ENPP1 蛋白在腫瘤微環境中免疫抑制的角色

ENPP1 Suppresses Immune Responses in the Tumor Microenvironment

ENPP1 蛋白在腫瘤微環境中免疫抑制的角色

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) 蛋白已知在軟組織和骨質礦化 (mineralization) 的生理調控中扮演重要的角色。

近年研究發現，ENPP1 蛋白在腫瘤細胞微環境 (tumor microenvironment) 中更有一直先天免疫反應 (innate immunity) 與促進腫瘤細胞生長與轉移 (tumor progression and metastasis) 的作用，成為新興抗癌標靶開發的重點蛋白 (1-2)！

ENPP1 蛋白對於旁分泌 STING 訊號傳遞路徑的抑制作用

在癌症細胞中，染色體的不穩定促使微核 (micronuclei) 的生成，導致雙股 DNA (double-stranded DNA, dsDNA) 滲出至細胞質中 (2-3)。在細胞質中，這些 dsDNA 進一步被DNA調控系統中的環鳥苷酸-腺苷酸合成酶 (cyclic GMP-AMP synthase, cGAS) 辨識出來，誘導2'3'-cyclic GMP-AMP (2'3'-cGAMP) 的生成 (Fig.1).

而2'3'-cGAMP作為免疫刺激的第二信使，進一步與干擾素刺激因子 (stimulator of interferon genes, STING) 結合並將其活化，從而促使下游轉錄因子：IRF3和NF-κB的磷酸化，引起第一型IFN促發炎細胞因子的分泌 (4)。

腫瘤細胞中染色體的不穩定性，引導下游STING 路徑更偏向於 NF-Κb誘導的免疫反應，造成dsDNA 的損傷，降低腫瘤細胞的轉移 (2)。

Fig. 1 cGAS/STING signaling within chromosomally unstable cancer cells favors metastatic phenotypes.

腫瘤細胞將溶解度高的2'3'-cGAMP 輸出到細胞質外，附近的宿主細胞 (host cell) 將其吞噬，進而活化典型 STING 訊號傳遞路徑 (canonical STING signaling)，加劇免疫反應。這一系列的免疫反應包含了：第一型 IFN 的生成、樹突狀細胞聚集 (dendritic cell recruitment) 以及毒殺型T細胞的活化 (Fig.2)，進一步惡化癌症疾病的進程 (2-3,5)。

反之，ENPP1 蛋白可以透過減弱上訴免疫風暴的訊號傳遞，改善預後不良的狀況 (2,5-6) 。

常見的癌症路徑包含：卵巢癌 (ovarian)、乳癌 (breast cancer)、肝癌 (HCC)、甲狀腺癌 (thyroid cancer) 以及胰臟癌 (pancreatic cancer) 等 (2,5-6)。

ENPP1 蛋白是一種穿膜蛋白 (transmembrane protein)，具有面向外部的催化結構域 (external-facing catalytic domain) 能夠將2'3'-cGAMP降解，進而抑制腫瘤微環境中，旁分泌STING訊號的傳遞。

透過這個機制可以有效的削弱免疫反應 (3,7)。

也只有在細胞外的2'3'-cGAMP降解時，能夠抑制腫瘤細胞的免疫活化反應，更能夠減少腫瘤細胞轉移的情況發生 (3)。

Figure 2. ENPP1 degrades extracellular 2'3'-cGAMP to inhibit paracrine STING signaling (upper panel). In the absence of ENPP1 (lower panel), 2'3'-cGAMP is taken up by healthy host cells in the tumor microenvironment, leading to activation of canonical STING signaling and the production of tumor-suppressive immune responses.

ENPP1蛋白在腺苷 (adenosine) 訊號傳遞的貢獻

ENPP1 蛋白在免疫脫逃 (immune evasion)中具有輔助的作用，協助腺苷(adenosine) 的生成。上著提到， ENPP1蛋白可以把2'3'-cGAMP水解，形成 GMP和 AMP (7)。ENPP1 蛋白同時也可以水解 ATP (Fig.3)。在ATP水解的過程中，同時也意味著腫瘤微環境壓力的增加。因為細胞損傷和凋亡不停地發生，並且促進發炎反應的發生，進而產出AMP和無機焦磷酸鹽 (inorganic pyrophosphate, PPi)。

AMP 的兩大來源：2'3'-cGAMP水解和ATP水解所得。

AMP進一步被ecto-5'-nucleotidase (NT5E; CD73) 水解，形成腺苷，與細胞外的腺苷受體結合，抑制促發炎細胞因子的生成分泌，促進抗發炎細胞因子的分泌，促進細胞遷移 (cell migration)，並促進腫瘤細胞的生長 (2,7)。

透過降解 ATP和 2'3'-cGAMP， ENPP1 蛋白不單抑制了促發炎的狀況，更刺激腺苷的分泌。而腺苷具有免疫抑制的功能性。

Figure 3. AMP produced from ENPP1-mediated hydrolysis of 2’3’-cGAMP or ATP is further degraded to adenosine, which suppresses immune responses in the tumor microenvironment.

推薦產品：

ENPP1 Fluorescent Inhibitor Screening Assay Kit Screen for inhibitors of human ENPP1 Includes recombinant human ENPP1 and positive control ENPP1 inhibitor C Assay Reagent Assay 45 samples in duplicate or 29 samples in triplicate Plate-based fluorometric measurement (Ex/Em = 485/520 nm)	702090
2'3'-cGAMP ELISA Kit Measure 2'3'-cGAMP in cell lysates, plasma, serum, and tissue samples Monitor the kinetics of 2'3'-cGAMP formation and hydrolysis in a biological setting Assay 24 samples in triplicate or 36 samples in duplicate LLOD = 9.6 pg/ml (0.01 pmol/ml) Run overnight or incubate for just 2 hours without compromising sensitivity	501700

ENPP1 蛋白作為癌症治療的重點

References

1. Roberts, F., Zhu, D., Farquharson, C., et al. ENPP1 in the regulation of mineralization and beyond. Trends Biochem. Sci. 44(7), 616-628 (2019).

2. Li, J., Duran, M.A., Dhanota, N., et al. Metastasis and immune evasion from extracellular cGAMP hydrolysis. Cancer Discov. 11(5), 1212-1227 (2021).

3. Carozza, J.A., Brown, J.A., Böhnert, V., et al. Structure-aided development of small-molecule inhibitors of ENPP1, the extracellular phosphodiesterase of the immunotransmitter cGAMP. Cell Chem. Biol. 27(11), 1347-1358 (2020).

4. Kumar, M. and Lowery, R.G. Development of a high-throughput assay to identify inhibitors of ENPP1. SLAS Discov. 26(5), 740-746 (2021).

5. Carozza, J.A., Böhnert, V., Nguyen, K.C., et al. Extracellular cGAMP is a cancer-cell-produced immunotransmitter involved in radiation-induced anticancer immunity. Nat. Cancer1(2), 184-196 (2020).

6. Wang, H., Ye, F., Zhou, C., et al. High expression of ENPP1 in high-grade serous ovarian carcinoma predicts poor prognosis and as a molecular therapy target. PLoS One 16(2), e0245733 (2021).

7. Onyedibe, K.I., Wang, M., and Sintim, H.O. ENPP1, an old enzyme with new functions, and small molecule inhibitors – a STING in the tale of ENPP1. Molecules 24(22), 4192 (2019).

8. Weston, A., Thode, T., Munoz, R., et al. Abstract 3077: Preclinical studies of SR-8314, a highly selective ENPP1 inhibitor and an activator of STING pathway. Cancer Res. 79(13 Suppl), 3077 (2019).

9. Weston, A.S., Thode, T.G., del Villar, R.R., et al. Abstract LB-118: SR8541A is a potent inhibitor of ENPP1 and exhibits dendritic cell mediated antitumor activity. Cancer Res. 80(16 Suppl), LB-118 (2020).

10. Baird, J., Dietsch, G., Florio, V., et al. MV-626, a potent and selective inhibitor of ENPP1 enhances STING activation and augments T-cell mediated anti-tumor activity in vivo. Society for Immunotherapy of Cancer 2018 Annual Meeting Posters. 7 (2018).

更多資訊，請洽 Cayman 台灣代理 - 昶安科技。