes

This work was supported by National Institutes of Health SPORE (Specialized Programs of Research Excellence) in Head and Neck Cancer Grant CA-96784 (to B.D.N. and D.W.B.).

Disclosure Summary: The authors have no relationships to disclose.

First Published Online September 1, 2009

Abbreviations: ATC, Anaplastic thyroid cancer; BID, twice a day; DMSO, dimethylsulfoxide; DTC, differentiated thyroid cancer; MEK, MAPK kinase; mTOR, mammalian target of rapamycin; p, phosphorylated; PI-3K, phosphatidylinositol 3-kinase; p70S6K, p70S6 kinase.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

[Supplemental Data]
jc.2009-0662_index.html (1.2KB, html)
jc.2009-0662_1.pdf (8.7KB, pdf)
jc.2009-0662_2.pdf (80KB, pdf)

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