This study found that histone H3 trimethylation status is closely associated with metastatic OS and that the broad-spectrum inhibitor of histone lysine demethylase, IOX-1, has a potent therapeutic effect on a wide range of metastatic properties, including cell migratory and invasive abilities, the EMT process, CSC development and the efficacy of cisplatin chemotherapy.
Previously published evidence supports our findings showing that increased KDM expression and decreased histone H3 lysine trimethylation is associated with metastatic OS progression. For instance, higher KDM6A and KDM6B expression is reportedly associated with greater H3K27me3 demethylation in human OS tissue and different OS cell lines 16. In another study that examined IHC-stained OS tissue samples from 53 patients, higher KDM4B levels were positively associated with shorter OS survival as well as OS progression, as determined by higher Enneking staging results, a higher likelihood of distant metastases and poor differentiation on histology 24. KDM4C silencing has been shown to increase H3K9me3 levels in MG63 and SaOS-2 human OS cells 25.
Hypoxia and chemotherapy are key upstream conditions that are linked to histone H3 demethylation in OS. Hypoxic environments occur in many solid tumors, including OS, and can trigger the release of hypoxia-inducible factors HIF-1α, HIF-2α, and HIF-3α, which modulate EMT master regulators such as SNAIL and TWIST to promote EMT and CSC progression 26 27 28. In OS, HIF-3α overexpression in hypoxic conditions can activate KDM3A to decrease H3K9me2 levels at the SOX9 mRNA promoter region, leading to greater OS proliferation and invasion while restricting apoptosis 26, while another study has found that higher levels of KDM4B and HIF-1α are associated with worse OS prognosis and progression to metastatic disease 24.
Chemotherapy exposure appears to alter H3 lysine methylation. In a study that treated OS cells with cisplatin, KDM6A and KDM6B levels were significantly elevated, while H3K27me3 levels were reduced in cisplatin-resistant cells 16. Similarly, in our study, MG63-CR cells exhibited reductions in histone H3 methylation, specifically of H3K4me3, H3K9me3, H3K27me3 and H3K36me3. Chemotherapy resistance may in part be due to hypoxia, whereby the reduction of HIF-1α levels in cisplatin-resistant OS cells makes them sensitive to cisplatin 28.
In relation to the effects of IOX-1 upon MMPs, previous research has reported that IOX-1 dosing of hepatic stellate cells significantly reduces levels of MMP1 expression by increasing H3K9me2 levels at the promoter region of MMP1 29. Our study demonstrates that IOX-1 reduces levels of MMP2, MMP3, and MMP9 expression. It is possible that IOX-1 reduces those MMP levels by modulating histone H3 methylation levels at the MMP promotor regions.
Chemotherapy resistance in OS has been linked to the upregulation of ABC transporters, which increase the efflux of chemotherapy drugs from a variety of cancer cells 30. In particular, the upregulation of three ABC transporters (ABCB1, ABCC1 and ABCG2) in both CSCs and tumor cells highlights their importance as candidate therapeutic markers in OS; ABCB1 and ABCC1 are most strongly associated with chemotherapy resistance in OS cells 21. Similarly, our study found elevated levels of these ABC transporters in MG63-CR cells. With reference to our evidence demonstrating that IOX-1 downregulates ABC transporters, we speculate that cisplatin may then accumulate to toxic levels and thus ‘sensitize’ MG63-CR cells to this chemotherapy.
IOX-1 30 µM effectively reduced MG63-CR cell viability, but had no such effect upon MG63 cell viability. One potential explanation is that IOX-1 may decrease the viability of MG63-CR cells by inhibiting programmed death-ligand 1 (PD-L1), which is expressed by tumor cells and binds with programmed death-1 (PD-1) on cytotoxic cells to stop immunogenic cancer cell death 31. Research suggests that immunogenic cell death may be stimulated by findings showing that IOX-1 inhibits KDM3A, which downregulates β-catenin and ultimately reduces PD-L1 expression 31. Moreover, IOX-1-induced downregulation of β-catenin inhibits P-glycoprotein (another ATP-dependent efflux transporter), which increases intracellular doxorubicin levels and induces cancer cell death 31.
A potential mechanism behind IOX-1 may be its inhibition of the CSC regulator, SOX2 25. Our study found that shRNA inhibition of SOX2 was associated with a wide range of metastatic markers, including the downregulation of ABC transporters and corresponding increases in cisplatin sensitivity of MG63-CR cells. SOX2 inhibition also reduced levels of metastatic markers MMP-9, N-cadherin, vimentin, SNAIL, and TWIST in MG63-M10 cells. The efficacy of IOX-1 is enhanced by its targeting of upstream metastatic regulators such as SOX2. Another potential mechanism that may explain the ability of IOX-1 to downregulate metastatic markers in OS may operate via the Wnt pathway. Recent evidence has shown that IOX-1 can reduce colorectal cancer tumorigenesis by suppressing the Wnt pathway 32. By inhibiting KDM3, IOX-1 prevents the demethylation of H3K9 at Wnt target gene promotor sites 32. Moreover, by inhibiting the Wnt pathway, IOX-1 suppresses cancer stem cell properties in colorectal tumor cells 32, which is similar to the findings of this study. Further studies are required to verify whether the effects of IOX-1 are mediated via SOX2, PD-1/PD-L1, P-glycoprotein, the Wnt pathway, or some other mechanism.