Borges Sahra, Döppler Heike, Perez Edith A, Andorfer Cathy A, Sun Zhifu, Anastasiadis Panos Z, Thompson E, Geiger Xochiquetzal J, Storz Peter
Breast Cancer Res. 2013 Aug 23;15(2):R66. doi: 10.1186/bcr3460.
DNA methylation-induced silencing of genes encoding tumor suppressors is common in many types of cancer, but little is known about how such epigenetic silencing can contribute to tumor metastasis. The PRKD1 gene encodes protein kinase D1 (PKD1), a serine/threonine kinase that is expressed in cells of the normal mammary gland, where it maintains the epithelial phenotype by preventing epithelial-to-mesenchymal transition.
The status of PRKD1 promoter methylation was analyzed by reduced representation bisulfite deep sequencing, methylation-specific PCR (MSP-PCR) and in situ MSP-PCR in invasive and noninvasive breast cancer lines, as well as in humans in 34 cases of "normal" tissue, 22 cases of ductal carcinoma in situ, 22 cases of estrogen receptor positive, HER2-negative (ER+/HER2-) invasive lobular carcinoma, 43 cases of ER+/HER2- invasive ductal carcinoma (IDC), 93 cases of HER2+ IDC and 96 cases of triple-negative IDC. A reexpression strategy using the DNA methyltransferase inhibitor decitabine was used in vitro in MDA-MB-231 cells as well as in vivo in a tumor xenograft model and measured by RT-PCR, immunoblotting and immunohistochemistry. The effect of PKD1 reexpression on cell invasion was analyzed in vitro by transwell invasion assay. Tumor growth and metastasis were monitored in vivo using the IVIS Spectrum Pre-clinical In Vivo Imaging System.
Herein we show that the gene promoter of PRKD1 is aberrantly methylated and silenced in its expression in invasive breast cancer cells and during breast tumor progression, increasing with the aggressiveness of tumors. Using an animal model, we show that reversion of PRKD1 promoter methylation with the DNA methyltransferase inhibitor decitabine restores PKD1 expression and blocks tumor spread and metastasis to the lung in a PKD1-dependent fashion.
Our data suggest that the status of epigenetic regulation of the PRKD1 promoter can provide valid information on the invasiveness of breast tumors and therefore could serve as an early diagnostic marker. Moreover, targeted upregulation of PKD1 expression may be used as a therapeutic approach to reverse the invasive phenotype of breast cancer cells.
DNA甲基化导致编码肿瘤抑制因子的基因沉默在多种癌症中很常见，但对于这种表观遗传沉默如何促进肿瘤转移却知之甚少。PRKD1基因编码蛋白激酶D1（PKD1），一种丝氨酸/苏氨酸激酶，在正常乳腺细胞中表达，通过防止上皮-间质转化维持上皮表型。
采用简化代表性亚硫酸氢盐深度测序、甲基化特异性PCR（MSP-PCR）和原位MSP-PCR分析侵袭性和非侵袭性乳腺癌细胞系以及34例“正常”组织、22例导管原位癌、22例雌激素受体阳性、人表皮生长因子受体2阴性（ER+/HER2-）浸润性小叶癌、43例ER+/HER2-浸润性导管癌（IDC）、93例HER2+ IDC和96例三阴性IDC中的PRKD1启动子甲基化状态。在MDA-MB-231细胞中体外以及在肿瘤异种移植模型中体内使用DNA甲基转移酶抑制剂地西他滨进行重新表达策略，并通过RT-PCR、免疫印迹和免疫组织化学进行检测。通过Transwell侵袭试验在体外分析PKD1重新表达对细胞侵袭的影响。使用IVIS Spectrum临床前体内成像系统在体内监测肿瘤生长和转移。
在此我们表明，PRKD1基因启动子在侵袭性乳腺癌细胞及其肿瘤进展过程中异常甲基化并表达沉默，且随着肿瘤侵袭性增加。使用动物模型，我们表明用DNA甲基转移酶抑制剂地西他滨逆转PRKD1启动子甲基化可恢复PKD1表达，并以PKD1依赖的方式阻断肿瘤向肺部的扩散和转移。
我们的数据表明，PRKD1启动子的表观遗传调控状态可为乳腺肿瘤的侵袭性提供有效信息，因此可作为早期诊断标志物。此外，靶向上调PKD1表达可作为一种治疗方法来逆转乳腺癌细胞的侵袭表型。
