PMC:13919 / 36490-43390 JSONTXT

{"project":"Colil","denotations":[{"id":"T5","span":{"begin":1146,"end":1147},"obj":"1851551"},{"id":"T6","span":{"begin":1148,"end":1149},"obj":"7705943"}],"namespaces":[{"prefix":"_base","uri":"http://pubannotation.org/docs/sourcedb/PubMed/sourceid/"}],"text":"Discussion\nThe progression of cells from the normal to neoplastic state is a \t\t multistep process that involves alterations in multiple signaling pathways. \t\t Both epidermal growth factor receptor and erbB-2 have been identified as \t\t signaling molecules that play a dominant role in breast cell transformation \t\t [28,29]. There is an extensive \t\t amount of evidence for erbB-2 as a breast cancer gene that is \t\t overexpressed in approximately 30% of breast cancer cases [13]. The identification of other breast oncogenes that \t\t function in the remaining 70% of cases is an ongoing challenge, as is \t\t establishing a causal role for such oncogenes in HME cell transformation.\nLarge regions of gene amplification in cancer cells can be detected by \t\t techniques such as comparative genomic hybridization and high-density arrays, \t\t which helps to localize areas that may contain functional oncogenes. FGFR1 and \t\t FGFR2, which were previously established as candidate breast cancer oncogenes, \t\t were found to be amplified within large amplicons on 8p11 and 10q26, \t\t respectively, in the breast cancer cell line SUM-52PE [14]. Previous studies [5,6,7] have shown that the FGFR2 gene is \t\t amplified in about 5-10% of cases. Because genes can be amplified without being \t\t overexpressed [14], we chose to examine whether FGFR2 \t\t may be an important oncogene in this breast cancer cell line by examining its \t\t expression at the mRNA and protein levels. Our results showed that SUM-52PE \t\t cells overexpressed many alternatively spliced forms of FGFR2 at both the \t\t transcript and protein level, as compared with normal mammary epithelial cells. \t\t By contrast, FGFR1 is not expressed in SUM-52 cells.\nIn contrast to SUM-52PE cells, FGFR2 expression at the message level \t\t is very low in HME cells. Indeed, even prolonged exposure of Northern blots to \t\t film did not allow the visualization of FGFR2 message in normal cells. However, \t\t Western blots did indicate the presence of FGFR2 protein in HME cells. To \t\t resolve this apparent paradox, two rounds of RT-PCR were performed using HME \t\t cell-derived RNA, which resulted in the isolation of three alternatively \t\t spliced forms of FGFR2 message, each of which expressed the IIIb exon. The \t\t predicted protein products of these clones correspond to that which was \t\t observed in Western blots.\nThe variability in FGFR2 isoform expression is complex and involves \t\t exon IIIb/c, which encodes the second half of the third Ig-like loop, \t\t variations in the carboxyl terminal end of the receptor involving the C1/C2 or \t\t C3 domains, and variable expression of the Ig-like loops and acid box in the \t\t extracellular portion of the receptor.\nAlternative splicing of the FGFR2 mRNA that encodes the carboxyl \t\t terminus has been shown to involve at least two different exons, which can \t\t produce at least three different variants. The C1- and C2-type carboxyl termini \t\t are encoded by the same exon, having two different splice acceptor sites, \t\t whereas the C3-type carboxyl terminus is encoded by a separate exon [4]. The biologic significance of the full-length carboxyl \t\t terminus (C1), as compared with the truncated variant found primarily in \t\t tumorigenic samples (C3), has been studied in NIH3T3 transfection assays. The \t\t IIIb variants KGF receptor (C1) and K-sam C3 were both able to produce \t\t transformed foci, growth in soft agar and tumorigenicity in nude mice as \t\t compared with full-length IIIc variants of FGFR2 and FGFR1, which were not \t\t transforming [4]. The question of whether C3 variants are \t\t more transforming than C1 variants remains to be determined conclusively, \t\t because the number of transformed foci obtained using K-sam C3 was only \t\t threefold greater than that obtained using KGF receptor (C1) variants. A \t\t significant difference between the C3 and C1 termini is that the former does \t\t not contain the binding site for phospholipase Cγ. Thus, the ability of \t\t the variants of FGFR2 containing the C3 terminus to transform 3T3 cells \t\t suggests that signaling through this pathway is not necessary for FGFR2 IIIb to \t\t act as an oncogene.\nThe panel of FGFR2 isoforms isolated from SUM-52PE includes several \t\t unique and previously unreported isoforms. The first of these unique variants, \t\t C1-#10, contains a large deletion of the 5' region that includes the \t\t first Ig-like domain as well as the signal sequence, which could have \t\t interesting cellular localization and cell signaling properties due to the \t\t absence of part of the signal sequence. Clones C3-#3 and C3-#5 have not \t\t previously been reported and are missing the first Ig-like domain and acid box. \t\t C3-#5 is also missing the second Ig-like domain. The characterization of these \t\t three unique isoforms may build upon the findings of others concerning the \t\t transforming potential of FGFR2 variants [4]. In \t\t particular, because it has been demonstrated that C3-IIIb variants may have \t\t more transforming activity than C1-IIIb variants, differences between the three \t\t C3 clones we have isolated may provide information on the influence of \t\t particular structural domains on transforming potential.\nPrevious studies that examined FGFR2 expression in prostate cancer \t\t have suggested that a change in the expression from the exon IIIb to IIIc \t\t isoform correlates with a progression from an androgen-sensitive to an \t\t androgen-insensitive state. RT-PCR analysis on the SUM-52PE breast cancer cell \t\t line showed that this cell line exclusively expressed the IIIb FGFR2 isoform \t\t (Fig. 3). Exon IIIb expression was also exclusively found \t\t in normal luminal HME cells (data not shown). This suggests that exon IIIb to \t\t IIIc switching is not necessary for FGFR2 to act as an oncogene when the gene \t\t is amplified. Rather, overexpression of one of the common IIIb isoforms or one \t\t of the novel variants may be important in driving transformation of HME cells. \t\t Ongoing studies are aimed at characterizing the transforming ability of \t\t individual FGFR2 isoforms obtained from SUM-52PE cells. These studies will \t\t directly test the hypothesis that specific FGFR2 isoforms have transforming \t\t activity towards HME cells and will compare variants with the different \t\t carboxyl termini. Overexpression of the C1-#38 and C3-#5 FGFR2 clones has been \t\t successfully accomplished in both the MCF-10A and H16N2 HME cell lines, and \t\t these cells have acquired phenotypes that distinguish them from parental cells \t\t (to be described in detail in a separate paper that is in preparation). Thus, \t\t by overexpressing FGFR2 isoforms in a physiologically relevant system, we hope \t\t to determine the isoform(s) that acts in a dominant way in the process of cell \t\t transformation, as well as to determine whether different regions present in \t\t individual clones drive specific phenotypes associated with transformation."}