sFRP1及sFRP2在人類乳癌檢體中之角色

Abstract

Wnt訊息傳導路徑在細胞的生長、發育、分化都扮演重要角色，此路徑受到嚴密的調控，當調控機制出現問題，就很可能使細胞的生長發育不受控制，促進腫瘤之形成。Wnt訊息傳導路徑不活化時，細胞質中β-catenin濃度很低，反之，當這個訊息傳導路徑開啟，會使細胞質中β-catenin濃度升高，並進一步進入細胞核，調控特定基因之轉錄。目前許多癌症中，都發現Wnt訊息傳導路徑有被不正常活化的現象。在此訊息傳導路徑中首先被定義為致癌基因（oncogene）的是Wnt1，它被發現在受老鼠乳腺腫瘤病毒（mouse mammary tumor virus；MMTV）感染而產生的老鼠乳腺腫瘤內有過量表現的情形，且其表現會導致細胞的癌化。接下來許多研究中，分別發現Wnt訊息傳導路徑中其他成員蛋白也在不同的腫瘤細胞中有被異常表現的情形，如在此路徑中扮演負向調控角色（negative regulator）的adenomatosis polyposis coli protein（APC）或AXIN已被發現在結腸癌、口腔癌、胃癌中的表現有被抑制的現象；高達85%以上的結腸癌中可以發現APC的表現情形改變，然而，雖然在60%以上的乳癌檢體中可以發現細胞質中之β-catenin濃度較高，表示Wnt訊息傳導路徑的活化，卻無法看到APC或AXIN等蛋白表現量之改變。因此推測，造成乳癌中Wnt訊息傳導路徑不正常活化的原因，乃發生於APC及AXIN這個複合體的上游。 Secreted frizzled related proteins（sFRPs）這個家族的蛋白與Wnt訊息傳導路徑中之受體Frizzled蛋白相似，其中sFRPs的N端具有cysteine rich domain（CRD），與Frizzled中可與Wnt配體結合而開啟下游訊息傳導路徑的CRD具有30%~50%的相似度，因此被認為具有調控Wnt訊息傳導路徑的潛力。在哺乳類中，已發現5種sFRPs（sFRP1至sFRP5），在這篇論文中我們以sFRP1及sFRP2為研究重點。在人類結腸癌細胞株中過量表現sFRP1及sFRP2皆能抑制Wnt訊息傳導路徑之活化，也有多篇研究指出，在人類乳癌及結腸癌檢體中，sFRP1的表現量都有被抑制的情形，另一方面，sFRP2的表現量卻在犬乳癌檢體中上升，至於sFRP2在人類乳癌中的表現情形，尚無相關研究。目前sFRPs與癌症的形成之間的關係及機制尚不明朗。 在本篇論文中，我們探討sFRP1及sFRP2在人類乳癌檢體中的表現情形，以及與術後其他器官的轉移復發（systematic recurrence）的相關性。我們收集146個乳癌檢體及其中8對之鄰近正常組織，利用反轉錄-聚合酶連鎖反應（RT-PCR），分析sFRP1及sFRP2的表現量，並做生物統計分析。結果顯示，sFRP1在乳癌細胞中具有較其鄰近正常組織為低的表現量，而sFRP2的表現則有較其鄰近正常組織為高的趨勢；經初步生統分析後，無法看出sFRP1或sFRP2與術後其他器官的轉移復發（systematic recurrence）相關。因此我們嘗試扣除與術後其他器官的轉移復發高度相關的因子-p53或TTK/hMps1。首先，若扣除p53蛋白突變的檢體後，sFRP1之表現與術後其他器官的轉移復發相關（P<0.05）；若扣除TTK/hMps1表現量較高的檢體，也可得類似結果（P<0.05）；但sFRP2經生統分析後，無論是否扣除p53蛋白突變或TTK/hMps1表現量較高的檢體，與術後其他器官的轉移復發（systematic recurrence）都沒有相關性。

Wnt signaling pathway is strictly regulated, and it plays important roles in cell growth, development and differentiation. Aberrant expression of any component in this pathway may lead to uncontrolled cell growth, thus resulting in the development of tumors. The key indicator of this pathway is β-catenin. When Wnt pathway is inactivated, cytosolic β-catenin is phosphorylated by glycogen synthase kinase 3β（GSK3β）, and forms complex with adenomatosis polyposis coli protein（APC）and AXIN, then is degraded through the ubiquitin-proteosome degradation pathway. The cytosolic β-catenin thus remains in low concentration. When Wnt pathway is activated, GSK3β is inactivated; the cytosolic β-catenin is not degraded by the ubiquitin-proteosome degradation pathway. The cytosolic β-catenin will enter the nucleus, where it acts as a transcription factor and promote the expression of certain genes. Aberrant activation of Wnt pathway has been found in several cancers. The first oncogene identified in this pathway was Wnt1, which was found to be upregulated in MMTV(mouse mammary tumor virus)-infected mice. Later, some other components in Wnt pathway were found to be aberrantly expressed. For instances, APC (adenomatosis polyposis coli protein) or AXIN, which are both negative regulators of the Wnt pathway, were mutated in colorectal cancer, oral cancer, and gastric cancer. More than 85% colon cancer specimens have APC mutation. On the other hand, few APC or AXIN mutation were seen in breast carcinomas although more than 60% of human breast cancer specimens have higher cytosolic β-catenin concentration, which indicated the activation of Wnt signaling pathway. Therefore, we hypostasized that Wnt pathway is altered by mutations upstream of the APC and AXIN complex. Secreted frizzled related proteins (sFRPs) have N-terminal cystein rich domain (CRD) which share 30~50% homology with the N-terminal CRD domain of the Wnt receptor—Frizzled proteins. Frizzled proteins use their CRD to interact with the Wnt ligands, thus turn on the Wnt pathway. It is known that sFRPs may interact with Wnts through their CRD, and regulate the Wnt signaling pathway. In mammals, 5 sFRPs (sFRP1 to 5) have been discovered. Here we emphasize on sFRP1 and sFRP2. sFRP1 is downregulated in human breast and colorectal cancers; however, sFRP2 is upregulated in canine breast cancer. Overexpressing sFRP1 and sFRP2 in human colorectal cancer cell lines reduce the activity of Wnt pathway. Until now, there is no research reported on human breast cancer about sFRP2. Here we investigate the expression pattern of sFRP1 and sFRP2 in 146 human breast cancer specimens and 8 normal counterparts of them. We found that sFRP1 show higher expression level in all 8 normal counterparts, and sFRP2 show lower expression in 5 out of 8 normal counterparts. Analyzing the expression level of sFRP1 or sFRP2, at first we found no correlation between the expression level of sFRP1 or sFRP2 and systematic recurrence. Then we tried to subtract the effect of 2 factors, p53 or TTK/hMps1, which strongly correlates with systematic recurrence. We then found correlation between systematic recurrence and the expression level of sFRP1 if we subtract the specimens with p53 mutation (p<0.05). Subtracting the specimens with higher TTK/hMps1 expression level leads to similar results (p<0.05). As for sFRP2, there is no correlation with systematic recurrence at all.