結締組織生長因子促進頭頸癌細胞的幹細胞特性

幹細胞龕(Stem cell niche)，可以維持幹細胞原有的特性，像是自我更新以及分化成不同細胞類型的能力，而一但幹細胞離開龕(Niche)就會喪失了這些幹細胞特性，開始進行分化。幹細胞龕是由一群基質細胞(Stromal cell)所組成，過去已知基質細胞所分泌的生長因子與維持幹細胞的特性有相關大的關係，而基質細胞的組成中又以纖維母細胞(Fibroblast)占絕大部分，纖維母細胞所分泌最多的生長因子就是結締組織生長因子(CTGF)。此外，從腫瘤的形成中也已經知道會有一群基質細胞包圍，同樣地，這一群基質細胞也會分泌生長因子支撐癌幹細胞(cancer stem cell)的幹細胞特性。另外，在之前的研究中發現頭頸癌病人的組織切片其CD44(+)細胞群大都鄰近基質細胞，這就暗示說癌幹細胞與基質細胞存在著一種重要的關係。因此，我們認為結締組織生長因子與幹細胞特性可能有相關。結締組織生長因子是屬於CCN(Cyr61/CTGF/Nov)家族的一員，是一種分泌型的蛋白質，目前已知參與了許多生物功能，如細胞分裂、細胞凋亡、細胞附著、細胞移動、細胞外基質製造與血管新生等，也常在許多不同的癌症中扮演不同的角色。在本篇研究中，將要探討結締組織生長因子與頭頸癌細胞的幹細胞特性之間的關聯性。實驗中我們利用結締組織生長因子的重組蛋白模擬外生性的結締組織生長因子蛋白，另外利用結締組織生長因子的質體外送入內生性結締組織生長因子表現量低的頭頸癌細胞中，使其大量表現內生性的結締組織生長因子蛋白，觀察外生性與內生性結締組織生長因子對於頭頸癌細胞幹細胞特性的影響。我們發現不論經由結締組織生長因子的重組蛋白或質體處理後，都會促進頭頸癌細胞的幹細胞基因(Nanog、Oct4、Sox2)表現與富裕幹細胞的標記(CD44、ALDH1、Side population)。此外，也利用細胞非貼附性生長實驗(Soft agar assay)與聚球實驗(Sphere formation assay)證實結締組織生長因子不僅促進幹細胞基因與標記的增加外，更使頭頸癌細胞獲得幹細胞的特性。最後，我們證實結締組織生長因子調控幹細胞基因表現是需要透過ESR1與JUN轉錄因子。綜合以上實驗結果，說明了結締組織生長因子與幹細胞特性的關係，同時證實不論外生性或內生性的結締組織生長因子皆可促進頭頸癌細胞的幹細胞特性。

關鍵字

結締組織生長因子；幹細胞特性；頭頸癌

並列摘要

Fibroblasts, the major population of stromal cells in microenvironment, compose niches as well as tumoral microenvironment and secrete many growth factors; one of the most growth factors is connective tissue growth factor (CTGF/CCN2). CTGF is a member of the CCN family, which exhibit diverse cellular functions in areas such as regulation of cell division, chemotaxis, apoptosis, adhesion, motility, and ion transport. CTGF is also involved in the development of many different types of cancers. In the study, we investigated the role of CTGF, a cytokine providing crucial impact in different types of human cancers, in regulating the stem-like properties of head and neck cancer cells. We found that Sox2, Nanog and Oct4, three of the most important stemness genes, were up-regulated in SAS cells treated by recombinant CTGF (rCTGF) or ectopic over-expressing CTGF. We subsequently analyzed the putative stem cell markers in SAS cells treated by rCTGF or overexpressing CTGF versus control. It revealed that SAS cells treated by rCTGF and SAS/CTGF stable clones enriched the side population, CD44-positive population and aldehyde dehydrogenase 1(ALDH1) activity, indicating the stem-like phenotype, of oral cancer cells significantly. Moreover, functional assays for evaluating the self-renew property showed a increase in spheroid-forming ability in FaDu cells treated with rCTGF. Over-expression of CTGF also enhanced the clonogenecity in SAS cells. Finally, we demonstrated the downstream critical factors, ESR1 and JUN, responsible for CTGF-induced Oct4, Nanog and Sox2 genes expression. Overall, excessive endogenous (over-expression) or exogenous (recombinant) CTGF both promote stem-like properties of head and neck cancer cells. These findings suggest that CTGF promote stemness in head and neck cancer cells.

並列關鍵字

CTGF ； Stem-like properties ； Head and neck cancer

參考文獻

Aikawa, T., Gunn, J., Spong, S. M., Klaus, S. J., and Korc, M. (2006). Connective tissue growth factor-specific antibody attenuates tumor growth, metastasis, and angiogenesis in an orthotopic mouse model of pancreatic cancer. Mol Cancer Ther 5, 1108-1116.

Baltus, G. A., Kowalski, M. P., Tutter, A. V., and Kadam, S. (2009). A positive regulatory role for the mSin3A-HDAC complex in pluripotency through Nanog and Sox2. J Biol Chem 284, 6998-7006.

Bork, P. (1993). The modular architecture of a new family of growth regulators related to connective tissue growth factor. FEBS Lett 327, 125-130.

Bradham, D. M., Igarashi, A., Potter, R. L., and Grotendorst, G. R. (1991). Connective tissue growth factor: a cysteine-rich mitogen secreted by human vascular endothelial cells is related to the SRC-induced immediate early gene product CEF-10. J Cell Biol 114, 1285-1294.

Brigstock, D. R. (1999). The connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed (CCN) family. Endocr Rev 20, 189-206.

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結締組織生長因子促進頭頸癌細胞的幹細胞特性

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