細胞和細胞以及細胞和細胞外基質之間的動態平衡，造成細胞和組織之間的體內平衡。這些獨特的組織特異性主要是經由上皮細胞和鄰近基質(stroma)的交互作用所型成的。近來的研究顯示攝護腺癌上皮細胞和其基質之間的調控作用非常重要。這樣的交互作用不但形成惡性循環(vicious cycle)並進而造成細胞轉成惡性及轉移的作用。在男性雄性激素非依賴型之攝護腺癌(androgen independent prostate cancer, AIPC)中主要的轉移部位在骨頭，然而、這樣的轉移機制是尚未被釐清的。在Karnoub等之團隊研究及本實驗室研究同時發現，骨基質細胞和癌症細胞共同培養之後CCL5的表現量會增加。而AIPC骨轉移病人的血清樣本中也偵測到同樣的結果，因此懷疑在攝護腺癌及乳癌中CCL5表現量的增加，對於癌症轉移具有組織特異性上扮演重要的角色。利用即時定量聚合?連鎖反應(quantitative PCR)偵測攝護腺癌中趨化素G蛋白關聯性蛋白接受器(chemokine G protein- coupled receptor)的基因表現量，我們發現CCRL2和CXCR4表現量分別增加86倍和45倍之多。在AIPC細胞株上降低CCRL2的表現，同時也降低了CCL5促使細胞移動及侵入的活性，因此懷疑在CCL5引起的攝護腺癌轉移上CCRL2扮演重要的角色。我們假設CCL5幫助攝護腺癌的生長、侵入及轉移需要經由和CCRL2的交互作用來完成。而本實驗的目的為研究CCL5和CCRL2對於癌症細胞移動、侵入及轉移的調控途徑。 本實驗的目標為研究CCRL2和CCL5對於攝護腺癌骨轉移的調控機制，我們將針對三個主要方向，第一：研究CCRL2和CCL5之間的交互作用，了解CCRL2是否活化CCL5，並經由交聯實驗（cross-linking）來證實CCRL2和CCL5之間的作用。藉由偵測其他G protein的活性來了解可能和CCRL2作用的G protein為何。第二：針對可能調節CCRL2表現量的調控機制進行研究，利用磷酸化抗體微陣列(phospho-antibody array)我們可以初步了解調控CCRL2的下游機制。經由啟動子序列分析(promoter assay)我們可以了解CCRL2促使細胞移動的調控機制為何。第三：我們將了解CCRL2和CCL5的交互作用和攝護腺癌轉移之間的關連性。在活體的動物實驗上，我們藉由阻斷CCL5和CCRL2間的作用，來觀察攝護腺癌的轉移能力是否有大幅度的下降。 本實驗的結果將幫助我們了解：1)趨化素和其受器之間的調節網絡，對於攝護腺癌組織特異性的轉移能力是重要的。2)了解趨化素受器在惡性的攝護腺癌及骨轉移機制中存在的角色。3)在相關的動物實驗中藉由CCL5和CCRL2對於骨轉移的重要性來發展治療策略。經由本實驗我們可以更加了解腫瘤和基質之間作用對於癌細胞轉移的重要性，並發展一個新的治療方向。
Cell and tissue homeostasis results from a dynamic balance of cell-cell and cell-extracellular matrices cross talk. These unique organ-specific phenotypes are conferred and maintained by interaction between epithelium and adjacent stroma in a reciprocal manner. Recent evidence suggests prostate tumor epithelium and stroma interactions are tightly regulated. These intimate interactions create a “vicious cycle” further drives tumor epithelium to develop malignant properties and metastasis potential. The most common metastatic site in men with androgen independent prostate cancer (AIPC) progression is bone. However, the mechanisms to induce organ specific prostate cancer bone metastasis still need to be elucidated. Previously, both Karnoub et al. and our group noticed the increasing of CCL5 in bone stromal cells after co-culture with cancer cells. We further confirmed the increased soluble stromal factors can be validated in serum of prostate cancer patients with AIPC bone metastases, suggest both prostate and breast could induce the elevation of CCL5 in bone stromal cells that may play a role in cancer organ specific homing mechanisms. To evaluate the expression level of chemokine G-protein coupled receptors in prostate cancer epithelial cells, quatitative PCR to evaluate the mRNA level of different receptors was performed. We noticed the significant increased of CCRL2 (86-fold) and CXCR4 (45-fold) in AIPC cell lines. Knockdown of CCRL2 expression in AIPC cell line decreased CCL5 induced migration and invasion activities, suggests CCRL2 involve in CCL5 induce prostate cancer metastasis. Hence, we hypothesized CCL5 induce prostate cancer progression, invasion and bone metastasis through the interaction with orphan chemokine receptor, CCRL2. The objective of this proposal is to determine the role of CCL5-CCRL2 in prostate cancer migration, invasion and metastasis, and their common regulatory pathways. The goals of this proposal are to study the regulatory mechanisms of CCRL2 and CCL5 in prostate cancer bone metastases. To reach these goals, we will focus on three aims. Aim 1: We will evaluate the interaction between CCL5 and CCRL2 and determine if CCRL2 is the active receptor of CCL5. Cross-linking of CCL5-CCRL2 interaction will be performed to confirm CCL5 binding to CCRL2. Measurement of G-protein activities to determine possible G-protein involved in CCRL2 downstream signals will be conducted. Aim 2: We will study the regulatory pathway(s) in prostate cancer cells to induce CCRL2 expression. Phospho-Antibody array will be applied first to determine the induction of signal downstream of CCRL2. Promoter assay will be performed to determine the possible regulatory signals and will determine which pathways downstream of CCRL2 involve in cell migration. Aim 3: We will determine if the interaction between CCL5 and CCRL2 is response to prostate cancer metastasis. Finally, we will determine if blocking CCL5-CCRL2 interaction could decrease prostate cancer metastasis at higher degree in the in vivo animal studies. The results of these studies will help us understand: 1) chemokine-chemokine receptor intercellular regulatory network modulated by stromal cells in supporting prostate cancer organ specific homing mechanisms. 2) The molecular mechanisms to induce novel chemokine receptor downstream signal pathways in malignant prostate cancer progression and bone metastasis; and 3) the developing of therapeutic strategies to targeting CCL5 and CCRL2 interaction of cancer bone metastasis in relevant animal models. With the studies will improve our understanding of tumor-stromal interaction in cancer metastasis progression and provide a new direction for the therapeutic opportunity.
