探討癌細胞與癌幹細胞中紡錘體調控因子與細胞分裂的關係

在生物演化的過程中，細胞產生變異以適應不同環境，而癌症的演化亦然。癌細胞中存在高度基因不穩定性（genomic instability），導致其對基因的限制降低，因而使基因突變或功能失調的機率提高，是令癌症具有基因異質性（heterogeneous）以及促進癌症演化的主要原因。本實驗室先前的研究發現，隨著癌症演化，紡錘體（spindle）的型態在細胞有絲分裂中期（metaphase）產生了極大的變化。侵略能力（invasive ability）較弱的人類肺腺癌細胞株CL1-0中紡錘體的型態較短胖；而透過人工篩選出具有較高侵略能力的惡性子細胞株（subline）CL1-5則具有較瘦長的紡錘體。本試驗目的乃找出癌症演化過程中，導致紡錘體型態差異的關鍵因子，並研究此一調控因子在細胞分裂中扮演的角色。首先在細胞的移動能力試驗（transwell migration assay）的結果顯示，CL1-5較CL1-0具有高移動能力。此外，應用微陣列分析（microarray）以及反轉錄即時定量聚合酶鏈鎖反應（RT-qPCR），我們發現一微管調控子驅動蛋白5（Kinesin-5）在CL1-5細胞中的表現量顯著提升，甚至為CL1-0的兩倍。本研究也設計在CL1-0細胞株中過量表現驅動蛋白5，結果造成CL1-0在分裂中期的紡錘體型態變得較為瘦長，並與CL1-5極為相似。此等結果證實驅動蛋白5在癌細胞演化過程中紡錘體型態的差異上扮演關鍵角色。近年來，癌症幹細胞（cancer stem cell）的假說在癌症演化的研究中佔有一席之地。癌症幹細胞被指出能夠自我更新（self-renewal）以及細胞分化（differentiation），並具備有形成腫瘤的潛力。其中癌症幹細胞調控不對稱分裂（asymmetric cell division ）的能力，被指出可能與腫瘤惡性程度相關。透過驅動蛋白5參與在細胞分裂中的機制為暗示，我們認為紡錘體及微管調控子是控制癌症幹細胞不對稱分裂的關鍵因素。最後本試驗擬以大腸癌幹細胞為模式，研究紡錘體參與不對稱分裂的關鍵機制。透過球形成試驗（sphere-forming assay），已成功自大腸癌細胞株HCT116及HT29培養出大腸癌症幹細胞（colorectal cancer stem cells, CCSCs），並發現相較於母細胞株，其幹細胞標記Sox2、Oct4、Nanog及Lgr5基因表現量均顯著上升。初步實驗也發現，少數紡錘體調控因子的基因表現量在大腸癌幹細胞與大腸癌細胞間存在顯著差異。未來將利用聚合酶連鎖反應微陣列分析（PCR array），探討大腸癌幹細胞與一般大腸癌細胞株的差異，進而找出紡錘體參與不對稱分裂的關鍵機制。

關鍵字

癌症演化；癌症幹細胞；細胞分裂；不對稱分裂；紡錘體；驅動蛋白5

並列摘要

During evolution, cells phenotypically change and optimize themselves to adapt to the different environment. This kind of phenomenon also happen in cancer evolution as a result of the heterogeneity within tumor. We were interested in the case of evolution of metastatic cancer, and we approached the question with a lung cancer cell line CL1-0 and it highly metastatic subline, CL1-5. Although CL1-5 has a smaller cell size, the spindle of the metastatic CL1-5 was longer than CL1-0, conflicting with the idea that spindle length should correlate with cell size. By transwell migration assay, we confirm that CL1-5 exists higher migration ability than CL1-0. Furthermore, to identify the key regulator involved, we employed microarray analysis and found that Kinesin-5, a microtubule regulator, was up-regulated in G2 phase CL1-5, and the near two-fold up-regulation was confirmed by RT-qPCR. To address whether Kinesin-5 play a role in metaphase spindle scaling in human cancer cells, we ectopically expressed Kinesin-5 in CL1-0 and found that metaphase spindle became CL1-5-like. Collectively, our data demonstrate that up-regulation of Kinesin-5 can lead to lengthened metaphase spindle. For the past few years, cancer stem cell (CSC), a population of cancer cells with stemness properties, took its place in the field of cancer evolution. We were also interested in how the expression of spindle regulators might be altered in CSCs. This was based on the reasoning that CSCs might be dysregulated in the balance of symmetric versus asymmetric cell division, in which spindle regulators might play a definitive role. By sphere-forming assay, we had successfully isolated colorectal CSCs from HCT116 and HT29 cell lines, and found escalated expression of Sox2, Oct4, Nanog and Lgr5, which are stemness-associated genes. Several spindle regulators also show different expression level between isolated CSCs and its parental cells. We will continue to compare the expression of spindle regulators in colorectal CSCs and non-CSCs, with the hope to identify key molecules whose dysregulation might lead to the most devastating aspect of cancer.

並列關鍵字

cancer evolution ； cancer stem cell ； cell division ； asymmetric cell division ； spindle ； Kinesin-5

參考文獻

1. Marusyk, A. and K. Polyak, Tumor heterogeneity: causes and consequences. Biochim Biophys Acta, 2010. 1805(1): p. 105-17.

2. Burrell, R.A., et al., The causes and consequences of genetic heterogeneity in cancer evolution. Nature, 2013. 501(7467): p. 338-45.

3. Shackleton, M., et al., Heterogeneity in cancer: cancer stem cells versus clonal evolution. Cell, 2009. 138(5): p. 822-9.

4. Gatenby, R.A., R.J. Gillies, and J.S. Brown, Of cancer and cave fish. Nat Rev Cancer, 2011. 11(4): p. 237-8.

5. Welch, J.S., et al., The origin and evolution of mutations in acute myeloid leukemia. Cell, 2012. 150(2): p. 264-78.

國際替代計量

探討癌細胞與癌幹細胞中紡錘體調控因子與細胞分裂的關係

全文下載

主題瀏覽