Src Homology-2-Containing Protein Tyrosine Phosphatase-1 Restrains Cell Proliferation in Human Medullary Thyroid Carcinoma
http://www.100md.com
内分泌学杂志 2005年第6期
Section of Endocrinology, Department of Biomedical Sciences and Advanced Therapies, University of Ferrara, 44100 Ferrara, Italy
Address all correspondence and requests for reprints to: Ettore C degli Uberti, M.D., Section of Endocrinology, Department of Biomedical Sciences and Advanced Therapies, University of Ferrara, Via Savonarola 9, 44100 Ferrara, Italy. E-mail: ti8@unife.it.
Abstract
Medullary thyroid carcinoma (MTC) is a rare tumor originating from thyroid parafollicular C cells, where, in the inherited form, constitutive activation of the RET protooncogene is responsible for unrestrained cell proliferation. We previously demonstrated that somatostatin (SRIF) reduces cell growth in the human MTC cell line TT, which expresses all SRIF receptor (SSTR) subtypes and responds differently to selective SSTR agonists. The antiproliferative mechanism of SRIF and its analogs in MTC is still unclear. Src homology-2-containing protein tyrosine phosphatase-1 (SHP-1), a cytoplasmic protein tyrosine phosphatase (PTP), is activated by somatotropin release-inhibiting factor and reduces mutated RET autophosphorylation in a heterologous system. In this study, we explore the role of PTP activation, in particular of SHP-1, in TT cells, where RET is constitutively activated. In TT cells, SRIF stimulated the PTP activity of SHP-1, which was associated with proliferation inhibition and with reduction in the MAPK pathway activation. Blockade of PTP activity with sodium orthovanadate induced cell proliferation and MAPK phosphorylation and blunted the inhibitory effects of SRIF. Moreover, SHP-1 associates with SSTR2 depending on its activation. By using a MAPK kinase inhibitor, we demonstrated that TT cell growth depends on MAPK pathway activation. Furthermore, in TT cells overexpressing SHP-1, cell proliferation and MAPK signaling were strongly down-regulated, whereas in TT cells transfected with a dominant negative form of SHP-1, cell proliferation and MAPK signaling were markedly induced.
Our data demonstrate that SRIF inhibitory effects on TT cell proliferation are mediated, at least in part, by SHP-1, which acts through a MAPK-dependent mechanism.
Introduction
MEDULLARY THYROID CARCINOMA (MTC), an aggressive tumor arising from neoplastic thyroid parafollicular C cells, can occur as part of the clinical manifestation of multiple endocrine neoplasia type 2 (MEN-2) or familial MTC because of different mutations of the RET protooncogene (1). The MEN-2A-associated RET mutation causes constitutive dimerization and activation of the RET kinase (2), which, in turn, induces multiple signaling pathways resulting in unrestrained cell growth. And indeed, it has been demonstrated that RET can activate the Ras/Erk pathway through recruitment of Grb2/Sos via the phosphorylated residue Tyr-1062 (3). It has recently been shown that Src homology-2-containing protein tyrosine phosphatase-1 (SHP-1), a cytoplasmic protein phospho-tyrosine phosphatase (PTP) containing two Src homology domains (4), can associate with mutated RET, reducing its autophosphorylation rate, with consequent suppression of the transformation potential (5). Moreover, recent evidence shows that both SHP-1 and SHP-2 are associated with a distinct multiprotein complex including RET mutants, which are not direct substrates of these phosphatases (6). SHP-2 has been demonstrated to mediate downstream signaling of the mutated receptor RET, suggesting that it may act as a limiting factor in RET-associated endocrine tumors (7).
SHP-1 is reported as a negative regulator of cytokine receptor-mediated signaling, because it dephosphorylates the cytokine receptor itself or receptor-associated phosphorylated mediators (8). Moreover, SHP-1 has an important role in terminating mitogenic signals induced by growth factors and may participate in the negative regulation of cell proliferation by somatostatin (SRIF) and its analogs, which are known to activate intracellular pathways involving PTP activation (9, 10, 11). SHP-1 has been documented to physically and functionally associate with SRIF receptor subtype 2 (SSTR2) and to mediate the growth-inhibitory signal transduction pathways triggered by SSTR2 activation. Furthermore, binding of SRIF to SSTR2 induces a rapid dissociation of SHP-1 from this receptor and an increase in its activity (9). We previously demonstrated that in the human MTC cell line TT (12), displaying a C634W RET mutation (13) and expressing all SSTR subtypes, treatment with SRIF or with SSTR2-selective agonists inhibits cell proliferation (14, 15). On the other hand, currently available SRIF analogs, mainly interacting with both SSTR2 and SSTR5, fail to control MTC growth progression, even if they can be sometimes useful to reduce calcitonin secretion in clinical settings (16, 17).
To understand whether PTPs could participate to the mechanisms that regulate parafollicular C cell proliferation, we investigated the effects of PTP activation in the TT cell line, mainly focusing on SHP-1, and verifying these effects in MTC primary cultures.
Materials and Methods
TT cell line
The TT cell line, expressing SSTR1 to SSTR5 (14), was obtained from the American Type Culture Collection (Manassas, VA) and maintained in culture as described previously (14). TT cells express and secrete calcitonin, carcinoembryonic antigen, chromogranin A, and many other peptides (18). TT cells harbor a MEN-2A-type mutation (19), with a cysteine-to-tryptophan substitution at the level of the RET codon 634 (13), and a glycine-to-serine at codon 691 in exon 11, reported to be a RET polymorphism (20).
Isolation of RNA, RT-PCR, and quantitative PCR for human SSTRs mRNA
Total RNA from TT cells was isolated with TRIzol reagent (Invitrogen, Milano, Italy), according to the manufacturer’s protocol, and subjected to RT with random hexamers, as previously described (21). Quantitative PCR was performed as previously described (21, 22). Primers and probes were designed using Primer Express Software (PE Applied Biosystems, Monza, Italy) and are described in Table 1.
TABLE 1. Sequences of primers and probes used in quantitative PCR experiments
Primary culture
To explore the effects of PTP activation on MTC primary culture, monolayer culture of tumor cells was performed from a portion of the fresh tissue as described before (23). Tissue samples were collected from MEN-2A patients undergoing total thyroidectomy for MTC in accordance with the guidelines of the local committee on human research. Briefly, tumor tissue was minced and enzymatically dissociated using 0.35% collagenase (Sigma-Aldrich, Milano, Italy) and 1% trypsin at 37 C for 60 min. Cell suspensions were filtered through double layers of gauze and washed twice with serum-free F-12 Ham’s modified medium (Euroclone Ltd., Wetherby, UK). Tumor cells were resuspended in F-12 with 10% fetal bovine serum and antibiotics, seeded in 96-well culture plates (2 x 104 cells per well), and incubated at 37 C in a humidified atmosphere of 5% CO2 and 95% air. After 24 h, cells were incubated overnight with serum-free F-12 medium. The day after, cells were treated with test substances, and cell viability was assessed.
Viable cell number assessment
Variations in cell number were assessed by the Cell Titer 96 AQueous nonradioactive cell proliferation assay (Promega, Milano, Italy), as previously described (24). TT cells or primary cultured cells were incubated with or without 10–8 M SRIF (Stilamin; Serono, Milano, Italy), 10–5 M sodium orthovanadate (V), a PTP inhibitor (Sigma-Aldrich), or 10–7 M PD98059, a MAPK kinase (MEK) inhibitor (Calbiochem, La Jolla, CA). The 10–8 M SRIF dose was selected on the basis of previous experience, showing the efficacy of this SRIF concentration in inhibiting TT cell growth (15). After incubation, the revealing solution was added and the absorbance at 490 nm was recorded using the Wallac Victor 1420 multilabel counter (PerkinElmer, Monza, Italy) in at least six experiments in eight replicates. The same assay was used for evaluating viable cell number in transfected TT cells.
Western blot analysis and immunoprecipitation
For cell extract preparations, subconfluent TT cells were lysed as previously described (24). Fifty micrograms of protein were fractionated on 12.5% SDS-PAGE and transferred by electrophoresis to polyvinylidene difluoride membrane (Invitrogen). The blots were incubated as previously described (25) with the primary antibody. Horseradish peroxidase-conjugated antibody IgG was used at 1:1000, and binding was revealed using enhanced chemiluminescence (Amersham Biosciences, Uppsala, Sweden). The blots were then stripped and used for further blotting. The primary antibodies used were the following: phosphoPlus p44/42 MAPK (Thr202/Tyr204) antibody, directed against phosphorylated MAPK (New England Biolabs Inc., Beverly, MA); p44/42 MAPK antibody, directed against both phosphorylated and unphosphorylated MAPK (New England Biolabs); SH-PTP1 (c-19) antibody, which recognizes SHP-1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA); and h-SSTR2B antibody (Gramsch Laboratories, Schwabhausen, Germany). Western blot analysis was also performed on previously immunoprecipitated samples by using antibodies against SHP-1 or SSTR2.
In immunoprecipitation experiments, protein G Sepharose beads (Amersham) were washed with 1 ml wash buffer (HEPES 30 mM, NaCl 30 mM, Triton X-100 0.1%, pH 7.45) and then tumbled for 4 h at 4 C with 1 μg primary antibody per sample. The beads were washed again, and 100 μg of protein extracts were added for an additional incubation for 12 h at 4 C, gently rocking. Then the beads were washed and resuspended in 50 μl sample buffer (Na2HPO4 5 mM, SDS 3%, glycerol 10%, ?-mercaptoethanol 5%), boiled, and used for Western blot analysis.
Tyrosine phosphatase assay
To evaluate the PTP activity of SHP-1 in TT cells, protein extracts immunoprecipitated with the SHP-1 antibody were subjected to PTP activity assay by using the tyrosine phosphatase assay system (Promega), following the manufacturer’s instructions. The immunoprecipitate was washed three times with 30 mM HEPES, 30 mM NaCl, and 0.1% Triton X-100 and then incubated with 60 mM sodium acetate and 100 μM phosphopeptide substrate (Tyr phosphopeptide-1) at room temperature for 10 min in a 96-well plate. To stop the reaction, 50 μl of molybdate dye/additive mixture was added to the wells, and the absorbance at 600 nm was recorded using the Wallac Victor 1420 multilabel counter (PerkinElmer) in at least three experiments in duplicate.
Cell transfections
Transfection of TT cells was performed by using the Effectene transfection reagent kit (QIAGEN, Milano, Italy), following the manufacturer’s instructions. The plasmids encoding the dominant negative (DN) SHP-1 mutant (pcDNA3/DN SHP-1), or the wild-type (wt) SHP-1 (pcDNA3/wt SHP-1) were kindly provided by C. Nahmias (Institut Cochin de Génétique Moléculaire, Paris, France) and C. Bousquet (INSERM U151, Institut Louis Bugnard, Toulouse, France). Briefly, 1.5 x 106 TT cells were plated in 100-mm dishes (85% confluence), and the next day, transfection was performed with 2 μg DNA per transfected plate. Stable transfectants were selected in culture medium containing geneticin at 500 μg/ml, and medium was renewed every 3 d. Transfection efficiency was estimated around 25%. Individual colonies were isolated for clonal expansion after 3 wk of incubation in selection medium. Cells thus selected from three independent transfections with each expression vector were tested for reproducibility.
Statistical analysis
Results are expressed as the mean ± SE. For cell viability experiments, a preliminary analysis was carried out to determine whether the datasets conformed to a normal distribution, and a computation of homogeneity of variance was performed using Bartlett’s test. The results were compared within each group and between groups using ANOVA. If the F values were significant (P < 0.05), Student’s paired or unpaired t test was used to evaluate individual differences between means. Concerning the other experiments, differences between means were evaluated by paired and unpaired Student’s t test, and P values < 0.05 were considered significant.
Results
Quantification of SSTRs in TT cells
Real-time quantitative PCR showed that SSTR2 was the most abundantly expressed receptor subtype in TT cells (2.82 x 105 molecules/μg of reverse-transcribed total RNA), being approximately 20 times more expressed than SSTR5 (1.15 x 104 molecules/μg of reverse-transcribed total RNA) (Fig. 1).
FIG. 1. SSTR mRNA levels in TT cells. SSTR expression was evaluated by quantitative PCR. The copy numbers of SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5 are presented as mean mRNA molecules/μg total RNA ± SE.
PTP activity in TT cells
To verify the effects of SRIF and V on SHP-1 protein levels and PTP activity, we evaluated SHP-1 protein levels in TT cells treated for 2 h with or without 10–8 M SRIF, 10–5 M V, or both by Western blot analysis. The 10–8 M SRIF concentration was chosen because it has been previously shown to significantly influence TT cell proliferation (15) and gene expression (24). Moreover, dose-response studies showed that the 10–5 M V concentration was the lowest V dose that could significantly induce cell proliferation and inhibit PTP activity in TT cells (data not shown).
No significant change was observed in SHP-1 protein levels (data nor shown). We then performed a PTP activity assay in parallel to verify SHP-1 activation under our experimental conditions. The same samples were therefore immunoprecipitated with the SHP-1 antibody (Ip SHP-1) and subjected to the PTP assay. We found that PTP activity of Ip SHP-1 in starved TT cells was quite high (0.27 pmol of inorganic phosphate released/min·μg of protein), being nearly 2-fold higher than the PTP activity previously reported in a human pancreatic cell line (10). As shown in Fig. 2A, PTP activity of Ip SHP-1 in TT cells was significantly induced (P < 0.01) by treatment with SRIF. On the other hand, as expected, treatment with V was capable of significantly reducing both basal (P < 0.01) and SRIF-stimulated (P < 0.05) Ip SHP-1 activity. Approximately the same amount of SHP-1 was contained in all Ip SHP-1 samples, as verified by Western blot (Fig. 2B).
FIG. 2. PTP activity of SHP-1 immunoprecipitated proteins. A, PTP activity assessed in total protein extracts immunoprecipitated with the SHP-1 antibody (Ip SHP-1) from TT cells incubated with 10–8 M SRIF, 10–5 M V, or both. Data from three individual experiments evaluated independently are expressed as the mean ± SE fold induction PTP activity vs. untreated control cells. *, P < 0.05 and **, P < 0.01 vs. control cells; +, P < 0.05 vs. SRIF-treated cells. B, Western blotting for SHP-1 of Ip SHP-1 material evaluated in the PTP assay.
SHP-1 activation inhibits MTC cell proliferation
After demonstrating that SRIF can indeed induce PTP activity of SHP-1 in TT cells, to analyze the effects of PTP activation on TT cell proliferation, we performed growth experiments by incubating the cells for 48 h either with or without 10–8 M SRIF, 10–5 M V, or both. As shown in Fig. 3A, treatment with SRIF caused a significant inhibition of cell growth (–50%; P < 0.01 vs. control untreated cells). On the contrary, treatment with V significantly induced cell growth (+80%; P < 0.01 vs. control untreated cells) and completely blocked the antiproliferative effects of SRIF on TT cells.
FIG. 3. Effects of SHP-1 activation on TT cell growth. A, Viability of TT cells incubated with or without 10–8 M SRIF, 10–5 M V, or both. Data are expressed as the mean ± SE percent cell proliferation inhibition vs. untreated control cells. *, P < 0.05 and **,P < 0.01 vs. untreated control cells; +, P < 0.01 vs. SRIF-treated cells. B, Viability of TT cells stably transfected with either pcDNA3/wt SHP-1 (wt SHP-1), pcDNA3/DN SHP-1 (DN SHP-1), or the empty vector (pcDNA3). Data are expressed as the mean ± SE percent cell proliferation inhibition vs. untreated control cells. #, P < 0.05 vs. nontransfected control cells.
To verify whether the inhibitory effect on TT cell proliferation was mediated by SHP-1, we assessed cell proliferation in TT cells stably transfected with pcDNA3/DN SHP-1, pcDNA3/wt SHP-1, or the empty vector. We verified that cell proliferation was greatly impaired in SHP-1-overexpressing cells, whereas it was markedly induced in cells transfected with DN SHP-1 compared with cells transfected with the empty vector (Fig. 3B).
To evaluate whether PTP modulation could affect MTC primary culture cell viability, we performed growth experiments by incubating the primary cultures for 48 h either with or without 10–8 M SRIF, 10–5 M V, or both. As shown in Fig. 4, treatment with SRIF caused a significant inhibition of cell growth (–17.4%; P < 0.05 vs. control untreated cells). On the contrary, treatment with V significantly induced cell growth (+20%; P < 0.05 vs. control untreated cells) and completely blocked the antiproliferative effects of SRIF on MTC primary cultured cells.
FIG. 4. Effect of SRIF and V on MTC primary culture cell viability: viability of MTC primary cultured cells incubated with or without 10–8 M SRIF, 10–5 M V, or both. Data from three individual experiments are expressed as the mean ± SE percent cell proliferation inhibition vs. untreated control cells. *, P < 0.05 vs. untreated control cells; +, P < 0.05 vs. SRIF-treated cells.
MAPK pathway blockade reduces TT cell proliferation.
To investigate whether the TT cell proliferation is dependent on the MAPK pathway, we performed cell viability experiments by using a specific MEK inhibitor, PD98059. TT cells were incubated with or without 10–7 M PD98059, 10–5 M V, or both for 48 h, and as previously shown, treatment with V significantly induced cell growth (+75%; P < 0.01 vs. control untreated cells). On the contrary, inhibition of MEK by treatment with PD98059 caused a significant reduction in cell growth and completely blocked the stimulatory effect of V on TT cell proliferation (Fig. 5).
FIG. 5. Effect of MAPK pathway blockade on TT cell proliferation: viability of TT cells incubated with or without 10–5 M V, 10–7 M PD98059, or both. Data are expressed as the mean ± SE percent cell proliferation inhibition vs. untreated control cells. *, P < 0.05 and **, P < 0.01 vs. untreated control cells; ##, P < 0.01 vs. cells treated with V.
SHP-1 activation inhibits MAPK phosphorylation
To investigate whether PTP activation could influence the MAPK pathway, we assessed the effects of 10–8 M SRIF or 10–5 M V or both on the phosphorylation status of ERK1/p44 and ERK2/p42. Time-course studies showed that the level of phosphorylated ERK1/2 did not change up to 30–60 min of incubation with SRIF or V; therefore, a longer incubation time was chosen. As shown in Fig. 6A, treatment with SRIF for 2 h caused a reduction in ERK1/2 phosphorylation, which, on the contrary, was induced by treatment with V, which in turn completely blocked the inhibitory effects of SRIF on MAPK phosphorylation in TT cells.
FIG. 6. Effects of SHP-1 activation on MAPK phosphorylation levels: Western blot analysis for phosphorylated MAPK (pMAPK) and total MAPK (tot MAPK) of protein extracts from TT cells incubated without or with 10–8 M SRIF, 10–5 M V, or both (A) and from TT cells stably transfected with pcDNA3/wt SHP-1 (wt SHP-1), pcDNA3/DN SHP-1 (DN SHP-1), or the empty vector (pcDNA3) (B).
To verify whether the inhibitory effect on the MAPK pathway was mediated by SHP-1, we assessed MAPK phosphorylation in TT cells stably transfected with pcDNA3/DN SHP-1, pcDNA3/wt SHP-1, or the empty vector. We verified that MAPK phosphorylation was reduced nearly 10-fold in SHP-1-overexpressing cells, whereas it was induced by 2-fold in cells transfected with DN SHP-1 compared with mock-transfected cells (Fig. 6B).
SHP-1 associates with SSTR2
To verify whether in our model SHP-1 interacted with SSTR2, we evaluated the presence of SSTR2 in TT cell protein extracts immunoprecipitated with the SHP-1 antibody (Ip SHP-1). As shown in Fig. 7, a band corresponding to SSTR2 was detected in SHP-1 immunoprecipitated protein extracts in TT cells incubated for 48 h in control medium, but not in starved cells. We then evaluated whether induction of PTP activity could modulate SHP-1 interaction with SSTR2, by investigating SSTR2 protein levels in the Ip SHP-1 complex from TT cells treated with or without 10–8 M SRIF or 10–5 M V. Figure 7 shows that the amount of SSTR2 in Ip SHP-1 was reduced by approximately 2-fold under SRIF treatment, whereas it was induced more than 10-fold by treatment with V, which in turn was capable of blocking the effect of SRIF.
FIG. 7. SHP-1 associates with SSTR2. Western blot analysis for SSTR2 and SHP-1 on protein extracts immunoprecipitated with the SHP-1 antibody (Ip SHP-1) derived from TT cells incubated with or without 10–8 M SRIF, 10–5 M V, or both.
Discussion
It has been previously demonstrated that activation of RET by its ligand glial cell line-derived neurotrophic factor stimulates the PI3K/Akt and the Ras/Erk pathway in a transfected subclone of the NIH3T3 mouse fibroblast cell line (3). In a similar model, mutated RET has been shown to associate with SHP-1, which in turn reduces the transforming potential of this kinase (5). However, there is no evidence so far that SHP-1 takes part in the control of proliferation in human cells expressing the constitutively active form of RET. In this study, we investigated the role of PTP activity, and in particular of SHP-1, in the control of cell growth in a cell line deriving from a human MTC, bearing a MEN-2A mutation, therefore displaying a constant RET tyrosine kinase activity. Mutated RET has been shown to induce constitutive activation of the MAPK pathway and to promote MAPK-dependent cell survival (26). Activation of the MAPK pathway is generally related to growth-promoting actions of growth factors, cytokines, and ligands for G protein-coupled receptors (27). However, MAPK activation can also be associated with differentiation or growth inhibition (28). Thus, the biological outcome of MAPK activation is dependent on cell types, extracellular factors, and their receptors. The MAPK pathway is also elicited by SSTR activation, but its modulation and the biological outcome of its activation differ according to the receptor subtypes and the cellular environment (28). Our data show that TT cell proliferation depends on MAPK pathway activation, which can be enhanced by PTP blockade and by SHP-1 silencing, resulting in additional increased cell proliferation. Moreover, MEK inhibition abrogates the effects of PTP blockade on cell proliferation, suggesting that the MAPK pathway might be involved in the downstream signaling of PTPs, including SHP-1. We can therefore hypothesize that SHP-1 restrains MAPK activation by the constitutively active RET-dependent pathway, suggesting an important role for SHP-1 in TT cell growth control. This hypothesis is further strengthened by the demonstration that SHP-1 overexpression in TT cells causes a dramatic reduction in cell proliferation rate, which is, on the contrary, markedly enhanced when SHP-1 is selectively silenced by a dominant negative construct. In addition, the role of PTPs in controlling MTC cell proliferation is confirmed by the demonstration that PTP blockade in MTC primary cultures results in enhanced cell viability.
Moreover, a negative regulatory role for SHP-1 on MAPK pathway is further substantiated by the demonstration that SHP-1 overexpression potently reduces MAPK phosphorylation in TT cells, which is, on the contrary, enhanced in TT cells with a silenced SHP-1.
Our results further support the hypothesis that SHP-1 may act as a tumor suppressor, because, as suggested by a recent review (29), it may play an important role in the pathogenesis of lymphoma/leukemia and of other cancers, including neuroendocrine tumors (30).
Concerning MTC, we demonstrated high levels of PTP activity of SHP-1 in TT cells, suggesting that, in these cells, growth rate is determined by a tight balance between the constitutive activation of the RET tyrosine kinase and the sustained PTP activity of SHP-1. We also demonstrated that native SHP-1 is still inducible in parafollicular C cells by exogenous stimuli, such as SRIF, resulting in a reduced cell proliferation rate. Previous evidence has shown that SHP-1 activation by SRIF is maximal after treatment for 5–30 min (9, 10). However, in our model, SHP-1 activation by SRIF is delayed, probably because of the capacity of SHP-1 to continuously associate with mutated RET (5).
In our experiments, cotreatment with SRIF and V on TT cells results in a net increase in SHP-1 activity compared with control cells, suggesting that V is not capable of completely abrogating SRIF-induced PTP activity of SHP-1. This slightly increased SHP-1 activity corresponds to slightly reduced proliferation capacity of TT cells treated with both SRIF and V compared with TT cells treated with V only. These results suggest that TT cell proliferation induction by V might be caused by inhibition of other phosphatases, different from SHP-1. The use of vanadate to inhibit TT cell PTP activity does not rule out the involvement of other phosphatases, such as SHP-2 or PTP1B, which could play a role in the regulation of TT cell proliferation. TT cell viability is indeed increased under cotreatment with SRIF and V, compared with control cells, suggesting that vanadate might inhibit also other phosphatases, different from SHP-1, whose activity is not accounted for in the PTP assay performed with SHP-1 immunoprecipitated material. SHP-2 has indeed been demonstrated to mediate intracellular signaling initiated by RET mutants (7), and PTP1B has been shown to restrain the proliferative effects of IGF-1 (31), a growth factor involved in the autocrine regulation of TT cell growth (32).
In the present study we demonstrated that in TT cells SSTR2 is predominantly expressed compared with the other receptor subtypes. It has been previously shown that SHP-1 is associated with SSTR2 and is tyrosine phosphorylated. Upon treatment with octreotide, a synthetic SRIF analog, preformed SSTR2/SHP-1 complexes transiently increase and subsequently dissociate, releasing an unphosphorylated form of SHP-1, which has an enhanced PTP activity (9). Our data showed that in TT cells SSTR2 coimmunoprecipitates with SHP-1 and that the SSTR2/SHP-1 complex can be modulated by PTP inhibition or activation. In fact, the amount of SSTR2 in the SSTR2/SHP-1 complex is reduced by PTP activation and, inversely, increased by PTP inhibition. We can therefore hypothesize that in TT cells SHP-1 is associated with SSTR2. Upon SRIF treatment, SHP-1 dissociates from activated SSTR2, being therefore capable of reducing the proliferative signals. Our data indeed show that SRIF reduces TT cell proliferation by involving SHP-1, most likely activated by SSTR2, which is the most expressed SSTR subtype in TT cells.
The role of the MAPK pathway in SSTR2-mediated cell growth inhibition and the mechanisms involved are not well established. In human SY5Y neuroblastoma cells, SSTR2 activation inhibits platelet-derived growth factor-dependent MAPK phosphorylation and platelet-derived growth factor-induced cell proliferation, whereas in human U343 glioma cells, stimulation of SSTR2 only slightly affects epidermal growth factor-promoted cell proliferation despite a strong inhibition of epidermal growth factor-dependent MAPK hyperphosphorylation (33, 34). In addition, SSTR2-induced transient MAPK activation has been reported in antiproliferative signaling by SRIF (28). However, in our hands, SRIF down-regulates the MAPK pathway, which seems to have a crucial role in TT cell proliferation.
We show that PTP activity inhibition induces an increase in the SSTR2/SHP-1 complex, suggesting that, when inactivated, SHP-1 fails to dissociate from SSTR2. As a consequence, an increased downstream proliferating signaling can be observed. We found indeed that PTP blockade and SHP-1 silencing induce cell proliferation and MAPK pathway activation. To further demonstrate that the MAPK transduction pathway is involved in these effects, we used a specific MEK inhibitor. We showed that the specific blockade of the MAPK pathway blunts TT cell proliferation, both in basal conditions and after treatment with V. We can therefore suggest that the enhanced cell proliferation induced by PTP inhibition is mediated by the MAPK pathway. These data are further confirmed by the demonstration that the MAPK pathway is up-regulated in TT cells transfected with a dominant negative form of SHP-1.
Our results indicate that PTPs act through a molecular event that occurs upstream of MAPK phosphorylation and activation and do not rule out a possible role in the control of parafollicular C cell growth for PTPs different from SHP-1, as well as for molecular pathways not involving SHP-1.
Altogether these findings suggest that MTC cell proliferation is controlled, at least in part, by PTPs, including SHP-1, which can actively restrain growth-promoting signals by the RET-induced MAPK pathway. SHP-1 could therefore represent a molecular target for future treatment of MTC, together with new drugs that block the action of tyrosine kinase (35, 36), because current chemotherapy agents offer little benefit.
References
Santoro M, Melillo RM, Carlomagno F, Visconti R, De Vita G, Salvatore G, Lupoli G, Fusco A, Vecchio G 1998 Molecular biology of the MEN2 gene. J Intern Med 243:505–508
Santoro M, Carlomagno F, Romano A, Bottaro DP, Dathan NA, Grieco M, Fusco A, Vecchio G, Matoskova B, Kraus MH, Di Fiore PP 1995 Activation of RET as a dominant transforming gene by germline mutations of MEN2A and MEN2B. Science 267:381–383
Besset V, Scott RP, Ibanez CF 2000 Signaling complex and protein-protein interactions involved in the activation of the Ras and phosphatidylinositol 3-kinase pathways by the c-RET receptor tyrosine kinase. J Biol Chem 275:39159–39166
Fauman EB, Saper MA 1996 Structure and function of the protein tyrosine phosphatases. Trends Biochem Sci 21:413–417
Hennige AM, Lammers R, Hoppner W, Arlt D, Strack V, Teichmann R, Machicao F, Ullrich A, Haring HU, Kellerer M 2001 Inhibition of RET oncogene activity by the protein tyrosine phosphatase SHP1. Endocrinology 142:4441–4447
Incoronato M, D’Alessio A, Paladino S, Zurzolo C, Carlomagno MS, Cerchia L, de Franciscis V 2004 The Shp-1 and Shp-2, tyrosine phosphatases, are recruited on cell membrane in two distinct molecular complexes including Ret oncogenes. Cellular Signalling 16:847–856
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
Klingmuller U, Lorenz U, Cantley LC, Neel BG, Lodish HF 1995 Specific recruitment of SH-PTP1 to the erythropoietin receptor causes inactivation of JAK2 and termination of proliferative signals. Cell 80:729–738
Lopez F, Esteve JP, Buscail L, Delesque N, Saint-Laurent N, Theveniau M, Nahmias C, Vaysse N, Susini C 1997 The tyrosine phosphatase SHP-1 associates with the sst2 somatostatin receptor and is an essential component of sst2-mediated inhibitory growth signalling. J Biol Chem 26:24448–24454
Douziech N, Calvo E, Coulombe Z, Muradia G, Bastien J, Aubin RA, Lajas A, Morisset J 1999 Inhibitory and stimulatory effects of somatostatin on two human pancreatic cancer cell lines: a primary role for tyrosine phosphatase SHP-1. Endocrinology 140:765–777
Bousquet C, Delesque N, Lopez F, Saint-Laurent N, Esteve JP, Bedecs K, Buscail L, Vaysse N, Susini C 1998 sst2 somatostatin receptor mediates negative regulation of insulin receptor signalling through the tyrosine phosphatase SHP-1. J Biol Chem 273:7099–7106
Leong SS, Horoszewicz JS, Shimaoka K, Firedman M, Kawinski E, Song MJ, Zeigel Z, Chu TM, Baylin SB, Mirand EA 1981 A new cell line for study of human medullary carcinoma. In: Andreoli M, ed. Advances in thyroid neoplasia. Rome: Field Educational; 95–108
Carlomagno F, Salvatore D, Santoro M, de Franciscis V, Quadro L, Pabariello L, Colantuoni V, Fusco A 1995 Point mutation of the RET proto-oncogene in the human TT medullary thyroid carcinoma cell line. Biochem Biophys Res Commun 207:1022–1028
Zatelli MC, Tagliati F, Taylor JE, Rossi R, Culler MD, degli Uberti EC 2001 Somatostatin receptor subtypes 2 and 5 differentially affect proliferation in vitro of the human medullary thyroid carcinoma cell line TT. J Clin Endocrinol Metab 86:2161–2169
Zatelli MC, Tagliati F, Taylor JE, Piccin D, Culler MD, degli Uberti EC 2002 Somatostatin, but not somatostatin receptor subtypes 2 and 5 selective agonists, inhibits calcitonin secretion and gene expression in the human medullary thyroid carcinoma cell line, TT. Horm Metab Res 34:229–233
Diez JJ, Iglesias P 2002 Somatostatin analogs in the treatment of medullary thyroid carcinoma. J Endocrinol Invest 25:773–778[Medline]
Vitale G, Caraglia M, Ciccarelli A, Lupoli G, Abbruzzese A, Tagliaferri P, Lupoli G 2001 Current approaches and perspectives in the therapy of medullary thyroid carcinoma. Cancer 91:1797–1808
Zabel M, Seidel J, Kaczmarek A, Surdyk-Zasada J, Grzeszkowiak J, Gorny A 1995 Immunocytochemical characterization of two thyroid medullary carcinoma cell lines in vitro. Histochem J 27:859–868
Mulligan LM, Kwok JB, Healey CS, Elsdon MJ, Eng C, Gardner E, Love DR, Mole SE, Moore JK, Papi L, Ponder MA, Telenius H, Tunnacliffe A, Ponder BA 1993 Germ-line mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2A. Nature 363:458–460
Ceccherini I, Hofstra RM, Luo Y, Stulp RP, Barone V, Stelwagen T, Bocciardi R, Nijveen H, Bolino A, Seri M, Ronchetti P, Pasini B, Bozzano M, Buys CH, Romeo G 1994 DNA polymorphisms and conditions for SSCP analysis of the 20 exons of the ret proto-oncogene. Oncogene 9:3025–3029[Medline]
Zatelli MC, Piccin D, Tagliati F, Ambrosio MR, Margutti A, Padovani R, Scanarini M, Culler MD, degli Uberti EC 2003 Somatostatin receptor subtype 1 selective activation in human growth hormone- and prolactin-secreting pituitary adenomas: effects on cell viability, growth hormone and prolactin secretion. J Clin Endocrinol Metab 88:2797–2802
Zatelli MC, Piccin D, Bottoni A, Ambrosio MR, Margutti A, Padovani R, Scanarini M, Taylor JE, Culler MD, Cavazzini L, degli Uberti EC 2004 Evidence for differential effects of selective somatostatin receptor subtype agonists on -subunit and chromogranin A secretion and on cell viability in human nonfunctioning pituitary adenomas in vitro. J Clin Endocrinol Metab 89:5181–5188
Zatelli MC, Piccin D, Bondanelli M, Tagliati F, De Carlo E, Culler MD, degli Uberti EC 2003 An in vivo Octreoscan-negative adrenal pheochromocytoma expresses somatostatin receptors and responds to somatostatin analogs treatment in vitro. Horm Metab Res 35:349–354
Zatelli MC, Tagliati F, Piccin D, Taylor JE, Culler MD, Bondanelli M, degli Uberti EC 2002 Somatostatin receptor subtype 1 selective activation reduces cell growth and calcitonin secretion in a human medullary thyroid carcinoma cell line. Biochem Biophys Res Commun 297:828–834
Bottoni A, Piccin D, Tagliati F, Luchin A, Zatelli MC, degli Uberti EC 12 January 2005 miR-15a and miR-16-1 down-regulation in pituitary adenomas. J Cell Physiol 10.1002/jcp.20282
De Vita G, Melillo RM, Carlomagno F, Visconti R, Castellone MD, Bellacosa, Billaud M, Fusco A, Tsichilis, Santoro M 2000 Tyrosine 1062 of RET-MEN2A mediates activation of Akt (protein kinase B) and mitogen-activated protein kinase pathways leading to PC12 cell survival. Cancer Res 60:3727–3731
Pierce KL, Luttrell LM, Lefkowitz RJ 2001 New mechanisms in heptahelical receptor signaling to mitogen activated protein kinase cascades. Oncogene 20:1532–1539
Lahlou H, Saint-Laurent N, Esteve JP, Eychene A, Pradayrol L, Pyronnet S, Susini C 2003 sst2 somatostatin receptor inhibits cell proliferation through Ras-, Rap1-, and B-Raf-dependent ERK2 activation. J Biol Chem 278:39356–39371
Wu C, Sun M, Liu L, Zhou GW 2003 The function of the protein tyrosine phosphatase SHP-1 in cancer. Gene 306:1–12
Shaw S, Bencherif M, Marrero MB 2003 Angiotensin II blocks nicotine-mediated neuroprotection against ?-amyloid (1–42) via activation of the tyrosine phosphatase SHP-1. J Neurosci 23:11224–11228
Buckley DA, Cheng A, Kiely PA, Tremblay ML, O’Connor R 2002 Regulation of insulin-like growth factor type I (IGF-I) receptor kinase activity by protein tyrosine phosphatase 1B (PTP-1B) and enhanced IGF-I-mediated suppression of apoptosis and motility in PTP-1B-deficient fibroblasts. Mol Cell Biol 22:1998–2010
Yang KP, Samaan NA, Liang YF, Castillo SG 1992 Role of insulin-like growth factor-I in the autocrine regulation of cell growth in TT human medullary thyroid carcinoma cells. Henry Ford Hosp Med J 40:293–295[Medline]
Cattaneo MG, Taylor JE, Culler MD, Nisoli E, Vicentini LM 2000 Selective stimulation of somatostatin receptor subtypes: differential effects on Ras/MAP kinase pathway and cell proliferation in human neuroblastoma cells. FEBS Lett 481:271–276
Held-Feindt J, Forstreuter F, Pufe T, Mentlein R 2001 Influence of the somatostatin receptor sst2 on growth factor signal cascades in human glioma cells. Brain Res Mol Brain Res 87:12–21[Medline]
Carlomagno F, Vitagliano D, Guida T, Basolo F, Castellone MD, Melillo RM, Fusco A, Santoro M 2003 Efficient inhibition of RET/papillary thyroid carcinoma oncogenic kinases by 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). J Clin Endocrinol Metab 88:1897–1902
Santoro M, Carlomagno F, Melillo RM, Fusco A 2004 Dysfunction of the RET receptor in human cancer. Cell Mol Life Sci 61:2954–2964(Maria Chiara Zatelli, Dan)
Address all correspondence and requests for reprints to: Ettore C degli Uberti, M.D., Section of Endocrinology, Department of Biomedical Sciences and Advanced Therapies, University of Ferrara, Via Savonarola 9, 44100 Ferrara, Italy. E-mail: ti8@unife.it.
Abstract
Medullary thyroid carcinoma (MTC) is a rare tumor originating from thyroid parafollicular C cells, where, in the inherited form, constitutive activation of the RET protooncogene is responsible for unrestrained cell proliferation. We previously demonstrated that somatostatin (SRIF) reduces cell growth in the human MTC cell line TT, which expresses all SRIF receptor (SSTR) subtypes and responds differently to selective SSTR agonists. The antiproliferative mechanism of SRIF and its analogs in MTC is still unclear. Src homology-2-containing protein tyrosine phosphatase-1 (SHP-1), a cytoplasmic protein tyrosine phosphatase (PTP), is activated by somatotropin release-inhibiting factor and reduces mutated RET autophosphorylation in a heterologous system. In this study, we explore the role of PTP activation, in particular of SHP-1, in TT cells, where RET is constitutively activated. In TT cells, SRIF stimulated the PTP activity of SHP-1, which was associated with proliferation inhibition and with reduction in the MAPK pathway activation. Blockade of PTP activity with sodium orthovanadate induced cell proliferation and MAPK phosphorylation and blunted the inhibitory effects of SRIF. Moreover, SHP-1 associates with SSTR2 depending on its activation. By using a MAPK kinase inhibitor, we demonstrated that TT cell growth depends on MAPK pathway activation. Furthermore, in TT cells overexpressing SHP-1, cell proliferation and MAPK signaling were strongly down-regulated, whereas in TT cells transfected with a dominant negative form of SHP-1, cell proliferation and MAPK signaling were markedly induced.
Our data demonstrate that SRIF inhibitory effects on TT cell proliferation are mediated, at least in part, by SHP-1, which acts through a MAPK-dependent mechanism.
Introduction
MEDULLARY THYROID CARCINOMA (MTC), an aggressive tumor arising from neoplastic thyroid parafollicular C cells, can occur as part of the clinical manifestation of multiple endocrine neoplasia type 2 (MEN-2) or familial MTC because of different mutations of the RET protooncogene (1). The MEN-2A-associated RET mutation causes constitutive dimerization and activation of the RET kinase (2), which, in turn, induces multiple signaling pathways resulting in unrestrained cell growth. And indeed, it has been demonstrated that RET can activate the Ras/Erk pathway through recruitment of Grb2/Sos via the phosphorylated residue Tyr-1062 (3). It has recently been shown that Src homology-2-containing protein tyrosine phosphatase-1 (SHP-1), a cytoplasmic protein phospho-tyrosine phosphatase (PTP) containing two Src homology domains (4), can associate with mutated RET, reducing its autophosphorylation rate, with consequent suppression of the transformation potential (5). Moreover, recent evidence shows that both SHP-1 and SHP-2 are associated with a distinct multiprotein complex including RET mutants, which are not direct substrates of these phosphatases (6). SHP-2 has been demonstrated to mediate downstream signaling of the mutated receptor RET, suggesting that it may act as a limiting factor in RET-associated endocrine tumors (7).
SHP-1 is reported as a negative regulator of cytokine receptor-mediated signaling, because it dephosphorylates the cytokine receptor itself or receptor-associated phosphorylated mediators (8). Moreover, SHP-1 has an important role in terminating mitogenic signals induced by growth factors and may participate in the negative regulation of cell proliferation by somatostatin (SRIF) and its analogs, which are known to activate intracellular pathways involving PTP activation (9, 10, 11). SHP-1 has been documented to physically and functionally associate with SRIF receptor subtype 2 (SSTR2) and to mediate the growth-inhibitory signal transduction pathways triggered by SSTR2 activation. Furthermore, binding of SRIF to SSTR2 induces a rapid dissociation of SHP-1 from this receptor and an increase in its activity (9). We previously demonstrated that in the human MTC cell line TT (12), displaying a C634W RET mutation (13) and expressing all SSTR subtypes, treatment with SRIF or with SSTR2-selective agonists inhibits cell proliferation (14, 15). On the other hand, currently available SRIF analogs, mainly interacting with both SSTR2 and SSTR5, fail to control MTC growth progression, even if they can be sometimes useful to reduce calcitonin secretion in clinical settings (16, 17).
To understand whether PTPs could participate to the mechanisms that regulate parafollicular C cell proliferation, we investigated the effects of PTP activation in the TT cell line, mainly focusing on SHP-1, and verifying these effects in MTC primary cultures.
Materials and Methods
TT cell line
The TT cell line, expressing SSTR1 to SSTR5 (14), was obtained from the American Type Culture Collection (Manassas, VA) and maintained in culture as described previously (14). TT cells express and secrete calcitonin, carcinoembryonic antigen, chromogranin A, and many other peptides (18). TT cells harbor a MEN-2A-type mutation (19), with a cysteine-to-tryptophan substitution at the level of the RET codon 634 (13), and a glycine-to-serine at codon 691 in exon 11, reported to be a RET polymorphism (20).
Isolation of RNA, RT-PCR, and quantitative PCR for human SSTRs mRNA
Total RNA from TT cells was isolated with TRIzol reagent (Invitrogen, Milano, Italy), according to the manufacturer’s protocol, and subjected to RT with random hexamers, as previously described (21). Quantitative PCR was performed as previously described (21, 22). Primers and probes were designed using Primer Express Software (PE Applied Biosystems, Monza, Italy) and are described in Table 1.
TABLE 1. Sequences of primers and probes used in quantitative PCR experiments
Primary culture
To explore the effects of PTP activation on MTC primary culture, monolayer culture of tumor cells was performed from a portion of the fresh tissue as described before (23). Tissue samples were collected from MEN-2A patients undergoing total thyroidectomy for MTC in accordance with the guidelines of the local committee on human research. Briefly, tumor tissue was minced and enzymatically dissociated using 0.35% collagenase (Sigma-Aldrich, Milano, Italy) and 1% trypsin at 37 C for 60 min. Cell suspensions were filtered through double layers of gauze and washed twice with serum-free F-12 Ham’s modified medium (Euroclone Ltd., Wetherby, UK). Tumor cells were resuspended in F-12 with 10% fetal bovine serum and antibiotics, seeded in 96-well culture plates (2 x 104 cells per well), and incubated at 37 C in a humidified atmosphere of 5% CO2 and 95% air. After 24 h, cells were incubated overnight with serum-free F-12 medium. The day after, cells were treated with test substances, and cell viability was assessed.
Viable cell number assessment
Variations in cell number were assessed by the Cell Titer 96 AQueous nonradioactive cell proliferation assay (Promega, Milano, Italy), as previously described (24). TT cells or primary cultured cells were incubated with or without 10–8 M SRIF (Stilamin; Serono, Milano, Italy), 10–5 M sodium orthovanadate (V), a PTP inhibitor (Sigma-Aldrich), or 10–7 M PD98059, a MAPK kinase (MEK) inhibitor (Calbiochem, La Jolla, CA). The 10–8 M SRIF dose was selected on the basis of previous experience, showing the efficacy of this SRIF concentration in inhibiting TT cell growth (15). After incubation, the revealing solution was added and the absorbance at 490 nm was recorded using the Wallac Victor 1420 multilabel counter (PerkinElmer, Monza, Italy) in at least six experiments in eight replicates. The same assay was used for evaluating viable cell number in transfected TT cells.
Western blot analysis and immunoprecipitation
For cell extract preparations, subconfluent TT cells were lysed as previously described (24). Fifty micrograms of protein were fractionated on 12.5% SDS-PAGE and transferred by electrophoresis to polyvinylidene difluoride membrane (Invitrogen). The blots were incubated as previously described (25) with the primary antibody. Horseradish peroxidase-conjugated antibody IgG was used at 1:1000, and binding was revealed using enhanced chemiluminescence (Amersham Biosciences, Uppsala, Sweden). The blots were then stripped and used for further blotting. The primary antibodies used were the following: phosphoPlus p44/42 MAPK (Thr202/Tyr204) antibody, directed against phosphorylated MAPK (New England Biolabs Inc., Beverly, MA); p44/42 MAPK antibody, directed against both phosphorylated and unphosphorylated MAPK (New England Biolabs); SH-PTP1 (c-19) antibody, which recognizes SHP-1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA); and h-SSTR2B antibody (Gramsch Laboratories, Schwabhausen, Germany). Western blot analysis was also performed on previously immunoprecipitated samples by using antibodies against SHP-1 or SSTR2.
In immunoprecipitation experiments, protein G Sepharose beads (Amersham) were washed with 1 ml wash buffer (HEPES 30 mM, NaCl 30 mM, Triton X-100 0.1%, pH 7.45) and then tumbled for 4 h at 4 C with 1 μg primary antibody per sample. The beads were washed again, and 100 μg of protein extracts were added for an additional incubation for 12 h at 4 C, gently rocking. Then the beads were washed and resuspended in 50 μl sample buffer (Na2HPO4 5 mM, SDS 3%, glycerol 10%, ?-mercaptoethanol 5%), boiled, and used for Western blot analysis.
Tyrosine phosphatase assay
To evaluate the PTP activity of SHP-1 in TT cells, protein extracts immunoprecipitated with the SHP-1 antibody were subjected to PTP activity assay by using the tyrosine phosphatase assay system (Promega), following the manufacturer’s instructions. The immunoprecipitate was washed three times with 30 mM HEPES, 30 mM NaCl, and 0.1% Triton X-100 and then incubated with 60 mM sodium acetate and 100 μM phosphopeptide substrate (Tyr phosphopeptide-1) at room temperature for 10 min in a 96-well plate. To stop the reaction, 50 μl of molybdate dye/additive mixture was added to the wells, and the absorbance at 600 nm was recorded using the Wallac Victor 1420 multilabel counter (PerkinElmer) in at least three experiments in duplicate.
Cell transfections
Transfection of TT cells was performed by using the Effectene transfection reagent kit (QIAGEN, Milano, Italy), following the manufacturer’s instructions. The plasmids encoding the dominant negative (DN) SHP-1 mutant (pcDNA3/DN SHP-1), or the wild-type (wt) SHP-1 (pcDNA3/wt SHP-1) were kindly provided by C. Nahmias (Institut Cochin de Génétique Moléculaire, Paris, France) and C. Bousquet (INSERM U151, Institut Louis Bugnard, Toulouse, France). Briefly, 1.5 x 106 TT cells were plated in 100-mm dishes (85% confluence), and the next day, transfection was performed with 2 μg DNA per transfected plate. Stable transfectants were selected in culture medium containing geneticin at 500 μg/ml, and medium was renewed every 3 d. Transfection efficiency was estimated around 25%. Individual colonies were isolated for clonal expansion after 3 wk of incubation in selection medium. Cells thus selected from three independent transfections with each expression vector were tested for reproducibility.
Statistical analysis
Results are expressed as the mean ± SE. For cell viability experiments, a preliminary analysis was carried out to determine whether the datasets conformed to a normal distribution, and a computation of homogeneity of variance was performed using Bartlett’s test. The results were compared within each group and between groups using ANOVA. If the F values were significant (P < 0.05), Student’s paired or unpaired t test was used to evaluate individual differences between means. Concerning the other experiments, differences between means were evaluated by paired and unpaired Student’s t test, and P values < 0.05 were considered significant.
Results
Quantification of SSTRs in TT cells
Real-time quantitative PCR showed that SSTR2 was the most abundantly expressed receptor subtype in TT cells (2.82 x 105 molecules/μg of reverse-transcribed total RNA), being approximately 20 times more expressed than SSTR5 (1.15 x 104 molecules/μg of reverse-transcribed total RNA) (Fig. 1).
FIG. 1. SSTR mRNA levels in TT cells. SSTR expression was evaluated by quantitative PCR. The copy numbers of SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5 are presented as mean mRNA molecules/μg total RNA ± SE.
PTP activity in TT cells
To verify the effects of SRIF and V on SHP-1 protein levels and PTP activity, we evaluated SHP-1 protein levels in TT cells treated for 2 h with or without 10–8 M SRIF, 10–5 M V, or both by Western blot analysis. The 10–8 M SRIF concentration was chosen because it has been previously shown to significantly influence TT cell proliferation (15) and gene expression (24). Moreover, dose-response studies showed that the 10–5 M V concentration was the lowest V dose that could significantly induce cell proliferation and inhibit PTP activity in TT cells (data not shown).
No significant change was observed in SHP-1 protein levels (data nor shown). We then performed a PTP activity assay in parallel to verify SHP-1 activation under our experimental conditions. The same samples were therefore immunoprecipitated with the SHP-1 antibody (Ip SHP-1) and subjected to the PTP assay. We found that PTP activity of Ip SHP-1 in starved TT cells was quite high (0.27 pmol of inorganic phosphate released/min·μg of protein), being nearly 2-fold higher than the PTP activity previously reported in a human pancreatic cell line (10). As shown in Fig. 2A, PTP activity of Ip SHP-1 in TT cells was significantly induced (P < 0.01) by treatment with SRIF. On the other hand, as expected, treatment with V was capable of significantly reducing both basal (P < 0.01) and SRIF-stimulated (P < 0.05) Ip SHP-1 activity. Approximately the same amount of SHP-1 was contained in all Ip SHP-1 samples, as verified by Western blot (Fig. 2B).
FIG. 2. PTP activity of SHP-1 immunoprecipitated proteins. A, PTP activity assessed in total protein extracts immunoprecipitated with the SHP-1 antibody (Ip SHP-1) from TT cells incubated with 10–8 M SRIF, 10–5 M V, or both. Data from three individual experiments evaluated independently are expressed as the mean ± SE fold induction PTP activity vs. untreated control cells. *, P < 0.05 and **, P < 0.01 vs. control cells; +, P < 0.05 vs. SRIF-treated cells. B, Western blotting for SHP-1 of Ip SHP-1 material evaluated in the PTP assay.
SHP-1 activation inhibits MTC cell proliferation
After demonstrating that SRIF can indeed induce PTP activity of SHP-1 in TT cells, to analyze the effects of PTP activation on TT cell proliferation, we performed growth experiments by incubating the cells for 48 h either with or without 10–8 M SRIF, 10–5 M V, or both. As shown in Fig. 3A, treatment with SRIF caused a significant inhibition of cell growth (–50%; P < 0.01 vs. control untreated cells). On the contrary, treatment with V significantly induced cell growth (+80%; P < 0.01 vs. control untreated cells) and completely blocked the antiproliferative effects of SRIF on TT cells.
FIG. 3. Effects of SHP-1 activation on TT cell growth. A, Viability of TT cells incubated with or without 10–8 M SRIF, 10–5 M V, or both. Data are expressed as the mean ± SE percent cell proliferation inhibition vs. untreated control cells. *, P < 0.05 and **,P < 0.01 vs. untreated control cells; +, P < 0.01 vs. SRIF-treated cells. B, Viability of TT cells stably transfected with either pcDNA3/wt SHP-1 (wt SHP-1), pcDNA3/DN SHP-1 (DN SHP-1), or the empty vector (pcDNA3). Data are expressed as the mean ± SE percent cell proliferation inhibition vs. untreated control cells. #, P < 0.05 vs. nontransfected control cells.
To verify whether the inhibitory effect on TT cell proliferation was mediated by SHP-1, we assessed cell proliferation in TT cells stably transfected with pcDNA3/DN SHP-1, pcDNA3/wt SHP-1, or the empty vector. We verified that cell proliferation was greatly impaired in SHP-1-overexpressing cells, whereas it was markedly induced in cells transfected with DN SHP-1 compared with cells transfected with the empty vector (Fig. 3B).
To evaluate whether PTP modulation could affect MTC primary culture cell viability, we performed growth experiments by incubating the primary cultures for 48 h either with or without 10–8 M SRIF, 10–5 M V, or both. As shown in Fig. 4, treatment with SRIF caused a significant inhibition of cell growth (–17.4%; P < 0.05 vs. control untreated cells). On the contrary, treatment with V significantly induced cell growth (+20%; P < 0.05 vs. control untreated cells) and completely blocked the antiproliferative effects of SRIF on MTC primary cultured cells.
FIG. 4. Effect of SRIF and V on MTC primary culture cell viability: viability of MTC primary cultured cells incubated with or without 10–8 M SRIF, 10–5 M V, or both. Data from three individual experiments are expressed as the mean ± SE percent cell proliferation inhibition vs. untreated control cells. *, P < 0.05 vs. untreated control cells; +, P < 0.05 vs. SRIF-treated cells.
MAPK pathway blockade reduces TT cell proliferation.
To investigate whether the TT cell proliferation is dependent on the MAPK pathway, we performed cell viability experiments by using a specific MEK inhibitor, PD98059. TT cells were incubated with or without 10–7 M PD98059, 10–5 M V, or both for 48 h, and as previously shown, treatment with V significantly induced cell growth (+75%; P < 0.01 vs. control untreated cells). On the contrary, inhibition of MEK by treatment with PD98059 caused a significant reduction in cell growth and completely blocked the stimulatory effect of V on TT cell proliferation (Fig. 5).
FIG. 5. Effect of MAPK pathway blockade on TT cell proliferation: viability of TT cells incubated with or without 10–5 M V, 10–7 M PD98059, or both. Data are expressed as the mean ± SE percent cell proliferation inhibition vs. untreated control cells. *, P < 0.05 and **, P < 0.01 vs. untreated control cells; ##, P < 0.01 vs. cells treated with V.
SHP-1 activation inhibits MAPK phosphorylation
To investigate whether PTP activation could influence the MAPK pathway, we assessed the effects of 10–8 M SRIF or 10–5 M V or both on the phosphorylation status of ERK1/p44 and ERK2/p42. Time-course studies showed that the level of phosphorylated ERK1/2 did not change up to 30–60 min of incubation with SRIF or V; therefore, a longer incubation time was chosen. As shown in Fig. 6A, treatment with SRIF for 2 h caused a reduction in ERK1/2 phosphorylation, which, on the contrary, was induced by treatment with V, which in turn completely blocked the inhibitory effects of SRIF on MAPK phosphorylation in TT cells.
FIG. 6. Effects of SHP-1 activation on MAPK phosphorylation levels: Western blot analysis for phosphorylated MAPK (pMAPK) and total MAPK (tot MAPK) of protein extracts from TT cells incubated without or with 10–8 M SRIF, 10–5 M V, or both (A) and from TT cells stably transfected with pcDNA3/wt SHP-1 (wt SHP-1), pcDNA3/DN SHP-1 (DN SHP-1), or the empty vector (pcDNA3) (B).
To verify whether the inhibitory effect on the MAPK pathway was mediated by SHP-1, we assessed MAPK phosphorylation in TT cells stably transfected with pcDNA3/DN SHP-1, pcDNA3/wt SHP-1, or the empty vector. We verified that MAPK phosphorylation was reduced nearly 10-fold in SHP-1-overexpressing cells, whereas it was induced by 2-fold in cells transfected with DN SHP-1 compared with mock-transfected cells (Fig. 6B).
SHP-1 associates with SSTR2
To verify whether in our model SHP-1 interacted with SSTR2, we evaluated the presence of SSTR2 in TT cell protein extracts immunoprecipitated with the SHP-1 antibody (Ip SHP-1). As shown in Fig. 7, a band corresponding to SSTR2 was detected in SHP-1 immunoprecipitated protein extracts in TT cells incubated for 48 h in control medium, but not in starved cells. We then evaluated whether induction of PTP activity could modulate SHP-1 interaction with SSTR2, by investigating SSTR2 protein levels in the Ip SHP-1 complex from TT cells treated with or without 10–8 M SRIF or 10–5 M V. Figure 7 shows that the amount of SSTR2 in Ip SHP-1 was reduced by approximately 2-fold under SRIF treatment, whereas it was induced more than 10-fold by treatment with V, which in turn was capable of blocking the effect of SRIF.
FIG. 7. SHP-1 associates with SSTR2. Western blot analysis for SSTR2 and SHP-1 on protein extracts immunoprecipitated with the SHP-1 antibody (Ip SHP-1) derived from TT cells incubated with or without 10–8 M SRIF, 10–5 M V, or both.
Discussion
It has been previously demonstrated that activation of RET by its ligand glial cell line-derived neurotrophic factor stimulates the PI3K/Akt and the Ras/Erk pathway in a transfected subclone of the NIH3T3 mouse fibroblast cell line (3). In a similar model, mutated RET has been shown to associate with SHP-1, which in turn reduces the transforming potential of this kinase (5). However, there is no evidence so far that SHP-1 takes part in the control of proliferation in human cells expressing the constitutively active form of RET. In this study, we investigated the role of PTP activity, and in particular of SHP-1, in the control of cell growth in a cell line deriving from a human MTC, bearing a MEN-2A mutation, therefore displaying a constant RET tyrosine kinase activity. Mutated RET has been shown to induce constitutive activation of the MAPK pathway and to promote MAPK-dependent cell survival (26). Activation of the MAPK pathway is generally related to growth-promoting actions of growth factors, cytokines, and ligands for G protein-coupled receptors (27). However, MAPK activation can also be associated with differentiation or growth inhibition (28). Thus, the biological outcome of MAPK activation is dependent on cell types, extracellular factors, and their receptors. The MAPK pathway is also elicited by SSTR activation, but its modulation and the biological outcome of its activation differ according to the receptor subtypes and the cellular environment (28). Our data show that TT cell proliferation depends on MAPK pathway activation, which can be enhanced by PTP blockade and by SHP-1 silencing, resulting in additional increased cell proliferation. Moreover, MEK inhibition abrogates the effects of PTP blockade on cell proliferation, suggesting that the MAPK pathway might be involved in the downstream signaling of PTPs, including SHP-1. We can therefore hypothesize that SHP-1 restrains MAPK activation by the constitutively active RET-dependent pathway, suggesting an important role for SHP-1 in TT cell growth control. This hypothesis is further strengthened by the demonstration that SHP-1 overexpression in TT cells causes a dramatic reduction in cell proliferation rate, which is, on the contrary, markedly enhanced when SHP-1 is selectively silenced by a dominant negative construct. In addition, the role of PTPs in controlling MTC cell proliferation is confirmed by the demonstration that PTP blockade in MTC primary cultures results in enhanced cell viability.
Moreover, a negative regulatory role for SHP-1 on MAPK pathway is further substantiated by the demonstration that SHP-1 overexpression potently reduces MAPK phosphorylation in TT cells, which is, on the contrary, enhanced in TT cells with a silenced SHP-1.
Our results further support the hypothesis that SHP-1 may act as a tumor suppressor, because, as suggested by a recent review (29), it may play an important role in the pathogenesis of lymphoma/leukemia and of other cancers, including neuroendocrine tumors (30).
Concerning MTC, we demonstrated high levels of PTP activity of SHP-1 in TT cells, suggesting that, in these cells, growth rate is determined by a tight balance between the constitutive activation of the RET tyrosine kinase and the sustained PTP activity of SHP-1. We also demonstrated that native SHP-1 is still inducible in parafollicular C cells by exogenous stimuli, such as SRIF, resulting in a reduced cell proliferation rate. Previous evidence has shown that SHP-1 activation by SRIF is maximal after treatment for 5–30 min (9, 10). However, in our model, SHP-1 activation by SRIF is delayed, probably because of the capacity of SHP-1 to continuously associate with mutated RET (5).
In our experiments, cotreatment with SRIF and V on TT cells results in a net increase in SHP-1 activity compared with control cells, suggesting that V is not capable of completely abrogating SRIF-induced PTP activity of SHP-1. This slightly increased SHP-1 activity corresponds to slightly reduced proliferation capacity of TT cells treated with both SRIF and V compared with TT cells treated with V only. These results suggest that TT cell proliferation induction by V might be caused by inhibition of other phosphatases, different from SHP-1. The use of vanadate to inhibit TT cell PTP activity does not rule out the involvement of other phosphatases, such as SHP-2 or PTP1B, which could play a role in the regulation of TT cell proliferation. TT cell viability is indeed increased under cotreatment with SRIF and V, compared with control cells, suggesting that vanadate might inhibit also other phosphatases, different from SHP-1, whose activity is not accounted for in the PTP assay performed with SHP-1 immunoprecipitated material. SHP-2 has indeed been demonstrated to mediate intracellular signaling initiated by RET mutants (7), and PTP1B has been shown to restrain the proliferative effects of IGF-1 (31), a growth factor involved in the autocrine regulation of TT cell growth (32).
In the present study we demonstrated that in TT cells SSTR2 is predominantly expressed compared with the other receptor subtypes. It has been previously shown that SHP-1 is associated with SSTR2 and is tyrosine phosphorylated. Upon treatment with octreotide, a synthetic SRIF analog, preformed SSTR2/SHP-1 complexes transiently increase and subsequently dissociate, releasing an unphosphorylated form of SHP-1, which has an enhanced PTP activity (9). Our data showed that in TT cells SSTR2 coimmunoprecipitates with SHP-1 and that the SSTR2/SHP-1 complex can be modulated by PTP inhibition or activation. In fact, the amount of SSTR2 in the SSTR2/SHP-1 complex is reduced by PTP activation and, inversely, increased by PTP inhibition. We can therefore hypothesize that in TT cells SHP-1 is associated with SSTR2. Upon SRIF treatment, SHP-1 dissociates from activated SSTR2, being therefore capable of reducing the proliferative signals. Our data indeed show that SRIF reduces TT cell proliferation by involving SHP-1, most likely activated by SSTR2, which is the most expressed SSTR subtype in TT cells.
The role of the MAPK pathway in SSTR2-mediated cell growth inhibition and the mechanisms involved are not well established. In human SY5Y neuroblastoma cells, SSTR2 activation inhibits platelet-derived growth factor-dependent MAPK phosphorylation and platelet-derived growth factor-induced cell proliferation, whereas in human U343 glioma cells, stimulation of SSTR2 only slightly affects epidermal growth factor-promoted cell proliferation despite a strong inhibition of epidermal growth factor-dependent MAPK hyperphosphorylation (33, 34). In addition, SSTR2-induced transient MAPK activation has been reported in antiproliferative signaling by SRIF (28). However, in our hands, SRIF down-regulates the MAPK pathway, which seems to have a crucial role in TT cell proliferation.
We show that PTP activity inhibition induces an increase in the SSTR2/SHP-1 complex, suggesting that, when inactivated, SHP-1 fails to dissociate from SSTR2. As a consequence, an increased downstream proliferating signaling can be observed. We found indeed that PTP blockade and SHP-1 silencing induce cell proliferation and MAPK pathway activation. To further demonstrate that the MAPK transduction pathway is involved in these effects, we used a specific MEK inhibitor. We showed that the specific blockade of the MAPK pathway blunts TT cell proliferation, both in basal conditions and after treatment with V. We can therefore suggest that the enhanced cell proliferation induced by PTP inhibition is mediated by the MAPK pathway. These data are further confirmed by the demonstration that the MAPK pathway is up-regulated in TT cells transfected with a dominant negative form of SHP-1.
Our results indicate that PTPs act through a molecular event that occurs upstream of MAPK phosphorylation and activation and do not rule out a possible role in the control of parafollicular C cell growth for PTPs different from SHP-1, as well as for molecular pathways not involving SHP-1.
Altogether these findings suggest that MTC cell proliferation is controlled, at least in part, by PTPs, including SHP-1, which can actively restrain growth-promoting signals by the RET-induced MAPK pathway. SHP-1 could therefore represent a molecular target for future treatment of MTC, together with new drugs that block the action of tyrosine kinase (35, 36), because current chemotherapy agents offer little benefit.
References
Santoro M, Melillo RM, Carlomagno F, Visconti R, De Vita G, Salvatore G, Lupoli G, Fusco A, Vecchio G 1998 Molecular biology of the MEN2 gene. J Intern Med 243:505–508
Santoro M, Carlomagno F, Romano A, Bottaro DP, Dathan NA, Grieco M, Fusco A, Vecchio G, Matoskova B, Kraus MH, Di Fiore PP 1995 Activation of RET as a dominant transforming gene by germline mutations of MEN2A and MEN2B. Science 267:381–383
Besset V, Scott RP, Ibanez CF 2000 Signaling complex and protein-protein interactions involved in the activation of the Ras and phosphatidylinositol 3-kinase pathways by the c-RET receptor tyrosine kinase. J Biol Chem 275:39159–39166
Fauman EB, Saper MA 1996 Structure and function of the protein tyrosine phosphatases. Trends Biochem Sci 21:413–417
Hennige AM, Lammers R, Hoppner W, Arlt D, Strack V, Teichmann R, Machicao F, Ullrich A, Haring HU, Kellerer M 2001 Inhibition of RET oncogene activity by the protein tyrosine phosphatase SHP1. Endocrinology 142:4441–4447
Incoronato M, D’Alessio A, Paladino S, Zurzolo C, Carlomagno MS, Cerchia L, de Franciscis V 2004 The Shp-1 and Shp-2, tyrosine phosphatases, are recruited on cell membrane in two distinct molecular complexes including Ret oncogenes. Cellular Signalling 16:847–856
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
Klingmuller U, Lorenz U, Cantley LC, Neel BG, Lodish HF 1995 Specific recruitment of SH-PTP1 to the erythropoietin receptor causes inactivation of JAK2 and termination of proliferative signals. Cell 80:729–738
Lopez F, Esteve JP, Buscail L, Delesque N, Saint-Laurent N, Theveniau M, Nahmias C, Vaysse N, Susini C 1997 The tyrosine phosphatase SHP-1 associates with the sst2 somatostatin receptor and is an essential component of sst2-mediated inhibitory growth signalling. J Biol Chem 26:24448–24454
Douziech N, Calvo E, Coulombe Z, Muradia G, Bastien J, Aubin RA, Lajas A, Morisset J 1999 Inhibitory and stimulatory effects of somatostatin on two human pancreatic cancer cell lines: a primary role for tyrosine phosphatase SHP-1. Endocrinology 140:765–777
Bousquet C, Delesque N, Lopez F, Saint-Laurent N, Esteve JP, Bedecs K, Buscail L, Vaysse N, Susini C 1998 sst2 somatostatin receptor mediates negative regulation of insulin receptor signalling through the tyrosine phosphatase SHP-1. J Biol Chem 273:7099–7106
Leong SS, Horoszewicz JS, Shimaoka K, Firedman M, Kawinski E, Song MJ, Zeigel Z, Chu TM, Baylin SB, Mirand EA 1981 A new cell line for study of human medullary carcinoma. In: Andreoli M, ed. Advances in thyroid neoplasia. Rome: Field Educational; 95–108
Carlomagno F, Salvatore D, Santoro M, de Franciscis V, Quadro L, Pabariello L, Colantuoni V, Fusco A 1995 Point mutation of the RET proto-oncogene in the human TT medullary thyroid carcinoma cell line. Biochem Biophys Res Commun 207:1022–1028
Zatelli MC, Tagliati F, Taylor JE, Rossi R, Culler MD, degli Uberti EC 2001 Somatostatin receptor subtypes 2 and 5 differentially affect proliferation in vitro of the human medullary thyroid carcinoma cell line TT. J Clin Endocrinol Metab 86:2161–2169
Zatelli MC, Tagliati F, Taylor JE, Piccin D, Culler MD, degli Uberti EC 2002 Somatostatin, but not somatostatin receptor subtypes 2 and 5 selective agonists, inhibits calcitonin secretion and gene expression in the human medullary thyroid carcinoma cell line, TT. Horm Metab Res 34:229–233
Diez JJ, Iglesias P 2002 Somatostatin analogs in the treatment of medullary thyroid carcinoma. J Endocrinol Invest 25:773–778[Medline]
Vitale G, Caraglia M, Ciccarelli A, Lupoli G, Abbruzzese A, Tagliaferri P, Lupoli G 2001 Current approaches and perspectives in the therapy of medullary thyroid carcinoma. Cancer 91:1797–1808
Zabel M, Seidel J, Kaczmarek A, Surdyk-Zasada J, Grzeszkowiak J, Gorny A 1995 Immunocytochemical characterization of two thyroid medullary carcinoma cell lines in vitro. Histochem J 27:859–868
Mulligan LM, Kwok JB, Healey CS, Elsdon MJ, Eng C, Gardner E, Love DR, Mole SE, Moore JK, Papi L, Ponder MA, Telenius H, Tunnacliffe A, Ponder BA 1993 Germ-line mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2A. Nature 363:458–460
Ceccherini I, Hofstra RM, Luo Y, Stulp RP, Barone V, Stelwagen T, Bocciardi R, Nijveen H, Bolino A, Seri M, Ronchetti P, Pasini B, Bozzano M, Buys CH, Romeo G 1994 DNA polymorphisms and conditions for SSCP analysis of the 20 exons of the ret proto-oncogene. Oncogene 9:3025–3029[Medline]
Zatelli MC, Piccin D, Tagliati F, Ambrosio MR, Margutti A, Padovani R, Scanarini M, Culler MD, degli Uberti EC 2003 Somatostatin receptor subtype 1 selective activation in human growth hormone- and prolactin-secreting pituitary adenomas: effects on cell viability, growth hormone and prolactin secretion. J Clin Endocrinol Metab 88:2797–2802
Zatelli MC, Piccin D, Bottoni A, Ambrosio MR, Margutti A, Padovani R, Scanarini M, Taylor JE, Culler MD, Cavazzini L, degli Uberti EC 2004 Evidence for differential effects of selective somatostatin receptor subtype agonists on -subunit and chromogranin A secretion and on cell viability in human nonfunctioning pituitary adenomas in vitro. J Clin Endocrinol Metab 89:5181–5188
Zatelli MC, Piccin D, Bondanelli M, Tagliati F, De Carlo E, Culler MD, degli Uberti EC 2003 An in vivo Octreoscan-negative adrenal pheochromocytoma expresses somatostatin receptors and responds to somatostatin analogs treatment in vitro. Horm Metab Res 35:349–354
Zatelli MC, Tagliati F, Piccin D, Taylor JE, Culler MD, Bondanelli M, degli Uberti EC 2002 Somatostatin receptor subtype 1 selective activation reduces cell growth and calcitonin secretion in a human medullary thyroid carcinoma cell line. Biochem Biophys Res Commun 297:828–834
Bottoni A, Piccin D, Tagliati F, Luchin A, Zatelli MC, degli Uberti EC 12 January 2005 miR-15a and miR-16-1 down-regulation in pituitary adenomas. J Cell Physiol 10.1002/jcp.20282
De Vita G, Melillo RM, Carlomagno F, Visconti R, Castellone MD, Bellacosa, Billaud M, Fusco A, Tsichilis, Santoro M 2000 Tyrosine 1062 of RET-MEN2A mediates activation of Akt (protein kinase B) and mitogen-activated protein kinase pathways leading to PC12 cell survival. Cancer Res 60:3727–3731
Pierce KL, Luttrell LM, Lefkowitz RJ 2001 New mechanisms in heptahelical receptor signaling to mitogen activated protein kinase cascades. Oncogene 20:1532–1539
Lahlou H, Saint-Laurent N, Esteve JP, Eychene A, Pradayrol L, Pyronnet S, Susini C 2003 sst2 somatostatin receptor inhibits cell proliferation through Ras-, Rap1-, and B-Raf-dependent ERK2 activation. J Biol Chem 278:39356–39371
Wu C, Sun M, Liu L, Zhou GW 2003 The function of the protein tyrosine phosphatase SHP-1 in cancer. Gene 306:1–12
Shaw S, Bencherif M, Marrero MB 2003 Angiotensin II blocks nicotine-mediated neuroprotection against ?-amyloid (1–42) via activation of the tyrosine phosphatase SHP-1. J Neurosci 23:11224–11228
Buckley DA, Cheng A, Kiely PA, Tremblay ML, O’Connor R 2002 Regulation of insulin-like growth factor type I (IGF-I) receptor kinase activity by protein tyrosine phosphatase 1B (PTP-1B) and enhanced IGF-I-mediated suppression of apoptosis and motility in PTP-1B-deficient fibroblasts. Mol Cell Biol 22:1998–2010
Yang KP, Samaan NA, Liang YF, Castillo SG 1992 Role of insulin-like growth factor-I in the autocrine regulation of cell growth in TT human medullary thyroid carcinoma cells. Henry Ford Hosp Med J 40:293–295[Medline]
Cattaneo MG, Taylor JE, Culler MD, Nisoli E, Vicentini LM 2000 Selective stimulation of somatostatin receptor subtypes: differential effects on Ras/MAP kinase pathway and cell proliferation in human neuroblastoma cells. FEBS Lett 481:271–276
Held-Feindt J, Forstreuter F, Pufe T, Mentlein R 2001 Influence of the somatostatin receptor sst2 on growth factor signal cascades in human glioma cells. Brain Res Mol Brain Res 87:12–21[Medline]
Carlomagno F, Vitagliano D, Guida T, Basolo F, Castellone MD, Melillo RM, Fusco A, Santoro M 2003 Efficient inhibition of RET/papillary thyroid carcinoma oncogenic kinases by 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). J Clin Endocrinol Metab 88:1897–1902
Santoro M, Carlomagno F, Melillo RM, Fusco A 2004 Dysfunction of the RET receptor in human cancer. Cell Mol Life Sci 61:2954–2964(Maria Chiara Zatelli, Dan)