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1 Department of Public Health and Cell Biology, University of Rome Tor Vergata, Via Montpellier, 1, 00133 Rome, Italy
2 Digestive and Liver Disease Unit, II Medical School, University of Rome La Sapienza, Rome, Italy
3 Institute of experimental Neuroscience, IRCCS Fondazione Santa Lucia, Rome, Italy
(Requests for offprints should be addressed to G D Fave who is now at Digestive and Liver Disease Unit, II Medical School, University of Rome La Sapienza, Rome, Italy; Email: gianfranco.dellefave{at}uniroma1.it); C Sette is now at Department of Public Health and Cell Biology, University of Rome Tor Vergata, Via Montpellier, 1, 00133 Rome, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The Src family of tyrosine kinases comprises nine members: Src, Lck, Fyn, Yes, Hck, Blk, Fgr, Lyn and Yrk (Thomas & Brugge 1997). These kinases act downstream of growth factor receptors and integrins and are implicated in several aspects of cell growth and metabolism, ranging from cell cycle regulation to cell adhesion and motility (Thomas & Brugge 1997, Playford & Schaller 2004). They contain two highly conserved protein interaction domains, the SH2 and SH3 domains, located upstream of the kinase lobe. The SH2 domain allows Src kinases to interact with tyrosine phosphorylated residues, whereas the SH3 domain binds to proline-rich sequences. Both domains participate in the regulation of Src family kinases activity and are required to interact with several substrates and to localize to discrete subcellular locations (Thomas & Brugge 1997). Following growth factor stimulation, protein–protein interactions with membrane receptor and/or downstream signalling effectors displace auto-inhibitory interactions of the SH2 and SH3 domain with the kinase lobe and release the fully active Src (Thomas & Brugge 1997). Dephosphorylation of the autoinhibitory tyrosine 527 by tyrosine phosphatases like PTP
contribute to maintain Src in its active state (Ponniah et al. 1999).
Adhesion to the extracellular matrix (ECM) is a fundamental process for both normal and neoplastic cells (Malliri & Collard 2003, Cavallaro & Christofori 2004). Cell adhesion and spreading is mediated by the interaction of transmembrane integrin molecules and the ECM (Giancotti & Tarone 2003). A complex array of proteins are then recruited to the cell membrane and are involved in the assembly of the actin cytoskeleton around the site of cell attachment. The catalytic activity of tyrosine kinases like Src, Fyn and the focal adhesion kinase FAK are required for these events (Playford & Schaller 2004). Interestingly, in several cell types, FAK directly recruits Src to the focal adhesion sites (Schaller et al. 1994, Thomas et al. 1998) and Src potentiates activation of FAK through phosphorylation of additional tyrosine residues (Schlaplafer et al. 1994, Calalb et al. 1995). Tyrosine phosphorylation of FAK and integrin molecules creates docking sites for other proteins involved in actin cytoskeleton remodelling, like the scaffold protein p130Cas, paxillin and p190RhoGAP (Playford & Schaller 2004). A role for Src in cell adhesion and motility of fibroblasts was indicated by the observation that transformation with the constitutively active v-Src causes a round morphology and detachment from the ECM (Rohrschneider 1980). On the other hand, fibroblasts harbouring null mutations in the Src, Fyn and Yes genes are defective in focal adhesion turnover and cell migration (Webb et al. 2004).
In line with its oncogenic potential, the activity of several Src family kinases is increased in a multitude of primary tumours and metastatic lesions (Irby et al. 1999, Irby & Yeatman 2000, Yeatman 2004). More recently, it has been shown that Src enhances pancreatic adenocarcinoma cell adhesion to the ECM through the vß3 integrin complex (Duxbury et al. 2004a). Furthermore, inhibition of Src was shown to sensitize pancreatic adenocarcinoma cells to chemotherapic agents both in culture and in vivo (Duxbury et al. 2004b, Yezhelyev et al. 2004). However, no data are currently available on the role of Src family kinases in PETs. Moreover, information on the interaction of PET cells with the ECM is also scarce.
Herein, we have investigated the expression and function of Src family kinases in two available PET cell line models and PET lesions. Our experiments show that Src is highly expressed in PETs and PET cell lines and is required for both PET cell adhesion to the ECM and their spreading and migration.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The human PET cell line QGP-1, derived from a somatostatinoma was obtained from Cancer Research UK Cell Services; and CM, which originates from an insulinoma, was kindly provided by Dr Marco Baroni (Rome, Italy). QGP-1 and CM cells were grown at 37 °C in a humidified 5% CO2 atmosphere in Roswell Park Memorial Institute (RPMI) 1640 medium (Bio-Whittaker Cambrex Bioscience, Belgium) supplemented with 10 and 5% fetal bovine serum (FBS; Gibco BRL, Invitrogen) respectively. For adhesion, assays were collected after application of trypsin/EDTA (Gibco BRL) for 1 min and seeded on non-coated or coated plates in complete medium or serum-free medium respectively. The cells were incubated with either 0.1% dimethyl sulfoxide (DMSO; Sigma-Aldrich) or 10 µM PP2 (4-amino-5-(4-chlorophenyl)-7(t-butyl)pyrazolo[3,4-d]pyrimidine; Calbiochem, San Diego, CA, USA) during adhesion.
Immunohistochemistry
Immunohistochemical analysis (IHC) was performed on formalin-fixed, paraffin-embedded tissue sections from 27 PETs (17 primaries, 10 metastases) from 24 individual patients. All samples, except for four insulinomas, were from patients with NF-PETs. According to the WHO guidelines (Solcia et al. 2000), there were six well-differentiated endocrine tumour (WDETs), twenty well-differentiated endocrine carcinoma (WDECs) and two poorly-differentiated endocrine carcinoma (PDECs). The expression of Src was also analysed in normal islet cells identified in the same samples and in three normal pancreatic samples negative for tumour invasion. All samples were first analysed histologically, and subsequently with several markers and Ki-67 as described previously (Capurso et al. 2005). Subsequent IHC analysis was performed with a rabbit anti-Src antibody (Cell Signalling Technology, Beverly, MA, USA) at working dilution in the ratio of 1:100. Src antibody was optimized on both human colorectal cancer specimens. To ensure antibody specificity, consecutive sections were incubated in the absence of primary antibody and showed no immunostaining. The immunoreactivity for Src was evaluated on a semiquantitative scale considering both the percentage of positive cells (score: 0–4 for respectively, <5, 5–20, 20–40, 40–80, >80%) and the intensity (score: 0–3) of staining. The product of both yield a final immunostaining score (range: 0–12). The immunostaining was visualized using the EnVision polymer method (DakoCytomation, Hamburg, Germany) followed by haematoxylin counterstaining.
Statistical analysis
Src immunostaining scores were continuous data expressed as the mean (95% confidence interval, CI) in different subgroups and evaluated by t-test. Correlation between the IHC score and other factors was evaluated with the Spearman’s rank correlation test; a P value <0.05 was considered statistically significant.
Flow cytometry
At the appropriate time, cells were fixed in 1% paraformaldehyde for 30 min, washed in PBS and incubated for 16 h with 70% ethanol. After washing, cells were incubated with RNAse A for 30 min at 37 °C and then stained with propidium iodide (10 µg/ml) for additional 30 min at 37 °C in the dark. Stained cells were analysed on a FACSCalibur Flow Cytometer (Becton Dickinson, San Josè, CA, USA).
Assay of cell growth
The cells were seeded at 1–3 x 104/ml in 35 mm dishes. After overnight incubation at 37 °C, PP2 inhibitor was added (or DMSO, as control) and cells were incubated at 37 °C for 24 h. Viable cells were examined using the trypan blue dye and counted in a Thoma’s chamber. Results are mean ± S.D. of three experiments performed in triplicate.
Adhesion assay
The assay was performed in 35 mm dishes. At 37 °C, 103 cells were incubated for 30 min in suspension, with 10 µM PP2 or equal volume of DMSO, then were plated and incubated at 37 °C for the indicated time. Suspended cells were collected by pipetting and rinsing, attached cells were collected by trypsinization. Collected cells were counted in a Thoma’s chamber. In the experiments with purified ECM proteins, dishes were pre-coated with 10 µg/ml collagen IV (Chemicon, Temecula, CA, USA), fibronectin (Sigma-Aldrich), laminin (Sigma-Aldrich) or 0.1 µg/cm2 of vitronectin (Sigma-Aldrich) or FBS 10% (Gibco BRL) or BSA 3% (Sigma-Aldrich) overnight at 37 °C or with 0.1 mg/ml poly-L-lysine for 5 min at room temperature. After twice brief washes in Sterile Water (endotoxin tested, Sigma-Aldrich), dishes were dried for 1 h to be used for the adhesion assay. Dishes were then washed twice in PBS and adsorbed with 1% BSA at 37 °C for 1 h. After a brief wash in PBS, pre-coated dishes were used for the adhesion assay as described above. Results are mean ± S.D. of three experiments performed in triplicate.
Scratch wound-healing motility assay
QGP-1 cells were plated at 60% of confluence and incubated at 37 °C until the plate was confluent. The confluent monolayer was incubated for 30 min at 37 °C with 10 µM PP2 or DMSO before creating a wound by scratching with a sterile pipette tip. The plate was photographed immediately and 16 and 24 h after scratching.
Immunoprecipitation assay
QGP-1 cells were plated and incubated at 37 °C for 75 min with 10 µM PP2 or 0.1% DMSO. Cells were resuspended in lysis buffer (100 mM NaCl, 10 mM MgCl2, 30 mM Tris–HCl (pH 7.5), 1 mM dithio-threitol, 2 mM Na-ortovanadate and protease inhibitor cocktail, (Sigma-Aldrich), supplemented with 1% Triton X-100) and kept on ice for 10 min. For immunoprecipitation of FAK, lysis buffer was supplemented with 0.1% SDS and 0.5% Na-deoxycolic acid. Soluble extracts were separated by centrifugation at 12 000 r.p.m. for 10 min. Extracts (500–850 µg total proteins) were pre-adsorbed to 1 µg mouse or rabbit preimmune IgGs bound to Protein A/G-sepharose beads (Sigma-Aldrich) for 1 h at 4 °C under constant shaking. At the end of incubation, supernatants were incubated for 2 h at 4 °C under constant shaking with 2 µg anti-phosphotyrosine antibody (PY20, Santa Cruz Biotechnology, INC., Santa Cruz, CA SC-508, USA) or 1 µg anti-p130Cas (Cell Signalling), or anti-FAK (Cell Signalling) or control IgGs and protein A/G-sepharose beads. All the beads with antibodies were pre-adsorbed with 0.05% BSA at 4 °C under constant shaking, before incubation with the extracts. Immunocomplexes were washed thrice with lysis buffer and adsorbed proteins were eluted in SDS-sample buffer (62.5 mM Tris–HCl (pH 6.8), 10% glycerol, 2% (wt/vol.) SDS, 0.7 M 2-mercaptoethanol and 0.0025% (wt/vol.) bromophenol blue) and resolved on a 8% SDS-PAGE for subsequent Western blot analysis.
Plasmid construction
The sequence encoding the SH3 domain of Src was amplified by PCR using pCMV5-Src containing the chicken Src sequence (accession number V00402 [GenBank] ) as template, the Pfu polymerase (Stratagene, La Jolla, CA, USA) and oligonucleotides 5'-AGGGATCCACCACTTCCGTGGCTCTCTACGAC-3' (forward) and 5'-AGGGATCCCTCGATGGAGTCTGAGGGCGC-3' (reverse). The PCR product was subcloned in-frame with glutathione-S-transferase (GST) into the pGEX-4X1 vector (Pharmacia) into the BamHI site. GST-SrcSH2 has been previously described (Sette et al. 2002). Expression and purification of GST fusion proteins was performed by standard procedures as described previously (Sette et al. 1998).
Pull-down assay
The cell extracts (250–300 µg) were pre-cleared on glutathione–sepharose beads (Sigma-Aldrich) for 1 h at 4 °C. Pre-cleared cell extracts were then incubated for 2 h at 4 °C under constant shaking with glutathione–sepharose beads (Sigma-Aldrich) coated with purified GST, or GST-SrcSH2 or GST-SrcSH3 fusion proteins. Hence, beads were washed thrice with lysis buffer and adsorbed proteins were eluted in SDS-sample buffer for Western blot analysis.
Tissue extracts
Donor islets were purified as previously reported (Capurso et al. 2006). Neuroendocrine tumors (NETs) were snap-frozen immediately after surgical removal from patients. Extracts were prepared by resuspending the samples in 500 µl lysis buffer (see above) and homogenized with a Dounce glass homogenator. After clarification of the extracts by centrifugation (12 000 r.p.m. for 10 min at 4 °C), soluble extracts were analysed for protein concentration by standard methods (Sette et al. 2002) and diluted in SDS-sample buffer before using them for Western blot analysis.
Western blot analysis
Cell extracts or immunoprecipitated proteins were diluted in SDS-sample buffer and boiled for 5 min. Proteins were separated on either 10 or 8% SDS-PAGE gels and transferred to PVDF Transfer Membrane Hybond-P (Amersham Bioscience). Membranes were saturated with 5% non-fat dry milk in PBS containing 0.1% Tween 20, or with TBS containing 0.1% Tween 20 and 5% BSA for 1 h at room temperature, and incubated with the following primary antibody: mouse -tubulin (1:1000, Sigma-Aldrich), rabbit -actin (1:1000, Sigma-Aldrich), rabbit -ERK2 (1:1000, SantaCruz Biotechnology), mouse -v-Src (1:500, Oncogene, Research Products, Calbiochem, Ab-1), rabbit -Fyn (1:1000, SantaCruz Biotechnology), rabbit -Lck (1:1000, Cell Signalling) rabbit, rabbit -FAK (1:1000, Cell Signalling), mouse -p130Cas (1:1000, BD Transduction Laboratories, Lexington, KY, USA), mouse -phosphotyrosine (PY20, 1:1000, SantaCruz Biotechnology), rabbit anti-pSrc418 (1:1000, Biosource, Calmarillo, CA, USA). Secondary -mouse or -rabbit IgGs conjugated to horseradish peroxidase (Amersham) were incubated with the membranes for 1 h at room temperature, at a 1:10 000 dilution in PBS or TBS containing 0.1% Tween 20. Immunostained bands were detected by chemiluminescent method (SantaCruz Biotechnology).
Immunofluorescence analysis
QGP-1 cells were fixed for 10 min in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and processed for immunofluorescence analysis using -phalloidin antibody (1:200, Sigma-Aldrich) or -p130Cas (1:100, BD Tranduction Laboratories) antibody and Hoechst DNA staining. Cy3-conjugate anti-mouse secondary antibody (1:300, Chemicon) was used to detect p130Cas localization.
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Results |
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To investigate the expression and the function of Src family kinases in PET cells, we have used two cell lines that were originated from metastatic insulinoma (CM cells) and somatostatinoma (QGP-1). These cell lines have been previously shown to maintain some characteristics of the original tumours (Iguchi et al. 1990, Baroni et al. 1999). Western blot analyses with antibodies specific for Src, Fyn and Lck, three of the more commonly activated Src family kinases in cancer (Irby & Yeatman 2000), indicated that all three isoforms were expressed. Src was the most readily detected by this technique in CM and QGP-1 cells, followed by Fyn and Lck (Fig. 1A
). To determine the relative activity of Src in these PET cell lines, we analysed cell extracts from QGP-1 and CM cells with an antibody that recognizes the active form of Src (phosphorylated on tyrosine 416; anti-pSrc) and compared it with that of cancer cell lines of different origins. We observed that QGP-1 cells display very high levels of Src activity, as elevated as that of Colo205 (Fig. 1B), a colon cancer cell line that is frequently used to study the role of up-regulation of Src in cancer cells (Golas et al. 2005). Although lower than in QGP-1, the activity of Src in CM cells was comparable with that of MCF-7, a breast cancer cell line in which Src is required for cell proliferation and invasiveness (Castoria et al. 2001). Quantitative densitometric analyses of Src expression level and activity are reported in Fig. 1C and represent the mean of two separate experiments.
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). In PETs, Src staining was positive in all of the 27 samples examined, with a mean score of 10.6 (95% CI 9.7–11.4; Table 1). The immunoreactivity was diffusely cytoplasmic, with some membrane reinforcement (Fig. 2B) and with a clearly stronger intensity and diffusion when compared with that seen in normal islet cells (Fig. 2B–D). Src immunoreactivity in primary lesions, mean score 11.7 (95% CI 11.3–12.2), was significantly higher than that in metastatic samples, mean score 8.5 (95% CI 6.6–10.3; P<0.0001). In the three cases for which we were able to analyse both primary and matched metastatic lesion, immunoreactivity was higher in the primary lesion in two patients and equal in the third. Moreover, Src immunoreactivity seemed stronger in WDETs (mean score 12) than in WDECs (mean score 10.5) and PDECs (mean score 6), albeit without a significant difference. To determine the activity of Src in islet cells and PETs, we performed Western blot analyses with the anti-pSrc antibody on cell extracts obtained from three purified fractions of normal islet cells or from PET lesions (one primary tumour and two liver metastases). As shown in Fig. 2E, Src activity was elevated in neoplastic tissues when compared with normal islets. These data indicate that Src expression and activity are elevated in human PETs.
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To investigate the role of Src family kinases in PET cells, we inhibited the catalytic activity using the specific inhibitor PP2 (Hanket et al. 1996; Fig. 3A). The effect of prolonged inhibition of Src family activity on cell cycle progression in QGP-1 cells was tested by FACS analysis of DNA content after 24–48-h incubation. We observed that PP2 caused a slight accumulation of cells in G1 after 24 h (Fig. 3B) and a decrease in mitotic figures (Fig. 3C). However, cell cycle retardation was transient and 48 h after plating the effect of Src inhibition on mitotic figures (Fig. 3C) and on accumulation in G1 (data not shown) was almost negligible. Similar results were obtained with CM cells (data not shown). Moreover, treatment with PP2 did not affect cell survival in both QGP-1 and CM cells, as determined by trypan blue staining (Fig. 3D) and TUNEL assay (data not shown). Interestingly, we observed that PP2 interfered with cell rounding in mitosis, indicating that inhibition of Src caused a defective reorganization of the cytoskeleton and may cause a retarded progression through mitosis (Fig. 3E).
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To investigate whether Src kinases play a role in the reorganization of the cytoskeleton, we measured adhesion of PET cells in the presence of the PP2 inhibitor. In the absence of inhibitors, approximately 70% of QGP-1 cells attached to the plate within 1 h and 90% were attached 2 h after plating, as evident from the flat shape of the attached cells (Fig. 4A, black arrows). Treatment with PP2 caused a dose-dependent inhibition of Src (Fig. 4B) that correlated with reduction in the number of cells attached, which remained with a rounded shape loosely bound to the plate even after 2 h (Fig. 4C). The effect of PP2 was more dramatic in QGP-1 cells, which display higher levels of Src activity (Fig. 1B
), than in CM cells (data not shown).
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). Non-integrin-mediated adhesion to poly-L-lysine-coated plates was unaffected by PP2 (Fig. 4D). Inhibition of Src kinases completely blocked also the spreading of attached cells on FBS- or vitronectin-coated plates, as shown by the impairment of the actin cytoskeleton and the round shape of the cells treated with PP2 (Fig. 4E).
To determine the role of Src family kinases in migration of QGP-1 cells, we performed a wound-healing assay in the presence or absence of PP2. As shown in Fig. 5, QGP-1 was capable to migrate and colonize the wounded region of the plate within 16–24 h. However, PP2 strongly impaired this process indicating that Src family activity is also required for their migration.
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Given the role of Src activity in adhesion, spreading and motility of QGP-1 cells, we searched for potential molecular targets. As a first approach, we determined if some proteins were phosphorylated in a Src kinases-dependent manner during adhesion. Cell extracts from QGP-1 cells in suspension displayed only weak tyrosine-phosphorylated bands. On the other hand, two tyrosine-phosphorylated bands of approximately 130 and 190 kDa were increased 30 or 75 min after the cells were seeded in the plate (Fig. 6A). Treatment with PP2 during this process strongly affected phosphorylation of these two proteins, indicating that their phosphorylation was dependent on Src kinases activity (Fig. 6A). To isolate and characterize these proteins, we performed an immunoprecipitation experiment using the anti-phosphotyrosine antibody PY20. Western blot analysis of proteins immunoprecipitated showed four major tyrosine-phosphorylated bands: p190, p130, p72 and p55 (Fig. 6B). All these phosphorylations were strongly affected by addition of PP2 during adhesion, confirming that their phosphorylation was mediated by Src kinases.
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, all phosphoproteins were efficiently depleted from the extracts (unbound) and bound to GST-SrcSH2 beads but not to GST beads, indicating that they can interact with this domain of Src.
The docking protein p130Cas and the tyrosine kinase FAK are known substrates of Src and Fyn that are involved in remodelling of the actin cytoskeleton. To determine whether these proteins were substrates of Src kinases, QGP-1 cells were collected 75 min after seeding in the presence or absence of PP2 and extracts were immunoprecipitated with either control IgGs or anti-p130Cas or anti-FAK antibodies (Fig. 7A). Tyrosine phosphorylation was detected by Western blot with the PY20 antibody. Both p130Cas and FAK were strongly phosphorylated during adhesion of QGP-1 cells; however, Src family activity was required only for phosphorylation of p130Cas. By contrast, phosphorylation of FAK was even increased in cells treated with PP2, indicating that this protein is not substrate of Src kinases under these conditions.
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). Inhibition of Src family activity completely blocked the re-localization of p130Cas, confirming that this protein is modulated by Src kinases during adhesion of QGP-1 cells. |
Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Since Src was strongly expressed and active in both QGP-1 and CM cells, we investigated its expression also in human PET samples by immunohistochemistry and Western blot. Our results indicate that Src, whose expression is low in normal islet cells, is strongly up-regulated in PETs, with positive staining in 100% of the samples. Moreover, Western blot analysis with the anti-pSrc antibody showed that the activity of the kinase is increased in the three PET samples examined compared with purified donor islets. Src is the family member most commonly up-regulated in cancer (Irby & Yeatman 2000) and it was suggested that it might be required in the initial phases of neoplastic transformation, whereas its function is substituted when the cancer cells become poorly differentiated (Weber et al. 1992). Our results in human PETs are in line with this hypothesis and suggest that the amount of Src protein is higher in primary lesions than in metastases.
The main defect caused by inhibition of Src family activity in PET cells was seen on cytoskeletal structures and organization. First of all, PP2 treatment caused a marked delay in QGP-1 and CM cell adhesion. The effect was more pronounced in QGP-1 cells, which express higher Src activity, than CM cells. The delay in adhesion was overcome after 4–5 h from seeding, indicating that although Src kinases were inactive, PET cells are able to use other less efficient pathways to adhere. Src activity was required for adhesion on several extracellular matrices, particularly on fibronectin, vitronectin and laminin, indicating its crucial role in several adhesion pathways. Interestingly, both fibronectin (Maitra et al. 2003, Capurso et al. 2006) and vitronectin (Hansel et al. 2004) are up-regulated in PETs and gene ontology analyses suggest that several genes for ECM constituents are aberrantly regulated in PETs, highlighting the possible role of cancer-stroma crosstalk in this cancer type (Capurso et al. 2006).
Although Src activity is involved in cell proliferation (Irby & Yeatman 2000), we found that its inhibition in QGP-1 cells does not interfere with their proliferation and survival. The reduced number of mitotic figures found 24 h after seeding QGP-1 cells in the presence of PP2 was already attenuated after 48-h treatment. Since the cells grown in PP2 were incompletely detached from the plate in mitosis, we hypothesized that the initial delay in cell divisions was due to altered cytoskeletal dynamics in the absence of Src activity, which may limit the initial attachment to the plate and delay the consequent onset of cell cycle progression.
Alterations in the expression or regulation of proteins involved in actin cytoskeleton turnover are often observed in cancer cells and contribute to their migration and invasiveness (Cavallaro & Christofori 2004). Focal adhesions, the sites where the actin cytoskeleton is linked to the ECM by integrin/receptor complexes, contribute to cell anchorage and to the recruitment of signalling complexes that are involved in a broad range of cellular processes, including migration, proliferation, transformation and apoptosis. Remarkably, Src family kinases have been reported to act downstream of the modifications of the integrin/ ECM complexes that contribute to neoplastic transformation (O’Neill et al. 2000, McLean et al. 2003, Cabodi et al. 2006). Although the role of adhesion molecules is also recognized in endocrine cancer cells (Ezzat & Asa 2005), no data are currently available in PETs. Herein, we have demonstrated that QGP-1 cells attached to the plates in the presence of PP2 were not capable to spread on the surface and could not reorganize the actin filaments when compared with control cells. In addition, wound-healing assays showed that Src activity was required also for migration of QGP-1, indicating a role of these kinases also in the actin cytoskeleton turnover. Our results suggest that pharmacological inhibition of Src family kinases might be useful to control the invasiveness of PET cells.
Cell adhesion, spreading and motility use common signalling pathways and many of the proteins involved in these pathways are substrates of Src. We observed that several proteins are phosphorylated in a Src-dependent manner during QGP-1 adhesion. In particular, we identified four proteins of apparent molecular weight of 190, 130, 72 and 55 kDa that were reproducibly phosphorylated by Src in these cells. Interestingly, 130 kDa is the molecular weight of two known substrate of Src strongly involved in adhesion, spreading and motility: p130Cas and the tyrosine kinase FAK (Hanks et al. 1992, O’Neill et al. 2000, Bouton et al. 2001). Immunoprecipitation and pull-down experiments demonstrate that p130Cas interacts with Src and it is phosphorylated by the kinase during the adhesion process of QGP-1. Cas plays a role as scaffold in the actin turnover pathway (Playford & Schaller 2004) and its involvement in neoplastic transformation of breast epithelial cells (Cabodi et al. 2006) and lymphoma cells (Ambrogio et al. 2005) has been reported. Our results suggest that p130Cas may also be involved in the physiology of PET cells.
Surprisingly, we found that tyrosine phosphorylation of FAK was independent of Src activity in QGP-1 cells. FAK was strongly phosphorylated during adhesion even in the presence of PP2, and its activity may account for the ability of PET cells to slowly attach to the ECM even in the absence of Src activity. A similar Src-independent regulation of FAK has been recently reported in colon carcinoma cells (Brunton et al. 2005), suggesting that QGP-1 is not unique in the regulation of this adhesion pathway. Although the nature of p190, p72 and p55 is still unknown, their molecular weights correspond to those of molecules involved in actin cytoskeleton like RhoGAP (p190), paxillin (p72) or Src kinases themselves (p55).
The results presented herein show for the first time the requirement of Src activity in PET cell adhesion, spreading and migration in culture conditions. Although these effects cannot reliably predict an in vivo efficacy of such a strategy, the pathway identified may help design therapeutic treatments aimed to limit the metastatic potential of these endocrine tumour cells and improve the outcome of patients affected by this rare disease.
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Acknowledgements |
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P<0.05 as determined by Student’s t-test). (D) Cell survival was analysed by Trypan blue staining in QGP-1 and CM cells treated with or without PP2 inhibitor for 24 h. The inhibition of Src kinases activity does not reduce cell survival. (E) Hoechst staining and phase images of prometaphase, metaphase, anaphase and telophase QGP-1 cells and metaphase and anaphase of QGP-1 cells treated with PP2 for 24 h. Inhibition of Src kinases activity reduces the ability of QGP-1 cells to become round and detach from the plate at mitosis, as shown by black arrows.
