SP110核蛋白的核仁轉位對化療藥物的影響

Abstract

SP110是一種受干擾素調控的核蛋白，屬於SP100/140家族。在人類中，其主要功能包括調控基因轉錄和調節免疫反應。目前已鑑定出三種主要亞型：SP110a、SP110b和SP110c。這些蛋白常被稱為“斑點蛋白”，因為它們會形成聚集體，並與核體共定位。核體是參與許多關鍵細胞反應的大分子結構，例如轉錄調控、細胞分裂、細胞凋亡、細胞老化以及對DNA損傷或感染的反應。在我們先前的研究中，我們鑑定了SP110中兩個潛在的核定位訊號NLS1和NLS2，並證實NLS1可以將SP110轉位到核仁，顯示NLS1具有隱藏的核仁定位訊號（NoLS）功能。儘管SP110b攜帶核仁定位訊號，但在常氧條件下，其在H1299肺癌細胞中表達時，僅極少部分（低於5%）被轉位於核仁。我們進一步證實，微氧顯著增強了SP110的核仁轉位。有趣的是，SP110b的突變體SP110b Mut1+3（NLS1發生突變）即使在常氧條件下也表現出強烈的核仁轉位。SP110b的核仁轉位似乎影響癌細胞的行為：它促進H1299細胞的克隆（細胞群）形成，並延緩微氧誘導的細胞死亡，暗示其可能在促進癌細胞存活和賦予其化療抗藥性方面發揮作用。因此，本研究探討了SP110b的核仁轉位如何影響細胞對化療藥物 （特別是Docetaxel、Cisplatin、Doxorubicin、5-FU和Etoposide）的反應，並探討了其潛在的機制。我們的研究結果顯示，SP110b驅動的核仁轉位增強了Docetaxel、Cisplatin及5-FU處理的H1299細胞存活率。在另一種肺癌細胞系PC9細胞中，SP110b也影響藥物反應；然而，與H1299細胞不同，這些治療並未導致總存活期的顯著差異。免疫螢光分析顯示，Cisplatin及Etoposide處理後，H1299細胞中核仁蛋白的分佈改變。在PC9細胞中，Cisplatin、Doxorubicin及5-FU處理後觀察到異常的核仁蛋白模式。此外，5-FU 治療導致部分的 SP110b Mut1+3 蛋白失去其核仁定位。這項研究顯示微氧或突變誘導的 SP110b 核仁轉位可能透過改變核仁組織來增強肺癌細胞的存活率並調節對化療藥物的敏感性。

SP110 is an interferon-regulated nuclear protein belonging to the SP100/140 family. In humans, its primary roles include regulating gene transcription and modulating immune responses. Three major isoforms have been identified: SP110a, SP110b, and SP110c. These proteins are often referred to as “speckle proteins” because they form aggregates that are co-localized with nuclear bodies, macromolecular structures involved in key cellular processes, such as transcriptional regulation, cell division, apoptosis, senescence, and responses to DNA damage or infection. In our previous studies, we identified two potential nuclear localization signals, NLS1 and NLS2, within SP110 and showed that NLS1 can direct SP110 to the nucleolus, indicating that NLS1 functions as a cryptic nucleolar localization signal (NoLS). Although SP110b carries this NoLS, it localizes to the nucleolus only infrequently (less than 5%) when expressed in H1299 lung cancer cells under normoxic conditions. We further demonstrated that hypoxia dramatically enhances SP110 nucleolar localization. Interestingly, a mutant form of SP110b, SP110b Mut1+3, which contains a mutation in NLS1, shows strong nucleolar localization even under normoxia. Nucleolar localization of SP110b appears to influence cancer cell behavior: it promotes colony formation in H1299 cells and delays hypoxia-induced cell death, suggesting a potential role in promoting cancer cell survival and conferring resistance to chemotherapeutic agents. Therefore, the present study investigates how SP110b’s nucleolar translocation affects cellular responses to anticancer drugs, specifically cisplatin, docetaxel, doxorubicin, 5-fluorouracil (5-FU), and etoposide, as well as explores potential underlying mechanisms. Our findings indicate that SP110b-driven nucleolar translocation enhances the survival of H1299 cells treated with docetaxel, cisplatin, or 5-FU. In PC9 cells, another lung cancer cell line, SP110b also influences drug responses; however, unlike in H1299 cells, these treatments do not lead to significant differences in overall survival. Immunofluorescence analysis shows that nucleolin distribution becomes altered in H1299 cells following cisplatin and etoposide exposure. In PC9 cells, abnormal nucleolin patterns are observed after treatment with cisplatin, doxorubicin, and 5-FU. Furthermore, 5-FU treatment causes a portion of the SP110b Mut1+3 protein to lose its nucleolar localization. This study suggests that hypoxia- or mutation-induced nucleolar localization of SP110b may enhance lung cancer cell survival and modulate sensitivity to chemotherapeutic agents by altering nucleolar organization.