Department of Oncology and Diagnostic Sciences, University of Maryland Dental School, 650 W Baltimore St., 7th floor (south), Rm7207, Baltimore, MD, 21201, USA.
Cell Commun Signal. 2019 Jul 22;17(1):80. doi: 10.1186/s12964-019-0395-6.
Expression of CD44 receptor is associated with the onset of several tumors. The intracellular domain of CD44 (CD44-ICD) has been implicated as a co-transcription factor for RUNX2 in the regulation of expression of MMP-9 in breast carcinoma cells. Previous studies from our laboratory demonstrated the role of CD44 in migration and invasion of PC3 prostate cells through activation of MMP-9. CD44 signaling regulates the phosphorylation and hence the localization of RUNX2 in the nucleus. The role of CD44-ICD has not been studied in prostate cancer cells. This study aimed to explore the role of CD44-ICD and RUNX2 in the regulation of expression of metastasis-related genes.
PC3 and PC3 cells overexpressing RUNX2 protein were analyzed for RUNX2/CD44-ICD interaction by immunoprecipitation, immunoblotting, and Immunofluorescence analyses. Wound healing and tumorsphere formation analyses were also done in these cells. The real-time PCR analysis was used to detect the expression levels of different genes.
Expression of CD44 and RUNX2 was observed only in PC3 cells (androgen receptor positive) and not in LNCaP or PCa2b cells (androgen receptor negative). Therefore, CD44-ICD fragment (~ 15-16 kDa) was observed in PC3 cells. Moreover, localization of CD44-ICD was more in the nucleus than in the cytoplasm of PC3 cells. Inhibition of cleavage of CD44 with a γ-secretase inhibitor, DAPT reduced the formation of CD44-ICD; however, accumulation of CD44-external truncation fragments (~ 20 and ~ 25 kDa) was detected. RUNX2 and CD44-ICD interact in the nucleus of PC3 cells, and this interaction was more in PC3 cells transfected with RUNX2 cDNA. Overexpression of RUNX2 augments the expression of metastasis-related genes (e.g., MMP-9 and osteopontin) which resulted in increased migration and tumorsphere formation.
We have shown here a strong functional relationship between CD44-ICD and RUNX2 in PC3 cells. RUNX2 forms a complex with CD44-ICD as a co-transcriptional factor, and this complex formation not only activates the expression of metastasis-related genes but also contributes to migration and tumorsphere formation. Therefore, RUNX2 and CD44-ICD are potential targets for anti-cancer therapy, and attenuation of their interaction may validate the regulatory effects of these proteins on cancer migration and progression.
CD44 受体的表达与几种肿瘤的发生有关。CD44 的细胞内结构域（CD44-ICD）已被认为是乳腺癌细胞中 RUNX2 调节 MMP-9 表达的共转录因子。我们实验室的先前研究表明，CD44 通过激活 MMP-9 在 PC3 前列腺细胞的迁移和侵袭中发挥作用。CD44 信号调节 RUNX2 在核内的磷酸化和定位。CD44-ICD 在前列腺癌细胞中的作用尚未得到研究。本研究旨在探讨 CD44-ICD 和 RUNX2 在调节转移相关基因表达中的作用。
通过免疫沉淀、免疫印迹和免疫荧光分析分析过表达 RUNX2 蛋白的 PC3 和 PC3 细胞中 RUNX2/CD44-ICD 相互作用。还对这些细胞进行了划痕愈合和肿瘤球形成分析。实时 PCR 分析用于检测不同基因的表达水平。
仅在 PC3 细胞（雄激素受体阳性）中观察到 CD44 和 RUNX2 的表达，而在 LNCaP 或 PCa2b 细胞（雄激素受体阴性）中未观察到。因此，在 PC3 细胞中观察到 CD44-ICD 片段（15-16 kDa）。此外，CD44-ICD 在 PC3 细胞中的核内定位多于细胞质。用 γ-分泌酶抑制剂 DAPT 抑制 CD44 的切割减少了 CD44-ICD 的形成；然而，检测到 CD44 外部截断片段（20 和~25 kDa）的积累。RUNX2 与 PC3 细胞核内的 CD44-ICD 相互作用，并且在转染了 RUNX2 cDNA 的 PC3 细胞中这种相互作用更多。RUNX2 的过表达增强了转移相关基因（例如 MMP-9 和骨桥蛋白）的表达，导致迁移和肿瘤球形成增加。
我们在这里显示了 CD44-ICD 和 RUNX2 在 PC3 细胞中之间存在很强的功能关系。RUNX2 与 CD44-ICD 形成复合物作为共转录因子，这种复合物的形成不仅激活了转移相关基因的表达，而且促进了迁移和肿瘤球形成。因此，RUNX2 和 CD44-ICD 是癌症治疗的潜在靶点，并且减弱它们的相互作用可能会验证这些蛋白质对癌症迁移和进展的调节作用。
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