利用免疫螢光進行乳癌mTOR 途徑藥物癌伏妥錠(Everolimus)敏感度評估研究

Abstract

在許多癌症腫瘤中，PI3K/AKT/mTOR路徑常被激勵活化並且掌控癌細胞的主要功能。PI3K/AKT/mTOR路徑的下游分子，磷酸化的4EBP1蛋白及磷酸化的S6K1激酶更在細胞的生化功能裡扮演重要的角色。除此之外，mTOR標靶治療更是在乳癌病人對於賀爾蒙治療或化學治療產生抗藥性時進行治療。而讓mTOR中的專一性鍵結失效來達到調降 PI3K/AKT/mTOR路徑的活化程度的癌伏妥錠(Everolimus)則為mTOR標靶治療中常見的藥物。但在某些mTOR標靶治療病人當中，癌伏妥錠卻沒有產生其應達到的治療效果。本論文研究係以免疫螢光方式對於Hs578T, MCF7, BT474, MDA-MB-231, 乳癌細胞株及ABC-82T及ABC-16TX1 PDCC細胞株進行mTOR抑制劑癌伏妥錠(Everolimus)之敏感度測定。先以標準MTT測試及細胞生長率測試判別其細胞株之藥物敏感度，而後則是利用磷酸化4EBP1 Thr37/46蛋白抗體及磷酸化S6K1 Ser424激酶抗體對細胞株進行免疫螢光亮值測定以和測出的敏感度加以比較。研究結果顯示利用免疫螢光之分析方法的確可指出其乳癌細胞株對於癌伏妥錠(Everolimus)之敏感度。單獨的磷酸化4EBP1 Thr37/46蛋白抗體及磷酸化S6K1 Ser424激酶抗體之螢光表現可將乳癌細胞株及PDCC細胞株進行分群，磷酸化4EBP1 Thr37/46及磷酸化S6K1 Ser424兩者綜合之螢光亮值則可代表其藥物敏感度程度。當細胞對癌伏妥錠具有抗藥性潛力時，磷酸化4EBP1 Thr37/46及磷酸化S6K1 Ser424亦會產生動態變化，並且由實驗結果得知mTOR藥物的抗藥性並不是只對於AKT/mTOR路徑有專一性，而是與其它特定蛋白如LKB1或其他路徑如MAPK/ERK路徑有關。

In many solid tumors, the PI3K/AKT/mTOR pathway is activated and is believed to play major roles in a range of cellular functions. Downstream of this pathway, pho-4EBP1 and pho-S6K1 perform critical role in controlling biological processes. In breast cancer, when patients start to exhibit resistance with hormonal therapy or chemotherapy, mTOR inhibitor, for example Everolimus, is often considered, which can deregulate the PI3K/AKT/mTOR pathway by allosteric binding the catalytic site of mTORC1. But in some mTOR-targeted therapy cases, Everolimus does not exhibit the desired efficacy. This work assesses the use of immunofluorescence analysis to predict the efficacy of the mTOR inhibitor Everolimus using breast cancer cell lines- Hs578T, MCF7, BT474, MDA-MB-231-and patient-derived cell culture (PDCC)-ABC-82T, and ABC-16TX1.These cells provided predictive information on the sensitivity of Everolimus using cell viability and MTT assays based on immunofluorescence intensities of pho-4EBP1 Thr37/46 and pho-S6K1 Ser424. Results show that the immunofluorescence analyses can be used to indicate the efficacy of mTOR inhibitor Everolimus on cells tested. Independently,pho-4EBP1 Thr37/46 and pho-S6K1 Ser424 immunofluorescence expression can classify into different groups based on their intensities for cell lines and PDCC. The combined immunofluorescence intensity of pho-4EBP1 Thr37/46 and pho-S6K1 Ser424 is representative of the efficacy of Everolimus. Results also suggest that dynamitic change for pho-4EBP1 and pho-S6K1 occur when cells have resistance characteristic of Everolimus. Further, mTOR resistance is not only consequence of AKT/mTOR but also by LKB1 or MAPK/ERK pathway.Furthermore, LKB1 and pho-GSK3β may also be potential markers to predict the efficacy of Everolimus therapy.