Introduction

Lung cancer is the leading cause of cancer-related deaths worldwide, accounting for approximately 25% of all cancer fatalities (Brody 2020; Nasim et al. 2019). Lung cancers can be classified into two main types: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), with NSCLC being the most prevalent, comprising about 85% of all cases (Chen et al. 2014). Within NSCLC, lung adenocarcinoma (LUAD) is the most common subtype, occurring frequently among non-smokers (Ma et al. 2023; Yang et al. 2020). Despite advancements in early detection and targeted therapies, the prognosis for LUAD remains poor due to its high potential for recurrence, metastasis, and drug resistance (Schenk et al. 2020; Uprety et al. 2024). Therefore, a more comprehensive understanding of the molecular mechanisms underlying LUAD tumorigenesis and progression, exploring the target molecules associated with poor prognosis of LUAD is crucial for developing more effective diagnostic and therapeutic strategies.

Ribosomal proteins play essential roles in protein synthesis by forming the core structural and functional components of ribosomes. Among these, the ribosomal P complex, formed by RPLP0, RPLP1 and RPLP2, is a key component of the ribosomal stalk, which is crucial for the interaction between ribosomes and translation factors during protein synthesis (Artero-Castro et al. 2015). Recent studies have indicated that RPLP0, RPLP1 and RPLP2 play essential roles in a various type of cancer development and progression (Artero-Castro et al. 2011a, b). For instance, RPLP0 has been associated with prostate cancer (Pérez-Gómez et al. 2023), in addition, RPLP0 is expected to become a new biomarker for the treatment, diagnosis and prognosis of hepatocellular carcinoma. While RPLP1 is overexpressed in multiple cancers and exerts an oncogenic function (Zhen et al. 2023; Xie et al. 2021; Xia et al. 2020; He et al. 2018; Du et al. 2023), among them, RPLP1 is upregulated in triple-negative breast cancer (TNBC) tissues and cells, and high expression levels are associated with increased risk of recurrence and metastasis. In addition, high expression of RPLP1 is associated with poor prognosis and is an independent prognostic marker in TNBC. Additionally, RPLP2 has been associated with the progression of hepatocellular carcinoma and the development of gynecologic tumors, elevated RPLP2 is closely related to advanced clinicopathological features and predicts poor prognosis in hepatocellular carcinoma patients (Artero-Castro et al. 2011a, b; Guo et al. 2023; Yang et al. 2023). However, up to now, no studies have shown the role of RPLP0, RPLP1, and RPLP2 in LUAD, and the prognosis of these proteins with LUAD remains unclear.

In our study, we utilized public resources to explore the function of RPLP0, RPLP1 and RPLP2 in LUAD. We initially examined their mRNA and protein expression in LUAD. Next, we assess the relationship between the survival of LUAD patients and RPLP0, RPLP1 or RPLP2 expression. Subsequently, we analyzed the impact of copy number variation and promoter methylation levels on the expression of these ribosomal proteins. Furthermore, we evaluated the correlation between their expression and the immune infiltration of LUAD. Finally, we screened the drugs that may affect the expression of these ribosomal proteins. In our study, we reported for the first time of the prognostic value of RPLP0, RPLP1 and RPLP2 in LUAD by bioinformatics analysis, high expression of RPLP0, RPLP1, or RPLP2 indicates a poor prognosis in patients with LUAD. RPLP0, RPLP1 and RPLP2 are overexpressed in LUAD, which are likely attributed to abnormal copy number variations and decreased methylation levels of their promoters. Our study aims to provide new target molecules for clinical treatment and prognosis prediction of LUAD.

Materials and methods

Analysis of RPLP0, RPLP1 and RPLP2 expression in LUAD with various clinical features

The expression of RPLP0, RPLP1 and RPLP2 mRNA in various cancers was examined using the Tumor Immune Estimation Resource (TIMER) database (Li et al. 2017). The differential expression of RPLP0, RPLP1 and RPLP2 mRNA was evaluated using The University of ALabama at Birmingham CANcer Data Analysis Portal (UALCAN) (Chandrashekar et al. 2022), DriverDBv4 (Liu et al. 2024) and TNMplot (Bartha and Győrffy 2021). The protein expression was determined by the Clinical Proteomic Tumor Analysis Consortium (CPTAC) feature of UALCAN. The association between the expression of these ribosomal proteins and LUAD patient outcomes with indicated clinical features was explored by UALCAN. The correlation among the expression of RPLP0, RPLP1 and RPLP2 was measured by TIMER, Gene Expression Profiling Interactive Analysis (GEPIA) (Tang et al. 2017) and cBioPortal (Gao et al. 2013).

Survival analysis

We utilized the Kaplan-Meier plotter platform (Győrffy 2024) to identify the correlation between the clinical outcomes of LUAD patients and RPLP0, RPLP1 or RPLP2 levels.

Copy number variation (CNV) and promoter methylation level analysis

cBioPortal and Gene Set Cancer Analysis (GSCA) (Liu et al. 2023) were employed to investigate the impact of CNV and promoter methylation level on the expression levels of RPLP0, RPLP1 or RPLP2.

Immune infiltration analysis

We assessed the association between RPLP0, RPLP1 or RPLP2 expression and immune cell infiltration status using the Integrated Repository Portal for Tumor-Immune System Interactions (TISIDB) (Ru et al. 2019) and GSCA.

Drug sensitivity analysis

We analyzed the correlation between the expression levels of RPLP0, RPLP1 or RPLP2 and drug sensitivity to identify potential inhibitors for these ribosomal proteins. This analysis was conducted using GSCA, based on data from the Genomics of Drug Sensitivity in Cancer (GDSC) database (Yang et al. 2013).

Statistical analysis

All statistical analyses were performed by the indicated software or databases. p < 0.05 is considered statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001. Information of all databases used in the article is supplemented in Table S1.

Results

RPLP0, RPLP1 and RPLP2 are overexpressed in LUAD

To explore the expression of RPLP0, RPLP1 and RPLP2 in various cancers, we performed a pan-cancer analysis using the TIMER database. The results revealed that RPLP0 exhibits significantly higher expression in 15 types of cancers relative to the corresponding control tissues, while overexpression of RPLP1 and RPLP2 is detected in 14 and 13 types of cancers, respectively. Notably, RPLP2 expression is downregulated in breast cancers compared to noncancerous breast tissues (Figure S1A-S1C). In LUAD, the expression of all three ribosomal proteins was upregulated (Figure S1A-S1C). To verify this finding, we utilized three additional bioinformatic tools: UALCAN, DriverDBv4, and TNMplot, based on the transcriptomic analysis data of LUAD. The results showed that the mRNA levels of RPLP0, RPLP1 and RPLP2 are indeed upregulated in LUAD samples (Fig. 1A, S2A and S2B). Furthermore, overexpression of these ribosomal proteins was observed in all LUAD samples, regardless of the individual cancer stages, nodal metastasis status or p53 mutations (Fig. 1B and D; Table 1). Consistent with these data, LUAD samples also have higher protein levels of RPLP0, RPLP1 and RPLP2 compared to control tissues (Fig. 2A and C; Table 2). Additionally, the overexpression of these ribosomal proteins exhibits a positive correlation in LUAD (Fig. 3A and C). Taken together, these results demonstrate that RPLP0, RPLP1 and RPLP2 are overexpressed in LUAD, indicating that the dysregulation of their expression may contribute to LUAD development and progression.

Fig. 1
figure 1

The mRNA expression of RPLP0, RPLP1 and RPLP2 is upregulated in LUAD. (A) The result from the UALCAN database shows that RPLP0, RPLP1 and RPLP2 mRNAs are overexpressed in LUAD tissues. (B–D) UALCAN analysis data indicate that overexpression of these ribosomal proteins is present in LUAD tissues independent of their individual cancer stages (B), nodal metastasis status (C) or p53 mutations (D)

Full size image
Table 1 P-values for mRNA expression comparisons
Full size table
Fig. 2
figure 2

RPLP0, RPLP1 and RPLP2 protein levels are elevated in LUAD. (A) The outcome from the UALCAN database reveals that RPLP0, RPLP1 and RPLP2 protein levels are increased in LUAD tissues. (B and C) UALCAN analysis results indicate that overexpression of these ribosomal proteins is identified in LUAD tissues regardless of their individual cancer stages (B) or tumor grades (C)

Full size image
Table 2 P-values for protein expression comparisons
Full size table
Fig. 3
figure 3

RPLP0, RPLP1, and RPLP2 expression are positively correlated in LUAD. (A–C) Correlation analysis by TIMER (A), GEPIA (B) and cBioPortal (C) uncovers a positive association between the expression of RPLP0 and RPLP1, RPLP0 and RPLP2, as well as RPLP1 and RPLP2

Full size image

Excessive RPLP0, RPLP1 and RPLP2 are linked to increased CNV and reduced promoter methylation levels

To elucidate the mechanisms underlying the overexpression of RPLP0, RPLP1 and RPLP2, we analyzed their CNV and promoter methylation levels using cBioPortal and GSCA. Spearman correlation analysis outcomes revealed that CNV is positively correlated with the expression of RPLP0 (cor = 0.39, FDR = 8.8e-19), RPLP1 (cor = 0.33, FDR = 7.6e-14), and RPLP2 (cor = 0.24, FDR = 1e-07) (Fig. 4A). Moreover, the expression of these genes is lower in samples with shallow deletions compared to those with diploid or gain (Fig. 4B). Conversely, the methylation levels of the promoters of these ribosomal proteins are negatively associated with their mRNA levels (Fig. 4C and D), fitting with the well-recognized notion that increased methylation of promoters inhibits gene expression (Dhar et al. 2021). These observations suggest that both genetic and epigenetic alterations contribute to the overexpression of RPLP0, RPLP1 and RPLP2 in LUAD tissues.

Fig. 4
figure 4

The levels of CNV and promoter methylation of RPLP0, RPLP1 and RPLP2 are dysregulated in LUAD. (A) cBioPortal analysis data show that the expression of RPLP0, RPLP1 and RPLP2 is positively linked to CNV levels. (B) GSCA data indicating the expression of RPLP0, RPLP1 and RPLP2 in LUAD samples with shallow deletion, diploid and gain. (C and D) cBioPortal (C) and GSCA (D) outcomes suggest that RPLP0, RPLP1 and RPLP2 expression is negatively associated with promoter methylation levels in LUAD

Full size image

Overexpression of RPLP0, RPLP1 and RPLP2 predicts poor clinical outcome of LUAD patients

To assess the prognostic value of RPLP0, RPLP1 and RPLP2 expression in LUAD, we conducted Kaplan-Meier survival analysis. The results revealed that LUAD patients expressing high levels of these genes had worse clinical outcomes in terms of overall survival (OS), first progression (FP) and post-progression survival (PPS) compared to those expressing low levels of RPLP0, RPLP1 or RPLP2, indicates a poor prognosis (Fig. 5A and C). This finding indicates that RPLP0, RPLP1 and RPLP2 may function as oncogenes in LUAD.

Fig. 5
figure 5

High expression of RPLP0, RPLP1 and RPLP2 is associated with poor survival of LUAD patients. (A–C) Kaplan-Meier plotter data show that LUAD patients with a high expression of RPLP0 (A), RPLP1 (B) or RPLP2 (C) had a worse overall survival (OS), first progression (FP) and post-progression survival (PPS). OS was defined as the time from randomization to death (for any causes). FP was defined as the time between the initiation of randomization and the first occurrence of tumor progression (any aspect). PPS was defined as the time between the occurrence of tumor progression (any aspect) to death (for any causes)

Full size image

RPLP0, RPLP1 and RPLP2 overexpression significantly correlates to immune infiltration of LUAD

To explore the effect of RPLP0, RPLP1 and RPLP2 expression on the immune infiltration status of LUAD, we performed analyses using TISIDB and GSCA. The results from TISIDB revealed high expression of these ribosomal genes across five immune subtypes (Fig. 6A). Next, we assessed the correlation between the expression of RPLP0, RPLP1 or RPLP2 and various infiltrative immune cell populations. GSCA analysis uncovered significant correlations between their expression and the infiltration of multiple types of immune cells (Fig. 6B and C and S3-S5). Specifically, while both RPLP1 and RPLP2 expression show the most significant positive association with CD8 + T cell infiltration and a negative association with iTreg infiltration, RPLP0 expression exhibits the most significant positive association with nTreg infiltration and a negative association with CD4 + T cell infiltration, indicating their slightly differential influence on infiltrative immune cell populations (Fig. 6C). These data collectively suggest that the overexpression of these ribosomal proteins actively affects immune cell infiltration into LUAD tissues.

Fig. 6
figure 6

RPLP0, RPLP1 and RPLP2 expression affects immune cell infiltration into LUAD tissues. (A) TISIDB analysis depicting the expression of RPLP0, RPLP1 and RPLP2 in different C subtypes. (B) GSCA analysis data showing the correlation between the expression of RPLP0, RPLP1 and RPLP2 and infiltration of various immune cells. (C) GSCA analysis results illustrate the significant correlation between the expression of RPLP0, RPLP1 or RPLP2 and the indicated infiltrative immune cell populations in LUAD

Full size image

The expression of RPLP1 and RPLP2 may be repressed by multiple existing drugs

Given the potential oncogenic role of RPLP0, RPLP1 and RPLP2 in LUAD, we aimed to screen drugs that may downregulate their expression. We employed the GSCA platform and the integrated GDSC drug sensitivity and expression correlation plugin. The results uncovered dozens of drugs that may target RPLP1 and RPLP2, but not RPLP0 (Fig. 7). These drugs include common anti-cancer chemicals like 5-Fluorouracil, Methotrexate, and Vorinostat (Fig. 7). Interestingly, compared with RPLP1, the screened drugs show more robust inhibitory effects on RPLP2 expression, indicating that RPLP2 may be a better target for anti-cancer treatment by these drugs. Thus, RPLP2 is highly sensitive to these drugs, followed by RPLP1, while RPLP0 is less sensitive to these drugs.

Fig. 7
figure 7

Existing drugs show potential inhibition of RPLP1 and RPLP2 expression. GSCA analysis outcomes showing the top 30 drugs that may exhibit inhibitory effects on the expression of RPLP1 and RPLP2

Full size image

Discussion

Protein synthesis is an essential and highly coordinated process facilitated by ribosomes. Ribosomal proteins, as critical components of ribosomes, are crucial for proper cellular function. When their expression and function are deregulated, it can lead to abnormal cell survival, growth and proliferation (Jiao et al. 2023). Malignant cells, with their high proliferation rates and active metabolism, often overexpress ribosomal proteins to meet increased demands for protein synthesis (Khoury and Nasr 2021). Among these, RPLP0, RPLP1 and RPLP2 have been found to be overexpressed in multiple cancer types as mentioned above. Our pan-cancer analysis further supports this observation, revealing elevated levels of these proteins across various cancers.

The mechanisms driving the overexpression of RPLP0, RPLP1 and RPLP2 in LUAD are not fully understood. Our data indicate significant associations between their high expression levels and both CNV and promoter methylation, suggesting that genomic alterations and epigenetic modifications may contribute to their upregulation. However, post-transcriptional and post-translational regulators could also play a role. For example, miR-4731-5p acts as a tumor suppressor in NSCLC by targeting RPLP0, implying that dysregulation of such miRNAs might lead to RPLP0 overexpression in LUAD (Chang and Xu 2022). Therefore, dysregulation of the factors that control the expression of miR-4735-5p may also cause RPLP0 overexpression in LUAD. Further investigations are required to explore these possibilities.

RPLP0, RPLP1 and RPLP2 are recognized for their oncogenic potential in various contexts. Consistent with this, our survival analysis shows that high expression of these ribosomal proteins correlates with poor prognosis in LUAD patients. At present, many studies have shown the prognostic value of RPLP0, RPLP1 and RPLP2 in tumorigenesis. In this study, LUAD patients with high expression of RPLP0, RPLP1 or RPLP had worse clinical prognosis in terms of OS, FP and PPS. These results highlight the importance of RPLP0, RPLP1 and RPLP2 as clinical prognostic molecules in LUAD, and detection of the expression of these proteins has certain reference significance for prognosis prediction. While their prognostic value is evident, the precise role of RPLP0, RPLP1 and RPLP2 in LUAD progression remains unclear. Our findings suggest that these proteins may influence immune cell infiltration within tumors, highlighting their regulatory roles in the immune microenvironment.

Although RPLP0, RPLP1 and RPLP2 form a complex, our correlation analysis data show that their expression is significantly linked to each other in LUAD. However, their expression seems to differentially affect infiltrative immune cell populations. Our bioinformatic analysis reveals positive correlations between the expression of RPLP0, RPLP1 or RPLP2 and infiltrative CD8 + T cells and effector memory T cells. This suggests that RPLP0, RPLP1 or RPLP2 might enhance anti-tumor immune responses, as these T cells are known to repress tumorigenesis (Pages et al. 2005; Durgeau et al. 2018). Conversely, the expression of these ribosomal proteins negatively correlates with infiltrative CD4 + T cells and natural killer cells, which also play roles in anti-cancer functions (Chu et al. 2022; Tay et al. 2021). These contradictory effects on immune cell infiltration make it premature to definitively assess their roles in regulating the tumor immune microenvironment. Functional assays using LUAD cellular and animal models are necessary to determine the impact of RPLP0, RPLP1 and RPLP2 on immune cell infiltration in LUAD. Immunotherapy has become a major breakthrough in the field of cancer treatment. In-depth understanding of the interaction between tumor and immune infiltration and exploring the molecular mechanism of immune response are of great significance for improving the effect of immunotherapy. Studies have shown that the tumor microenvironment rich in T cells, especially CD8 + T cells, generally predicts a favorable response to immune checkpoint inhibitor therapy. Regulation of proteins targeting these immune infiltrating cells is essential for the treatment of diseases. In addition, comprehensive analysis of the immune subtypes of tumors can more accurately predict the response of patients to specific immunotherapy, and then guide more personalized treatment strategies.

Given the potential oncogenic roles of RPLP0, RPLP1 and RPLP2, targeting these proteins with therapeutic agents could be beneficial in treating LUAD. Through drug screening, we identified several compounds that inhibit the expression of RPLP1 and RPLP2, though they have minimal impact on RPLP0. Interestingly, the inhibition of RPLP2 by these drugs is more robust compared to RPLP1. This discrepancy underscores the need to better understand the regulatory mechanisms governing the expression of these ribosomal proteins and which is necessary to guide the specific targeted therapy of tumors. Moreover, the efficacy of these compounds must be validated through experimental studies beyond our computational predictions.

Despite the comprehensive nature of our bioinformatic analysis, this study has limitations, such as a limited cohort size and the lack of experimental validation. Further research is essential to confirm our findings and to provide a more detailed understanding of the roles of RPLP0, RPLP1 and RPLP2 in LUAD.

Conclusion

In summary, our study reveals that RPLP0, RPLP1 and RPLP2 are overexpressed in LUAD and negatively associated with the clinical outcomes of LUAD patients. The oncogenic potential of these ribosomal proteins underscores their value as prognostic biomarkers and therapeutic targets, potentially aiding in the diagnosis, prediction, and treatment of LUAD.