help button home button Endocrine Society JCEM JCEM Call for Nominations for EIC
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liu, Z.
Right arrow Articles by Nwariaku, F. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, Z.
Right arrow Articles by Nwariaku, F. E.
The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 7 3503-3509
Copyright © 2004 by The Endocrine Society

Antiproliferative Effects of Src Inhibition on Medullary Thyroid Cancer

Zijuan Liu, Joy Falola, Xudong Zhu, Ying Gu, Lawrence T. Kim, George A. Sarosi, Thomas Anthony and Fiemu E. Nwariaku

Departments of Surgery and Pulmonary Medicine (Z.L., J.F., X.Z., Y.G., G.A.S., T.A., F.E.N.), University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390; Dallas Veterans Affairs Medical Center (Z.L., Y.G., G.A.S., T.A., F.E.N.), Dallas, Texas 75216; and University of Arkansas for Medical Sciences (L.T.K.), Little Rock, Arkansas 72205

Address all correspondence and requests for reprints to: Fiemu E. Nwariaku M.D., Department of Surgery, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9156. E-mail: Fiemu.Nwariaku{at}UTSouthwestern.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
There is no effective treatment for recurrent or metastatic medullary thyroid cancer (MTC). Hereditary MTC is associated with mutations in the RET protooncogene, which encodes for a tyrosine kinase. We postulated that Src tyrosine kinases regulate MTC proliferation.

Proliferation of the human MTC cell line, TT, was examined in the presence of a Src-specific tyrosine kinase inhibitor, PP2, or genistein. Cell counts were performed with a Coulter counter or by flow cytometry. DNA synthesis was evaluated by bromodeoxyuridine incorporation. A cell death ELISA was used to assess apoptosis. Akt phosphorylation was determined by Western immunoblot. MAPK activity was measured using an immunoprecipitation kinase assay, and MAPK inhibition was achieved with SB202190 (p38 MAPK) and PD098059 (MAPK kinase). Data were analyzed by ANOVA.

Compared with controls, PP2 reduced DNA synthesis, abolished Akt phosphorylation, and increased apoptosis. The MAPK kinase inhibitor, PD098059, attenuated DNA synthesis, whereas genistein caused modest declines in cell count and DNA synthesis and minimal changes in apoptosis.

We conclude that Src-dependent MTC proliferation occurs via increased DNA synthesis and reduced apoptosis. The latter effect may be mediated by Akt survival signals. Modulation of Src activity is a potential therapeutic target in MTC.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THERE IS NO effective therapy for advanced-stage or recurrent medullary thyroid cancer (MTC). Because germline RET mutations are detected in over 95% of hereditary MTC and somatic RET mutations are present in up to 25% of sporadic MTC, considerable work has focused on the potential to manipulate RET function in MTC (1). The RET gene has been mapped to chromosome 10; however, the downstream targets of RET are still being studied. All hereditary forms of MTC contain a gain-of-function mutation in the RET protooncogene, which activates intracellular tyrosine kinases and causes uncontrolled proliferation or prolonged survival (2, 3). Both point mutations and gene rearrangements in RET are responsible for familial thyroid cancer syndromes such as multiple endocrine neoplasia (MEN) type 2 (MEN2A, MEN2B, and familial MTC) (4). Thus the identification of downstream targets involved in RET-mediated oncogenesis could potentially lead to development of targeted treatment.

Tyrosine kinases regulate numerous intracellular processes including cell proliferation and apoptosis (5). Src family tyrosine kinases are one of the most studied group of intracellular protein tyrosine kinases and are potential downstream targets of RET signaling as they appear to be in other cell types. Src kinases are major mediators of growth factor signaling and mitogenesis (6, 7, 8, 9, 10, 11) and thus serve as attractive targets for modulating uncontrolled mitosis as is seen in cancer cells. Several investigators have linked RET-mediated proliferation with Src activation in a variety of cell types (6, 8, 12). Studies performed in NIH3T3 cells suggest that Src may be a mediator of RET signaling in nontumorigenic cells stimulated with glial cell line-derived neurotrophic factor, GDNF (6). Similarly, Kato and associates (13) demonstrated that inactive RET mutations that prevent cellular proliferation could be rescued by c-Src and v-Src. Src family kinases have also been implicated in prolonged cell survival (14, 15). However, few studies have examined the role of c-Src in proliferation of a thyroid cancer cell line with endogenous RET activity causing uncontrolled proliferation (6, 7). Similarly, there are no studies to our knowledge that examine the mechanism of uncontrolled cellular proliferation or cell death in such a cell line. Thus we sought to determine the role of Src pathways in a human MTC cell line that harbors a gain-of-function RET mutation. The human MTC cell line, TT cells, was initially derived from a patient with MTC. These cells harbor a codon 634 mutation (cys-trp) in RET (16) that is associated with constitutively active tyrosine kinase activity. We examined the effects of c-Src inhibition on proliferation and apoptosis in TT cells.


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

The human MTC cell line, TT was purchased from American Type Culture Collection (Manassas, VA) and cultured in endothelial cell growth medium with 2% fetal bovine serum (EGM-2; Bio-Whitaker, Walkersville, MD). As previously mentioned, these cells harbor a codon 634 mutation (cys-trp) in RET (16). This mutation constitutively activates RET tyrosine kinase.

Inhibitors

c-Src inhibition was accomplished using PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4-d]pyrimidine), a potent and selective Src inhibitor (17). Genistein (4',5,7-trihydroxyisoflavone), a natural isoflavonoid phytoestrogen, is a nonspecific inhibitor of protein tyrosine kinases and was used at a concentration of 20 µM (18, 19). p38 and ERK MAPKs were inhibited using the compounds SB202190 and PD098059 (Calbiochem, San Diego, CA), respectively. SB202190, 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl) 1H-imidazole is a potent cell-permeable inhibitor of p38-induced phosphorylation of activating transcription factor 2(ATF-2). It has no inhibitory effect on activity of JNK or ERK MAPKs. PD98059 (2'-amino-3'-methoxyflavone) is a selective MAPK kinase (MEK) inhibitor (20, 21). SB202190 was added to the cells at a concentration of 10 µM, whereas PD098059 was used at 20 µM. Orthophenyl acetate and retinoic acid were used as nonspecific controls. Resiniferonol 9,13,14-orthophenylacetate binds with high potency to protein kinase C, and trans-retinoic acid is a potent modulator of growth and differentiation. Both agents have been shown to regulate growth and differentiation of thyroid cancer cells (22, 23).

Kinase assays

We examined activation of p38 and ERK MAPKs using a nonradioactive immunoprecipitation kinase assay (Cell Signaling Technology, Inc., Beverly, MA). TT cell monolayers were exposed to culture medium or inhibitors for up to 24 h. Lysis buffer [20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerophosphate, 1 mM Na3VO4, and 10 mM MgCl2] was added for 5 min, and cells were scraped, sonicated, and centrifuged for 10 min at 14,000 rpm. Phosphorylated MAPK was immunoprecipitated from the resulting supernatant using immobilized phosphospecific p38 or ERK MAPK monoclonal antibodies. This was achieved by overnight incubation at 4 C and centrifugation. The resulting immunoprecipitates were resuspended in kinase buffer [25 mM Tris (pH 7.5), 5 mM ß-glycerophosphate, 2 mM dithiothreitol, 0.1 mM Na3VO4, and 10 mM MgCl2) supplemented with 200 µM ATP and the respective MAPK substrate, i.e. ATF-2 or Elk-1. Substrate phosphorylation was then detected by Western blotting for phospho-ATF-2 or phospho-Elk-1.

Cell proliferation

Cell proliferation was assessed using a colorimetric immunoassay kit to quantify incorporation of 5-bromo-2'-deoxyuridine (BrdU) during DNA synthesis (Roche Diagnostics, Mannheim, Germany). Briefly, the cells were serum starved for 24 h before assessment of proliferation, and BrdU was added to the cells for 24 h. Fixation and partial DNA denaturation was performed before staining with anti-BrdU antibody. Immune complexes were detected by subsequent substrate reaction, and the absorbance was measured using a multiwell spectrophotometer.

Cell cycle analysis

TT cells were synchronized by serum starvation for 24 h, and then serum was added in the presence or absence of PP2 (10 µM). At varying time points (15, 24, 48, and 72 h), cells were trypsinized, washed with PBS, centrifuged at 900 rpm for 5 min, fixed with 5 ml of cold 85% ethanol, and resuspended in PBS. Cell suspensions were stained with propidium iodide solution (15 µg/ml propidium iodide, 0.1% Triton, 1 µg/ml EDTA, and 100 µg/ml DNA-free RNase) for 30 min at room temperature. Cell cycle analysis was then performed by flow cytometry (FACSCalibur; BD Biosciences, San Jose, CA). Cell cycle data were analyzed using Modfit LT software, version 3.1 (Verity Software House Inc., Topsham, ME).

Cell death detection

Cell death was assessed using a photometric enzyme immunoassay kit (Roche Diagnostics) using antibodies against DNA histone complexes. TT cells (1 x 104) were cultured in 96-well plates in the presence of 10 µM PP2. Serum-free medium was added 24 h before performing experiments. After 24 h, the supernatant was removed and cells were lysed (centrifugation at 1600 rpm for 10 min) and left at 4 C. A 20-µl volume of cell lysate was transferred to a streptavidin-coated microtiter plates, and 80 µl of immunoreagent solution (antihistone and anti-DNA antibodies) was added to each well and incubated for 2 h. After washing, 100 µl of the 2,2'-azino-di-[3-ethylbenzthiazoline sulfate (6)] (colorimetric) solution is added to each well and incubated in a shaker for 15 min. Colorimetry is measured at 405 nm against 2,2'-azino-di-[3-ethylbenzthiazoline sulfate (6)] solution as blank and reference wavelength of 492 nm.

Akt phosphorylation

One pathway mediating survival signals is the phosphoinositol-3-Akt pathway. As such, phosphorylation of Akt was examined to determine the role of Src in activating survival pathways. TT cells were incubated with vehicle, PP2 (10 µM), or PD098059 (20 µM) for 6 or 24 h and washed twice with ice-cold PBS. Cells were incubated on ice for 10 min with lysis buffer. Cell suspensions were scraped and sonicated four times for 5 sec and centrifuged (14,000 rpm for 15 min at 4 C). SDS sample buffer (3x) was added to supernatants and boiled for 5 min. Samples were then loaded onto 10% SDS-PAGE gels. After transfer, membranes were incubated with antibodies against either phospho-Akt or total Akt. Phospho-Akt was detected using a polyclonal antibody raised against phosphorylated serine 473 of all three isoforms of Akt, Akt1, Akt2, and Akt3 (Biosource, Camarillo, CA). Phosphorylation of Akt at Ser473 is required for full activation of Akt (24). Total Akt was detected using a polyclonal antibody raised against the carboxy terminus of human Akt (Santa Cruz Biotechnology, Santa Cruz, CA). Detection was achieved by enhanced chemiluminescence.

Statistical analysis

Data are expressed as mean ± SE of the mean. Statistical comparisons were performed using ANOVA with the Bonferroni post hoc test. Differences between groups were considered statistically significant at a P value of <0.05. The Western blot experiments were performed in triplicate. The sample sizes for experiments involving cell proliferation and counting were six to eight per experimental group.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Src inhibition reduces TT cell number

Proliferation of TT cell cultures was assessed for up to 6 d in the presence or absence of a specific inhibitor of Src, PP2. Src inhibition with PP2 reduced cell counts at all time points (Fig. 1Go), although the effect became statistically significant at 72 h. Cell number was also examined in the presence of genistein and the MEK inhibitor PD098059. PP2 caused a 75% reduction in TT cell counts. In contrast, genistein and PD098059 reduced TT cell proliferation by 50 and 27%, respectively (Fig. 2Go). The differences in cell proliferation between the genistein and PD098059 groups were not statistically significant. However, PP2 caused a statistically significant decrease in cell number compared with genistein or PD098059.



View larger version (10K):
[in this window]
[in a new window]
  		FIG. 1. Cell numbers after exposure to vehicle (open bar) or PP2 (black bar) for up to 6 d. PP2 significantly reduced cell numbers on d 3 and 6. *, P < 0.05; n > 8 per group.

 


View larger version (15K):
[in this window]
[in a new window]
  		FIG. 2. Cell numbers after 72 h of exposure to vehicle (open bar), PP2 (black bar), genistein (dark shade), or PD098059 (light shade). All inhibitors reduced cell counts compared with controls (*, P < 0.05); however, PP2 cell counts were significantly less than genistein or PD 098059 (#, P < 0.05 vs. all other groups). n > 8 per group.

 
Src inhibition prevents ERK activation

The effect or Src inhibition on MAPK activity was examined because inhibition of Src and ERK decreased cell counts to different extents. Orthophenylacetate and trans-retinoic acid were used as controls because both agents have been shown to modulate thyroid cancer differentiation in both clinical and laboratory studies (22, 25, 26). ERK and p38 MAPK were constitutively activated in proliferating TT cells in culture (Fig. 3Go). Addition of PP2 to TT cells in culture induced a 10-fold decrease in ERK activity 6 and 24 h after exposure. Genistein had no effect on baseline ERK activation. However, phenylacetate and trans-retinoic acid increased ERK activity 6 h after exposure, but ERK activity returned to baseline by 24 h (Fig. 3Go). In contrast, PP2, phenylacetate, retinoic acid, and genistein had no effect on p38 activity (Fig. 4Go).



View larger version (48K):
[in this window]
[in a new window]
  		FIG. 3. Effect of PP2 on ERK activity. PP2 significantly prevented constitutive ERK activity; P < 0.05. Genistein (Gen) demonstrated no change in ERK activity, whereas orthophenylacetate (PA) and trans-retinoic acid (RA) increased ERK activation compared with controls.

 


View larger version (25K):
[in this window]
[in a new window]
  		FIG. 4. p38 activity in TT cells exposed to inhibitors. There was no significant difference in p38 activity between the groups.

 
Tyrosine kinase inhibition prevents DNA synthesis and causes cell cycle arrest

To determine whether the effect of PP2 on TT cell number was a result of decreased proliferation, we exposed serum-starved TT cells to vehicle (control), PP2, genistein, or PD098059. DNA synthesis was measured by BrdU incorporation after 24 h of culture. Compared with vehicle, PP2 and genistein reduced DNA synthesis by 80 and 70%, respectively (Fig. 5Go). PD098059 reduced DNA synthesis by 64%. The differences in DNA synthesis between the PP2, genistein, and PD098059 groups were not statistically significant. Inhibition of p38 using SB202190 resulted in no significant change in DNA synthesis (data not shown). In separate experiments to determine the effect of PP2 on the cell cycle, we examined the cell cycle by flow cytometry. Compared with vehicle, PP2 effectively prevented the increase in S-phase fraction at 48 h after culture, suggesting that these cells were arrested before the S phase in the G0/G1 phase of the cell cycle (Fig. 6Go). To confirm that the MEK inhibitor was effective against ERK activation in this cell line, we examined ERK activation in the presence of PD098059 (Fig. 7Go) and observed a 75% reduction in ERK activity. Interestingly, the p38 inhibitor SB202190 caused a mild reduction of ERK activity at 6 h.



View larger version (9K):
[in this window]
[in a new window]
  		FIG. 5. DNA synthesis was measured by incorporation of BrdU. Data are expressed as a percentage of control (vehicle). All inhibitors significantly reduced new DNA synthesis; however, PP2 caused the most reduction of new DNA synthesis. *, P < 0.05.

 


View larger version (21K):
[in this window]
[in a new window]
  		FIG. 6. Cell cycle analysis was performed by flow cytometry. A, Flow cytometry of TT cells 48 h after exposure to vehicle (left panel) or PP2 (right panel). PP2 reduced the percentage of cells in the S phase and increased the G0/G1 cell fraction. B, Graph shows the total percentage of cells in S phase in cells exposed to vehicle ({diamondsuit}) or PP2 ({circ}) over 72 h. PP2 caused cell cycle arrest in the G0/G1 phase, an effect that was most apparent 48 h after exposure. *, P < 0.05.

 


View larger version (41K):
[in this window]
[in a new window]
  		FIG. 7. ERK MAPK activity was assessed using a kinase assay with Elk (p-Elk) as a substrate. A histogram is shown of ERK activity at 6 and 24 h and a representative immunoblot. PD098059 (gray bars) caused marked reduction of ERK activity compared with SB202190 (black bars) or vehicle controls (open bars).

 
Src inhibition increases TT cell apoptosis and Akt phosphorylation

We examined the effect of tyrosine kinase and ERK MAPK activity on cell death by the use of a cell death ELISA for histone-DNA complexes. Treatment of TT cells with PP2, genistein, or PD098059 resulted in a 60-, 14-, and 8-fold increase in cell death, respectively (Fig. 8Go). Concordantly, Akt phosphorylation in TT cells was abolished by exposure to PP2 at 6 and 24 h after exposure, whereas inhibition of ERK with PD098059 did not affect Akt phosphorylation (Fig. 9Go).



View larger version (9K):
[in this window]
[in a new window]
  		FIG. 8. Apoptosis was measured in cells exposed to PP2 (black bars), genistein (dark shade), or PD098059 (light shade). Data are normalized for cell counts and expressed as percentage of vehicle controls.

 


View larger version (21K):
[in this window]
[in a new window]
  		FIG. 9. Phosphorylation of Akt at 6 and 24 h in presence or absence of inhibitors. PP2 prevented Akt phosphorylation at both time points.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we show that interruption of tyrosine kinase signaling effectively prevents cell proliferation and activation of growth signals in MTC cells. Specifically, the inhibition of the non-receptor tyrosine kinase, c-Src, effectively prevented TT cell proliferation, ERK activation, and DNA synthesis. In addition, Src inhibition markedly increased cellular apoptosis.

The RET protooncogene encodes for a tyrosine kinase that activates multiple intracellular signals. Gain-of-function mutations of the RET protooncogene result in uncontrolled growth and cellular proliferation in a variety of neoplasms (1, 4, 27, 28, 29). Such mutations have been identified in the majority of patients with MEN2A, MEN2B, and familial MTC. Moreover, approximately 25% of tumor samples from patients with sporadic MTC harbor RET mutations (30). A high frequency of RET gene rearrangement has also been described in patients with sporadic papillary thyroid carcinoma (31, 32). Despite the knowledge that RET mutations are prevalent in the majority of patients with hereditary MTC, identification of intracellular proliferative pathways downstream of RET has been elusive. Because RET encodes for a tyrosine kinase, considerable interest exists in elucidating which tyrosine kinases are activated by RET mutations, because these kinases are potential targets for adjuvant therapy in MTC (33, 34, 35). A particularly promising group of intracellular mediators are the Src family of tyrosine kinases (36, 37), in part because of evidence suggesting a direct interaction between RET and Src. For example, Melillo and associates (6) demonstrated direct binding of RET to the SH2 domain of c-Src, a process that resulted in stimulation of c-Src activity in NIH3T3 cells. In those studies, RET expression resulted in higher Src kinase activity compared with wild-type controls. Furthermore, microinjection of a kinase-inactive c-Src mutant blocked RET-mediated mitogenesis. Although these studies provide evidence for Src as mediator in RET-induced mitogenesis, the mechanisms that culminate in cellular proliferation are less clear. However, recent evidence suggests that Src may be capable of restoring proliferative signals in cells with loss-of-function RET mutations (13), suggesting that Src is indeed downstream of RGT.

This study provides evidence that Src increases proliferation and activates survival pathways in cells harboring a gain-of-function RET mutation. The reduction in TT cell proliferation induced by PP2 is in keeping with observations which indicate that c-Src is a potent mediator of cellular proliferation and activation of growth signals in other cell types (36, 38, 39). The effects of Src inhibition appear to be dependent on constitutive RET activation due to the C634W RET mutation present in TT cells. This study did not address the role of somatic M918T RET mutations that occur in the majority of patients with sporadic MTC. However, recent evidence suggests that both cysteine missense mutations (common in MEN2A) and methionine-to-threonine mutations (MEN2B) in RET, signal through similar pathways during cellular proliferation (40). In fact, Murakami et al. (41) showed that both categories of mutations increase phosphatidylinositol-3 kinase activity, although the RETM918T mutation showed stronger activation.

Many intracellular proliferative signals are mediated by MAPKs such as ERK. Others have demonstrated RET-mediated ERK activation in neuroectodermal-derived cell lines (42, 43). Although we observed constitutive ERK activity within TT cells in this study, ERK inhibition using PD098059 caused only a modest decrease in cell number. Our observation that PP2 markedly reduced ERK activation is supported by previous studies that suggest that RET-mediated MAPK activation may be Src dependent (38, 44, 45). This suggests a proximal and divergent pathway for Src signaling. Notably, PD098059 significantly decreased proliferation (DNA synthesis) yet had only a modest effect on cell number, suggesting that Src may also activate survival pathways that are ERK independent.

The observation of increased ERK activity during trans-retinoic acid exposure is similar to findings by Yen and others (46). Retinoic acid is a regulator of thyroid cancer differentiation; thus it is not unexpected that it activates growth signals in TT cells. Unlike ERK, basal p38 MAPK activity was lower and did not differ between groups, suggesting that ERK may be the dominant MAPK signal in proliferating TT cells. However, the mild decrease in ERK activity induced by PD098059 at the early time point suggests cross-talk between p38 and ERK pathways. Such a mechanism has been previously suggested in a model of corneal wound healing (47) but remains poorly defined.

The net effect of alterations in proliferation and cell death are reflected in changes in cell number. Given the observation that PP2 decreased TT cell counts, we examined the potential mechanisms of DNA synthesis and apoptosis. PP2 caused profound decreases in DNA synthesis and increased apoptosis. Marked TT cell cycle arrest was observed in the G0/G1 phase upon PP2 exposure. Thus, Src effects appear to involve both apoptosis and the cell cycle. Indeed, c-Src is a recognized mediator of growth factor-induced DNA synthesis in many cell types (9, 48, 49). Touyz et al. (9) clearly showed an important role of the Src to ERK pathway in angiotensin II-mediated DNA and protein synthesis in vascular smooth muscle cells. However, the majority of the effect of PP2 on cell number could not be attributed to its effect on ERK-dependent proliferation. PP2 also caused a dramatic increase in cell death, thus explaining the very low cell counts observed in cells exposed to PP2 compared with genistein or PD098059. In fact, Src activation has been shown to render colon cancer cell lines resistant to anoikis (15, 50). Together, these observations suggest that c-Src delays or prevents cellular apoptosis; thus Src inactivation may be a potential target to increase MTC cell death.

Such effects on survival may be accomplished by decreased apoptosis. Indeed, the observation that PP2 prevented constitutive Akt phosphorylation is highly suggestive that Src-mediated TT cell proliferation occurs through the interruption of survival signals involving the Akt pathway. In fact, c-Src has been shown to prolong survival by activation of the phosphatidylinositol-3 kinase pathway, a known regulator of Akt activity (36). The observation that ERK inhibition had no effect on Akt phosphorylation is in concordance with the minimal effect of PD098059 on cellular apoptosis. These observations indicate that TT cell proliferation is mainly dependent on c-Src activity. Inhibition of c-Src markedly diminishes cellular proliferation by reducing DNA synthesis and increasing apoptosis. The latter effect appears to be dependent on the activation of the Akt survival pathway. ERK activation, although downstream of Src, appears to play a lesser role in TT cell growth signaling.

We conclude that Src family tyrosine kinases regulate MTC cellular proliferation in vitro. c-Src appears to mediate growth signals by increasing DNA synthesis and decreasing apoptosis (Fig. 10Go). One potential clinical application of tyrosine kinase inhibitors would be as postsurgical therapy in patients with large or locally advanced MTCs. Although these observations require in vivo confirmation of efficacy before clinical application, c-Src may be a potential target for modulating growth signals in MTCs with RET mutations.



View larger version (14K):
[in this window]
[in a new window]
  		FIG. 10. Our proposed schematic for the activity of PP2.

 


   Footnotes
 
This work was supported in part by a Robert Wood Johnson Minority Medical Faculty Development Award and the VA Merit Review Entry Program Grant.

Abbreviations: ATF, Activating transcription factor; BrdU, bromodeoxyuridine; MEK, MAPK kinase; MEN, multiple endocrine neoplasia; MTC, medullary thyroid cancer.

Received November 6, 2003.

Accepted March 19, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Cote GJ, Wohllk N, Evans D, Goepfert H, Gagel RF 1995 RET proto-oncogene mutations in multiple endocrine neoplasia type 2 and medullary thyroid carcinoma. Baillieres Clin Endocrinol Metab 9:609–630[CrossRef][Medline]
  2. Jhiang SM 2000 The RET proto-oncogene in human cancers. Oncogene 19:5590–5597[CrossRef][Medline]
  3. Ponder BA 1999 The phenotypes associated with ret mutations in the multiple endocrine neoplasia type 2 syndrome. Cancer Res 59:1736s–1741s; discussion 1742s
  4. Santoro M, Melillo RM, Carlomagno F, Fusco A, Vecchio G 2002 Molecular mechanisms of RET activation in human cancer. Ann NY Acad Sci 963:116–121[Abstract/Free Full Text]
  5. Summy JM, Gallick GE 2003 Src family kinases in tumor progression and metastasis. Cancer Metastasis Rev 22:337–358[CrossRef][Medline]
  6. Melillo RM, Barone MV, Lupoli G, Cirafici AM, Carlomagno F, Visconti R, Matoskova B, Di Fiore PP, Vecchio G, Fusco A, Santoro M 1999 Ret-mediated mitogenesis requires Src kinase activity. Cancer Res 59:1120–1126[Abstract/Free Full Text]
  7. Nakaigawa N, Weirich G, Schmidt L, Zbar B 2000 Tumorigenesis mediated by MET mutant M1268T is inhibited by dominant- negative Src. Oncogene 19:2996–3002[CrossRef][Medline]
  8. Pandey A, Liu X, Dixon JE, Di Fiore PP, Dixit VM 1996 Direct association between the Ret receptor tyrosine kinase and the Src homology 2-containing adapter protein Grb7. J Biol Chem 271:10607–10610[Abstract/Free Full Text]
  9. Touyz RM, He G, Wu XH, Park JB, Mabrouk ME, Schiffrin EL 2001 Src is an important mediator of extracellular signal-regulated kinase 1/2-dependent growth signaling by angiotensin II in smooth muscle cells from resistance arteries of hypertensive patients. Hypertension 38:56–64[Abstract/Free Full Text]
  10. Boney CM, Sekimoto H, Gruppuso PA, Frackelton Jr AR 2001 Src family tyrosine kinases participate in insulin-like growth factor I mitogenic signaling in 3T3–L1 cells. Cell Growth Differ 12:379–386[Abstract/Free Full Text]
  11. Zhao WQ, Alkon DL, Ma W 2003 c-Src protein tyrosine kinase activity is required for muscarinic receptor-mediated DNA synthesis and neurogenesis via ERK1/2 and c-AMP-responsive element-binding protein signaling in neural precursor cells. J Neurosci Res 72:334–342[CrossRef][Medline]
  12. Hennige AM, Lammers R, Arlt D, Hoppner W, Strack V, Niederfellner G, Seif FJ, Haring HU, Kellerer M 2000 Ret oncogene signal transduction via a IRS-2/PI 3-kinase/PKB and a SHC/Grb-2 dependent pathway: possible implication for transforming activity in NIH3T3 cells. Mol Cell Endocrinol 167:69–76[CrossRef][Medline]
  13. Kato M, Takeda K, Kawamoto Y, Iwashita T, Akhand AA, Senga T, Yamamoto M, Sobue G, Hamaguchi M, Takahashi M, Nakashima I 2002 Repair by Src kinase of function-impaired RET with multiple endocrine neoplasia type 2A mutation with substitutions of tyrosines in the COOH-terminal kinase domain for phenylalanine. Cancer Res 62:2414–2422[Abstract/Free Full Text]
  14. Karni R, Levitzki A 2000 pp60(cSrc) is a caspase-3 substrate and is essential for the transformed phenotype of A431 cells. Mol Cell Biol Res Commun 3:98–104[CrossRef][Medline]
  15. Boudny V, Nakano S 2003 Src tyrosine kinase but not activated Ras augments sensitivity to taxanes through apoptosis in human adenocarcinoma cells. Anticancer Res 23:7–12[Medline]
  16. Carlomagno F, Salvatore D, Santoro M, de Franciscis V, Quadro L, Panariello L, Colantuoni V, Fusco A 1995 Point mutation of the RET proto-oncogene in the TT human medullary thyroid carcinoma cell line. Biochem Biophys Res Commun 207:1022–1028[CrossRef][Medline]
  17. Hanke JH, Gardner JP, Dow RL, Changelian PS, Brissette WH, Weringer EJ, Pollok BA, Connelly PA 1996 Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor. J Biol Chem 271:695–701[Abstract/Free Full Text]
  18. Spinozzi F, Pagliacci MC, Migliorati G, Moraca R, Grignani F, Riccardi C, Nicoletti I 1994 The natural tyrosine kinase inhibitor genistein produces cell cycle arrest and apoptosis in Jurkat T-leukemia cells. Leuk Res 18:431–439[CrossRef][Medline]
  19. Migita K, Eguchi K, Kawabe Y, Mizokami A, Tsukada T, Nagataki S 1994 Prevention of anti-CD3 monoclonal antibody-induced thymic apoptosis by protein tyrosine kinase inhibitors. J Immunol 153:3457–3465[Abstract]
  20. Niwa K, Inanami O, Ohta T, Ito S, Karino T, Kuwabara M 2001 p38 MAPK and Ca2+ contribute to hydrogen peroxide-induced increase of permeability in vascular endothelial cells but ERK does not. Free Radic Res 35:519–527[CrossRef][Medline]
  21. Wheeler-Jones CP, May MJ, Houliston RA, Pearson JD 1996 Inhibition of MAP kinase kinase (MEK) blocks endothelial PGI2 release but has no effect on von Willebrand factor secretion or E-selectin expression. FEBS Lett 388:180–184[CrossRef][Medline]
  22. Eigelberger MS, Wong MG, Duh QY, Clark OH 2001 Phenylacetate enhances the antiproliferative effect of retinoic acid in follicular thyroid cancer. Surgery 130:931–935[CrossRef][Medline]
  23. Kebebew E, Wong MG, Siperstein AE, Duh QY, Clark OH 1999 Phenylacetate inhibits growth and vascular endothelial growth factor secretion in human thyroid carcinoma cells and modulates their differentiated function. J Clin Endocrinol Metab 84:2840–2847[Abstract/Free Full Text]
  24. Persad S, Attwell S, Gray V, Mawji N, Deng JT, Leung D, Yan J, Sanghera J, Walsh MP, Dedhar S 2001 Regulation of protein kinase B/Akt-serine 473 phosphorylation by integrin-linked kinase: critical roles for kinase activity and amino acids arginine 211 and serine 343. J Biol Chem 276:27462–27469[Abstract/Free Full Text]
  25. Gruning T, Tiepolt C, Zophel K, Bredow J, Kropp J, Franke WG 2003 Retinoic acid for redifferentiation of thyroid cancer: does it hold its promise? Eur J Endocrinol 148:395–402[Abstract]
  26. Schmutzler C, Kohrle J 2000 Retinoic acid redifferentiation therapy for thyroid cancer. Thyroid 10:393–406[Medline]
  27. Fusco A, Santoro M, Grieco M, Carlomagno F, Dathan N, Fabien N, Berlingieri MT, Li Z, De Franciscis V, Salvatore D 1995 RET/PTC activation in human thyroid carcinomas. J Endocrinol Invest 18:127–129[Medline]
  28. Cetta F, Chiappetta G, Melillo, RM, Petracci M, Montalto G, Santoro M, Fusco A 1998 The ret/ptc1 oncogene is activated in familial adenomatous polyposis-associated thyroid papillary carcinomas. J Clin Endocrinol Metab 83:1003–1006[Abstract/Free Full Text]
  29. Santoro M, Grieco M, Melillo RM, Fusco A, Vecchio G 1995 Molecular defects in thyroid carcinomas: role of the RET oncogene in thyroid neoplastic transformation. Eur J Endocrinol 133:513–522[Abstract]
  30. Wohllk N, Cote GJ, Bugalho MM, Ordonez N, Evans DB, Goepfert H, Khorana S, Schultz P, Richards CS, Gagel RF 1996 Relevance of RET proto-oncogene mutations in sporadic medullary thyroid carcinoma. J Clin Endocrinol Metab 81:3740–3745[Abstract]
  31. Fabien N, Paulin C, Santoro M, Berger N, Grieco M, Galvain D, Barbier Y, Dubois PM, Fusco A 1992 Detection of RET oncogene activation in human papillary thyroid carcinomas by in situ hybridisation. Br J Cancer 66:1094–1098[Medline]
  32. Barone MV, Sepe L, Melillo RM, Mineo A, Santelli G, Monaco C, Castellone MD, Tramontano D, Fusco A, Santoro M 2001 RET/PTC1 oncogene signaling in PC Cl 3 thyroid cells requires the small GTP-binding protein Rho. Oncogene 20:6973–6982[CrossRef][Medline]
  33. Arighi E, Alberti L, Torriti F, Ghizzoni S, Rizzetti MG, Pelicci G, Pasini B, Bongarzone I, Piutti C, Pierotti MA, Borrello MG 1997 Identification of Shc docking site on Ret tyrosine kinase. Oncogene 14:773–782[CrossRef][Medline]
  34. Bongarzone I, Pierotti MA, Monzini N, Mondellini P, Manenti G, Donghi R, Pilotti S, Grieco M, Santoro M, Fusco A, Vecchio G, Della Porta G1989 High frequency of activation of tyrosine kinase oncogenes in human papillary thyroid carcinoma. Oncogene 4:1457–1462
  35. Durick K, Yao VJ, Borrello MG, Bongarzone, I., Pierotti MA, Taylor SS 1995 Tyrosines outside the kinase core and dimerization are required for the mitogenic activity of RET/ptc2. J Biol Chem 270:24642–24645[Abstract/Free Full Text]
  36. Encinas M, Tansey MG, Tsui-Pierchala BA, Comella JX, Milbrandt J, Johnson Jr EM 2001 c-Src is required for glial cell line-derived neurotrophic factor (GDNF) family ligand-mediated neuronal survival via a phosphatidylinositol-3 kinase (PI-3K)-dependent pathway. J Neurosci 21:1464–1472[Abstract/Free Full Text]
  37. Poteryaev D, Titievsky A, Sun YF, Thomas-Crusells J, Lindahl M, Billaud M, Arumae U, Saarma M 1999 GDNF triggers a novel ret-independent Src kinase family-coupled signaling via a GPI-linked GDNF receptor {alpha}1. FEBS Lett 463:63–66[CrossRef][Medline]
  38. Bonacchi A, Romagnani P, Romanelli RG, Efsen E, Annunziato F, Lasagni L, Francalanci M, Serio M, Laffi G, Pinzani M, Gentilini P, Marra F 2001 Signal transduction by the chemokine receptor CXCR3: activation of Ras/ERK, Src, and phosphatidylinositol 3-kinase/Akt controls cell migration and proliferation in human vascular pericytes. J Biol Chem 276:9945–9954[Abstract/Free Full Text]
  39. Blake RA, Broome MA, Liu X, Wu J, Gishizky M, Sun L, Courtneidge SA 2000 SU6656, a selective Src family kinase inhibitor, used to probe growth factor signaling. Mol Cell Biol 20:9018–9027[Abstract/Free Full Text]
  40. D’Alessio A, Califano D, Incoronato M, Santelli G, Florio T, Schettini G, Carlomagno MS, Cerchia L, de Franciscis V 2003 The tyrosine phosphatase Shp-2 mediates intracellular signaling initiated by Ret mutants. Endocrinology 144:4298–4305[Abstract/Free Full Text]
  41. Murakami H, Iwashita T, Asai N, Shimono Y, Iwata Y, Kawai K, Takahashi M 1999 Enhanced phosphatidylinositol 3-kinase activity and high phosphorylation state of its downstream signalling molecules mediated by ret with the MEN 2B mutation. Biochem Biophys Res Commun 262:68–75[CrossRef][Medline]
  42. Mograbi B, Bocciardi R, Bourget I, Busca R, Rochet N, Farahi-Far D, Juhel T, Rossi B 2001 Glial cell line-derived neurotrophic factor-stimulated phosphatidylinositol 3-kinase and Akt activities exert opposing effects on the ERK pathway: importance for the rescue of neuroectodermic cells. J Biol Chem 276:45307–45319[Abstract/Free Full Text]
  43. van Weering DH, Medema JP, van Puijenbroek A, Burgering BM, Baas PD, Bos JL 1995 Ret receptor tyrosine kinase activates extracellular signal-regulated kinase 2 in SK-N-MC cells. Oncogene 11:2207–2214[Medline]
  44. Abe J, Takahashi M, Ishida M, Lee JD, Berk BC 1997 c-Src is required for oxidative stress-mediated activation of big mitogen-activated protein kinase 1. J Biol Chem 272:20389–20394[Abstract/Free Full Text]
  45. Klint P, Kanda S, Kloog Y, Claesson-Welsh L 1999 Contribution of Src and Ras pathways in FGF-2 induced endothelial cell differentiation. Oncogene 18:3354–3364[CrossRef][Medline]
  46. Yen A, Roberson MS, Varvayanis S, Lee AT 1998 Retinoic acid induced mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase-dependent MAP kinase activation needed to elicit HL-60 cell differentiation and growth arrest. Cancer Res 58:3163–3172[Abstract/Free Full Text]
  47. Kumar A, Middleton A, Chambers TC, Mehta KD 1998 Differential roles of extracellular signal-regulated kinase-1/2 and p38(MAPK) in interleukin-1ß- and tumor necrosis factor-{alpha}-induced low density lipoprotein receptor expression in HepG2 cells. J Biol Chem 273:15742–15748[Abstract/Free Full Text]
  48. Roche S, Koegl M, Barone MV, Roussel MF, Courtneidge SA 1995 DNA synthesis induced by some but not all growth factors requires Src family protein tyrosine kinases. Mol Cell Biol 15:1102–1109[Abstract]
  49. Taylor SJ, Shalloway D 1996 Src and the control of cell division. Bioessays 18:9–11[CrossRef][Medline]
  50. Windham TC, Parikh NU, Siwak DR, Summy JM, McConkey DJ, Kraker AJ, Gallick GE 2002 Src activation regulates anoikis in human colon tumor cell lines. Oncogene 21:7797–7807[CrossRef][Medline]



This article has been cited by other articles:


Home page
 FASEB J.Home page
A. Spreafico, S. Schenone, T. Serchi, M. Orlandini, A. Angelucci, D. Magrini, G. Bernardini, G. Collodel, A. Di Stefano, C. Tintori, et al.
Antiproliferative and proapoptotic activities of new pyrazolo[3,4-d]pyrimidine derivative Src kinase inhibitors in human osteosarcoma cells
FASEB J, May 1, 2008; 22(5): 1560 - 1571.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
T. Chen, Y. Pengetnze, and C. C. Taylor
Src inhibition enhances paclitaxel cytotoxicity in ovarian cancer cells by caspase-9-independent activation of caspase-3
Mol. Cancer Ther., February 1, 2005; 4(2): 217 - 224.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liu, Z.
Right arrow Articles by Nwariaku, F. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, Z.
Right arrow Articles by Nwariaku, F. E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals