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Eukaryotic Translation Initiation Factor 3b is both a Promising Prognostic Biomarker and a Potential Therapeutic Target for Patients with Clear Cell Renal Cell Carcinoma

Eukaryotic translation initiation factors (eIFs) constitute a new class of therapeutic cancer targets. EIF3b is the major scaffold protein of eIF3 (the largest core of eIFs). We sought to define the role played by and the mechanism of action of eIF3b in patients with clear cell renal cell carcinoma (ccRCC). We found that high-level eIF3b expression in tumors was not only associated with an aggressive tumor phenotype, but was also independently prognostic for patients with ccRCC. Knockdown of eIF3b impaired the action of the Akt pathway, thus inhibiting cell proliferation by disrupting the cell cycle and triggering apoptosis. Furthermore, the epithelial-to-mesenchymal transition was impaired after eIF3b depletion, via suppression of cell migration and invasion. Additionally, eIF3b knockdown significantly inhibited the growth of subcutaneous xenografts in mice. Together, these data show that eIF3b is both a promising prognostic biomarker and a potential therapeutic target for patients with ccRCC.


Introduction
In 2017, it is predicted that 63,990 patients will be diagnosed with kidney cancer in the USA and 14,400 inpatients will die [1]. Renal cell carcinoma (RCC) is the predominant form of kidney cancer, accounting for 2-3% of all adult malignancies [2]. Clear cell RCC (ccRCC) is the most common (75%) and most lethal subtype of RCC [3]. Although recent surgical developments have improved the overall survival of patients with ccRCC, the long-term prognosis remains unsatisfactory. Therefore, there is an urgent need for a better understanding of the molecular mechanisms underlying ccRCC development.
In recent years, deregulated protein synthesis and degradation have been shown to contribute to cancer development and progression [4,5].
Translation initiation is the critical rate-limiting process in the synthesis of most proteins [6]. Research has shown that inhibition of eukaryotic translation initiation factors (eIFs) is key in terms of reducing tumor initiation and progression, and improving prognosis; eIFs are promising therapeutic targets [4,5].
Of the 12 known eIFs, the multi-subunit complex known as eukaryotic translation initiation factor 3 (eIF3) is the largest, with 13 putative subunits [7]. Of the 13 subunits, eIF3b forms a central core to which the other components bind with varying affinities [6,8]. After binding to the 40S ribosomal subunit [9], eIF3 serves as a platform that recruits other protein components to form the 43S pre-initiation complex Ivyspring International Publisher [10]. Furthermore, eIF3b interacts with eIF4G, resulting in formation of the 48S pre-initiation complex, which recognizes the AUG start codon [11]. The contribution made by the eIF3 subunits eIf3a, eIf3b, eIF3c, eIF3e, eIF3h, eIF3i, and eIF3m to malignant transformation and progression have been defined over the past few years [12][13][14][15][16][17]. These factors are potentially prognostic biomarkers of several types of cancer, are easy to measure, and may form the basis of a new class of cancer therapeutics. Within the eIF3 complex, subunit b (eIF3b) is a major scaffold protein with which the other core subunits interact [18].
Earlier research showed that eIF3b played an important role in colon cancer [13], glioblastoma [12], esophageal squamous carcinoma [19], and bladder cancer [20]. However, the role of eIF3b in ccRCC remains unclear. In the present study, we show that eIF3b is both a promising prognostic biomarker and a potential therapeutic target in patients with ccRCC.

Patients and specimens
A total of 82 cancer specimens and tumor-adjacent renal tissues were obtained from patients with pathologically confirmed ccRCC who underwent radical or partial nephrectomy in 2009 or 2010 at the Department of Urology, Shandong University Qilu Hospital, Jinan, China. No patient underwent preoperative radiotherapy, chemotherapy, or immunotherapy. All specimens were re-evaluated using the TNM staging system [21]; the nuclear grades were scored using the Fuhrman criteria [22]. Patients with incomplete follow-up records were excluded. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed on random tissue samples obtained during radical, partial, or cytoreductive nephrectomy; the tissues were stored in liquid nitrogen prior to use. The samples included 27 normal kidney (NK) tissues taken >2 cm distant from tumors; 30 adjacent NK tissues (ANKs) taken <1 cm distant from tumors; 25 non-metastatic tumor tissues; 5 pairs of primary tumor tissues and metastatic tumor tissues obtained from patients with metastatic ccRCC. Written informed consent was obtained from all patients. Our study was approved by the Ethics Board of Qilu Hospital and was performed in accordance with all relevant principles of the Declaration of Helsinki.

RNA preparation and reverse transcription PCR
Total RNA was extracted from frozen tissues and cells using an RNeasy® Mini Kit (catalog no. 74104; Qiagen, USA). cDNA was synthesized from 2-µg amounts of total RNA using a ReverTra Ace® qPCR RT Kit (catalog no. FSQ-101; Toyobo, Japan) according to the manufacturer's instructions. All cDNA was stored at -20°C prior to analysis.

Scratch migration assay
A498 and CAKI-2 cell monolayers were transfected with negative-control or eIF3b-targeting siRNA for 48 h, scratched with a 100-µL pipette tip, washed twice with phosphate buffer saline (PBS) to remove detached cells, and incubated further in growth medium without FBS to ensure that the cells did not proliferate extensively and that most distributional changes would reflect cell migration. Wound closure (reflected by cell migration into the initially cell-free scratch area) was measured after 0, 8, 16, and 24 h. All experiments were performed in triplicate.

Transwell migration and invasion assays
We used Corning Matrigel® Invasion Chambers (8-μM pore size; BD Biosciences, USA). Cells (5.0 × 10 4 /500 μL serum-starved medium) were added to the upper chambers and complete medium to the bottom wells. After 16 h, 5% (w/v) glutaraldehyde was added to both chambers, followed by 0.1% (w/v) crystal violet. The upper faces of the filters were then wiped with cotton swabs. Images of five 200× fields were captured from each membrane and the numbers of invading cells were counted. In the invasion assay, the upper faces of the membranes were coated with 60-µL amounts of a 1:6 (w/v) dilution of Matrigel®. After incubation at 37°C for 1 h, the chambers were used in the invasion assay. The subsequent steps were the same as described above.

Cell cycle analysis and measurement of apoptosis
Seventy-two hours after transfection, cells were harvested and subjected to flow cytometry, for evaluation of cell cycle status using a Bestbio (China) kit (catalog no. BB-4104-50T) and measurement of apoptosis status using a BD (USA) kit (catalog no. 556547). We employed a BD FACSCalibur TM cytometer. Cell cycle status was evaluated using Modifit LT 4.1 software (Verity Software House, USA) and apoptosis status analyzed with the aid of FCS version V3 software (De Novo, USA).

Lentivirus transfection
A lentivirus-mediated eIF3b siRNA-delivery system was constructed by Genechem, China. The GV248 vector contained the elements hU6-MCS-ubiquitin-EGFP-IRES-puromycin; the clone was transfected with the aid of Enhanced Infection Solution (Genechem, China) with 5 µg/mL polybrene. After 12 h of incubation, the transfection medium were changed to normal culture medium with 10% (v/v) FBS.

Colony formation
Lentivirus-transfected cells were seeded at 100 cells/well into 6-well culture dishes, incubated for 8 d, and fixed and stained with 0.1% (w/v) crystal violet. Colonies containing > 50 cells were counted. All experiments were performed in triplicate.

Subcutaneous xenografts in nude mice
Tumor xenografts were established by injecting 5 × 10 6 A498-LV-eIF3b-RNAi cells in 200-µL amounts of PBS into the right flanks of 6-week-old female BALB/c nude mice (n = 5 per group; Biomedical Research Institution of Nanjing University, China). The two greatest tumor diameters were measured every 4 days with calipers and tumor volumes were calculated as V = L × l 2 × 0.5, where L and l represent the larger and smaller diameters. All animal care followed our institutional guidelines. Mice were sacrificed before the volume of the largest tumor attained 1,000 mm 3 . All animal experiments followed the dictates of the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the work was approved by the Committee on the Use and Care of Animals of Shandong University.

Statistical Analysis
Statistical analyses were performed with the aid of SPSS version 20 (IBM, USA) and GraphPad Prism® version 5.01 (GraphPad Software, USA). The immunohistochemically measured eIF3b levels and the clinicopathological parameters were compared using chi-squared and Fisher's exact tests. Kaplan-Meier analysis followed by log-rank testing was employed in the univariate analysis. Multivariate analyses were performed using Cox's proportional hazards regression model. A p-value < 0.05 was considered to indicate statistical significance. Migration and invasion levels were compared using the Student's t-test. Real-time PCR data were compared with the aid of the Mann-Whitney U test.

Patient characteristics
The 82 patients with ccRCC had a mean age of 57 years and included 55 males (66.7%) and 27 females (33.3%). Twenty patients (24.7%) died during the follow-up period. The estimated 5-year overall survival (OS) was 84.1%. The numbers of patients with TNM grades I, II, III, and IV were 64, 7, 7, and 4, respectively. The numbers of patients with Fuhrman nuclear grades 1, 2, 3, and 4 were 7, 25, 28, and 22, respectively. The median follow-up time was 76 months. Table 1 lists the details.

Depletion of eIF3b inhibited cell proliferation, migration, and invasion
A498 and CAKI-2 cells (which express high levels of eIF3b) were used to conduct in vitro experiments (Supplementary Figure 1A). Cell proliferation was significantly inhibited after eIF3b knockdown ( Figure 3A and 3B; Supplementary Figure  1B). Additionally, cell colony numbers fell after eIF3b depletion ( Figure 3C). Furthermore, migration capacity was significantly impaired after eIF3b knockdown ( Figure 3D). In the transwell assay, eIF3b depletion reduced the number of cells traversing the membrane (compared with control cells) ( Figure 3E). Therefore, eIF3b depletion impaired both cell migration and invasion. Notably, eIF3b depletion caused the cells to become smaller and rounded, suggesting that both migration and adhesion were impaired compared with control cells ( Figure 3F).

eIF3b depletion triggered G1/S and G2/M arrest
We explored the effects of eIF3b depletion on the cell cycle. The proportion of cells in S-phase was lower in eIF3b-depleted cells ( Figure 4A) and the proportion in the G1 phase was higher ( Figure 4A) than that in the negative control. Western blotting showed that the G1/S arrest was caused by eIF3b depletion ( Figure 4B). During the G1/S transition, cyclins D and E combine with cyclin-dependent kinases (CDK) to form the cyclin/CDK complexes required for the transition. The complexes phosphorylate the retinoblastoma protein (Rb), releasing the E2F transcription factor that activates expression of G1/S progression genes. We found that the levels of both cyclins D and E decreased after eIF3b knockdown. Cyclins D and E, Rb, and the inactivated form of Rb (p-Rb) were downregulated after eIF3b knockdown. In addition, the levels of p27 Kip1 and p21 Cip1, inhibitors of the cyclin/CDK complexes, significantly increased after eIF3b knockdown. Interestingly, Western blotting of G2/M-related proteins indicated that the G2/M transition was also inhibited after eIF3b knockdown ( Figure 4C). Cyclin A, which accumulates steadily during the G2 phase and is abruptly destroyed at mitosis, was upregulated after eIF3b depletion. Cyclin B is required for the G2/M transition; we found that the cyclin B level fell. Also, the levels of Myt1 and Wee1 increased after eIF3b depletion; these proteins inhibit cell entry into mitosis. The observed decrease in histone H3 phosphorylation indicated that chromosome condensation was reduced by eIF3b depletion; fewer cells were in the mitotic phase.

Knockdown of eIF3b promoted apoptosis of renal cancer cells
As eIF3b depletion reduced cell proliferation in vitro, we explored whether apoptosis was involved. The levels of apoptosis increased slightly after eIF3b depletion ( Figure 4D). Further analysis ( Figure 4E) showed that, after eIF3b knockdown, the pro-apoptotic factors Bax, caspase-3, and the activated form thereof (cleaved caspase-3) were all upregulated and the pro-survival factor Bcl-2 was downregulated. More importantly, cleavage of poly-ADP-ribose polymerase (c-PARP), a marker of apoptosis, was also increased. Therefore, apoptosis increased after eIF3b knockdown. However, interestingly, knockdown reduced the level of cleaved caspase-12, which is required for endoplasmic reticulum-stress-induced apoptosis ( Figure 4E).

Impairment of the β-catenin pathway inhibited the epithelial-to-mesenchymal transition (EMT) after eIF3b depletion
The EMT is a key event in tumor invasion and metastasis, including RCC [23]. We found that the epithelial marker E-cadherin was upregulated and the levels of repressors thereof (Slug and Snail) were downregulated after eIF3b depletion. Additionally, the mesenchymal markers N-cadherin and vimentin were downregulated. Therefore, the EMT was inhibited by eIF3b depletion ( Figure 5A). The β-catenin pathway is involved in regulation of the EMT [24]. We found that β-catenin expression was significantly downregulated after eIF3b depletion ( Figure 5A). The level of cyclin D1 ( Figure 4B), a target of β-catenin, was also downregulated after eIF3b depletion. Together, our data showed that eIF3b depletion inactivated the β-catenin pathway and inhibited the EMT of ccRCC.

The Akt pathway was impaired after eIF3b depletion
The serine/threonine kinase Akt (also termed protein kinase B or PKB) has attracted a great deal of attention because Akt plays critical roles in the regulation of many cellular functions including metabolism, growth, proliferation, survival, transcription, and protein synthesis [25][26][27][28][29]. We found that eIF3b depletion was not associated with any significant change in Akt levels; however, the level of the activated form, p-Akt, fell in parallel with the extent of eIF3b depletion, indicating that the Akt signaling pathway was involved in such depletion.
The significant morphological changes in cells after eIF3b depletion ( Figure 3F) suggested that the integrin pathway might be affected; integrin links the extracellular matrix to the intracellular cytoskeleton to facilitate focal adhesion [30]. After eIF3b knockdown, the levels of integrins α2 and α5 fell significantly, as did the level of the upstream phosphorylated Focal adhesion kinase (p-FAK) protein, suggesting that integrin/FAK/Akt signaling was inhibited ( Figure  5B).
Akt plays a critical role in cell growth by directly phosphorylating the mechanistic target of rapamycin (mTOR) [31]. We found that p-mTOR was downregulated after eIF3b knockdown, indicating that the Akt/mTOR pathway was impaired ( Figure  5B). Furthermore, the observed downregulation of HIF-1α, HIF-2α, and p-NF-κB after eIF3b depletion may indicate that the Akt/mTOR/HIF [29] and Akt/mTOR/NF-κB [28] pathways were also downregulated ( Figure 5B), compromising cell proliferation and inducing apoptosis. The observed reduction in HIF-1α and HIF-2α levels suggested that the cells would be less able to endure hypoxia, reducing cell survival. Vascular Endothelial Growth Factor Receptor (VEGFR), which is often targeted in patients with RCC, was also downregulated ( Figure  5B). Akt promotes cell survival by inhibiting apoptosis via phosphorylation (inactivation) of several proteins [32], including Bcl-2 and Bax ( Figure  4E). Apart from the roles played in survival and apoptosis, the Akt pathway is also involved in cell cycle regulation, preventing GSK-3β-mediated phosphorylation and degradation of cyclin D1 [25,33] and negatively regulating the actions of p27 Kip1 [26] and p21 Waf1/Cip1 [27]. We found that GSk-3β and cyclin D1 were upregulated and p27 Kip1 and p21 Cip1 were downregulated after eIF3b depletion ( Figures 4B and 5B), indicating that the Akt/GSK-3β pathway was involved in cell cycle regulation of A498 and CAKI-2 cells.

EIF3b depletion inhibited the growth of subcutaneous xenografts in mice
To further investigate the roles played by eIF3b and to validate eIF3b as a potential therapeutic target in vivo, we injected nude mice with A498 cells transfected with the eIF3b-RNAi-containing lentivirus (Supplementary Figure 1C). Control cells yielded could grow into significantly larger tumors than did eIF3b-RNAi-lentivirus transfected cells (p < 0.001; Figure 6A and 6B). Therefore, eIF3b knockdown inhibited the growth of subcutaneous xenografts in mice. And about 2 months after transfection, cells transfected with eIF3b-RNAi lentivirus was found in a worse situation with more cell debris than control cells ( Figure 6C).
We found that higher-level tumor eIF3b expression was associated with a more aggressive phenotype and, more importantly, that the tumor eIF3b expression level was independently prognostic of ccRCC progression. This carcinogenic role of eIF3b was confirmed by in vitro experiment that eIF3b knockdown inhibited tumor cell proliferation, migration, and invasion.
We then showed that eIF3b depletion inhibited cell proliferation by compromising the cell cycle and inducing cell apoptosis. The G1/S arrest after eIF3b depletion has also been noted in bladder cancer [20], glioblastoma [12], and esophageal squamous carcinoma [19]. Wang et al. found that reduction in overall protein synthesis by eIF3b depletion lead to the smaller size of cells which cannot pass the major size regulatory "R or restriction point" checkpoint, which is also a G1/S arrest reason [20]. We also found, in vitro, that the G2/M transition was impaired after eIF3b depletion. However, such impairment may be weak; it was not apparent on flow cytometry. In addition, apoptosis increased after eIF3b depletion. Similar pro-apoptotic effects have been reported in colon cancer [13], glioblastoma [12], and esophageal squamous carcinoma [19]. Interestingly, ER-stress-induced apoptosis seemed to be downregulated after eIF3b depletion. However, the overall effect of eIF3b depletion was pro-apoptotic in ccRCC. Cell proliferation, migration, and invasion were reduced after eIF3b knockdown. The EMT is a key event in renal tumor invasion and metastasis [23]. We found that the EMT was impaired after eIF3b knockdown, which was characterized by upregulation of E-cadherin and downregulation of N-cadherin, vimentin, and repressors of E-cadherin. We also found that inhibition of the β-catenin pathway contributed to the EMT impairment after eIF3b depletion. eIF3b knockdown inhibited many activities of the Akt pathway network in RCC cells, including the integrin/FAK/Akt, Akt/mTOR/HIF/VEGF, Akt/mTOR/NF-κB, Akt/Bcl-2/Bax, and Akt/GSK-3β pathways. Inhibition of the integrin/FAK/Akt pathway changed the cellular morphology and impaired adhesion. Inhibition of the Akt/mTOR/HIF/VEGF and Akt/mTOR/NF-κB pathways compromised cell proliferation and increased apoptosis. Akt pathway inhibition also induced apoptosis via downregulation of Bcl-2 and Bax. Inhibition of the Akt/GSK-3β pathway contributed to G1/S arrest.
Dysregulation of protein synthesis has been implicated in the impaired oncogenicity after eIF3b depletion. The inherent "translatability" of different mRNAs varies widely [43,44]. "Weak" mRNAs encoding proteins involved in cell proliferation are translated after activation of the protein synthesis apparatus [14,45]. Not all mRNAs are translated to an equal extent, as we also found. mRNA encoding oncogenic proteins seem to be poor competitors (compared with other mRNAs) of the translational complex. Therefore, after eIF3b depletion, the translation of oncogenic proteins in the EMT and Akt signaling pathways is significantly inhibited. In other words, these pathways are stimulated when the level of overall protein synthesis is high (e.g., in tumors expressing high levels of eIF3b). Activation of protein synthesis triggers overproduction of oncogenic proteins and rapid cell growth; downregulation of protein synthesis preferentially impairs the expression of oncogenic proteins, restoring controls on proliferation. This explains why patients with high levels of tumor eIF3b experience poor OS.
Many clinical analyses and in vitro experiments have indicated that tumor eIF3b plays a role in tumor progression. EIF3b is highly expressed in certain tumors [12,13,19,20]. It is thus of considerable interest that we found eIF3b to be downregulated in ccRCC, as evident both immunohistochemically and upon qRT-PCR. Furthermore, we found that high-level eIF3b expression in ANK tissue correlated with longer OS and a less aggressive tumor phenotype; eIF3b thus seems to play a totally different role in normal kidney cells. It is well-known that, transforming growth factor beta (TGF-β) plays different roles in cancer initiation and progression, both suppressing and promoting such progression [46]. We suggest that eIF3b may, like TGF-β, play different roles in kidney cancer. In tumor cells, eIF3b contributes to tumor progression but in non-tumor cells, it plays a protective role in patients with ccRCC. Further in vitro research and study of a larger sample of tumor and normal tissues are required. eIF3b knockdown inhibited the growth of subcutaneous tumors in mice, confirming eIF3b as a potential therapeutic target for patients with ccRCC. As the high expression in normal kidney tissues, the impact on normal kidney tissues cannot be ignored and it is difficult to design specific therapeutic agents to target this protein. However, after we uncover the role of eIF3b in normal kidney in the future, it is possible to apply this protein to the clinic. Nowadays, RNA-based therapeutics, such as small-interfering (siRNA), microRNAs, antisense oligonucleotides (ASOs), CRISPR-Cas9, have great potential to target a large part of the currently undruggable genes to treat cancers [47]. Besides, recent advances has helped to overcome the lipid bilayer to deliver the RNA into cells cytoplasm and nucleus and enabled RNA-based therapeutics directed to other cancer targets to progress into clinical trials for cancer [48,49]. There is also research showing that intratumoral administration of ASOs can antagonize pancreatic cancer growth in vivo [50], which points out a promising way of for clinic application research of RNA-based therapeutics targeting eIF3b.
There were certain limitations to our study. We failed to explore the reason why eIF3b expression is higher in ANK than that in tumors. Regulation of involved protein in cancer progression is very complicated. Methylation is probably one of them. A research on breast cancer showed that the status of methylation of genes in tumor is not exactly the same as the adjacent normal [51]. The difference of methylation of eIF3b gene may contribute to the discrepancy of eIF3b protein expression in tumor compared to adjacent tissue. So more work need to be done to uncover the methylation and gene status of ANK and NK tissue.
Together, our results suggest that high-level expression of eIF3b in cancer cells may be essential for expression of the transformed phenotype, and that tumor eIF3b expression is independently prognostic for patients with ccRCC. Furthermore, in vitro eIF3b knockdown effectively impaired the Akt pathway, inhibiting cell proliferation by disrupting the cell cycle, and triggering apoptosis. eIF3b depletion also impaired the EMT, suppressing cell migration and invasion. More importantly, the in vivo work showed that eIF3b may be a useful therapeutic target for patients with ccRCC. More work is needed to understand what role eIF3b plays in non-tumor kidney cell and how eIF3b interacts with other eIFs in terms of cancer development.