利用三維組織培養模式探討Haspin 在人類大腸癌的角色

Abstract

大腸癌是造成癌症致死率及發病率最高的人類癌症之一。Haspin是一種Histone 3的磷酸化激酶，可透過磷酸化Histone 3上threonine 3的位點去幫助染色體運送複合體的形成，而促進細胞有絲分裂的進行。然而，Haspin在大腸癌中的功能仍不清楚。在本研究中，我們採用三維組織培養的模式去探討Haspin在人類大腸癌中所扮演的角色。首先，人類大腸癌組織被培養在含Matrigel的培養基中，觀察癌症幹性是否與Haspin 蛋白的表現有所關聯性。有趣的是，我們發現人類大腸癌在三維組織培養的情況下Haspin 的蛋白表現量較低，相反地CD133蛋白則是大量表現。Haspin的下游分子之磷酸化H3T3蛋白表現的也較低。我們也利用免疫螢光染色發現有相同的結果。此外，當在人類大腸癌組織細胞中轉殖Haspin表現載體，大量表現Haspin蛋白時，CD133的蛋白表現會減少。此外，我們透過免疫組織化學染色來比較臨床大腸癌病人的大腸正常組織及腫瘤中Haspin 蛋白、磷酸化H3T3蛋白Survivin蛋白，大部分的大腸腫瘤相較於正常的大腸組織會大量表現Haspin蛋白及Survivin蛋白。在我們結果顯示，在三維組織培養的情況下，Haspin蛋白會減少癌幹特性，但Haspin 蛋白在大腸癌組織中則大量表現，我們推測在三維組織培養中Haspin表現量低時可以維持癌症的幹性，然而，在臨床病人的大腸癌組織中Haspin大量表現時則會促進腫瘤的生長。我們認為Haspin具有雙重功能在調控癌症的幹性與腫瘤的生長，Haspin是一個具有潛力的癌症標靶，可以作為未來治療大腸癌的新策略。

Colorectal cancer (CRC) is one of the leading causes of cancer-related morbidity and mortality in the world. Haspin is a histone H3 kinase that can phosphorylate H3 on threonine 3 (p-H3T3), which recruits chromosomal passenger complex (CPC) and promotes cell division. However, the functions of Haspin in CRC is still unclear. In this study, we used the 3D tissue culture model to investigate the role of Haspin in the patient CRC tissues. The CRC tissues were cultured with Matrigel to observe the cancer stemness properties whether correlated to Haspin protein expression. Interestingly, we found these tissue cells that expressed the lower protein levels of Haspin but highly expressed CD133 proteins by using the 3D tissue culture system. The p-H3T3 protein levels also expressed at the low level. We also verified the similar results by immunofluorescence staining and confocal microscopy. Moreover, overexpression of Haspin proteins by transfection with a Hapsin-expressed vector reduced the protein levels of CD133. In addition, we compared the Haspin and p-H3T3 protein levels in colorectal normal tissues and tumors from CRC patients. Most CRC tumor tissue samples highly expressed Haspin proteins by comparing normal tissues using immunohistochemistry staining. These results suggest that lower expression of Haspin may maintain the cancer stemness property under 3D tissue culture condition; however, higher Haspin proteins promote tumor growth in patient CRC tumors. We propose that Haspin can display the bi-functional effects in regulating the cancer stemness and tumorigenesis of CRC. Our findings demonstrate that Haspin is a potential target and novel strategy for CRC therapy.