| 摘要: | 大腸直腸癌為全球在男性及女性發生率皆為第三名的癌症。在台灣長年來皆位居兩性發生率和死亡率的第三名。近期研究針對台灣族群指出STIM1 (Stromal Interaction Molecule 1)這種參與在鈣池調控鈣離子流入(Store-Operated Calcium Entry, SOCE)的分子在大腸直腸癌上的高表現量對於腫瘤的生長和轉移有很大的影響。近期研究發現1,4,5-三磷酸肌醇激酶-C(Inositol 1, 4,5 -trisphosphate 3-kinase C, ITPKC)為SOCE之負調控子，並參與SOCE所誘導的NFAT訊息傳遞。為探討ITPKC是否經由抑制鈣池調控鈣離子流入而達到抑制大腸直腸癌的轉移作用，本研究將ITPKC大量表現於Caco2細胞中，並觀察其遷移、侵入與細胞鈣離子內流量。結果顯示ITPKC的過量表現會抑制經由鈣池調控鈣離子通道(Store-Operated Calcium Channel, SOC)之鈣離子內流，進而抑制大腸直腸癌的遷移和侵襲。本研究亦納入418名台灣大腸直腸癌患者，並篩選出7個ITPKC的標籤單一核苷酸基因多型性，探討ITPKC基因多型性與大腸直腸癌腫瘤生長轉移之相關性。結果顯示，有3個ITPKC單一核苷酸多型性(Single Nucleotide Polymorphism, SNP)- rs10420685、rs11673492、rs28493229分別與淋巴結轉移(Lymph Node Involvement)(rs10420685)、神經轉移(Perineurial Invasion)(rs28493229)和癌症分期(TNM Stage)(rs11673492、rs28493229)有顯著相關，rs10420685和rs11673492攜帶高危險基因型者(G/G與T/T)相較於低危險型(A/A與C/C)分別會導致淋巴結轉移和癌症分期末期化的風險增加；而rs28493229攜帶G/G基因型時，在神經轉移和癌症分期末期化的風險都會降低。
近年來，越來越多研究發現腫瘤的發生和發炎環境的相關性，PTX3基因已被證實在體內發炎免疫系統扮演了重要的角色，且在多種癌症上發顯其高表現量，而鈣池調控鈣離子通道(SOC)和腫瘤的生長和發炎反應也是息息相關。為探討PTX3基因的活化和鈣池調控鈣離子通道(SOC)是否相關，本研究透過細胞實驗利用Thapsigargin(TG)誘導PTX3基因的活化並使用鈣池調控鈣離子通道抑制劑，SKF96365，確認PTX3基因的活化和SOC開啟的相關性。結果顯示，TG刺激六小時後可以看到PTX3基因活化，而使用SKF96365前處理後，PTX3基因活化情形被抑制了。並且發現，在PTX3啟動子區域中的AP1結合位點是TG誘導活化PTX3表現路徑中最為重要的轉錄因子結合位。同時也針對PTX3基因多型性進行分析，在篩選出的4個PTX3標籤單一核苷酸基因多型性中，發現rs3845978和癌症分期(TNM Stage)有顯著相關，當攜帶C/T和T/T基因型時癌症分期末期化的風險會降低。
本研究顯示在SOC訊息傳遞路徑中ITPKC基因為一保護性的角色，會抑制大腸直腸癌腫瘤的生長和轉移，且鈣池調控鈣離子通道(SOC)的開啟會誘導發炎基因PTX3活化，在未來的研究中可能可當成在臨床癌症診斷和治療上之具潛力的生物標記。
Colorectal cancer is a major cause of morbidity and mortality worldwide. In Taiwan, it is the third leading cause of cancer death and incidence in both men and women. Previous study reported that Stromal Interaction Molecule 1 (STIM1), which is involved in store-operated calcium entry (SOCE), play important roles in colorectal tumor size, depth of invasion, lymph node metastasis status in Taiwanese population. Recent studies have indicated that 1, 4, 5 -trisphosphate 3-kinase C, ITPKC, acts as a negative regulator of SOC participates in SOC-mediated NEAT signal pathway. To investigate whether ITPKC inhibits colorectal cancer metastasis via attenuation of SOCE, this study tested the roles of ITPKC in a cell-based model using a colorectal cancer cell line – Caco2 cells and assessed the cancer migration, invasion and calcium influx. My results showed that overexpression of ITPKC attenuated the calcium influx through store-operated calcium entry (SOCE) and inhibited the migration and invasion of colorectal cancer. In addition, my study analyzed the genotypic distribution of sevent genetic variation of ITPKC among 418 Taiwanese patients with colorectal cancer to identify the association bwtween ITPKC genetic polymorphisms and colorectal cancer development. My results revealed that three single nucleotide polymorphisms (SNPs), rs10420685, rs11673492 and rs28493229, were correlated with lymph node involvement (rs10420685), perineurial invasion (rs28493229) and TNM stage ( rs11673492、rs28493229); G/G and T/T genotypes of rs10420685 and rs11673492 significantly increased the risk of lymph node involvement and progressed to TNM stageⅢ&Ⅳ. Additionally, G/G genotype of rs28493229 was found to have significant correlation with decreasing the risk in perineurial invasion and progressed to TNM stageⅢ&Ⅳ.
Recent studies have indicated that inflammatory molecules are an essential component of the tumor development, and PTX3 is an essential component of the innate immunity and inflammation reactions. Overexpression of PTX3 has been observed in lung cancer, breast cancer and protate cnacer and SOC is an important requirement for the regulation of malignant progression. In this study, I identified that the activation of SOC signal pathway induces PTX3 expression in colorectal cancer cell line – Caco2 cells. I used thapsigargin (TG) (a SOC activator) to explore the mechanism of how calcium regulated PTX3 gene expression, and pharmacological inhibitors of SOC channels (2-APB and SKF-96365) to test the association between the SOC channel and PTX3 expression. My results suggest that activation of SOC channel mediated PTX3 gene expression. A promoter-mutation assay revealed that AP1 binding site is the critical calcium-responsive area. Mutation of AP1 binding site resulted in the decrease of the PTX3 gene expression. Additionally, we analyzed the genotypic distribution of four genetic variation of PTX3 gene, and found that rs3845978 with C/T and T/T genotypes were correlated with decreasing the risk in progressing to TNM stageⅢ&Ⅳ.
The study demonstrated the potential biomarker to diagnosis, treatment and drug development of cancer in the future. |
