摘要:骨骼基質內富含許多成長因子及細胞激素,經由與腫瘤細胞互動,導引該腫瘤細胞移行至骨骼增殖,造成癌症的骨轉移,臨床研究發現約有80%癌症會在治療過程中轉移至骨骼,常見腫瘤包括肺癌、乳癌、攝護腺癌、腎細胞癌、甲狀腺癌等。目前許多腫瘤在診斷時可能已出現骨轉移,但有些未必發生,而是在癌症經過治療之後才出現,針對此種情形,必須具備多項條件,即腫瘤細胞經化學治療後會產生抗藥性乃得以存活,骨骼內具有特殊生長或趨化因子,容易吸引這些產生抗藥性後存活下來的腫瘤細胞移行到骨骼,進一步增殖,產生骨轉移病灶,因此本研究計畫擬探討癌症治療過程中,骨骼內的特殊因子與腫瘤細胞抗藥性對骨轉移的相關性。先前研究發現,許多發炎因子會參與腫瘤轉移過程,引起腫瘤細胞周圍血管新生、促使腫瘤侵入組織,再轉移至遠端器官。研究指出,造骨蛋白(osteopontin,OPN)及基質細胞源性生長因子1 (stromal cell-derived factor-1,SDF-1)都是參與誘導轉移的重要細胞激素。造骨蛋白是一種存在於骨骼胞外基質的非膠原性黏附性醣蛋白,1989年在成骨細胞中被發現,參與骨再塑功能,其結構上具有Arg-Gly-Asp (RGD)結合功能區,可與細胞膜上的v3 integrin 結合,造骨蛋白也可與CD44 結合,但此與RGDdomain 無關。研究發現造骨蛋白與惡性轉化也有密切關係,許多人類腫瘤細胞株都會誘導造骨蛋白的表現,且在多種人類腫瘤常見合成大量造骨蛋白,其中罹患具有高度骨骼轉移性如攝護腺癌病患,其血液中含有大量的造骨蛋白表現。近年來造骨蛋白具有3 種同種型(isoform),分別是OPNa,OPNb,OPNc,不同的isoform 對腫瘤的血管新生及牛微血管內皮細胞微管形成(bovine capillary endothelial cells (BCE) tube)和血管内皮生長因子(vascular endothelial growth factor,VEGF),具有不同影響,其中造骨蛋白isoform c 與腫瘤的生長、癌症嚴重程度相關,OPNc 可視為預後指標。因此,腫瘤轉移至骨骼的治療策固上,可針對造骨蛋白同種型進行分析研究。此外,發生癌症轉移到骨骼時,骨骼會大量表現CXCL12 (stromal cell-derived factor-1, SDF-1),此為驅化素的一種,會與其接受器─趨化因子受體(CXC-chemokine receptor 4,CXCR4)結合,促使腫瘤生長、轉移及血管新生,當腫瘤細胞在周圍環境刺激下,可自行大量表現CXCR4,此時CXCL12 (SDF-1)與表現CXCR4 的腫瘤細胞結合,令其移行具有方向性,造成腫瘤轉移至骨骼的特定現象。本研究計畫擬進一步探討造骨蛋白isoform 對腫瘤轉移相關性,並探討造骨蛋白與CXCR4 之調控機制。2009 年9 月,Meads 等人(Nature Review Cancer, 9, 665-674)提出一個腫瘤抗藥性相關的理論環境媒介抗藥性(Environment-mediated drug resistance,EMDR),敘述關於腫瘤相關顯微環境中的SDF-1、IL-6、VEGF 及integrin 如何影響腫瘤細胞週期及誘發細胞凋亡,造成de novo drug resistance。基於前述現象,可見即使投予有強效的化學治療藥物,腫瘤細胞產生抗藥性仍是臨床上化學治療過程中的必然現象,也是影響治療成果預後的瓶頸。當腫瘤細胞受到威脅時會啟動自我保護機制,活化ABC transporter,將化療藥物排出細胞外,令化療藥物失效,此現象即為抗藥性。病患產生抗藥性時,由於腫瘤的再度快速生長,發生多處轉移,會促使其病症加速惡化,增加死亡率。癌症幹細胞也很容易對化療藥物產生抗藥性。本研究小組的初步實驗結果顯示,造骨蛋白會增加ABC 轉運蛋白(ATP-binding cassette(ABC) transporter)的表現,而抗癌藥物會增加造骨蛋白的表現,此外,造骨蛋白也會增加CXCR4 之表現,這些情形的互動,更會造成惡性循環。因此,本研究計畫擬探討造成腫瘤轉移骨骼的前述相關因子與抗藥性產生的關聯性,並希望所得研究結果可供發展有用的生物製劑,供未來治療癌症骨轉移的參考。本研究為三年期計畫,擬採用細胞模式和動物模式,擬進一步探討造骨蛋白isoform對腫瘤轉移相關性,和造骨蛋白與CXCR4 對於腫瘤幹細胞之調控,同時擬探討造成腫瘤轉移骨骼的前述相關因子與抗藥性產生的關聯性,三年期間的研究項目如下:第一年:探討在容易轉移至骨骼的腫瘤細胞株及其腫瘤幹細胞上,造骨蛋白與CXCR4對抗藥性ABC 轉運蛋白(ATP-binding cassette (ABC) transporter)的影響及作用機轉。第二年:利用活體動物實驗,探討造骨蛋白與CXCR4 對容易轉移至骨骼的腫瘤之抗癌藥物敏感性及產生抗藥性所扮演的角色。第三年:利用OPN 及v3 抗體探討OPN、CXCR4 及其他腫瘤影響因子在活體動物所扮演的角色,並探討具抗藥性腫瘤癌症病患之腫瘤抗藥性與骨轉移的關聯性經由本研究計畫的執行,希望所得研究成果可以闡明癌症治療過程中,造成腫瘤發生骨骼抗藥性和癌細胞轉移骨骼的關聯性,並希望所得研究結果可供發展有用的生物製劑,供未來治療癌症骨轉移的參考。
Abstract: cancer cells may mediate the migration of cancer cells to bone, local proliferation ofcancer cells and results in bone metastasis. Clinical investigation showed about 80% ofcancers may develop bone metastasis during the treatment, common malignancies associatedwith bone metastasis including cancer from breast, prostate, lung, kidney, and thyroid, etc.Many cancers may present bone metastasis at dignosis whereas some cancers do not developbone metastasis initially. The late development of bone metastasis after treatment needsinteractions of many conditions, i.e., development of drug resistance in the survival cancercells after chemotherapy, specific growth factors or chemokines in bone attracting thesesurvivors to bone, followed by proliferation of cells to result in metastatic lesions. Thereforewe aim to investigate the relationship between such specific factors in bone and the cancercells with drug resistance during treatment of cancers.Previous studies have shown that many inflammatory factors are involved in the tumormetastasis process, including neoangiogenesis in microenvironment around the cancer cells,tumor invasion, and distant metastasis. The researches showed osteopontin (OPN) and stromalcell-derived factor-1 (SDF-1) are both i paramount cytokines involved in tumor metastasis.OPN, a bone extracellular matrix noncollagen protein, is found in osteoblasts in 1989. It isinvolved in bone remodeling. Osteopontin has a functional Arg-Gly-Asp (RGD) bindingdomain, which interacts with the αvβ3 integrin. In addition, OPN also interacts with CD44receptor in a RGD-independent manner. OPN has been reported to be associated withmalignant transformation, upregulated in many kinds of human cancer cell lines andoverexpression of OPN confers as a malignant marker in a variety of human tumors. Amongthem in patients with prostate cancer, high serum OPN levels correlate with high rate ofmetastasis to bone. Three human mRNA splice variants (isoforms) of OPN are identified inthe recent years, i.e., OPNa,OPNb,OPNc. They exert different properties in angiogenesiswith increased bovine capillary endothelial cells (BCE) tube formation and vascularendothelial growth factor (VEGF) secretion. OPNc expression profile is associated withtumor progression and severity of cancer. OPNc is likely to be of particular utility as aprognostic marker. Therapeutic strategies may be focused on the development agents thatselectively inhibit OPN isoforms. On the other hand, CXCL12 (stromal cell-derived factor-1,SDF-1) overexpreesion has been found at bone metastasis. CXCL12, a chemokine, caninteract with CXC-chemokine receptor 4 (CXCR4), and induce tumor growth, metastasis andangiogenesis. Cancer cells may self-secret abundant CXCR4 under the stimulation by themicroenvironment, then interact with high SDF-1 level in bone marrow, thus set up distinctpatterns of cancer metastasis to bone. Therefore we aim to focus on the investigation of therelatiosnship betweem the OPN isoforms and the development of bone metastasis, and themechanism involved in the regulation between the OPN and CXCR4.Recently, Meads et al., indicate the environment-mediated drug resistance (EMDR) todescribe the mechanism how tumor-associated microenvironment (SDF-1、IL-6、VEGF andintegrin, etc.) regulates tumor cell cycle and apoptosis and induces de novo drug resistance(Nature Review Cancer, 9, 665-674, 2009, Sep.). Based on such theory, the cancer cells willdevelop drug resistance during the chemotherapy course even under administration of strongchemotherapeutic agents. Therefore the prognosis will be affected. Under the threatening ofchemotherapy, the cancer cells will turn on self-protection mechanism and overexpress theATP-binding cassette (ABC) drug transporters that actively pump out a variety ofamphipathic compounds from cells and decrease the therapeutic effects of chemotherapeuticagents, i.e., multidrug resistance (MDR). The patients who develop MDR will presentrecurrent rapid growth of tumor and multiple metastases that incur poor high morbidity andmortality.Cancer stem cells are reported to resist chemotherapy. Our preliminary results showthat OPN upregulates the expression of ABC drug transporter and anticancer drugs furtheincreased the expression of OPN, thus inducing a vicious cycles. In addition, OPN alsoenhances the expression of CXCR4. Therefore in this project, we will further investigate theroles of OPN and CXCR4 in the development of drug resistance during chemotherapy, andhop that the results can be used for the development of potential bioagents used as cancertherapy in the future.This 3-year project will adopt both cell culture models and animal models to deepinvestigate the roles of OPN isoforms on the development of bone metastasis, the mechanisminvolved in the regulation of OPN and CXCR4 on the cancer stem cells, and their relationshipbetween the development of drug resistance and the aforementioned factors related to bonemetastasis. The research works in these 3 years is as following:Year 1: Investigate the effects and mechanism of OPN and CXCR4 on the development ofdrug resistance using ABC transporter as a marker in cancer cell line and cancer stem cellswith high potential metastasis to bone.Year 2: Use animal models to investigate the effects and mechanism of OPN and CXCR4 onthe chemotherapy drug sensitivity and drug resistance in cancers with high potentialmetastasis to bone.Year 3: Investigate the role of OPN antibody and v3 antibody on the expression ofOPN, CXCR4 and other associated factors in the animal models, as well as investigatethe relastionship between their expression and effects in the serum and tumor tissuesfrom patients with development of drung resistance and bone metastasis.Through this research project, we hope that the relationship between the development ofdrug resistance and bone metastasis during treatment of cancer can be understood. We alsohope that the reseach results can be used for the development of bioagents used as basis fortreatment of cancers with bone metastasis.