Zhang Yue, Zhang Qingyuan, Cao Zhongru, Huang Yuanxi, Cheng Shaoqiang, Pang Da
Department of Clinical Oncology, Harbin Medical University Cancer Hospital, Harbin, China.
Department of Breast Surgery, Harbin Medical University Cancer Hospital, Harbin, China.
Cell Physiol Biochem. 2018;46(4):1737-1747. doi: 10.1159/000489249. Epub 2018 Apr 23.
BACKGROUND/AIMS: Homeobox D3 (HOXD3) is a member of the homeobox family of genes that is known primarily for its transcriptional regulation of morphogenesis in all multicellular organisms. In this study, we sought to explore the role that HOXD3 plays in the stem-like capacity, or stemness, and drug resistance of breast cancer cells.
Expression of HOXD3 in clinical breast samples were examined by RT-PCR and immunohistochemistry. HOXD3 expression in breast cancer cell lines were analyzed by RT-PCR and western blot. Ability of drug resistance in breast cancer cells were elevated by MTT cell viability and colony formation assays. We examined stemness using cell fluorescent staining, RT-PCR and western blot for stem cell marker expression. Finally, activity of wnt signaling was analyzed by FOPflash luciferase assays. RT-PCR and western blot were performed for downstream genes of wnt signaling.
We demonstrated that HOXD3 is overexpressed in breast cancer tissue as compared to normal breast tissue. HOXD3 overexpression enhances breast cancer cell drug resistance. Furthermore, HOXD3 upregulation in the same cell lines increased sphere formation as well as the expression levels of stem cell biomarkers, suggesting that HOXD3 does indeed increase breast cancer cell stemness. Because we had previously shown that HOXD3 expression is closely associated with integrin β3 expression in breast cancer patients, we hypothesized that HOXD3 may regulate breast cancer cell stemness and drug resistance through integrin β 3. Cell viability assays showed that integrin β 3 knockdown increased cell viability and that HOXD3 could not restore cancer cell stemness or drug resistance. Given integrin β 3's relationship with Wnt/β-catenin signaling, we determine whether HOXD3 regulates integrin β 3 activity through Wnt/β-catenin signaling. We found that, even though HOXD3 increased the expression of Wnt/β-catenin downstream genes, it did not restore Wnt/β-catenin signaling activity, which was inhibited in integrin β3 knockdown breast cancer cells.
We demonstrate that HOXD3 plays a critical role in breast cancer stemness and drug resistance via integrin β3-mediated Wnt/β-catenin signaling. Our findings open the possibility for improving the current standard of care for breast cancer patients by designing targeted molecular therapies that overcome the barriers of cancer cell stemness and drug resistance.
背景/目的：同源盒D3（HOXD3）是同源盒基因家族的成员，主要因其在所有多细胞生物中对形态发生的转录调控而闻名。在本研究中，我们试图探讨HOXD3在乳腺癌细胞的干细胞样能力（即干性）和耐药性中所起的作用。
通过逆转录聚合酶链反应（RT-PCR）和免疫组织化学检测临床乳腺样本中HOXD3的表达。通过RT-PCR和蛋白质免疫印迹法分析乳腺癌细胞系中HOXD3的表达。通过MTT细胞活力和集落形成试验提高乳腺癌细胞的耐药能力。我们使用细胞荧光染色、RT-PCR和蛋白质免疫印迹法检测干细胞标志物表达来检测干性。最后，通过FOPflash荧光素酶试验分析Wnt信号通路的活性。对Wnt信号通路的下游基因进行RT-PCR和蛋白质免疫印迹分析。
我们证明，与正常乳腺组织相比，HOXD3在乳腺癌组织中过表达。HOXD3的过表达增强了乳腺癌细胞的耐药性。此外，同一细胞系中HOXD3的上调增加了球体形成以及干细胞生物标志物的表达水平，这表明HOXD3确实增加了乳腺癌细胞的干性。因为我们之前已经表明，HOXD3的表达与乳腺癌患者中整合素β3的表达密切相关，所以我们假设HOXD3可能通过整合素β3调节乳腺癌细胞的干性和耐药性。细胞活力试验表明，整合素β3的敲低增加了细胞活力，并且HOXD3不能恢复癌细胞的干性或耐药性。鉴于整合素β3与Wnt/β-连环蛋白信号通路的关系，我们确定HOXD3是否通过Wnt/β-连环蛋白信号通路调节整合素β3的活性。我们发现，尽管HOXD3增加了Wnt/β-连环蛋白下游基因的表达，但它并没有恢复在整合素β3敲低的乳腺癌细胞中被抑制的Wnt/β-连环蛋白信号通路活性。
我们证明HOXD3通过整合素β3介导的Wnt/β-连环蛋白信号通路在乳腺癌干性和耐药性中起关键作用。我们的研究结果为通过设计克服癌细胞干性和耐药性障碍的靶向分子疗法来改善乳腺癌患者的当前护理标准开辟了可能性。
