ARTICLE

Role of MEN2A-Derived RET in Maintenance and Proliferation of Medullary Thyroid Carcinoma

Matthias Drosten, Gero Hilken, Miriam Böckmann, Florian Rödicker, Nikica Mise, Aaron N. Cranston, Uta Dahmen, Bruce A. J. Ponder, Brigitte M. Pützer

Affiliations of authors: Department of Vectorology and Experimental Gene Therapy, University of Rostock, Rostock, Germany (MD, MB, FR, NM, BMP); Central Animal Facility (GH), Department of General and Transplantation Surgery (UD), University of Essen Medical School, Essen, Germany; University of Cambridge and Cancer Research UK, Department of Oncology, Cambridge, United Kingdom (ANC, BAJP)

Correspondence to: Brigitte M. Pützer, MD, PhD, Department of Vectorology and Experimental Gene Therapy, University of Rostock, Schillingalle 70, D-18055 Rostock, Germany (e-mail: brigitte.puetzer{at}med.uni-rostock.de)


    ABSTRACT
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Dominant-activating mutations in the RET protooncogene, a receptor tyrosine kinase, have been identified as a cause of medullary thyroid carcinoma. Such oncogenic RET mutations induce its ligand-independent constitutive trans-autophosphorylation. We investigated the role of endogenous oncogenic RET autophosphorylation in maintaining the neoplastic phenotype in medullary thyroid carcinoma cells and orthotopic medullary thyroid carcinomas in RET transgenic mice. Methods: We constructed adenoviral vectors expressing a dominant-negative truncated form of RET, termed RET{Delta}TK, and analyzed its effect on cell viability, apoptosis, and proliferation of TT medullary thyroid carcinoma cells. We investigated the effect of RET{Delta}TK on downsteam signaling by assessing alterations in phosphorylation or in gene expression. The effect of RET{Delta}TK in primary medullary thyroid carcinomas in transgenic mice was assessed by monitoring tumor growth. All statistical tests were two-sided. Results: Cell viability was reduced. Phosphorylation of Akt and extracellular signal-regulated kinase (ERK), components of downstream signal transduction pathways, was abolished, and cell cycle progression was reduced. Expression of cell cycle regulator cyclin D1 was decreased, and expression of cell cyle regulators p21CIP1/WAF1 and p27KIP1 was increased. Apoptosis was stimulated and concurrently the expression of BCL-2 was decreased. All in vitro experiments compared TT cells expressing RET{Delta}TK with untreated control cells or control vector-treated cells. Furthermore, 2 weeks after injecting adenovirus-carrying RET{Delta}TK into thyroid glands of transgenic mice with orthotopic medullary thyroid carcinoma, tumors were statistically significantly smaller than their initial size in mice treated with RET{Delta}TK (43.6%, 95% confidence interval [CI] = 30.7% to 56.5%; P = .010; two-sided unpaired Student's t test), whereas tumors in mice treated with a control vector were larger than their initial size (139.8%, 95% CI = 120.3% to 159.3%; P<.001). Conclusion: Selective disruption of oncogenic RET signaling in medullary thyroid carcinoma in vitro and in vivo is associated with loss of the neoplastic phenotype of medullary thyroid carcinoma and should be investigated further as the basis for new therapeutic approaches for this disease.



    INTRODUCTION
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Thyroid malignancies occur with an incidence of three per 100 000 people in Europe and the United States. Medullary thyroid carcinoma accounts for 5%–10% of such thyroid carcinomas, and it originates from the parafollicular C cells, whose main function is to produce and secrete calcitonin. About 1000 people are diagnosed with medullary thyroid carcinoma each year in the United States alone, and the 10-year survival rate of patients with medullary thyroid carcinoma is estimated at 60%–70% (1,2). The current treatment is characterized by surgical removal of neoplastic tissue. However, radiotherapy and chemotherapy have produced inconsistent results, indicating the need for alternative therapies (3).

Dominant activating mutations in the RET protooncogene have been identified as the key cause for the development of medullary thyroid carcinoma; these mutations confer dominant ligand-independent constitutive trans-autophosphorylation activity on the mutant RET protein (4,5). The RET gene encodes a receptor tyrosine kinase that binds neurotrophic factors of the glial cell line–derived neurotrophic factor family and is involved in the development of the kidney and in the survival of various neurons of the enteric nervous system (6,7).

Medullary thyroid carcinoma occurs in sporadic (approximately 75%) or hereditary (approximately 25%) forms. Hereditary medullary thyroid carcinoma is inherited in an autosomal dominant manner with the following three clinical manifestations: multiple endocrine neoplasia types 2A (MEN2A) and 2B (MEN2B) and familial medullary thyroid carcinoma. The MEN2A subtype accounts for approximately 90% of all inherited medullary thyroid carcinomas; most mutations affect one of the cysteine residues in the cysteine-rich domain (i.e., 609, 611, 618, 620, or 634). These mutations and those identified in familial medullary thyroid carcinoma lead to the formation of permanent receptor dimers with constitutive autophosphorylation activity. The active dimers stimulate downstream signal transduction pathways and thus promote cell transformation. The most aggressive disease is associated with the MEN2B subtype; mutations identified with this subtype appear to induce a conformational change in the tyrosine kinase domain of RET that alters substrate specificity (810).

Oncogenic RET proteins activate a complex network of signal transduction pathways that contributes to cellular transformation (11,12), including the Raf/Mek/extracellular signal-regulated kinase (ERK) cascade, which can regulate cell proliferation (13), and the phosphatidylinositol 3-kinase (PI3K)/Akt signal transduction pathway, which regulates cellular survival (14). Activation of the PI3K/Akt pathway is required for oncogenic RET-mediated transformation (15).

To investigate the mechanism used by the most common oncogenic RET protein in medullary thyroid carcinoma type MEN2A cells to maintain neoplastic properties, we constructed RET{Delta}TK, a dominant-negative truncated RET protein that lacks the entire cytoplasmic tyrosine kinase domain, to inhibit intrinsic oncogenic RET autophosphorylation. Baldassarre et al. (16) reported that a related dominant negative RET mutant efficiently inhibits glial cell line–derived neurotrophic factor–mediated RET activation and the activity of MEN2A-derived RET by dimerizing with endogenous RET to form an inactive dimer that lacks trans-autophosphorylation activity. We used adenoviral vectors to insert the RET{Delta}TK gene into cells and tumors and investigated which signal transduction pathways are altered by the inhibition of oncogenic RET activity with RET{Delta}TK in vivo and in vitro.


    MATERIALS AND METHODS
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cell Lines

The human medullary thyroid carcinoma cell line TT (product CRL-1803, American Type Culture Collection, Manassas, VA) was derived from a patient with aggressive medullary thyroid carcinoma and carries a frequent RET mutation (C634W) identified in patients with MEN2A disease (17). TT cells were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum. The TT cell line has been extensively studied and is a well-established in vitro model for medullary thyroid carcinoma. Human H1299 lung cancer cells and 293 cells (human embryonic kidney cells transformed with E1A from adenovirus type 5; used to propagate adenoviral vectors) were grown in Dulbecco's Modified Eagle Medium supplemented with 10% fetal calf serum. All medium contained 2 mM L-glutamine, penicillin at 100 µg/mL, and streptomycin at 100 U/mL.

Construction of Adenoviral Vectors

Adenoviral vectors expressing truncated RET were constructed by first using the polymerase chain reaction (PCR) to obtain a cDNA fragment corresponding to amino acids 1–718 of wild-type RET with the primer pair 5'-CCGCCCAAGCTTATGGCGAAGGCGACGTCCGGTGCC-3' and 5'-CGGCGGAAGCTTTTAGAGGCTAGCATAATCAGGAACATCATACGGATATTCCCACTTTGGATCCTCCAGGATCTTGAAG-3'. These primers gave rise to the RET cDNA lacking the complete intracellular region with the tyrosine kinase domain but including an in-frame hemagglutinin tag and followed by a stop codon (Fig. 1, A). This cDNA fragment—designated RET{Delta}TK—was then cloned into pShuttleCMV via HindIII restriction endonuclease cleavage sites that have been added to the primer sequences to generate an adenoviral vector expressing RET{Delta}TK (Ad.RET{Delta}TK) under control of the cytomegalovirus (CMV) promoter (Fig. 1, B). We used the AdEasy System (18) as previously decribed (19) for this cloning. Adenoviral vectors expressing green fluorescent protein (GFP; designated Ad.GFP) or wild-type human p53 (designated Ad.p53), used as control vectors, have also been described (20). Adenovirus maintenance, propagation, and titration in 293 cells were described previously by Pützer et al. (21).



View larger version (39K):
[in this window]
[in a new window]
 
Fig. 1. Construction of adenoviral vector Ad.RET{Delta}TK. A) Wild-type RET (top) and truncated RET{Delta}TK (bottom). HA-Tag = hemagglutinin tag. B) Detail of adenoviral vector Ad.RET{Delta}TK. The transgene is located in the E1 region, and expression is driven by the cytomegalovirus (CMV) immediate early promoter. Transcription is terminated by a simian virus 40 polyadenylation signal. ITR = inverted terminal repeats; Ad5 = adenovirus type 5. C) Western blot showing RET{Delta}TK expression in H1299 cells. Cells were treated with Ad.RET{Delta}TK at the multiplicities of infection (moi) indicated. Forty-eight hours after infection, cells were lysed, and extracts were probed with an anti-hemagglutinin ({alpha}-HA) antibody (upper panel) or an anti-RET antibody ({alpha}-RET H-300) recognizing an extracellular RET epitope (lower panel). Molecular masses are indicated at the left in kilodaltons.

 
Western Blot Analysis

To prepare whole cell extracts, cells were lysed in ice-cold RIPA buffer (150 mM NaCl, 10 mM Tris–HCl [pH 7.2], 0.1% sodium dodecyl sulfate, 0.1% Triton X-100, 1% deoxycholate, 5 mM EDTA, supplemented with the protease inhibitor mixture Complete Mini [Roche, Mannheim, Germany]) as previously described (19). In brief, proteins in whole-cell extracts were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and subsequently transferred to nitrocellulose membranes (Amersham Pharmacia, Braunschweig, Germany) with the Trans Blot SD semi-dry transfer system (Bio-Rad, München, Germany). Membranes were subsequently probed, according to the manufacturer’s guidelines, with antibodies against hemagglutinin (product sc-805), RET51 (product sc-1290), RET H-300 (product sc-13104), {beta}-actin (product sc-1616), or phosphorylated RET [Tyr-1062] (product sc-20252) from Santa Cruz Biotechnology, Heidelberg, Germany; with antibodies against Akt (product 9272), phosphorylated Akt (product 9271), p44/42 mitogen-activated protein kinase (i.e., ERK1/2; product 9102), phosphorylated p44/42 mitogen-activated protein kinase (product 9101), cleaved caspase 3 (product 9661), or cleaved poly(ADP-ribose) polymerase (product 9541) from Cell Signaling Technologies, Frankfurt, Germany; or with antibodies against BCL-2 (product OP60-100UG) from Calbiochem, Bad Soden, Germany. Primary antibodies were detected with appropriate secondary antibody–horseradish peroxidase conjugates (Amersham Pharmacia).

Semiquantitative Reverse Transcription–PCR

Total RNA, prepared with the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol, was transcribed into cDNAs by use of oligodeoxythymidine primers (Applied Biosystems, Darmstadt, Germany) and an Omniscript Reverse Transcription Kit (Qiagen). To obtain a semiquantitative result within the linear range, PCR was performed with the minimum number of cycles necessary to acquire a clear signal. Precise PCR conditions and primer sequences are shown in Table 1. PCR products were stained with SYBR Gold nucleic acid gel stain (Molecular Probes, Leiden, Netherlands) and quantitated in relative software units using a Bio-Imaging-Analyzer (Fuji, Düsseldorf, Germany) with the TINA program, version 2.09. All mRNA levels were normalized to the mRNA level of the ribosomal S9 gene.


View this table:
[in this window]
[in a new window]
 
Table 1. Primer pairs used for reverse transcription–polymerase chain reaction

 
5-Bromo-2'-Deoxyuridine (BrdU) Incorporation Assay and Determination of Sub-G1 DNA Content

To determine the cell cycle distribution, cells were treated with adenoviral vectors as indicated. Cells in S phase were labeled with BrdU at a final concentration of 10 µM for 1 hour by use of the FLUOS In Situ Cell Proliferation Kit (Roche), according to the manufacturer's protocol. BrdU-labeled cells were measured by flow cytometry with a FACSVantage flow cytometer (BD Biosciences Immunocytometry Systems, Heidelberg, Germany).

For flow cytometry analysis to detect the population of cells with a sub-G1 DNA content, cells were harvested 72 hours after infection and fixed in 70% ethanol, and DNA was stained with propidium iodide. Flow cytometry analysis was carried out in a FACSVantage flow cytometer using CellQuest Software.

3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyl Tetrazolium Bromide (MTT) Assay

Cells were placed in 96-well plates and allowed to adhere for 48 hours before infection. Cell viability in triplicate wells was determined 2 days, 4 days, and 6 days after infection by use of the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Mannheim, Germany), an MTT-based assay, as described by the manufacturer.

Animal Experiments

Transgenic mice expressing the germline MEN2A-derived mutant RET (C634R) have been described previously (22). Because medullary thyroid carcinoma cells produce and secrete calcitonin, serum calcitonin levels can be used to indicate tumor progression. We measured serum calcitonin with the Calcitonin Chemiluminescence Assay Kit (Nichols Institute Diagnostics, Bad Vilbel, Germany) according to the manufacturer's protocol.

Transgenic mice were infected with adenoviral vectors as follows: Mice were fully anesthetized with ketamine (100 mg/kg of body weight) and xylazine (2 mg/kg). Thyroid glands were surgically exposed, and their volumes were measured in two perpendicular diameters with calipers. A total of 1 x 109 plaque-forming units of adenoviral vectors (5 x 108 plaque-forming units per thyroid lobe) were injected into multiple sites on different sides of each thyroid gland to ensure uniform distribution, and the incision was sutured. We treated a total of 13 animals with Ad.RET{Delta}TK and another 12 with Ad.GFP. Fourteen days later the animals were killed, and the thyroid glands were exposed and measured with calipers. Tumor volumes were calculated from the longest diameter and average width by assuming a prolate spheroid shape (23). Previous comparison of thyroid and tumor volumes revealed these volumes were statistically significantly correlated with each other, thus, we used thyroid volume as an indication of tumor size (24). Tumor tissue was fixed in phosphate-buffered formalin, embedded in paraffin, and sectioned. Sections were stained with hematoxylin and eosin for histologic examination. Phosphorylated ERK and phosphorylated Akt proteins in tumor sections were immunohistochemically labeled by use of antibodies specific for phosphorylated p44/42 mitogen-activated protein kinase (Thr-202/Tyr-204; product 9101), phosphorylated Akt (Ser-473; product 9277), unphosphorylated Akt (product 9272), and unphosphorylated p44/42 mitogen-activated protein kinase (ERK1/2; product 9102) (all from Cell Signaling Technologies). All animal experiments were approved by and carried out according to the guidelines set forth by the Animal Research Ethics Board of the University of Essen, Essen, Germany.

Statistical Analysis

Variations in phosphorylation and/or expression levels of various cellular proteins, BrdU incorporation, DNA content in the sub-G1 population of cells, and tumor size were compared with an unpaired Student's t test. All statistical tests were two-sided.


    RESULTS
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
RET{Delta}TK Protein Expression

To confirm that the adenoviral vector Ad.RET{Delta}TK can express RET{Delta}TK, we infected RET-negative H1299 cells with Ad.RET{Delta}TK vectors, and 48 hours later, we measured the expression of RET{Delta}TK protein in these cells or in uninfected cells by use of western blot analysis. As shown in Fig. 1, C, RET{Delta}TK protein was expressed in a viral load-dependent manner, as detected by use of antibodies against either the hemagglutinin tag attached to the recombinant protein or the extracellular RET epitope by the use of the RET antibody H-300, which recognizes an extracellular epitope. The truncated RET proteins were identified in bands of 135 and 115 kd, corresponding to RET{Delta}TK proteins located at the cell surface and the endoplasmic reticulum, respectively. RET proteins are generally detected as fully glycosylated proteins located in the cell membrane and immature glycosylated proteins present in the endoplasmic reticulum (6).

RET Autophosphorylation and Cell Viability

To determine whether the expression of RET{Delta}TK interferes with RET-mediated signal transduction, we used the TT cell line, a well-established in vitro model for medullary thyroid carcinoma. Expression of RET{Delta}TK in TT cells decreased RET-mediated trans-autophosphorylation 1 and 2 days after transduction (Fig. 2), as measured by the phosphorylation of tyrosine residue 1062, a multifunctional docking site for numerous downstream effector proteins (16). The level of total endogenous RET protein remained constant, indicating that RET{Delta}TK interfered with endogenous RET-mediated trans-autophosphorylation but not with RET expression. Because oncogenic RET stimulates cell proliferation and survival (16,17), we next investigated whether the expression of RET{Delta}TK and the correspondingly reduced endogenous RET trans-autophosphorylation altered TT cell viability. We infected TT cells with adenoviral vectors expressing GFP (Ad.GFP), wild-type p53 (Ad.p53), or RET{Delta}TK (Ad.RET{Delta}TK) and assayed cell viability over a 6-day period. Infection with Ad.GFP had no effect on cell viability, whereas decreased cell survival was detected after infection with Ad.p53 (survival rate of 39.8% after 6 days) or with Ad.RET{Delta}TK (survival rates of 74.7% after 4 days and 45.9% after 6 days) compared with the viability of untreated control TT cells (Fig. 3, A). To investigate whether RET{Delta}TK alone inhibited cell viability, we infected RET-negative H1299 cells with the same vectors used in TT cells above and investigated the viability of these cells. Infection with Ad.p53 decreased the viability of H1299 cells (survival rates of 28.3% after 4 and 27.4% after 6 days; Fig. 3, B) compared with that of untreated control H1299 cells, but infection with Ad.GFP or with Ad.RET{Delta}TK did not alter the viability of H1299 cells. Thus, RET{Delta}TK appears to act by specifically blocking the action of oncogenic RET.



View larger version (61K):
[in this window]
[in a new window]
 
Fig. 2. A) Endogenous RET trans-autophosphorylation status in TT cells after infection with the adenoviral vector Ad.RET{Delta}TK. Phosphorylation of RET at tyrosine residue 1062 was determined by probing extracts with antibody against RET phosphorylated at tyrosine 1062 (phospho-RETTyr1062) (upper panel). Phosphorylated RETTyr1062 bands from three independent experiments were measured with the Bio-Imaging-Analyzer (Fuji) using the TINA program, version 2.09 (shown as relative software units). The value in untreated control was set as 1 in the bar graph, and mean relative amounts of phosphorylated RET are shown for days 1 and 2 after infection with error bars showing upper 95% confidence intervals. B) Western blot analysis of total RET protein in whole cell extracts. Total RET protein was determined by probing extracts with a RET antibody (RET51) recognizing the long isoform expressed endogenously. The 175- and 155-kd bands identified to the right represent the mature cell surface receptor and the immature intracellular form, respectively. The relative amounts of the 175-kd isoform representing the fully glycosylated proteins at the cell membrane in uninfected cells (day 0) and in infected cells 1 and 2 days after infection are shown above the blot, and the relative amounts of the 155-kd isoform representing the immature glycosylated form are shown below the blot. To verify the presence of RET{Delta}TK in the extracts, the blots were also probed with an anti-hemagglutinin (HA) antibody (lower panel).

 


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 3. Analysis of cell viability with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Relative viability of RET-positive TT cells (A) or RET-negative H1299 cells (B) was assayed 2, 4, and 6 days after infection with adenoviral vectors. Open circles = untreated cells; solid circles = cells infected with the adenoviral vector Ad.GFP; solid squares = cells infected with the adenoviral vector Ad.RET{Delta}TK; open triangles = cells infected with the adenoviral vector Ad.p53. Percentage of viable cells is shown relative to that of untreated cells (set as 100%). Data were obtained from three replicates of one experiment; error bars show 95% confidence intervals.

 
Phosphorylation of Downstream Effectors ERK1/2 and Akt

We further characterized the mechanism of the loss of cell viability by determining whether transformation-relevant signal transduction pathways were inhibited. TT cells were infected with Ad.RET{Delta}TK or with Ad.GFP vectors (as a control to exclude virus-related effects), and then the phosphorylation status of ERK1/2 and Akt was assessed. ERK1/2 and Akt are kinases with major roles in neoplastic transformation because they are part of two important signal transduction pathways activated by oncogenic RET (1315); both kinases are activated by phosphorylation. Infection with the control Ad.GFP virus vector did not alter the endogenous constitutive phosphorylation of ERK1/2 or Akt, whereas 2 days after infection with Ad.RET{Delta}TK vectors, phosphorylation of ERK1/2 was substantially decreased, and 2 days after such an infection, phosphorylation of Akt was not detected (Fig. 4). Thus, expression of RET{Delta}TK inhibits the phosphorylation of both ERK1/2 and Akt in TT cells.



View larger version (59K):
[in this window]
[in a new window]
 
Fig. 4. Phosphorylation status of endogenous extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt proteins. TT cells were left untreated or infected with the adenoviral vectors Ad.GFP or Ad.RET{Delta}TK as indicated. Levels of total and phosphorylated (phospho-) ERK1/2 and Akt protein were determined by probing extracts with antibodies specific for the indicated proteins. Bar graphs show results from three experiments as relative software units normalized to levels in untreated cells. Data are the mean, and error bars show the upper 95% confidence intervals.

 
Regulation of Cell Proliferation and Survival

To investigate how reduced levels of phosphorylated ERK and Akt may contribute to the loss of TT cell viability, we analyzed the regulation of cell proliferation and apoptosis by these two pathways and determined whether changes in the expression of various factors regulated by activated ERK and/or Akt were associated with reduced cell viability. Because Akt and ERK participate in the control of cell proliferation (2527), we first assessed the fraction of actively proliferating cells by determining the percentage of TT cells in S phase 72 hours after infection with Ad.RET{Delta}TK or Ad.GFP. The percentage of TT cells in S phase after the expression of RET{Delta}TK was statistically significantly reduced (3.9%, 95% CI = 2.3% to 5.5%) compared with that of untreated TT cells (10.2%; difference = 6.3%, 95% CI = 7.9% to 12.5%; P = .039) or that of TT cells infected with Ad.GFP (9.4%; difference = 5.5%, 95% CI = 8.0% to 10.8%; P = .004) (Fig. 5, A, upper panel). Second, we investigated whether oncogenic RET influences cell cycle progression by activating ERK and/or Akt, by measuring changes in the expression of the cell cycle regulators cyclin D1, p21CIP1/WAF1, and p27KIP1. Thirty-six hours after treatment with Ad.RET{Delta}TK, when endogenous RET trans-autophosphorylation was inhibited, expression of mRNAs for the cyclin-dependent kinase inhibitors p21CIP1/WAF1 and p27KIP1 increased, and expression of cyclin D1 mRNA decreased substantially, indicating that oncogenic RET enhances cell cycle progression in medullary thyroid carcinoma cells. Because Akt enhances cell survival and inhibits apoptosis (25,26), we investigated whether oncogenic RET inhibited apoptosis by a pathway involving Akt phosphorylation and BCL-2, an antiapoptotic protein that is regulated by Akt (28,29), as well as other factors. We found reduced levels of BCL-2 protein 48 hours after infection with Ad.RET{Delta}TK (Fig. 5, C), indicating that oncogenic RET may suppress the induction of apoptosis by activating pro-survival pathways. To test this possibility, we explored whether the RET{Delta}TK-mediated decreased cell viability is accompanied by the induction of apoptosis by determining the percentage of apoptotic TT cells (i.e., the population of cells with a sub-G1 DNA content) 72 hours after infection. Cultures infected with Ad.RET{Delta}TK contained more apoptotic cells (18.1%, 95% CI = 12.4% to 23.8%) than uninfected cultures (5.3%, 95% CI = 4.1% to 6.5%; P = .006) or cultures infected with Ad.GFP (mock infected) (8.5%, 95% CI = 8.4% to 8.6%; P = .04) (Fig. 5, A, bottom panel). We also detected cleaved products of procaspase 3 and poly(ADP-ribose) polymerase, which are also indicative of apoptosis (30), 48 and 72 hours after infection with Ad.RET{Delta}TK but not after infection with Ad.GFP (Fig. 5, C). Thus, inhibition of oncogenic RET activity with RET{Delta}TK inhibits cell proliferation by increasing the expression of genes for the cyclin-dependent kinase inhibitors p21CIP1/WAF1 and p27KIP1 as well as by decreasing the expression of cyclin D1 and induces apoptosis by suppressing the activity of antiapoptotic proteins.



View larger version (47K):
[in this window]
[in a new window]
 
Fig. 5. Disruption of RET trans-autophosphorylation in TT cells. A) Determination of percentage of cells in S phase and percentage of apoptotic cells. To determine the percentage of cells in S phase, cells were treated with the adenoviral vector Ad.RET{Delta}TK or the control adenoviral vector Ad.GFP; 5-bromo-2'-deoxyuridine was applied to the cells for 1 hour, and then cells were fixed and processed for flow cytometric analysis as indicated. Upper panel: The mean percentage of cells resembling actively proliferating cells in S phase are indicated (untreated versus treated with Ad.RET{Delta}TK, P = .039; treated with Ad.GFP versus treated with Ad.RET{Delta}TK, P = .004). Error bars = upper 95% confidence intervals. Lower panel: To determine the mean percentage of apoptotic cells, the percentage of cells with a sub-G1-phase DNA content, indicative of apoptosis, was determined from three experiments by flow cytometry (untreated versus treated with Ad.RET{Delta}TK, P = .006; treated with Ad.GFP versus treated with Ad.RET{Delta}TK, P = .04). Error bars = upper 95% confidence intervals. B) Analysis of cyclin D1, p21CIP1/WAF1, and p27KIP1 mRNA expression in TT cells. Reverse transcription–polymerase chain reaction (RT-PCR) analysis was performed on total RNA prepared from Ad.RET{Delta}TK-treated cells, as indicated, after infection. Expression levels of RNA from the ribosomal S9 gene served as the control. PCR products were quantitated in relative software units by the Bio-Imaging-Analyzer (Fuji) using the TINA program, version 2.09 (shown as fold induction or reduction, respectively), data were normalized to S9 values, and the untreated control was set as 1. Data are shown below the bands. C) Analysis of BCL-2, cleaved caspase 3, and cleaved poly(ADP-ribose) polymerase (PARP) protein expression levels. TT cells were infected with Ad.GFP or Ad.RET{Delta}TK, and whole-cell extracts were prepared as indicated and subjected to western blot analysis with specific antibodies against the indicated proteins. Extracts were probed with an anti-{beta}-actin antibody as a loading control.

 
Treatment of Medullary Thyroid Carcinoma In Vivo

To investigate the effects of RET{Delta}TK in vivo, we used a medullary thyroid carcinoma model, the RET C634R transgenic mouse that develops orthotopic bilateral medullary thyroid carcinoma with almost complete penetrance at 6–8 months of age (22,24). Because medullary thyroid carcinomas secrete calcitonin, we monitored serum calcitonin levels between 6 and 12 months after birth to determine when the tumors developed. After calcitonin levels increased, we confirmed the presence of tumors immunohistochemically (data not shown). After serum calcitonin levels increased to more than 1000 pg/mL, usually after 10–12 months, we exposed the thyroid glands of 13 mice by surgery and injected 5 x 108 plaque-forming units of Ad.RET{Delta}TK into each lobe. We also injected each lobe of 12 other mice with 5 x 108 plaque-forming units of Ad.GFP, for a total of 1 x 109 plaque-forming units of Ad.RET{Delta}TK or Ad.GFP per mouse. Fourteen days after infection, we measured tumor volumes and found a statistically significant difference in tumor volume between mice treated with Ad.RET{Delta}TK (43.6% of initial volume, 95% CI = 30.7% to 56.5%) and mice treated with Ad.GFP (139.8% of initial volume, 95% CI = 120.3% to 159.3%; P<.001, two-sided unpaired Student's t test) (Fig. 6, A). In support of our in vitro data, Ad.RET{Delta}TK-induced tumor growth inhibition was characterized by decreased phosphorylation of ERK and Akt compared with that in Ad.GFP-treated tumors, as shown by western blot and immunohistochemistry analyses (Fig. 6, B). Thus, primary medullary thyroid carcinomas require oncogenic RET activity, and expression of RET{Delta}TK efficiently blocks the activity of oncogenic RET by inhibiting the stimulation of downstream signal transduction pathways required for tumor maintenance and progression, thereby reducing the volume of primary medullary thyroid carcinomas in vivo.



View larger version (75K):
[in this window]
[in a new window]
 
Fig. 6. Antitumor activity of the adenoviral vector Ad.RET{Delta}TK against primary medullary thyroid carcinomas in RET C634R transgenic mice. A) Tumor volumes were assessed on the day of virus injection and 14 days after treatment. The difference in the relative increase of the size of Ad.GFP- versus Ad.RET{Delta}TK-treated tumors at day 14 was statistically significant (P<.001). Tumor volume before treatment was statistically significantly larger than that after treatment with Ad.RET{Delta}TK (Ad.RET{Delta}TK [day 0] versus Ad.RET{Delta}TK [day 14]; P = .010). B) Total and phosphorylated (phospho-) extracellular signal-regulated kinase 1/2 (ERK1/2) (44/42 kd) and Akt (62 kd) levels in Ad.GFP- and Ad.RET{Delta}TK -treated tumors 14 days after injection. Left: Whole-cell protein extracts were prepared from tumor tissue and analyzed by western blot. Right: Immunohistochemical staining of paraffin-embedded tissue for phosphorylated Akt and phosphorylated ERK after RET{Delta}TK treatment. Micrographs show the cytoplasmic, nuclear, and membrane localization of phosphorylated Akt, and the nuclear localization of phosphorylated ERK1/2 in tumors treated with Ad.GFP or Ad.RET{Delta}TK. Scale bar = 100 µm. Arrowheads indicate phosphorylated Akt and ERK1/2 proteins in tumor tissue.

 

    DISCUSSION
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In medullary thyroid carcinoma, activated oncogenic RET plays a major role in cellular transformation (810). In this study, we disrupted signaling by oncogenic RET to investigate the mechanisms used by oncogenic RET to contribute to tumor development and to begin a foundation for an alternative therapeutic approach to medullary thyroid carcinoma.

We used a dominant negative truncated RET mutant, designated RET{Delta}TK, to block oncogenic RET activity in TT cells, a line derived from a patient with medullary thyroid carcinoma. Our results and those from Baldassarre et al. (16) indicate that co-expression of RET{Delta}TK is capable of blocking endogenous RET trans-autophosphorylation, most likely by forming an inactive dimer with oncogenic RET, because RET{Delta}TK lacks the entire cytoplasmic tyrosine kinase domain, which is required to bind and activate signaling adapter molecules when phosphorylated (11) thereby ablating RET trans-autophosphorylation activity. The constitutive tyrosine kinase activity of oncogenic RET appears to be a prerequisite for cellular transformation, at least in the MEN2A type of medullary thyroid carcinoma, in which mutations in the cysteine-rich region of RET lead to the constitutive formation of active dimers.

We found that disrupting oncogenic RET activity decreased cell viability and cell proliferation, indicating that oncogenic RET stimulates proliferation and blocks apoptosis. Results of previous reports (1315) indicate that the PI3K/Akt and Raf/ERK signaling pathways contribute to RET-mediated transformation. Our results also indicate that both pathways are involved, because the activities of Akt and ERK1/2 are eliminated after the expression of the dominant negative RET inhibitor RET{Delta}TK. Both pathways regulate proliferation and/or survival by different but overlapping mechanisms (25,27).

Consistent with another report (31), we detected elevated transcription of cyclin D1 mRNA in cells expressing oncogenic RET. Cyclin D1 mRNA is tightly regulated by the Raf/Mek/ERK cascade through phosphorylation of the AP1 transcription factor complex (32). In addition, the PI3K/Akt pathway has also been implicated in the regulation of cyclin D1 by sustaining protein stability with a mechanism involving interference with protein degradation (25). Furthermore, we observed increased expression of mRNAs for cyclin-dependent kinase inhibitors p21CIP1/WAF1 and p27KIP1 after oncogenic RET activity was inhibited by RET{Delta}TK. Because Akt inhibits the expression of p21CIP1/WAF1 and p27KIP1 by phosphorylating the transcription factor AFX/FKHR (25,33), oncogenic RET may also contribute to enhanced cell cycle progression, possibly by inhibiting the expression of p21CIP1/WAF1 and p27KIP1 mRNA through the PI3K/Akt pathway. However, because RET, ERK, and Akt have many downstream targets, other targets could also be responsible for the observed decrease in cell number in TT cells infected with Ad.RET{Delta}TK. In addition to the Raf/ERK and PI3K/Akt cascades, RET activates a complex network of signal transduction pathways, including the c-Jun amino-terminal kinase pathway, the p38 mitogen-activated protein kinase pathway, the extracellular signal-regulated kinase 5 pathway, or the signal transducer and activator of transcription 3 pathway (11,34). Akt can also modulate cell cycle progression via many other mechanisms, including the regulation of proliferating-cell nuclear antigen or phosphorylation of glycogen synthase kinase 3{beta} that leads to inhibition of cyclin D1 protein degradation (35,36). The variety of pathways potentially regulated by RET and their contribution to cancer have still to be discovered in detail.

After oncogenic RET activity was disrupted, cells actively underwent apoptosis, indicating that oncogenic RET appears to inhibit apoptosis. Other oncogenic tyrosine kinases, including MET, BCR-ABL, HER-2/neu, or RON, have antiapoptotic activity that is involved in cellular transformation. This activity has also been attributed to enhanced PI3K/Akt signaling, although Raf/ERK activation may be involved as well (3740). Thus, stimulation of antiapoptotic signaling might be a common mechanism used by oncogenic tyrosine kinases in the multistage process of tumorigenesis.

Because BCL-2 plays a major role in medullary thyroid carcinoma progression and because the level of BCL-2 protein expression is generally elevated in medullary thyroid carcinomas (41), we examined the expression of BCL-2 and found that inhibition of oncogenic RET activity decreased the level of BCL-2 protein. BCL-2 strongly inhibits apoptosis by maintaining mitochondrial membrane integrity and thus preventing caspase activation (42). Therefore, our results provide an explanation of how oncogenic RET stimulates cell survival. BCL-2 expression, among other mechanisms, is tightly regulated by nuclear factor-{kappa}B transcription factors (28), which in turn are stimulated by enhanced Akt activity by the phosphorylation of I{kappa}B kinase (29). Thus, stimulation of Akt may be one mechanism that links oncogenic RET activity with pro-survival signaling mediated by enhanced BCL-2 expression.

Our results suggest that orthotopic medullary thyroid carcinomas in transgenic mice also develop a state of dependence on signals mediated by activated oncogenic RET. Injection of RET{Delta}TK into primary tumors led to a statistically significantly decreased tumor volume after 2 weeks compared with that in mice injected with the appropriate control vectors. Our results indicate that inhibition of oncogenic RET activity might be a viable target for the development of new treatments for medullary thyroid carcinomas. Results of previous studies have demonstrated that oncogenic RET activity can be nonspecifically blocked by small-molecule compounds (43,44) or the expression of tyrosine phosphatases (45). In this study, we describe phenotypic changes induced by specifically inhibiting RET trans-autophosphorylation with RET{Delta}TK.

In summary, oncogenic RET increases cell proliferation by regulating the expression of cyclin D1, p21CIP1/WAF1, and p27KIP1 mRNAs through the PI3K/Akt and Raf/ERK signaling pathways. Oncogenic RET increases antiapoptotic signaling by increasing the expression of BCL-2 (Fig. 7). Although each of these results alone could have important implications for future medullary thyroid carcinoma therapy, we propose that disrupting oncogenic RET may be the most promising target.



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 7. Schematic illustration of mechanisms leading to apoptosis suppression and enhanced proliferation triggered by a MEN2A-derived RET oncogene. RET mutations lead to sustained oncogenic signal transduction in MEN2A cells. This is accompanied by enhanced S-phase progression via constitutive activation of the extracellular signal-regulated kinase 1/2 and cyclin D1 mRNA induction. Repression of mRNA of the cyclin-dependent kinase inhibitors p21CIP1/WAF1 and p27KIP1 also confers to elevated cell cycle progression. Oncogenic RET directly stimulates protein expression of the antiapoptotic factor BCL-2 via a pathway involving Akt phosphorylation thereby suppressing apoptosis.

 


    NOTES
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Present address: Matthias Drosten, Department of General Surgery and Transplantation, University Hospital Essen, Essen, Germany.

Supported by grant PU188/3-1/3-2/3-3 from the Deutsche Forschungsgemeinschaft. Bruce A. J. Ponder is a Gibb Fellow of Cancer Research UK.

We thank Carmen Theseling, Barbara Pollmeier, and Jutta Hunke for technical assistance.


    REFERENCES
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

1 Gimm O. Thyroid cancer. Cancer Lett 2001;163:143–56.[CrossRef][ISI][Medline]

2 Hundahl SA, Fleming ID, Fremgen AN, Menck HR. A National Cancer Data Base report on 53,856 cases of thyroid carcinoma treated in the U.S., 1985–1995. Cancer 1998;83:2638–48.[CrossRef][ISI][Medline]

3 Vitale G, Caraglia M, Ciccarelli A, Lupoli G, Abbruzzese A, Tagliaferri P, et al. Current approaches and perspectives in the therapy of medullary thyroid carcinoma. Cancer 2001;91:1797–808.[CrossRef][ISI][Medline]

4 Mulligan LM, Kwok JB, Healey CS, Elsdon MJ, Eng C, Gardner E, et al. Germ-line mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2A. Nature 1993;363:458–60.[CrossRef][ISI][Medline]

5 Asai N, Iwashita T, Matsuyama M, Takahashi M. Mechanism of activation of the ret proto-oncogene by multiple endocrine neoplasia 2A mutations. Mol Cell Biol 1995;15:1613–9.[Abstract]

6 Manie S, Santoro M, Fusco A, Billaud M. The RET receptor: function in development and dysfunction in congenital malformation. Trends Genet 2001;17:580–9.[CrossRef][ISI][Medline]

7 Schuchardt A, D'Agati V, Larsson-Blomberg L, Costantini F, Pachnis V. Defects in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret. Nature 1994;367:380–3.[CrossRef][ISI][Medline]

8 Ponder BA, Smith D. The MEN II syndromes and the role of the ret proto-oncogene. Adv Cancer Res 1996;70:179–222.[ISI][Medline]

9 Eng C. RET proto-oncogene in the development of human cancer. J Clin Oncol 1999;17:380–93.[Abstract/Free Full Text]

10 Hansford D, Mulligan LM. Multiple endocrine neoplasia type 2 and RET: from neoplasia to neurogenesis. J Med Genet 2000;37:817–27.[Abstract/Free Full Text]

11 Takahashi M. The GDNF/RET signaling pathway and human diseases. Cytokine Growth Factor Rev 2001;12:361–73.[CrossRef][ISI][Medline]

12 van Weering DH, Bos JL. Signal transduction by the receptor tyrosine kinase Ret. Recent Results Cancer Res 1998;154:271–81.[Medline]

13 Melillo RM, Santoro M, Ong SH, Billaud M, Fusco A, Hadari YR, et al. Docking protein FRS2 links the protein tyrosine kinase RET and its oncogenic forms with the mitogen-activated protein kinase signaling cascade. Mol Cell Biol 2001;21:4177–87.[Abstract/Free Full Text]

14 Besset V, Scott RP, Ibanez CF. Signaling complexes and protein–protein interactions involved in the activation of the Ras and phosphatidylinositol 3-kinase pathways by the c-RET receptor tyrosine kinase. J Biol Chem 2000;275:39159–66.[Abstract/Free Full Text]

15 Segouffin-Cariou C, Billaud M. Transforming ability of MEN2A-RET requires activation of the phosphatidylinositol 3-kinase/Akt signaling pathway. J Biol Chem 2000;275:3568–76.[Abstract/Free Full Text]

16 Baldassarre G, Bruni P, Boccia A, Salvatore G, Melillo RM, Motti ML, et al. Glial cell line-derived neurotrophic factor induces proliferative inhibition of NT2/D1 cells through RET-mediated up-regulation of the cyclin-dependent kinase inhibitor p27(kip1). Oncogene 2002;21:1739–49.[CrossRef][ISI][Medline]

17 Carlomagno F, Salvatore D, Santoro M, de Franciscis V, Quadro L, Panariello A, et al. Point mutation of the RET proto-oncogene in the TT human medullary thyroid carcinoma cell line. Biochem Biophys Res Commun 1995;207:1022–8.[CrossRef][ISI][Medline]

18 He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, Vogelstein B. A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci U S A 1998;95:2509–14.[Abstract/Free Full Text]

19 Drosten M, Stiewe T, Putzer BM. Antitumor capacity of a dominant-negative RET proto-oncogene mutant in a medullary thyroid carcinoma model. Hum Gene Ther 2003;14:971–82.[CrossRef][ISI][Medline]

20 Rodicker F, Putzer BM. p73 is effective in p53-null pancreatic cancer cells resistant to wildtype TP53 gene replacement. Cancer Res 2003;63:2737–41.[Abstract/Free Full Text]

21 Putzer BM, Stiewe T, Crespo F, Esche H. Improved safety through tamoxifen-regulated induction of cytotoxic genes delivered by Ad vectors for cancer gene therapy. Gene Ther 2000;7:1317–25.[CrossRef][ISI][Medline]

22 Reynolds L, Jones K, Winton DJ, Cranston A, Houghton C, Howard L, et al. C-cell and thyroid epithelial tumours and altered follicular development in transgenic mice expressing the long isoform of MEN 2A RET. Oncogene 2001;20:3986–94.[CrossRef][ISI][Medline]

23 Addison CL, Braciak T, Ralston R, Muller WJ, Gauldie J, Graham FL. Intratumoral injection of an adenovirus expressing interkeukin 2 induced regression and immunity in murine breast cancer model. Proc Natl Acad Sci U S A 1995;92:8522–6.[Abstract]

24 Cranston AN, Ponder BA. Modulation of medullary thyroid carcinoma penetrance suggests the presence of modifier genes in a RET transgenic mouse model. Cancer Res 2003;63:4777–80.[Abstract/Free Full Text]

25 Vivanco I, Sawyers CL. The phosphatidylinositol 3-kinase AKT pathway in human cancer. Nat Rev Cancer 2002;2:489–501.[CrossRef][ISI][Medline]

26 Chang F, Lee JT, Navolanic PM, Steelman RS, Shelton JG, Blalock WL, et al. Involvement of PI3K/Akt pathway in cell cycle progression, apoptosis, and neoplastic transformation: a target for cancer chemotherapy. Leukemia 2003;17:590–603.[CrossRef][ISI][Medline]

27 Roovers K, Assoian RK. Integrating the MAP kinase signal into the G1 phase cell cycle machinery. Bioessays 2000;22:818–26.[CrossRef][ISI][Medline]

28 Romashkova JA, Makarov SS. NF-kappaB is a target of AKT in anti-apoptotic PDGF signalling. Nature 1999;401:86–90.[CrossRef][ISI][Medline]

29 Grossmann M, O'Reilly LA, Gugasyan R, Strasser A, Adams JM, Gerondakis S. The anti-apoptotic activities of Rel and RelA required during B-cell maturation involve the regulation of Bcl-2 expression. EMBO J 2000;19:6351–60.[Abstract/Free Full Text]

30 Reed JC. Mechanisms of apoptosis. Am J Pathol 2000;157:1415–30.[Abstract/Free Full Text]

31 Watanabe T, Ichihara M, Hashimoto M, Shimono K, Shimoyama Y, Nagasaka T, et al. Characterization of gene expression induced by RET with MEN2A or MEN2B mutation. Am J Pathol 2002;161:249–56.[Abstract/Free Full Text]

32 Shaulian E, Karin M. AP-1 as a regulator of cell life and death. Nat Cell Biol 2002;4:E131–6.[CrossRef][ISI][Medline]

33 Medema RH, Kops GJ, Bos JL, Burgering BM. AFX-like Forkhead transcription factors mediate cell cycle regulation by Ras and PKB through p27kip1. Nature 2000;404:782–7.[CrossRef][ISI][Medline]

34 Schuringa JJ, Wojtachnio K, Hagens W, Vellenga E, Buys CH, Hofstra R, et al. MEN2A-RET-induced cellular transformation by activation of STAT3. Oncogene 2001;20:5350–8.[CrossRef][ISI][Medline]

35 Rossig L, Jadidi AS, Urbich C, Badorff C, Zeiher AM, Dimmeler S. Akt-dependent phophorylation of p21 (Cip1) regulates PCNA binding and proliferation of endothelial cells. Mol Cell Biol 2001;21:5644–57.[Abstract/Free Full Text]

36 Diehl JA, Cheng M, Roussel MF, Sherr CJ. Glycogen synthase kinase-3beta regulates cyclin D1 proteolysis and subcellular localization. Genes Dev 1998;12:3499–511.[Abstract/Free Full Text]

37 Xiao GH, Jeffers M, Bellacosa A, Mitsuuchi Y, Vande Woude GF, Testa JR. Anti-apoptotic signaling by hepatocyte growth factor/Met via the phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase pathways. Proc Natl Acad Sci U S A 2001;98:247–52.[Abstract/Free Full Text]

38 Faderl S, Talpaz M, Estrov Z, O'Brien S, Kurzrock R, Kantarjian HM. The biology of chronic myeloid leukemia. N Engl J Med 1999;341:164–72.[Free Full Text]

39 Yakes FM, Chinratanalab W, Ritter CA, King W, Seelig S, Arteaga CL. Herceptin-induced inhibition of phosphatidylinositol 3-kinase and Akt is required for antibody-mediated effects on p27, cyclin D1, and antitumor action. Cancer Res 2002;62:4132–41.[Abstract/Free Full Text]

40 Danilkovitch-Miagkova A. Oncogenic signaling pathways activated by RON receptor tyrosine kinase. Curr Cancer Drug Targets 2003;3:31–40.[Medline]

41 Wang DG, Liu WH, Johnston CF, Sloan JM, Buchanan KD. Bcl-2 and c-Myc, but not bax and p53, are expressed during human medullary thyroid carcinoma tumorigenesis. Am J Pathol 1998;152:1407–13.[Abstract]

42 Cory S, Adams JM. The Bcl-2 family: regulators of the cellular life-or-death switch. Nat Rev Cancer 2002;2:647–56.[CrossRef][ISI][Medline]

43 Carlomagno F, Vitagliano D, Guida T, Napolitano M, Vecchio G, Fusco A, et al. The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes. Cancer Res 2002;62:1077–82.[Abstract/Free Full Text]

44 Strock CL, Park JI, Rosen M, Dionne C, Ruggeri B, Jones-Bolin S, et al. CEP-701 and CEP-751 inhibit constitutively activated RET tyrosine kinase activity and block medullary thyroid carcinoma cell growth. Cancer Res 2003;63:5559–63.[Abstract/Free Full Text]

45 Hennige AM, Lammers R, Hoppner W, Arlt D, Strack V, Teichmann R, et al. Inhibition of Ret oncogene activity by the protein tyrosine phosphatase SHP1. Endocrinology 2001;142:4441–7.[Abstract/Free Full Text]

Manuscript received February 25, 2004; revised June 8, 2004; accepted June 17, 2004.



             
Copyright © 2004 Oxford University Press (unless otherwise stated)
Oxford University Press Privacy Policy and Legal Statement