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，但目前尚未有针对生物钟基因与乳腺癌预后相关性的系统性研究。为此，我们对766例未接受新辅助及辅助治疗的淋巴结阴性乳腺癌患者的肿瘤组织中17种生物钟组分的表达情况进行了检测。此外，我们还分析了其与无转移生存期（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，我们通过相关性分析探究了生物钟基因的表达关联。单因素Cox回归分析（Cox regression analyses）显示，多种生物钟基因（如<i>CLOCK</i>、<i>PER1</i>、<i>PER2</i>、<i>PER3</i>、<i>CRY2</i>、<i>NPAS2</i>及<i>RORC</i>）的高表达与更长的无转移生存期相关（风险比（hazard ratio, HR）<1，错误发现率（False Discovery Rate, FDR）校正后<i>P</i> < 0.05）。按分子亚型（molecular subtype）分层分析后发现，<i>PER1</i>、<i>PER3</i>、<i>CRY2</i>及<i>NFIL3</i>在雌激素受体阳性/人表皮生长因子受体2阴性（ER+/HER2-）亚型中具有预后价值，<i>CLOCK</i>与<i>NPAS2</i>在雌激素受体阴性/人表皮生长因子受体2阴性（ER-/HER2-）亚型中具有预后价值，而<i>ARNTL2</i>则在人表皮生长因子受体2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<i>PER3</i>（HR = 0.66; <i>P</i> = 0.016）与<i>RORC</i>（HR = 0.42; <i>P</i> = 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（如<i>PER2</i>-<i>PER3</i>与<i>CRY2</i>-<i>PER3</i>）之间的成对相关性在ER+、HER2-及低级别癌组织中更强；而在ER-、HER2+肿瘤、高级别肿瘤及发生转移的肿瘤组织中，相关系数则较弱。综上，生物钟基因的表达缺失与乳腺癌较差的预后相关。生物钟协同共表达特征（提示功能性昼夜节律系统的存在）在ER+、HER2-、低级别及未发生转移的肿瘤中得以维持，但在更具侵袭性的癌组织中则遭到破坏。