Abstract B26: Roles of NgBR in promoting Ras translocation and Ras signaling in human breast cancer cells

NgBR is a type I receptor with a single transmembrane domain. Our previous work has shown that axon guidance gene family Nogo-B and its receptor (NgBR) are essential for chemotaxis and morphogenesis of endothelial cells in vitro and blood vessel formation in Zebrafish. Here, we demonstrate that NgBR binds and translocates farnesylated Ras to plasma membranes, which results in the activation of PI3K-Akt and Raf-MEK-ERK signaling pathways in human breast cancer cells. Comparing NgBR to other sequences in the NCBI database reveals that regions of the cytoplasmic domain have a high degree of similarity (49%) to the cis-lsoprenyl Disphosphate Synthases (cis-IPPS) family of lipid modifying enzymes. However, NgBR lacks key residues required for catalytic activity, but contains a conserved hydrophobic tunnel for shuttling prenyl lipids and/or prenylated proteins. Farnesylation of Ras is essential for Ras translocation and activation. Direct interaction between NgBR and farnesylated Ras has been demonstrated by co-immunoprecipitation of MBP-NgBR from incubation of purified recombinant MBP-NgBR and farnesylated Ras protein. Knockdown of NgBR in MDA-MB-231 human breast cancer cells remarkably reduces the amount of membrane-associated Ras. These results suggest that NgBR is a potential modulator for Ras translocation. Immunohistochemistry analysis of human breast tumor tissue arrays revealed that NgBR is highly expressed in human breast cancer tissues. We further used real-time PCR to determine the copy number of NgBR transcripts in human breast tumor tissues. Accompanying pathology reports were used to categorize the 84 breast tumor tissues into four different grades from I to IV. Real-time PCR results show that NgBR expression is significantly higher in Grade II and Grade III-IV breast tumors when compared with normal breast samples (12 samples) and Grade I breast tumor samples (21 samples). 62% of Grade II (34 samples) and 55% of Grade III-IV (29 samples) breast tumors had increased NgBR transcripts (>50% higher) when compared with normal breast tissues. These data suggest that NgBR expression is strongly associated with advanced breast cancers. Interestingly, NgBR is highly expressed in several breast cancer cells such as MDA-MB-231 cells as compared to immortalized mammary epithelial cells MCF-10A. Knockdown of NgBR expression in MDA-MB-231 breast cancer cells decreases cancer cell colony formation, and abolishes EGF-stimulated Ras activation and phosphorylation of Akt in MDA-MB-231 cells. In addition, overexpression of NgBR caused the transformation of NIH-3T3 cells via enhancing Ras plasma membrane translocation and EGF-stimulated Ras signaling. Consequently, our preliminary results further demonstrated that inducible knockdown of NgBR in tumor xenograft of MDA-MB-231 cells in nude mice significantly retards tumor growth. Thus, these data demonstrate that NgBR is a unique receptor that recruits farnesylated Ras to the plasma membrane via its hydrophobic cytoplasmic domain and then activates both PI3K-Akt and Raf-MEK-ERK pathways in human breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B26.