Al-Rakan Maha A, Hendrayani Siti-Faujiah, Aboussekhra Abdelilah
Present address: Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Saud University, Riyadh, 11211, Kingdom of Saudi Arabia.
Department of Molecular Oncology, King Faisal Specialist Hospital and Research Center, MBC# 03, PO BOX 3354, Riyadh, 11211, Kingdom of Saudi Arabia.
BMC Cancer. 2016 Aug 2;16:575. doi: 10.1186/s12885-016-2614-5.
Active fibroblasts, the predominant and the most active cells of breast cancer stroma, are responsible for tumor growth and spread. However, the molecular mediators and pathways responsible for stromal fibroblast activation, and their paracrine pro-carcinogenic effects are still not well defined. The CHEK2 tumor suppressor gene codes for a protein kinase, which plays important roles in the cellular response to various genotoxic stresses.
Immunoblotting, quantitative RT-PCR and Immunofluorescence were used to assess the expression of CHEK2 in different primary breast fibroblasts and in tissues. The effect of CHEK2 on the expression and secretion of SDF-1 and IL-6 was evaluated by immunoblotting and ELISA. The WST-1 colorimetric assay was used to assess cell proliferation, while the BD BioCoat Matrigel invasion chambers were utilized to determine the effects of CHEK2 on the migratory and the invasiveness capacities of breast stromal fibroblasts as well as breast cancer cells.
We have shown that CHEK2 is down-regulated in most cancer-associated fibroblasts (CAFs) as compared to their corresponding tumor counterpart fibroblasts (TCFs) at both the mRNA and protein levels. Interestingly, CHEK2 down-regulation using specific siRNA increased the expression/secretion of both cancer-promoting cytokines SDF-1 and IL-6, and transdifferentiated stromal fibroblasts to myofibroblasts. These cells were able to enhance the proliferation of non-cancerous epithelial cells, and also boosted the migration/invasion abilities of breast cancer cells in a paracrine manner. The later effect was SDF-1/IL-6-dependent. Importantly, ectopic expression of CHEK2 in active CAFs converted these cells to a normal state, with lower migration/invasion capacities and reduced paracrine pro-carcinogenic effects.
These results indicate that CHEK2 possesses non-cell-autonomous tumor suppressor functions, and present the Chk2 protein as an important mediator in the functional interplay between breast carcinomas and their stromal fibroblasts.
活化的成纤维细胞是乳腺癌基质中占主导地位且最活跃的细胞，负责肿瘤的生长和扩散。然而，负责基质成纤维细胞活化的分子介质和途径及其旁分泌促癌作用仍未明确界定。CHEK2肿瘤抑制基因编码一种蛋白激酶，其在细胞对各种基因毒性应激的反应中发挥重要作用。
采用免疫印迹、定量逆转录聚合酶链反应和免疫荧光法评估CHEK2在不同原发性乳腺成纤维细胞和组织中的表达。通过免疫印迹和酶联免疫吸附测定法评估CHEK2对基质细胞衍生因子-1（SDF-1）和白细胞介素-6（IL-6）表达和分泌的影响。采用WST-1比色法评估细胞增殖，同时利用BD生物包被基质胶侵袭小室来确定CHEK2对乳腺基质成纤维细胞以及乳腺癌细胞迁移和侵袭能力的影响。
我们已经表明，与相应的肿瘤对应成纤维细胞（TCF）相比，大多数癌症相关成纤维细胞（CAF）中的CHEK2在mRNA和蛋白质水平均下调。有趣的是，使用特异性小干扰RNA（siRNA）下调CHEK2可增加促癌细胞因子SDF-1和IL-6的表达/分泌，并使基质成纤维细胞转分化为肌成纤维细胞。这些细胞能够增强非癌上皮细胞的增殖，还能以旁分泌方式增强乳腺癌细胞的迁移/侵袭能力。后一种作用依赖于SDF-1/IL-6。重要的是，在活化的CAF中异位表达CHEK2可将这些细胞转变为正常状态，其迁移/侵袭能力降低，旁分泌促癌作用减弱。
这些结果表明CHEK2具有非细胞自主性肿瘤抑制功能，并表明Chk2蛋白是乳腺癌与其基质成纤维细胞之间功能相互作用中的重要介质。
