To Study the Inhibitory Mechanisms of Nm23-H1 in Human Head and Neck Squamous Cell Carcinoma: the Role of Sonic Hedgehog Signaling

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Project title

Code/計畫編號

MOST104-2314-B075-055

Translated Name/計畫中文名

探討Nm23-H1抑制頭頸鱗狀上皮癌細胞惡化之作用機制---Sonic Hedgehog 訊息之角色

Funding Organization/主管機關

National Science and Technology Council

Co-Investigator(s)/共同執行人

王怡芬(計畫主持人)

Department/Unit

Taipei Veterans General Hospital Department of Otorhinolaryngology-Head and Neck Surgery

Website

https://www.grb.gov.tw/search/planDetail?id=11587455

Year

2015

Start date/計畫起

01-08-2015

Expected Completion/計畫迄

31-07-2016

Co-Investigator(s)

Chun-Ju Chang

Bugetid/研究經費

950千元

ResearchField/研究領域

臨床醫學
藥學
基礎醫學

研究背景：頭頸癌的放射線及化學治療抗性及高度轉移能力是其預後不佳的主因。我們最近的研究顯示患者腫瘤組織Nm23-H1 (non-metastatic clone 23 type 1) 低表現時，容易遠端轉移、化放療效果不佳、存活期短。頭頸鱗狀上皮細胞癌SAS細胞模式證實，減低Nm23-H1表現造成細胞入侵及化放療抗性；而Nm23-H1過表現能改善cisplatin敏感性、促進細胞週期S期累積、增加細胞周期蛋白cyclin E1及A表現，可能因此抑制頭頸癌細胞增生、提升化療效果 (Wang et al., 2008 & 2014)。患者腫瘤組織Sonic Hedgehog (SHH)之下游訊息分子PTCH過表現及細胞核GLI1增加也與淋巴轉移有關；細胞核GLI1過表現患者之腫瘤較大、容易復發 (Wang et al., 2012)，但SHH訊息如何影響頭頸癌惡化特徵仍缺乏明確證據。我們的先期實驗發現，SAS細胞Nm23-H1低表現時其SHH大量增加，因此我們有興趣探討SHH訊息路徑在Nm23-H1調節頭頸癌預後之重要性。本研究目標為：一、鑑別Nm23-H1所抑制之SHH訊息分子；二、探討SHH訊息分子對Nm23-H1所調節之化學放射治療敏感性的作用機轉；三、研究SHH訊息分子對Nm23-H1所抑制之細胞轉移的作用機轉。研究方法：以SAS細胞株所建立的Nm23-H1高度或低度表現之穩定轉殖株(stable clones)共五株，第一年：以免疫染色法觀察Nm23-H1表現量對於SHH、細胞膜PTCH及SMO、細胞核GLI的影響。第二年：以small interfering RNA (siRNA)、單株抗體或拮抗劑抑制SHH訊息因子之基因表現或活化，評估其對放射線及化學治療效果的影響（包括：群落形成能力、48小時存活率及-H2AX）、細胞凋亡現象（包括：細胞週期、TUNEL及Annexin V-FITC assays）、SHH訊息分子之基因表現分析(即時定量聚合酶連鎖反應及免疫轉漬法)。第三年：以siRNA或單株抗體抑制SHH訊息因子之基因表現或活化，觀察對於細胞轉移之影響及作用機轉（包括：轉移盤遷徙/入侵試驗、MMP-9及E-cadherin等細胞移動相關蛋白表現）。預期結果：我們的研究將提供Nm23-H1調控頭頸癌惡化過程中SHH訊息的新穎角色的證據與分子機制。我們的結果將推論SHH訊息去活化可能成為治療人類癌症的新策略。 Background: Chemo-/Radio-resistance and highly metastasis are poorly prognostic factors of head and neck cancer. Our previous studies demonstrated that low tumor Nm23-H1 (non-metastatic clone 23 type 1) expression correlated with locoregional recurrence of HNSCC following postoperative cisplatin-based therapy and poor patient prognosis. In HNSCC SAS cell line, knockdown of Nm23-H1 renders in vitro invasion and chemo-/radio-resistance. Besides, overexpression of Nm23-H1 improved the chemosensitivity of SAS cells to cisplatin, which was associated with increased cisplatin-induced S-phase accumulation and upregulation of cyclin E1 and A. Meanwhile, overexpression of Sonic Hedgehog (SHH) signaling molecules, PTCH, SMO and nuclear GLI1, were associated with lymphatic metastasis. Moreover, nuclear GLI1 overexpression correlated with primary tumor size, and tumor recurrence. However, the effects of SHH signaling activation on cancer malignancies of HNSCC are still unclear. Our preliminary results revealed that knockdown of Nm23-H1 induced SHH induction in SAS cells. Therefore, we main focus is to investigate the role of SHH signaling in Nm23-H1-mediated prognosis of HNSCC. This study has the following three specific aims. Aim 1: To identify Nm23-H1-targeted SHH signaling molecule; Aim 2: To study the effects and mechanisms of SHH signaling molecule on Nm23-H1-regulated chemo-/radio-sensitivity; Aim 3: To investigate the mechanisms of SHH signaling molecule on Nm23-H1-suppressed cancer migration/invasion in HNSCC. Methods: By the established in vitro models of HNSCC cell lines with stable Nm23-H1 overexpression and knockdown, we will research into the effects of Nm23-H1 expression on SHH, PTCH and SMO, as well as nuclear GLI in the first year. In the second year, using siRNAs and antagonists of SHH signaling molecules, the effects of SHH signaling on chemothearpy and radiation will be assessed by gamma-H2AX (DNA double strand breaks) assay, trypan blue exclusion assay and clonogenic assay in each cell lines, respectively. Using cell cycle analysis, TUNEL assay, and Annexin V-FITC assay, we will investigate the effects of SHH signaling molecules on cell cycle regulation and classic apoptosis. Using real-time quantitativey PCR and immunoblotting, the gene expression will be analyzed. In the third year, the regulatory mechanisms of SHH signaling molecules on cell migration/invasion will be studied using in vitro transwell assays. Using Western blot analysis, the expression levels of tumor metastatic proteins, such as MMP-9 and E-cadherin, will be measured in each SAS cell lines. Anticipated results: Our studies will provide the evidence and molecular mechanism to identify novel roles of SHH signaling in Nm23-H1 regulating cancer malignance of HNSCC cells. Our results will suggest that inactivating SHH signaling may be an important therapeutic strategy for this disease.

Keyword(s)

化學放射治療抗性
細胞遷徙
SHH
PTCH
GLI
chemo-/radio-resistance
cell migration

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To Study the Inhibitory Mechanisms of Nm23-H1 in Human Head and Neck Squamous Cell Carcinoma: the Role of Sonic Hedgehog Signaling

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