口腔鱗狀細胞癌 (Oral squamous cell carcinoma; OSCC) 是一種惡性腫瘤，分佈在 世界各地。儘管已經對 OSCC 的診斷和治療進行了深入研究，但其致癌機制仍需進一 步探討。先前的研究顯示蛋白激酶在 OSCC 腫瘤中有異常表現量，並在促進 OSCC 細 胞增殖中起關鍵作用。在這項研究中，我們探討 ACK1 調控 OSCC 細胞生長的分子 機制。我們先前的研究發現，以 shRNA 基因刪減 (knockdown) 抑制 OSCC 細胞中 ACK1 表現量，導致 p27 表現量上高，G1 週期停滯和細胞生長變慢。以基因刪減口腔 癌細胞中 ACK1 所引發之 p27 表現量增加，也因重新大量表現 ACK1 而後使 p27 表現量降低且細胞生長增快。本次研究中，我們最初探討了哪些 E3 泛素連接酶負責 OSCC 細胞中 p27 的降解。我們發現在 OECM-1 或 SAS 細胞中刪減 Skp2 會導致 p27 表現量增加，而刪減 COP1 或 KPC1 會導致 p27 表現量降低。但 ACK1 表現量 對 OSCC 細胞中 Skp2 表現量並沒有影響。透過在 HEK-293T 細胞中共同表現 p27 和 ACK1，其 p27 的酪氨酸磷酸化程度增加，而僅表現 p27 的表現量則沒有磷酸化。 此外，p27 在 OECM-1 細胞中的表現量與控制組相比，有較高的酪氨酸磷酸化。更重 要地，我們的結果顯示 ACK1 可以與 p27 相互結合，並主要磷酸化 p27 在酪氨酸 88 的位點上，此磷酸化對於 p27 與 ACK1 或和 Skp2 的結合致關重要。此外，我們以 PI3K 抑制劑 LY294002 及 AKT shRNA 證明 ACK1 會藉由活化 AKT 而導致 p27 蛋白酶體降解。以上實驗證明，在口腔癌細胞中，ACK1 促進 p27 的降解可透過直接 與 p27 結合並磷酸化 p27 於酪氨酸 88 的位點上，導致 Skp2 與 p27 的結合，或間 接藉由活化 AKT 進而導致 p27 的降解。

Oral squamous cell carcinoma (OSCC) is a malignant tumor that is distributed around the world. Although the diagnosis and treatment of OSCC have been thoroughly studied, the carcinogenic mechanism still needs to be further explored. Previous studies have shown that protein kinases are aberrantly expressed or activated in OSCC tumors and play a key role in promoting OSCC cell proliferation. In this study, we explored the molecular mechanism by which activated Cdc42-associated kinase 1 (ACK1), a non-receptor tyrosine kinase, regulates the growth of OSCC cells. Our previous study showed that ACK1 could promote the proteasomal degradation of p27, enhancing OSCC cell proliferation. To examine how ACK1 could regulate p27 degradation, we initially explored which E3 ubiquitin ligase is responsible for p27 degradation in OSCC cells. We found that knockdown of Skp2, a p27-associated E3 ubiquitin ligase, in OECM-1 or SAS cells, resulted in elevated p27 expression, whereas depletion of COP1 or KPC1, two p27-associated E3 ubiquitin ligases, caused a decrease in p27 expression. Interestingly, manipulating ACK1 expression did not affect in Skp2 expression in OSCC cells. The co-expression of p27 and mouse ACK1 (mACK1) in HEK-293T cells resulted in a great level of tyrosine phosphorylation of p27, while p27 alone showed no phosphorylation. Moreover, overexpression of p27 in OECM-1 cells exhibited a high tyrosine phosphorylation level, higher than that in vector control cells. Importantly, our results showed that ACK1 could bind p27 and mainly phosphorylated p27 at the tyrosine 88 site. This phosphorylation was crucial for the binding of p27 to ACK1 or Skp2. In addition, our data revealed that PI3K inhibitors LY294002 or AKT shRNA could rescue ACK1-suppressed p27 expression in OSCC cells. The results suggested that ACK1 could promote the degradation of p27 by directly binding p27 and phosphorylating it at the tyrosine 88 site or indirectly activating AKT in OSCC cells.

中文摘要 I
Abstract III
縮寫對照表 V
目錄 VII
第一章、背景簡介 1
1. 口腔癌 1
2. Activated Cdc42-associated kinase 1 (ACK1) 1
3. ACK1 與癌細胞生長之關係 2
4. Cyclin-dependent kinase inhibitor 1B (p27Kip1) 訊息傳遞路徑 2
第二章、研究目的 5
第三章、實驗設計 7
第四章、實驗材料與方法 9
1.細胞株之繼代培養 9
2.西方墨點法 10
3.病毒製備與感染 12
第五章、結果 15
1. 調控 E3 泛素連接酶表現量對 OSCC 細胞中 p27 之表現量影響。 15
2. OECM-1 及 SAS 細胞中先降解 ACK1 表現量再大量表現 ACK1 對 p27 及 E3 泛素連接酶之影響。 15
3. ACK1 可磷酸化 p27。 16
4. p27 的野生型或突變體在 HEK-293T 細胞中的表現量。 16
5. p27 在酪氨酸 88 的磷酸化對於 p27 與 ACK1 或 Skp2 的相互結合致關重要。 16
6. LY294002 對 HSC-3 與 SAS 細胞中先降解 ACK1 表現量後再大量表現 ACK1 對 p27 表現量之影響。 17
7. OECM-1 與 SAS 細胞中先降解 ACK1 表現量後再大量表現 ACK1，再降解 AKT 表現量對 p27 之影響。 17
第六章、結論 19
第七章、討論 21
第八章、圖表 23
圖一、在 OECM-1 及 SAS 細胞中，以降解 COP1、Skp2 及 KPC1 對 p27 表現量的影響。 23
圖二、改變 OECM-1 及 SAS 細胞中 ACK1 表現量對 p27 表現量之影響。 24
圖三、ACK1 與 p27 的酪氨酸磷酸化有關。 25
圖四、p27 的野生型或點突變在 HEK-293T 細胞中的表現。 26
圖五A、HEK-293T 細胞中 ACK1 磷酸化 p27 並與其結合。 27
圖五B、SAS 細胞中 ACK1 磷酸化 p27 並與其結合。 28
圖六A和B、PI3K 抑制劑 (LY294002) 對 HSC-3 和 SAS 細胞中 p27 表現量之影響。 29
圖六C和D、降解 ACK1-reexpressing OECM-1 或 SAS 細胞中之 AKT 表現量對 p27 之影響。 30
第九章、參考文獻 31
第十章、附表 35

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