Department of Surgery, Samuel Oschin Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA.
Key Laboratory for Breast Cancer Diagnosis and Treatment, Shantou University Medical College Cancer Hospital, Shantou 515041, China.
Int J Biol Sci. 2022 Jun 27;18(10):4203-4218. doi: 10.7150/ijbs.70583. eCollection 2022.
Triple-negative breast cancer (TNBC) does not respond to anti-estrogen and anti-HER2 therapies and is commonly treated by chemotherapy. TNBC has a high recurrence rate, particularly within the first 3 years. Thus, there is an urgent clinical need to develop more effective therapies for TNBC. Topoisomerase I (TOP1) inhibitors cause DNA damage, making these drugs desirable for TNBC treatment since DNA repair machinery is defective in this subtype of breast cancer. Among the main molecular subtypes of breast cancer, the TNBC cell lines exhibited the highest TOP1 inhibition sensitivity. However, clinically used TOP1 inhibitors, such as topotecan and irinotecan, have shown limited clinical applications and the reasons remain unclear. Understanding the mechanism of differential responses to TOP1 blockade and identifying the predictive markers for cancer cell sensitivity will help further TOP1-targeted therapy for TNBC treatment and improve the clinical use of TOP1 inhibitors. Viability assays were used to evaluate breast cancer cell sensitivity to topotecan and other TOP1 inhibitors as well as TOP2 inhibitors. An -derived topotecan-resistant TNBC cell model and TNBC xenograft models were employed to confirm cancer cell response to TOP1 blockade. RNA-seq was used to identify potential predictive markers for TNBC cell response to TOP1 blockade. Western blotting and qRT-PCR were performed to measure the protein levels and RNA expression. ATAC-seq and luciferase reporter assays were used to examine transcriptional regulations. The effects of MYC and JNK in cancer cell response to TOP1 inhibition were validated via loss-of-function and gain-of-function experiments. We observed two distinct and diverging cancer cell responses - sensitive versus resistant to TOP1 inhibition, which was confirmed by TNBC xenograft mouse models treated by topotecan. TNBC cells exhibited bifurcated temporal patterns of ATR pathway activation upon TOP1 inhibitor treatment. The sensitive TNBC cells showed an "up then down" dynamic pattern of ATR/Chk1 signaling, while the resistant TNBC cells exhibited a "persistently up" profile. On the contrary, opposite temporal patterns of induced expression of MYC, a key regulator and effector of DNA damage, were found in TNBC cells treated by TOP1 inhibitors. Mechanistically, we showed that TOP1-induced JNK signaling upregulated MYC expression. Furthermore, pharmacological inhibition of ATR reversed TNBC cell resistance to topotecan, whereas knockdown and JNK inhibition reduced cancer cell sensitivity. Dynamic temporal profiles of induced ATR/Chk1 and JNK activation as well as MYC expression, may predict cancer cell response to TOP1 inhibitors. JNK activation-mediated constitutive elevation of MYC expression may represent a novel mechanism governing cancer cell sensitivity to TOP1-targeting therapy. Our results may provide implications for identifying TNBC patients who might benefit from the treatment with TOP1 inhibitors.
三阴性乳腺癌（TNBC）对抗雌激素和抗 HER2 治疗无反应，通常采用化疗治疗。TNBC 的复发率较高，尤其是在最初的 3 年内。因此，迫切需要开发更有效的 TNBC 治疗方法。拓扑异构酶 I（TOP1）抑制剂会导致 DNA 损伤，这使得这些药物成为 TNBC 治疗的理想选择，因为在这种乳腺癌亚型中，DNA 修复机制存在缺陷。在乳腺癌的主要分子亚型中，TNBC 细胞系表现出对 TOP1 抑制的最高敏感性。然而，临床上使用的 TOP1 抑制剂，如拓扑替康和伊立替康，其临床应用有限，原因尚不清楚。了解对 TOP1 阻断的不同反应的机制并确定癌症细胞敏感性的预测标志物将有助于进一步进行针对 TOP1 的治疗，并改善 TOP1 抑制剂的临床应用。
采用细胞活力测定法评估了 TNBC 细胞对拓扑替康和其他 TOP1 抑制剂以及 TOP2 抑制剂的敏感性。使用源自 - 的拓扑替康耐药 TNBC 细胞模型和 TNBC 异种移植模型来证实癌症细胞对 TOP1 阻断的反应。RNA-seq 用于鉴定 TNBC 细胞对 TOP1 阻断反应的潜在预测标志物。采用 Western blot 和 qRT-PCR 检测蛋白水平和 RNA 表达。ATAC-seq 和荧光素酶报告基因测定用于检查转录调控。通过失活和过表达实验验证了 MYC 和 JNK 在癌症细胞对 TOP1 抑制反应中的作用。
我们观察到两种截然不同的癌细胞反应——对 TOP1 抑制敏感和耐药，这在接受拓扑替康治疗的 TNBC 异种移植小鼠模型中得到了证实。TNBC 细胞在接受 TOP1 抑制剂治疗后，ATR 通路激活呈现出两种不同的、发散的时间模式。敏感的 TNBC 细胞表现出 ATR/Chk1 信号的“先升后降”动态模式，而耐药的 TNBC 细胞则表现出持续升高的模式。相反，在接受 TOP1 抑制剂治疗的 TNBC 细胞中，诱导表达的 MYC（DNA 损伤的关键调节因子和效应因子）表现出相反的时间模式。
在机制上，我们表明 TOP1 诱导的 JNK 信号上调了 MYC 的表达。此外，ATR 的药理学抑制逆转了 TNBC 细胞对拓扑替康的耐药性，而 knockdown 和 JNK 抑制降低了癌症细胞的敏感性。
诱导的 ATR/Chk1 和 JNK 激活以及 MYC 表达的动态时间谱可能预测癌症细胞对 TOP1 抑制剂的反应。JNK 激活介导的 MYC 表达的组成性升高可能代表一种新的机制，控制着癌症细胞对 TOP1 靶向治疗的敏感性。我们的结果可能为鉴定可能受益于 TOP1 抑制剂治疗的 TNBC 患者提供依据。
