研究c-KIT-ETV1路徑對胃腸道基質瘤的癌化與進程之影響

Abstract

胃腸道基質瘤(gastrointestinal stromal tumors, 簡稱GISTs)是腸胃道中最常見的間葉細胞 (mesenchyme) 瘤， 它可能發生在腸胃道中的任何一處。這些腫瘤並非源自於上皮細胞，可能由Cajal氏間質細胞 (interstitial cells of Cajal, ICCs)造成。 世界各地的研究顯著地顯示出一致的結果，每年每百萬人口中有11到20例，胃與小腸是最常出現的地方。在外科切除後，這些腫瘤通常局部復發，擴散分佈於腹部的漿膜表面，而且經常轉移到肝臟。GIST晚期會轉移到遠處器官，包括肺臟與骨頭。 約95%利用免疫組織化學法檢測細胞表面受體KIT(CD117)為陽性，是GIST非常重要的診斷標記。c-kit 基因可以編譯出 145-kDa細胞表面糖蛋白。它是Ⅲ型受體酪氨酸激酶的家族 [type III receptor tyrosine kinase (RTK) family]成員之一，此家族其中包括platelet-derived growth factor receptor-α and -β (PDGFRA與PDGFRB)。 大部分的GISTs (約75%) 在KIT近膜區 (juxtamembrane region) 的發生突變。而導致受體本質的活化。因此突變的KIT仍然是這些腫瘤非常重要的治療標的。 這些KIT的突變與GIST的發展高度相關而且突變率的提高也被視為GIST的診斷標記。而SCF-KIT 的交互作用在許多人類細胞中包含ICCs的發展是非常重要。 雖然多數GISTs的發生是具偶發性，但有少數發生在罕見家族型 (familial forms) c-kit遺傳疾病基因特定的遺傳突變有關。家族型GISTs是體染色體顯性遺傳。 此外，由於在自然生物體中存在(GNNK+, GNNK-) 之c-KIT的兩種異構體(isoforms)。相較於轉染c-KIT-GNNK+ 之293T細胞，含有c-KIT-GNNK- isoform 之293T細胞不論在形成細胞聚落或是在soft agar中生長的能力都比較具有優勢。 另外，也有大量的文獻報導GIST的發生與ETS家族成員的ETV1的高度表現相關，被視為在這類型癌症的腫瘤生成中扮演重要的角色。另一方面，當使用KIT或MEK抑制劑治療GISTs cell lines，透過蛋白酶体降解，ETV1蛋白質含量降低。 這些報告，證明了KIT signal transduction pathway是透過MAPK路徑，穩定了ETV1。 在本篇研究，我們利用microarray技術分析了不同惡化能力的GISTs 極低 (VL)、低(L)、中(I)、高(H) 並抽取其RNA去釐清miRNA在GIST癌化的進程所扮演的角色。我們發現在GIST癌化進程中有一些miRNA表現有變化(包括miR-296, miR-627, miR-1237)，而且會使ETV1的蛋白表現減少。因此我們預測這些miRNA有潛力成為 target c-KIT 3’UTR。

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. They can occur anywhere along the gastrointestinal tract. These tumors are of non-epithelial origin potentially arising from the interstitial cells of Cajal (ICC). Worldwide studies show strikingly consistent annual incidences of 11 to 20 per million populations with the stomach and small intestine being the most common sites. After surgical resection, these tumors usually recur locally, spread diffusely throughout the serosal surfaces of the abdomen and usually the most common site of metastases is the liver. Advanced disease is associated with metastases to more distant sites, including the lungs and bones. Most GISTs (~95%) are immunohistochemically positive for the cell surface receptor KIT (also known as CD117), which remains as a very important diagnostic marker for these tumors. The c-kit gene encodes for a receptor tyrosine kinase (RTK), the cell surface receptor for a growth factor termed stem cell factor (SCF). KIT is a 145-kDa cell surface glycoprotein. It is a member of the type III receptor tyrosine kinase family that includes platelet-derived growth factor receptor-α and -β (PDGFRA, and PDGFRB) among others. The majority of GISTs (~75%) contain activating mutations within the KIT juxtamembrane region. These mutations lead to the constitutive activation of the receptor and mutant KIT remains a very important therapeutic target for these tumors. These gain-of-function mutants of the c-kit proto-oncogene have been tightly associated with the development of GISTs and their elevated expression in these tumors has also been regarded as a diagnostic marker for GISTs. SCF-KIT interaction is crucial for the development of various cell types such as melanocytes, erythrocytes, germ cells, mast cells and ICCs. Although the vast majority of GISTs incidences are sporadic, rare familial forms exist that are associated with the characteristic heritable mutations in the KIT gene. The familial form of GIST is autosomal dominant. Knowing the existence of several naturally occurring isoforms of c-KIT (GNNK+ and GNNK－) protein we decided to investigate their effect on tumorigenesis of HEK-293T cells. We have found that, c-KIT-GNNK- is better at supporting the growth of HEK-293T cell colonies than its counterpart, c-KIT-GNNK+. Besides, c-KIT GNNK- was also stronger at promoting the anchorage-independent growth of these cells in soft agar. Besides the very important role of c-KIT in GISTs, the ETS family member ETV1 has also been shown to be universally highly expressed in all GISTs and suggested to play an crucial role in the tumorigenesis of this type of cancer. On the other hand, when GIST cell lines are treated with either a KIT or a MEK inhibitor ETV1 protein levels can be reduced through proteasomal degradation. These reports support the current belief that KIT signaling through the MAPK pathway stabilizes ETV1 thus maintaining its activity. In the second part of this study, we performed microRNA (miRNA)-expression profiling analyses on the isolated RNA samples of GISTs with different clinical risk schemes [very low (VL), low (L), intermediate (I) and high (H)] to gain insight of the potential role(s) of miRNAs in GIST tumorigenesis and progression. We have found that the expression of several miRNAs change during GIST tumor progression. Furthermore, a few of these miRNAs are able to down-regulate the expression of ETV1 at a protein level. These results suggest that our candidate miRNAs could be targeting ETV1 3’UTR leading to its degradation.