Artificial Downregulation of Ribosomal Protein L34 Restricts the Proliferation and Metastasis of Colorectal Cancer by Suppressing the JAK2/STAT3 Signaling Pathway

Abstract

The highly conserved ribosomal protein L34 (RPL34) has been reported to play an essential role in the progression of diverse malignancies. RPL34 is aberrantly expressed in multiple cancers, although its significant in colorectal cancer (CRC) is currently unclear. Here, we demonstrated that RPL34 expression was higher in CRC tissues than in normal tissues. Upon RPL34 overexpression, the ability of proliferation, migration, invasion, and metastasis of CRC cells were significantly enhanced in vitro and in vivo. Furthermore, high expression of RPL34 accelerated cell cycle progression, activated the JAK2/STAT3 signaling pathway, and induced the epithelial-to-mesenchymal transition (EMT) program. Conversely, RPL34 silencing inhibited the CRC malignant progression. Utilizing immunoprecipitation assays, we identified the RPL34 interactor, the cullin-associated NEDD8-dissociated protein 1 (CAND1), which is a negative regulator of cullin-RING ligases. CAND1 overexpression reduced the ubiquitin level of RPL34 and stabilized RPL34 protein. CAND1 silencing in CRC cells resulted in a decrease in the ability of proliferation, migration, and invasion. CAND1 overexpression promoted CRC malignant phenotypes and induced EMT, and RPL34 knockdown rescued CAND1-induced CRC progression. In summary, our study indicates that RPL34 acts as a mediator, is stabilized by CAND1, and promotes proliferation and metastasis, in part, through the activation of the JAK2/STAT3 signaling pathway and induction of EMT in CRC.

INTRODUCTION

Colorectal cancer (CRC) is one of the most commonly diagnosed cancers in adults and ranks fourth among cancer-related death causes.¹ Besides family genetic factors, this phenomenon is also attributed to chronic stress, unhealthy lifestyle, and diet.² Although widespread CRC screening and surgical resection treatment have improved the survival of CRC patients, therapy for advanced CRC remains a challenge. The development in secondary resistance of standard therapies and metastatic disease results in nearly 50% of deaths in CRC patients with advanced disease.³ Discovery of new CRC targets and elucidation of related mechanisms are essential to improve the therapeutic outcome of CRC patients.

Ribosomal proteins (RPs) are important assembly components of the ribosome, and mutations in them result in ribosomopathies, which is associated with an increased risk of developing malignancies.⁴ In recent years, researchers have increasingly discovered that in addition to their role in ribosome biogenesis, RPs also exhibit some extraribosomal functions in other cellular processes.⁵ As a member of the L34E family of RPs, ribosomal protein L34 (RPL34) has been revealed to be involved in cellular functions such as cell proliferation,⁶ cell cycle,⁷ and biosynthesis.⁸ Notably, aberrant expression pattern of RPL34 has been described in various cancer entities, and RPL34 has both oncogenic and tumor suppressive properties in different malignancies.

For instance, Zhu et al. reported that overexpression of RPL34 inhibited cervical cancer cell proliferation and the ability to migrate and invade,⁹ while another study indicated that knockdown of RPL34 attenuated malignant proliferation of nonsmall cell lung cancer cells.¹⁰ In addition, RPs were shown to be degraded by the ubiquitin-proteasome system.^11,12 Whether RPL34 is involved in the process of CRC tumorigenesis and metastasis and its upstream regulatory mechanisms have not been elucidated.

Cullin-RING ligases (CRLs) are the largest group of E3 ubiquitin ligases involved in ubiquitin-proteasome system-mediated protein ubiquitination. CRLs are responsible for post-translational modifications of many targets with an essential role in regulating various biological processes, including cell cycle, apoptosis, and transcription.¹³ The cullin-associated NEDD8-dissociated protein 1 (CAND1) is a negative regulator of CRLs.¹⁴ During the process of neddylation, the deneddylation enzyme COP9 signalosome (CSN) is displaced and CRL is activated upon covalent binding of cullin to the ubiquitin-like protein NEDD8, and subsequently, the substrate is ubiquitinated. Similar to the ubiquitination process, the neddylation process is also reversible. Under the catalysis of CSN, NEDD8 is dissociated out of this binding.^14,15 Deneddylated cullin allows binding to CAND1, and is sequestered and maintained in an inactive state.¹⁶

In this way, CAND1 functions as a mediator that regulates protein degradation and stabilization.^17,18 Accumulated evidence highlights the importance of CAND1 in several human cancers.^16,19 However, the significance of CAND1 in CRC and the associated mechanisms remain to be determined. This study was to explore the function and mechanism of RPL34 in CRC and its regulatory relationship with CAND1 through gene interference.

MATERIALS AND METHODS

Gene interference

For upregulation of RPL34 or CAND1 expression, RPL34 or CAND1 overexpressing vector and corresponding negative control vector were constructed and transfected into CRC cells. Two knockdown approaches were used in this study. For most cell experiments, the siRNA pool was used for RPL34 or CAND1 gene silencing. shRNA plasmid targeting RPL34 with GFP (shRPL34) were used for in vivo experiments. Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) were used for transfection following the manufacturer's protocol. Stable transfected cells were selected with G418 (250 mg/mL; Invitrogen) for 1 week and then cultured in completed medium. Additional materials and methods for this study are described in Supplementary Methods and Supplementary Table S1. This study was approved by our institutional ethics committee (Shengjing Hospital of China Medical University; No. 2022PS1003K) and informed consent was waived.

RESULTS

Differentially expressed genes identification and function annotation

To investigate the differentially expressed genes (DEGs) between normal and CRC tumor tissues, the microarray data of three GEO datasets were analyzed. A total of 83 DEGs were identified (Fig. 1a), and their logFC values were presented in Fig. 1b. To explore the potential functions and related pathways of these DEGs, we performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis to obtain functional and pathway enrichment results. As indicated in Fig. 1c, GO analysis on ClueGO software revealed that the DEGs were mainly enriched in the cytosolic ribosome, cyclin-dependent protein kinase holoenzyme complex. Figure 1d listed the top 20 enriched GO terms, including structural constituent of ribosome, cytoplasmic translation, and amide biosynthetic process. KEGG analysis revealed enrichment pathways for these DEGs (Fig. 1e), most of which were associated with ribosome, cell cycle, and DNA replication pathways.

Figure 1.

DEGs identification and high expression of RPL34 in CRC. (a) Venn diagram illustrating the overlapped DEGs among three GEO datasets (GSE47063, left; GSE185770, right; GSE77953, below), with p < 0.05 and |log FC| > 1 as the DEGs screening criteria. (b) The heatmap showing the logFC values of 83 overlapping genes in the three datasets. (c) The GO analysis of the 83 DEGs using ClueGO software. (d) Joyplot showing the top 20 significantly enriched terms for GO analysis with the R package clusterProfiler. (e) KEGG pathway analysis for the overlapped DEGs by R package clusterProfiler. (f, g) The chord graph displaying the genes enriched in the top terms of GO and KEGG pathway. The left of the circle showed the enriched genes and the right of the circle represented the corresponding GO or KEGG pathway term. (h) Real-time PCR analysis of RPL34 expression in 30 paired CRC tissues and paracancerous tissues. (i) Western blot analysis of RPL34 expression in six paired CRC tissues and paracancerous tissues. (j) Representative images of IHC staining for RPL34 in distinct TNM stages of CRC. Scale bar, 50 μm. **p < 0.01. CRC, colorectal cancer; DEG, differentially expressed gene; FC, fold change; GEO, Gene Expression Omnibus; GO, Gene Ontology; IHC, immunohistochemistry; KEGG, Kyoto Encyclopedia of Genes and Genomes; RPL34, ribosomal protein L34; TNM, Tumor Node Metastasis.

The chord diagrams of GO and KEGG pathway illustrated the genes enriched in the top terms of GO and KEGG (Fig. 1f, g). Of note, many genes of the RPL family were enriched. In addition, we note that studies on the biological role of RPL34 in cancer have been gradually increasing recently. RPL34 is involved in multiple cellular functions, including cell proliferation, cell cycle, and biosynthesis. However, the role of RPL34 in CRC has not been reported, thus we preferred RPL34 as the target in this study.

Upregulation of RPL34 in CRC

The expression pattern of RPL34 in CRC progression was first evaluated in public databases. TIMER2.0 online tool was used to assess RPL34 expression in different types of cancer. As labeled in Supplementary Fig. S1a, RPL34 expression was remarkably elevated in COAD (colon adenocarcinoma). UALCAN portal revealed that RPL34 was highly expressed in colon cancer tissues both at transcriptional and translational levels, across cancer stages, nodal metastasis, and histological types (Supplementary Fig. S1b, c). Real-time PCR and Western blotting analysis demonstrated that RPL34 expression was increased in tumor tissues (T) compared to paracancerous tissues (P) (Fig. 1h, i).

The high RPL34 expression was associated with tumor node metastasis stages of CRC (Supplementary Table S2), and the representative images of immunohistochemistry (IHC) staining were displayed in Fig. 1j. Moreover, the expression of RPL34 was detected in five CRC cell lines (Supplementary Fig. S1d). We noticed that the RPL34 expression level was highest in SW620 cell line and lowest in SW480 cell line.

RPL34 promotes the proliferation of CRC cells

To investigate the biological functions of RPL34 in CRC cells, we downregulated the expression of RPL34 in SW620 cells using the siRNA-pool method and transfected RPL34 overexpression vector into SW480 cells. The transfection efficiency was confirmed by real-time PCR and Western blot. The mRNA and protein expression of RPL34 was significantly downregulated in SW620 transfected with siRPL34 (Fig. 2a), and significant upregulated in SW480 transfected with RPL34 overexpression vector. The impact of RPL34 on CRC cell proliferation was evaluated by Cell Counting Kit-8 (CCK-8), cell cycle, and 5-ethynyl-2′-deoxyuridine (EdU) assays. The viability of SW620 cells was reduced after silencing RPL34 (Fig. 2b), while the opposite results was true in SW480 cells after RPL34 overexpression.

Figure 2.

RPL34 promotes the proliferation and metastasis of CRC in vitro. (a) RPL34 knockdown or overexpression efficiency in CRC cells was verified by real-time PCR and Western blot analysis. (b) CRC cell proliferation was determined by CCK-8 assay. (c) The proliferation of CRC cells was monitored by EdU staining. Scale bar, 50 μm. (d) Cell cycle analysis of CRC cells on flow cytometry. Histogram showing the distribution of PI-positive stained cells in each phase. Statistics of the percentage of cells in each phase. (e) Wound healing assay to evaluate the migratory capacity of CRC cells, and cell migration ratio was calculated. Scale bar, 200 μm. (f) Transwell assay was used to assess cell invasion ability, and the invaded cells was counted. Scale bar, 100 μm. (g) Changes in the EMT-related protein levels were detected by Western blotting. *p < 0.05, **p < 0.01. CCK-8, Cell Counting Kit-8; EMT, epithelial-to-mesenchymal transition; PI, propidium iodide.

EdU-positive stained cells were decreased by RPL34 knockdown and increased by RPL34 overexpression, indicating that RPL34 accelerated CRC cells into the DNA replication phase of the cell cycle (Fig. 2c). The cell cycle distribution of CRC cells was analyzed by flow cytometry. RPL34-silenced SW620 cells were blocked in G1 phase, and fewer cells were in S phase (Fig. 2d). RPL34 overexpression had the opposite effect in SW480 cells. These findings support that RPL34 promotes proliferation of CRC cells.

Expression of RPL34 is associated with migration, invasion, and epithelial-to-mesenchymal transition of CRC cells

The ability of migration and invasion of CRC cells was reduced after downregulating RPL34 and enhanced after upregulating RPL34 (Fig. 2e, f). Considering that epithelial-to-mesenchymal transition (EMT) is a key process in tumor metastasis, the expression levels of EMT markers were detected. RPL34-silenced SW620 cells experienced an increase in the expression level of the epithelial marker E-cadherin and a decrease in the levels of the mesenchymal marker N-cadherin and the transcription factor ZEB1 (Fig. 2g; Supplementary Fig. S2a). In contrast, E-cadherin was decreased in SW480 cells with RPL34 overexpression, while N-cadherin and ZEB1 were increased. The above data demonstrated the potential of RPL34 to enhance the metastasis of CRC cells.

RPL34 contributes to the malignant progression of CRC via the JAK2/STAT3 signaling pathway

Given the close relationship between dysregulated ribosome biogenesis and tumor development,²⁰ the effect of RPL34 on ribosomal stress was assessed. As shown in Supplementary Fig. S2b, RPL34 knockdown decreased the expression of 18S rRNA and pre-rRNA, which are involved in ribosome biogenesis. RPL34 overexpression caused the opposite results, suggesting that RPL34 knockdown may reduce ribosome biogenesis. It is known that the activation of the JAK2/STAT3 signaling pathway is essential in CRC progression,^21,22 therefore we assessed whether it is involved in RPL34-mediated CRC. RPL34 silencing caused diminished expression of p-JAK2 and p-STAT3 in SW620 cells; it was upregulated in RPL34-overexpressed SW480 cells (Fig. 3a). To further block the JAK2/STAT3 signaling pathway, SW480 cells were treated with JAK2 inhibitor AG490 after transfection of RPL34 overexpression vector.

Figure 3.

RPL34 activates the JAK2/STAT3 signaling pathway during CRC cell proliferation and migration. (a) Effect of RPL34 on the JAK2/STAT3 signaling pathway was analyzed by Western blotting. (b) Cell proliferation of RPL34-overexpressed CRC cells treated with or without the JAK2/STAT3 signaling pathway inhibitor AG490. (c, d) Cell migration ability was determined in CRC cells with or without AG490 treatment. Scale bar, 200 μm. *p < 0.05, **p < 0.01.

The proliferation of SW480 cells was promoted by overexpression of RPL34 (Fig. 3b), while suppressed after incubation with AG490. Furthermore, AG490 treatment alleviated the enhanced cell migration ability induced by RPL34 (Fig. 3c, d). These results suggest that RPL34 promotes CRC proliferation and migration ability, may be in part, through activation of the JAK2/STAT3 signaling pathway.

RPL34 promotes CRC tumorigenesis and metastasis in vivo

As the above results demonstrate that RPL34 expression is associated with the acquisition of malignant phenotype in CRC cells, we next validated the biological function of RPL34 in vivo. Stably transfected CRC cells were constructed and injected subcutaneously into nude mice, and then the tumor growth was monitored periodically. As presented in Fig. 4a, xenograft tumors formed by shRPL34-transfected SW620 cells grew more slowly than those formed by shNC-transfected cells. The opposite trend was found in xenograft tumors formed by RPL34 overexpressing SW480 cells (Fig. 4b). Accordingly, RPL34 expression in xenograft tumors was suppressed by RPL34 knockdown and upregulated by RPL34 overexpression (Fig. 4c). IHC analysis confirmed that Ki67-positive staining cells were decreased in RPL34 knockdown tumors and increased in RPL34 overexpression tumors compared to the corresponding controls (Fig. 4d).

Figure 4.

RPL34 promotes CRC tumorigenesis and metastasis in vivo. (a, b) Photographs and tumor growth curves of xenograft tumors formed by RPL34 silencing SW620 cells and RPL34 overexpressing SW620 cells. (c) RPL34 protein expression in CRC cells-derived xenograft tumors. (d) Representative IHC images of Ki-67, p-JAK2, and p-STAT3 expression in xenograft tumors. Scale bar, 50 μm. (e) Western blot analysis of proteins in the JAK2/STAT3 signaling pathway. (f) RPL34 knockdown SW620 cells and RPL34 overexpressing SW480 cells were injected into the distal spleen of nude mice. Bioluminescence images taken at week 0 and 15 after CRC cell injection into mice. (g) Mice liver and spleen were photographed to observe CRC metastasis in liver. Arrows point to metastatic nodules on the liver surface. Scale bar, 1 cm. (h) The CRC metastatic nodes in the liver of mice were counted. (i) Representative H&E-stained sections of mouse liver. Metastatic lesions were marked with stars. Scale bar, 500 μm. **p < 0.01. H&E, hematoxylin-eosin.

In addition, RPL34 silencing resulted in a reduction in the expression of p-JAK2 and p-STAT3, whereas RPL34 overexpression showed the opposite effect (Fig. 4d, e; Supplementary Fig. S2c).

As with the tumorigenesis assay, we also performed investigations for the effect of RPL34 on CRC metastasis in a liver metastasis mouse model. Bioluminescence images displayed that liver metastasis was repressed by RPL34 silencing and facilitated by RPL34 overexpression (Fig. 4f). By macroscopic analysis, we found that RPL34 knockdown induced mice to form fewer liver tumor nodules, and vice versa for RPL34 overexpression (Fig. 4g, h). We further verified the occurrence of liver metastases by hematoxylin-eosin staining and confirmed that RPL34-knockdown cells formed fewer metastatic foci in the liver, in contrast to RPL34-overexpressing cells (Fig. 4i). Overall, these experiments provide in vivo evidence that RPL34 promotes tumor growth, JAK2/STAT3 signaling activation, and liver metastasis in CRC.

The regulatory between CAND1 and RPL34

We predicted the interactors of RPL34 using the GeneMANIA database and constructed a protein-protein interaction network using Cytoscape software (Fig. 5a). Based on the results of GO analysis (Fig. 5b), these genes were mainly enriched in protein ubiquitination, cytosolic ribosome, and cullin-RING ubiquitin ligase complex. KEGG pathway analysis revealed that these genes were associated with ubiquitin-mediated proteolysis, hedgehog signaling pathway, cell cycle, and pathways in cancer (Fig. 5c). These results demonstrated the potential involvement of these RPL34-associated genes in ubiquitin modifications and cancer. The proteomics-based research showed that CAND1 interacts with RPL34.²³ Thus, we further explored the interaction between CAND1 and RPL34 in CRC.

Figure 5.

CAND1 interacts with RPL34. (a) The PPI network of RPL34 based on GeneMANIA database analysis. (b) GO enrichment analysis for RPL34 interactors. (c) KEGG analysis for RPL34 interactors. (d, e) The RPL34 protein and mRNA expression were measured in CRC cells with or without CAND1 knockdown and overexpression. (f) Immunofluorescent double-staining to determine the expression of CAND1 and RPL34 in SW620 cells. Scale bar, 50 μm. (g) Co-IP assay was conducted to analyze the interaction between CAND1 and RPL34. (h) RPL34 expression was detected by Western blotting in CAND1-silenced cells with CHX (100 μg/mL) treatment. (i) Immunoprecipitation assays for ubiquitination levels of RPL34 in CAND1 knocked down and overexpressed cells. (j) RPL34 expression was detected by Western blotting in CAND1-silenced cells with and without MG132 treatment. **p < 0.01. CAND1, cullin-associated NEDD8-dissociated protein 1; CHX, cycloheximide; PPI, protein-protein interaction.

To explore the regulatory role of CAND1 on RPL34, the CAND1 was downregulated in SW620 cells and upregulated in SW480 cells by transfection with CAND1 siRNA or overexpression vectors (Supplementary Fig. S2d). RPL34 protein expression was decreased or increased upon CAND1 knockdown or overexpression (Fig. 5d). However, real-time PCR assays showed no significant changes in RPL34 mRNA expression by CAND1 knockdown or overexpression (Fig. 5e). The immunofluorescence double-staining assay revealed that RPL34 and CAND1 were coexpressed in CRC cells (Fig. 5f). Co-IP assay confirmed the binding of RPL34 and CAND1 (Fig. 5g). Therefore, we hypothesized that CAND1 may regulate the protein level of RPL34 through post-translational modifications. Then, whether CAND1 impacts the expression of RPL34 by regulating its protein stability was investigated.

Knockdown of CAND1 decreased the half-life of RPL34 protein in the presence of the protein synthesis inhibitor cycloheximide (Fig. 5h). Given the essential function of CAND1 in protein ubiquitination, we examined the impact of CAND1 on RPL34 ubiquitination level and found that CAND1 inhibited RPL34 ubiquitination degradation (Fig. 5i). Furthermore, downregulation of RPL34 caused by CAND1 silencing was upregulated in the context of proteasome inhibition by MG132 (Fig. 5j; Supplementary Fig. S2e). Our findings revealed that CAND1 upregulates RPL34 expression by inhibiting ubiquitination-mediated degradation of RPL34.

RPL34 is essential for CAND1-mediated malignant progression of CRC cells

We further investigated the expression and biological functions of CAND1 in CRC. The transcriptional and translational levels of CAND1 were both elevated in tumor tissues (Fig. 6a–c). CAND1 expression was higher in late stage than in early stage of CRC (Fig. 6d). The knockdown of CAND1 inhibited the proliferation of SW620 cells (Fig. 6e), as measured by CCK-8 assay. Following silencing of CAND1, the migratory and invasive ability of SW620 cells were detected to be reduced (Fig. 6f–i). Furthermore, the expression of p-JAK2 and p-STAT3 was upregulated by CAND1 overexpression and downregulated by CAND1 knockdown (Fig. 6j; Supplementary Fig. S2f). These results indicate that downregulation of CAND1 impairs the proliferation, the ability of invasion and migration, and the activation of the JAK2/STAT3 signaling pathway in CRC cells.

Figure 6.

CAND1 promotes the malignant progression of CRC via RPL34. (a) Gene expression of CAND1 in 30 pairs of CRC and paracancerous tissues. (b, c) Protein expression of CAND1 in 6 pairs of CRC and paracancerous tissues. (d) Representative images of IHC staining for CAND1 in distinct TNM stages of CRC. (e) SW620 cells were transfected with CAND1 siRNA, followed by detection of cell proliferation. (f, g) Would healing assay was conducted to evaluate CRC cell migratory capacity. (h, i) Invasive capacity of CRC cells was tested by transwell assay. (j) Western blot analysis of proteins in the JAK2/STAT3 signaling pathway. (k) Cell proliferation was examined in SW480 cells with CAND1 overexpression and RPL34 knockdown. (l–o) Migration and invasive capacity of SW480 cells. (p) The protein level of E-cadherin, N-cadherin, and ZEB1 in SW480 cells. *p < 0.05, **p < 0.01 ,^¶ P < 0.05, ^¶¶ P < 0.01. CRC, colorectal cancer.

To test whether RPL34 mediates the function of CAND1 on CRC progression, a rescue experiment was designed where CAND1 was overexpressed and RPL34 was knocked down in SW480 cells. Knockdown of RPL34 inhibited the enhanced proliferation of SW480 cells induced by CAND1 overexpression (Fig. 6k). CAND1 overexpression enhanced the ability of migration and invasion, while downregulation of RPL34 restored this trend (Fig. 6l–o). Knockdown of RPL34 reduced the occurrence of EMT induced by CAND1 overexpression, as evidenced by elevated E-cadherin expression, and decreased N-cadherin and ZEB1 expression (Fig. 6p; Supplementary Fig. S2g). All together, these findings suggest that RPL34 mediates the function of CAND1 on proliferation, metastasis, and EMT of CRC cells.

DISCUSSION

Most CRC patients develop distant metastatic disease, especially liver metastases, within 5 years of diagnosis of the primary tumor, which is the leading cause of death from CRC.^24,25 Therefore, it is crucial to better understand the mechanism of liver metastasis in CRC and to find new targets for suppressing the malignant progression of CRC. Our study reported that CAND1 interacted with RPL34 and stabilized RPL34 by inhibiting RPL34 ubiquitination degradation. RPL34 and CAND1 were overexpressed in CRC and enhanced cell proliferation and metastasis, while RPL34 knockdown reversed the malignant phenotypes and EMT processes in CRC induced by CAND1 overexpression. RPs are generally considered to be general-purpose cellular proteins dedicated to the maintenance of ribosome integrity.

However, accumulating evidence suggests that RPs, in addition to their role in ribosome biogenesis, exhibit a number of extra-ribosomal functions in other cellular processes and are involved in the development and progression of some cancers, such as proliferation, apoptosis, and chemoresistance.^26

–29 Altered expression of individual RPs may affect ribosome biogenesis, leading to the condition known as ribosomal stress. In response to this stress, some cytosolic RPs are transferred to the nucleoplasm as free forms of ribosomes to exert extraribosomal functions.⁵ For RPL34, it has been shown that RPL34 promote cell proliferation and reduce apoptosis to facilitate the progression of nonsmall cell lung cancer.¹⁰ Knockdown of RPL34 inhibited the proliferation and metastasis of esophageal cancer cells.³⁰ In addition, dysregulation of ribosome biogenesis is closely associated with the development and progression of cancer.²⁰ Cancer cell proliferation could be slowed down by reducing ribosome biogenesis.³¹

Our study suggests that knockdown of RPL34 may suppress the malignant phenotypes of CRC, in part, through reducing ribosome biogenesis. Considering that the role of RPs on ribosome biogenesis is widely recognized, our current work uncovered a novel mechanism regarding the extraribosomal function of RPL34, which is that knockdown of RPL34 inhibits CRC progression possibly, in part, through the JAK2/STAT3 signaling pathway. On the basis of these findings, selective modulators or inhibitors that control the activity or expression of RPL34 may be promising strategies for CRC treatment.

Abnormal expression of RPL34 has been reported in several tumor malignancies.^30,32 RPL34 was first shown to be oncogenic in nonsmall cell lung cancer, and RPL34 expression was higher in nonsmall cell lung cancer tissues than in paraneoplastic tissues.¹⁰ Knockdown of RPL34 inhibited cell proliferation by inducing apoptosis and cell cycle arrest.¹⁰ RPL34 was also significantly upregulated in glioblastoma, and its overexpression was significantly associated with poor survival in glioblastoma patients.³² Through downregulation of RPL34, the migratory and invasive ability of esophageal cancer cells was inhibited.³³ Consistent with these reports, our current study demonstrated that RPL34 is a potential oncogene in CRC. We found that RPL34 was abundantly expressed in CRC tissues, and that silencing of RPL34 caused impairments in cell proliferation, tumor growth, migration, and invasion, and induced cell cycle arrest.

An in vivo study showed that knockdown of RPL34 inhibited metastasis of pancreatic cancer cells.³⁴ Similar to this study, we observed that RPL34 promoted liver metastasis of CRC. In contrast to the above mentioned cancers, RPL34 acts as a tumor suppressor in cervical cancer.⁹ It implies the differences in the molecular regulatory mechanisms of RPL34 in different cancers.

Metastasis is a complex and multifactor triggered process and is the main cause of CRC-related death.³⁵ EMT is considered as one of the drivers of primary tumors to develop metastasis. During this process, cells lose the epithelial marker E-cadherin, gain mesenchymal markers, and induce different transcription factors expression.³⁶ The EMT program has been shown to enhance the migration, invasion, and metastatic potential in CRC cells.²² The present study suggested that after overexpressing RPL34, E-cadherin expression was decreased and N-cadherin and ZEB1 expression was increased, promoting migration and invasion of CRC cells. ZEB1 as an EMT-induced transcription factor, its deletion significantly reduced the invasiveness of highly aggressive tumor cells and intensely inhibited metastasis.³⁷

The importance of aberrant activation of the JAK2/STAT3 signaling pathway in promoting the progression of various tumors has been widely recognized. Activation of JAK2 and STAT3 signaling and its downstream pathways promote invasion and migration of gastric cancer cells.³⁸ Augmented JAK2/STAT3 signaling was found in CRC and was associated with poor prognosis of CRC patients.³⁹ In addition, repression of the JAK2/STAT3 signaling pathway resulted in restraint of the EMT process in lung cancer cells.⁴⁰ Given the significance of the JAK2/STAT3 signaling pathway, blocking it may be a promising strategy for CRC treatment. In the present study, RPL34 overexpression increased the expression of p-JAK2 and p-STAT3 in SW480 cells and its derived xenograft tumors, and the JAK2/STAT3 pathway inhibitor AG490 decreased the RPL34-mediated proliferation and migration of SW480 cells. It indicates that the JAK2/STAT3 signaling pathway is activated as downstream of RPL34, as previously reported,⁴¹ advancing the proliferation and metastasis of CRC.

Substantial evidence highlights the importance of CAND1 in many kinds of malignancies as an oncogene.^16,42,43 In recent years, many studies have identified that CAND1 is upregulated in various cancers and the high CAND1 expression is associated with the tumor progression.^44
–46 CAND1 exerts multidimensional functions in cancer, involving cell proliferation, apoptosis, cell cycle, cell migration, and signal transduction.^42,44 In our study, CAND1 expression was increased in CRC tissues and cells, and it promoted CRC cell proliferation, migration, invasion, and EMT, while silencing of RPL34 rescued this malignant progression. As substrate receptor exchange factor, CAND1 influences ubiquitination-dependent degradation of many proteins by regulating the assembly of Skp1-Cul1-F-box complexes.⁴⁷ CAND1 participates in tumorigenesis through regulation of protein stability. For example, CAND1 suppressed the ubiquitinated degradation of NRF2 and promoted gastric carcinogenesis.⁴⁶ CAND1 also prevented breast cancer-associated estrogen receptor α from proteasome-mediated degradation.⁴⁸ In this work, RPL34 was regulated by CAND1 and proved the ability of CAND1 to block the ubiquitin-proteasome system-mediated degradation of RPL34. Thus, we conclude that CAND1 exerts a tumor-promoting effect by stabilizing RPL34, and targeting CAND1 could be considered in the development of new drugs against CRC.

CONCLUSION

RPL34 was upregulated in CRC tissues and cells. RPL34 enhanced the ability of proliferation and metastasis of CRC cells in vitro and in vivo, in part, by inducing the EMT program and activating the JAK2/STAT3 signaling pathway. Furthermore, CAND1 was identified to stabilize RPL34 protein in CRC cells via deubiquitylation function. Blocking the CAND1/RPL34 axis and thus inhibiting the associated oncogenic pathways may be developed as an effective therapeutic strategy for CRC patients.

Footnotes

AUTHORs' CONTRIBUTIONS

Y.-C.L.: conceptualization, writing—original draft, formal analysis, writing—review and editing, and investigation; R.L. and S.-R.B.: investigation, formal analysis; and writing—review and editing; Z.-L.L.: investigation; H.-Z.Y.: conceptualization, supervision, and writing—review and editing. C.-L.D.: supervision and writing—review and editing.

AUTHOR DISCLOSURE

No competing financial interests exist.

FUNDING INFORMATION

No funding was received for this article.

SUPPLEMENTARY MATERIAL

Supplementary Methods

Supplementary Figure S1

Supplementary Figure S2

Supplementary Table S1

Supplementary Table S2

References

Dekker

, Tanis

, Vleugels

JLA

, et al. Colorectal cancer. Lancet, 2019; 394(10207):1467–1480; doi: 10.1016/S0140-6736(19)32319-0

Siegel

, Miller

, Fedewa

, et al. Colorectal cancer statistics, 2017. CA Cancer J Clin, 2017; 67(3):177–193; doi: 10.3322/caac.21395

La Vecchia

, Sebastián

. Metabolic pathways regulating colorectal cancer initiation and progression. Semin Cell Dev Biol, 2020; 98:63–70; doi: 10.1016/j.semcdb.2019.05.018

Kang

, Brajanovski

, Chan

, et al. Ribosomal proteins and human diseases: Molecular mechanisms and targeted therapy. Signal Transduct Target Ther, 2021; 6(1):323; doi: 10.1038/s41392-021-00728-8

Pecoraro

, Pagano

, Russo

, et al. Ribosome biogenesis and cancer: Overview on ribosomal proteins. Int J Mol Sci, 2021; 22(11):5496; doi: 10.3390/ijms22115496

Akanuma

, Nanamiya

, Natori

, et al. Inactivation of ribosomal protein genes in Bacillus subtilis reveals importance of each ribosomal protein for cell proliferation and cell differentiation. J Bacteriol, 2012; 194(22):6282–6291; doi: 10.1128/JB.01544-12

Devitt

, Stafstrom

. Cell cycle regulation during growth-dormancy cycles in pea axillary buds. Plant Mol Biol, 1995; 29(2):255–265; doi: 10.1007/BF00043650

Panagiotidis

, Huang

, Canellakis

. Relationship of the expression of the S20 and L34 ribosomal proteins to polyamine biosynthesis in Escherichia coli. Int J Biochem Cell Biol, 1995; 27(2):157–168; doi: 10.1016/1357-2725(94)00068-m

Zhu

, Ren

, Jiang

, et al. RPL34-AS1-induced RPL34 inhibits cervical cancer cell tumorigenesis via the MDM2–P53 pathway. Cancer Sci, 2021; 112(5):1811–1821; doi: 10.1111/cas.14874

10.

Yang

, Cui

, Yang

, et al. Over-expressed RPL34 promotes malignant proliferation of non-small cell lung cancer cells. Gene, 2016; 576(1 Pt 3):421–428; doi: 10.1016/j.gene.2015.10.053

11.

Watanabe

, Fujiyama

, Takafuji

, et al. GRWD1 regulates ribosomal protein L23 levels via the ubiquitin-proteasome system. J Cell Sci, 2018; 131(15):jcs213009; doi: 10.1242/jcs.213009

12.

Sung

M-K

, Reitsma

, Sweredoski

, et al. Ribosomal proteins produced in excess are degraded by the ubiquitin-proteasome system. Mol Biol Cell, 2016; 27(17):2642–2652; doi: 10.1091/mbc.E16-05-0290

13.

Nakayama

, Nakayama

. Ubiquitin ligases: Cell-cycle control and cancer. Nat Rev Cancer, 2006; 6(5):369–381; doi: 10.1038/nrc1881

14.

Zemla

, Thomas

, Kedziora

, et al. CSN- and CAND1-dependent remodelling of the budding yeast SCF complex. Nat Commun, 2013; 4:1641; doi: 10.1038/ncomms2628

15.

Lyapina

, Cope

, Shevchenko

, et al. Promotion of NEDD-CUL1 conjugate cleavage by COP9 signalosome. Science, 2001; 292(5520):1382–1385; doi: 10.1126/science.1059780

16.

Eigentler

, Tymoszuk

, Zwick

, et al. The impact of Cand1 in prostate cancer. Cancers (Basel), 2020; 12(2):428; doi: 10.3390/cancers12020428

17.

Nakashima

, Takeuchi

, Iwama

, et al. Cullin-associated NEDD8-dissociated protein 1, a novel interactor of rabphilin-3A, deubiquitylates rabphilin-3A and regulates arginine vasopressin secretion in PC12 cells. Endocr J, 2018; 65(3):325–334; doi: 10.1507/endocrj.EJ17-0399

18.

Kajiho

, Yamamoto

, Sakisaka

. CAND1 regulates lunapark for the proper tubular network of the endoplasmic reticulum. Sci Rep, 2019; 9(1):13152; doi: 10.1038/s41598-019-49542-x

19.

Tanaka

, Nakatani

, Kamitani

. Negative regulation of NEDD8 conjugation pathway by novel molecules and agents for anticancer therapy. Curr Pharm Des, 2013; 19(22):4131–4139; doi: 10.2174/1381612811319220017

20.

Bursać

, Prodan

, Pullen

, et al. Dysregulated ribosome biogenesis reveals therapeutic liabilities in cancer. Trends Cancer, 2021; 7(1):57–76; doi: 10.1016/j.trecan.2020.08.003

21.

Zhang

, Hu

, Li

, et al. Human colorectal cancer-derived mesenchymal stem cells promote colorectal cancer progression through IL-6/JAK2/STAT3 signaling. Cell Death Dis, 2018; 9(2):25; doi: 10.1038/s41419-017-0176-3

22.

Wei

, Yang

, Wang

, et al. Crosstalk between cancer cells and tumor associated macrophages is required for mesenchymal circulating tumor cell-mediated colorectal cancer metastasis. Mol Cancer, 2019; 18(1):64; doi: 10.1186/s12943-019-0976-4

23.

Bennett

, Rush

, Gygi

, et al. Dynamics of cullin-RING ubiquitin ligase network revealed by systematic quantitative proteomics. Cell, 2010; 143(6):951–965; doi: 10.1016/j.cell.2010.11.017

24.

, Yang

, Ma

, et al. Spatiotemporal immune landscape of colorectal cancer liver metastasis at single-cell level. Cancer Discov, 2022; 12(1):134–153; doi: 10.1158/2159-8290.CD-21-0316

25.

LeGolvan

, Resnick

. Pathobiology of colorectal cancer hepatic metastases with an emphasis on prognostic factors. J Surg Oncol, 2010; 102(8):898–908; doi: 10.1002/jso.21817

26.

, Ge

, Yin

, et al. Silencing expression of ribosomal protein L26 and L29 by RNA interfering inhibits proliferation of human pancreatic cancer PANC-1 cells. Mol Cell Biochem, 2012; 370(1–2):127–139; doi: 10.1007/s11010-012-1404-x

27.

Luo

, Zhao

, Fowdur

, et al. Highly expressed ribosomal protein L34 indicates poor prognosis in osteosarcoma and its knockdown suppresses osteosarcoma proliferation probably through translational control. Sci Rep, 2016; 6:37690; doi: 10.1038/srep37690

28.

Kim

J-H

, You

K-R

, Kim

, et al. Over-expression of the ribosomal protein L36a gene is associated with cellular proliferation in hepatocellular carcinoma. Hepatology, 2004; 39(1):129–138; doi: 10.1002/hep.20017

29.

Shen

D-W

, Liang

X-J

, Suzuki

, et al. Identification by functional cloning from a retroviral cDNA library of cDNAs for ribosomal protein L36 and the 10-kDa heat shock protein that confer cisplatin resistance. Mol Pharmacol, 2006; 69(4):1383–1388; doi: 10.1124/mol.105.017525

30.

Fan

, Li

, Jia

, et al. Silencing of ribosomal protein L34 (RPL34) inhibits the proliferation and invasion of esophageal cancer cells. Oncol Res, 2017; 25(7):1061–1068; doi: 10.3727/096504016X14830466773541

31.

Bublik

, Bursać

, Sheffer

, et al. Regulatory module involving FGF13, miR-504, and p53 regulates ribosomal biogenesis and supports cancer cell survival. Proc Natl Acad Sci U S A, 2017; 114(4):E496–E505; doi: 10.1073/pnas.1614876114

32.

, Wang

, Jia

, et al. Suppression of RPL34 inhibits tumor cell proliferation and promotes apoptosis in glioblastoma. Appl Biochem Biotechnol, 2022; 194(8):3494–3506; doi: 10.1007/s12010-022-03857-0

33.

Gong

, Li

, Cang

, et al. RPL34-AS1 functions as tumor suppressive lncRNA in esophageal cancer. Biomed Pharmacother, 2019; 120:109440; doi: 10.1016/j.biopha.2019.109440

34.

Wei

, Ding

, Wei

, et al. Ribosomal protein L34 promotes the proliferation, invasion and metastasis of pancreatic cancer cells. Oncotarget, 2016; 7(51):85259–85272; doi: 10.1863/2/oncotarget.13269

35.

Pantel

, Brakenhoff

. Dissecting the metastatic cascade. Nat Rev Cancer, 2004; 4(6):448–456; doi: 10.1038/nrc1370

36.

Pastushenko

, Blanpain

. EMT transition states during tumor progression and metastasis. Trends Cell Biol, 2019; 29(3):212–226; doi: 10.1016/j.tcb.2018.12.001

37.

Krebs

, Mitschke

, Lasierra Losada

, et al. The EMT-activator Zeb1 is a key factor for cell plasticity and promotes metastasis in pancreatic cancer. Nat Cell Biol, 2017; 19(5):518–529; doi: 10.1038/ncb3513

38.

Liu

, Li

, Zhang

, et al. RBMS1 promotes gastric cancer metastasis through autocrine IL-6/JAK2/STAT3 signaling. Cell Death Dis, 2022; 13(3):287; doi: 10.1038/s41419-022-04747-3

39.

Schulz-Heddergott

, Stark

, Edmunds

, et al. Therapeutic ablation of gain-of-function mutant p53 in colorectal cancer inhibits Stat3-mediated tumor growth and invasion. Cancer Cell, 2018; 34(2):298; doi: 10.1016/j.ccell.2018.07.004

40.

Wang

L-N

, Zhang

Z-T

, Wang

, et al. TGF-β1/SH2B3 axis regulates anoikis resistance and EMT of lung cancer cells by modulating JAK2/STAT3 and SHP2/Grb2 signaling pathways. Cell Death Dis, 2022; 13(5):472; doi: 10.1038/s41419-022-04890-x

41.

, Wang

, Liu

, et al. Knockdown of RPL34 inhibits the proliferation and migration of glioma cells through the inactivation of JAK/STAT3 signaling pathway. J Cell Biochem, 2019; 120(3):3259–3267; doi: 10.1002/jcb.27592

42.

Che

, Liu

, Zhang

, et al. Targeting CAND1 promotes caspase-8/RIP1-dependent apoptosis in liver cancer cells. Am J Transl Res, 2018; 10(5):1357–1372.

43.

Alhammad

. Bioinformatics analysis of the prognostic significance of CAND1 in ERα-positive breast cancer. Diagnostics (Basel), 2022; 12(10):2327; doi: 10.3390/diagnostics12102327

44.

Kang

, Li

, Zhao

, et al. miR-33a inhibits cell proliferation and invasion by targeting CAND1 in lung cancer. Clin Transl Oncol, 2018; 20(4):457–466; doi: 10.1007/s12094-017-1730-2

45.

Korzeniewski

, Hohenfellner

, Duensing

. CAND1 promotes PLK4-mediated centriole overduplication and is frequently disrupted in prostate cancer. Neoplasia, 2012; 14(9):799–806; doi: 10.1593/neo.12580

46.

Chen

, Jinlin

, Wang

, et al. GSTM3 deficiency impedes DNA mismatch repair to promote gastric tumorigenesis via CAND1/NRF2-KEAP1 signaling. Cancer Lett, 2022; 538:215692; doi: 10.1016/j.canlet.2022.215692

47.

Pierce

, Lee

, Liu

, et al. Cand1 promotes assembly of new SCF complexes through dynamic exchange of F box proteins. Cell, 2013; 153(1):206–215; doi: 10.1016/j.cell.2013.02.024

48.

ASN

, Zhang

, Mak

VCY

, et al. AKTIP loss is enriched in ERα-positive breast cancer for tumorigenesis and confers endocrine resistance. Cell Rep, 2022; 41(11):111821; doi: 10.1016/j.celrep.2022.111821

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