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標題: Src可藉由活化Cdk5促進乳癌細胞骨架建構與細胞遷移
Src Might Promotes Cytoskeletal Organization and Cell Migration of Breast Cancer Cells through Regulating Cdk5 Activation
作者: 周敬唐
Jou, Jing-Tang
關鍵字: 乳癌
Breast cancer
Rho GTPase
Focal adhesion
Signal transduction
Rho GTPase
Stress fiber
Focal adhesion
出版社: 生命科學系所
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摘要: 癌症轉移(Cancer Metastasis)是罹癌病患死亡的關鍵原因之一,因此預防及診斷癌症轉移是極為重要的治療手段。癌細胞遷移(Migration)的產生是癌症轉移的第一步,過去已有許多研究針對癌細胞遷移的細胞分子機制進行探討,文獻指出細胞內之Rho GTPases family家族成員是細胞遷移的訊息的匯集者,掌控由細胞外傳遞之訊息、如生長因子;至細胞內訊息、如致癌基因蛋白(Oncoprotein)等,而調控細胞骨架的動態改變(Dynamic)、影響細胞型態與細胞遷移。在細胞遷移機制中,細胞藉由Rho GTPases調控前端(Leading edge)形成細胞偽足(Lamellipodia)而向前延展(Protrusion)、Rac GTPase調控後端(Rare-end)產生張力纖維(Stress fiber)而收縮(Retraction)、以及c-Src與Focal adhesion kinase(FAK)調控前後Focal Adhesion消長(Turnover)而改變貼附能力(adhesion)等Subcellular events,影響整體細胞的遷移。 Cyclin-dependent kinase 5為serine/threonine kinase,雖與Cdk家族成員結構相似,但並不參與細胞週期調控。Cdk5透過結合p35/p39參與神經細胞的發育和功能調節,也在神經退化性疾病中扮演重要角色,近年來研究也陸續指出Cdk5在許多種類癌細胞皆有活化,而對癌細胞的惡化(Progression)有相當程度的貢獻。此外,過去在神經系統、血管內皮細胞與眼組織上皮細胞的研究指出Cdk5與Rho GTPases family及Src、FAK等關鍵細胞骨架調控者皆有交互作用,並藉此影響神經發育時細胞之遷移延展、表皮傷口癒合以及血管新生(Angiogenesis)等作用,然而Cdk5在癌細胞遷移機制上的調控目前了解的十分有限;因此本研究即探討Cdk5在癌細胞中是否經由類似的訊息傳遞路徑,即探討Cdk5透過Rho GTPases family調控細胞骨架動態改變影響細胞偽足或張力纖維結構、及細胞貼附能力,並藉此影響癌細胞的細胞遷移。 本研究首先藉由傷口癒合分析法(Wound healing assay)證實Cdk5具有促進乳癌、肺癌、膀胱癌細胞遷移的能力,並利用細胞免疫染色法發現活化Cdk5有集中分布於細胞偽足與Focal adhesion的現象、且抑制Cdk5能使結構消失而暗示Cdk5可能對細胞偽足與Focal adhesion結構形成有貢獻。接著本研究藉由促進或抑制Cdk5活性發現Cdk5正向調控著下游Rho GTPase的effector – ROCK而進一步促進張力纖維結構的形成,並藉此影響細胞的遷移。另一方面,本研究也發現Cdk5與致癌基因蛋白c-Src存在交互作用關係,兩者共免疫沉澱(Co-immunopercipitation)與共位(Co-localization)於細胞偽足顯示著兩者的間接交互作用關係。後續實驗也釐清乳癌細胞中c-Src居於調控路徑上游,藉由直接磷酸化Cdk5而調控著Cdk5的活性以及Cdk5活化分布位置;同時Cdk5也能夠反向藉由促進c-Src蛋白降解而抑制c-Src活性,顯示c-Src促進Cdk5活性、而活化Cdk5反過去抑制過度活化c-Src的相互調控模式。最後本研究證實c-Src能夠藉由正向調控Cdk5 – ROCK訊息傳遞路徑而促進張力纖維結構形成與細胞遷移,顯示c-Src – Cdk5 – ROCK訊息傳遞鍊調控張力纖維結構為Cdk5影響癌細胞遷移能力的路徑之一,彰顯了Cdk5在癌細胞遷移中機制的重要性;本研究之成果或可提供Cdk5作為癌症治療評估指標之標的,為癌症治療盡一份貢獻。
Cancer cell migration, the first step of cancer metastasis which causes poor diagnosis of patients with cancer, was extensively studied in recent decades. It is now widely accepted that the major subcellular mechanisms of migration is the leading edge protrusion by Rac GTPase regulated lamellipodium, the rare-end retraction by Rho GTPase regulated stress fiber contraction, and adhesions dynamic between front and rare by Src-FAK(Focal adhesion kinase) mediated focal adhesion turnover. All these steps must be coordinated both in space and time to generate productive, net forward movement, and the Rho GTPases family member – Rac and Rho GTPases, take center stage of upstream signaling to downstream cytoskeleton regulations. Cyclin-dependent kinase 5 (Cdk5), which belongs to Cdk family but not involves cell cycle regulation, plays roles ubiquitously in proliferation, apoptosis, cytoskeleton organization and motility of many cell types. Previous study shows that Cdk5 promotes metastasis of prostate cancer, glioblastoma, and pancreatic cancer, which suggest that Cdk5 regulates cancer metastasis across cancer types, but general principle still remains unclear. Several reports focus on angiogenesis and corneal wound healing that demonstrates Cdk5 promotes endothelial cell migration through regulating Rac GTPases mediated lamellipodia protrusion, regulates stress fiber formation and migration of cornel and lens epithelial cell through Rho GTPases, and interacts with oncoprotein c-Src to maintain stress fibers and undifferentiated state of len epithelial cells. These findings may also present in cancer cells, and still need provement. Here we show that Cdk5 interacted with oncoprotein c-Src and regulated Rho GTPases effector – ROCK mediated stress fiber formation to promote cancer cell migration. First we confirmed the post-effect of Cdk5 on cell migration of breast, lung and bladder cancers. Next we found pY15-Cdk5 localized at lamellipodia and focal adhesion, and these Cdk5 localization as well as stress fiber structure was distrupted by Cdk5 inihibition, suggesting Cdk5 might play roles on these structures. We next confirmed that Cdk5 promoted cell migration through up-regulating ROCK protein level and ROCK mediated stress fiber formation, indicating Cdk5 might regulate cancer cell migration through Rho/ROCK pathway and stress fiber organization. Futhermore, we presented evidence that oncoprotein c-Src directly phosphorylated, then up-regulated Cdk5 activity, and regulated stress fiber formation and cancer cell migration through Cdk5-Rho/ROCK pathway; meanwhile Cdk5 reversly down-regulated activated-c-Src protein level through proteasome-dependent degradation, suggesting a feed-back loop. Taken together, our present data suggests that Cdk5 plays important roles in cytoskeletal organization and cell migration of cancer cells, and might involve in oncoprotein c-Src regulated cancer cell migration by direct interacted with c-Src.
其他識別: U0005-1508201313314100
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