HOXD3 Plays a Critical Role in Breast Cancer Sternness and Drug Resistance

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 sternness, and drug resistance of breast cancer cells. Methods: 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 sternness 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. Results: 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 stern cell biomarkers, suggesting that HOXD3 does indeed increase breast cancer cell sternness. Because we had previously shown that HOXD3 expression is closely associated with integrin beta 3 expression in breast cancer patients, we hypothesized that HOXD3 may regulate breast cancer cell sternness and drug resistance through integrin beta 3. Cell viability assays showed that integrin beta 3 knockdown increased cell viability and that HOXD3 could not restore cancer cell sternness or drug resistance. Given integrin beta 3's relationship with Wnt/beta-catenin signaling, we determine whether HOXD3 regulates integrin beta 3 activity through Wnt/beta-catenin signaling. We found that, even though HOXD3 increased the expression of Wnt/beta-catenin downstream genes, it did not restore Wnt/beta-catenin signaling activity, which was inhibited in integrin beta 3 knockdown breast cancer cells. Conclusion: We demonstrate that HOXD3 plays a critical role in breast cancer sternness and drug resistance via integrin beta 3-mediatedWnt/beta-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 sternness and drug resistance. (C) 2018 The Author(s) Published by S. Karger AG, Basel.