c-jun 之抑制性磷酸化作用在乳癌的機制探討

Mechanistic study of inhibitory phosphorylation of c-jun in breast cancer

吳莉葳(Li-Wei Wu)

指導教授 ： 侯明鋒

高雄醫學大學/醫學院/醫學研究所/碩士(2013年)

摘要

絲胺酸/酥胺酸分裂原活化蛋白質激酶 (serine/threonine mitogen-activated protein kinases, MAPKs) 已被認定和癌症的發展有極其密不可分的關係。部份發炎細胞激素、營養因子的缺乏及某些細胞壓力可導致 c-jun NH2-terminal kinase (JNK) 與 p38 的活化。JNK 可在細胞處於紫外線照射 (UV irradiation) 下或致癌蛋白質表現時磷酸化 c-jun之 N 端轉活化功能區 (N-terminal transactivation domain) 進而活化其轉錄活性。同時，c-jun 為原致癌基因 (proto-oncogene) ，具有多功能性的 (pleiotropic) 功能可幫助腫瘤的發展。c-jun為AP-1 (activation protein) 家族的組成成員之一，調控了細胞凋亡、血管新生、細胞侵襲以及腫瘤轉移等功能。一般認為，c-jun 欲執行其轉錄作用時，而 N 端 Ser-63 及 Ser-73位置必須被磷酸化才會具有轉錄活性，而這兩個位置的磷酸化通常是由 JNK 來執行。然而，此二處胺基酸亦可被 ERK1/2 所磷酸化。ERK1/2也能在 c-jun 蛋白質Ser-243 (p-ser-243-c-jun) 的位置增加磷酸基團，進而降低c-jun 之穩定性，並且其形成 c-jun/AP-1 蛋白質複合體的 DNA 結合能力也會被抑制。
初步分析乳癌組織病理切片後，發現細胞質中 p-ser-243-c-jun 表現強度與病患有無淋巴結轉移及復發呈正相關 (p<0.001 及 p=0.044)；然而，細胞核中 p-ser-243-c-jun 的表現頻率與 Her2/neu 表現狀況呈負相關 (p=0.047)。同時，結果也發現細胞質中 p-ser243-c-jun 的表現強度與同在細胞質中的 p-ERK1/2 表現強度呈正相關 (p=0.047)；而細胞核中 p-ser-243-c-jun 出現的頻率則與核中 COX-2 出現的頻率成正比 (p=0.008)。
進一步活體外的研究結果發現， p-ser-243-c-jun 對於細胞生長能力並無明顯影響；而在 MCF-7 及 Her2 穩定表現的細胞株中，S243A-c-jun 突變蛋白質的大量表現會增加細胞移行及侵襲能力並明顯高於對照組及野生型 c-jun。同時，大量表現 S243A-c-jun 能誘導 MMP9 蛋白質及 mRNA 的表現。因此，根據以上的觀察 p-ser243-c-jun 可能具有抑制乳癌病程發展的能力，並可做為一個乳癌的預後因子。

關鍵字

乳癌 ； 絲胺酸/酥胺酸分裂原活化蛋白質激酶前致癌基因 c-jun ； 激活蛋白-1

並列摘要

Serine/threonine mitogen-activated protein kinases (MAPKs) have been closely associated with cancer. Inflammatory cytokines, trophic factor deprivation and a number of cell stresses lead preferentially to activation of c-jun NH2-terminal kinase (JNK) and p38 MAP kinases. JNK activates c-jun through the phosphorylation of the N-terminal transactivation domain of c-jun upon UV irradiation and oncoprotein expression. The c-jun proto-oncogene has pleiotropic effects on tumor development and is one of the AP-1 family members, which has been demonstrated in the regulation of apoptosis, angiogenesis, invasion and metastasis. In general, N-terminal phosphorylation of c-jun is necessary for its transcriptional activity, whereas the JNK signaling phosphorylates the Ser-63 and Ser-73 residues of the c-jun. Other MAPKs target these sites; the Ser-63 and/or Ser-73 residues are phosphorylated by the ERK1/2. Intriguingly, ERK1/2 is able to phosphorylate the C-terminal inhibitory site Ser-243 of the c-jun, and results in decreasing the stability and DNA-binding activity of c-jun. Unlike N-terminal phosphorylation, phosphorylation of the C-terminal sites inhibits the DNA-binding activity of c-jun/AP-1 family.
Our results showed that cytoplasmic intensity of p-ser-243-c-jun was inversely correlated with lymph node metastasis, recurrence (p<0.001, p=0.044) and the nuclear p-ser-243-c-jun was inversely correlated with Her2/neu status (p=0.047). Increased cytoplasmic intensity of p-ser-243-c-jun was positively correlated with those of p-ERK1/2 (p=0.047). In addition, the nuclear frequency of p-ser-243-c-jun and COX-2 were positively correlated (p=0.008).
We further found that the phosphorylation of c-jun at the Ser-243 residue did not affect cell growth. Nevertheless, S243A-c-jun mutant dramatically increased the invasion and migration of the MCF-7 and Her2 stable overexpressing MCF-7 clone cells as compared to the control and wild-type c-jun. In addition, ectopic overexpression of S243A-c-jun induced both protein and mRNA levels of MMP9. Taken together, our results suggested that p-ser243-c-jun might play a tumor suppressive role and serve as a prognosis biomarker in breast cancer.

並列關鍵字

Breast cancer ； mitogen-activated protein kinases ； c-jun ； activation protein-1

參考文獻

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