The Roles of K+ Channel Interacting Protein 4 in Breast Cancer Progression

目的: 鉀離子通道作用蛋白四號屬於EF-hand protein 家族的一員，主要功能為調控A型鉀離子通道的表現，先前文獻已報導鉀離子通道在許多細胞都會有表現，也包含了癌細胞，然而這些鉀離子通道作用蛋白與癌症的關係從未釐清，本論文目標於探討鉀離子通道作用蛋白四號調控乳癌之進程及其分子機制。 實驗設計: 利用五株人類乳癌細胞株(MCF7、T47D、MDA-MB-361、SkBr-3 以及MDA-MB-231)來探討因鉀離子通道作用蛋白四號表現量被小髮夾型核醣核酸抑制或過度表現後之浸襲能力及生長能力是否受到影響。細胞移動能力之測定係利用博登細胞移行實驗分析；生長能力則使用細胞存活率分析以及流式細胞儀做測定。利用甲基化逆轉錄聚合酶反應觀察在雌激素受體的啟動子上甲基化的表現，並進一步使用染色質免疫共沉澱法來分析在雌激素受體的啟動子上相關組蛋白修飾作用的表現。動物模式實驗則是利用原位注射於乳腺脂肪墊方式來觀察抑制鉀離子通道作用蛋白四號的表現是否會影響乳癌細胞之轉移能力。 結果:實驗結果顯示鉀離子通道作用蛋白四號之表現量與乳癌病人存活率成負相關。細胞實驗發現鉀離子通道作用蛋白四號與細胞浸襲與移動能力成正相關，抑制T47D 與MDA-MB-231 中鉀離子通道作用蛋白四號的表現量會使細胞移動力明顯下降，但卻不影響細胞增生。我們進一步建立了抑制鉀離子通道作用蛋白四號之穩定MDA-MB-231 細胞株，證明抑制鉀離子通道作用蛋白四號可降低癌細胞之浸襲力。在分子機制層面，我們發現抑制鉀離子通道作用蛋白四號能顯著提升雌激素受體(ER)陰性乳癌細胞株之ER mRNA 及蛋白表現量，並且有意義提高賀爾蒙藥物之治療效果。經由核醣核酸微陣列分析找到下游分子PRMT6，過度表現該蛋白可顯著恢復細胞因鉀離子通道作用蛋白四號表現量抑制而造成移動能力的下降。利用甲基化逆轉錄聚合酶反應法，顯示在抑制鉀離子通道作用蛋白四號表現之穩定轉殖株中，雌激素受體的啟動子上甲基化的表現有明顯下降，而使用染色質免疫共沉澱法則可見雌激素受體啟動子上H3K4 trimethylation 有明顯提升。於抑制鉀離子通道作用蛋白四號之細胞株轉殖PRMT6 可回複雌激素受體之甲基化和H3K4 trimethylation。動物實驗方面，抑制鉀離子通道作用蛋白四號可明顯抑制乳癌細胞之轉移能力。 結論: 鉀離子通道作用蛋白四號可以當作乳癌臨床的癒後指標，並搭配荷爾蒙療法可作為未來治療雌激素受體陰性乳癌之治療方針Purpose: K+ channel interacting protein 4 (KCNIP4) belongs to EF-hand protein superfamily which modulates A type potassium channels and regulates their surface expression. Previous studies have showed that K+ channels are expressed in a variety of cells, including cancer cells. However, the regulatory mechanism of KCNIP4 in breast cancer progression was completely unknown. Method: Human breast cancer cell lines (MCF7, T47D, MDA-MB-361, SkBr-3, and MDA-MB-231) were used as in vitro model to investigate cell invasion and proliferation by KCNIP4 expression plasmid and silenced-KCNIP4 (shKCNIP4) transfection. Boyden chamber assay was performed to check migration and invasion abilities. Proliferation ability was identified by MTT assay and flow cytometry. Methylation PCR and chromatin immunoprecipitation (ChIP) assay were performed to verify the methylation status on ER promoter and H3K4 trimethylation expression in shKCNIP4 transfectants. Mice were orthotopic injection with stable transfected KCNIP4 or vector control in MDA-MB-231 cells to measure metastasis. Results: K+ channel interacting protein 4 was negatively correlated with survival rate in breast cancer patients. KCNIP4 expression was positively correlated with invasion ability in breast cancer cells. Transiently transfected T47D and MDA-MB-231 cells with shKCNIP4 knockdown plasmids significantly reduced invasion ability (P< 0.05) but did not affect proliferation ability. Furthermore, we established shKCNIP4-stable transfectants in MDA-MB-231 cells, and found that silencing KCNIP4 could significantly down-regulated migration and invasion ability (P< 0.05) but did not affect proliferation ability. In mechanically level, silenced-KCNIP4 significantly up-regulated estrogen receptor (ER) in ERα-negative breast cancer cell lines both in mRNA level and protein level, which resulted in re-sensitizing to ER inhibitors treatment. Additionally, we performed high throughput mRNA microarray analysis and identified a downstream effecter, PRMT6. Moreover, transiently transfected PRMT6 expression plasmid could restore invasion ability in shKCNIP4 transfectants (P< 0.05). We also found that silenced-KCNIP4 decreased the methylation status on ERα promoter but overexpressed- PRMT6 in shKCNIP4 transfectants could reverse the effect. In ChIP assay, we verified that silenced-KCNIP4 could up-regulate H3K4 trimethylation expression but overexpressed-PRMT6 in shKCNIP4 transfectants turned down the effect (P< 0.05). Tumor metastasis ability was decreased in orthotopic injection in mammary fat pad with shKCNIP4 transfectants, compared with control mice in vivo