Abstract 1235: Inducible overexpression of bHLH transcriptional regulator Id4 abrogates tumorigenicity of prostate cancer xenografts
Authors: Patel Divya , Morton Derrick J. , Korang-Yeboah Maxwell , Gorantla Yamini , Bhosle Sushma , Nagappan Perri , Bowen Nathen , Chaudhary Jaideep 
Published: August 2015

Introduction: Inhibitor of DNA binding/differentiation 4 (Id4) has been shown to be highly expressed during prostate development. However, in many cancers including prostate, Id4 is epigenetically silenced due to promoter hyper-methylation. In this study we developed a doxycycline inducible system for Id4 overexpression and show that controlled increase of Id4 in an aggressive metastatic prostate cancer cell line DU145, which lacks Id4 expression results in attenuation of tumor forming ability of this cell line and confirming its role as a tumor suppressor.

Methods: Tetracycline inducible system was used to stably overexpress Id4 in DU145 cells. Empty vector backbone was used as a control. The Id4 overexpressed (DU(+)iId4) and vector control (DU(+)iVC) cells were used to investigate migration (scratch wound assay) and anchorage independent growth (soft agar assay). Tumorigenicity of DU(+)iId4 and DU(+)iVC cells was investigated in Scid beige mice. The gene expression profile between DU(+)iId4 and DU(+)iVC cells was analyzed by RNAseq. The DU(+)iId4 and DU(+)iVC tumors from mice were used for immunohistochemistry to validate prostate cancer pathways and RNAseq data.

Results: Id4 was cloned in pcDNA4/TO/myc-His version A and stably transfected and clonally selected for overexpression in DU145 cells stably expressing pcDNA6/TR. The overexpression of Id4 on induction by doxycycline (a tetracycline analogue) was confirmed by qRT-PCR and western blotting. A clone with maximum expression was selected for further studies. On inducing the DU145 cells with doxycycline, an increase in apoptosis was observed as compared to un-induced DU145 as well as cells expressing the empty control vector. Decrease in migration and anchorage independent colony forming ability was also observed upon induction of Id4 in scratch wound and soft agar assay, respectively. Subcutaneous injection of DU145 cells in Scid beige mice resulted in formation of tumors, however upon induction of Id4 expression by doxycycline from day 0 or 2 weeks after injection of the cells showed a significant decrease in the tumor forming ability of the DU145 cells as evident from the reduction in the tumor weight and volume. Immunohistochemistry on tumor tissues was found to be in support of the RNAseq data.

Conclusion: This study reports for the first time that conditional overexpression of tumor suppressor Id4 in prostate cancer cell lines DU145 results in diminution of tumor formation. The data describes use of a system that allows for the temporal regulation of Id4 expression in vivo which could act as a preliminary step in mimicking controlled delivery of Id4 for therapeutic purpose.

Acknowledgement: This study was supported by NIH/NCI RO1 CA128914 and NIH/NCRR/RCMI G12MD007590.

Citation Format: Divya Patel, Derrick J. Morton, Maxwell Korang-Yeboah, Yamini Gorantla, Sushma Bhosle, Perri Nagappan, Nathen Bowen, Jaideep Chaudhary. Inducible overexpression of bHLH transcriptional regulator Id4 abrogates tumorigenicity of prostate cancer xenografts. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1235. doi:10.1158/1538-7445.AM2015-1235