Malignant Melanoma of Soft Parts (MMSP) is an aggressive tumor of soft tissue. This tumor occurs due to the function of dominant oncogene named EWS/ATFl, which is created by the chromosomal translocation fusing of Ewings Sarcoma oncogene (EWS) to the DNA binding (bZIP) domain of the cellular transcription factor ATFl . In normal cells, ATFl is directly phosphorylated by protein kinase A (PKA) and functions as a cAMP regulated transcriptional activator, upon directly binding to ATFl binding sites present in cAMP inducible promoter. In contrast, when assayed in a transient transfection, the EWS/ATFl fusion protein is a strong constitutive activator of several cAMP-inducible promoters, including the somatostatin promoter. Based on the transcriptional and tumor specific properties of EWS/AT...[ Read more ]

Malignant Melanoma of Soft Parts (MMSP) is an aggressive tumor of soft tissue. This tumor occurs due to the function of dominant oncogene named EWS/ATFl, which is created by the chromosomal translocation fusing of Ewings Sarcoma oncogene (EWS) to the DNA binding (bZIP) domain of the cellular transcription factor ATFl . In normal cells, ATFl is directly phosphorylated by protein kinase A (PKA) and functions as a cAMP regulated transcriptional activator, upon directly binding to ATFl binding sites present in cAMP inducible promoter. In contrast, when assayed in a transient transfection, the EWS/ATFl fusion protein is a strong constitutive activator of several cAMP-inducible promoters, including the somatostatin promoter. Based on the transcriptional and tumor specific properties of EWS/ATFl, my project aims to develop a cytotoxic gene therapy for MMSP, using the somatostatin promoter to achieve toxic gene expression specifically in tumor cells. In addition, we evaluated recombinant adenovirus as a gene delivery system for MMSP cells.

The major findings are as follows, the MMSP-derived cell line, DTCl (D7ltk cells) expressing the herpes simplex virus thymidine kinase (HSV-tk gene) under the control of the somatostatin promoter showed high sensitivity to ganciclovir (GCV). The sensitivity was dependent on the ATF binding site present in the somatostatin promoter indicating that it is EWS/ATFl dependent. Several recombinant adenoviruses containing EWS/ATFl responsive promoters were constructed and used to infect two MMSP derived cell lines, DTCl and Su-ccs-1. Surprisingly, the viral borne promoters were not responsive to EWS/ATFl in either cell type. These promoters could be activated by adenovirus ElA protein, indicating that the promoters were successfully delivered to the nucleus of MMSP cells and can be activated, at least by ElA.

In conclusion, our results show that the somatostatin promoter can be exploited as tumor specific promoter for cytotoxic pro-drug gene therapy in MMSP, using HSV-tk as the cytotoxic gene and GCV as a pro-drug. However, the commonly used recombinant adenovirus system for cytotoxic gene delivery may not be useful in MMSP cells due to loss of responsiveness to the endogenous EWS/ATFl protein.