Abstract B56: Notch signaling in prostate cancer progression

Abstract Background: The Gleason grading system provides the most consistently valuable information on the lethal potential of prostate cancers. Therefore, molecular correlates of higher Gleason grade should serve both as biomarkers of aggressive disease and as therapeutic targets. A recently published meta-analysis indicated that members of the Notch signaling pathway, particularly the receptor NOTCH3 and the pathway target Hairy and Enhancer Split family (HES6), distinguish prostate cancers with high Gleason grade. Thus, Notch signaling may play a distinctive role in aggressive prostate cancers. Here we use prostate cancer cell lines and transgenic mouse models to explore the function Notch signaling in prostate cancer progression. Methods: Using a custom TaqMan real time reverse transcription–polymerase chain reaction (qRT-PCR) assay, we compared expression of all Notch pathway components in benign prostate epithelial cells to those in prostate cancer cell lines - 22Rv1, LNCaP, DU145, PC3. We further confirmed differential expression using additional qRT-PCR assays. To understand how NOTCH3 receptor regulates the expression of pathway9s targets HES1, HES4 and HES6, we up- or down-regulated NOTCH3 levels using plasmid-mediated gene or small interfering RNA (siRNA) transfer. We also studied how HES6 protein levels affected the expression of the oncogene c-MYC and its effects in cell proliferation and migration in a wound-healing assay. We performed immunohystochemical staining for HES6 in prostate tumors and metastasis from Hi-Myc and TRAMP transgenic mice, as well as in human benign, low-grade and high-grade tumors. Results: Three members of the pathway were significantly overexpressed in prostate cancer cells compared with benign prostate epithelial cells: the receptor NOTCH3 and the two target genes HES4 and HES6. siRNA-mediated NOTCH3 silencing did not change HES4 levels, but decreased the expression of HES1 and HES6, implicating these genes as Notch response genes in the prostate. However, compared to controls, PC3 and LNCaP cells overexpressing HES6 showed no change in proliferation but migrated dramatically faster. Contrary to previous studies in neural development, HES6 overexpression in most prostate cancer cell lines induced HES1 expression indicating that rather than inhibiting, HES6 stimulates Notch signaling. We confirmed the importance of HES6 in prostate cancer progression through immunohistochemical analysis on tissues from mouse models (Hi-Myc and TRAMP) and human patients with prostate cancer. In each case, we confirmed increased strong nuclear HES6 expression with increased cancer grade or stage. Conclusion: Our results confirm differential expression of Notch pathway members in aggressive prostate cancer and indicate a role for HES6, one target of the pathway, in cancer cell migration. This effect may result from a direct action of HES6 or cross-talk with other members of the Notch pathway. These data suggest that Notch signaling, and particularly HES6, may be useful in distinguishing indolent from lethal prostate cancers and that Notch pathway blockade may be a useful therapeutic strategy in those cases that require treatment. Citation Format: Filipe L.F. Carvalho, Brian Simons, David M. Berman. Notch signaling in prostate cancer progression [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B56.