新多功能蛋白質PX對腫瘤發展之調節

Abstract

惡性腫瘤是全球十大死因之一，每年造成約960萬人死亡。在腫瘤發展過程中，會獲得數個所需的特徵，包含維持細胞不斷增殖、逃避細胞死亡訊號、忽視生長所受限制、維持遺傳物質複製機制、誘導血管生成和侵襲周邊組織、轉移至遠端器官等能力。由於遺傳變異，讓腫瘤細胞積累這些調控缺陷，而不受正常細胞增殖和體內平衡的調節所控制。

蛋白質雙硫鍵異構酶家族是一群作用於蛋白質雙硫鍵結、幫助蛋白質折疊的酵素，可以改變受質蛋白的雙硫鍵位置，並具有伴隨蛋白的功能。其家族成員分別與病原體的感染過程、受精作用、凝血機制、免疫反應、神經相關疾病發生、細胞生存能力或腫瘤產生有關。近期研究顯示部分成員在腫瘤發展、轉移和血管生成中扮演重要角色。然而，其在腫瘤發展中的分子機制和做為治療目標的潛力尚待研究。解析蛋白質雙硫異構酶家族在腫瘤中的作用有助於治療惡性腫瘤。

本實驗發現蛋白質雙硫鍵異構酶家族中的PX，在腫瘤細胞株和臨床人類肺癌組織中有高度表達。利用干擾RNA減少或利用慢病毒載體過表達腫瘤細胞中PX之實驗顯示，PX藉由降低凋亡蛋白酶3和7的活性，達到促進細胞生長之效果。和體外實驗相符，荷瘤小鼠實驗顯示過表達PX的Lewis肺癌細胞比其親代細胞在小鼠宿主發展更快，造成存活率降低、腫瘤增大、轉移增加以及細胞死亡與凋亡蛋白酶3和7活性降低等現象，而PX基因敲落則導致相反的結果。此外，自發性肝癌模式小鼠實驗結果顯示PX的缺乏顯著減少肝癌發生和囊腫形成，並增加小鼠存活率、腫瘤死亡與凋亡蛋白酶3和7活性。機制探討顯示PX透過用其活性結構CGHC結合並抑制凋亡蛋白酶3和7前驅物的降解和活化，從而減少腫瘤細胞死亡訊號。最後，藥物實驗發現PX的抑製劑DHTT，可以通過增強TS/A乳癌細胞凋亡蛋白酶3和7活化，誘使腫瘤細胞死亡，進而減緩腫瘤之發展。

本實驗還探討了PX在腫瘤微環境中的作用。臨床數據顯示肺癌組織中的PX表達量與患者的存活率呈負相關，且PX在腫瘤基質細胞和腫瘤浸潤白血球中高度表達。此外，實驗發現PX在正常基質細胞中表達較低，但該表達在腫瘤組織的基質細胞中的提高，此高度表達似乎為受到腫瘤細胞的刺激造成。這些發現證實PX是腫瘤發展的正調節因子，並顯示PX是治療惡性腫瘤的潛在目標。

Cancer is a leading cause of death worldwide, accounting for 9.6 million deaths each year. Cancer may acquire the capabilities to sustain proliferation, resist cell death, evade growth suppression, enable replicative immortality, induce angiogenesis, and activate invasion/metastasis. As a result of genetic alterations, cancer cells can accumulate defects in regulatory circuits that govern normal cell proliferation and homeostasis.

Protein disulfide isomerases (PDIs) are implicated in pathogen infection, fertilization, coagulation, immunity, neuropathy, cell viability and growth of tumors. Emerging evidence suggests that PDI family plays important role in tumor development, metastasis and angiogenesis. However, its molecular mechanism and therapeutic potential in cancer are unclear. Therefore, deciphering the role of PDIs in cancers may help cancer therapy.

Here, we discovered that PX expression was upregulated in a variety of tumor cell lines and human lung adenocarcinoma tissues. Knockdown and overexpression of PX in tumor cells showed that PX facilitated cell growth via the reduction of caspase 3 and 7 activity. Consistently, Lewis lung carcinoma (LLC) cells overexpressing PX grow faster than parental cells did in tumor-bearing mice, as shown by a reduced survival rate, increased tumor size and metastasis, and decreased cell death and caspase 3 and 7 activities. PX knockdown resulted in opposite outcomes. Moreover, results obtained in mice with spontaneous hepatoma indicated that PX deficiency significantly reduced hepatic tumorigenesis and cyst formation and increased mouse survival, tumor death, and caspase 3 and 7 activity. Mechanistic studies illustrated that PX negatively regulated tumor cell death by inhibiting degradation and activation of procaspases 3 and 7 by their mutual interaction in a CGHC-dependent manner. Finally, we found that 1,2-dihydroxytrideca-5,7,9,11-tetrayne (DHTT), a PX inhibitor, reduced tumor development via enhancement of caspase-mediated cell death in TS/A tumor-bearing mice.

We also investigated the impact of PX in tumor microenvironment. We found that PX expression in lung cancer tissues was negatively correlated with survival outcomes of patients. Consistently, PX was highly expressed in cancer stromal cells and tumor infiltrating leukocytes. Moreover, we found PX was expressed at a low level in stromal cells and this expression was upregulated akin to its expression in cancer cells. This upregulation seem to be stimulated by tumor cell-derived stimuli. These findings characterize PX as a positive regulator of cancer development and suggest that PX is a potential therapeutic target for cancer.