Clinical Research Institute, National Hospital Organization Kyushu Cancer Center, Fukuoka 811-1395, Japan.
Department of Pharmacy, Fukuoka University Hospital, Fukuoka 814-0180, Japan.
Int J Oncol. 2014 Apr;44(4):1316-24. doi: 10.3892/ijo.2014.2293. Epub 2014 Feb 10.
To investigate the molecular mechanisms of lung cancer-induced bone metastasis, we established a bone-seeking subclone (HARA-B4) from a human squamous lung cancer cell line (HARA) using an in vivo selection method. We compared comprehensive gene expression profiles between HARA and HARA-B4, and identified the critical factors for the formation of bone metastasis using in vitro and in vivo assays. The number of bone metastatic colonies in the hind legs was significantly higher in HARA-B4-inoculated mice than in HARA-inoculated mice at 4 weeks after inoculation. In addition, visceral (adrenal) metastases were not found in HARA-B4-inoculated mice at autopsy, suggesting an increase in cancer cell tropism to bone in HARA-B4. Based on a comprehensive gene expression analysis, the expression level of CXC chemokine ligand 14 (CXCL14) was 5-fold greater in HARA-B4 than in HARA. Results of a soft agar colony formation assay showed that anchorage-independent growth ability was 4.5-fold higher with HARA-B4 than with HARA. The murine pre-osteoblast cell line MC3T3-E1 and the pre-osteoclast/macrophage cell line RAW264.7 migrated faster toward cultured HARA-B4 cells than toward HARA cells in a transwell cell migration assay. Interestingly, CXCL14 was shown to be involved in all events (enhancement of cancer cell tropism to the bone, anchorage-independent growth and/or recruitment of bone marrow cells) based on siRNA experiments in HARA-B4 cells. Furthermore, in clinical specimens of lung cancer-induced bone metastasis, expression of CXCL14 was observed in the tumor cells infiltrated in bone marrow in all specimens examined. CXCL14 was able to promote bone metastasis through enhancement of cancer cell tropism to the bone and/or recruitment of bone marrow cells around metastatic cancer cells.
为了研究肺癌诱导骨转移的分子机制，我们使用体内选择方法从人鳞状肺癌细胞系（HARA）中建立了一个骨趋向性亚克隆（HARA-B4）。我们比较了 HARA 和 HARA-B4 之间的综合基因表达谱，并使用体外和体内试验鉴定了形成骨转移的关键因素。在接种后 4 周，接种 HARA-B4 的小鼠后腿骨转移灶的数量明显高于接种 HARA 的小鼠。此外，在尸检中未发现接种 HARA-B4 的小鼠有内脏（肾上腺）转移，这表明 HARA-B4 中的癌细胞对骨骼的趋向性增加。基于综合基因表达分析，HARA-B4 中的 CXC 趋化因子配体 14（CXCL14）的表达水平是 HARA 的 5 倍。软琼脂集落形成试验的结果表明，HARA-B4 的无锚定生长能力比 HARA 高 4.5 倍。在 Transwell 细胞迁移试验中，鼠前成骨细胞系 MC3T3-E1 和前破骨细胞/巨噬细胞系 RAW264.7 向培养的 HARA-B4 细胞迁移的速度比向 HARA 细胞迁移的速度快。有趣的是，基于 HARA-B4 细胞中的 siRNA 实验，发现 CXCL14 参与了所有事件（增强癌细胞对骨骼的趋向性、无锚定生长和/或骨髓细胞的募集）。此外，在肺癌诱导骨转移的临床标本中，在所有检查的标本中都观察到 CXCL14 在浸润骨髓的肿瘤细胞中表达。CXCL14 能够通过增强癌细胞对骨骼的趋向性和/或募集转移性癌细胞周围的骨髓细胞来促进骨转移。
