- 學位論文
探討integrin β3和纖維母細胞在OPN-a剪接變異型對於CL1-5肺癌細胞生長調控所扮演的角色
The role of integrin β3 and fibroblasts in OPN-a isoform mediated growth regulation in CL1-5 lung cancer cell line
摘要
Osteopontin (OPN)是ㄧ個分泌型的磷酸化醣蛋白，在許多癌症中有過度表現的情形。OPN的表現與腫瘤細胞的轉移能力具有明顯的正相關性。OPN有三種剪接變異型，OPN-a、OPN-b和OPN-c，在肺癌病人組織及癌細胞株中發現全長的OPN (OPN-a)的表現量最高。我們發現不同OPN剪接變異型的蛋白對肺癌細胞株的生長及侵犯有著不同的影響，其中令人感到訝異的是，表現量最高的全長OPN-a會抑制CL1-5細胞的生長以及侵襲能力，而對A549細胞只抑制侵襲能力但不影響生長能力。我們進而證實了OPN-a對於CL1-5及A549之間生長影響的差異性，主要是因為這兩株細胞中integrin β3 (ITGβ3)表現量的差異性所致。抑制了CL1-5的ITGβ3表現後，OPN-a即失去了抑制細胞生長的能力;反之，當ITGβ3過度表現於A549細胞株後，OPN-a則可抑制A549細胞的生長。有趣的是，在沒有ITGβ3的CL1-5細胞中，OPN-a不但無法抑制細胞生長，反而可以透過NF-κB促進生長。我們更進一步發現，在不表現OPN-a的CL1-5細胞中抑制了ITGβ3的表現後，細胞幾乎停止生長。這結果顯示ITGβ3是CL1-5細胞生長所需要的受體，但是有OPN存在時，細胞則不需要ITG 3。因此，CL1-5肺癌細胞的生長速度可受到OPN-a及ITGβ3的表現量所調控。在肺癌中表現高的OPN-a會抑制腫瘤生長的事實，與OPN普遍被認為是一個促進腫瘤發展的蛋白是極為矛盾的。我們因而假設腫瘤表現的OPN可能會刺激腫瘤微環境中的其他細胞，像是免疫細胞、纖維母細胞等，再間接地經由免疫細胞、纖維母細胞等促進腫瘤細胞之惡化。我們的結果顯示，腫瘤細胞所分泌的OPN-a會刺激纖維母細胞，使得纖維母細胞所產生的條件培養液具有促進CL1-5細胞的生長，但對細胞的侵犯能力沒有影響。因此，在體內雖然肺癌細胞分泌的OPN-a會透過autocrine的機制抑制CL1-5細胞的生長，但是分泌出來的OPN-a則會透過paracrine活化纖維母細胞，進而返過來促進細胞生長。更有甚者，如果OPN-a的受體，ITGβ3，表現降低之後，細胞的生長則不但不會被OPN-a抑制，反而會增加。這個研究顯示了OPN-a對癌細胞生長的影響可以分兩個層面考慮，其一是OPN影響腫瘤與腫瘤微環境之間的作用，其二是會因為不同的OPN受體表現量的高低而對癌細胞的生長有不同的影響。因此，想要抑制過度表現OPN之肺癌細胞的生長，可能需要同時抑制好幾個不同的受體才能達到效果。
並列摘要
Osteopontin (OPN) is a glyco-phosphoprotein which is overexpressed in many cancers. The expressin of OPN is significantly correlated with tumor metastasis. Three OPN splicing variants, OPN-a, OPN-b and OPN-c, were identified and the expression of OPN-a is the highest among them in patients with lung cancer tissue and cancer cell lines. Different splicing variants of OPN have distinct impact on the growth and invasion of lung cancer cell lines. It is surprising that the full-length OPN (OPN-a) inhibited CL1-5 cell growth and invasion, and also inhibited invasion of A549 cells but had no effect on cell growth. We further confirmed that the differences of the effect on cell growth by OPN-a between CL1-5 and A549 was due to the diferential expression level of integrin β3 (ITGβ3). Knock-down the expression of ITGβ3 in CL1-5, blocked the effect of OPN-a mediated growth inhibition. On the other hand, overexpression of ITGβ3 in A549, exhibited OPN-a induced growth inhibition. Interestingly, in the absence of ITGβ3, OPN-a not only lost growth inhibition ability but promoted cell growth through NFκB pathway. Although ITGβ3 is required for OPN-a mediated growth inhibition, knock-down expression of ITG 3 almost completely inhibited growth of CL1-5 in the absence of OPN-a. Our results suggested that ITGβ3 is essential for the growth of CL1-5; however, ITGβ3 is no longer required if cells express OPN-a. Thus, the growth rate of CL1-5 may be subjected to the balance between OPN-a and ITGβ3 levels. The growth and invasion inhibitory roles of OPN-a in lung cancer cells are contradictory to the well recognized oncogenic role of OPN. Therefore, we hypothesized that the expression of OPN may promote cross-talk between cancer cells and its microenvironment. Our results showed that conditioned medium collected from fibroblasts treated with tumor secreted OPN-a greatly enhanced growth of CL1-5 cells, but had no effect on the ability of invasion. Thus, although OPN-a secreted by lung cancer cells would inhibit CL1-5 cell growth through autocrine pathway, the secreted OPN-a can activate fibroblast cells through paracrine pathway to secrete growth factor or cytokines that in turn promotes cancer cell growth. Moreover, cancer cells may reverse OPN-a mediated growth inhibition to growth enhancement by down-regulation the expression of ITGβ3. This study reveals two aspects on OPN-a regulated cancer cell growth: one is OPN-a mediated cross-talk between tumor and microenvironment; the other is regualated by the expression levels of different OPN receptors. Therefore, simutaneouly targeting several different receptors may be required in order to inhibit growth of lung cancer cells with overexperssed OPN-a.