sFRPs基因在人類乳癌中所扮演的角色

Abstract

Wnt訊息傳導路徑在細胞的生長和分化上都扮演重要角色，當Wnt配體(ligands)與細胞膜上的Frizzled受體(receptors)結合後，會促使細胞質中beta-catenin增加並進入細胞核中調控特定基因之轉錄，例如：c-myc、c-jun和cyclin D1等與細胞生長有關之基因。目前在許多癌細胞中都發現參與負調控Wnt訊息傳導路徑的APC (adenomatosis polyposis coli protein)或AXIN等因子有變異的情形，因而造成此路徑有不正常的活化；然而乳癌檢體中也被發現有β-catenin增加的情形，不過並無APC或AXIN等蛋白的異常，因此推測造成乳癌中Wnt訊息傳導路徑不正常活化的原因，應該發生於APC及AXIN這個複合體的上游。由於過去的研究，造成癌細胞中此訊息傳導路徑持續被活化的基因異常，往往是受體或此訊息傳導路徑中的其他成員，而非配體本身，因此突顯了在上游調控Wnt配體與細胞膜上的Frizzled受體結合的蛋白質sFRPs(secreted frizzled related proteins；包含 sFRP1至sFRP5五個成員)的重要性。我們因而有系統性的分析sFRPs在乳癌中的表現情形以及與臨床症狀的相關性，並進一步以乳癌細胞株做模式研究其分子機制。 本論文中，我們以半定量聚合酶連鎖反應(semi-quantitative RT-PCR)或是real time PCR等方法分析sFRP3、4和5在144個乳癌檢體中的表現情形，結合過去實驗室對於sFRP1和sFRP2已完成的研究，結果發現低表現量的sFRP1或sFRP4皆與乳癌的較差愈後有關，如與術後其他器官的轉移復發(systematic recurrence)具相關性，其生統分析得到的P值分別為0.0224和0.0099。進ㄧ步我們建立了sFRP1或sFRP4穩定表現的乳癌細胞株進而分析其癌化情形。結果發現無論是sFRP1或sFRP4均會減低癌細胞的生長速度，調高癌細胞對胎牛血清的需求量，甚至減少soft agar 群落的生成能力。接下去則分析細胞中與Wnt訊息傳導路徑相關分子的表現情形，結果發現在sFRP1或sFRP4的表現下會促進GSK-3beta的活性，進而beta-catenin蛋白質在細胞質和細胞核的表現量減少，因此調控轉錄的能力下降，致使其下游基因的表現，包括：c-myc和cyclin D1都有下降的情形。另外進一步以siRNA將sFRP1抑制後，則相反地會促進Wnt訊息傳導路徑的活化，以上實驗結果皆應證了sFRP1或sFRP4皆具有抑制Wnt訊息傳導路徑的活化的功能。 綜合以上的研究結果，我們提出一個重要的乳癌致癌機制，那就是sFRP1和sFRP4會抑制Wnt訊息傳導路徑的活化，若失去正常sFRP1和sFRP4的表現量時，Wnt訊息傳導路徑則會過度的活化，而使乳癌的癌化程度增加。至於sFRP1和sFRP4是否會透過其他生理功能參與調控乳癌之癌化過程，則有待深入的研究。

Wnt signaling pathway plays an important role in normal embryonal development of different tissues by regulating growth, apoptosis and differentiation. Binding of a particular Wnt ligand to its target Frizzled (Fz) receptor involves formation of a membrane-located complex and a key output of this process is the stabilization of intracellular beta-catenin. As a result, increase level of beta-catenin is targeted to the nucleus, and it enables transcriptional activation of a panoply of target genes, such as c-myc, c-jun, cyclin D1 and etc. Aberrant activation of Wnt pathway has been found in several cancers. For instances, APC (adenomatosis polyposis coli protein) or AXIN, which are both negative regulators of the Wnt pathway, is mutated in colorectal cancer, oral cancer, and gastric cancer. A few of APC or AXIN mutations are found in breast carcinomas, while more than 60% of human breast cancer specimens have higher beta-catenin concentration. Therefore, it is possible that, in breast cancer, Wnt signaling is activated by components of the pathway in the upstream of the Axin complex. The recent reports indicate sFRPs (secreted frizzled related proteins) are modulators of the Wnt pathways through influencing the binding of Wnts and frizzled receptors. Hence, we are interested in exploring the roles of sFRPs in breast cancer carcinogenesis. First, we investigate the expression pattern of sFRPs in 144 human breast cancer specimens by either semi-quantitative RT-PCR or real time PCR. After statistical data analysis, lower expression level of sFRP1 or sFRP4 correlates with systematic recurrence (p=0.0224 and p= 0.0099, respectively). Ectopic expression of sFRP1 or sFRP4 in breast cancer cells has important implications in tumorigenesis, including inhibition of growth rate and soft agar colony formation. Saturation density and serum requirement assay are slightly changed. The migration activity in vitro is influenced as well. Besides, we find that expression of sFRP1 or sFRP4 in breast cancer cells attenuates Wnt signaling, including GSK-3beta activation, down-regulation of beta-catenin, inhibition of beta-catenin transcriptional activity and down-regulation of its target genes such as c-myc and cyclin D1. These data indicate that sFRP1 or sFRP4 plays an important role in negatively regulation of Wnt signaling pathway. In conclusion, we discover an important mechanism in human breast cancer carcinogenesis. The mechanism is that sFRP1 or sFRP4 inhibits Wnt signaling pathway, and loss of sFRP1 or sFRP4 will enhance breast cancer carcinogenesis. However, we don’t know if sFRP1 or sFRP4 modulates breast cancer carcinogenesis through other signaling pathway, more detail investigation will be needed.