核苷二磷酸激酶 3 藉由雙重功能抑制乳癌細胞的轉移

Abstract

核苷二磷酸激酶3(NME3) 是屬於苷二磷酸激酶 (NDPK) 家族的其中一員，已知 NME3存在粒線體外膜上，也是一個潛在的組氨酸激酶 (Histidinekinase)。從乳癌 患者臨床數據中發現核苷二磷酸激酶 3 的表現量與患者的預後呈負相關。我們發 現在高度轉移性乳癌細胞中，例如 MDA-MB-231 細胞，具有較低的 NME3 表現 量。在 MDA-MB-231 細胞中增加完整的 NME3 表現量，會抑制 MDA-MB-231 細 胞的移動、侵襲與轉移，然而增加 N 端去除的 NME3 表現量卻無效果。我們證明 了 NME3 會降低粒線體的短桿狀 (fragmentation)、絲狀偽足 (filopodia) 的生成 以及肌凝蛋白輕鏈 2 (MLCII) 的磷酸化。為了釐清 NME3 如何同時影響這些分子 事件，我們比較表現不同型的 NME3 在細胞中，包含野生型 (wild-type) NME3、 酵 素 活 性 抑 制 型 (catalytic-dead) NME3 oligomerization defective) NME3。在 MDA-MB-231 中，酵素活性抑制的 NME3 會 filopodia 的生成，但維持了 RhoA-ROCK 所調控的 MLCII 磷酸化。在 NME3 表現的細胞中，阻止粒線體融合 (fusion) 而干擾 NME3 調 控的粒線體延長可以恢復細胞的移動和侵襲能力，但在野生型 NME3 表現的細胞 中則無此現象。相反地， 組氨酸磷酸化 (histidine phosphorylation) 的 filopodia 的生 成和 MLCII 磷酸化皆下降，進而抑制細胞移動及侵襲的能力。根據這些結果，我 們推測 NME3 存在兩種不同的功能以抑制細胞移動及侵襲所需的 filopodia 的生 成，其一是藉由 NME3 的聚合促進粒線體的延長，另外則是藉由 NME3 的組氨酸 激酶功能抑制 RhoA-ROCK-MLC II 路徑。

NME3, termed as non-metastasis enzyme (NME), is a nucleoside diphosphate kinase (NDPK). Given the presence of high-energy-phosphate intermediate at the catalytic histidine residue, NME3 is also a potential histidine kinase located on mitochondrial outer membrane. Data mining from databases of clinical specimens of breast cancer patients demonstrated that the expression level of NME3 was correlated with poor prognosis. We found that highly metastatic breast cancer cell lines such as MDA-MB-231 cells express lower level of NME3. Increased expression of full-length of NME3 inhibited MDA-MB- 231 cells in migration, invasion and in vivo metastasis, while NME3 deleted of N- terminus region had no effect. We showed NME3-mediated reduction in mitochondrial fragmentation, filopodia formation, and myosin light chain II (MLCII) phosphorylation. Expression of wild-type, catalytic-dead, and oligomerization defective of NME3 all caused the loss of invasion capability in MDA-MB231. The expression of catalytic-dead mutant of NME3 led to mitochondrial elongation with decreased amount of filopodia while retaining RhoA-ROCK-mediated MLCII phosphorylation in MDA-MB-231 cells. Blocking mitochondrial fusion to interfere NME3-mediated mitochondrial elongation was able to restore migration and invasion in cells expressing catalytic-dead mutant but not wild-type. As for the cells expressing oligomerization mutant of NME3 defective in stimulation of mitochondrial elongation, the abilities in migration and invasion were still suppressed with reduction in filopodia formation and MLCII phosphorylation . According to these results, we proposed that NME3 exerts two different functions to suppress filopodia formation for migration and invasion, one by stimulating mitochondrial elongation via oligomerization and the other by suppressing RhoA-ROCK-MLCII pathway via the histidine kinase function.