Tymoszuk Piotr, Charoentong Pornpimol, Hackl Hubert, Spilka Rita, Müller-Holzner Elisabeth, Trajanoski Zlatko, Obrist Peter, Revillion Françoise, Peyrat Jean-Philippe, Fiegl Heidi, Doppler Wolfgang
Division of Medical Biochemistry, Biocenter, Innsbruck Medical University, Innrain 80-82, 6020 Innsbruck, Austria.
BMC Cancer. 2014 Apr 12;14:257. doi: 10.1186/1471-2407-14-257.
STAT1 has been attributed a function as tumor suppressor. However, in breast cancer data from microarray analysis indicated a predictive value of high mRNA expression levels of STAT1 and STAT1 target genes belonging to the interferon-related signature for a poor response to therapy. To clarify this issue we have determined STAT1 expression levels and activation by different methods, and investigated their association with tumor infiltration by immune cells. Additionally, we evaluated the interrelationship of these parameters and their significance for predicting disease outcome.
Expression of STAT1, its target genes SOCS1, IRF1, CXCL9, CXCL10, CXCL11, IFIT1, IFITM1, MX1 and genes characteristic for immune cell infiltration (CD68, CD163, PD-L1, PD-L2, PD-1, CD45, IFN-γ, FOXP3) was determined by RT-PCR in two independent cohorts comprising 132 breast cancer patients. For a subset of patients, protein levels of total as well as serine and tyrosine-phosphorylated STAT1 were ascertained by immunohistochemistry or immunoblotting and protein levels of CXCL10 by ELISA.
mRNA expression levels of STAT1 and STAT1 target genes, as well as protein levels of total and serine-phosphorylated STAT1 correlated with each other in neoplastic tissue. However, there was no association between tumor levels of STAT1 mRNA and tyrosine-phosphorylated STAT1 and between CXCL10 serum levels and CXCL10 expression in the tumor. Tumors with increased STAT1 mRNA amounts exhibited elevated expression of genes characteristic for tumor-associated macrophages and immunosuppressive T lymphocytes. Survival analysis revealed an association of high STAT1 mRNA levels and bad prognosis in both cohorts. A similar prognostically relevant correlation with unfavorable outcome was evident for CXCL10, MX1, CD68, CD163, IFN-γ, and PD-L2 expression in at least one collective. By contrast, activation of STAT1 as assessed by the level of STAT1-Y701 phosphorylation was linked to positive outcome. In multivariate Cox regression, the predictive power of STAT1 mRNA expression was lost when including expression of CXCL10, MX1 and CD68 as confounders.
Our study confirms distinct prognostic relevance of STAT1 expression levels and STAT1 tyrosine phosphorylation in breast cancer patients and identifies an association of high STAT1 levels with elevated expression of STAT1 target genes and markers for infiltrating immune cells.
信号转导和转录激活因子1（STAT1）被认为具有肿瘤抑制功能。然而，在乳腺癌微阵列分析数据中，STAT1及其属于干扰素相关特征的靶基因的高mRNA表达水平对治疗反应不佳具有预测价值。为了阐明这一问题，我们采用不同方法测定了STAT1的表达水平和激活情况，并研究了它们与免疫细胞肿瘤浸润的相关性。此外，我们评估了这些参数之间的相互关系及其对预测疾病转归的意义。
在两个由132例乳腺癌患者组成的独立队列中，通过逆转录聚合酶链反应（RT-PCR）测定STAT1及其靶基因细胞因子信号转导抑制因子1（SOCS1）、干扰素调节因子1（IRF1）、CXC趋化因子配体9（CXCL9）、CXCL10、CXCL11、干扰素诱导蛋白1（IFIT1）、干扰素诱导跨膜蛋白1（IFITM1）、Mx蛋白1（MX1）以及免疫细胞浸润特征性基因（CD68、CD163、程序性死亡受体配体1（PD-L1）、PD-L2、程序性死亡受体1（PD-1）、白细胞共同抗原（CD45）、干扰素-γ（IFN-γ）、叉头框P3（FOXP3））的表达。对于部分患者，通过免疫组织化学或免疫印迹法确定总STAT1以及丝氨酸和酪氨酸磷酸化STAT1的蛋白水平，并通过酶联免疫吸附测定法（ELISA）确定CXCL10的蛋白水平。
肿瘤组织中，STAT1及其靶基因的mRNA表达水平以及总STAT1和丝氨酸磷酸化STAT1的蛋白水平相互关联。然而，肿瘤中STAT1 mRNA水平与酪氨酸磷酸化STAT1之间以及CXCL10血清水平与肿瘤中CXCL10表达之间均无关联。STAT1 mRNA量增加的肿瘤表现出肿瘤相关巨噬细胞和免疫抑制性T淋巴细胞特征性基因表达升高。生存分析显示，两个队列中STAT1 mRNA高水平均与不良预后相关。在至少一个队列中，CXCL10、MX1、CD68、CD163、IFN-γ和PD-L2表达与不良转归也存在类似的预后相关相关性。相比之下，通过STAT1-Y701磷酸化水平评估的STAT1激活与良好转归相关。在多变量Cox回归分析中，当将CXCL10、MX1和CD68的表达作为混杂因素纳入时，STAT1 mRNA表达的预测能力丧失。
我们的研究证实了STAT1表达水平和STAT1酪氨酸磷酸化在乳腺癌患者中具有不同的预后相关性，并确定了STAT1高水平与STAT1靶基因表达升高以及浸润免疫细胞标志物之间的关联。
