We have previously demonstrated that after KRP-DOX injection, OS xenograft-bearing mice showed almost no dissociated DOX in their circulation. This indicated that normal tissues are protected from the toxic side effects of DOX in the KRP-DOX treatment group. Additionally, KRP-DOX could change the biodistribution pattern of the small molecule DOX from simply spreading into multiple organs to targeted accumulation through enhanced permeability in solid tumors and the retention effect (EPR effect). Binding to tumor cells is achieved through electrostatic interaction between the positively charged KRP and the cell’s negative surface charge, allowing KRP-DOX to be internalized by cancer cells and avoiding lysosomal degradation. KRP-DOX can also enter the nuclei of tumor cells, triggering their death (Yu et al., 2019).
In the present work, gene expression was analyzed in three groups of microarray data of OS tissues obtained from mice treated with KRP-DOX, DOX-HCl, or saline. Comparison of the KRP-DOX and DOX-HCl groups identified a total of 790 common DEGs, including 463 upregulated and 327 downregulated genes, with P < 0.05 and [log 2 FC] > 1. Subsequently, both upregulated and downregulated DEGs were subjected to GO term enrichment analysis for functional annotation. The GO analysis classified the DEGs into three groups: BP, CC, and MF. These analyses showed that the functions of the DEGs are related to ribosomes. Ribosome biogenesis is essential for the survival, growth, and proliferation of cells. Numerous studies have confirmed that the hyperactivation of ribosome biogenesis plays a vital role in the development and progression of cancer. Ribosome biogenesis is generally found to be more active in tumor cells due to the need to adapt to the increased metabolic demands of neoplastic cells. In addition, the ribosome acts as a central information hub, regulated by many oncogenic signaling pathways (Catez et al., 2019; Pelletier et al., 2018; Penzo et al., 2019). Several studies on OS have demonstrated an association between increased ribosomal biogenesis and poor prognosis(Montanaro et al., 2007). The DEG analysis showed that RPS6KA2 (one of the major DEGs) was closely related to ribosome biogenesis and, therefore, may be involved in the anti-tumor effect of the KRP-DOX complex(Gianferante et al., 2017; Kim et al., 2013; Mitchell et al., 2015).
The analysis of the interrelationships between pathways showed that most DEGs were associated with pathways related to ribosomes, glycolysis/gluconeogenesis, or biosynthesis of amino acids. The important role of increased ribosome biogenesis and protein synthesis in maintaining tumor cell growth and proliferation has been discussed previously in the literature (Prakash et al., 2019). Furthermore, recent studies have shown that tumorigenesis is driven not only by an increased number but also by modifications of ribosomes (de Las et al., 2014; Kim et al., 2017; Sulima et al., 2017). Consistent with the current results, the ribosomal pathway was found to be one of the most significant pathways in an OS polygenic interaction study. The KRP-DOX complex may inhibit the growth of OS by affecting ribosome biogenesis.
In addition, the analysis of differentially expressed genes has shown that RPS6KA2 is closely related to ribosome biogenesis and, therefore, may be involved in the anti-tumor effect of the KRP-DOX complex(Montanaro et al., 2007). In combination with a review of the literature, it was concluded that the downregulated RPS6KA2 was closely related to ribosomal processes. The RPS6KA2 gene encodes ribosomal protein S6 kinase A2, which is an important member of the ribosomal protein S6 kinase (S6Ks) family. Biochemical and genetic studies have established that S6Ks are major kinases responsible for the phosphorylation of ribosomal protein S6 (RPS6) in vivo(Pende et al., 2004). Studies of the phosphorylation levels of RPS6 in rps6ka2-deficient mouse cells have found that RPS6KA2 is an essential S6K responsible for the phosphorylation of RPS6(Chauvin et al., 2014).RPS6 is an important component of the 40S subunit of ribosomes, and RPS6 post-translational modification plays an important role in the biogenesis of ribosomes. In addition, studies have reported that over 75% of the transcription factors associated with ribosomal biogenesis are regulated by S6Ks(Orsolic et al., 2016), including Nop14, Nop56, Gar1, Rrp9, Rrp15, Rrp12, and Pwp2 nucleoli proteins. Thus, current studies suggest that RPS6KA2 gene expression plays an important role in ribosomal biogenesis. On the other hand, qRT-PCR and Western blot analysis verified that the expression of RPS6KA2 was significantly downregulated by KRP-DOX treatment in OS. The finding that the ribosomal biogenesis-related oncogene RPS6KA2 was differentially and significantly downregulated in the KRP-DOX group raises the hypothesis that the KRP-DOX complex may inhibit ribosome biogenesis by downregulating RPS6KA2, thus enhancing the anti-tumor effect. In the past decade, studies have indicated that the over-activation of ribosomal biogenesis plays an important role in tumor development, and drugs that inhibit ribosome biogenesis may provide a new means for tumor treatment(Orsolic et al., 2016). including Nop14, Nop56, Gar1, Rrp9, Rrp15, Rrp12, and Pwp2 nucleoli proteins. Thus, current studies suggest that RPS6KA2 gene expression plays an important role in ribosomal biogenesis. On the other hand, qRT-PCR and Western blot analysis verified that the expression of RPS6KA2 was significantly downregulated by KRP-DOX treatment in OS. The finding that the ribosomal biogenesis-related oncogene RPS6KA2 was differentially and significantly downregulated in the KRP-DOX group raises the hypothesis that the KRP-DOX complex may inhibit ribosome biogenesis by downregulating RPS6KA2, thus enhancing the anti-tumor effect. In the past decade, studies have indicated that the over-activation of ribosomal biogenesis plays an important role in tumor development, and drugs that inhibit ribosome biogenesis may provide a new means for tumor treatment (Ma et al., 2011; Park et al., 2016). The activation of RPS6KA2 leads to the phosphorylation of functionally diverse p90RSK substrates in the cytoplasm and nucleus, thereby promoting the occurrence and development of carcinomas. The members of the P90RSK family have been shown to be overexpressed or hyper-activated in several cancers including breast cancer, lung cancer, and OS. Given that RPS6KA2 increases cell apoptosis, enhances cell chemosensitivity, inhibits proliferation and migration, and suppresses tumor formation, this protein might be a potential target of biotherapy for OS(Hirashita et al., 2016; Poomakkoth et al., 2016). Additionally, the downregulation of RPS6KA2 affects the activity of human OS cells through inactivation of the AKT/mTOR signaling pathways (Qiu et al., 2016). Pathway cards revealed that the RPS6KA2 upstream of AKT/ mTOR is ERK1/2, and that downstream is mTORC1.
Thus, it is hypothesized that the KRP-DOX complex downregulates RPS6KA2, inhibiting the MAPK-RPS6KA2-mTOR signaling pathway, and consequently blocks ribosomal biogenesis, ultimately inhibiting tumor growth (Fig. 6).
In summary, RPS6KA2 is significantly associated with an anti-OS effect and can serve as a potential biomarker and therapeutic target for OS. Further studies are necessary to identify the specific molecular mechanisms by which RPS6KA2 affects the initiation and progression of OS.
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