Abstract A13: β2-adrenergic receptor as a potential differentiation marker in prostate cancer
Authors: Ramberg Håkon , Grytli Helene H. , Braadland Peder R. , Wang Wanzhong , Nielsen Heidi K. , Krobert Kurt A. , Levy Finn Olav , Bjartell Anders , Svindland Aud , Taskén Kristin Austlid 
Published: May 2016

Background and purpose: It has previously been shown that the level of β2-adrenergic receptor (ADRB2) in prostate cancer tissue is correlated with biochemical recurrence (BCR) after radical prostatectomy. Here, we investigated this association in an independent cohort, and utilized stable ADRB2 knockdown in LNCaP cells to identify potential mechanisms.

Experimental procedures and results: The protein expression of ADRB2 in prostate cancer tissue from 63 prostate cancer patients (Skåne University Hospital, Malmø, Sweden) was measured by immunohistochemical analysis. Time to BCR was analyzed by Cox regression modeling. The ADRB2 protein level in RP tissue material was inversely associated with Gleason grade, which is often equated with the degree of differentiation of prostate cancer cells (p-value &lt; 0.001). Weak ADRB2 staining intensity also correlated with progression to BCR (HR 2.31, 95% CI 1.06-5.03, p-value 0.035). This association was no longer evident when adjusting for Gleason grade, indicating that Gleason grade is the stronger predictor of the two.

To look into potential mechanisms by which the ADRB2 is involved in differentiation of prostate cancer cells, we stably transfected the prostate cancer cell line LNCaP with shRNA targeting the ADRB2 (shADRB2) or a non-targeting shRNA (shCtrl). Stable knockdown of the receptor in the resulting shADRB2-LNCaP cell lines was verified by Real-Time RT-PCR, radioligand binding assay and adenylyl cyclase activity measurements. Knockdown of ADRB2 in LNCaP cells significantly down-regulated the expression of the luminal markers CD24 and slightly reduced the level of NKX3.1. ADRB2 and NKX3.1 mRNAs in radical prostatectomy specimens from 22 patients were measured using Nanostring technology, and the correlation coefficient between the two was 0.61 (p-value 0.003).

Proliferation was measured using Incucyte technology and MTS- assay, which showed a small reduction in growth rate in shADRB2-LNCaP cell lines compared to shCtrl cells.

One of the shADRB2 LNCaP cell lines and the shCtrl LNCaP cell line were injected into NON SCID mice, and tumor volume was measured every week for 8 weeks. The shADRB2 LNCaP cells did not display any difference in growth rate; however, the time to growth initiation was prolonged for these cells compared to the shCtrl LNCaP cells. In soft-agar colony formation assay, we observed that shADRB2 cell lines produced fewer colonies than the shCtrl LNCaP cells. Further analyses of the animal experiment and the ADRB2 knockdown cell lines are ongoing as of September 2015, and novel findings will be presented at the conference.

Conclusion: Low protein level of ADRB2 in prostate cancer tissue is correlated with high Gleason grade, and is also associated with biochemical recurrence after radical surgery. Knockdown of ADRB2 in the cell line LNCaP induced reduction in the expression of differentiation markers. Our observations suggest ADRB2 as a marker of differentiation in prostate cancer cells, and indicate that ADRB2 down-regulation might be one of the drivers of this process.

Citation Format: Håkon Ramberg, Helene H. Grytli, Peder R. Braadland, Wanzhong Wang, Heidi K. Nielsen, Kurt A. Krobert, Finn Olav Levy, Anders Bjartell, Aud Svindland, Kristin Austlid Taskén. β2-adrenergic receptor as a potential differentiation marker in prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr A13.