2017-08-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/704607摘要：膀胱癌是最常見的泌尿癌。相較於膀胱根除手術，膀胱癌病人接受放射為基礎的治療，長期預後是比較差的。而且對於復發的膀胱或腎盂泌尿上皮癌，放射治療的效果是很不好的。我在之前的科技部計畫顯示：afatinib，一個同時阻斷EGFR 和HER2 的酪胺酸激酶抑制劑，可以在膀胱癌細胞增加放射治療的效果。然而從病人經驗中我們猜測：放射治療在某些情況下是可以促進膀胱癌惡化的。Afatinib 放射增敏的機轉除了增加細胞毒殺與凋亡的效果外，也可能抑制了這種放射治療引發的膀胱癌惡化。本計畫的目的是澄清放射線對細胞間質中蛋白質的影響，同時找到有效可以增加放射治療效果的藥物。在前驅研究中我們發現次致死劑量的放射線可以促進膀胱癌細胞的侵犯。由於膀胱癌細胞以Matrigel 和Gelatin 塗層的Transwell 侵犯實驗有類似的效果，意味著可與Gelatin 結合的MMP-2 和MMP-9 可能在這個過程扮演重要角色。我們證明了MMP-9 在細胞內的表現量和在細胞培養基的酵素活性都可以在放射線照射後增加。因此我們假設MMP-9 這種放射治療引發的膀胱癌侵犯和轉移有關。本計畫的目標：目標一：探討MMP-9 在放射引發的膀胱癌侵犯和轉移中扮演的角色。我們將用不同的方法調控膀胱癌細胞株MMP-9 的表現，同時引進細胞共同培養的方法去檢驗癌症相關纖維母細胞(CAF)的影響。免疫功能正常和免疫功能不足的老鼠都將接受膀胱癌細胞的接種。我們也將使用MMP-9 基因剔除的老鼠來除去膀胱癌細胞以外的MMP-9 來源。初步的結果顯示：Transwell 實驗中，放射線促進癌細胞的侵犯在MMP-9 表現降低的膀胱癌細胞株就看不到了。我們也顯示了放射可以增加活體中膀胱癌老鼠的肺轉移。目標二：了解MMP-9 在放射引發的膀胱癌侵犯和轉移中確切的機轉。初步的結果顯示：p-EGFR, pHER2 和MMP9 的表現會在膀胱癌細胞接受放射線照射後增加，而在EGFR/ HER2 的雙重抑制劑使用後減少這樣的效果。EGFR/HER2 的雙重抑制劑也減少了放射引發的膀胱癌侵犯。我們的研究也顯示HER2 可能藉由影響ERK/c-Jun 或NF-kB 來改變MMP-9 的表現。目標三：發現PD-1/PD-L1 免疫治療可能在放射引發的膀胱癌侵犯和轉移扮演的治療角色。我們將收集放射治療前的病人與老鼠手術與血液檢體。細胞間質蛋白(例如MMP-9)與免疫檢查點(例如PD-L1)是分析的重點。這些結果將提供將來實驗室研究與臨床試驗的重要參考。我希望這個研究的結果可以幫助晚期泌尿上皮癌的病人有更好的臨床結果。<br> Abstract: Bladder cancer is the most common urinary tract cancer. However, the long-term prognosis of radiationbasedtherapy is inferior to radical cystectomy in bladder cancer patients. Also, for recurrent urothelialcarcinoma from bladder or renal pelvis, the effect of radiotherapy is poor. In previous MOST grant we haveshowed that afatinib, a tyrosine kinase inhibitor with EGFR and HER2 dual blockade effect, can enhancesradiation effect in bladder cancer cells. From patient experience we speculated that under certain conditionsradiation can promote bladder cancer progression. The mechanism underlying afatinib radiosensitizing effectmay not only increase cancer cell killing and apoptosis but also inhibit such radiation-promoted bladdercancer progression.The objective of current study is to clarify the influence of radiotherapy on stroma protein and finduseful drugs to enhance the effect of radiotherapy. In pilot study we found that after sublethal dose ofirradiation, the percentage of bladder cancer cell invasion increased. The results of Transwell invasion assayin gelatin-coating wells were similar to Matrigel-coating wells, meaning that gelatin-binding matrixmetalloproteinases-2 (MMP-2) and matrix metalloproteinases-9 (MMP-9) might play a role in this process.We demonstrated that MMP-9 intracellular protein expression and enzyme activity in culture media increasedafter irradiation in different bladder cancer cells. Therefore, we hypothesize that MMP-9 is related toradiation-induced invasion and metastasis in bladder cancer cells.In this project we have three aims:Aim 1: To explore the role of MMP-9 in radiation-induced invasion and metastasis in bladder cancercells. We will manipulate the in vitro expression of MMP-9 in bladder cancer cells by different ways andintroduce a co-culture system to examine the effect of cancer-associated fibroblast (CAF). Both immunocompetentand immunocompromised mice will be used in in vivo study. We will also use MMP-9 knock-outmice to eliminate non-cancer sources of MMP-9. The preliminary data showed that radiation-inducedinvasion in Transwell invasion assay can be blocked in MMP-9 knock-down cells. We also observed thatlung metastasis in murine bladder cancer model can be promoted by irradiation.Aim 2: To understand the mechanism how MMP-9 mediates radiation-induced invasion andmetastasis in bladder cancer cells. The preliminary data showed that that expression of phospho-EGFR,phospho-HER2 and MMP-9 increased after irradiation in bladder cancer cell lines but was decreased aftertreatment with EGFR/HER2 dual inhibitor. EGFR/HER2 dual inhibitor could also abrogate radiation-inducedtumor invasion. Our study also showed that HER2 may affect MMP-9 expression by ERK/c-Jun or NF-kBpathways.Aim 3: To find the therapeutic potential of PD-1 checkpoint inhibitor in radiation-induced invasionand metastasis in bladder cancer cells. We will collect patient as well as mouse tissue and blood samplebefore and after radiotherapy. The pathway analysis will focus on stroma protein (like MMP-9) and immunecheckpoint (like PD-L1) and provide important clue for further bench work and clinical trial.I hope that the results of this study can help patient with advanced urothelial carcinoma to have better clinicaloutcomes.