蛋白質精胺酸甲基化抑制與核仁壓力對FUS蛋白分布之影響及協力降低乳癌細胞存活可能性之探討

蛋白質精胺酸甲基轉移酶(PRMTs)在許多癌症中都是過表達的狀態，調節蛋白質精胺酸甲基化可以作為一種治療方向。研究顯示，RNA結合蛋白FUS經PRMT1精胺酸甲基化後，會調節其於「無膜次胞器」的分布，當處於低甲基化環境時，觀察到在細胞核內FUS的大顆粒狀結構生成。根據核仁蛋白質體動力學，FUS在核仁壓力藥物的處理下會往核仁的位置聚集。本研究透過慢病毒感染下調人類口腔癌細胞HSC-3的甲基化表現，或是選擇具有MTAP基因缺失特性使得細胞本身處於低甲基化狀態的乳腺癌細胞MCF-7，進行核仁壓力藥物處理。免疫螢光染色的結果顯示，低甲基化會導致內生性的FUS更易形成細胞核內細小granule，但是這些FUS granules不是聚集在核仁而是在paraspeckle。然而，在核仁壓力藥物處理與甲基轉移酶抑制劑處理的試驗中，都觀察到乳腺癌細胞的存活可能受到抑制。因此本研究繼續探討癌細胞在受到「蛋白質精胺酸甲基化抑制」與「核仁壓力」共處理後，癌細胞的「細胞存活率」以及細胞核內「無膜次胞器」會受到的影響，甲基轉移酶抑制劑與核仁壓力藥物對於抑制乳腺癌細胞的存活是否具有協同作用。選擇同為MTAP基因缺失的乳腺癌細胞MDA-MB-231，MTAP基因正常的乳腺癌細胞MDA-MB-435、MDA-MB-468以及抗輻射乳腺癌細胞231-RR進行實驗。結果顯示，廣效型的甲基轉移酶抑制劑AdOx在低劑量時即可抑制MTAP基因缺失的乳腺癌細胞，但與核仁壓力藥物CX-5461共處理無法更有效抑制乳腺癌細胞生長。然而，CX-5461與PRMT1特異性抑制劑K313共處理卻可以有效抑制MDA-MB-231和231-RR的細胞存活率，其中又以231-RR細胞的抑制效果最佳。後續免疫螢光染色顯示，經K313處理後，231-RR細胞內DNA損傷的情形顯著增加，顯示K313增強了231-RR細胞對輻射線的敏感度。根據研究結果，我們認為「蛋白質精胺酸甲基轉移酶PRMT1的活性抑制」與「核仁壓力」具協同抑制乳腺癌細胞生長的作用，而PRMT1特異性抑制劑K313還可能作為231-RR細胞的放射增敏劑，這對具有抗輻射特性的乳腺癌細胞而言可能會是一個有效的治療策略。

關鍵字

蛋白質精胺酸甲基化

並列摘要

Protein arginine methyltransferases (PRMTs) are often upregulated in various cancers, thus modulation of protein arginine methylation can be a therapeutic strategy. The RNA-binding protein FUS can be arginine methylated by PRMT1 to modulate the subcellular distribution. FUS appeared to form a few large nuclear granules in a hypomethylated environment and according to the studies of nucleolar proteome dynamics, FUS might move to nucleoli when treated with nucleolar stress inducers. In this study, we first treated a PRMT1 knocked-down oral cancer HSC-3 cell line, or a MTAP-deficient hypomethylated breast cancer cell line MCF-7, with a nucleolar stress drug. Immunofluorescent staining showed that, endogenous FUS formed many small granules in these cells under hypomethylation or normal conditions. However, these granules did not co-localize with the nucleolar marker fibrillarin but co-localize with a paraspeckle marker NONO. Moreover, we observed that treatments of breast cancer cells with nucleolar stress drugs and methyltransferase inhibitors might reduce the viability. We thus further investigated whether methyltransferase inhibitors would work with nucleolar stress drugs to suppress the growth of breast cancer cells MCF-7, together with another MTAP-deficient MDA-MB-231, two MTAP normal MDA-MB-435 and MDA-MB-468 cell lines and also a radio-resistant MDA-MB-231 cell (named as 231-RR) were included in the study. MTT assay revealed that though an indirect methyltransferase inhibitor AdOx significantly reduced the survival of MTAP-deficient cells, co-treatment with nucleolar stress drug CX-5461 could not suppress breast cancer cells growth further. However, a PRMT1-specific inhibitor K313 worked synergistically with CX-5461 in MDA-MB-231, especially in 231-RR cells. We demonstrated that the DNA damage level in 231-RR cells with radiation treatment was significantly increased when pre-treated with K313, indicating that K313 might enhance the radiation sensitivity in 231-RR cells. Our study suggests that inhibition of PRMT1 and nucleolar stress can suppress breast cancer cell growth synergistically and K313 might function as a radiosensitizer to 231-RR cells. The treatment might be an effective therapeutic strategy to radio-resistant breast cancer cells.

並列關鍵字

protein arginine methylation

參考文獻

Adriaens, C., Standaert, L., Barra, J., Latil, M., Verfaillie, A., Kalev, P., Boeckx, B., Wijnhoven, P. W., Radaelli, E., Vermi, W., Leucci, E., Lapouge, G., Beck, B., van den Oord, J., Nakagawa, S., Hirose, T., Sablina, A. A., Lambrechts, D., Aerts, S., . . . Marine, J. C. (2016). p53 induces formation of NEAT1 lncRNA-containing paraspeckles that modulate replication stress response and chemosensitivity. Nat Med, 22(8), 861-868. https://doi.org/10.1038/nm.4135

Google Scholar

Andersen, J. S., Lam, Y. W., Leung, A. K., Ong, S. E., Lyon, C. E., Lamond, A. I., Mann, M. (2005). Nucleolar proteome dynamics. Nature, 433(7021), 77-83. https://doi.org/10.1038/nature03207

Google Scholar

Andersen, J. S., Lyon, C. E., Fox, A. H., Leung, A. K., Lam, Y. W., Steen, H., Mann, M., Lamond, A. I. (2002). Directed proteomic analysis of the human nucleolus. Curr Biol, 12(1), 1-11. https://doi.org/10.1016/s0960-9822(01)00650-9

Google Scholar

Anderson, P., Kedersha, N. (2006). RNA granules. J Cell Biol, 172(6), 803-808. https://doi.org/10.1083/jcb.200512082

Google Scholar

Arun, B., Akar, U., Gutierrez-Barrera, A. M., Hortobagyi, G. N., Ozpolat, B. (2015). The PARP inhibitor AZD2281 (Olaparib) induces autophagy/mitophagy in BRCA1 and BRCA2 mutant breast cancer cells. Int J Oncol, 47(1), 262-268. https://doi.org/10.3892/ijo.2015.3003

Google Scholar

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