探討前列腺癌骨轉移作用分子及其臨床應用

Abstract

前列腺癌 (prostate cancer, PCa) 特別傾向於轉移至骨骼，且骨轉移為其死亡的相關主要原因，主導前列腺癌細胞與成骨細胞 (osteoblast) 間交互作用的黏附分子 (adhesion molecular) 可能扮演了重要角色。Cadherin-11，又稱為OB-cadherin，即為黏附分子之一，且在前列腺癌細胞株上有高度的表現。 Cadherin-11已被證實在前列腺癌骨轉移扮演重要角色，但其藉由何種機轉而在此癌細胞轉移的過程中作用則尚不清楚。本研究利用未表現有cadherin-11的 C4-2B4 細胞株使其表面表現cadherin-11，可促使其細胞擴展作用 (spreading) 並嵌入 (intercalation) 成骨細胞層，並刺激C4-2B4細胞的移行 (migration) 及侵襲 (invasion) 現象。對於已表現有cadherin-11之PC3細胞株進行cadherin-11負調控 (downregulation) 則減少其細胞的活動及侵襲作用。更進一步證實，cadherin-11胞質區域尾端的juxtamembrane及β-catenin結合區域皆為細胞移行與侵襲作用所必需。這些結果顯示，cadherin-11不僅提供前列腺癌細胞與成骨細胞間的連結，也透過其細胞質區域的訊息傳遞增加細胞活動及侵襲作用，且可能促使前列腺癌細胞骨轉移後的移生 (colonization)。 本研究也同時發現cadherin-11的黏附基序 (adhesion motif) 在其同類親合性(homophilic) 的細胞間黏附扮演重要角色，透過抑制此基序可干擾cadherin-11介導的前列腺癌細胞與成骨細胞黏附作用，並避免或減緩前列腺癌細胞發生骨轉移。 Cadherin 介導的細胞黏附作用為鈣離子依賴性，本研究發現cadherin-11的黏附基序主要在其細胞外區域的第三區塊 (EC3)。針對cadherin-11細胞外區域製備21株單株抗體，其中mAb 2C7 與 1A5 主要結合至EC3，並可抑制細胞與細胞間的聚集作用，當2C7在EC3的抗原決定位 (epitope) 發生突變時，cadherin-11促使的細胞黏附作用則大幅降低。 在前列腺癌細胞中，cadherin-11的異常表達提高了其在體外成骨細胞的黏附作用，且在活體試驗也增加發生骨轉移的機率。過去對於阻擋此黏附作用是否會抑制前列腺癌細胞的骨轉移並不清楚，但透過癌細胞轉移的實驗模式，將前列腺癌細胞直接注入老鼠心臟， mAb 2C7全身性給藥可顯著的減少擴散的前列腺癌細胞株PC3-mm2發生骨轉移的機率。綜合以上結果，透過干擾cadherin-11介導的黏附作用，可預防前列腺癌或其它癌細胞發生骨轉移。

Prostate cancer (PCa) has propensity to metastasize to bone and bone metastasis is the major cause of PCa related mortality. Cell adhesion molecules that mediate the interactions between metastatic PCa cells and osteoblasts, a major cell type in bone, may play a role in the metastasis of PCa cells to bone. Cadherin-11, also known as OB-cadherin, is one such adhesion molecule and highly expressed in a PCa cell line. Cadherin-11 has been shown to play a role in the metastasis of PCa cells to bone but the mechanism by which cadherin-11 is involved in this process is not known. In this study, we show that expression of cadherin-11 in cadherin-11-negative C4-2B4 cells increases their spreading and intercalation into an osteoblast layer, and stimulates C4-2B4 cell migration and invasiveness. Downregulation of cadherin-11 in cadherin-11-expressing metastatic PC3 cells decreases cell motility and invasiveness. Further, both the juxtamembrane and β-catenin binding domains in the cytoplasmic tail of cadherin-11 are required for cell migration and invasion. These observations suggest that cadherin-11 not only provides a physical link between PCa cells and osteoblasts but also increases PCa cell motility and invasiveness through its cytoplasmic domain that may facilitate the metastatic colonization of PCa cells in bone. We also identify a novel adhesion motif that mediates cadherin-11 homophilic cell-cell adhesion. We show that interfering cadherin-11-mediated PCa cell and osteoblast adhesion through inhibition of this motif may be developed for preventing or delaying prostate cancer bone metastasis. The cadherin family of cell adhesion molecules mediates Ca2+-dependent cell-cell adhesion. We identified a novel adhesion motif in the EC3 domain in cadherin-11. We generated 21 monoclonal antibodies against cadherin-11 EC domains. Among them, mAb 2C7 and 1A5 were found to inhibit cadherin-11-mediated cell aggregation. Mutation of the mAb 2C7 epitope in the EC3 domain abolished cadherin-11-mediated adhesion. In PCa cells, the aberrant expression of cadherin-11 increases their adhesion to osteoblasts in vitro and metastasis to bone in vivo. Whether blocking this adhesion will inhibit PCa metastasis to bone was previously unknown. Using an experimental metastasis model by injecting PCa cells intracardially, we showed that systemic delivery of mAb 2C7 significantly reduced the metastasis of disseminated PC3-mm2 to bone. Our studies suggest that perturbing cadherin-11-mediated adhesion may prevent bone metastasis from prostate or other cancers.