Xie FenFen, Wang LiLi, Liu YaJing, Liu ZhenBang, Zhang ZuoYang, Pei Jing, Wu ZhengSheng, Zhai MuXin, Cao YunXia
Reproductive Medicine Center, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, Hefei, China.
NHC Key Laboratory of Study on Abnormal Gametes and Reproductive Tract (Anhui Medical University), Hefei, China.
Front Oncol. 2020 Oct 21;10:537247. doi: 10.3389/fonc.2020.537247. eCollection 2020.
Triple-negative (PR, ER, HER-2) breast cancer (TNBC) is regarded as more aggressive and more likely to recur after medical care. Emerging evidence has demonstrated that the circadian clock system regulates cell-signaling pathways critical to cancer cell proliferation, survival and metastasis, meaning that it could be a good candidate for TNBC treatment. As such, the aim of the current study was to examine the molecular mechanism by which the circadian clock system contributes to cancer progression in TNBC.
Cancer cells and primary breast cancer tissues were immunostained for the measurement of circadian clock proteins (CLOCK, BMAL1 and PER1) and acetylserotonin methyltransferase (ASMT). The association between ASMT and clock proteins was assessed using siRNA and Western blot. Transwell assays were used to detect cancer cell migration and invasion while MTT assays were utilized to evaluate cell proliferation.
Circadian clock proteins (CLOCK, BMAL1, and PER1) and ASMT expression were higher in TNBC and triple positive breast cancer (TPBC) compared with para-carcinoma tissues (PCTs). Intriguingly, there was an obvious correlation between circadian clock proteins and ASMT expression in both TPBC and TNBC. Similarly, circadian clock proteins and ASMT were expressed to a greater extent in BT-474 (triple-positive) cells than in MDA-MB-231 (triple-negative) cells. The inhibition of ASMT reduced circadian clock protein levels in both breast cancer cell lines. Further analysis showed that the expression levels of ASMT and circadian clock proteins did not correlate with clinical parameters such as age, tumor size, histologic grade and CK5/6, but increased significantly with lymphatic invasion in TNBC. In agreement with this finding, knockdown of ASMT significantly leads to reductions in migration and invasion in MDA-MB-231 cells. However, over-expression of CLOCK reversed the decreases seen in ASMT inhibited cells.
Our study suggests that ASMT regulates the circadian clock system in breast cancer and inhibition of ASMT reduces the invasiveness of triple-negative breast cancer cells by downregulating clock protein in a certain extent, indicating the potential value of ASMT as a drug target for TNBC treatment.
三阴性（PR、ER、HER-2）乳腺癌（TNBC）被认为更具侵袭性，且在治疗后更易复发。新出现的证据表明，生物钟系统调节对癌细胞增殖、存活和转移至关重要的细胞信号通路，这意味着它可能是TNBC治疗的一个良好候选靶点。因此，本研究的目的是探讨生物钟系统促进TNBC癌症进展的分子机制。
对癌细胞和原发性乳腺癌组织进行免疫染色，以检测生物钟蛋白（CLOCK、BMAL1和PER1）和乙酰血清素甲基转移酶（ASMT）。使用小干扰RNA（siRNA）和蛋白质免疫印迹法评估ASMT与生物钟蛋白之间的关联。采用Transwell实验检测癌细胞迁移和侵袭能力，同时利用MTT实验评估细胞增殖能力。
与癌旁组织（PCT）相比，TNBC和三阳性乳腺癌（TPBC）中生物钟蛋白（CLOCK、BMAL1和PER1）及ASMT表达更高。有趣的是，在TPBC和TNBC中，生物钟蛋白与ASMT表达之间存在明显相关性。同样，与MDA-MB-231（三阴性）细胞相比，生物钟蛋白和ASMT在BT-474（三阳性）细胞中的表达程度更高。抑制ASMT可降低两种乳腺癌细胞系中生物钟蛋白水平。进一步分析表明，ASMT和生物钟蛋白的表达水平与年龄、肿瘤大小、组织学分级和CK5/6等临床参数无关，但在TNBC中随淋巴浸润显著增加。与此发现一致，敲低ASMT可显著降低MDA-MB-231细胞的迁移和侵袭能力。然而，过表达CLOCK可逆转ASMT抑制细胞中出现的降低现象。
我们的研究表明，ASMT在乳腺癌中调节生物钟系统，抑制ASMT可通过一定程度下调生物钟蛋白来降低三阴性乳腺癌细胞的侵袭性，表明ASMT作为TNBC治疗药物靶点的潜在价值。
