OS is the commonly aggressive tumor in children and adolescents with poor survival rates. Recently, prognostic models for improving the prognosis of OS have attracted considerable attention. In this study, we constructed and verified the RMWs patterns and prognosis signature in OS and shed light on strophanthidin as a novel therapeutic drug for OS patients.
RNA adenosine modification is the most common type of RNA modification, and it was reported to involve various pathophysiological processes, including tumorigenesis15, 16. m6A modification is the RNA modification with dynamic and reversible post-transcriptional characters by affecting RNA processing, degradation and translation. Studies showed that the m6A modification levels were dysregulated in tumor tissues, which is associated with the occurrence and progression of various tumors 17 18 19 20. The “writers” play a key role in these RNA modifications, and some of the writers were reported as therapeutic targets of tumors. For example, METTL3, the m6A writer, has been related to the prognosis and progression of acute myeloid leukemia (AML), and the inhibition of METTL3 was reported as a therapeutic strategy for myeloid leukaemia21. A-to-I RNA editing is another abundant RNA modification event affecting adenosines in mammals, playing a critical role in the pathogenesis of various tumor 22. Xu et al. revealed the role of A-to-I-edited miRNA cancer progression and highlighted the translational potential of edited miRNAs as a new class of cancer therapeutics23. Moreover, other adenine-related RNA modifications (m1A methylation and APA) have recently also emerged as key players in cancer pathophysiology by regulating the expression of cancer-related genes24. In OS, m6A modification of YAP was involved in the progression of OS20. The relationship between other adenine-related RNA modification with OS remains unknown. RNA modification plays an important role in various biological progressions through interaction with various “writers” (RMWs). Recent studies have focused on RNA modification signatures to evaluate the prognosis of various tumors. Our study explored RMWs patterns and RNA modification DEGs patterns which were associated with survival of OS patients. Next, the RMWs prognostic signature was constructed and identified as an independent prognostic factor in OS. In conclusion, these results indicated the important role RMWs in OS.
The immune microenvironment (TME) was related to the prognosis of various cancers, including OS25–27. And the recruiting stromal cells were reported to be associated with a poor patient prognosis. Infiltrating myeloid cells are the common and important components of TME, and they are reported to drive OS progression28. Tumor-infiltrating myeloid cells include monocytes, dendritic cells (DCs), tumor-associated macrophages (TAMs), and neutrophils. In OS lesions, the monocytes and the macrophages are the most common myeloid cells, and DCs account for < 5% of myeloid cells29. M2 TAMs were anti-inflammatory macrophages, and often associated with a worse prognosis30. M1 TAMs are antitumor immune cells that express proinflammatory cytokines. Anti-PD-1 is an effective therapeutic strategy for OS due to the regulation of the infiltration of M1 and M2 macrophages in tumor tissue31. Recently, DCs were identified as therapeutic targets of immunotherapy due to their powerful antigen-presenting features. Zhou et al. showed that the infiltration of CD1c+ DC was significantly increased in metastatic OS29. In this study, we used xcell to reveal the correlations between risk signature and immune infiltration. We found that Macrophages M1, M2 and cDCs were significantly increased in the OS patients with high-risk. Recent studies shed new light on the regulation of RNA modification on the immune system32. Furthermore, RNA adenosine modifications have been associated with immunoregulation in tumor tissues32. METTL3-mediated m6A modification was reported to promote tumor growth and metastasis by macrophage reprogramming33. METTL3-mediated m6A modification also affect resistance to chemotherapy by regulating M2-TAMs infiltration34. METTL3-mediated m6A methylation regulates dendritic cell activation35. These results highlighted that the RNA adenosine modifications might affect prognosis of OS partly by regulating immune microenvironment.
DGIdb and CMap, the drug prediction database, are widely used to screen drugs that could regulate certain target genes. In this study, we utilized these two databases to obtain the possible RMWs-related drugs for OS. 10 drugs were identified, including dyclonine, resveratrol, zaprinast, abamectin, thioguanosine, cefotaxime, mesoridazine, clioquinol, physostigmine and strophanthidin. Dyclonine, an ALDH3A1 inhibitor, was widely used as a topical antipruritic agent36. Recent studies reported that dyclonine could enhance MG132-induced cell cytotoxic on breast cancer37. Resveratrol, an estrogenic, was reported as a treatment for osteoporosis with anti-inflammatory and antioxidant properties38. Recent studies also revealed the therapeutic potentials of resveratrol on various tumors, including OS39–41. Zaprinast, a synthetic GPR35 agonist, was reported to rescue OVX-induced bone42. It also has an anti-tumor character in colon cancer, lung cancer, and melanoma 43–45. Abamectin, an anthelmintic agent in animals, was reported to induced oxidative stress in cerebral and hepatic tissues46. Thioguanosine has anti-tumor activities for hepatocellular carcinoma 47. Mesoridazine, an antipsychotic drug, has cardiac conduction side-effects with fatal consequences in patients48. Clioquinol has been reported as an anti-tumor drug for OS48. Physostigmine has been reported to repress pancreatic cancer growth with an anticholinergic toxicity49. Strophanthidin, a cardiac glycoside, was reported as a promising anticancer agent by regulating cell cycles of breast, lung and liver cancer cells13. It is also an immune cell activator by activating ROR T receptors14. So, we selected strophanthidin for further functional analysis. In this study, vivo and vitro experiments showed that strophanthidin elicited the anti-tumor activity against OS via repressing cell proliferation and cell cycle. As shown in Fig. 8, Smad3 was predicted as the target of strophanthidin. Smad3 is a key mediator of TGF-β signalling, an important signal pathway in tumorigenesis50. Smad3 was also reported to involve in the regulation of cell cycle via induction of CDK inhibitors51, 52. Based on the above research, we speculate that strophanthidin regulates cell cycle of OS cells via targeting Smad3.
In summary, we analyzed RNA modification “writers” (RMWs) in OS, and revealed a RMWs patterns and RMWs signature associated with cell cycle and immune infiltration, and strophanthidin was identified and verified as a candidate therapy for OS via repressing growth and cell cycle of OS cells.