利用微陣列晶片分析辨識與食道鱗狀上皮癌預後相關之創新生物標記並探討其分子生物機轉之轉譯醫學研究

Identification of A Novel Biomarker Using Microarray Analysis and Investigation of Its Molecular Mechanisms in Esophageal Squamous Cell Carcinoma: A Translational Cancer Study

許峰銘(Feng-Ming Hsu)

指導教授 ： 莊曜宇

國立臺灣大學/電機資訊學院/生醫電子與資訊學研究所/博士(2016年)

https://doi.org/10.6342/NTU201602635

摘要

論述重點：
臺灣食道癌之發生率與死亡率逐年上升,是國內男性前十大惡性腫瘤之一。多數病患因吞嚥困難而就醫確診，診斷時多已為局部晚期，因此預後不佳。此外百分之九十以上的病患罹患的食道癌組織類型為鱗狀上皮細胞癌，其與西方國家多為好發於中下段食道及胃食道交接處的腺癌，不論在致病機轉、危險因子、預後與治療反應等均有很大的不同。針對局部晚期食道癌，前瞻性隨機分派臨床研究顯示包含化學治療、放射治療以及根除性手術之治癒性整合治療能夠為病患帶來較佳的預後。儘管如此，病患整體的無病存活期中位數只達約 20 到 25 個月。同步化學放射治療不論是作為根除性手術前之前導性治療 或是治癒性治療，均能夠增加食道癌病患之整體存活率，因此同步化學放射治療已成為食道癌之標準方式治療之一。目前已知食道腫瘤能否在化學放射治療後達到病理完全緩解是重要的預後因子，但是目前臨床上仍然缺乏有效的方式來預測病患對於化學放射治療之反應與預後，從而從而擬定個人化的治療方式，以最大化治療效益並且最小化治療相關毒性。除此以外，食道鱗狀上皮細胞癌亦尚未有適當的標靶治療藥物能夠進一步增進治療反應率。因此我們試圖藉由微陣列晶片分析辨識與食道鱗狀上皮癌預後相關之創新生物標記並探討所發現生物標記之致癌機轉，藉由此轉譯醫學研究尋找具備預測與治療潛力的分子生物標靶，以期提升食道鱗狀上皮細胞癌之預後。

方法：
本研究分兩部分進行。第一部分收集食道鱗狀上皮細胞癌局部晚期病患於前導性同步化學放射治療前後周邊血液檢體，抽取其循環訊息核醣核酸(messenger RNA)，進行微陣列晶片實驗取得表現體學資料，其後藉由生物資訊與生物統計方法辨識與病患接受根除性手術後達病理完全緩解最具關聯性的生物標記，並以反轉錄聚合酶鏈式反應來驗證發現結果，且在獨立病患群組中驗證其蛋白表現與預後之關聯性。同時我們亦在病理檢體與食道鱗狀上皮細胞癌細胞株偵測該創新生物標記是否過度表現，以證實其具有生物功能。第二部分，我們利用已建立的國人食道鱗狀上皮細胞癌細胞株，藉由調控該創新生物標記基因之表現量，探討該基因對細胞株在致癌能力與對化學放射治療反應之影響，並利用皮下異種腫瘤小鼠模式探討該基因在腫瘤生長與分化所扮演的角色。此外亦進行微陣列晶片實驗取得細胞株在有無抑制該基因之表現體學資料，尋找可能與該基因相關聯的基因。依據這些實驗，我們能夠更深入了解所辨識的創新生物標記在食道鱗狀上皮細胞癌所扮演的角色。

結果：
在第一部分研究方面，我們證實循環基因表現譜在化學放射治療前後會有顯著的變化，並且假設循環基因表現變化可用以預測治療結果。利用資訊學與統計學方法，我們成功辨識出一個食道鱗狀上皮細胞癌的創新生物標記，FAM84B。我們發現FAM84B此一循環訊息核醣核酸與蛋白在同步化學放射治療前後表現量的變化具有顯著的改變。該循環訊息核醣核酸表現量在前導性治療後下降的程度可以用來預測食道腫瘤對化學放射治療的反應程度。此外人類FAM84B蛋白在食道鱗狀上皮細胞癌病理切片與細胞株均呈現過度表現之現象，但在正常食道上皮組織與細胞則為低度表現。在第二部分研究方面，我們發現在食道癌細胞株將人類FAM84B蛋白表現以短髮夾核醣核酸干擾抑制後，其細胞之群落存活率與遷移能力均會下降。相反的，若細胞株過度表現FAM84B則會增加細胞株的群落存活率。而在小鼠異種移植腫瘤生長延遲實驗亦證實抑制FAM84B可以減少食道腫瘤的分裂與增殖能力，並且促使細胞分化較為良好。我們進一步研究發現，人類FAM84B對細胞致腫瘤性的機轉，可能與控制上皮細胞分化蛋白KRT15的表現有關。此外在細胞表現體學實驗分析部分，我們發現人類FAM84B會顯著降低細胞週期基因CCND2表現，並在細胞實驗證實抑制FAM84B會影響細胞週期在G1至S過渡期的進展。最後，我們也證實抑制人類FAM84B會影響食道鱗狀上皮癌細胞株對游離輻射與細胞毒殺藥物順鉑的存活率。

討論：
我們利用高通量生物標記檢測以及生物資訊學分析，發現了一個與食道鱗狀上皮細胞癌相關的周邊循環創新生物標記，人類FAM84B。值得注意的是，和其他類似研究不同，我們所發現的循環生物標記確實在食道鱗狀上皮細胞癌具有致癌特性。人類FAM84B基因位在第八對染色體長臂8q24.21的位置上。目前已知8q24.21該位置的拷貝數變異和許多癌症具有相關性，但由於其鄰近另一個重要的致癌基因，人類C-MYC，因此目前文獻上對於人類FAM84B在細胞功能與致癌機轉的瞭解仍相當的缺乏。我們的研究與其他學者的獨立研究，目前都證實了人類FAM84B在華裔食道鱗狀上皮細胞癌病患的致病機轉應該具有特定的角色。我們的實驗雖然證實人類FAM84B會影響食道癌細胞株的群落存活率、遷移能力、細胞週期、細胞分化與對去氧核醣核酸傷害之反應等，但在分子生物機轉細節方面，尚有待更深入的研究。而在我們的前期研究中更發現，人類FAM84B在食道鱗狀上皮細胞癌的主要病理變化可能是來自於拷貝數變異而非蛋白過度表現，並且在其他具有8q24.21拷貝數變異的惡性腫瘤，如大腸癌細胞株等證實抑制FAM84B能夠明顯降低細胞存活率。因此我們的研究發現僅是一個開端，值得後續的臨床檢體研究與實驗室基礎研究探索人類FAM84B在癌症生物學的功能。

關鍵字

人類FAM84B ； 生物標記 ； 同步化學放射治療 ； 食道鱗狀細胞癌 ； 基因靶向 ； 致腫瘤性 ； 微陣列晶片分析

並列摘要

Background:
The incidence of esophageal cancer has increased year by year to become one of the ten most common and lethal cancers in Taiwanese males. Most patients are diagnosed with initial presentation of dysphagia and therefore have poor prognosis due to the locally advanced disease. Furthermore, the majority of patients have a histology of squamous cell carcinoma (SCC), which quite different in pathogenesis, risk factors, outcomes, and treatment responses, from the western countries, where adenocarcinoma of the lower esophagus and esophagogastric junction is predominant. For patients with locally advanced esophageal cancer, prospective randomized trials have shown that combined modality therapy, including chemotherapy, radiotherapy, and radical esophagectomy, provides the best treatment results. However, median progression free survival remains unsatisfactorily around 20 to 25 months. Concurrent chemoradiotherapy (CCRT), either as neoadjuvant or definitive therapy, improves the overall survival in patients with esophageal SCC (ESCC). It has been recognized that pathological complete response to CCRT is the most important prognostic factor. At present, there is no reliable factor which predicts response to chemoradiation, and thus enables the individualization of treatment strategies. Furthermore, there is no molecular target therapy to improve outcomes for ESCC. In the present study, we utilized microarray analysis to identify novel biomarkers in association with outcomes for ESCC and investigated underlying mechanisms in tumorigenicity. Our aim in conducting the present translational research was to discover biomarkers with the potential to predict outcomes and which may be used as therapeutic targets, to help improve the prognosis of ESCC.

Methods:
In the first part of this study, we collected blood samples in ESCC patients before and after neoadjuvant CCRT and extracted the messenger RNA. We performed microarray experiments and identified candidate circulating biomarkers using bioinformatics and biostatistics tools. We validated our findings using reverse-transcriptase polymerase chain reaction and proximal ligation assay to detect serum protein levels in an independent cohort. We also investigated whether the identified biomarker was overexpressed in cancerous tissues using immunohistochemistry (IHC) analysis, and in ESCC cell lines using western blot. In the second part, we used established ESCC cell lines to study the biological properties of the candidate gene by manipulating its expression levels. We performed in vitro cell assay and in vivo subcutaneous xenograft animal models. We also used a gene expression microarray analysis to identify candidate genes associated with our gene of interest.

Results:
In the first part, we showed that circulating expression profiles were significantly altered before and after CCRT. We therefore hypothesized that changes in gene expression may be associated with treatment outcome and successfully identified a novel candidate biomarker, human FAM84B. Expression levels of FAM84B were significantly down-regulated after CCRT and its fold change was predictive of pathological response to CCRT. Furthermore, we demonstrated that the human FAM84B protein was overexpressed in ESCC cancerous tissue and cell lines but not in the normal esophageal epithelium and epithelial cells. In the second part, we discovered that FAM84B knockdown by short hairpin RNA impaired the clonogenic survival and migration of ESCC cell lines, while FAM84B knock-in promoted clonogenic survival. Inhibition of FAM84B in ectopic xenografts also delayed tumor growth with reduced Ki-67 proliferation index, mitotic index, and promoted cell differentiation. We showed that the tumorigenicity of FAM84B may be associated with expression of the squamous cell differentiation protein KRT15. In addition, microarray analysis revealed that CCND2 expression was significantly associated with FAM84B expression. We demonstrated that inhibition of FAM84B delayed G1/S phase progression in the cell cycle by downregulating CCND2. In the last part, we showed that FAM84B was associated with sensitivity to ionizing radiation and cytotoxic cisplatin in ESCC cell lines.

Discussion
Unlike similar research, we identified a novel circulating biomarker with biological properties using high-throughput technology. Human FAM84B is located at chromosome 8q24.21, which is known to associate with different cancer types due to copy number alterations. The function of FAM84B is understudied due to its proximity to the well-known oncogene, MYC. However, we and other independent investigators have shown that FAM84B is associated with pathogenesis in ESCC in the Han population. While our earlier findings showed the oncogenic properties of FAM84B in ESCC, its molecular mechanisms required further investigation. In our preliminary research, we found that copy number variations in FAM84B may have a more important role in ESCC. Furthermore, inhibition of FAM84B in 8q24.21 amplified cancers, such as colorectal cancer cells, and significantly impaired cell proliferation. Our finding is only the beginning, and we believe it worth further investigation in both clinical specimens and laboratory work to study the role of FAM84B in cancer biology.

並列關鍵字

Biomarkers ； Carcinogenesis ； Concurrent chemoradiotherapy ； Esophageal squamous cell carcinoma ； Gene targeting ； Human FAM84B ； Microarray analysis

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