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表皮生長因子誘導血管生成素樣蛋白 4 表現對頭頸癌細胞轉移所扮演的角色

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國立成功大學「邁向頂尖大學計畫」

延攬優秀人才工作報告表

NCKU’s “Aim for the Top University Project”

Work Report Form for Distinguished Scholars

□續聘 continuation of employment ■離職 resignation

100 年 7 月 13 日更新

受聘者姓名

Name of the Employee

許晉源 ■男 女

Male Female

聘 期

Period of Employment

from 104 年(y) 10 月(m) 31 日(d)

to 104 年(y) 11 月(m) 30 日(d)

研究或教學或科技研發與

管理計畫名稱

The project title of research,

teaching, technology development and management

表皮生長因子誘導血管生成素樣

蛋白 4 表現對頭頸癌細胞轉移

所扮演的角色

計畫主持人

(申請單位主管)

Project Investigator (Head of Department/Center)

陳炳焜

補助延聘編號

Grant Number

HUA 104 – 3 - 22 - 228

一、 研究、教學、科技研發與管理工作全程經過概述。 (由受聘人填寫)

Please summarize the entire research, teaching, or science and technology R&D and management work process (To be completed by the employee)

Aim 1. To study mechanisms involved in ARNT-regulated PDK1 expression in EGF-treated HNSCC cells.

Here we seek to examine the control of PDK1 expression that possibly associates with tumor metastasis (also in those of Aim 2). In these studies we focus on defining the effects of EGF on these phenomena, in particular its correlation with various cancer types.

[1-1]: Identify the expression of EGF-induced PDK1 in various cancers.

First, we want to clarify whether EGF-induced PDK1 expression in different cancer cell lines. The EGF-induced PDK1 expression was dramatically increased in head and neck squamous cell carcinoma (HNSCC) cell lines (Fig. 1A). To clarify that EGF-induced PDK1 expression is mediated by transcriptional activation of the promoter and/or mRNA stability, we will study the change of PDK1 promoter activity and mRNA degradation rate in EGF-treated cells, respectively. Preliminary results indicated that EGF enhanced PDK1 promoter activity in dose-dependent manner (Fig. 1B). Consideration of cell type specific effects and the degree of malignancy are likely to affect the overall metabolic phenotype of a cancer cell, the activation of PDK1 promoter activity, gene expression and protein level will be further confirmed by treating differential cancer cells with various concentrations of EGF. In addition, the possibility that growth factors such as PDGF, VEGF, IGF and TGFβ, as well as EGF may regulate their physiological effects in tumors by altering the expression of PDK1 will be examined. These issues are also interesting for us to study in the future.

A. B.

Figure 1. EGF induces PDK1 expression in HNSCC cells. (A) HNSCC cells were treated with 50 ng/ml EGF in serum-free medium for 24 h. Cell lysates were prepared and subjected to SDS-PAGE and western blot analysis using antibodies against PDK1 and β-actin. (B) Cells were transfected with PDK1 promoter construct using lipofection. Cells were treated with varius concentration of EGF in serum-free medium for 24 h. Luciferase activity and protein concentrations were then determined and normalized. Values represent means ± S.E.M. of three determinations.

[1-2]: Study the mechanisms involved in the regulation of PDK1 promoter activity

To investigate the mechanism involved in the regulation of promoter activity, the responsive elements for promoter activity will be clarified by a series of 5’-deletion of PDK1 promoter. According to our preliminary result, the first candidate of transcription factor that for

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transactivation of PDK1 promoter was ARNT. Functionally, siARNT and overexpression of ARNT in cells for a period of time will be performed to examine their effects on PDK1 promoter activity, gene and protein expression. In preliminary data, EGF-induced PDK1 promoter activity was block by siARNT (Fig. 2). The binding of ARNT to PDK1 promoter will be examined using DAPA and ChIP assays.

We will further study whether the potential binding sites of transcription factors are essential for EGF-induced PDK1 promoter activity by using the mutants of PDK1 promoter such as PDK1-ARNTm. These experiments will allow us to identify the mechanisms involved in the regulation of EGF-induced PDK1 gene expression, and how ARNT participates in EGF-induced gene expression, thus greatly expanding the overall impact and potential implications of the project.

It is important to consider that the promoter regulation of PDK1 is not only dependent on ARNT factor in EGF-treated cells. This cannot impair our ability to identify factors involved in the regulation of PDK1, because we will not be mired in difficulties in lacking reports about the promoter regulation of PDK1. Instead, we will screen potential binding sites located on PDK1 promoter using TF search program. Although it is too early to describe that the study on these factors are our targets in this step, we have experienced to clarify the association of ARNT and these factors with their functions (e.g. posttranslational modifications vs. protein-protein interaction) in the regulation of gene expression. We will also dig out potential factors that have not been discovered now by using DAPA-pull down assay.

Factors binding to the promoter region that is responsible for PDK1 induction will be identified using proteomics analysis. This approach will give us an opportunity to fish out the candidates, as well as ARNT bound to COX-2 promoter by the same approach used in our previous study (1), and clarify their functions in EGF-regulated PDK1 promoter activation.

TU183 FaDu

Figure 2: EGF promotes PDK1 promoter activity via activation of ARNT. Cells were transfected with 20 nM ARNT siRNA oligonucleotides and 0.5μg of pPDK1 promoter construct by lipofection and then treated with 50 ng/ml EGF for 24 h. The luciferase activities and protein concentrations were then determined and normalized.Values represent means ± S.E.M. of three determinations.

[1-3]: Clarify signal transduction pathways that mediate EGF-induced PDK1 expression

Although we have shown that EGF induced PDK1 expression, we do not know the precise signal pathways of this response. Since the response of protein induction by EGF was observed (Fig. 1A), we will first perform experiments to determine the effects of EGF-activated kinases, such as ERK, JNK, p38 and Akt on PDK1 mRNA expression. We will repeat this entire procedure (i.e. by detecting protein levels) and use kinase inhibitors to clarify signal pathways involved in the regulation of EGF-induced PDK1 expression. The efficacy of inhibitors can be confirmed by detecting the reduced kinase phosphorylation (i.e. phospho-ERK, phospho-JNK, phospho-Akt and phospho-p38). To further confirm that kinases activation stimulated by EGF is associated with regulation of PDK1 induction, siERK, siJNK and other (i.e.

shp38 and siAkt, if they are required) cell lines with deficiency of kinases expression will be selected. The knockdown efficiency of kinases expression can be characterized using PCR and Western blot analyses. This will include at least 5 individual cell lines for each kinase knockdown cells. Induction of PDK1 gene promoter activity, mRNA and protein will be examined by comparing parental cells with kinase knockdown cells. The downstream (i.e. AP1, STAT3 and NF-κB) and upstream (i.e. EGFR and Ras) regulators linked to kinase activation, and induction of PDK1 expression will be further studied in detail.

Aim 2. To clarify PDK1 expression that in the correlation with EGF-induced tumor metastasis.

The data generated in Aim 1 will provide mechanisms, including promoter activation and signal transduction pathways in the

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regulation of EGF-induced PDK1 expression. In this Aim, we will identify the biological functions of PDK1 induced by EGF in cells. At first, we will focus on the switch of EGF-regulated aerobic glycolysis in tumor cells that is determined by PDK1 or not. Since EGFR clearly and undeniably contributes to tumorigenesis, alternatively, we proposed that the EGF-regulated PDK1 associated with tumor formation must exist. Previous report also showed that glucose consumption and lactate production were increased in the high number of EGF receptors MDA-468 human breast cancer cells (2). EGF stimulates the expression and activation of lactate dehydrogenase (LDH), resulting in the up-regulation of lactate production (3). However, the precise mechanisms involved in ARNT-regulated PDK1 in EGF-promoted cancer cell metastasis in unclear.

[2-1]: Study the effect of EGF-induced PDK1 on tumor growth

To investigate the effects of EGF-induced PDK1 on the switch of aerobic glycolysis, the rate of cell proliferation, expression of mitochondria markers, productions of glucose and lactate will be examined. Here, we will assess the importance of EGF-induced PDK1 expression for cell proliferation via small interfering RNA (siRNA) knockdown of PDK1 (siPDK1). The cell proliferation rate and tumor growth of stable knockdown of PDK1 in EGF-treated conditions will be studied by using cell proliferation assay and colony formation assay. To address whether it is the PDK1 that is specifically critical for cell proliferation, the stable cell lines expressing flag-tagged PDK1 (f-PDK1) will be used in siPDK1 conditions. The differential growth rate (parental vs. PDK1 knockdown cells) caused by interrupting cell cycle could be studied using flowcytometry assays. These studies will provide us evidence whether PDK1 participates in EGF-regulated tumor growth. Although the mechanisms by which PDK1 regulate cell cycle to promote tumor growth are unknown, we will also devote to clarify this issue following the experimental results mentioned above in the future.

[2-2]: Examine the role of PDK1 in EGF-enhanced tumor metastasis

Interestingly, we found that PDK inhibitor dichloroacetate (DCA) significantly inhibited EGF-induced cancer cell migration (Fig. 3).

In our preliminary results, EGF significantly enhanced TU183 and FaDu cells invasion and significantly blocked by siPDK1 (Fig. 4). It is well known that activation of EGFR signaling regulates EMT-associated invasion and migration in malignant mammary epithelial cells by disrupting the adhesion junctions including catenin-E-cadherin complexes through tyrosine phosphorylation of catenin components (4). The tight junction protein, claudin-1 can up-regulate MMP-1 and MMP-2, which in turn induce the cleavage of the laminin-5 gamma 2-chain, followed by the binding of the laminin-5 gamma 2-chain to EGFR and/or integrin and the activation of intracellular cascades that promote cell growth and the motility of cancer cells (5). It was reported that PKM2 knockdown inhibits EGF-enhanced expression of the β-catenin target gene cyclin D1, resulting in promoting cell proliferation and tumorigenesis (6). These results suggest that EGFR signaling has dual roles that are essential for tumorigenesis: regulating cancer cell metabolism and gene transcription required for cell proliferation via activating the metabolic enzyme. Even though the role of glycolysis in providing proliferative and survival advantages to cancer cells has been described, the impact of aberrant expression of metabolic enzymes on cancer progression remains unknown. It was reported that inhibition of lipogenic enzyme (e.g. fatty acid synthase) decreases the metastatic potential of colorectal cancer cells (7). We proposed that EGF-induced PDK1 expression determine progression of cancer to a metastatic phenotype. In this study, we will utilize approaches, including transwell migration assay, transendothelial invasion assay, cell adhesion assay to investigate the cellular mechanisms by which EGF-induced PDK1 in the regulation of metastasis of tumor cells. We will also clarify EMT-associated marker (e.g. E-cadherin, N-cadherin, fibronectin, MMP-2, MMP-9 and Rac1/cdc42). In the future, we will use mouse model to clarify whether EGF-induced PDK1 expression is involved in cancer cell metastasis in vivo.

Figure 3. DCA inhibits EGF-induced cell migration. A431 cells were treated with DCA (0.2~5 mM) and 50 ng/ml EGF for 15 h. Migrated cells were stained with Giemsa and observed by microscope camera.

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TU183 FaDu

Figure 4: PDK1 regulates EGF-induced HNSCC cell invasion. The invasive properties of tumor cells were examined using the invasion assay as described in the “Materials and methods”. Parental and siPDK1 TU1831 and FaDu cells were treated with 50 ng/ml EGF in serum-free medium for 48 h. Images were captured under a microscope

References:

1. K.-Y. Chang et al., Epidermal growth factor-activated aryl hydrocarbon receptor nuclear translocator/HIF-1 signal pathway up-regulates cyclooxygenase-2 gene expression associated with squamous cell carcinoma. J Biol Chem 284, 9908-9916 (2009).

2. O. Kaplan et al., Toxicity and effects of epidermal growth factor on glucose metabolism of MDA-468 human breast cancer cells.

J. Biol. Chem. 265, 13641-13649 (1990).

3. F. Boussouar, M. Benahmed, Epidermal growth factor regulates glucose metabolism through lactate dehydrogenase A messenger ribonucleic acid expression in cultured porcine sertoli cells. Biol. Reprod. 61, 1139-1145 (1999).

4. M. Cozzolino et al., p120 Catenin Is Required for Growth Factor-dependent Cell Motility and Scattering in Epithelial Cells. Mol.

Biol. Cell 14, 1964-1977 (2003).

5. R. Largillier et al., Prognostic factors in 1038 women with metastatic breast cancer. Ann. Oncol. 19, 2012-2019 (2008).

6. W. Yang et al., Nuclear PKM2 regulates beta-catenin transactivation upon EGFR activation. Nature 480, 118-122 (2011).

7. Y. Y. Zaytseva et al., Inhibition of fatty acid synthase attenuates CD44-associated signaling and reduces metastasis in colorectal cancer. Cancer Res. 72, 1504-1517 (2012).

二、 研究或教學或科技研發與管理成效評估( 由計畫主持人或單位主管填寫 )

Please evaluate the performance of research, teaching or science and technology R&D and management Work: (To be completed by Project Investigator or Head of Department/Center)

(1)是否達到延攬預期目標?

Has the expected goal of recruitment been achieved?

是。

(2)研究或教學或科技研發與管理的方法、專業知識及進度如何?

What are the methods, professional knowledge, and progress of the research, teaching, or R&D and management work?

受延攬人在延攬期間,有效協助計畫進行,並執行後續的相關實驗。

(3)受延攬人之研究或教學或科技研發與管理成果對該計畫(或貴單位)助益如何?

How have the research, teaching, or R&D and management results of the employed person given benefit to the project (or your unit)?

受延攬人在延攬期間,使計畫能夠順利進行,對本計畫有顯著的助益。

(4)受延攬人於補助期間對貴單位或國內相關學術科技領域助益如何?

How has the employed person, during his or her term of employment, benefited your unit or the relevant domestic academic field?

受延攬人所研究的丙酮酸脫氫激酶 1 在表皮生長因子促進頭頸鱗狀癌轉移中扮演的角色,是目前國內

相關學術領域中尚未探討的領域,對於未來相關學術領域的研究有顯著的助益。

(5)具體工作績效或研究或教學或科技研發與管理成果:

Please describe the specific work performance, or the results of research, teaching, or R&D and management work:

受延攬人在延攬期間,研究丙酮酸脫氫激酶 1 在表皮生長因子促進頭頸鱗狀癌轉移中扮演的角色。

(6) 是否續聘受聘人?

Will you continue hiring the employed person?

■ 續聘 Yes □不續聘 No

※ 此報告表篇幅以三~四頁為原則。 This report form should be limited to 3-4 pages in principle.

※ 此表格可上延攬優秀人才成果報告繳交說明網頁下載。

This report form can be downloaded in http://scholar.lib.ncku.edu.tw/explain/

數據

Figure 1. EGF induces PDK1 expression in HNSCC cells. (A) HNSCC cells were treated with 50 ng/ml EGF in serum-free medium for  24  h
Figure  2:  EGF  promotes  PDK1  promoter  activity  via  activation  of  ARNT.  Cells  were  transfected  with  20  nM  ARNT  siRNA  oligonucleotides  and  0.5μg  of  pPDK1  promoter  construct  by  lipofection  and  then  treated  with  50  ng/ml  EGF  f

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