Investigating the Importance of Her2 and Cdk5-Dependent Pathways to Androgen Receptor Stability and Prostate Cancer Cell Growth

正常攝護腺發育及攝護腺癌演進皆受到雄性激素受體之調控。對於抑制攝護腺癌演進之策略，最常見者為雄性激素阻斷法。然而，部分腫瘤細胞終將擺脫對雄性激素的依賴並發展為雄性激素非依賴型攝護腺癌。欲研究雄性激素阻斷前後細胞內分子機制之變化，林赫老師實驗室從LNCaP細胞株篩選並建立出雄性激素非依賴的LNCaPdcc細胞株。篩選結果發現，LNCaPdcc細胞呈現了神經內分泌性(neuroendocrine)之型態及較緩慢的生長速率。即使細胞週期分佈與母系(parental) LNCaP細胞相似，但LNCaPdcc之細胞週期相關蛋白的表現卻明顯低於母系LNCaP細胞。有趣的是，在LNCaPdcc細胞中，雄性激素受體表現量及其絲胺酸(serine, Ser, S) 81位點的磷酸化有明顯增加之情形。而雄性激素受體下游調控基因—攝護腺特異抗原(prostate-specific antigen, PSA)之表現量亦有顯著增加之趨勢。此外，雄性激素受體於細胞核內之分佈及蛋白穩定性同樣有上升之現象。而雄性激素對於細胞增生的實驗則出現了相異的結果，低濃度(0.1及1 nM)的R1881(人工合成雄性激素)刺激了母系LNCaP細胞之增生，卻抑制了LNCaPdcc細胞之增生。另一方面，本人也發現比起母系LNCaP細胞，在LNCaPdcc細胞中，Her2 (ErbB2)、ErbB3、ErbB4受體之表現及Her2酪胺酸(tyrosine, Tyr, Y) 1221/1222兩個位點的磷酸化有上升之情形；heregulin (ErbB3配體)處理所導致的Her2磷酸化較高並較延遲；而Her2抑制劑(AG825及Herceptin)的處理，亦對LNCaPdcc的細胞增生造成較強的抑制效果。再者，Her2抑制劑較有效地降低LNCaPdcc細胞中雄性激素受體之穩定性和絲胺酸81位點之磷酸化。以上結果說明，在攝護腺癌細胞由雄性激素依賴轉變為非依賴的過程中，Her2對於雄性激素受體之穩定性扮演了重要角色。另一方面，Cdk5 (cyclin-dependent kinase 5)激酶與其活化蛋白p35在癌症研究中是新興的目標蛋白。第二部份的研究結果顯示在低濃度的雄性激素(0.1 nM R1881)下，Cdk5激酶會透過與雄性激素受體進行生化交互作用而磷酸化雄性激素受體上絲胺酸81的位點，進而導致雄性激素受體穩定性增加。Cdk5激酶導致的雄性激素受體穩定，造成了雄性激素受體於細胞核內的累積並活化，更進而正向調控細胞in vitro及in vivo之生長。將雄性激素受體上絲胺酸81的位點突變成丙胺酸(alanine, Ala, A)，則實驗結果顯示，雄性激素受體與Cdk5激酶的交互作用受阻，於細胞核內的分佈減少，其蛋白穩定性亦降低，進而減緩攝護腺癌細胞之增生。此外，林赫老師實驗室收集了177位有雄性激素受體表現之攝護腺癌組織切片(tissue array)。免疫組織化學染色的結果指出，雄性激素受體分別與Cdk5激酶和p35之蛋白表現呈現顯著的正相關性。以上的發現說明了Cdk5激酶對於雄性激素受體活化及攝護腺癌生長之調控扮演了重要角色。綜合上述結果，本人提出了雄性激素受體蛋白穩定性及攝護腺癌細胞生長會受到Her2及Cdk5相關訊息路徑所調控之證據。期盼這些發現能為荷爾蒙抗性的攝護腺癌治療提供新的思維。The normal prostate development and prostate cancer progression are mediated by androgen receptor (AR). Androgen ablation therapy is the most common strategy for suppressing prostate cancer progression; however, tumor cells eventually escape androgen dependence and progress to an androgen-independent phase. We screened and generated an androgen-independent prostate cancer cell line (LNCaPdcc) from androgen-dependent LNCaP cell line to investigate changes of molecular mechanisms before and after androgen withdrawal. We found that LNCaPdcc cells display a neuroendocrine morphology, less aggressive growth, and lower expression levels of cell cycle-related factors, although the cell cycle distribution is similar to parental LNCaP cells. Notably, higher protein expressions of AR, phospho-Ser81-AR, and PSA in LNCaPdcc cells are observed. The nuclear distribution and protein stability of AR increase in LNCaPdcc cells. In addition, cell proliferation results exhibit the biphasic nature of the androgen effect in two cell lines. Parental LNCaP cell proliferation is sensitive to synthetic androgen R1881 at limiting concentrations (0.1 and 1 nM) whereas LNCaPdcc cell proliferation is inhibited at low concentrations. On the other hand, LNCaPdcc cells express higher levels of Her2, phospho-Y1221/1222-Her2, ErbB3, and ErbB4 proteins than parental LNCaP cells. These two cell lines exhibit distinct responses to Her2 activation on Her2 phosphorylation and Her2 inhibition on cell proliferation, respectively. Heregulin-induced Her2 activation in LNCaPdcc cells is stronger and delayed. LNCaPdcc cell proliferation declines more significantly in response to Her2 inhibitors (AG825 or Herceptin). In addition, the Her2 inhibitor (AG825) more effectively cause AR degradation and diminish AR Ser81 phosphorylation in LNCaPdcc cells. Taken together, our data demonstrate that Her2 plays an important role in the support of AR protein stability in the transition of androgen requirement in prostate cancer cells. On the other hand, Cdk5 and its activator, p35, are oncoming targets in cancer research. In the second part, Cdk5 enables to phosphorylate AR at Ser81 site through direct biochemical interaction and therefore results in the stabilization of AR proteins at the low concentration of androgen (0.1 nM R1881). The Cdk5-dependent AR stabilization causes nuclear accumulation and subsequent activation of AR proteins. Besides, the positive regulations of Cdk5-AR on cell growth are also determined in vitro and in vivo. S81A mutant of AR diminishes its interaction with Cdk5, reduces its nuclear localization, fails to stabilize its protein level, and therefore decreases prostate cancer cell proliferation. Prostate carcinoma specimens collected from 177 AR-positive patients (tissue array) indicate the significant correlations between the protein levels of AR and Cdk5 or p35. These findings demonstrate that Cdk5 is an important modulator of AR activation and contributes to prostate cancer growth. In conclusion, we display the modulations of AR stability and prostate cancer growth by Her2- and Cdk5-dependent signal pathways and hope these findings will provide novel insight into the treatment of hormone-refractory prostate cancer.誌謝辭 i 中文摘要 iii Abstract v Contents vii List of Figures and Tables x Abbreviation xiii Introduction 1 1. Prostate cancer 1 2. Androgen receptor 2 3. Molecular events during prostate cancer transition 4 4. Serine phosphorylation of AR 5 5. ErbB family 6 6. Growth factors/receptors-dependent AR activation 8 7. Cyclin-dependent kinase 5 /p35 9 8. Cdk5/p35 in cancers 11 9. Cdks and AR activation 13 10. Reviews of androgen-inde pendent LNCaP sublines 14 Hypothesis 17 Materials and Methods 18 Materials 18 Cell culture 19 Cell viability assay 21 Trypan blue assay 21 Analysis of cell cycle distribution 21 Immunoblotting, immunoprecipitation and fractionation analyses 22 In vitro kinase assay 23 Immunocytochemistry 24 Transfection 25 Reporter assay 25 Immunohistochemistry 26 Xenografted tumor growth in nude mice 27 Semi-quantitative RT-PCR 28 Statistics 28 Chapter I The significance of Her2 on androgen receptor protein stability in the transition of androgen requirement in prostate cancer cells 29 Results 30 Comparisons of characteristics of LNCaPdcc versus parental LNCaP cells 30 AR-related features in two cell lines 30 Her2-related features in two cell lines 31 AR stability in LNCaPdcc cells depends on high Her2 activation 32 Discussion 34 Figures 39 Supplemental Figures 47 Chapter II Chapter II Regulation of androgen receptor activation and prostate cancer growth by cyclin-dependent kinase 5 51 Results 52 The biochemical relationship between Cdk5 and AR 52 Cdk5 increases AR stability through phosphorylation 53 Cdk5 promotes AR activation 54 Cdk5 activity regulates the growth of prostate cancer cells through phosphorylating AR 55 Clinical evidence 56 Discussion 58 Figures 63 Supplemental Figures 73 Tables 81 Conclusion 83 Acknowledgements 85 References 8