研究背景: 許多文獻指出，Kirsten 鼠肉瘤病毒癌基因 (Kirsten sarcoma viral oncogene, Kras) 的突變會促進癌細胞轉移及生長，並且使臨床病人產生不良預後；此外，研究也發現Kras基因突變會促進下游轉錄因子的表現。本研究室過去發現致癌性鋅指蛋白 (zinc finger protein) ZNF322A會藉由調控下游基因轉錄進而加速癌症發展。我們進一步利用基因轉殖鼠 (transgenic mice) 模式發現ZNF322A能加成由Kras誘導的肺腫瘤生成，暗示Kras也許會透過促進ZNF322表現，進而加速癌症惡化。
研究目的: 本研究旨在探討於肺癌細胞及動物模式中，Kras基因突變是否能促進ZNF322A表現以及促癌特性，並更進一步尋找由Kras誘導ZNF322A轉錄活化的可能機制。我們並以肺癌病人檢體闡明ZNF322A以及其上游調節因子的關係。
研究結果: 在基因轉殖鼠中，kras/znf322a 雙轉殖鼠較znf322a單轉殖鼠有較高的腫瘤發生率。我們利用在肺癌細胞中大量表現活化態Kras (KrasG13V)，發現Kras的突變會促進ZNF322A的表現，並且進一步促進癌細胞遷移。接著我們利用冷光啟動子分析 (luciferase promoter activity)、染色質免疫沉澱-即時聚合酶連鎖反應 (ChIP-qPCR)、即時逆轉錄聚合酶連鎖反應 (qRT-PCR) 驗證在Kras突變下，陰陽1 (Ying Yang 1, YY1) 透過正向調控ZNF322A基因啟動子區域進而上調ZNF322A表現，並促進由ZNF322A介導的癌細胞遷移、癌細胞增生以及周圍人類臍帶血管內皮細胞 (human umblical vein endothelail cell, HUVEC) 的遷移。在動物實驗中，我們利用Matrigel plug angiogenesis assay發現若抑制肺癌細胞中YY1或ZNF322A表現，會減少由Kras促使之血管新生現象。以上結果證明Kras-YY1-ZNF322A路徑確實能促使腫瘤進程及腫瘤血管新生。在臨床檢體研究，我們透過免疫組織染色化學法發現ZNF322A的表現與YY1以及代表血管新生標記 CD31的表現具有正相關。另一方面，除了YY1之外，我們也發現轉錄訊息傳遞活化子3 (Signal transducer and activator of transcription 3, STAT3) 可能藉由與核因子活化B细胞輕鍊增強子-p65單元 (Nuclear factor kappa-light-chain-enhancer of activated B cells p65 subunit, NF-B p65) 合作，或是藉由調控ZNF322A 基因啟動子區域的過度甲基化，進一步降低ZNF322A的轉錄表現。
結論： 我們的研究深入探討ZNF322A上游的轉錄調控機制，並由細胞及動物實驗發現Kras/YY1藉由正向調控ZNF322A轉錄而促進癌症腫瘤特性。另外，STAT3可能藉由促進ZNF322A基因啟動子區域的過度甲基化而降低ZNF322A的表現。Kras/YY1、STAT3/ZNF322A路徑在未來也許是具有潛力的治療目標。
關鍵字：肺癌、ZNF322A鋅指蛋白、Kirsten鼠肉瘤病毒癌基因(Kirsten rat sarcoma viral oncogene)、陰陽1 (Ying Yang 1)、轉錄訊息傳遞活化子3 (Signal transducer and activator of transcription 3)、腫瘤血管新生。

Background: Accumulating evidence has revealed that mutation of Kirsten rat sarcoma viral oncogene (Kras) contributes to tumor metastasis, growth, and poor prognosis. In addition, previous studies showed that Kras mutation promoted expression of downstream transcription factors. Our previous study identified a novel zinc finger transcription factor ZNF322A, which could promote lung tumorigenesis through transcriptional dysregulation of downstream genes. Notably, our transgenic mice study demonstrated that ZNF322A synergized KrasG12D mutation-driven lung tumorigenesis, suggesting that Kras may upregulate ZNF322A expression and thus promote tumor malignancy.
Purpose: This study aims to investigate whether Kras mutation enhances ZNF322A expression and tumorigenicity, and to further dissect underlying mechanisms of Kras mutation-driven ZNF322A transcriptional activation. In addition, clinical study will elucidate the relationship between ZNF322A and its upstream transcriptional regulators.
Results: Our transgenic mice study revealed that mice harbored with KrasG12D/Znf322a possessed higher tumor initiation ability compared to those with KrasG12D single transgene. We next adapted in vitro KrasG13V inducible model and found that KrasG13V mutation promoted ZNF322A transcription, and Kras-ZNF322A axis contributed to cancer cell migration. Among Kras downstream transcription factors, we validated that Ying Yang 1 (YY1) upregulated ZNF322A expression through targeting its promoter in the context of Kras mutation by quantitative reverse transcriptase-PCR, luciferase promoter assay and chromatin immunoprecipitation-PCR. Reconstitution experiments by knocking down of YY1 under KrasG13V activation decreased KrasG13V-promoted cancer cell migration and proliferation. Furthermore, knockdown of YY1 or ZNF322A attenuated angiogenesis in vitro and in vivo by HUVEC endothelial cell migration assay and matrigel plug assay, respectively. These cell and animal data depicted the role of Kras-YY1-ZNF322A axis in lung tumorigenesis and neo-angiogenesis. In clinical patients, ZNF322A expression correlated with angiogenesis marker CD31, and YY1 also showed boarder line correlation with CD31. On the contrary, we identified that signal transducer and activator of transcription 3 (STAT3) may downregulate ZNF322A transcription by cooperating with NF-B or by enhancing ZNF322A promoter hypermethylation.
Conclusions: Our study dissects the novel upstream transcriptional regulation of ZNF322A. Kras/YY1 axis enhanced ZNF322A transcription and promoted ZNF322A tumorigenicity in vitro and in vivo, while active STAT3 attenuated ZNF322A transcription by enhancing ZNF322A promoter hypermethylation. Targeting Kras/YY1 or STAT3/ZNF322A axis may be potential for lung cancer treatment.
Key words: Lung cancer, ZNF322A, Kras, YY1, STAT3, angiogenesis.

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