[Objectives] Clear cell carcinoma is an important chemotherapy-resistant subtype in epithelial ovarian carcinomas, and associated with poor prognosis of the patients. Understanding of molecular carcinogenesis of this tumor is mandatory to develop effective treatments, however, gene abnormalities of clear cell carcinoma have not been fully understood. Recently, novel oncogenic genes or genetic alterations in various malignancies were identified using the full-length cDNA expression library on a retroviral vector and subsequent functional screening, such as the EML4-ALK fusion gene in lung cancer. To identify the genes responsible for clear cell carcinogenesis, we performed a functional screening using a constructed retroviral expression library of the human ovarian clear cell carcinoma cell.

[Methods] 1) cDNA library in pDON-AI/Neo retroviral plasmid was constructed from mRNA extracted from clear cell carcinoma RMG-1 cells. NIH3T3 cells were seeded and transfected with the retroviral supernatant. Transformed foci were identified and the genomic DNA from transformed foci was extracted for sequencing. 2) The mRNA and protein levels of candidate genes were examined using real time RT-PCR and immunohistochemistry in 62 cases of ovarian carcinoma tissues. 3) The PCR-direct sequencing was performed in 24 cases of clear carcinoma cell samples. 4) Transforming activity of isolated genes was evaluated using NIH3T3 cells.

[Results] 1) Among 20 genes isolated from the transformed cells, we focused on two genes, Sec61B (protein translocation regulator in ER) and C1ORF38 (Toll-like receptor modulator). In addition, mutations in DVL1 (Wnt signal modulator) were found in C-terminal region. 2). The mRNA levels of three candidate genes and the protein expression of Sec61B and

C1ORF38 were two to three-fold increased (p<.005) in clear cell carcinoma compared with other histological subtypes including serous, mucinous endometrioid carcinomas. 3) Only one case had the point mutation in DVL1 gene in clear cell carcinoma samples. 4) Forced expression of Sec61B or C1ORF38 using pMEI/Neo vector resulted in plaque formation of NIH3T3 cells.

[Conclusions] We identified C1ORF38 and Sec61B as possible functional genes in ovarian clear cell carcinogenesis using retrovirus-based cDNA expression library. C1ORF38 and Sec61B were over-expressed in ovarian clear cell carcinoma compared with other histological subtypes. Further study is warranted to characterize the functions of these genes in clear cell carcinoma.

Citation Format: Tanri Shiozawa, Tsutomu Miyamoto, Akihisa Suzuki, Ryo-ichi Asaka. Identification of novel genes involved in clear cell carcinogenesis of the ovary using retrovirus-based cDNA expression library. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3002. doi:10.1158/1538-7445.AM2013-3002