Abstract 4438: Changes in Nrdp1 regulation of ErbB3 in androgen-dependent vs. independent prostate cancer
Authors: Rosalinda M. Savoy, Salma Siddiqui, William H. Fry, Kermit L. Carraway, Paramita Ghosh
Journal: Cancer Research (2014)

Abstract In prostate cancer, the androgen receptor (AR) is a major regulator of gene transcription. We previously demonstrated that the E3 ubiquitin ligase Nrdp1 is transcriptionally regulated by the AR [Chen, L., Cancer Res, 2010]. The effect of the AR on ErbB3 is mediated by Nrdp1, which regulates the degradation of ErbB3. Therefore, we investigated the function of the various domains of Nrdp1 on ErbB3 in prostate cancer. ErbB3 is a major factor in cell growth and proliferation so finding ways to regulate it would be beneficial for patient survival. We found that the Nrdp1 protein had two splice variants, a full length 36kDa protein and a C-terminal 28kDa protein. In addition, the full-length protein remained in the cytoplasm of LNCaP cells, which are androgen dependent, while the 28kDa protein went into the nucleus of C4-2 cells, an androgen independent subline of LNCaP cells. To further investigate the subcellular difference and role of these two splice variants on ErbB3, we used 4 flag tagged vectors that contain either full-length Nrdp1 (Nrdp1), an N-terminal Nrdp1 containing amino acids 1-169 (Zn), a C-terminal Nrdp1 containing amino acids 169-317 (32) , and an Nrdp1 lacking the coiled coil domain (ΔCC). We found that the ΔCC vector caused an increase of cell proliferation in LNCaP cells, an androgen dependent cell line, with no to little change in cell proliferation in its androgen independent sub cell lines, LNCaP R273H (LNCaP stably transfected with a mutated p53), C4-2, and C4-2B, and PC3 and PC3 WT-AR cells. Despite this, we found that the ΔCC protein caused a decrease of ErbB3 protein in all cell lines. We also performed subcellular fractionation on these cell lines transfected with the Nrdp1 flag vectors. All of the Nrdp1 proteins were found in the cytoplasm of the cell lines. In addition, the ΔCC protein was also found in the nuclear fraction of LNCaP R273H, C4-2, and C4-2B, while in LNCaP cells the Zn protein was also found in the nuclear fraction and in PC3 cells the 32 protein was also found in the nuclear fraction. In LNCaP cells, ErbB3 was found in the nucleus when transfected with Nrdp1 protein, Zn protein, and 32 protein, but found in the cytoplasm when transfected with the ΔCC protein. However, in LNCaP R273H, C4-2, and C4-2B cell lines, ErbB3 was found in the nucleus when transfected with all of the Nrdp1 vectors. Based on available data, we concluded that the coiled coil domain is important for ErbB3 protein regulation, but it had opposing effects in castration sensitive and resistant cells. In castration sensitive LNCaP cells, the coiled coil domain of Nrdp1 likely suppresses ErbB3-mediated cell proliferation by preventing ErbB3 translocation from the nucleus to the cytoplasm. However, in castration resistant cells, the coiled coil domain prevents the degradation of ErbB3 by Nrdp1. Further studies are required to determine the mechanism of action of the Nrdp1 coiled coil domain in these two phenotypes. Citation Format: Rosalinda M. Savoy, Salma Siddiqui, William H. Fry, Kermit L. Carraway, Paramita Ghosh. Changes in Nrdp1 regulation of ErbB3 in androgen-dependent vs. independent prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4438. doi:10.1158/1538-7445.AM2014-4438