探討Osteopontin剪接變異型對人類肺癌細胞株CL1-5生長與侵襲之影響

Osteopontin (OPN) 是一個磷酸化的醣蛋白，許多文獻皆指出，OPN在病理與生理上扮演多種角色。OPN存在於細胞內與細胞外，目前已知OPN mRNA可經轉錄後剪接形成三種剪接變異型，全長OPN full-length (OPN-a)、OPN-exon5- (OPN-b)、 OPN-exon4- (OPN-c)。在許多癌症中發現OPN有過度表現的情形，包括乳癌、前列腺癌、大腸癌、頭頸癌、肝癌與肺癌等等。在多數轉型細胞、動物實驗模式、以及人類癌細胞實驗皆證實了OPN的表現與腫瘤細胞的轉移具有明顯的正相關。這些結果表示，OPN在腫瘤發展上扮演著重要的角色。然而在肺癌中各個OPN剪接變異型所扮演的角色仍未清楚。本論文將探討OPN剪接變異型對肺癌細胞株的生長與侵犯所扮演的角色。 OPN在多數的肺癌細胞株中表現量極低，除了CL1-0、H460、H1355外。OPN高表現量的CL1-0，侵犯能力是低的；OPN低表現量的CL1-5，侵犯能力卻是高的。在這裡，我們利用CL1-5及A549肺癌細胞株過度表現OPN剪接變異型，觀察細胞有何影響。我們的實驗結果顯示，過度表現OPN-exon4-、OPN- exon5-的細胞株的生長速度和帶有空載體的控制組相似，而內生性的全長OPN可抑制細胞生長。有趣的是，OPN-exon4-會增加CL1-5細胞的侵犯能力，而OPN- exon5-則不影響CL1-5細胞的侵犯能力。令人意外的是，全長的OPN會抑制細胞侵犯能力，甚至含有全長OPN的條件培養液也會抑制細胞侵犯的情形。這結果令人相當訝異，因為先前文獻指出，全長OPN會促進而非抑制A549細胞侵犯能力。由我們的實驗結果發現，在體外實驗中，過度表現全長OPN會抑制肺癌細胞株的生長與侵犯能力。過度表現OPN-exon4-會增加CL1-5細胞的侵犯能力，而OPN-exon5-對肺癌細胞的生長與侵犯則是沒什麼影響。由於最近的趨勢是將OPN當成癌症治療的標的基因，而不同剪接變異型的OPN對癌細胞的影響很不一樣，因此，我們應進一步分析肺癌組織以及其微環境中，是表現哪一種剪接變異型的OPN。所以探討腫瘤生長與轉移上OPN剪接變異型所扮演的角色，以及剖析這些機制所參與的訊息傳遞路徑都是相當重要的。

關鍵字

骨橋蛋白；細胞侵犯

並列摘要

Osteopontin (OPN) is a glyco-phosphoprotein independently discovered by investigators from diverse scientific backgrounds and implicated in a broad array of physiological and pathological processes. OPN exists both intra- and extracellularly and in numerous post-translational isoforms. OPN is subject to alternative splicing, which yields 3 messages, osteopontin-a (full-length), osteopontin-b (exon 5-) and osteopontin-c (exon 4-). High level of OPN expression has been found in a number of cancer types including breast, prostate, colon, head and neck, and hepatic cancers and lung cancer. Strong association of OPN expression with tumor metastasis has been established in various transformed cell lines, animal tumor models, and human cancers. These findings suggest that OPN is a key extracellular molecule involved in tumor development and progression. However, OPN splice variants have not been extensively evaluated in lung cancer. This study aimed to explore the roles of various splice forms of osteopontin in cell growth and invasion in lung cancer cell lines. The expression of OPN in lung cancer cell lines is very low except in the CL1-0,H460 and H1355 cell lines. The low invasive cell line CL1-0 expresses high level of OPN while OPN expression in high invasive cell line CL1-5 is very low. In this study, we tested the effect of OPN splice variants on growth and invasion in CL1-5 and A549 lung cancer lines. Our data showed that CL1-5 cells stably expressed OPN with deletion of exon 4 (OPN-exon4- ) or exon 5 (OPN-exon5-) had similar growth activity compared with vector control, while exogenous full-length OPN had the ability to inhibit cell growth. Interestingly, stable expression of exogenous OPN-exon4- enhanced in vitro invasive ability of CL1-5 cell compared to vector control, while OPN-exon5- did not change invasive ability. Unexpectedly, overexpression of full-length OPN inhibited CL1-5 invasion ability, and this ability is retained in contioned medium treatment. This result is rather surprising, since most studies showed that OPN enhanced, but not inhibited cell invasion. In summary, overexpression of full-length OPN reduced lung cancer cell growth and inhibited cell invasion. Overexpression of OPN-exon4- enhanced CL1-5 cell invasive ability, while OPN-exon5- has little effect on lung cancer cell growth and invasion. It is important to resolve which isoform of OPN is/are overexpressed in lung cancer and its microenvironment. Since OPN becomes a noticeable target for cancer therapy, it is important to explore the functional role of OPN splice variants in tumor growth and metastasis, and to dissect signaling pathways that involved in these mechanisms.

並列關鍵字

Osteopontin ； invasion

參考文獻

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Asad Junaid et al. (2007) Osteopontin localizes to the nucleus of 293 cells and associates with polo-like kinase-1.Am J Physiol Cell Physiol. 292, C919-C926

Agnihotri R. et al. (2001) Osteopontin, a novel substrate for matrix metalloproteinase-3 (stromelysin-1) and matrix metalloproteinase-7 (matrilysin). J. Biol. Chem. 276, 28261–28267

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探討Osteopontin剪接變異型對人類肺癌細胞株CL1-5生長與侵襲之影響

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