探討丙烯醛與泌尿上皮癌治療抗藥性和致癌性的生物學特徵

Abstract

臨床上發現末期腎病患者罹患泌尿上皮癌的機率頗高，而慢性腎病變是末期腎病的前身，其亦與泌尿上皮癌相關，然，慢性腎病變與泌尿上皮癌間之相互作用仍缺乏確切證據。 研究發現尿毒症的「毒素」之一即多胺（polyamines）產生的丙烯醛，其濃度與慢性腎功能衰竭密切相關。從先前研究發現丙烯醛能誘導DNA損傷並抑制尿路上皮細胞中的DNA修復，進而導致膀胱癌的形成。 據此，本研究假設丙烯醛參與了慢性腎病變患者泌尿上皮癌的形成，在研究中收集62名泌尿上皮癌患者及43名健康對照受試者。藉由分析受試者其腫瘤組織中的丙烯醛-DNA (Acr-dG)加合物(adduct)和p53基因突變、血漿丙烯醛-蛋白質偶聯物 (plasma acrolein-protein conjugates, Acr-PC) 以及 3-HPMA濃度、尿液Acr代謝物來驗證研究假設。 本研究結果發現Acr-dG組織濃度與泌尿上皮癌患者的慢性腎病變分期有統計上的顯著關聯性。研究中，大多數受試者的p53突變是G到A和G到T的突變，其中更有50%的G:C對突變發生在CpG位點，形成Acr-dG加合物誘導的突變譜，慢性腎病變泌尿上皮癌受試者血漿中的 Acr-PC濃度顯著高於健康對照受試者 (p<0.001)。此外，與健康受試者相比，患有慢性腎病變的泌尿上皮癌受試者，其尿液3-HPMA亦發生了改變。 綜合以上結果顯示，丙烯醛可能為內源性物質，即尿毒症的毒素藉由體內誘發，導致慢性腎病變患者的泌尿上皮細胞癌化。 在驗證假設一丙烯醛可能與泌尿上皮癌化具正相關後，我們更進一步探討丙烯醛是否和泌尿上皮癌治療的抗藥性有關。 臨床上使用順鉑(Cisplatin)為第一線化學治療泌尿上皮癌的藥物。然而，順鉑的抗藥性與膀胱癌復發及預後相關，而過去的研究指出，成纖維細胞生長因子受體 (FGFR)和人類表皮生長因子受體2(HER2)基因被激活與上皮癌細胞增生及抗藥性有關，另外，臨床上已知吸煙亦為上皮癌最常見的危險因子之一，並且有實驗證據顯示吸煙與順鉑抗藥性有關。然而，兩者背後潛在的機制仍未清楚。 丙烯醛為一種高活性醛，除了烹飪油煙和汽車廢氣，也大量存在於菸草煙霧中，本研究為了驗證丙烯醛與順鉑抗藥性有關，利用非肌肉浸潤性和肌肉浸潤性兩類膀胱癌細胞株RT4和T24，分別利用丙烯醛暴露後，分析其誘發之順鉑抗藥性和下游信號調控變化。利用Cell viability assay、Cell proliferation assay、Soft agar colony formation assay、Cell migration assay、Quantitative real-time RT-PCR及蛋白質轉漬法(Western blot analysis)相關細胞實驗來做確認，此外，亦利用異種移植小鼠模型來確認腫瘤抗藥性和藥物逆轉可行性。 本研究結果發現丙烯醛在非肌肉浸潤性和肌肉浸潤性膀胱癌細胞株 RT4 和 T24 中，分別透過HER2和FGFR3信號的激活，引起RT4和T24細胞中丙烯醛誘導的順鉑抗藥性。而抗 HER2 抗體曲妥珠單抗和 FGFR 抑製劑 PD173074 分別逆轉了 RT4 和 T24 細胞的順鉑抗藥性。此外，透過丙烯醛誘導的順鉑抗性T24 克隆的異種移植小鼠模型，我們發現與單獨使用順鉑相比，順鉑與 PD173074 合併使用可顯著減小腫瘤大小。綜合以上結果顯示，順鉑抗藥性背後的不同訊號調控顯著改變了標靶治療合併化療的有效性。而本研究為順鉑抗藥性之膀胱癌的治療提供可行的策略。 泌尿上皮細胞脫落之細胞urinary exfoliated cell (UEC)，此類細胞中之癌細胞根據先前腔內植入或區域癌化之理論，在疾病進展或復發過程具有其特殊角色，以往多利用其細胞型態或免疫螢光標記作為腫瘤之偵測應用。而臨床已知上泌尿道上皮癌接受根治性腎輸尿管切除術後有高達20-50%於膀胱復發，顯示urinary exfoliated cell (UEC) 可能是癌細胞用以攜帶原位腫瘤細胞資訊及促進癌化。 為了分析尿液中相關細胞包括UEC或腫瘤細胞周圍細胞，以進行細胞特性鑑定和分析，以更全面了解癌細胞如何促進正常表皮細胞癌化與腫瘤復發之過程。本研究收集21位上泌尿道上皮癌病患接受腎臟輸尿管根除或膀胱根除術前後，及7位健康對照組之自解尿液，經由慢速離心後分離並利用多重免疫螢光染色(multiplex immunofluorescence)鑑別出UEC(Pan CK+/ CD45-/ DAPI+)。本研究發現，泌尿上皮癌患者的 UEC 高於健康對照組，且嚴重期別組別顯著高於早期期別與健康對照組，顯示UEC 與上皮癌間具正關聯性。此外，本研究也發現手術後三個月UEC與手術前UEC相比，其下降幅度降低 75% 之患者，其無復發存活期較長(HR 0.091, 95% CI 0.0009-0.912, p= 0.0415)，可能可做為區分疾病復發的生物標記。 本研究由三個層面切入—分析丙烯醛在泌尿上皮癌癌化角色和化療順鉑藥物抗藥性、以及尿液脫落細胞對癌化起源與復發之初步探索，全面性的解構泌尿上皮癌從初始癌化之因素以及治療過程中復發、惡化及抗藥性形成。為泌尿上皮癌癌化危險因子，治療抗藥性和腫瘤復各議題提出相關之見解。

There is high prevalence of urothelial carcinoma (UC) in patients with end-stage renal disease (ESRD). Although chronic kidney disease (CKD), a precursor to ESRD, is associated with urothelial carcinoma2, definitive evidence for an interaction between chronic kidney disease and urothelial carcinoma remains lacking.Acrolein, which is known to be produced from polyamines, is considered a uremic "toxin". Previous studies have found that acrolein induces DNA damage and inhibits DNA repair in urothelial cells, leading to bladder cancer. Therefore, we hypothesized that acrolein is involved in the formation of UC in patients with CKD. Sixty-two patients with UC and 43 healthy control subjects were collected and analyzed in the study. We analyzed acrolein-DNA (Acr-dG) adducts and p53 gene mutations in tumor tissues, plasma acrolein-protein conjugates (Acr-PC) and 3-HPMA levels, urine Acr metabolites of these patients to verify this hypothesis. We found a statistical correlation (p<0.01) between the level of Acr-dG and the stage of CKD in patients with UC. In these patients, the majority of p53 mutations were G to A and G to T mutations, and 50% of G:C pair mutations occurred at CpG sites, which was similar to the mutation spectrum induced by Acr-dG adducts. Plasma Acr-PC levels in UC patients with CKD were significantly higher than those in control subjects (p<0.001). Furthermore, urinary 3-HPMA was altered in UC patients with CKD compared with controls. Taken together, acrolein may act as an endogenous uremic toxin and promote the formation of UC in patients with CKD. In addition to that acrolein may be related to carcinogenesis of UC, we further explored whether acrolein is related to drug resistance of cancer treatment. Currently, cisplatin-based chemotherapy is the first-line therapy for metastatic UC. However, cisplatin resistance is associated with tumor recurrence and prognosis. Previous studies have linked activation of fibroblast growth factor receptor (FGFR) and HER2 signaling to proliferation and drug resistance in UC. In addition, smoking is known to be one of the most common risk factors for UC, and there is evidence that smoking is associated with cisplatin resistance. However, the underlying mechanism remains unclear. The key substance of interest in this study, acrolein, is a highly reactive aldehyde, which is present in large quantities in tobacco smoke in addition to cooking fumes and vehicle exhaust. To test this hypothesis, we analyzed acrolein-induced cisplatin resistance and downstream signaling regulation using both non-muscle invasive bladder UC and muscle invasive bladder UC cell lines —RT4 and T24, respectively. Cell experiments including cell viability assay, cell proliferation assay, soft agar colony formation assay, cell migration assay, Quantitative real-time RT-PCR and Western blot analysis were used to evaluate. We also use xenograft mouse model to validate tumor resistance and drug test. We found that acrolein induced cisplatin resistance and tumor progression in RT4 and T24, respectively. Activation of signaling through HER2 and FGFR3 causes acrolein-induced cisplatin resistance in RT4 and T24 cells, respectively. Furthermore, anti-HER2 antibody trastuzumab and FGFR inhibitor PD173074 reversed cisplatin resistance in RT4 and T24 cells, respectively. Using a xenograft mouse model with an acrolein-induced cisplatin-resistant T24 clone, we found that the combination of cisplatin and PD173074 significantly reduced tumor size compared to cisplatin alone. Taken together, these results suggest that differential signaling regulation behind cisplatin resistance significantly alters the effectiveness of targeted therapy combined with chemotherapy. This study provides a feasible strategy for the treatment of cisplatin-resistant UC. Urinary exfoliated cell (UEC)is the exfoliated cell of urinary tract. According to the previous theory of intraluminal implantation or field effect of regional canceration17,18, some of these cell has a special role in the process of disease progression or recurrence. Based on this, cell morphology or immunofluorescence labeling as a tumor detection was its most widely application. Up to 20-50% of upper urinary tract cancers recur in the bladder after radical nephroureterectomy, indicating that urinary exfoliated cell (UEC) may carry tumor cell information from primary tumors and may promote urothelial carcinogenesis. In order to more fully understand how cancer cell promotes the process of normal epidermal cell carcinogenesis and tumor recurrence, we characterize and investigate the urinary cells in urine. In this study, urine of patients with urothelial carcinoma before and after radical nephroureterectomy or radical cystectomy, as well as 7 healthy controls were collected. The urine was processed with slow centrifugation for cell pallet and stained with multiplex immunofluorescence. UEC was identified as Pan-CK+/CD45-/DAPI+ cells. The current study revealed that the UEC of patients with UC was higher than that of healthy controls, and the UEC level in more advanced stage was significantly higher than that of early stage and healthy controls, indicating a positive association between UEC and UC. In addition, this study also found that patients whose UEC decreased by 75% at three months after surgery compared with the initial UEC before operation had a significantly longer recurrence-free survival (HR 0.091, 95% CI 0.0009-0.912, p= 0.0415). This study takes the investigation from three aspects - analyzing the role of acrolein in carcinogenesis and cisplatin resistance as well as the effect of urine exfoliated cells on cancer development and recurrence. Based on the above research results, a preliminary analysis is achieved for the biological characteristics of acrolein and urothelial cancer drug resistance and carcinogenicity.