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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2007-0942
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The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 12 4845-4852
Copyright © 2007 by The Endocrine Society

Bcl-2 Overexpression in Thyroid Carcinoma Cells Increases Sensitivity to Bcl-2 Homology 3 Domain Inhibition

Constantine S. Mitsiades, Patrick Hayden, Vassiliki Kotoula, Douglas W. McMillin, Ciaran McMullan, Joseph Negri, Jake E. Delmore, Vassiliki Poulaki and Nicholas Mitsiades

Department of Medical Oncology (C.S.M., P.H., D.W.M., C.M., J.N., J.E.D., N.M.), Dana-Farber Cancer Institute, and Massachusetts Eye and Ear Infirmary (V.P.), Harvard Medical School, Boston, Massachusetts 02114; and Department of Pathology (V.K.), School of Medicine, Aristotle University of Thessaloniki, Thessaloniki 54006, Greece

Address all correspondence and requests for reprints to: Constantine S. Mitsiades, M.D., Ph.D., Department of Medical Oncology, Dana Farber Cancer Institute, Mayer Building, Room M555, 44 Binney Street, Boston, Massachusetts 02115. E-mail: constantine_mitsiades{at}dfci.harvard.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Context: The Bcl-2 family of proteins regulates apoptosis in various models and may represent a promising therapeutic target in human malignancies.

Objective/Methods: We evaluated the sensitivity of thyroid carcinoma cell lines (two papillary, one follicular, two anaplastic, three medullary) in vitro to BH3I-1 and BH3I-2', two cell-permeable inhibitors of the Bcl-2 homology (BH)-3 domain-mediated interaction between proapoptotic and antiapoptotic Bcl-2 family members. The thyroid carcinoma cell line FRO was stably transfected with cDNA for Bcl-2 or constitutively active Akt and evaluated for sensitivity to BH3-domain inhibition.

Results: BH3-domain inhibition disrupted the mitochondrial membrane potential in thyroid carcinoma cells, induced caspase-dependent apoptosis, and potently sensitized them to sublethal concentrations of doxorubicin and the proteasome inhibitor bortezomib (Velcade). Overexpression of constitutively active Akt suppressed BH3I-1-induced cell death. Bcl-2-overexpressing FRO cells were more resistant to conventional chemotherapeutic agents (such as doxorubicin) but significantly more sensitive to BH3I-1 than control cells and were found to overexpress caspase-9, caspase-8, Bmf, Bok, and Bik transcripts and express less A1, BRaf, and FLIP transcripts.

Conclusions: Bcl-2 expression protects thyroid carcinomas against chemotherapy-induced apoptosis. Nevertheless, overexpression of Bcl-2 may result in "oncogene addiction" of the cancer cell, which can be exploited by using BH3-domain inhibitors alone or in combination with other agents, including conventional chemotherapeutics (such as doxorubicin) or novel targeted therapies (such as the proteasome inhibitor bortezomib), for the treatment of aggressive thyroid cancer, including the medullary and anaplastic types.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID CARCINOMA, the most prevalent endocrine neoplasia, is diagnosed annually in approximately 33,550 new patients in the United States (1). Poorly differentiated, anaplastic, and medullary thyroid cancers exhibit an aggressive biological behavior, leading to 1500 deaths annually. Conventional chemotherapeutic agents, such as doxorubicin, have been used against aggressive thyroid carcinomas with overall poor results, suggesting that thyroid carcinoma cells are resistant to chemotherapy-induced apoptosis.

The Bcl-2 family of proteins is a major intracellular regulator of apoptotic signaling, with at least 20 members in mammalian cells (2). The prototypic member, Bcl-2, as well as Bcl-xL, Bcl-w, Al, and Mcl-1, promote carcinogenesis not by accelerating proliferation but by protecting cells from a wide range of apoptotic stimuli and triggers, including chemotherapeutic drugs and DNA-damaging irradiation (2, 3). Other members of the Bcl-2 family play a proapoptotic role. Specifically, Bax, Bak, and Bok have structure very similar to Bcl-2, with three conserved Bcl-2 homology (BH) domains (BH1, BH2, and BH3) and, when activated, can undergo conformational change and insert into the outer mitochondrial membrane as homooligomers. The resulting permeabilization of the outer mitochondrial membrane allows the release of apoptotic mediators normally sequestered inside the mitochondria, such as cytochrome c (which activates Apaf-1 and leads to caspase-9 activation), second mitochondria-derived activator of caspases (Smac), and apoptosis-inducing factor (a mitochondrial flavoprotein that, on release from the mitochondria, translocates to the nucleus, in which it contributes to chromatin degradation) (4, 5). Bcl-2 (or other antiapoptotic Bcl-2 family members) can block this apoptotic pathway via BH3 domain-mediated heterodimerization with Bax and Bak.

Another group of proapoptotic Bcl-2 family members, which includes Noxa, PUMA, Bim, Bad, Bmf, and Bik, share only the BH3 domain with Bcl-2. These BH3-only proteins are transcriptionally or posttranslationally activated by extracellular proapoptotic signals and intracellular damage and, conversely, inhibited by prosurvival mediators, such as the kinase Akt (2, 4, 5): Noxa and PUMA expression is induced at the transcription level by p53 after DNA damage and treatment with several chemotherapeutic agents (6, 7). Bim expression can be up-regulated in cytokine-deprived cells by the transcription factors of the forkhead family, homolog in rhabdomyosarcoma (FoxO1a)-L1, which is inhibited in the presence of various cytokines via phosphorylation by the Akt (8). Bad is constitutively expressed in many cell types, yet its phosphorylation by Akt triggers its binding to the 14-3-3 scaffold proteins and resulting inactivation (9). Bmf is a cytoskeleton-associated member of the BH3-only family that has been implicated in induction of anoikis (cell death that occurs on detachment from extracellular matrix) (10). The finding that these BH3-only proteins cannot induce apoptosis in the absence of both Bax and Bak (11, 12) suggests that their primary role is to allow for activation of Bax and/or Bak, probably by inactivating the antiapoptotic members of the Bcl-2 family. The BH3 domain is an amphipathic {alpha}-helix that interacts with the hydrophobic cleft formed by the BH1, BH2, and BH3 domains of the multidomain members of the Bcl-2 family (13). The balance between pro- and antiapoptotic Bcl-2 family members controls, via the interaction of their BH domains, the activation of the mitochondrial/cytochrome c/caspase-9 pathway and decides the cell’s apoptotic fate (2, 5).

In agreement with their proposed antiapoptotic role, Bcl-2, Bcl-xL, Bcl-w, Al, and Mcl-1 have been found to be overexpressed in various types of malignancies, suggesting that their antiapoptotic function confers an advantage to the neoplastic cell and that they may be valid targets for novel anticancer therapeutics. Small organic molecules or peptides that structurally mimic BH3 domains have been designed with the goal to competitively block the interaction between a BH3 domain and the prosurvival molecule (e.g. Bcl-2), thus inhibiting its antiapoptotic activity (14, 15, 16, 17, 18, 19), with the hope of antineoplastic effect. From a library consisting of 16,320 chemicals, the compounds BH3 domain inhibitors (BH3Is)-1 and -2' demonstrated the highest potency in disrupting the BH3-Bcl-xL interaction (14).

Bcl-2 is present in normal and neoplastic thyroid tissue, and its expression is dramatically reduced in thyrocytes of Hashimoto’s thyroiditis that undergo apoptosis (20). We previously reported that Bcl-2 overexpression in thyroid carcinoma cells confers protection against several novel anticancer agents, such as the histone deacetylase inhibitor suberoylanilide hydroxamic acid (21), the proteasome inhibitor bortezomib (PS-341, Velcade; Millennium Pharmaceuticals, Cambridge, MA) (22), and the heat shock protein 90 inhibitors 17-AAG and 17-DMAG (23). In the present study, we examined the effect of BH3Is, BH3I-1 and BH3I-2', on a panel of thyroid carcinoma cell lines. Both BH3I-1 and BH3I-2' exerted an antineoplastic effect in thyroid carcinoma cells in vitro and potently sensitized them to sublethal concentrations of doxorubicin and the proteasome inhibitor bortezomib (PS-341, Velcade). Interestingly, overexpression of Bcl-2 may result in oncogenic addiction of the cancer cell, which can be exploited for therapeutic purposes by using BH3Is, alone or in combination with other chemotherapeutic agents. This may represent a promising approach for the treatment of thyroid cancer, including the aggressive medullary and anaplastic types.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

Doxorubicin and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were from Sigma Chemical Co. (St. Louis, MO). The cell-permeable inhibitors BH3I-1 and BH3I-2', and the pan-caspase inhibitor ZVAD-FMK were from Calbiochem (La Jolla, CA). Bortezomib (PS-341, Velcade; Millennium Pharmaceuticals) was dissolved in dimethylsulfoxide and stored at –20 C until use.

Cell lines

The papillary thyroid carcinoma cell line BHP2 was a generous gift of Dr. Jerome M. Hershman (West Los Angeles Veterans Affairs Medical Center, Los Angeles, CA). The SW579 (papillary thyroid carcinoma) and the TT (medullary thyroid carcinoma) cell lines were purchased from American Type Culture Collection (Manassas, VA). The follicular carcinoma cell line WRO and the anaplastic thyroid carcinoma lines FRO and ARO were generous gifts of Dr. James A. Fagin (Memorial Sloan Kettering Cancer Center, New York, NY) (24). The DRO81–1 and HRO85–1 medullary lines were generous gifts of Dr. Guy J. F. Juillard (University of California, Los Angeles, School of Medicine, Los Angeles, CA). All cells were grown in DMEM (BioWhittaker, Walkersville, MD) with 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% fetal bovine serum (Invitrogen, Carlsbad, CA), unless stated otherwise.

MTT colorimetric survival assay

Cells were plated in 48-well plates at 70–80% confluence and then treated as indicated. Cell survival was examined using the MTT colorimetric assay, as previously described (25). Cell viability was estimated as a percentage of the value of untreated controls. All experiments were repeated at least three times, and each experimental condition was repeated at least in quadruplicate wells in each experiment. Data reported are average values ± SD of representative experiments.

Assessment of the effect of BH3 inhibition on the mitochondrial membrane potential

FRO cells were treated in serum-free medium with BH3I-1 (50 or 100 µM) or vehicle for 3, 6, or 12 h. The cells were then collected (both from the supernatant and the adherent cells; the latter were removed using trypsin) and stained with the MitoProbe DiOC2 (3) assay kit for flow cytometry (Molecular Probes/Invitrogen) following the manufacturer’s protocol. Cells treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP) served as a positive control. The samples were analyzed using a BD Canto II flow cytometer (BD Biosciences, Franklin Lakes, NJ).

Immunoblotting analysis

Immunoblotting was performed as previously described (25). Antibodies for poly(ADP-ribose) polymerase (PARP), cleaved caspase-3, and cleaved caspase-9 were from Cell Signaling (Danvers, MA). Monoclonal antibodies against Bcl-2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were from Dako Corp. (Carpinteria, CA), and Santa Cruz Biotechnology (Santa Cruz, CA), respectively.

Effect of BH3 domain inhibition on thyroid carcinoma cells overexpressing Bcl-2 or constitutively active Akt

To evaluate further the role of constitutive Bcl-2 overexpression and the impact of Akt signaling on cell death induced by BH3 domain inhibitors, anaplastic carcinoma FRO cells were stably transfected with a vector carrying the Bcl-2 cDNA or encoding the myc-tagged, myristoylated, constitutively active form of Akt (both from Upstate Biotechnologies, Lake Placid, NY) or the empty (neo) vector using Lipofectamine 2000 according to the instructions of the manufacturer. Forty-eight hours later, the cells were incubated in growth medium containing G418 (500 µg/ml; Invitrogen, Carlsbad, CA) to select stable transfectants, which were subsequently treated with BH3I-1 (0–200 µM). The overexpression of Bcl-2 or Akt, respectively, in transfected cells has been confirmed by immunoblotting.

RNA extraction and relative quantification of selected transcripts

RNA was extracted with TRIzol-LS (Invitrogen), further treated with DNase I, and purified with the RNeasy MinElute cleanup kit (QIAGEN, Valencia, CA). Reverse transcription was accomplished with random hexamers and SuperScript II, followed by incubation with RNase H (Invitrogen). Amplification reactions (25 µl, 100 ng cDNA/reaction) with Taqman FAM/MGB probes were performed in a 7500 real-time PCR system (Applied Biosystems, Foster City, CA) for the 23 apoptosis-related transcripts listed in Table 1Go. Runs were repeated at least twice. Relative quantification for each target vs. a reference gene transcript (glucuronidase-β) was assessed automatically with the SDS version 1.3 software (Applied Biosystems). For all assessments, the evaluation threshold was set at 0.3. Expression of Bcl-2 mRNA was assessed by RT-PCR with the following primers: Bcl-2F, 5'-CGGGGTGAACTGGGGGAGGATTGT-3' and Bcl-2R, 5'-CACCAGGGCCAAACTGAGCAGAGTCTT-3'. The 259-bp PCR product was visualized on a 2% agarose gel.


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  		TABLE 1. Relative expression of transcripts for 23 antiapoptotic and proapoptotic molecules in the four individually isolated FRO-Bcl-2 clones in comparison with that in FRO cells transfected with empty vector (FRO-neo cells, which were arbitrarily assigned a value of 1), as quantified by real-time RT-PCR

 
Statistical analysis

To evaluate the differences across various experimental conditions in the viability experiments (e.g. with or without BH3 mimetics and with or without ZVAD-FMK), one-way ANOVA was performed, and post hoc tests (Duncan and Dunnett’s T3 tests) served to evaluate differences between individual pairs of experimental conditions. The effect of BH3I1 in FRO-neo cells vs. the various Bcl-2 transfected clones was evaluated, across different doses of BH3I1, with two-way ANOVA (followed by Duncan and Dunnett’s T3 post hoc tests to evaluate differences between individual experimental conditions). The additive or synergistic nature of the interaction between BH3I1 and bortezomib or doxorubicin was evaluated by two-way ANOVA (GraphPad Prism 4.03; GraphPad Inc., San Diego, CA). In all analyses, P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The cell-permeable BH3Is BH3I-1 and BH3I-2' induce apoptosis in thyroid carcinoma cell lines in vitro

We examined the effect of BH3I-1 and BH3I-2' on the viability of a panel of thyroid carcinoma cell lines in vitro. Both BH3Is resulted in significant decrease in viability in all cell lines, including medullary and anaplastic cells (Fig. 1AGo). Treatment with BH3-I1 resulted in a dose- and time-dependent decrease in mitochondrial membrane potential (Fig. 1BGo). We confirmed that cell death induced by BH3Is in thyroid carcinoma cells is apoptotic in nature by demonstrating cleavage of caspase-9, caspase-3, and PARP (Fig. 1CGo). Moreover, the pan-caspase inhibitor ZVAD-FMK had a strong attenuating effect on cell death induced in FRO cells treated with BH3I-1 for 48 h (Fig. 1DGo), indicating functional involvement of caspases in apoptosis induced by BH3Is. Due to the stronger activity of BH3I-1 than BH3I-2', our study focused primarily on BH3I-1.


Figure 1
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  		FIG. 1. Induction of apoptosis in thyroid carcinoma cells by BH3I-1 and BH3I-2'. A, Dose-response curves of a panel of thyroid carcinoma cell lines (papillary: SW579, BHP2; follicular: WRO; anaplastic: FRO, ARO; and medullary: TT, HRO85-1, and DRO81-1) treated with the cell-permeable selective BH3Is BH3I-1 (solid bars; 0, 25, 50, and 100 µM) and BH3I-2' (empty bars, 50 and 100 µM) for 48 h. Cell survival (average ± SD) was quantified using the MTT assay, and values are expressed as percentages over those of vehicle-treated controls. The treatment was repeated three times with similar results. All P values were < 0.05, with the exception of WRO and TT cells treated with the lowest concentration of BH3I-1 (25 µM), which is not statistically significant. The expression of Bcl-2 in these cell lines is shown by immunoblotting. GAPDH is shown for loading control. B, Disruption of the mitochondrial membrane potential by BH3 inhibition. Cationic cyanine dyes, such as DiOC2(3 ) (3,3'-diethyloxacarbocyanine iodide) accumulate in mitochondria with active membrane potentials. DiOC2(3 ) staining intensity decreases when cells are treated with reagents that disrupt mitochondrial membrane potential. FRO cells were incubated in serum-free medium containing BH3I-1 (50 or 100 µM) or vehicle for 3, 6, or12 h. The cells were then collected (both from the supernatant and the adherent cells; the latter were removed using trypsin) and stained with the MitoProbe DiOC2(3 ) assay kit for flow cytometry (Molecular Probes/Invitrogen) following the manufacturer’s protocol. Cells treated with CCCP, which is a known disruptor of the mitochondrial membrane potential, served as a positive control. The samples were analyzed using a BD Canto II flow cytometer. The histograms represent the distribution of the intensity of cell fluorescence: green, unstained cells [no DiOC2(3 )]; red, vehicle-treated control cells; blue, positive control (cells treated with CCCP); orange, BH3I-1 (50 µM); and cyan, BH3I-1 (100 µM). The mean fluorescence is also reported in the accompanying table. Treatment with BH3-I1 resulted in a dose- and time-dependent decrease in mitochondrial membrane potential, indicating mitochondrial membrane permeabilization. C, FRO cells were treated with BH3I-1 (50 µM) for 0, 24, and 48 h. Immunoblotting revealed cleavage of caspase-9, caspase-3, and its substrate PARP, confirming the apoptotic nature of this type of cell death. D, Percent cell survival of FRO cells treated with BH3I-1 (50 µM) for 48 h in the presence or absence of the pan-caspase inhibitor ZVAD-FMK (20 µM). ZVAD-FMK had a strong attenuating effect on apoptosis (P < 0.05), indicating functional involvement of caspases in apoptosis induced by BH3 domain inhibitors.

 
Overexpression of Bcl-2 protects thyroid carcinoma cells against the DNA-damaging chemotherapeutic agent doxorubicin

We next investigated the role of Bcl-2 and BH3Is in apoptosis induced by conventional chemotherapeutic agents, such as doxorubicin, in thyroid carcinoma cells. A pool of Bcl-2-overexpressing FRO cells were more resistant to doxorubicin than control cells (Fig. 2AGo), confirming an antiapoptotic role for Bcl-2 in this model. Moreover, this finding raises the hypothesis that inhibition of endogenous Bcl-2 function may increase the efficacy of DNA-damaging chemotherapeutic agents such as doxorubicin.


Figure 2
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  		FIG. 2. Involvement of Bcl-2 in regulation of doxorubicin-induced cell death in thyroid carcinoma cells. A, Overexpression of Bcl-2 partially attenuated doxorubicin-induced cell death in thyroid carcinoma cells (P < 0.05 at all drug concentrations). FRO cells transfected with the empty vector (empty squares) and a pool of Bcl-2-transfected FRO cells (solid black squares) were treated with doxorubicin (Doxo; 0–1000 ng/ml) for 48 h. Percent cell viability (mean ± SD) was quantified by MTT. Data reported are average values ± SD of representative experiments. The expression of Bcl-2 in these cells (by immunoblotting) is shown. GAPDH is shown for loading control. B and C, Sensitizing effect of BH3 domain inhibitors to conventional cytotoxic chemotherapy in thyroid carcinoma cells. SW579 (papillary, B) and FRO (anaplastic, C) cells were treated with doxorubicin (0.125 µg/ml) for 48 h in the presence or absence of a subtoxic concentration of BH3I-1, BH3I-2', or vehicle. Cell viability was quantified by MTT and expressed as a percentage of the viability of control cells (treated with vehicle only). The bars represent cell death, which was calculated as 100% (percent cell viability). Control cells had by definition 0% cell death and the bar was omitted. All experiments were repeated at least three times, and each experimental condition was repeated at least in quadruplicate wells in each experiment. Data reported are average values ± SD of representative experiments. Thyroid carcinoma cells are relatively resistant to cytotoxic chemotherapy, but treatment with a subtoxic concentration of BH3 domain inhibitors had a strong sensitizing effect on doxorubicin-induced cell death (P < 0.001 for both cell lines by two-way ANOVA).

 
BH3Is sensitize thyroid carcinoma cells to conventional cytotoxic chemotherapy

We treated SW579 (papillary) and FRO (anaplastic) cells with doxorubicin for 48 h in the presence or absence of subtoxic concentrations of BH3I-1, BH3I-2' or vehicle. We found that both BH3Is sensitized both cell lines to doxorubicin (Fig. 2BGo). These data suggest that inhibition of endogenous antiapoptotic Bcl-2 family members can increase the efficacy of DNA-damaging chemotherapeutic agents such as doxorubicin and support the combined use of BH3Is with conventional chemotherapeutic agents for the treatment of aggressive thyroid carcinoma.

BH3Is sensitize thyroid carcinoma cells to cell death induced by the proteasome inhibitor bortezomib (PS-341, Velcade)

We recently reported that anaplastic and medullary thyroid carcinoma cells are very sensitive to the proteasome inhibitor bortezomib (22), an agent approved for the treatment of multiple myeloma (26, 27). We next treated FRO cells with a subtoxic concentration of bortezomib for 24 h in the presence or absence of BH3I-1 or vehicle. We found that BH3I-1 had a strong sensitizing effect on bortezomib-induced cell death (Fig. 3Go). These data are in agreement with our previous finding that Bcl-2 overexpression in thyroid carcinoma cells confers protection against bortezomib (22) and suggest that inhibition of endogenous antiapoptotic Bcl-2 family members can increase the efficacy of proteasome inhibitors, supporting the combined use of BH3Is with proteasome inhibitors, such as bortezomib, for the treatment of aggressive thyroid carcinoma.


Figure 3
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  		FIG. 3. Sensitizing effect of BH3 domain inhibitors to the proteasome inhibitor bortezomib in thyroid carcinoma cells. FRO cells were treated with bortezomib (4 and 6 nM) for 24 h in the presence or absence of BH3I-1 (40 µM). Cell viability was quantified by MTT and expressed as a percentage of the viability of control cells (treated with vehicle only). The bars represent cell death, which was calculated as 100% (percent cell viability). Control cells had by definition 0% cell death and the bar was omitted. All experiments were repeated at least three times, and each experimental condition was repeated at least in quadruplicate wells in each experiment. Data reported are average values ± SD of representative experiments. The apoptotic effect of the combination BH3I-1+bortezomib in thyroid carcinoma cells was greater than additive (P < 0.005 for both cell lines by two-way ANOVA).

 
Overexpression of myristoylated, constitutively active Akt (myrAkt) protects thyroid carcinoma cells from cell death induced by the BH3 domain inhibitor BH3I-1

We investigated the effect of the Akt pathway on cell death induced by BH3 domain inhibitors. FRO cells transfected with myrAkt were less sensitive to BH3I-1 than cells transfected with the empty vector (Fig. 4Go), suggesting that activation of the Akt pathway can attenuate cell death induced by BH3Is.


Figure 4
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  		FIG. 4. Activation of Akt protects thyroid carcinoma cells from cell death induced by the BH3 domain inhibitor BH3I-1. FRO cells transfected with the empty vector (empty squares) and myrAkt-transfected FRO cells (solid black squares) were treated with BH3I-1 for 48 h. Experiments were repeated at least three times, and each experimental condition was repeated at least in quadruplicate wells in each experiment. Percent cell viability (mean ± SD) was quantified by MTT. Data reported are average values ± SD of representative experiments. Overexpression of myristoylated, constitutively active Akt protected thyroid carcinoma cells from BH3I-1 (P < 0.05 at all drug concentrations). The expression of phosphorylated Akt in these cells is shown by immunoblotting. GAPDH is shown for loading control.

 
Paradoxical sensitivity of Bcl-2-overexpressing thyroid carcinoma cells to BH3 domain inhibition

We next investigated the effect of Bcl-2 overexpression on sensitivity to BH3Is in thyroid carcinoma cells. Four distinct (independently isolated at the time of the original transfection) Bcl-2-overexpressing clones (no. 1, 5, 8, and 14, selected for high Bcl-2 expression) were treated with BH3I-1. Somewhat unexpectedly, we found that all four Bcl-2-overexpressing clones were significantly more sensitive to BH3I-1 than control cells transfected with the empty vector (Fig. 5Go).


Figure 5
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  		FIG. 5. Overexpression of Bcl-2 cDNA sensitizes thyroid carcinoma cells to cell death induced by the BH3 domain inhibitor BH3I-1. A, FRO cells transfected with the empty vector (empty squares) and four individually isolated Bcl-2-transfected FRO clones were treated with BH3I-1 for 24 h. Percent cell viability (mean ± SD) was quantified by MTT. Experiments were repeated at least three times, and each experimental condition was repeated at least in quadruplicate wells in each experiment. Data reported are average values ± SD of representative experiments. P values were < 0.05 for all drug concentrations in all clones. B, The expression of Bcl-2 protein in the empty vector- and Bcl-2-transfected FRO cells, as evidenced by immunoblotting, has been previously reported (22 ) and is also shown here for comparison. GAPDH protein is shown as a loading control. C, The expression of Bcl-2 mRNA in the empty vector- and Bcl-2-transfected FRO cells is shown, as evidenced by RT-PCR. Actin was used as a housekeeping gene.

 
Transcriptional profile of apoptosis modulators in Bcl-2-overexpressing thyroid carcinoma clones

We attempted to explain this paradoxical sensitivity of our Bcl-2-overexpressing thyroid carcinoma clones to BH3I1 by assessing the expression of a panel of transcripts corresponding to several key apoptosis modulators (Table 1Go). The transcriptional profile varied within clones (confirming their distinct origin) and between Bcl-2-overexpressing clones and the empty vector-transfected pool. Although the transcriptional variability in Bcl-2-overexpressing clones included occasional differences that could lead to the opposite effect (i.e. apoptosis resistance), certain patterns that would promote sensitivity to apoptosis were identified. Specifically, we found that FRO cells stably transfected with the Bcl-2 cDNA were expressing higher levels of caspase-9, caspase-8, Bmf, Bok, and Bik transcripts and lower levels of A1, BRaf, and FLIP transcripts. These findings may help explain the molecular background of the oncogenic addiction of these cells to Bcl-2 and their paradoxical sensitivity to BH3 mimetics.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Thyroid carcinomas are, in general, resistant to conventional DNA-damaging chemotherapeutic agents, suggesting the presence of apoptosis inhibitors in excess of apoptosis inducers. We now report that BH3I-1 and BH3I-2', two cell-permeable inhibitors of the BH3 domain-mediated interaction between proapoptotic and antiapoptotic members of the Bcl-2 family, exerted an antineoplastic effect in thyroid carcinoma cells in vitro and sensitized them to sublethal concentrations of doxorubicin and bortezomib. Interestingly, stable overexpression of Bcl-2 in thyroid carcinoma cells resulted in increased sensitivity to BH3 domain inhibition, suggesting a phenomenon of oncogene addiction of the cancer cell.

The prominent role of Bcl-2 and the other antiapoptotic members of this family in several malignancies has generated intense interest in this field and several ongoing efforts to target these molecules pharmacologically. Oblimersen sodium (Genasense, G3139), an 18-mer phosphorothioate antisense oligonucleotide targeted to the initiation codon region of the Bcl-2 mRNA, down-regulates the expression of Bcl-2 mRNA and protein, can induce caspase-dependent apoptosis via the intrinsic mitochondrial pathway in cancer cells, and has entered phase III clinical trials in a number of human cancers (28), although its exact mechanism of action is still debated (29, 30). Other approaches designed to target Bcl-2 family members have included cell permeable Bcl-2- and Bcl-xL-binding peptides (13, 16, 17, 31), nonpeptidic small molecules (15, 18, 19), and RNA interference (30). The wide variability in expression of the various Bcl-2 family members among different types of cancer raises the hypothesis that selective targeting of these individual proteins may prove more efficacious and, hopefully, less toxic to nonmalignant cells (13).

In the present study, we report that the BH3Is BH3I-1 and BH3I-2' induce apoptotic cell death in a panel of thyroid carcinoma cell lines. Furthermore, they potentiated the antineoplastic effect of the conventional chemotherapeutic doxorubicin, an agent frequently used for the treatment of very aggressive and anaplastic thyroid carcinomas, and the proteasome inhibitor bortezomib (Velcade), an agent approved for the treatment of multiple myeloma (26, 27) which has potent in vitro activity against thyroid carcinoma cells (22). These findings support the clinical evaluation of BH3 mimetics for use in thyroid carcinomas, alone or in combination with other anticancer agents, such as doxorubicin and proteasome inhibitors.

We previously reported that Bcl-2 overexpression in thyroid carcinoma cells confers protection against several novel anticancer agents, such as the histone deacetylase inhibitor, suberoylanilide hydroxamic acid (21); the proteasome inhibitor bortezomib (22); and the heat shock protein 90 inhibitors, 17-AAG and 17-DMAG (23). In the present study, Bcl-2-overexpressing FRO cells were also more resistant to the conventional chemotherapeutic agent doxorubicin than control cells. All these findings are in agreement with the well-reported antiapoptotic function of Bcl-2 in many other models and raise important questions for the rational design of targeted anticancer therapies. Specifically, if cancer cells overexpressing Bcl-2 (or other apoptosis inhibitors of the same family) are less sensitive to cancer chemotherapy, then novel Bcl-2 targeting therapies may also prove more toxic to the normal cells, which express lower levels of the apoptosis inhibitors than the malignant ones.

Fortunately and somewhat unexpectedly, the opposite was true in our study because thyroid carcinoma cells stably overexpressing Bcl-2 were more sensitive to BH3I-1 than control cells. Overexpression of Akt had, as expected, a protective effect, suggesting that sensitization to BH3Is is specifically associated with overexpression of Bcl-2-related molecules. This finding suggests a form of addiction of these cells to the high levels of Bcl-2 and has obvious therapeutic implications. In agreement, Enyedy et al. (18) reported that compound 6, a novel, nonpeptide small-molecule inhibitor of the binding of the Bak BH3 domain to Bcl-2, potently inhibited cell viability in human myeloid leukemia and breast carcinoma cells expressing high levels of Bcl-2 but not in other cell lines that expressed low Bcl-2 levels. Similarly, Tzung et al. (19) demonstrated that antimycin A, an inhibitor of mitochondrial electron transfer, also binds to the BH3 domain of Bcl-2 and Bcl-xL and induces apoptosis in hepatocyte cell lines, with those that overexpress Bcl-xL paradoxically exhibiting higher sensitivity. Moreover, oncogene addiction to Bcl-2 family members has been described in leukemic and non-small cell lung carcinoma cells (13).

The concept of oncogene addiction has been proposed to describe the phenomenon in which cancer cells are often completely dependent on the continued function of certain activated or overexpressed oncogenes for maintenance of their malignant phenotype and viability, to the point at which specific inhibition of this particular oncogene product results in massive cancer cell apoptosis and tumor regression (32, 33). This extreme dependence represents an Achilles heel for tumors that can be exploited clinically (32) and has already been described in several models with significant clinical importance. For example, inhibitors of the epidermal growth factor receptor tyrosine kinase domain, such as gefitinib and erlotinib, have been found to be clinically more effective against those non-small cell lung carcinoma cells that harbor activating somatic mutations of the epithelial growth factor receptor tyrosine kinase (34, 35). In a similar manner, we now propose that thyroid carcinoma cells that overexpress Bcl-2 may be more sensitive to Bcl-2-targeting approaches, such as the small molecules used in our study, used either alone or in combination with other chemotherapeutic agents.

The molecular basis of oncogene addiction has not been elucidated. We investigated the expression of a panel of transcripts corresponding to several key apoptosis modulators and found that these clones express higher levels of caspase-9, caspase-8, Bmf, Bok, and Bik transcripts and lower levels of A1, BRaf, and FLIP transcripts. BRaf is frequently mutated in thyroid carcinomas (including FRO cells) and promotes proliferation and cell survival (36), whereas FLIP inhibits caspase-8 activation and plays an antiapoptotic role in thyroid carcinomas (37, 38). Based on these findings, we propose that the overexpression of Bcl-2 in our model permitted the emergence of clones with this molecular profile, which would be expected to be more prone to apoptosis, had it not been for the high expression of Bcl-2, thus leading to paradoxical sensitivity to BH3 mimetics.

The synergy between doxorubicin and BH3I-1 and between bortezomib and BH3I-1, which we detected in this study, confirms that, in the absence of BH3Is, the antiapoptotic members of the Bcl-2 family exert a suppressive effect over the apoptotic signaling triggered by doxorubicin and bortezomib, respectively, in thyroid carcinoma cells. More importantly, however, the antineoplastic activity of BH3Is when used as monotherapy against thyroid carcinoma cells suggests that these cells are primed to undergo apoptosis, even in the absence of an exogenous noxious stimulus or sensitizer. It has been suggested that the dysregulated cell cycle checkpoints and genomic instability of cancer cells can lead them to a fragile state of increased sensitivity to apoptosis (13, 39), which they avoid due to tonic antiapoptotic stimulation by one or more oncogenes, such as those of the Bcl-2 family. Neutralization of these life-sustaining oncogene products, e.g. by the BH3Is in our study, exposes this oncogene addiction and unleashes the repressed apoptotic program. This view of cancer cell biology has obvious clinical implications because it suggests that therapeutic interventions should be tailored to target the specific oncogene involved in and sustaining the viability of each individual cancer. Moreover, it leads to the hypothesis (and hope) that nonmalignant cells, being genetically stable and thus unprimed for apoptosis, may prove more resistant to these novel therapies, thus providing a therapeutic window for cancer treatment.

In conclusion, we have shown that BH3 domain inhibition is a promising novel approach for induction of apoptosis in thyroid carcinomas, including the anaplastic and medullary forms, alone or in combination with other chemotherapeutic agents.


   Acknowledgments
 
The authors thank Dr. Johanna Pallotta for her support and mentorship. The authors dedicate this work to the memory of Professor Michael J. Stephen (1933–2007).


   Footnotes
 
Disclosure Statement: The authors have nothing to disclose.

First Published Online September 11, 2007

Abbreviations: BH, Bcl-2 homology; BH3I, BH3 domain inhibitor; CCCP, carbonyl cyanide 3-chlorophenylhydrazone; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; myrAkt, myristoylated, constitutively active Akt; PARP, poly(ADP-ribose) polymerase.

Received April 26, 2007.

Accepted September 5, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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