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Expression of Ligand-Activated KIT and Platelet-Derived Growth Factor Receptor ß Tyrosine Kinase Receptors in Synovial Sarcoma

¹ Experimental Molecular Pathology, Department of Pathology and Departments of ² Experimental Oncology, ³ Soft Tissue Surgery, and ⁴ Radiotherapy, Istituto Nazionale per lo Studio e al Cura dei Tumori, Milan, Italy, and ⁵ Istituto Federazione Italiana Ricerca Cancro Institute of Molecular Oncology, Milan, Italy

	ABSTRACT

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Coexpression of KIT and... Molecular Analysis: Expression... ICC Analysis for KIT... DISCUSSION REFERENCES

 
Purpose: The use of tyrosine kinase receptor inhibitors is increasingly becoming a valuable therapeutic alternative in tumors carrying activated tyrosine kinase receptors. In a previous study, we described a coexpression of KIT and stem cell factor (SCF) mRNA in Synovial sarcomas, (SS) and in a limited number of cases, we demonstrated the presence of an activated receptor. Here, in a wider number of cases, we investigated the expression level and phosphorylation status of two structurally related tyrosine kinase receptors, KIT and platelet-derived growth factor receptor ß (PDGFRß), at the light of their role as possible targets of tyrosine kinase receptors inhibitor molecules.

Experimental Design: Forty-three SS cases were analyzed for KIT and PDGFRß expression/activation by immunoprecipitation/Western blotting experiments. The cognate ligands, SCF and PDGFB, were detected by reverse transcription-PCR.

Results: KIT was observed in 48 and 41% (45% total) whereas PDGFRß in 54 and 33% (45% total) of monophasic and biphasic SS cases, respectively. With respect to the fusion transcript type SYTSSX1 and SYTSSX2, KIT was more expressed in SYTSSX1 carrying cases (48 versus 38%), whereas PDGFRß resulted more frequently expressed in SYTSSX2 ones (54 versus 37%). When expressed, the receptors were phosphorylated. Their ligands were detected in all of the activated cases.

Conclusions: About 70% of the cases express one of the two activated tyrosine kinase receptors with a mutually exclusive expression trend. Coexpression is not frequent and seems to be restricted to monophasic subtype. These data indicate that a consistent fraction of this tumor type could represent a good candidate for kinase inhibitor molecules effective on KIT and PDGFRß where their activation is sustained by an autocrine loop.

	INTRODUCTION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Coexpression of KIT and... Molecular Analysis: Expression... ICC Analysis for KIT... DISCUSSION REFERENCES

 
Receptor tyrosine kinases (RTKs) translate signals into biological events through the reversible phosphorylation of specific substrata, regulating most of the process linked to cell growth or cell death. The deregulation of these receptors is associated to diseases, including neoplastic growth. Several mechanisms could be responsible for a pathological RTK activation such as gain of function mutations, autocrine/paracrine loop, and translocations. In the last case, the chromosomal rearrangements can directly drive the constitutive expression/activation of RTKs or, involving transcription factors, deregulate their expression and/or that of their cognate ligands.

Among sarcomas, activating mutations are described only in KIT receptor and platelet-derived growth factor receptor (PDGFR) in gastrointestinal stromal tumors (1 , 2) . Concerning structural alterations, in inflammatory myofibroblastic tumors, the rearrangement between chromosome 2 and 5 generates the chimeric activated kinase TPM3-ALK (3) , whereas in congenital fibrosarcoma, the cytogenetic aberration between chromosome 12 and 15 creates the chimeric activated-rearranged receptor ETV6-NTRK3 (4) . Otherwise, as in the case of dermatofibrosarcoma protuberans, the translocation event involves the receptor’s ligand, PDGFB, resulting in its unscheduled expression. In this context, the activation of PDGFR is sustained by an autocrine loop (5) . Furthermore, in some sarcomas, aberrant transcription factors, derived from the tumor-specific translocations, may induce the expression of RTKs or their ligands. Experimental evidence is reported regarding the expression of PDGFR, which is induced by the aberrant fusion transcript PAX3-FKHR in rhabdomyosarcomas (6) , whereas PDGFA has demonstrated that it is induced by the chimera EWS-WT1 gene in desmoplastic small round-cell tumor (7) . In other sarcomas instead, activated RTKs are only described associated with the presence of specific aberrant transcription factor, but a direct functional demonstration that the latter are able to transactivate RTKs is still lacking. This is the case of KIT receptor, which was observed activated in Ewing’s tumors cell lines (8) and in primary tumors (9) , and in Synovial sarcoma (SS). In particular, in the latter tumor type, previous gene by gene investigations demonstrated overexpression of insulin growth factor I receptor (10) , Met, strictly associated to epithelial glandular structures in biphasic tumors (11 , 12) , and KIT (13) . Subsequently, an up-regulation of several tyrosine kinase receptors, including ErbB2, IGFBP2, IGF-II, and KIT, was confirmed by global gene expression profile experiments performed through the microarray technology (14 , 15) .

We have previously observed a coexpression of KIT and SCF mRNA in SS, and in a limited number of cases, we demonstrated the presence of an activated receptor (13) . Here, by extending our previous observations, we have analyzed 43 cases of SSs by immunoprecipitation (IP) and Western blot (WB) experiments for the expression/activation status of KIT and PDGFRß receptors, whereas SCF and PDGFB ligands were investigated through reverse transcription-PCR (RT-PCR) experiments. We demonstrate that these two structurally related tyrosine kinase receptors are activated, most likely through an autocrine loop, in SSs.

These data indicate this tumor type as possible candidate for kinase inhibitor molecules effective on KIT and PDGFRß where their activation is sustained by an autocrine loop.

	MATERIALS AND METHODS

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Coexpression of KIT and... Molecular Analysis: Expression... ICC Analysis for KIT... DISCUSSION REFERENCES

 
Patients.
Forty-three cases of SS were analyzed in this study. Histologically according to permanent section sampling, 25 of them were monophasic, and 18 were biphasic in subtype.

Twenty-seven cases showed the fusion transcript SYTSSX1, 14 cases the SYTSSX2, and 2 the SYTSSX4. None of the cases showed coexpression of both SYTSSX1 and SYTSSX2 fusion transcripts. All molecular analyses were performed after written informed consent.

Positive Controls.
The human magakaryoblastic leukemia M07e cell line was kindly provided by Prof. Luigi Pegoraro and was kept in culture as described by Brizzi et al. (16) , and it was used as positive control for SCF expression.

For PDGFB expression, the cell line 5A transformed with DNA from a dermatofibrosarcoma protuberans patient (5) was used.

In IP and WB experiments, as positive control for KIT phosphorylation and expression, a cell line 559 overexpressing a mutated KIT receptor was used, whereas the above described cell line 5A was used for phosphorylation and expression of PDGFRß.

Protein Extraction.
Proteins were extracted from tissue samples stored at -80°C, by homogenization at 4°C in lysis buffer (50 mM HEPES, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl₂, 1 mM EGTA, 10 mM Na₄ P₂O ₇, and 100 mM NaF) supplemented with protease and phosphatase inhibitors (mixture inhibitors I and II; Sigma, St. Louis, MO). Lysis was performed by frequent vortexing. Proteic lysates were then cleared by centrifugation at 13000 rpm at 4°C for 30 min and quantitated through Bio-Rad protein assay.

IP and WB.
c-KIT protein was immunoprecipitated from 500 µg of proteic lysate using 1.8 µl of the monoclonal antibody Ab-3 (K-45, Neomarkers-Union City, Fremont, CA) directed against the receptor. The KIT/559 cell line lysate was used as positive control.

PDGFRß protein was immunoprecipitated from 500 µg of proteic lysate derived from the not bound proteins of the previously described anti-KIT immunoprecipitation using 3 µl of the polyclonal antibody 958 catalogue no. sc-432 (Santa Cruz Biotechnology, Santa Cruz, CA). In this case, the cell line 5A was used as positive control. Some cases (nos. 2, 3, 7, 8, 15, and 30) were firstly immunoprecipitated with anti-PDGFRß and subsequently, by using the not bound proteins, with anti KIT.

After 2 h of incubation with respective antibodies and protein A-Sepharose (Sigma) at 4°C with gentle agitation, the protein complexes were washed three times with HNTG buffer (20 mM HEPES, 150 mM NaCl, 10% glycerol, and 0.1% Triton X-100) supplemented with antiprotease and antiphosphatase mixtures. After 10 min of denaturation at 95°C in Laemli buffer [62 mM Tris-HCl (pH 6.8), 10% glycerol, 2% SDS, and 0.003% BBF), the samples were loaded on a 8% acrylamide minigel (Bio-Rad, Richmond, CA) and blotted to a polyvinylidene difluoride membrane. This was saturated with 4% BSA (Amersham-Pharmacia, Piscataway, NJ) for 1 h at room temperature. To reveal the phosphorylation status of c-KIT and PDGFRß receptors, the membrane was incubated with the antiphosphotyrosine mouse monoclonal antibody (clone 4G; Upstate Biotechnology, Lake Placid, NY) diluted 1:3000 in Tris-buffered saline buffer [20 mM Tris-HCl (pH 7.5) and 154 mM NaCl].

For the expression of c-KIT and PDGFRß proteins, we stripped and restained the same blots with the following rabbit polyclonal antibodies: c-19 (Santa Cruz Biotechnology) diluted 1:200 in Tris-buffered saline for the first receptor and 958 catalogue no. sc-432 (Santa Cruz Biotechnology) diluted 1:1000 in Tris-buffered saline for the second one.

The secondary antibodies were used at the recommended dilution (Sigma). Enhanced chemiluminescence detection system (Amersham-Pharmacia) was always used as a chemoluminescent technique to investigate specific bands.

RNA Extraction and RT-PCR.
Total RNA was extracted with the RNAzol method (Life Technologies, Inc.), and 1 µg was used for RT-PCR according to the manufacturer’s recommendations.

To check the integrity of cDNA the amplification of the housekeeping ß-actin gene was performed. One µl of cDNA was used as template for each PCR reaction.

SCF and PDGFB Amplification.
The detection of SCF, the c-KIT ligand, was performed with the following primers: SCF forward, 5'-ATTCAAGAGCCCAGAACCCA-3'; and SCF reverse, 5'-CTGTTACCAGCCAATGTACG-3'.

PCR conditions were 40 cycles of denaturation at 94°C for 30 s, annealing at 63°C for 1 min, and elongation at 72°C for 1 min, using the AmpliTaq Gold (Roche).

For the detection of PDGFB, the primers used were as follows: PDGFB forward, 5'-GTCCAGGTGAGAAAGATCGAG-3'; and PDGFB reverse, 5'-TGCGTGTGCTTGAATTTCCGG-3'. PCR conditions were 35 cycles at 94°C for 30 s, 58°C for 30 s, and 72°C for 1 min.

Immunocytochemistry (ICC).
Immunoperoxidase phenotyping was performed on representative sections of paraffin-embedded tumoral specimens, fixed in neutral buffered formalin, using CD117 antibodies provided by Dako (A4502, 1:50 diluted, epitope 963-973; Dako, Carpinteria, CA) and by Santa Cruz Biotechnology (C-19, 1:100 diluted; epitope 959-973; Santa Cruz Biotechnology) with heat-induced epitope retrieval.

	RESULTS

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Coexpression of KIT and... Molecular Analysis: Expression... ICC Analysis for KIT... DISCUSSION REFERENCES

 
Biochemical Analysis: Immunoprecipitation and Phosphorylation Status of the Tyrosine Kinases
KIT.
Total protein extracts obtained from 40 cases were analyzed for c-KIT receptor expression and phosphorylation by IP and subsequent WB experiments. Case nos. 2, 3, and 30 of Table 1 were not analyzed because of the lack of proteic extracts. The hybridization with the -Kit antibody revealed two bands of 145 KDa and 125 KDa corresponding to the fully and partially glycosylated forms of the receptor (Fig. 1A) . KIT resulted expressed in 18 of 40 cases (45%; Table 1 ), in particular, in 7 of 17 cases (41%) of biphasic tumors and in 11 of the 23 monophasic cases (48%; Table 2A ).

View larger version (63K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. KIT and platelet-derived growth factor receptor ß (PDGFRß) expression and activation. For each sample, total protein extracts were immunoprecipitated with KIT or PDGFRß antibodies, run on gel and blotted with pTyr antibody in the top panels for receptor tyrosine kinase (RTK) phosphorylation status and with KIT or PDGFRß in the bottom panels for RTK expression. A, top panel: KIT phosphorylation; lower panel: KIT expression. Lane 1: positive control, KIT 559 cell line expressing an activated KIT receptor. Lanes 2–4: cases expressing an activated KIT receptor (case nos. 4, 8, and 32 of Table 1 ). Lanes 5 and 6: cases negative for the receptor (case nos. 16 and 31 of Table 1 ). Lane 7: negative control. B, top panel: PDGFRß phosphorylation; bottom panel: PDGFR ß expression. Lane 1: positive control, 5A cell line expressing and activated PDGFRß receptor. Lanes 2–4: cases expressing an activated PDGFRß receptor (case nos. 1, 13, and 31 of Table 1 ). Lane 5 and 6: cases negative for the receptor (case nos. 9 and 34 of Table 1 ). Lane 7: negative control.

The hybridization of the same membrane with the -pTyr antibody revealed that in all of the cases in which KIT receptor was detected, it was phosphorylated (Fig. 1A) .

PDGFRß.
All cases but one (no. 32, Table 1 , where sufficient frozen material was not available) were analyzed for the PDGFRß activation and expression by IP and WB experiments.

Hybridization with a polyclonal -PDGFRß antibody revealed that 19 cases of 42 (45%) expressed the receptor (Tables 1 and 2) . These cases showed two bands of 180 KDa, corresponding to the mature form of the receptor, and 165 KDa, corresponding to the partially glycosylated one (Fig. 1B) . In particular, 6 of 18 (33%) of the biphasic and 13 of 24 (54%) of the monophasic subtype expressed PDGFRß (Tables 1 and 2A) .

Ten of 27 cases (37%) carried the SYTSSX1 fusion transcript type, 7 of 13 cases (54%) the SYTSSX2, and 2 of 2 cases (100%) the SYTSSX4 one (Table 2B) . The hybridization of the same blots with the -pTyr antibody revealed that 18 of 19 cases expressed a phosphorylated PDGFRß.

	Coexpression of KIT and PDGFRß Receptors

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Coexpression of KIT and... Molecular Analysis: Expression... ICC Analysis for KIT... DISCUSSION REFERENCES

 
Coexpression of both receptors was detected in 7 cases only (16%). These cases are reported in Table 1 (case nos. 19, 21, 25, 26, 36, 41, and 43). There were 6 monophasic and 1 biphasic SSs. Four cases expressed the SYTSSX1 fusion transcript, 2 the SYTSSX2, and 1 the SYTSSX4.

In 14 cases (7, 9, 10, 11, 12, 15, 16, 18, 23, 24, 33, 37, 38, and 39; 32.5%), neither KIT or PDGFRß resulted to be expressed. There were 10 cases of SYTSSX1 and 4 cases of SYTSSX2; 7 were biphasic and 7 were monophasic in subtype. Overall, 67.5% of the entire set expressed at least one of the activated receptors.

	Molecular Analysis: Expression of the Tyrosine Kinase Ligands

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Coexpression of KIT and... Molecular Analysis: Expression... ICC Analysis for KIT... DISCUSSION REFERENCES

 
SCF.
The expression SCF was analyzed by RT-PCR as described in "Materials and Methods" using as positive control the M07e cell line.

The data are summarized in Table 1 . All of the cases with an activated KIT receptor expressed SCF. Eight cases resulted negative for both SCF and KIT receptor expression, whereas only SCF expression was detected in 14 cases. In 3 cases (nos. 2, 3, and 30; Table 1 ), the expression of SCF could not be associated to any biochemical data because of the lack of material.

Two specific SCF bands (494 and 409 bp, named L and S, respectively) were detected and correspond to two SCF isoforms, which are both biologically active.

In all of the cases where SCF expression was not observed, the amplification of the housekeeping ß-actin gene was performed twice as additional control.

PDGFB.
The expression of PDGFRß ligand was analyzed by RT-PCR as described in "Materials and Methods" using as positive control the 5A cell line.

All cases with an activated PDGFRß resulted positive for its ligand. The analysis extended to all samples demonstrated PDGFB expression in 16 tumors in which the receptor was not detected (Table 1) . Seven cases resulted negative for both the ligand and the receptor.

	ICC Analysis for KIT Receptor

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Coexpression of KIT and... Molecular Analysis: Expression... ICC Analysis for KIT... DISCUSSION REFERENCES

 
We complemented our study with two antibodies provided by Dako and Santa Cruz Biotechnology, respectively, which recognize very similar epitopes in the COOH terminus of the receptor. No positive cases were detected by ICC screening with the Dako antibody. The Santa Cruz Biotechnology antibody identified other 12 positive cases (15 in total), of which, 6 fit with IP/WB experiments (Table 1) .

	DISCUSSION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Coexpression of KIT and... Molecular Analysis: Expression... ICC Analysis for KIT... DISCUSSION REFERENCES

 
In this study, we describe a biochemical and molecular analysis of KIT and PDGFRß in 43 cases of SS surgically treated in our institute, of which, cryopreserved material was available. Expression and phosphorylation status of these receptors were assessed by IP and WB experiments, whereas RT-PCR was used to demonstrate the presence of their ligands SCF and PDGFB.

KIT receptor was observed in 48 and 41% (45% total), whereas PDGFRß in 54 and 33% (45% total) of monophasic and biphasic SS cases, respectively.

With respect to fusion transcript type and excluding 2 cases with SYTSSX4 fusion transcript, KIT was more expressed in cases carrying the SYTSSX1 (48 versus 38%), whereas PDGFRß resulted more frequently expressed in SSs carrying the SYTSSX2 (54 versus 37%). However, we do not speculate on these differences because the low number of cases do not allow any reliable statistical evaluation.

KIT, when expressed, resulted phosphorylated and thus activated (Table 2) , whereas for PDGFRß, this occurred in all but 1 case. However, in this case (no. 20), PDGFB was detected, leading us to suppose that the autocrine loop presence and the lack of phosphorylation could be due to the experimental procedure we followed because we used the unbound protein extracts derived from KIT immunoprecipitation, most likely influencing the phosphatase activity.

Because previous studies indicate the autocrine loop as the most frequent mechanism for KIT and PDGFRß activation in sarcomas, we investigated the presence of their ligands, SCF and PDGFB, in our cases collection. Their detection in all of the samples expressing phosphorylated receptors led us to suppose the presence of an autocrine loop. This hypothesis is supported by our sequencing data performed in two cases of SSs (data not shown), which, showing a complete wild-type c-Kit cDNA, exclude the presence of activating mutations. For PDGFRß, to our knowledge, activating mutations have not been thus far described (17) . Nevertheless, we cannot exclude that a paracrine loop might coparticipate in RTK activation.

Overall, 70% of the cases expressed at least one of these receptors, KIT or PDGFRß. More in detail, most of the cases expressing KIT were negative for PDGFRß, suggesting that a mutually exclusive expression of the two tyrosine kinases is present in this tumor type. Only 7 cases showed a coexpression of the both activated tyrosine kinases. The coexpression did not correlate with the SYTSSX fusion transcript type but was mostly restricted to the monophasic subtype (6 cases of 7).

The present data are in keeping with our previous observations (13) , where c-Kit cDNA was detected in almost all of the cases analyzed and where KIT immunoreactivity, using the Santa Cruz Biotechnology antibody, was observed in biphasic SS mainly restricted to the epithelial component. A similar immunophenotype was recently reported in a survey of 14 cases of SSs, 6 biphasic and 8 monophasic (18) , and in 1 primary SS pulmonary tumor (19) . Because the literature data report a low percentage of KIT immunoreactivity in SS (20 , 21) and our previous and present IP/WB data strongly support the presence of KIT protein, we broadened our study with ICC using two antibodies. No case turned out to be positive with the Dako antibody in keeping with the literature. By contrast, 9 of 15 Santa Cruz Biotechnology-positive cases did not match the IP/WB data. The poor correlation strongly speaks in favor of lack of specificity of the Santa Cruz Biotechnology antibody. Instead, the apparent inconsistency between Dako/ICC and IP/WB data is ascribable to the different recognized epitope and to the different sensitivity of the methodologies applied.

Pathological activation of RTKs may be achieved by mutation (as with gastrointestinal stromal tumors), by chimeric TK translocation (as with TPM3-ALK and ETV6-NTRK3), or by autocrine/paracrine stimulation, which may be produced by chromosomal translocations not directly involving chimeric RTKs. Recently, the use of inhibitory molecules targeting activated RTKs has become a valid therapeutic alternative in RTK-positive tumors, alone or in association with the traditional chemotherapy. More in detail, Imatinib (Glivec, Novartis) has shown to be effective in patients with gastrointestinal stromal tumor carrying activating KIT mutations. In preclinical setting, this drug has revealed a growth inhibition on small cell lung cancer cell lines activated by autocrine loop, although at a lower level than a new compound, SU11248 (22) , and has been shown to be able to inhibit KIT autocrine loop activation sustained by a EWS-ETS family translocation in ES/pPNETs (23) .

Here, we provide the molecular and biochemical foundation for application of RTK inhibitor-based treatment in at least 70% of SS cases where, according with what observed in ES/pPNET preclinical model and in surgical specimens, KIT activation is sustained by a translocation mediated autocrine/paracrine loop. After the evidence of a therapeutic response in dermatofibrosarcoma protuberans, we can foresee a successful usage of Imatinib also in the PDGFRß-positive SSs. Regarding KIT-positive cases, a development of new drugs, able to effectively inhibit all of the different pathogenetically activated c-Kit forms such as PP1 (24) but safely useable in a clinical setting, is envisaged. The mechanism used by these new ATP competitor molecules, although less specific, may allow a wider possibility of applications in a multitarget situation such as in tumors carrying more than one activated RTK, a not infrequent event in SSs according to microarray data (14 , 15) or showing evidence of activation of alternative RTK receptors (25) .

	FOOTNOTES

 
Grant support: Ministero della Sanità, Ricerca Finalizzata 2001 Italy, and Associazione Italiana per la Ricerca sul Cancro 2001 Grant 420.198.122.

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

Notes: E. T. and L. B. contributed equally to this work. M. A. P. and S. P. are senior coauthors.

Requests for reprints: Silvana Pilotti, Unit of Experimental Molecular Pathology, Department of Pathology, Istituto Nazionale per lo Studio e la Cura dei Tumori, via G. Venezian, 1, 20133 Milan, Italy. Phone: 39-02-23902260; Fax: 39-02-23902756; E-mail: silvana.pilotti{at}istitutotumori.mi.it

Received 8/19/03; revised 10/10/03; accepted 10/16/03.

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Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Coexpression of KIT and... Molecular Analysis: Expression... ICC Analysis for KIT... DISCUSSION REFERENCES

 

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