藉由調控乳癌細胞 MDA-MB-231 之C1GALT1 可抑制其所導致之蝕骨細胞生成

C1GALT1 knockdown in MDA-MB-231cells suppresses breast cancer cell-induced osteoclatogenesis.

林佳宣(Jia-Hsuan Lin)

指導教授 ： 林能裕

國立臺灣大學/醫學院/解剖學暨細胞生物學研究所/碩士(2018年)

https://doi.org/10.6342/NTU201801943

摘要

乳癌為女性最常見的惡性腫瘤，而有近七成的乳癌會發生骨轉移。一旦發生骨轉移，病人會產生劇烈疼痛、病理性骨折、神經壓迫與運動功能障礙，因此乳癌骨轉移一直是一個棘手的問題。近年研究指出，溶骨性的乳癌骨轉移中，巨噬細胞集落刺激因子(macrophage colony-stimulating factor ; M-CSF)及核因子κ-B配體受體致活劑（Receptor activator of nuclear factor kappa-B ligand ; RANKL）可促進蝕骨細胞的分化，進而加速骨質溶解。黏蛋白種類O-醣基化為最常見的蛋白質轉譯後修飾之一，在合成O-glycan結構的過程中，Galβ1,3GalNAc稱為T antigen 或Core 1 structure，在正常組織中可被醣化轉換酵素延伸成更長的結構。 core 1 β1,3-galactosyltransferase (C1GALT1)為黏蛋白種類O-醣基化作用中可形成Core 1 structure (Gal-GalNAc-ser/The)的醣類轉移酶，而C1GALT1的分子伴侶chaperone Cosmc為C1GALT1活化所必須的。過去研究指出，C1GALT1會在乳癌細胞中大量表現，並且可以調控乳癌細胞的惡性行為。因此我們想知道，醣類轉移酶C1GALT1與其分子伴侶Cosmc在乳癌所誘導的蝕骨細胞生成中所扮演的角色。當乳癌發生骨轉移時，TGF-β(transforming growth factor-β)會因蝕骨作用而從bone matrix中釋出，然而大量的TGF-β也會促使乳癌細胞生長，形成一個惡性循環。因此我們想探討TGF-β與C1GALT1在乳癌骨轉移中是否有調控的機制。
在我們的研究中發現，C1GALT1會受TGF-β細胞激素調控。當MDA-MB-231下調C1GALT1與Cosmc後，其細胞表面的Tn抗原有累積的現象。為了更進一步了解C1GALT1與蝕骨細胞分化的關係，我們將小鼠骨髓細胞與乳癌細胞的培養基共培養。從TRAP staining與Bone Resorption assay的結果顯示，C1GALT1下調(knockdown)後，可抑制蝕骨細胞的分化。另外，由即時聚合酶連鎖反應及西方點墨法的實驗結果也可發現，下調C1GALT1與Cosmc後，RANKL及M-CSF的表現量皆下降。另外，在動物模式中，我們對小鼠進行乳癌細胞心臟注射，並將小鼠的骨頭以Micro CT分析，發現C1GALT1 Knockdown的組別其骨破壞的程度較低。因此綜合上述實驗結果，我們認為在乳癌細胞中下調C1GALT1可以抑制由乳癌細胞所誘導的蝕骨細胞分化，希望未來可以找到治療的標的，進而減緩乳癌骨轉移的發生。

關鍵字

乳癌 ； 醣類轉換酵素 ； 蝕骨細胞 ； 黏蛋白型 O-聚醣

並列摘要

Breast cancer is the most diagnosed malignancy among women worldwide, and bones are a common place for breast cancer cells to metastasize. More recently, several studies have concluded the potential mechanisms of osteolytic bone metastasis in breast cancer is referred to high level of cancer cell secreted Macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa B ligand (RANKL), activators important for osteoclast differentiation. Mucin-type O-glycosylation is one of the most common post-translational modifications of proteins and it is associated with many important biological functions. Aberrant glycosylation of cell surface is associated with malignant transformation of breast cancer. Core 1 ß1,3 galactosyltransferase (C1GALT1) controls the biosynthesis of core 1 O-glycan structure, which is called T antigen. Cosmc is a molecular chaperone thought to be required for expression of active T-synthase, the only enzyme that galactosylates the Tn-antigen to form T antigen. In previous study, overexpression of C1GALT1 has recently been shown to be related to the malignant phenotypes of breast cancer cells. In the bone matrix, transforming growth factor-β (TGF-β) is one of the most abundant growth factors, which is released in active form upon tumor-induced osteolytic bone resorption. TGF-β stimulates bone metastatic tumor cells to secrete factors that further drive osteolytic bone destruction adjacent to the tumor. Thus, TGF-β is a crucial factor responsible for driving the feed-forward vicious cycle of cell growth in bone. This study aims to determine the correlation between T-antigen and breast cancer bone metastasis. Our data show that knockdown C1GALT1 and Cosmc in MDA-MB-231 suppressed the mRNA and protein level of RANKL and M-CSF. Downexpression of C1GALT1 in breast cancer cell prevented osteoclast differentiation in vitro and increased the bone density by intra-cardic injection of breast cancer cells. These findings suggests that exposure of Tn-antigen in breast cancer cell suppress bone metastasis and prevented osteoclast differentiation.

並列關鍵字

Breast cancer ； C1GALT1 ； Mucin-type O-glycosylation ； osteoclastogenesis

參考文獻

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