The role of PDGFB in dermatofibrosarcoma protuberans and giant cell fibroblastoma

Sammanfattning: Dermatofibrosarcoma protuberans (DFSP) and giant cell fibroblastoma (GCF) are rare invasive tumors of the dermis. GCF is considered the juvenile form of DFSP. Both DFSP and GCF are characterized by the presence of chromosomal aberrations involving chromosomes 17 and 22. Supernumerary ring chromosomes are more common in DFSP, while the translocation t(17;22)(q22;q13) is the most common rearrangement observed in GCF. The aim of this thesis was to determine the molecular events behind these chromosomal aberrations. Chromosomal breakpoints were characterized by FISH walking and Southern blot analysis, which revealed that the platelet derived growth factor B-chain gene (PDGFB proto-oncogene or c-sis) on 22q was rearranged in DFSP/GCF. Using 5' RACE, the collagen type I alpha 1 gene (COL1A1) was demonstrated to be the 17q gene involved in the rearrangement. RT-PCR of the COL1A1/PDGFB fusion genes in 20 tumors, demonstrated that each fusion gene consisted of an exon of the COL1A1 alpha-helical domain fused in-frame to PDGFB exon 2, in all tumors analyzed. This is remarkably similar to Simian sarcoma virus and Parodi-Irgens feline sarcoma virus. These viruses consists of viral genes fused to the exon 2 of a PDGFB gene, thus removing all elements negatively regulated transcription and translation of the PDGFB gene. The COL1A1/PDGFB fusion gene has not been found in any other tumor type and was present in all tumors studied by RT-PCR. This fusion gene may thus be considered diagnostic for DFSP/GCF. Two DFSP case studies were also performed; In one DFSP tumor, quadruplicate copies of the fusion gene were found on a large marker chromosome. In another DFSP case, RT-PCR demonstrated the presence of a fusion gene with a COL1A1 exon 37 to PDGFB exon 2 on the mRNA level, a fusion previously only seen in a GCF tumor. This indicates that the location of the fusion breakpoint in COL1A1 has little effect on tumor type. In order to study the fusion gene in vitro, a COL1A1/PDGFB expressing cell line was constructed by transfection of NIH-3T3 cells with a cosmid containing a tumor-derived fusion gene. These cells were transformed, displaying increased growth, loss of contact inhibition and formation of tumors in nude mice. Using pulse-chase analysis and immunoprecipitations the production of a PDGFB-like protein was demonstrated. It was dimerized, localized and processed in a similar way to normal PDGFB. All COL1A1 segments were removed from the COL1A1/PDGFB protein during this processing. Upon addition of a PDGF receptor tyrosine kinase-specific inhibitor, the transformed COL1A1/PDGFB expressing cells reverted back to a normal phenotype. Thus, in a collagen expressing cell, the presence of a COL1A1/PDGFB gene results in the production of mature PDGF-BB dimers. Since DFSP/GCF cells express both collagen and PDGF receptors, then a similar mechanism is likely to be occurring in these cells. In DFSP/GCF tumor cells, it is likely that COL1A1 acts solely as a production vehicle for PDGFB, the end product of the fusion gene being mature PDGF-BB dimers which stimulate the cell in an autocrine fashion. Interruption of this autocrine stimulation using PDGF receptor inhibitors could be a possible treatment of DFSP/GCF, but is complicated by the presence of other genetic changes which tumor cells have likely undergone.