Purpose

Triple negative breast cancers (TNBCs) are enriched in cells bearing stem-like features, i.e., cancer stem cells (CSCs), which underlie cancer progression. Thus, targeting stemness may be an interesting treatment approach. The epigenetic machinery is crucial for maintaining the stemness phenotype. Bromodomain and extra-terminal domain (BET) epigenetic reader family members are emerging as novel targets for cancer therapy, and have already shown preclinical effects in breast cancer. Here, we aimed to evaluate the effect of the BET inhibitor JQ1 on stemness in TNBC.

Methods

Transcriptomic, functional annotation and qRT-PCR studies were performed on JQ1-exposed TNBC cells in culture. The results obtained were confirmed in spheroids and spheroid-derived tumours. In addition, limiting dilution, secondary and tertiary tumour sphere formation, matrigel invasion, immunofluorescence and flow cytometry assays were performed to evaluate the effect of JQ1 on CSC features. For clinical outcome analyses, the online tool Kaplan-Meier Plotter and an integrated response database were used.

Results

We found that JQ1 modified the expression of stemness-related genes in two TNBC-derived cell lines, MDA-MB-231 and BT549. Among these changes, the CD44 Antigen/CD24 Antigen (CD44/CD24) ratio and Aldehyde Dehydrogenase 1 Family Member A1 (ALDH1A1) expression level, i.e., both classical stemness markers, were found to be decreased by JQ1. Using a validated spheroid model to mimic the intrinsic characteristics of CSCs, we found that JQ1 decreased surface CD44 expression, inhibited self-renewal and invasion, and induced cell cycle arrest in G0/G1, thereby altering the stemness phenotype. We also found associations between four of the identified stemness genes, Gap Junction Protein Alpha 1 (GJA1), CD24, Epithelial Adhesion Molecule (EPCAM) and SRY-related HMG-box gene 9 (SOX9), and a worse TNBC patient outcome. The expression of another two of the stemness-related genes was found to be decreased by JQ1, i.e., ATP Binding Cassette Subfamily G Member 2 (ABCG2) and RUNX2, and predicted a low response to chemotherapy in TNBC patients, which supports a role for RUNX2 as a potential predictive marker for chemotherapy response in TNBC.

Conclusions

We identified a stemness-related gene panel associated with JQ1 and describe how this inhibitor modifies the stemness landscape in TNBC. Therefore, we propose a novel role for JQ1 as a stemness-targeting drug. Loss of the stem cell phenotype via JQ1 treatment could lead to less aggressive and more chemo-sensitive tumours, reflecting a better patient prognosis. Thus, the identified gene panel may be of interest for the clinical management of patients with aggressive TNBC.

中文翻译：

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在三阴性乳腺癌中与BET抑制相关的干性相关基因组的鉴定。

目的

三阴性乳腺癌（TNBC）富含具有干细胞样特征的细胞，即癌症干细胞（CSC），这是癌症进展的基础。因此，针对茎干可能是一种有趣的治疗方法。表观遗传机制对于维持茎表型至关重要。Bromodomain和末端外域（BET）表观遗传阅读器家族成员正在成为癌症治疗的新靶标，并且已经在乳腺癌中显示出临床前作用。在这里，我们旨在评估BET抑制剂JQ1对TNBC茎干的作用。

方法

在培养物中，JJ1暴露的TNBC细胞进行了转录组学，功能注释和qRT-PCR研究。在球状体和球状体衍生的肿瘤中证实了获得的结果。此外，进行了有限稀释，二级和三级肿瘤球形成，基质胶侵袭，免疫荧光和流式细胞术测定，以评估JQ1对CSC功能的影响。对于临床结果分析，使用了在线工具Kaplan-Meier Plotter和一个综合响应数据库。

结果

我们发现，JQ1修饰了两个TNBC衍生的细胞系MDA-MB-231和BT549中干性相关基因的表达。在这些变化中，发现CD44抗原/ CD24抗原（CD44 / CD24）的比率和醛脱氢酶1家族成员A1（ALDH1A1）的表达水平，即两个经典的干性标记，均被JQ1降低。使用经过验证的球体模型模拟CSC的固有特征，我们发现JQ1降低了表面CD44的表达，抑制了自我更新和侵袭，并诱导了G0 / G1的细胞周期停滞，从而改变了茎表型。我们还发现了四个已鉴定的干性基因，Gap Junction Protein Alpha 1（GJA1）之间的关联。，CD24，上皮粘附分子（EPCAM）和SRY相关的HMG-box基因9（SOX9），以及TNBC患者的预后较差。发现JQ1降低了另外两个与茎相关的基因的表达，即ATP结合盒亚家族G成员2（ABCG2）和RUNX2，并且预测TNBC患者对化疗的反应低，这支持了RUNX2作为TNBC化疗反应的潜在预测指标。

结论

我们鉴定了与JQ1相关的茎相关基因组，并描述了该抑制剂如何修饰TNBC中的茎景观。因此，我们提出了JQ1作为干性靶向药物的新作用。通过JQ1治疗导致干细胞表型的丧失可能导致侵略性降低和对化学敏感的肿瘤增多，从而反映出更好的患者预后。因此，鉴定的基因组对于侵略性TNBC患者的临床管理可能是感兴趣的。