Characterization of fibroblast growth factor receptor 2 overexpression in the human breast cancer cell line SUM-52PE
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DescriptionINTRODUCTION: The FGFR family of receptor tyrosine kinases includes four members, all of which are highly alternatively spliced and glycosylated. For FGFR2, alternative splicing of the second half of the third Ig-like domain, involving exons IIIb and IIIc, is a mutually exclusive choice that affects ligand binding specificity and affinity [1,2,3]. It appears that the second half of the third Ig-like domain can dictate high affinity for FGF-2 or keratinocyte growth factor (KGF), whereas affinity for FGF-1 appears to remain the same . Alternative splicing of the carboxyl terminus has been shown to involve at least two different exons that can produce at least three different variants. The C1-type and C2-type carboxyl termini are encoded by the same exon, and have two different splice acceptor sites, whereas the C3-type carboxyl terminus is encoded by a separate exon . The biologic significance of the C1 carboxyl terminus, as compared with the shorter C3 variant found primarily in tumorigenic samples, has been studied in NIH3T3 transfection assays, in which C3 variants were able to produce three times more transformed foci in soft agar than C1 variants (both IIIb), whereas full length FGFR2 and FGFR1 (both IIIc variants) showed no transforming activity . Previous studies [5,6] have found amplification and overexpression of FGFR2 in 5-10% of primary breast cancer specimens. A recent study  done using a tissue array consisting of 372 primary breast cancer specimens found a 5% incidence of FGFR2 amplification. To our knowledge, none of the HBC cell lines studied thus far have an FGFR2 gene amplification, although overexpression of FGFR2 message and protein has been documented for some breast cancer cell lines [6,8,9]. SUM-52PE is a breast cancer cell line previously isolated in our laboratory that grows under serum-free and epidermal growth factor-free conditions, has high levels of tyrosine-phosphorylated membrane proteins, and has the capacity to invade and grow under anchorage-independent conditions [10,11,12]. This cell line exhibits all of the important hallmarks of transformed, highly malignant cells. Therefore, SUM-52PE was used as a model to study the diversity of FGFR2 expression in a breast cancer cell line that has true amplification and overexpression of FGFR2. OBJECTIVES: This study was conducted to examine the degree of FGFR2 amplification and overexpression in the breast cancer cell line SUM-52PE. Subsequent sequencing and characterization of individual FGFR2 variants cloned from the SUM-52PE cell line was completed to determine the complexity of FGFR2 alternative splicing in the context of a highly metastatic breast cancer cell line. METHODS: Southern, Northern and Western blot analyses were done in order to determine the degree of FGFR2 amplification and overexpression in the breast cancer cell line SUM-52PE. Individual FGFR2 variants were cloned out of SUM-52PE using FGFR2-specific primers in a reverse transcription (RT) polymerase chain reaction (PCR). FGFR2 cDNAs were characterized by restriction fragment analysis, sequencing and transient transfection into 293 cells to examine the protein expression of each FGFR2 clone. RESULTS: The results of the Southern blot showed that there was a 12-fold amplification of FGFR2 in the SUM-52PE cell line. Northern blot analysis of SUM-52PE showed FGFR2 transcripts to be highly overexpressed compared with other breast cancer cell lines and normal HME cells. Several overexpressed bands of approximately 6.3, 5.0, 4.0, and 2.8kb were observed in SUM-52PE cells. The most prominent band, at 2.8kb, was so abundant that it was difficult to discern other individual bands clearly. Western blot analysis showed that both normal HME and HBC cells expressed two FGFR2 variants of 95 and 135kDa. The SUM-52PE cell line greatly overexpressed not only these two polypeptides, as compared with HME and HBC cells, but also overexpressed two unique variants of FGFR2 - 85 and 109kDa polypeptides - as well as several smaller polypeptides in the 46-53kDa range. The antibody used in Western blot analysis only recognizes FGFR2 isoforms that express the C1 carboxyl termini, therefore greatly underestimating the actual number of different FGFR2 variants that are overexpressed in this cell line. PCR was performed to determine the proportion of C1/C2 variants as compared with C3 variants in the SUM-52PE cell line. Results of this analysis indicated the presence of all three types of variants in this cell line, although the C1/C2 variants were predominant as compared with the C3 variants in SUM-52PE. Four different FGFR2-C1 clones were isolated and sequenced from SUM-52PE cells, which differed in their signal sequence, first Ig-like loop, and acid box. Two FGFR2-C2 clones were isolated from the SUM-52PE cell line, which were identical to each other except for the variable expression of the number of Ig-like domains (two or three). Three C3 clones were isolated and sequenced, two of which have not previously been described in the literature. Clone C3-#3 contained two Ig-like domains, but no acid box. C3-#5 was missing the first two Ig-like domains and the acid box, but did contain the third Ig-like domain. DISCUSSION: There is an extensive amount of evidence implicating erbB-2, a gene that is overexpressed in approximately 30% of breast cancer cases, as a breast cancer gene . The identification of other breast oncogenes that function in the remaining 70% of cases is an ongoing challenge, as is establishing a causal role for such oncogenes in HME cell transformation. FGFR1 and FGFR2, previously established oncogenes, were found to be amplified within large amplicons on 8p11 and 10q26, respectively, in the breast cancer cell line SUM-52PE . Previous studies have shown that the FGFR2 gene is amplified in about 5-10% of breast cancer cases. Our results showed that SUM-52PE cells overexpressed many alternatively spliced isoforms of FGFR2 at both the transcript and protein level as compared with normal HME cells. The variability in FGFR2 isoform expression is complex and involves exon IIIb/c, which encodes the second half of the third Ig-like loop; variations in the carboxyl terminal end of the receptor, involving the C1/C2 or C3 domains; and variable expression of the Ig-like loops and acid box in the extracellular portion of the receptor. The characterization of three unique FGFR2 isoforms that were cloned from SUM-52PE may build on the findings of others concerning the transforming potential of FGFR2 variants . In particular, because it has been demonstrated that expression of C3-IIIb variants may have more transforming activity than C1-IIIb variants, differences between the three C3 clones we have isolated may provide information regarding the influence of particular structural domains on transforming potential. Ongoing studies are aimed at characterizing the transforming ability of FGFR2 isoforms obtained from SUM-52PE cells by transducing these genes into normal HME cells. By overexpressing FGFR2 isoforms in a physiologically relevant system, we hope to determine the isoform(s) that acts in a dominant way in the process of cell transformation, and to determine whether different regions present in individual clones drive specific phenotypes associated with transformation.
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