Chemotherapy is especially important for cancer treatment, and it is the preferred systemic treatment for almost all cancers . For OS, high-dose chemotherapeutic treatments work well, but strong reverse effect are usually accompanied, limiting their efficacy on the treatment of OS patients . Therefore, the more effective and economical approaches are combination therapy, which increase the sensitivity and reduce the revers effect of single drug alone.
BET inhibitors including OTX015, have anticancer effects through the epigenetic repression of multiple pro-oncogene. Although BET inhibitors have been used as tumor-targeted tents, early clinical trial findings have been mixed, and drug susceptibility has restricted the therapeutic efficacy of BET inhibitors. However, several preclinical data indicated that the combination of BET inhibitors and HDAC inhibitors has a broader prospect[22, 23]. It was reported in several studies that combining BET inhibitors with specific HDAC inhibitor improved their efficacy. BRD4 inhibitors and histone deacetylase inhibitors can prevent the proliferation and metastasis of gallbladder cancer cells through the PI3K/Akt and MAPK/ERK pathways, and induce cell apoptosis, according to Liu et al . Shahbazi et al. found that JQ1 and panobinostat synergistically inhibited the growth of neuroblastoma cells and induced apoptosis by reducing the expression of LIN28B and N-Myc . Fiskus et al. reported that JQ1 and the HDAC inhibitor panobinostat could induce apoptosis in acute myeloid leukemia cells and have a synergistic anti-tumor effect .
Our results indicated that the combination of OTX015 and WT-161 synergistically inhibited cell proliferation and induced apoptosis, and resulted in a cell cycle arrest in OS cells. By the sphere-forming assay, we discovered that OTX015/WT-161 could worked together to inhibit OSCs' self-renewal ability. Mechanistical exploration showed that WNT and PTEN/PI3K/Akt signaling pathways were linked in the synergistic effect of OTX015/WT-161. Besides, overexpression of β-catenin recovered the killing effect of OTXWT-161, indicating β-catenin is responsible for the synergistic efficacy of OTX/WT-161 on OS cells. Finally, in vivo experiments revealed that OTX015/WT-161 had a strong inhibitory effect on the tumor xenografts.
Metastasis is a critical factor that affects cancer treatment . OS is a kind of highly aggressive cancer usually with distant metastases, especially lung metastases, at the time of diagnosis. To date, the 5-year survival rate for OS with pulmonary metastasis is as low as 20–30%, making metastasis as a significant challenge in OS therapy [26, 27]. EMT refers to a biological process in which epithelial cells lose their polarity, weaken cell-to-cell connections, and become more aggressive . As EMT makes cancer cells more aggressive, resulting in distant metastasis, inhibiting EMT can increase cancer patients' survival. We found that both OTX015 and WT-161 inhibited OS cells migration and invasion, reduced the mesenchymal marker proteins vimentin and N-cadherin and increased the epithelial marker E-cadherin. Notably, the efficacy were more pronounced when combination of OTX015 with WT-161.
Multiple genes balance control the dead and alive of cells during apoptosis, a physiological process. Caspase family proteins are essential determinants of apoptosis signaling and are required for the initiation and progression of apoptosis. Caspase-3 and caspase-6, which carry out the apoptotic process, and caspase-9, which is activated by signaling factors, are members of the caspase family of proteins . In our research, we found that treating OS cells with OTX015 and WT-161 together promoted apoptosis while also increasing cleaved caspase-3 and cleaved caspase-9 proteins. The expression of apoptosis-related proteins corroborated the flow cytometry findings. As for the phenomenon that OTX015/WT-161 can arrest the cell cycle in the G1/S phase, it has been previously reported that BET inhibitors can arrest the cell cycle in the G1 phase of a variety of malignant tumors [22, 30]; HDAC inhibitors induced apoptosis and blocked cells in the G1 phase , which was consistent with the effect of OTX015/WT-161 on the cell cycle of OS in this study. Furthermore, treatment with OTX015/WT-161 resulted in a decrease in CDK2 protein and an increase in p21 protein, confirming the role of OTX015/WT-161 on the cell cycle arrest.
The synergistic mechanism of OTX015/WT-161 on the repression of OS cells was explored in this study. We found that OTX015 mediated the degradation of β-catenin through inhibiting the expression of FZD2, a WNT receptor, whose activation increases the activity of the degradation complex against β-catenin. In addition, HDAC6 inhibitors have been demonstrated to cause the activation and membrane translocation the phosphatase and tensin homologue (PTEN), a well-known tumor suppressor gene inhibiting PI3K/ Akt signaling . Both the WNT and PI3K/Akt pathways can promote the development of cancer, and they are often interacted . Therefore, we suspected that WT-161 could also inhibit the growth of OS cells through the PTEN/PI3K/ Akt signaling pathway to participate in the synergistic therapeutic effect . Indeed, we found WT-161 treatment increased the expression of PTEN, an upstream regulator of GSK-3β/β-catenin. In a word, the WNT and PI3K/Akt pathways complemented each other to degrade β-catenin and thus play an anti-cancer role in the treatment of OTX015/WT-161 (Fig. 6C).
It's worth noting that OTX015/WT-161 has a synergistic inhibitory effect on OSCs. At the present, CSCs are considered to be a subtype of tumor cells that can facilitate tumor proliferation, metastasis, and self-renewal . The molecular mechanism of the synergistic effect of OTX015/WT-161 on OSCs needs to be analyzed further, as it will be promise the development of novel OS therapeutic strategies.