Loss of circadian clock gene expression is associated with tumor progression in breast cancer

Several studies suggest a link between circadian rhythm disturbances and tumorigenesis. However, the association between circadian clock genes and prognosis in breast cancer has not been systematically studied. Therefore, we examined the expression of 17 clock components in tumors from 766 node-negative breast cancer patients that were untreated in both neoadjuvant and adjuvant settings. In addition, their association with metastasis-free survival (MFS) and correlation to clinicopathological parameters were investigated. Aiming to estimate functionality of the clockwork, we studied clock gene expression relationships by correlation analysis. Higher expression of several clock genes (e.g., <i>CLOCK</i>, <i>PER1</i>, <i>PER2</i>, <i>PER3</i>, <i>CRY2</i>, <i>NPAS2</i> and <i>RORC</i>) was found to be associated with longer MFS in univariate Cox regression analyses (HR&lt;1 and FDR-adjusted <i>P</i> &lt; 0.05). Stratification according to molecular subtype revealed prognostic relevance for <i>PER1</i>, <i>PER3</i>, <i>CRY2</i> and <i>NFIL3</i> in the ER+/HER2- subgroup, <i>CLOCK</i> and <i>NPAS2</i> in the ER-/HER2- subtype, and <i>ARNTL2</i> in HER2+ breast cancer. In the multivariate Cox model, only <i>PER3</i> (HR = 0.66; P = 0.016) and <i>RORC</i> (HR = 0.42; P = 0.003) were found to be associated with survival outcome independent of established clinicopathological parameters. Pairwise correlations between functionally-related clock genes (e.g., PER2-PER3 and CRY2-PER3) were stronger in ER+, HER2- and low-grade carcinomas; whereas, weaker correlation coefficients were observed in ER- and HER2+ tumors, high-grade tumors and tumors that progressed to metastatic disease. In conclusion, loss of clock genes is associated with worse prognosis in breast cancer. Coordinated co-expression of clock genes, indicative of a functional circadian clock, is maintained in ER+, HER2-, low grade and non-metastasizing tumors but is compromised in more aggressive carcinomas.

多项研究表明，昼夜节律（circadian rhythm）紊乱与肿瘤发生（tumorigenesis）存在关联。然而，目前尚未有研究系统探讨生物钟基因与乳腺癌预后之间的关联。为此，本研究对766例未接受新辅助治疗（neoadjuvant therapy）及辅助治疗（adjuvant therapy）的淋巴结阴性乳腺癌（node-negative breast cancer）患者的肿瘤组织中17种生物钟组件的表达水平进行了检测。此外，还分析了这些基因的表达与无转移生存期（metastasis-free survival, MFS）的相关性，以及与临床病理参数（clinicopathological parameters）的关联。为评估生物钟系统的功能状态，本研究通过相关性分析探究了生物钟基因的表达关联模式。单因素Cox回归分析（Cox regression analysis）显示，多种生物钟基因（如CLOCK、PER1、PER2、PER3、CRY2、NPAS2及RORC）的高表达与更长的无转移生存期相关，风险比（Hazard Ratio, HR）<1，且经错误发现率（False Discovery Rate, FDR）校正后的P值<0.05。按分子亚型（molecular subtype）进行分层分析后发现：PER1、PER3、CRY2及NFIL3在雌激素受体阳性（Estrogen Receptor, ER）/人表皮生长因子受体2阴性（Human Epidermal Growth Factor Receptor 2, HER2）亚型（ER+/HER2-）中具有预后价值；CLOCK与NPAS2在雌激素受体阴性（ER-）/HER2阴性（HER2-）亚型中具有预后价值；而ARNTL2则在HER2阳性（HER2+）乳腺癌中具有预后价值。多因素Cox模型（multivariate Cox model）分析显示，仅PER3（HR=0.66；P=0.016）与RORC（HR=0.42；P=0.003）的表达与患者生存结局独立相关，不受已确立的临床病理参数影响。功能相关的生物钟基因间的两两相关性（如PER2-PER3与CRY2-PER3）在ER+、HER2-及低分级癌组织中更强；而在ER-、HER2+肿瘤组织、高分级癌组织以及发生转移的肿瘤组织中，相关系数则相对较弱。综上，生物钟基因的表达缺失与乳腺癌不良预后密切相关。生物钟基因的协调共表达（提示功能性昼夜节律钟的存在）在ER+、HER2-、低分级及未发生转移的肿瘤组织中得以维持，但在更具侵袭性的癌组织中则遭到破坏。