肝細胞癌中ASPM基因之臨床病理與功能研究

Abstract

肝細胞癌是一種世界性常見的惡性腫瘤，在台灣更是男性癌症死亡原因的第一位。肝細胞癌通常發生在長期的慢性肝臟疾病之後，因為肝細胞反覆的壞死性發炎反應造成伴隨著基因變異的細胞再生與不正常的DNA複製。這些不正常的基因變異包含有致癌基因與腫瘤抑制基因的差異性表現。為了更進一步瞭解肝細胞癌腫瘤發生與腫瘤進行過程的分子機制，分析並且辨別差異性表現的基因是非常重要的步驟。基因分示法是一種用來辨別差異性表現基因的方法，此方法有經由使用簡單的反轉錄聚合酶連鎖反應以及可同時比較多組檢體來減少個別差異的優點。利用基因分示法，我們發現人類ASPM基因在肝細胞癌中常會有過量的表現。 人類ASPM基因 (abnormal spindle-like microcephaly associated) 位於一種造成胎兒腦部發育缺陷的基因遺傳疾病MCPH (autosomal recessive primary microcephaly)的第五號染色體基因座位置上。ASPM基因的同型合子突變是造成MCPH最常見的原因，會造成病患的小腦症且心智發育遲緩。人類ASPM是一個很大的蛋白質，包括有一個預測性的微小管結合位置、一個calponin-homology區域、74個重複的calmodulin-binding isolucine-glutamine區域以及最後的carboxy端結尾。比較各物種生物間的ASPM同源基因，可發現其蛋白質結構帶有很高度的保留性，也因此各物種間的ASPM同源基因可能有相同的生物功能。果蠅的ASPM同源基因Abnormal spindle (Asp)蛋白質座落在中央紡綞體微小管的負端，對於微小管聚集在紡綞體極端的功能有重大的影響。因此果蠅Asp蛋白質的突變會造成不正常的紡綞體組織而使得果蠅在發育的過程於幼蟲至蛹期間發生死亡。此外，老鼠的ASPM同源基因Aspm，則被發現會於胎兒時期高度的表現在有旺盛神經發育的大腦神經皮質區，而在出生後由於神經發育停止而迅速減少表現。綜合這些研究，我們認為ASPM蛋白質可能是一個細胞分裂時與紡綞體相關且與大腦發育相關的蛋白質。 在本篇研究中，我們發現在252個原發性單一性肝細胞癌腫瘤病患中，有136人有ASPM基因的過量表現 (54%)。同時在數種常見的人類惡性腫瘤中也有過量的表現，但是良性腫瘤則不會表現或表現量很低。ASPM基因同時也表現在多個胎兒器官與數種癌症細胞株之中，但在成人器官中則表現量很低。經由臨床病理學分析，我們發現肝細胞癌中ASPM基因的過量表現會與病人血清中高於200ng/ml的甲型胎兒蛋白、分化差與高分期的肝細胞癌腫瘤相關，P值都小於0.0001。有ASPM過量表現的肝細胞癌病人，常是分化較差且伴隨著血管的侵犯，而且病患的手術後十年存活率較低，P值等於0.00001。我們使用nocodazole藥物來同步化細胞進行，進而檢測細胞週期，發現ASPM基因在細胞週期進行中會有不同的表現。當細胞從M期走向G0/G1期時，ASPM表現量減少但在S期則逐漸增加，於M期表現量最高。在老鼠的肝臟部分切除及肝臟再生實驗中，發現在肝細胞再生的第48至72小時（DNA複製期間），老鼠的Aspm基因表現量會升高。另一方面，在使用全反式retinoic acid處理造成分化的NT2細胞與不繼代處理造成分化的HepG2細胞中，ASPM基因的表現量會減少。在免疫螢光染色方面，使用合成peptide製造的兔子多株抗體，我們發現人類ASPM蛋白質染色於細胞核內，且在細胞分裂時聚集於紡綞體的極端。這些結果顯示ASPM基因與細胞分裂，細胞增殖以及細胞分化有著相關性。藉由siRNA降低基因表現的系統，我們發現減少ASPM表現的細胞株比起對照組細胞，在soft agar中會降低細胞的非附著性生長能力，且在膠原蛋白膠（3-dimensional type I collagen gel）中細胞的偽足會減少。ASPM表現降低的細胞同時也有p27表現增多與stathmin表現減少的現象，此二個蛋白質都與細胞活動性相關。這些結果顯示減少ASPM的基因表現會降低腫瘤細胞的生長與侵犯，而這樣的腫瘤細胞侵犯降低可能是經由p27的增加與stathmin的減少來調控。這些結果還需要更進一步的實驗來加以證明。 總而言之，我們的研究結果顯示人類ASPM基因參與了細胞的DNA複製、細胞分裂的週期、以及細胞的增殖與分化。而且肝細胞癌病患中ASPM基因的過量表現是很常見的現象。ASPM基因的過量表現在腫瘤的細胞增殖、細胞分化以及腫瘤侵犯與轉移扮演著重要的角色。另外，我們也認為人類ASPM基因的過量表現可以作為一個有效的手術後腫瘤早期再發及預後較差的預測因子。

Hepatocellular carcinoma (HCC) is a common malignancy worldwide. In Taiwan, it is the leading cause of cancer death in man. HCC usually occurs after a prolonged course of chronic liver disease with repeated necroinflammatory liver cell followed by cell regeneration, accompanied by DNA replication and accumulation of genetic and epigenetic alternations including oncogenes and tumor suppressor genes. To identify differentially expressed genes is an important step to better understand the molecular mechanisms in the tumorigenesis and tumor progression of HCC. Differential display (DD) is one of the methods to verify differentially expressed genes with advantages of using the simple RT-PCR and comparing multiple samples of different characters at the same time to minimize the individual variation. By differential display method, we identified the human ASPM gene that was frequently overexpresses in HCC. The human ASPM (abnormal spindle-like microcephaly associated) gene is at the 5th locus of MCPH (autosomal recessive primary microcephaly), a genetic disorder of fetal brain growth. The homozygous mutations of ASPM gene are the most common cause of MCPH and lead to microcephaly and mental retardation. The human ASPM is a large protein containing a putative amino terminal microtubule-binding region, a calponin-homology domain, 74 repeated calmodulin-binding isolucine-glutamine (IQ) domains and a carboxy terminal region. Comparisons of the ASPM homologue proteins show high conservations among the species and are therefore proposed to share biochemical functions. The Drosophila homologue abnormal spindle (Asp) protein localizes to the minus ends of central spindle microtubules and is required for the aggregation of microtubules into focused spindle poles. Therefore, mutations of Asp cause abnormal spindle structure and lead to larval-pupal lethality of Drosophila. Moreover, mouse homologue of human ASPM, Aspm, is highly expressed in embryonic days in the neuroepithelium at sites of active neurogenesis and decreased postnatally after the completion of neurogenesis. Taking these researches together, we suggest that ASPM protein is proposed to be a mitotic spindle related and brain growth required protein. In our study, we found a frequent overexpression of human ASPM gene in 136 out of 252 unifocal primary HCCs (54%), and in several common human malignancies but not in benign tumors. ASPM gene was also expressed in multiple fetal tissues and several cancer cell lines, but not adult tissues. By clinicopathological analysis, we showed that the overexpression of ASPM gene in HCC was associated with high AFP level (>200 ng/ml), high-grade, high-stage tumor HCC, all Ps<0.0001. The patients of HCCs with ASPM overexpression, which had poor differentiated tumors and more frequent vascular invasion, had poor prognosis and lower 10-years survival rate, P=0.00001. During the cell cycle progression after release from nocodazole block, ASPM gene showed differential expression. ASPM mRNA levels decreased from M phase to G0/G1 phase, but increased dramatically upon the entry into the S phase, and then reached the peak at M phase. The mouse Aspm gene was upregulated during the period of DNA synthesis in liver regeneration after partial hepatectomy. On the other hand, human ASPM mRNA level decreased after differentiation of NT2 cells induced by prolonged all trans-retinoic acid treatment and HepG2 cells induced by prolonged cultivation without passage. By immunofluorescence staining using polyclonal anti-ASPM antibody against synthetic peptide, we showed that ASPM was located in nuclei, with enrichment at the mitotic spindle poles during mitosis. These results suggest that ASPM gene is involved in cell mitosis, proliferation and differentiation. With siRNA knockdown system, we found that ASPM stable knockdown cells showed reduced anchorage-independent colony formation in soft agar assay and reduced tumor cell processes in 3D gel culture compared with control cells. ASPM knockdown cells also showed increased p27 and decreased stathmin, both are related to cell motility. These results indicate that decreased ASPM gene expression would lead to reduce tumor cell growth and tumor cell invasion in vitro, probably via the upregulation of p27 and downregulation of stathmin. Further studies are warranted for further clarification. In conclusion, our results indicate that human ASPM gene is involved in the DNA synthesis, cell cycle progression, and cell proliferation and differentiation, and the overexpression is a common event in HCC. ASPM overexpression plays important role in the tumor cell proliferation, differentiation, and invasion/metastasis, probably via the downregulation of p27 and upregulation of stathmin. It is also concluded that human ASPM overexpression is a useful predictive factor for early tumor recurrence after tumor resection, and a prognostic factor associated with poor prognosis.