探討SERPINE1在三陰性乳癌之DNA雙股斷裂修復中扮演的角色

Abstract

乳癌可依其受體的表達分為不同分子亞型，三陰性乳癌因其荷爾蒙受體與HER2受體表現量低，缺乏有效的治療靶點，目前早期臨床治療仍以切除手術搭配化學治療或放射線治療為主，然而三陰性乳癌接受放射線治療後的局部復發率高，為目前亟需解決之問題。 SERPINE1(serpin family E member 1)是在肥胖者血液循環中經常升高的一種脂肪素，在某些癌症中也會高度表達，包括三陰性乳癌基因亞型中的basal b 亞型，並與癌症對治療的抗性相關聯。實驗室先前透過飲食誘導肥胖之三陰性乳癌同源小鼠模型，觀察到腫瘤和腫瘤微環境中的Serpine1都有所升高，且使用tiplaxtinin抑制Serpine1可以顯著降低腫瘤之放射線抗性，表明Serpine1會促進腫瘤放射線抗性。後續在細胞實驗中發現放射線照射會促進乳癌細胞內SERPINE1表現量上升且細胞核內的SERPINE1含量也會增加。除此之外，內源或外源性的SERPINE1都會促進DNA雙股斷裂修復，但仍不清楚其背後機制。 本篇研究透過DNA雙股斷裂修復試驗初步發現SERPINE1可能會促進非同源末端接合(NHEJ)修復，以提升細胞整體DNA雙股斷裂修復效率。另外為了探討細胞核中的SERPINE1是否位於DNA損傷處直接參與修復，透過免疫螢光染色發現放射線照射後SERPINE1並不會移動到損傷位點。透過西方墨點法分析γH2AX含量也發現rSERPINE1可以促進DNA雙股斷裂修復，若額外加入AKT抑制劑則可以部分消除此效果，代表SERPINE1可能會透過活化AKT訊息傳遞路徑來促進DNA雙股斷裂修復。為了更進一步研究SERPINE1在DNA雙股斷裂修復路徑的選擇中所扮演的角色，我們建立了帶有另一種DNA雙股斷裂修復報導基因(pLCN-DRR)之報導基因細胞株。現階段對於SERPINE1促進DNA雙股斷裂修復之機制仍未明瞭，後續我們將繼續研究SERPINE1對修復路徑選擇的影響以及其促進DNA雙股斷裂修復之機制。

Breast cancer can be classified into different molecular subtypes based on its receptor expression. Triple-negative breast cancer (TNBC) lacks effective therapeutic targets due to the low expression of hormone receptors and HER2, so the mainstay of treatment for early TNBC is still surgery combined with chemotherapy or radiotherapy. However, the high local recurrence rate of TNBC after radiation therapy is a problem that needs to be solved urgently. SERPINE1 (serpin family E member 1), an adipokine often found elevated in the circulation of obese individuals, is also highly expressed in certain cancers, including the basal b subtype of TNBC, and is associated with treatment resistance. In our diet-induced obesity syngeneic mouse model of TNBC, we observed an increase in Serpine1 levels in both the tumor and the tumor microenvironment. Moreover, inhibition of Serpine1 by tiplaxtinin significantly reduced tumor radioresistance, suggesting that Serpine1 plays a role in promoting radioresistance in tumors. In cell-based assays, we found that ionizing radiation induced the expression and nuclear localization of SERPINE1 in TNBC cells. Furthermore, both endogenous and exogenous SERPINE1 promoted DNA double-strand break (DSB) repair in breast cancer cells after exposure to ionizing radiation, but the underlying mechanism is still unclear. In this study, the DSB repair assay revealed that SERPINE1 may promote non-homologous end joining (NHEJ) to enhance the overall DNA DSB repair efficiency. In addition, we investigated whether SERPINE1 of the nucleus is located at the DSB site and directly participates in the repair. Immunofluorescence staining revealed that SERPINE1 was not localized to the DNA break site after IR. Analysis of γH2AX level by the Western blotting also showed that rSERPINE1 promoted DNA DSB repair, and this effect was partially reversed by the addition of AKT inhibitor, suggesting that SERPINE1 might promote DSB repair by activating the AKT signaling pathway. To further investigate the role of SERPINE1 in DSB repair pathway choice, we have established reporter cell lines with another DSB repair reporter gene (pLCN-DRR). The mechanism by which SERPINE1 promotes DSB repair is still unclear at this stage, and we will continue to investigate the repair pathway choice affected by SERPINE1 and the mechanism by which it promotes DNA DSB repair.