Metastasis at the advanced stage is the leading cause of HCC-related mortality[22]. Despite landmark progress in HCC diagnosis in recent years, the prognosis of HCC patients remains poor[23]. Thus, identification of novel biomarkers is critical to enhance individualized therapies. RPLP2, an integral part of ribosomal stalk, plays a critical role in promoting translation of subsets of cellular mRNAs[24]. Many studies indicate that RPLP2 has been linked closely to the tumorigenesis, progression and malignant behavior of various cancers such as breast, lung and ovarian cancers[19, 20]. However, the specific role of RPLP2 in HCC has not been studied before. Here, we systematically explored the expression profile, clinicopathological, prognostic and diagnostic significance, DNA methylation level, immunomodulatory effects, the effect on ferroptosis and potential targeted therapy strategies of RPLP2 in HCC.
As a candidate oncogene, RPLP2 is overexpressed and closely associated with occurrence and progression of various cancers including but not limited to breast, ovarian, colon, lung cancer and AML[18–21], while there is no reports about the effect of RPLP2 on HCC. At present study, we first analyzed the expression level of RPLP2 in HCC. Using TCGA + GTEx, HCCDB, GEO and HPA, we found that RPLP2 mRNA was highly expressed in HCC compared with normal liver tissue, and the results of IHC and western blot further demonstrated it on the protein level. In addition, RPLP2 expression was correlated with clinical characteristics including age, histological grade, histological type, race, AFP levels, OS and tumor status, and logistic regression analysis showed that RPLP2 expression was significantly associated with age, race, histological grade, tumor status, weight and AFP levels. Moreover, Kaplan-Meier test demonstrated that HCC patients with elevated RPLP2 expression had undesirable OS, DSS and PFI, which was also exhibited in many clinical categories, according to subgroup survival analyses. The multivariate Cox regression analysis indicated high RPLP2 expression was an independent risk factor for OS and PFI in HCC. What’ more, the ROC curve analysis showed that RPLP2 had a great diagnostic performance for HCC.
DNA methylation, a common epigenetic mechanism, generally silencing gene expression by altering chromatin structure and DNA stability, plays a significant role in tumorigenesis[25, 26]. However, studies on the methylation level of RPLP2 in cancers are rare. To our knowledge, it has only been reported that RPLP2 is hypermethylated in CRC[27]. Here, we found that the high expression of RPLP2 in HCC may be linked to its promoter hypomethylation, and the survival analysis indicated that hypomethylated level of RPLP2 correlated significantly with the prognosis of HCC patients. Additionally, the meaningful correlation between RPLP2 expression and different immune subtypes, suggesting us to explore the function of RPLP2 in cancer deeply by targeting specific immune subtypes.
Tumor immune microenvironment (TIME), mainly composed of immune cells, plays a significant role in the occurrence and progression of cancer, which affects clinical prognosis and immunotherapy effect greatly[28, 29]. Previous study indicated that recombinant RPLP2 with Toll-like receptor 4 (TLR4) could induce the maturation and activation of DCs, and pulse tumor-specific antigen, to induce the activation of tumor-specific CD8IFN-γ T cells followed by tumor clearance[15]. In our study, we found that RPLP2 positively correlated with NK CD56 bright, TFH and Th2 cells, and especially NK CD56 bright cells, and strongly negatively correlated with neutrophils and Tcm cells, which was also verified by IHC results. NK CD56 bright cells have been proved to have high infiltration levels and promote tumorigenesis in breast, colorectal and lung cancers[30–32]. And Th2 cells was reported to be associated with immunosuppression and poor prognosis in various cancer types[33]. According to a recent study, strategies aimed at inducing Tcm in TLSs/E-TLSs combined with immune checkpoint inhibitors, might represent promising avenues for cancer treatment[34]. Conclusively, these results indicated that the elevated level of RPLP2 may affect the tumorigenesis of HCC by regulating the immune cell infiltration levels.
Ferroptosis, a newly defined form of iron-dependent non-apoptotic cell death caused by lipid peroxidation[35]. Triggering ferroptosis is considered to be a effective strategy on suppressing tumor progression[36, 37]. As a crucial regulator of ferroptosis, GPX4 plays a significant role of phospholipid hydroperoxides, and inactivation of GPX4 could accelerate the progression ferroptosis[38, 39]. Studies have demonstrated that the silencing of RPLP2 could lead to the accumulation of ROS in gynecological tumor[16], and RPLP2 has an obvious effect on the critical ferroptosis-related pathway “Oxidative Phosphorylation” in AML showed by GSEA analysis[21]. However, the specific role of RPLP2 in HCC has not been studied, and its molecular mechanism remains unclear. In this study, we first found that RPLP2 played a critical role in ferroptosis-related pathways including “Oxidative Phosphorylation”, “Regulation of Lipid Catabolic Process”, “Iron Ion Homeostasis” and “WP Ferroptosis” using GSEA. Then the correlation analysis showed that RPLP2 significantly positively linked with GPX4 in HCC, which was further confirmed by western blot and IHC. Additionally, the CCK8 analysis indicated that inhibition of RPLP2 accelerated ferroptosis of HCC cells. Moreover, RPLP2 knockdown lead to the decrease of GSH and increase of lipid ROS. These results demonstrated that RPLP2 is an important regulator of ferroptosis, which could be a potential target to eradicate HCC cells.
Silencing of RPLP2 may result in inhibition of proliferation of several cancer types of cells[20, 40]. However, the concrete effect of RPLP2 expression on the malignant phenotype of HCC remains unclear. Here, we discovered RPLP2 knockdown significantly suppressed the proliferation, migration and colony formation of HCC cells via in vitro experiments, and xenograft nude mice model further proved that tumor growth was inhibited after knockdown of RPLP2. Combined with the role of RPLP2 in ferroptosis of HCC cells, these results suggest that RPLP2 could promote cell proliferation and tumor growth of HCC by inhibiting ferroptosis.
The key role of RPLP2 in HCC development prompts us to explore highly sensitive drugs that specifically target RPLP2. Using the RNAactDrug database[41], we noticed that some commonly used anticancer drugs such as, methylundecylpiperidine, trans, iyomycin b1, and destruxin b showed high sensitivity in patients with high RPLP2 mRNA levels. In addition, patients with elevated methylation level of RPLP2 were significantly sensitive to tp4ek-k6, Ibrutinib and indole-2,3-dione, 3-[(o-chlorophenyl)hydrazone]. These findings suggested that RPLP2 may have the potential to be targeted to bring new advances in HCC therapy after well-established preclinical and clinical trials.
Generally, our present study revealed that high expression of RPLP2 is an independent adverse prognostic factor in HCC, and is significantly correlated with aggressive clinical characteristics, tumor growth and inhibition of ferroptosis. Mechanically, RPLP2 positively links to anti-ferroptosis protein GPX4, which then protects cells from ferroptosis to promote the progression of HCC. Our findings suggest that targeting RPLP2 to promote the ferroptosis of HCC cells could be a potential therapeutic strategy for HCC patients. However, the concrete regulatory mechanism of RPLP2 on GPX4 has not been explored in this study. And although we found that RPLP2 is strongly associated with immune infiltration, the underlying molecular mechanism and signaling pathways are not further investigated.