摘要:皮質類固醇(GC)常與抗癌化學藥物併用以預防化學藥物引起之噁心、嘔吐及過敏反應等副作用。一般認為GC不會直接影響上皮細胞癌之生長或死亡。然而,我們先前的研究發現GC確實對癌細胞株的生長以及化學藥物感受性有顯著的影響,且其影響因癌細胞而異。延續此研究,我們進一步探索GC對乳癌細胞的作用。我們發現,GC會增加七株三陰性(triple negative)乳癌細胞株的腫瘤形成能力(tumorigenicity),卻降低另外五株非三陰性(non-triple negative)乳癌細胞株的腫瘤形成能力。而且,GC會增加三陰性乳癌細胞株 MDA–MB-231細胞的幹細胞表面標記的表現。在原位異種移植的小鼠動物試驗中,我們發現GC會提早MDA–MB-231腋下淋巴結的轉移,並增加肝臟及肺臟的轉移病灶數目。鼠尾靜脈注射癌細胞,GC的治療亦會顯著增進MDA–MB-231肺臟轉移。綜合以上,我們假設GC會促進三陰性乳癌的腫瘤生成及轉移。特定目的:(1)探討GC影響三陰性乳癌腫瘤生成能力的分子機轉,(2)探討GC影響三陰性乳癌腫瘤轉移能力的分子機轉,(3)藉釐清三陰性乳癌與其他乳癌亞型之間對GC反應的差異。研究方法:選擇六株basal-like三陰性乳癌細胞株及五株post EMT(上皮細胞-間質細胞移轉型)三陰性乳癌細胞株,六株非三陰性乳癌細胞株及兩株三陰性乳癌細胞轉殖有荷爾蒙受體或HER2受體的細胞株進行研究。(1)以mammosphere形成效率,限制性稀釋法小鼠腫瘤生成能力試驗來鑑定GC對於腫瘤生成之影響。以流式細胞儀研究皮質類固醇對於乳癌幹細胞腫瘤標記表現的影響,並研究GC對於調控腫瘤生成及腫瘤幹細胞相關的基因/訊息傳導路徑,包括Wnt, Notch, Hedgehog, beta-catenin, and c-Myc等的影響及作用機轉。(2) 以傷痕癒合分析法,Boyden chamber 運動力分析,小鼠腫瘤肺臟轉移能力試驗來鑑定GC對於腫瘤轉移之影響。我們將進一步研究GC對於調控腫瘤轉移及 (EMT)相關的基因/訊息傳導路徑,包括MMP9, MMP2, ANGPTL4, cadherins, Fak, NFkB,以及 snail, twist, slug, vimentin, foxc2等的影響及作用機轉。(3) 以受體-Ligand 結合分析實驗檢驗GC受體表現量以及分析其共同調節因子的表現差異,釐清不同乳癌亞型之間對GC反應的差異性表現的原因。(4)應用調控基因表現的實驗技術以及致活性或抑制性化學藥品的處理,我們將針對上述實驗所找到相關的基因/訊息傳導路徑進行驗證。另外,將運用基因微陣列研究找尋其他未知的相關基因/訊息傳導路徑,並進行相關驗證之實驗。我們預期此一研究將加深我們三陰性乳癌生物學的了解,有助於發展新的有效治療策略。
Abstract: Glucocorticoid (GC) is commonly co-administered with chemotherapy to prevent drug-induced allergic reaction, nausea, and vomiting. They are not supposed to have any direct effect on the carcinoma cells. However, we have recently reported that GC may affect growth and chemosensitivity of some carcinoma cells via diverse mechanisms. Along this line, we further explored the effect of GC on breast cancer. Treatment of GC significantly increased tumorigenicity in seven triple negative breast cancer (TNBC) cells, while decreased tumorigenicity in five non-TNBC cell lines. Further, GC significantly increased the expression of TNBC stem cell markers in a fraction of TNBC cells. In MDA-MB-231 (a TNBC cell) orthotopic xenograft model, treatment of GC resulted in early axillary lymph node metastases, and increased number of lung and liver metastases. In tail vein injection assay of cancer metastasis, treatment of GC dramatically enhanced lung metastases of MDA-MB-231. Taken together, we hypothesized that GC has a detrimental effect on TNBC by inducing tumorigenicity and metastasization.Specific aims: (1) to explore the mechanism underlying the effects of GC on tumorigenicity in TNBC, (2) to explore the mechanism underlying the effects of GC on metastasization in TNBC; (3) to clarify the mechanisms underlying the deferential effect of GC between TNBC and non-TNBC subtype.Research methods: Six basal-like TNBC cell lines and 5 post epithelial-to-mesenchymal transition (EMT) TNBC cell lines, 6 non-TNBC cell lines, and 2 TNBC cell lines transfected with estrogen receptor or HER2 receptor will be used in this study. (1) To characterize the effect of GC on tumorigenicity, we will apply in vitro mammosphere forming efficiency assay, and in vivo limited dilution mouse tumorigenicity assay. To explore the underlying mechanisms, we will examine the expression of stem cell markers, and the regulation of signaling pathway related to tumorigenicity and/or stem cell regulation, such as Wnt, Notch, Hedgehog, beta-catenin, c-Myc. (2) To characterize the effect of GC on metastasization, we will apply in vitro wound healing assay and Boyden chamber motility assays, and in vivo mouse lung metastases study. We will examine the regulation of invasion/migration related genes, such as MMP9, MMP2, ANGPTL4, cadherins, Fak, and NFkB, as well as EMT-related genes and signal transduction pathways, such as snail, twist, slug, vimentin, and foxc2. (3) To clarify the mechanisms underlying the deferential effect of GC, we will examine the role of GC receptor content by ligand binding assay, and examine the differential co-regulator expression level in TNBC and non-TNBC cells. (4) To verify the role of genes / signaling pathways identified by above methods, we will conduct confirmation studies by using gene over-expression and/or suppression experiment, as well as chemical activators and/or inhibitors. To find un-identified, non-specific signaling pathway which may also responsible for the effect of GC on the tumorigenicity and metastasization, we will apply gene microarray study, followed by confirmation experiments.Result of this study will elucidate novel biology of TNBC, and help develop new strategy for the treatment of TNBC.