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Cancer Research 67, 11769, December 15, 2007. doi: 10.1158/0008-5472.CAN-07-1997
© 2007 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Cytosolic Phospholipase A2{alpha} Regulates Cell Growth in RET/PTC-Transformed Thyroid Cells

Stefania Mariggiò1, Beatrice Maria Filippi1, Cristiano Iurisci1, Luana Katia Dragani2, Valentina De Falco3, Massimo Santoro3 and Daniela Corda1

1 Department of Cell Biology and Oncology and 2 Environmental Sciences Center, Consorzio Mario Negri Sud, Santa Maria Imbaro, Italy and 3 Istituto di Endocrinologia ed Oncologia Sperimentale del CNR "G. Salvatore," c/o Dipartimento di Biologia e Patologia Cellulare e Molecolare, University "Federico II," Naples, Italy

Requests for reprints: Stefania Mariggiò or Daniela Corda, Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Via Nazionale 8/A, 66030 Santa Maria Imbaro (Chieti), Italy. Phone: 39-0872-570338; Fax: 39-0872-570412; E-mail: mariggio{at}negrisud.it or corda{at}negrisud.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Modulation of cytosolic phospholipase A2 (PLA2) expression levels and production of its metabolites have been reported in several tumor types, indicating involvement of arachidonic acid and its derivatives in tumorigenesis. Following our demonstration that the PLA2 group IV isoform {alpha} (PLA2IV{alpha}) controls TSH-independent growth of normal thyroid (PCCl3) cells, we have investigated the mitogenic role of PLA2IV{alpha} in rat thyroid cells transformed by the RET/PTC oncogenes (PC-PTC cells). We now report that PLA2IV{alpha} acts downstream of the RET/PTC oncogenes in a novel pathway controlling RET-dependent cell proliferation. In addition, we show that PLA2IV{alpha} is in its phosphorylated/active form not only in RET/PTC-transformed cells and in cells derived from human papillary carcinomas but also in lysates from tumor tissues, thus relating constitutive activation of PLA2IV{alpha} to RET/PTC-dependent tumorigenesis. Moreover, p38 stress-activated protein kinase is the downstream effector of RET/PTC that is responsible for PLA2IV{alpha} phosphorylation and activity. In summary, our data elucidate a novel mechanism in the control of thyroid tumor cell growth that is induced by the RET/PTC oncogenes and which is distinguishable from that of other oncogenes, such as BRAF. This mechanism is mediated by PLA2IV{alpha} and should be amenable to targeted pharmacologic intervention. [Cancer Res 2007;67(24):11769–78]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The different forms of phospholipase A2 (PLA2) thus far identified are all recognized regulators of cell function, whereby their phospholipid-hydrolyzing activities lead to formation of lipid metabolites that are directly involved in processes such as inflammation, apoptosis, proliferation, and carcinogenesis (1). Activation of the PLA2s results in formation of two main products: free fatty acids (with arachidonic acid the most common in mammals; ref. 2) and lysolipids. These products can lead to similar activities in different cell systems. However, the localization of the different PLA2 isoforms and their modes of activation, substrate specificities, and relative abundances, as well as the coordinated activation of arachidonic-acid–metabolizing enzymes, such as the cyclooxygenases (COX), lipooxygenases (LOX), and cytochrome P450, all determine the final, highly specific activities of the PLA2s (2–4). Accordingly, different secretory and cytosolic isoforms of PLA2 serve different functions, whereby some behave mainly as housekeeping enzymes devoted to phospholipid remodeling and others are rapid responders to receptor activation and cytosolic calcium modulation (4, 5).

We have recently shown that PLA2 group IV isoform {alpha} (PLA2IV{alpha}) controls TSH-independent cell proliferation in PCCl3 rat thyroid epithelial cells (6). This pathway is regulated by adrenergic and purinergic receptors, and most importantly, it leads to preferential hydrolysis of phosphatidylinositol to form lysophosphatidylinositol, glycerophosphoinositol (GroPIns), and arachidonic acid. PLA2IV{alpha} activation is downstream of the receptor-activated extracellular signal-regulated kinase (ERK) 1/2 mitogen-activated protein kinases (MAPK) and p38 stress-activated protein kinase (SAPK), and the phosphoinositide metabolites that promote cell proliferation are arachidonic acid and GroPIns [as shown by specific inhibitors, RNA interference (RNAi), and a dominant-negative PLA2IV{alpha}1-522; ref. 6]. Although present, the mitogenic role of PLA2IV{alpha} in normal thyroid tissue is minor compared with the known modulation by physiologic concentrations of TSH, which is mediated via the adenylyl cyclase/cyclic AMP (cAMP) cascade (7). However, in a different context, such as oncogenic cell transformation that results in loss of the TSH/cAMP control in the thyroid, the role of PLA2IV{alpha} might predominate. In support of this, a PLA2 activity associated with Ras-induced transformation has previously been reported in thyroid cells (8); in addition, this activity is specific for the phosphoinositides and can be detected by increases in the glycerophosphoinositols (6, 8–12).

To directly investigate the role of PLA2IV{alpha} in thyroid tumor growth, we have used thyroid cells transformed by the RET/PTC oncogenes: PC-PTC cells (13). The RET proto-oncogene encodes a tyrosine kinase receptor for growth factors of the glial-derived neurotrophic factor family (14). RET activation results in phosphorylation of 12 tyrosine residues in its cytoplasmic tail, which initiates multiple signaling cascades that involve phospholipase C{gamma}, c-Src, and adaptors such as the Shc proteins, IRS1/2, FRS2, and DOK1/4/5 (15). Germ-line point mutations convert RET into a dominantly transforming oncogene in multiple endocrine neoplasia 2A and 2B and in familial medullary thyroid carcinoma, whereas chromosomal aberrations cause recombination of the intracellular kinase-encoding domain of RET with heterologous genes, generating the constitutively active RET/PTC oncogene chimeras that are responsible for papillary thyroid carcinomas (PTC; refs. 16, 17).

Although to date there is no evidence that a PLA2 is part of RET signaling, the activation of Ras and Ras/ERK (by the Shc-Grb2-Sos complex; refs. 15, 16) is suggestive of such an involvement because it is known that, together with other kinases, such as c-Jun NH2-terminal kinase (JNK) and p38 SAPK, ERK1/2 MAPKs can also promote phosphorylation and activation of PLA2, which could initiate a mitogenic pathway parallel to that in normal thyroid cells (6).

We have analyzed this possibility and now show that PLA2IV{alpha} and arachidonic acid are indeed major regulators of cell proliferation in RET/PTC-transformed cells. This pathway is also active in human papillary carcinoma cells and in thyroid tumor tissues, indicating an essential role for PLA2IV{alpha} in promoting the mitogenic activity of this oncogene.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and antibodies. RPMI 1640 and Medium 199 were from Life Technologies. The anti-phosphorylated MAPK-activated protein (MAPKAP) kinase-2 (Thr222) antibody was from Cell Signaling Technology, the anti-MAPKAP kinase-2 antibody was from Upstate, and the anti-actin antibody was from Sigma. Pyrrophenone and ZD6474 were kindly provided by Dr. K. Seno (Shionogi Research Laboratories, Osaka, Japan; ref. 18) and AstraZeneca Pharmaceuticals (19), respectively. SP600125 was from Calbiochem. All other cell culture reagents, antibodies, and inhibitors were obtained as in ref. 6.

Cell lines and RNAi. PCCl3 rat thyroid cells were grown in Coon's modified Ham's F12 medium, 5% calf serum, and a six-hormone mix (6H), including TSH (7, 20). The RET/PTC3- and RET/PTC1-transformed PCCl3 cells (PC-PTC3 and PC-PTC1 cells, respectively) and the BRAF-transformed PCCl3 cells (PC-BRAF cells) were grown under the same conditions but in the absence of hormones (13). The BHP10-3 and TPC1 cells were grown as described previously, 10% fetal bovine serum in RPMI 1640 and DMEM, respectively (21, 22). Although BHP10-3 and TPC1 cells might have originally derived from the same cell population,4 they display distinct phenotypes perhaps due to in vitro evolution (morphology, growth rate, RET/PTC1 protein levels, and PLA2IV{alpha} phosphorylation levels; see [3H]thymidine incorporation and Results).

Sequence-specific PLA2IV{alpha} silencing in rat PC-PTC3 cells was as described previously (6) using an electroporated small interfering RNA (siRNA; ref. 23), with a siCONTROL nontargeting duplex (Dharmacon, Inc.) as control. For sequence-specific silencing in human BHP10-3 cells, the human PLA2IV{alpha} siGENOME SMARTpool siRNAs (Dharmacon) and the nontargeting siCONTROL duplex (Dharmacon) were used with Oligofectamine reagent (Invitrogen) following the manufacturer's instructions.

Analysis of [3H]inositol-containing metabolites. PC-PTC3, PCCl3, and PC-BRAF cells were labeled for 72 h in Medium 199, 1% calf serum, containing [3H]myo-inositol (5 µCi/mL), with (PCCl3 cells) or without (PC-PTC3 and PC-BRAF) 6H. Cell treatment, extraction, and high-performance liquid chromatography (HPLC) separation of [3H]inositol-labeled water-soluble metabolites were as described previously (24, 25). HPLC cpm are expressed as percentages of their individual controls for calculations of means in figures.

[3H]Thymidine incorporation. PC-PTC3 and PCCl3 cells were starved for 48 h in Coon's modified Ham's F12 medium with 0.3% bovine serum albumin and 2 mmol/L L-glutamine, with treatments and subsequent [3H]thymidine incorporation in the same medium (for PCCl3 cells, hormones were added, except for TSH) as described previously (6). In nonstimulated cells and across the full experimental data, the total [3H]thymidine incorporation levels under these conditions were 9,787 ± 1,522 cpm/well for PC-PTC3 cells and 4,258 ± 1,088 cpm/well for PCCl3 cells.

BHP10-3 and TPC1 cells were grown under normal growth conditions for 48 h and stimulated for a further 48 h in normal growth medium. [3H]Thymidine (0.5 µCi/well; Perkin-Elmer Life Sciences) was added 4 h before the end of this incubation. Incubations were ended as reported previously (6). As above, in nonstimulated cells, total [3H]thymidine incorporation levels under these conditions were 85,664 ± 9,927 cpm/well for BHP10-3 cells and 21,780 ± 1,563 cpm/well for TPC1 cells. Growth rates for BHP10-3 cells under RNAi were evaluated by blinded cell counting in a Neubauer chamber.

Sample preparation for LC-MS/MS. Cells were grown to near confluence in their respective medium and then detached, washed, and resuspended in 0.9% NaCl solution. Samples were used to measure relative cell volumes in comparison with PCCl3 cells by flow cytometry. The PCCl3 cell volume was taken as 0.97 ± 0.03 pL (12).

Parallel cell sample had 6 mL methanol/ 1 mol/L HCl (1:1; –20°C) added per 100-mm petri dish, with 5.5 mL chloroform added following cell scraping; this provided a two-phase acid extraction (24). The upper (aqueous) phase was lyophilized and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS; ref. 26). Each cell line was assessed in triplicate from at least three independent Petri dishes of each cell type.

Immunoblotting, intracellular cAMP, and [3H]arachidonic acid release. These were performed as reported previously (6, 27). Frozen thyroid tissue samples from patients affected by PTCs, assessed and processed as described (13), were obtained from Prof. F. Basolo (Department of Surgery, Division of Anatomic Pathology, University of Pisa, Pisa, Italy).

Statistical analysis. The data are presented as mean ± SE from at least three independent experiments. Statistical analysis was by the Mann-Whitney U test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PLA2IV{alpha} is involved in the control of cell proliferation in RET/PTC3-transformed cells. Following our recent demonstration that receptor-activated PLA2IV{alpha} is involved in TSH-independent (cAMP-independent) proliferation of PCCl3 thyroid cells (6), we investigated the role of PLA2IV{alpha} in growth control of transformed thyroid cells (13, 17, 20).

[3H]Thymidine incorporation was evaluated in thyroid cells transformed by the chimeric RET/PTC3 oncogene (PC-PTC3 cells; Fig. 1A ; ref. 13). The specific PLA2IV{alpha} inhibitor pyrrophenone (0.1–0.5 µmol/L, 48 h) was used to assess involvement of PLA2IV{alpha} in control of cell growth; pyrrophenone significantly inhibited basal PC-PTC3 cell proliferation at concentrations as low as 0.1 µmol/L. As shown in Fig. 1A, basal cell proliferation of normal PCCl3 cells was also inhibited by pyrrophenone (0.5 µmol/L), although to a lesser extent. As previously shown (28), the RET kinase inhibitor ZD6474 (1 µmol/L, 16 h) also reduced basal [3H]thymidine incorporation in transformed PC-PTC3 cells (Fig. 1A). These data indicate that PLA2IV{alpha}-dependent signaling is involved in growth stimulation induced by expression of the RET/PTC3 oncogene.


Figure 1
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  		Figure 1. Functional role of PLA2IV{alpha} in PC-PTC3 and PCCl3 cells. A, [3H]thymidine incorporation (see Materials and Methods) in PC-PTC3 and PCCl3 cells in medium deprived of serum (Ctrl) and with pyrrophenone (48 h; 0.1, 0.3, and 0.5 µmol/L) and ZD6474 (16 h; 1 µmol/L) treatment. Columns, mean percentages of their respective controls (PC-PTC3/PCCl3 cells) of three independent experiments, each performed in quadruplicate; bars, SE. Basal [3H]thymidine incorporation levels were 9,787 ± 1,522 and 4,258 ± 1,088 cpm/well for PC-PTC3 and PCCl3 cells, respectively. *, P < 0.02, two-tailed Mann-Whitney U test, significantly different from their respective controls; **, P < 0.0002, two-tailed Mann-Whitney U test, significantly different from their respective controls. B, representative Western blot of endogenously expressed PLA2IV{alpha} (6). PC-PTC3 cell lysate (100 µg) was compared with indicated amounts of PCCl3 cell lysates following separation by 10% SDS-PAGE. C, representative Western blot showing different levels of PLA2IV{alpha} phosphorylation with the indicated treatments, by gel shift, following separation by 8% SDS-PAGE analysis (6) in the two cell lines. Top and bottom, different exposure times of the same blot. These blots are representative of at least six independent experiments. p-PLA2IV{alpha}, phosphorylated PLA2IV{alpha}; M, molecular weight (kDa) marker.

 
Next, the expression and activation levels of PLA2IV{alpha} in PC-PTC3 cells were evaluated. Western blotting using a specific anti-PLA2IV{alpha} antibody [anti-cytosolic PLA2 (cPLA2; N-216); ref. 6] revealed that PLA2IV{alpha} expression levels in PC-PTC3 cells were lower than normal cells (~10-fold; Fig. 1B). Surprisingly, however, in these PC-PTC3, RET/PTC3-transformed cells, PLA2IV{alpha} was in its phosphorylated, and hence active, state (Fig. 1C). To analyze the relationships between PLA2IV{alpha} phosphorylation and RET/PTC3 transformation, we again used the RET kinase inhibitor ZD6474 (28). At 1 µmol/L ZD6474, PLA2IV{alpha} phosphorylation was reduced by 20% after 10 min (data not shown), by 40% to 50% after 30 min (Fig. 1C), and by >90% after 2 h of treatment (data not shown). This inhibition was still evident after 24 h (~30%; 1 µmol/L ZD6474; Fig. 1C).

The activity of phosphorylated PLA2IV{alpha} was then analyzed. Following the observation that, in normal PCCl3 cells, PLA2IV{alpha} preferentially hydrolyzes membrane phosphoinositides, thus releasing glycerophosphoinositols and arachidonic acid (see Introduction and Materials and Methods; ref. 6), formation of these phosphatidylinositol metabolites was measured in cells incubated with radiolabeled inositol and arachidonic acid, respectively (see Materials and Methods). First, basal levels of GroPIns and arachidonic acid release were analyzed: these were higher in PC-PTC3 cells compared with PCCl3 cells (Fig. 2A ). Then, stimulation of purinergic receptors (known to be present in PCCl3 cells; ref. 6) was followed by addition of the agonist ATP (100 µmol/L): this significantly increased intracellular levels of GroPIns (Fig. 2B) and release of arachidonic acid (Fig. 2C) in both PCCl3 and PC-PTC3 cells. However, whereas the increase in GroPIns was completely blocked by the specific PLA2IV{alpha} inhibitor pyrrophenone (0.5 µmol/L; Fig. 2B; ref. 18), arachidonic acid release was inhibited only in part (Fig. 2C). Thus, this pyrrophenone-sensitive PLA2IV{alpha} is active in both normal and RET/PTC3-transformed cells, and arachidonic acid, but not GroPIns, can also be released by other PLA2 isoforms.


Figure 2
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  		Figure 2. Basal and ATP-induced production of GroPIns and arachidonic acid in PC-PTC3, PCCl3, and PC-BRAF cells. A, [3H]GroPIns levels (left) and [3H]arachidonic acid release (right) were measured under basal conditions (see Materials and Methods). Columns, mean of at least four independent experiments, each performed in duplicate; bars, SE. **, P < 0.002, two-tailed Mann-Whitney U test, significantly different from PC-PTC3 cells. B and C, cells preincubated for 15 min without (–) and with (+) pyrrophenone (Pyr.; 0.5 µmol/L) and then treated without (–) and with (+) ATP (100 µmol/L) for an additional 15 min (see Materials and Methods). Columns, mean percentages of their respective controls within each cell type of three independent experiments, each performed in duplicate; bars, SE. B, stimulation of [3H]GroPIns production is expressed as percentages of untreated cells. C, stimulation of [3H]arachidonic acid release is expressed as percentages of untreated cells. All of the increases shown in (B) and (C) are statistically significant compared with their respective controls (P < 0.02, two-tailed Mann-Whitney U test).

 
In summary, in PC-PTC3 cells, PLA2IV{alpha} is phosphorylated and thus active under basal conditions; it can be further activated on agonist stimulation, probably as a consequence of increases in Ca2+, an essential cofactor of PLA2IV{alpha} activity. In addition, GroPIns levels can be used to specifically monitor PLA2IV{alpha} enzymatic activity.

In parallel experiments, intracellular GroPIns concentrations were also quantitatively determined by LC-MS/MS (see Materials and Methods). Under normal growth conditions, the GroPIns concentration was 420 ± 20 µmol/L in PC-PTC3–transformed cells and 270 ± 20 µmol/L in PCCl3 cells. Therefore, RET/PTC3-induced cell transformation is associated with an increase in PLA2 activity that leads to increased levels of GroPIns, as has also been reported for Ras-transformed cells (9, 12).

In addition, because activating point mutations of the BRAF serine-threonine kinase are frequent genetic events in PTCs (29), the same assays were performed in a PCCl3 cell line transformed by the BRAF oncogene (BRAF V600E; ref. 13). In these PC-BRAF cells, both basal (Fig. 2A) and ATP-stimulated (Fig. 2B) GroPIns levels were lower than in normal PCCl3 cells. ATP-stimulated release of arachidonic acid was also less pronounced than in PCCl3 cells relative to their controls; however, similar to PCCl3 cells, this was not fully inhibited by pyrrophenone in PC-BRAF cells (Fig. 2C), indicating that this specific PLA2IV{alpha} isoform is not downstream of the BRAF oncogene.

To further analyze the relationships between RET/PTC3 expression and PLA2IV{alpha} activity, we monitored the specific formation of GroPIns. After metabolic [3H] inositol labeling (see Materials and Methods), PC-PTC3 cells were exposed to the RET kinase inhibitor ZD6474 (1 µmol/L for 24 h). Here, basal intracellular GroPIns levels significantly decreased by ~30% while remaining unchanged in normal PCCl3 cells (Fig. 3A, left ). This was also confirmed by mass spectrometry: GroPIns intracellular concentrations were reduced by ~45% after a similar 24-h treatment with ZD6474 in PC-PTC3 cells. These data are consistent with increased activity of PLA2IV{alpha} in PC-PTC3 cells that correlates with expression of the RET/PTC3 oncogene, indicating that PLA2IV{alpha} is an important component of the signaling cascade downstream of this oncogene.


Figure 3
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  		Figure 3. PLA2IV{alpha} is basally activated in RET/PTC-transformed cells through p38 SAPK-mediated phosphorylation. A, PC-PTC3 and PCCl3 cells were incubated with 1 µmol/L ZD6474 for 24 h (left) and 0.1 µmol/L pyrrophenone (right) in the [3H]inositol labeling medium (see Materials and Methods). Columns, basal [3H]GroPIns levels are mean percentages of their respective controls (PC-PTC3/PCCl3 cells) of three independent experiments, each performed in duplicate; bars, SE. The untreated, control cells showed basal [3H]GroPIns levels of 7,967 ± 965 and 4,696 ± 559 cpm/well for PC-PTC3 and PCCl3 cells, respectively. *, P < 0.02, two-tailed Mann-Whitney U test, significant difference between samples. B, representative Western blots (left) following separation by 10% SDS-PAGE of siRNA-treated PC-PTC3 cells showing a decrease of PLA2IV{alpha} in PLA2IV{alpha}-siRNA duplex-treated cells, whereas actin levels were not significantly affected compared with siCONTROL-treated cells (see Materials and Methods). Basal [3H]GroPIns levels (right) in PC-PTC3 cells treated with siCONTROL and PLA2IV{alpha}-siRNA duplexes. Columns, mean percentages of siCONTROL cells of two independent experiments, each performed in triplicate; bars, SE. **, P < 0.002, two-tailed Mann-Whitney U test, significant difference between samples. C, SB203580 inhibition of p38 SAPK, MAPKAP kinase-2, and PLA2IV{alpha} phosphorylation levels in PC-PTC3 and PCCl3 cells. Cells were grown in the absence or presence of 25 µmol/L SB203580 for 24 h (top left and right) or 2 h (bottom left) in normal growth medium and then lysed (see Materials and Methods). Left, representative Western blots following separation of 100 µg cell lysates by 10% SDS-PAGE, showing phosphorylation levels [phosphorylated p38 SAPK (p-p38 SAPK) and phosphorylated MAPKAP kinase-2 (p-MAPKAP kinase-2)] and total levels of p38 SAPK (p38 SAPK) and MAPKAP kinase-2 (MAPKAP kinase-2); right, representative Western blot following separation of 70 µg cell lysates by 8% SDS-PAGE, showing different exposure of the same blot. All blots are representative of at least three independent experiments. D, representative Western blot (top) following separation of PC-PTC1 (100 µg) and PCCl3 (25 µg) cell lysates by 8% SDS-PAGE, showing complete phosphorylation of PLA2IV{alpha} in PC-PTC1 cells. Middle and bottom, representative Western blots following separation of 100 µg cell lysates by 10% SDS-PAGE, showing phosphorylated p38 SAPK and p38 SAPK levels and indicating that p38 SAPK is basally activated in RET/PTC1-transformed cells.

 
To further support this, we used both pharmacologic and molecular approaches. Treatment with pyrrophenone at a concentration specific for PLA2IV{alpha} (0.1 µmol/L; ref. 18) inhibited basal, intracellular GroPIns levels in PC-PTC3 cells by up to 50% (and by up to 30% in PCCl3 cells; Fig. 3A, right). Inhibitors known to specifically act on secretory PLA2 (50 µmol/L SPLA2IIA inhibitor-I; 5 h; ref. 30) and on Ca2+-independent PLA2 [1 µmol/L bromoenol lactone (BEL); 5 h; ref. 31] also had no effects on basal GroPIns levels in PC-PTC3 cells (Table 1 ).


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  		Table 1. Modulation of basal GroPIns levels in PC-PTC3 and PCCl3 cells in the presence of different PLA2 and kinase inhibitors

 
As an alternative approach, PLA2IV{alpha} was down-regulated by RNAi (by ~70%, see Materials and Methods and Fig. 3B, left). This led to a reduction in GroPIns levels of ~40% in PC-PTC3 cells compared with PC-PTC3 cells treated with nontargeting duplexes (Fig. 3B, right), in agreement with data using the specific inhibitor of PLA2IV{alpha} (Table 1; Fig. 3A, right).

Altogether, these data identify PLA2IV{alpha} as a component of the signaling pathway of the RET/PTC3 oncogene in the control of cell proliferation in PC-PTC3 thyroid cells.

Molecular cascade leading to PLA2IV{alpha} activation. The mechanisms involved in the RET/PTC3-dependent activation of PLA2IV{alpha} were then analyzed by specifically following hydrolysis of phosphoinositides and the consequent formation of GroPIns. RET/PTC3 is known to result in phosphorylation of several effectors (15). More specifically, activated RET has been shown to stimulate p38 SAPK activation (32). Under normal growth conditions, PC-PTC3 cells had an increased level of phosphorylated p38 SAPK compared with PCCl3 cells (Fig. 3C, top left).

ERK1/2 MAPKs are downstream of RET/PTC3 (33), and ERK1/2 MAPK phosphorylation of PLA2IV{alpha} at Ser505 produces an increase in enzymatic activity of this phospholipase (34). However, when PC-PTC3 cells were pretreated with the specific ERK1/2 MAPK inhibitor U0126 (30 µmol/L, 5 h), GroPIns levels were not affected (Table 1). Similarly, inhibitors of JNK (10 µmol/L SP600125) and protein kinase C (PKC; 5 µmol/L RO31-8220), two other potential regulators of PLA2IV{alpha} downstream of RET/PTC3, were ineffective (Table 1; similar data were obtained also in normal PCCl3 cells). In contrast, the phosphoinositide 3-kinase (PI3K) inhibitor wortmannin (1 µmol/L) inhibited GroPIns levels in PC-PTC3 cells by 20% (this effect was, however, not correlated to RET/PTC3 expression because similar inhibition was seen in normal PCCl3 cells; Table 1). Finally, the inhibitor of p38 SAPK (25 µmol/L SB203580), a kinase known to regulate phosphorylation of PLA2IV{alpha} (3), selectively decreased basal GroPIns levels in RET/PTC3-transformed cells (Table 1). This inhibition was evident from 5 to 48 h of SB203580 treatment, reaching a maximum of ~30% (Table 1). In PCCl3 cells, SB203580 treatment was either ineffective (5 h) or induced a significant increase in intracellular levels of GroPIns, again relating the effects reported above to RET/PTC oncogenic transformation (48 h; Table 1).

The role of p38 SAPK in this signaling cascade was confirmed by evaluating phosphorylation levels of p38 SAPK by Western blotting. As indicated above, these were higher (by 2.5-fold) in RET/PTC3-transformed cells compared with PCCl3 cells (Fig. 3C, top left). Interestingly, the specific p38 SAPK inhibitor SB203580 (25 µmol/L, 24 h) increased p38 SAPK phosphorylation (Fig. 3C, top left). This is not surprising considering that SB203580 blocks the kinase activity of p38 SAPK and not the actual phosphorylation itself (see Discussion and ref. 35). In addition, SB203580 treatment decreased the phosphorylation of the p38 SAPK substrate MAPKAP kinase-2, as seen by Western blotting with an anti-phosphorylated MAPKAP kinase-2 antibody (Fig. 3C, bottom left). This was paralleled by a decrease of up to 50% in PLA2IV{alpha} phosphorylation in RET/PTC3-transformed cells (Fig. 3C, right). In line with this, activation of PLA2IV{alpha} was also seen in cells transformed by the RET/PTC1 oncogene (PC-PTC1; Fig. 3D, top), where increased activation of p38 SAPK was seen (Fig. 3D, middle), as with PC-PTC3 cells (Fig. 3C, top left).

These data are consistent with a signaling cascade that links active RET/PTC oncogenes to p38 SAPK phosphorylation, which in turn phosphorylates and activates PLA2IV{alpha}, leading to release of phosphoinositide metabolites that have been previously shown to affect cell proliferation in normal thyroid cells (6, 32).

PLA2IV{alpha}-dependent regulation of cell proliferation in RET/PTC3-transformed cells. As with other thyroid cancer cells and transformed cell lines, PC-PTC3 cells proliferate in a TSH- and cAMP-independent manner (13). Use of the inhibitor pyrrophenone, which inhibited basal cell proliferation (see above and Fig. 1A), indicated involvement of PLA2IV{alpha} in control of cell growth in these RET/PTC3-transformed cells.

Thus, the correlation between the oncogenic RET/p38 SAPK/PLA2IV{alpha} signaling cascade that regulates GroPIns and arachidonic acid formation and cell proliferation was analyzed. The p38 SAPK inhibitor SB203580 (25 µmol/L, 48 h) significantly reduced basal [3H]thymidine incorporation in PC-PTC3 cells (Fig. 4A ), in line with effects of ZD6474 and pyrrophenone shown above (Fig. 1A). Other inhibitors of PLA2 activities, such as BEL (1 µmol/L), used at a concentration that specifically inhibits calcium-independent PLA2 (PLA2VI), did not affect [3H]thymidine incorporation, which was instead partially inhibited by specific inhibitor-I of secretory PLA2IIA (Fig. 4A).


Figure 4
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  		Figure 4. Cell growth regulation and levels of PLA2IV{alpha} phosphorylation in RET/PTC-transformed cells and tissues. A, [3H]thymidine incorporation (see Materials and Methods) during basal cell growth of PC-PTC3 cells treated with 25 µmol/L SB203580, 1 µmol/L BEL, and 25 µmol/L sPLA2IIA inhibitor-I for 48 h in medium deprived of serum (Ctrl; basal [3H]thymidine incorporation was 9,787 ± 1,522 cpm/well). Columns, mean percentages of the control of three independent experiments, each performed in quadruplicate; bars, SE. B, representative Western blots (top) following separation of 100 µg BHP10-3 and TPC1 cell lysates by 8% SDS-PAGE, showing phosphorylation levels of PLA2IV{alpha} (top) and total levels of RET/PTC3 (bottom). [3H]Thymidine incorporation (bottom graph; see Materials and Methods) during basal cell growth of BHP10-3 and TPC1 cells treated with ZD6474 (1 µmol/L, 16 h), SB203580 (25 µmol/L, 48 h), or pyrrophenone (0.5 µmol/L, 48 h) in growth medium. Columns, mean percentages of their respective controls within each cell type of three independent experiments, each performed in quadruplicate; bars, SE. The basal [3H]thymidine incorporation levels here were 85,664 ± 9,927 and 21,780 ± 1,563 cpm/well for BHP10-3 and TPC1 cells, respectively. A and B, **, P < 0.002, two-tailed Mann-Whitney U test, significantly different from their respective controls. C, representative Western blot (top) following separation of 100 µg lysate of siRNA-treated BHP10-3 cells by 10% SDS-PAGE, showing a decrease in PLA2IV{alpha} in PLA2IV{alpha}-siRNA duplex-treated cells at 72 h (representative of 48–96 h). Bottom, cell growth of siCONTROL-treated and PLA2IV{alpha}-siRNA–treated BHP10-3 cells. Points, mean percentages of cell numbers in the siCONTROL cells at 48 h of three independent experiments, each performed in quadruplicate; bars, SE. *, P < 0.02, two-tailed Mann-Whitney U test, significant difference between samples. D, densitometric analysis of Western blots of 150 µg lysates from RET/PTC-positive thyroid tumors from two different patients (A and B) with their respective controls (normal) following separation by 8% SDS-PAGE, indicating increased PLA2IV{alpha} phosphorylation in tumor tissues. The amounts of nonphosphorylated (PLA2IV{alpha}) and phosphorylated (p-PLA2IV{alpha}) enzyme are expressed as percentages of total PLA2IV{alpha} taken as 100 for each sample.

 
The results with this panel of inhibitors further support the involvement of the oncogenic RET/p38 SAPK/PLA2IV{alpha} signaling cascade in the control of cell growth in RET/PTC3-transformed cells because inhibition of each of the components hampered the cascade and in parallel prevented cell growth (Fig. 4A). The effect of inhibitor-I for secretory PLA2IIA (Fig. 4A) correlated with residual arachidonic acid release seen on pyrrophenone treatment (Fig. 2C), suggesting potential involvement of secretory PLA2IIA either downstream of (3), or independently from, PLA2IV{alpha} activity.

To further elucidate this mechanism, the products of PLA2IV{alpha} catalytic activity (i.e., GroPIns, arachidonic acid, and some arachidonic acid metabolites produced by COX activities) were added directly to growth medium under basal conditions or after inhibiting PLA2IV{alpha} with pyrrophenone (0.5 µmol/L). These data are summarized in Table 2 and indicate that, in PC-PTC3 cells, direct addition of prostaglandin E1 (PGE1; 30 nmol/L), PGE2 (30 nmol/L), and thromboxane B2 (TBXB2; 30 nmol/L) did not affect [3H]thymidine incorporation; similarly, the general COX inhibitor lysinate acetylsalicylic acid (300 µmol/L) was ineffective, excluding autocrine modulation of PC-PTC3 cell growth by this pathway (Table 2). Instead, stimulation of [3H]thymidine incorporation was seen with GroPIns (300 µmol/L) and arachidonic acid (0.1–1 µmol/L), although to different levels (115% and 132–212%, respectively). This was also evident on cell pretreatment with pyrrophenone (0.5 µmol/L), which significantly decreased [3H]thymidine incorporation (by 50%; Table 2); this decrease could be rescued partially by addition of arachidonic acid (1 µmol/L, by 44%; Table 2). Similar experiments were also performed in PCCl3 cells and indicated that arachidonic acid and GroPIns stimulate [3H]thymidine incorporation, in agreement with previously published data (Table 2; ref. 6).


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  		Table 2. Effects of different PLA2 metabolites on PC-PTC3 and PCCl3 cell growth without and with pyrrophenone treatment

 
These data are consistent with a role for RET/PTC3-activated PLA2IV{alpha} in the control of cell proliferation in PC-PTC3 cells and they identify PLA2IV{alpha} as an important effector of the mitogenic activity of the RET/PTC3 oncogene.

Role of PLA2IV{alpha} in human tumor cell proliferation. Several cell lines derived from thyroid papillary carcinomas are now available. Among these, some express activated versions of the RET/PTC1 oncogene (21, 36). In principle, in these human cancer cells, proliferation should be regulated by the oncogenic RET/p38 SAPK/PLA2IV{alpha} signaling cascade reported above. We analyzed this possibility using the BHP10-3 and TPC1 thyroid papillary carcinoma cell lines, which have been characterized for expression of the RET/PTC1 oncogene, which was some 2-fold higher in BHP10-3 cells over TPC1 cells (Fig. 4B, bottom).

These cells were incubated with inhibitors of RET, p38 SAPK, and PLA2IV{alpha} that were effective in PC-PTC3 cells to determine whether this cascade also controls tumor cell proliferation. [3H]Thymidine incorporation in both BHP10-3 and TPC1 cells was inhibited by ~70% by ZD6474 (1 µmol/L, 16 h), in line with the reported action of the RET/PTC oncogene (37). Inhibition of p38 SAPK (25 µmol/L SB203580, 48 h) also reduced cell proliferation (~40% and ~60% in BHP10-3 and TPC1 cells, respectively), and inhibition of PLA2IV{alpha} by pyrrophenone (0.5 µmol/L, 48 h) clearly reduced [3H]thymidine incorporation in BHP10-3 cells, whereas it had no significant effect in TPC1 cells (Fig. 4B, bottom). Interestingly, oncogenic RET/p38 SAPK/PLA2IV{alpha} modulation seemed more evident in BHP10-3 cells, which express higher levels of RET/PTC oncogene (~2-fold) and of phosphorylated PLA2IV{alpha} (100% versus 80% of total PLA2IV{alpha} in BHP10-3 and TPC1 cells, respectively; Fig. 4B, top). Inhibitors of arachidonic acid metabolism were also analyzed, and although the general COX inhibitor lysinate acetylsalicylic acid (300 µmol/L; 48 h) was ineffective, the inhibitor of thromboxane synthase and 5-LOX (50 µmol/L ketoconazole; 48 h) inhibited BHP10-3 cell proliferation by up to 90%, suggesting that either a thromboxane or a leukotriene is the active mitogenic metabolite.

This pharmacologic demonstration of the involvement of PLA2IV{alpha} in BHP10-3 cell growth was also confirmed with a molecular approach. PLA2IV{alpha} was knocked down by 90% by RNAi (Fig. 4C, top) and cell growth was monitored by cell counting; this was significantly inhibited in the presence of siRNAs for PLA2IV{alpha} (Fig. 4C, bottom).

The involvement of PLA2IV{alpha} in thyroid cell transformation was at this point examined in thyroid tissues obtained from surgery. PLA2IV{alpha} activation was analyzed by Western blotting of lysates from RET/PTC-positive thyroid tumors and their respective normal tissues (see Materials and Methods). In line with data reported for transformed cell lines, tumor samples also showed high levels of phosphorylated PLA2IV{alpha} (compared with nonphosphorylated PLA2IV{alpha}, which was virtually undetectable), whereas normal tissues showed comparable levels of these two PLA2IV{alpha} forms (Fig. 4D).

These data indicate that the oncogenic RET/p38 SAPK/PLA2IV{alpha} pathway described in PC-PTC3 cells is also active in these human thyroid carcinoma cells. In addition, they indicate that loss of thyroid differentiation (and associated loss of cAMP-dependent growth) can lead to an alternative pathway for cell proliferation that involves RET/PTC activation of PLA2IV{alpha}, with release of active lipid metabolites of the arachidonic acid cascade.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we report novel components of the mitogenic signaling cascade initiated by the RET/PTC oncogenes: a specific isoform of PLA2, PLA2IV{alpha}, and arachidonic-acid–derived lipid metabolites, which are shown here to be essential mediators of RET/PTC-induced thyroid tumor cell growth. More importantly, this mitogenic pathway is also relevant in human thyroid tumors characterized by oncogenic RET/PTC expression.

In human thyroid papillary carcinomas, cell proliferation is often promoted by phosphorylation cascades such as those induced by products of chimeric genes, such as RET/PTC (17, 38), or NTRAK1-derived oncogenes (39) or by rearrangements or mutations that constitutively activate the proto-oncogene BRAF, a kinase acting upstream of MAPKs (40).

Our recent finding that PLA2IV{alpha} is specifically involved in TSH-independent growth control in normal rat thyroid cells (6) led to our analysis of the role of this cascade in tumor cell growth. Quite surprisingly, PLA2IV{alpha} expression in PC-PTC3 and PC-PTC1 cells was relatively low, although PLA2IV{alpha} was fully phosphorylated (in a RET/PTC-dependent manner) and active (see Results). This is in line with a study reporting the gene expression profile in PC-PTC3 cells, which showed that, among other signaling enzymes, the PLA2IV{alpha} gene is also down-regulated 2.5-fold (13). Thus, on the one hand, expression of the RET/PTC3 oncogene results in decreased PLA2IV{alpha} gene transcription and hence lower expression of PLA2IV{alpha}, and on the other hand, it causes direct activation of PLA2IV{alpha} by stimulating its phosphorylation and thus increases production of its lipid products.

Although these two effects on protein levels and activation may seem contradictory, there is evidence that although tumorigenesis correlates with PLA2IV{alpha} activity, it does not necessarily correlate with increased PLA2 levels (41). A clear example of this is seen in the ApcMin and Apc{Delta}716 mouse models of colon tumor (42, 43), where cytosolic PLA2 expression and function are completely different within specific regions of the gut and small intestine, and in colon tumors, indicating that the tumorigenic process is driven by different pathophysiologic processes in different cells (41). The role of cytosolic PLA2 should thus be considered in a more general context that takes into account the levels and activities of the enzymes acting downstream of, and on the products of, PLA2, such as the COXs (44). In addition, besides being a substrate of COX, PLA2-derived arachidonic acid is also an active molecule per se, as it is involved in induction of apoptosis (45). For example, in mouse and human colon tumors, cPLA2 is greatly decreased at the mRNA and protein levels compared with normal tissues, whereas levels of COX-2 are increased. This leads to decreased production of arachidonic acid (due to lower PLA2 activity) coupled to increased metabolism (due to higher COX-2 activity). The final result is thus higher PGE2 levels, which enhance tumorigenesis and angiogenesis, and lower free arachidonic acid, which protects tumor cells from apoptosis (44).

As with normal PCCl3 cells, we also see that, in the PC-PTC3 cells, PLA2IV{alpha} preferentially hydrolyzes membrane phosphoinositides (6),5 which made possible this detailed study of the cascade leading from the RET/PTC3 oncogene to the specific PLA2IV{alpha} product GroPIns, through use of a panel of inhibitors. Thus, although oncogenic RET/PTC3 is clearly associated with increased phosphorylation of ERK1/2 MAPKs, p38 SAPK, MAPKAP kinase-2, and finally PLA2IV{alpha}, only when p38 SAPK phosphorylation and activation was inhibited did basal intracellular levels of GroPIns decrease, indicating that PLA2IV{alpha} is downstream of p38 SAPK phosphorylation. Consequently, thymidine incorporation by PC-PTC3 cells is also inhibited by p38 SAPK and PLA2IV{alpha} inhibitors, indicating that these enzymes act downstream of oncogenic RET/PTC in cell growth regulation (see Results).

Increased p38 SAPK phosphorylation in the presence of the inhibitor SB203580 has also been seen in other cell lines (Raw264.7 and CHO cells; ref. 45),5 and it probably arises from a block of p38 SAPK-dependent phosphatase activity, which results in increased p38 SAPK phosphorylation. Nevertheless, SB203580 inhibited p38 SAPK activity, as indicated by reduced phosphorylation of MAPKAP kinase-2.

According to the data herein reported, cell proliferation in RET/PTC3-transformed cells should be regulated by arachidonic acid or an arachidonic acid metabolite formed by COX, LOX, or cytochrome P450 activities. As the only effective inhibitor was ketoconazole, thromboxanes or leukotrienes are thus far the best candidates in this tumor cell growth (see Results).

Work is in progress to better define the specific mitogenic compound of this arachidonic acid cascade. This aspect is of particular interest because the possibility of inhibiting a specific intracellular mitogen could help to delineate a targeted therapy for thyroid and other tumors that involve PLA2IV{alpha}. In support of this proposal, we show here that the same regulatory cascade operating in rat thyroid cells regulates human thyroid tumor cell growth. Thus, oncogenic RET/PTC expression promotes thyroid cell growth (15), which can be inhibited by preventing the PLA2IV{alpha} cascade, either by specific inhibitors or by knocking down of the PLA2IV{alpha} protein by RNAi (see Results). Interestingly, although the human tumor cell lines examined respond equally well to the RET kinase inhibitor (37), they have a differential response to p38 SAPK and PLA2IV{alpha} inhibitors. This could be related to different levels of expression of RET/PTC and phosphorylated PLA2IV{alpha} in these cells (see Results). It can thus be hypothesized that RET/PTC-induced cell proliferation is activated through different pathways, the choice of which is determined by the level of expression of specific downstream effectors. Thus, Rap1 (46), PI3K/Akt (47), Ras-MAPK (48), p27Kip1 (37), phospholipase C{gamma} (49), or PLA2IV{alpha} (this study) can all be crucial mitogenic agents depending on levels of their expression/activation or concomitant expression of cofactors. It is of note, however, that the active PLA2IV{alpha} pathway in tumor tissues (see Results) further strengthens the involvement of PLA2IV{alpha} in RET/PTC-mediated tumorigenesis in vivo.

Altogether, these data further increase the complexity of signaling pathways present in thyroid tumors that can be promoted by different oncogenes linked to distinct signaling cascades. This is indeed the case for the RET/PTC and BRAF oncogenes; while activating common signaling pathways, such as the MAPK/ERK kinase–dependent cascade (40), they differ in their ability to initiate lipid signaling driven by PLA2IV{alpha}, which is peculiar of RET/PTC.

These data also lead to the proposal that thyroid and other types of tumors that are regulated by PLA2IV{alpha} signaling can be treated with already available agents that are targeted to elements of the PLA2IV{alpha} cascade. A more specific therapy could emerge from the identification of the arachidonic acid metabolite that regulates tumor growth (e.g., in thyroid or colon cancers), which would then allow the development and use of highly specific, targeted drugs.


   Acknowledgments
 
Grant support: Italian Association for Cancer Research (Milan, Italy), Telethon Italia (Italy), and Ministero dell'Università e della Ricerca (Italy). B.M. Filippi was a fellow of the Italian Foundation for Cancer Research (Milan, Italy).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank A. Luini, M. Di Girolamo, R.M. Melillo, F. Carlomagno, and C.P. Berrie for useful discussions; Dr. K. Seno for pyrrophenone; A.J. Ryan for ZD6474; J.M. Hershman for BHP10-3 cells; Prof. F. Basolo for the tumor samples; and E. Fontana for preparation of the figures.


   Footnotes
 
Note: Current address for B.M. Filippi: College of Life Sciences, University of Dundee, Dundee, Scotland.

4 J.M. Hershman, personal communication. Back

5 Unpublished results. Back

Received 5/30/07. Revised 9/19/07. Accepted 10/24/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Diaz BL, Arm JP. Phospholipase A2. Prostaglandins Leukot Essent Fatty Acids 2003;69:87–97.[CrossRef][Medline]
  2. Leslie CC. Regulation of arachidonic acid availability for eicosanoid production. Biochem Cell Biol 2004;82:1–17.[CrossRef][Medline]
  3. Kudo I, Murakami M. Phospholipase A2 enzymes. Prostaglandins Other Lipid Mediat 2002;68–9:3–58.
  4. Hirabayashi T, Shimizu T. Localization and regulation of cytosolic phospholipase A2. Biochim Biophys Acta 2000;1488:124–38.[Medline]
  5. Schaloske RH, Dennis EA. The phospholipase A2 superfamily and its group numbering system. Biochim Biophys Acta 2006;1761:1246–59.[Medline]
  6. Mariggiò S, Sebastià J, Filippi BM, et al. A novel pathway of cell growth regulation mediated by a PLA2{alpha}-derived phosphoinositide metabolite. FASEB J 2006;20:2567–9.[Abstract/Free Full Text]
  7. Kimura T, Van Keymeulen A, Golstein J, et al. Regulation of thyroid cell proliferation by TSH and other factors: a critical evaluation of in vitro models. Endocr Rev 2001;22:631–56.[Abstract/Free Full Text]
  8. Valitutti S, Cucchi P, Colletta G, Di Filippo C, Corda D. Transformation by the k-ras oncogene correlates with increases in phospholipase A2 activity, glycerophosphoinositol production and phosphoinositide synthesis in thyroid cells. Cell Signal 1991;3:321–32.[CrossRef][Medline]
  9. Corda D, Iurisci C, Berrie CP. Biological activities and metabolism of the lysophosphoinositides and glycerophosphoinositols. Biochim Biophys Acta 2002;1582:52–69.[Medline]
  10. Alonso T, Morgan RO, Marvizon JC, Zarbl H, Santos E. Malignant transformation by ras and other oncogenes produces common alterations in inositol phospholipid signaling pathways. Proc Natl Acad Sci U S A 1988;85:4271–5.[Abstract/Free Full Text]
  11. Berrie CP, Iurisci C, Corda D. Membrane transport and in vitro metabolism of the Ras cascade messenger, glycerophosphoinositol 4-phosphate. Eur J Biochem 1999;266:413–9.[Medline]
  12. Berrie CP, Dragani LK, van der Kaay J, et al. Maintenance of PtdIns45P2 pools under limiting inositol conditions, as assessed by liquid chromatography-tandem mass spectrometry and PtdIns45P2 mass evaluation in Ras-transformed cells. Eur J Cancer 2002;38:2463–75.[CrossRef][Medline]
  13. Melillo RM, Castellone MD, Guarino V, et al. The RET/PTC-RAS-BRAF linear signaling cascade mediates the motile and mitogenic phenotype of thyroid cancer cells. J Clin Invest 2005;115:1068–81.[CrossRef][Medline]
  14. Santoro M, Melillo RM, Carlomagno F, Vecchio G, Fusco A. Minireview: RET: normal and abnormal functions. Endocrinology 2004;145:5448–51.[Abstract/Free Full Text]
  15. Santoro M, Carlomagno F, Melillo RM, Fusco A. Dysfunction of the RET receptor in human cancer. Cell Mol Life Sci 2004;61:2954–64.[CrossRef][Medline]
  16. Ichihara M, Murakumo Y, Takahashi M. RET and neuroendocrine tumors. Cancer Lett 2004;204:197–211.[CrossRef][Medline]
  17. Fagin JA. How thyroid tumors start and why it matters: kinase mutants as targets for solid cancer pharmacotherapy. J Endocrinol 2004;183:249–56.[Abstract/Free Full Text]
  18. Ono T, Yamada K, Chikazawa Y, et al. Characterization of a novel inhibitor of cytosolic phospholipase A2{alpha}, pyrrophenone. Biochem J 2002;363:727–35.[CrossRef][Medline]
  19. Ryan AJ, Wedge SR. ZD6474—a novel inhibitor of VEGFR and EGFR tyrosine kinase activity. Br J Cancer 2005;92 Suppl 1:S6–13.[CrossRef][Medline]
  20. Fusco A, Berlingieri MT, Di Fiore PP, et al. One- and two-step transformations of rat thyroid epithelial cells by retroviral oncogenes. Mol Cell Biol 1987;7:3365–70.[Abstract/Free Full Text]
  21. Ohta K, Endo T, Haraguchi K, Hershman JM, Onaya T. Ligands for peroxisome proliferator-activated receptor {gamma} inhibit growth and induce apoptosis of human papillary thyroid carcinoma cells. J Clin Endocrinol Metab 2001;86:2170–7.[Abstract/Free Full Text]
  22. Ishizaka Y, Ushijima T, Sugimura T, Nagao M. cDNA cloning and characterization of ret activated in a human papillary thyroid carcinoma cell line. Biochem Biophys Res Commun 1990;168:402–8.[CrossRef][Medline]
  23. Pettus BJ, Bielawska A, Subramanian P, et al. Ceramide 1-phosphate is a direct activator of cytosolic phospholipase A2. J Biol Chem 2004;279:11320–6.[Abstract/Free Full Text]
  24. Falasca M, Silletta MG, Carvelli A, et al. Signalling pathways involved in the mitogenic action of lysophosphatidylinositol. Oncogene 1995;10:2113–24.[Medline]
  25. Berrie CP, Iurisci C, Piccolo E, Bagnati R, Corda D. Analysis of the phosphoinositides and their aqueous metabolites. Methods Enzymol 2007;434:187–232.
  26. Dragani LK, Berrie CP, Corda D, Rotilio D. Analysis of glycerophosphoinositol by liquid chromatography-electrospray ionisation tandem mass spectrometry using a β-cyclodextrin-bonded column. J Chromatogr B 2004;802:283–9.[CrossRef]
  27. Brondello JM, Brunet A, Pouyssegur J, McKenzie FR. The dual specificity mitogen-activated protein kinase phosphatase-1 and -2 are induced by the p42/p44MAPK cascade. J Biol Chem 1997;272:1368–76.[Abstract/Free Full Text]
  28. Carlomagno F, Vitagliano D, Guida T, et al. ZD6474, an orally available inhibitor of KDR tyrosine kinase activity, efficiently blocks oncogenic RET kinases. Cancer Res 2002;62:7284–90.[Abstract/Free Full Text]
  29. Kimura ET, Nikiforova MN, Zhu Z, et al. High prevalence of BRAF mutations in thyroid cancer: genetic evidence for constitutive activation of the RET/PTC-RAS-BRAF signaling pathway in papillary thyroid carcinoma. Cancer Res 2003;63:1454–7.[Abstract/Free Full Text]
  30. Church WB, Inglis AS, Tseng A, et al. A novel approach to the design of inhibitors of human secreted phospholipase A2 based on native peptide inhibition. J Biol Chem 2001;276:33156–64.[Abstract/Free Full Text]
  31. Ackermann EJ, Conde-Frieboes K, Dennis EA. Inhibition of macrophage Ca2+-independent phospholipase A2 by bromoenol lactone and trifluoromethyl ketones. J Biol Chem 1995;270:445–50.[Abstract/Free Full Text]
  32. Hayashi H, Ichihara M, Iwashita T, et al. Characterization of intracellular signals via tyrosine 1062 in RET activated by glial cell line-derived neurotrophic factor. Oncogene 2000;19:4469–75.[CrossRef][Medline]
  33. Castellone MD, Cirafici AM, De Vita G, et al. Ras-mediated apoptosis of PC CL 3 rat thyroid cells induced by RET/PTC oncogenes. Oncogene 2003;22:246–55.[CrossRef][Medline]
  34. Gordon RD, Leighton IA, Campbell DG, et al. Cloning and expression of cystolic phospholipase A2 (cPLA2) and a naturally occurring variant. Phosphorylation of Ser505 of recombinant cPLA2 by p42 mitogen-activated protein kinase results in an increase in specific activity. Eur J Biochem 1996;238:690–7.[Medline]
  35. Kumar S, Jiang MS, Adams JL, Lee JC. Pyridinylimidazole compound SB 203580 inhibits the activity but not the activation of p38 mitogen-activated protein kinase. Biochem Biophys Res Commun 1999;263:825–31.[CrossRef][Medline]
  36. Basolo F, Giannini R, Monaco C, et al. Potent mitogenicity of the RET/PTC3 oncogene correlates with its prevalence in tall-cell variant of papillary thyroid carcinoma. Am J Pathol 2002;160:247–54.[Abstract/Free Full Text]
  37. Vitagliano D, Portella G, Troncone G, et al. Thyroid targeting of the N-ras(Gln61Lys) oncogene in transgenic mice results in follicular tumors that progress to poorly differentiated carcinomas. Oncogene 2006;25:5467–74.[CrossRef][Medline]
  38. Pierotti MA. Chromosomal rearrangements in thyroid carcinomas: a recombination or death dilemma. Cancer Lett 2001;166:1–7.[CrossRef][Medline]
  39. Alberti L, Carniti C, Miranda C, Roccato E, Pierotti MA. RET and NTRK1 proto-oncogenes in human diseases. J Cell Physiol 2003;195:168–86.[CrossRef][Medline]
  40. Fusco A, Viglietto G, Santoro M. A new mechanism of BRAF activation in human thyroid papillary carcinomas. J Clin Invest 2005;115:20–3.[CrossRef][Medline]
  41. Nakanishi M, Rosenberg DW. Roles of cPLA2{alpha} and arachidonic acid in cancer. Biochim Biophys Acta 2006;1761:1335–43.[Medline]
  42. Hong KH, Bonventre JC, O'Leary E, Bonventre JV, Lander ES. Deletion of cytosolic phospholipase A2 suppresses ApcMin-induced tumorigenesis. Proc Natl Acad Sci U S A 2001;98:3935–9.[Abstract/Free Full Text]
  43. Takaku K, Sonoshita M, Sasaki N, et al. Suppression of intestinal polyposis in ApcD716 knockout mice by an additional mutation in the cytosolic phospholipase A2 gene. J Biol Chem 2000;275:34013–6.[Abstract/Free Full Text]
  44. Dong M, Guda K, Nambiar PR, et al. Inverse association between phospholipase A2 and COX-2 expression during mouse colon tumorigenesis. Carcinogenesis 2003;24:307–15.[Abstract/Free Full Text]
  45. Mariggiò S, Bavec A, Natale E, et al. Ga13 mediates activation of the cytosolic phospholipase A2{alpha} through fine regulation of ERK phosphorylation. Cell Signal 2006;18:2200–8.[CrossRef][Medline]
  46. De Falco V, Castellone MD, De Vita G, et al. RET/papillary thyroid carcinoma oncogenic signaling through the Rap1 small GTPase. Cancer Res 2007;67:381–90.[Abstract/Free Full Text]
  47. 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]
  48. Melillo RM, Santoro M, Ong SH, 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]
  49. Borrello MG, Alberti L, Arighi E, et al. The full oncogenic activity of Ret/ptc2 depends on tyrosine 539, a docking site for phospholipase C{gamma}. Mol Cell Biol 1996;16:2151–63.[Medline]



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