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

Eukaryotic initiation factor 3b (el3b) is an RNA-binding component of the eukaryotic translation initiation factor 3 (eif-3) complex, which is required for several steps in the initiation of protein synthesis. The elf-3 complex associates with the 40s ribosome and facilitates the recruitment of elf-1, elf-1A, elf-2:GTP:methionyl-tRNAi and elf-5 to form the 43s pre-initiation complex (43S PIC). The elf-3 complex is also required for disassembly and recycling of post-termination ribosomal complexes and subsequently prevents premature joining of the 40S and 60S ribosomal subunits prior to initiation. The elf-3 complex specifically targets and initiates translation of a subset of mRNAs involved in cell proliferation, including cell cycling, differentiation and apoptosis, and uses different modes of RNA stem-loop binding to exert either translational activation or repression. Studies showed that cell proliferation was significantly inhibited after eIF3b Knockdown. Additionally, cell colony numbers fell after eIF3b depletion. Furthermore, migration capacity was significantly impaired after eIF3b knockdown. EIF3b depletion reduced the number of cells traversing the membrane. 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. In a study conducted to explore the effects of eIF3b depletion on the cell cycle, the proportion of cells in S-phase was lower in eIF3b-depleted cells and the proportion in the G1 phase was higher  than that in the negative control. Western blotting showed that the G1/S arrest was caused by eIF3b depletion. 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. It was 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 p27Kip1 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 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. The levels of apoptosis increased slightly after eIF3b depletion. Further analysis 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-ribosepolymerase (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. The EMT is a key event in tumor invasion and metastasis, including RCC. It was 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. It was found that β-catenin expression was significantly downregulated after eIF3b depletion. The level of cyclin D1, 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 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. Studies 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 suggested that the integrin pathway might be affected; integrin links the extracellular matrix to the intracellular cytoskeleton to facilitate focal adhesion. After eIF3b knockdown, studies showed that 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 Akt plays a critical role in cell growth by directly phosphorylating the mechanistic target of rapamycin (mTOR). Studies has shown that p-mTOR was downregulated after eIF3b knockdown, indicating that the Akt/mTOR pathway was impaired. Furthermore, the observed downregulation of HIF-1α, HIF-2α, and p-NF-κB after eIF3b depletion may indicate that the Akt/mTOR/HIF and Akt/mTOR/NF-κB pathways were also downregulated, compromising cell proliferation and inducing apoptosis. Akt promotes cell survival by inhibiting apoptosis via phosphorylation (inactivation) of several proteins, including Bcl-2 and Bax. 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 and negatively regulating the actions of p27 Kip1  and p21 Waf1/Cip1. Research showed that GSk-3β and cyclin D1 were upregulated and p27 Kip1 and p21. Cip1 were downregulated after eIF3b depletion indicating that the Akt/GSK-3β pathway was involved in cell cycle regulation of A498 and CAKI-2 cells.

Clear cell renal cell carcinoma (CCRCC) is a renal cortical tumor typically characterized by malignant epithelial cells with clear cytoplasm and a compactalveolar (nested) or acinar growth pattern interspersed with intricate, arborizing vasculature. A variable proportion of cells with granulareosinophilic cytoplasm may be present. CCRCC is characterized genetically by alterations to chromosome 3p. CCRCC is proposed to arise from epithelial cellsof the proximal convoluted tubules of the nephron, within the renal cortex. Extension into the renal sinus is the most common pathway of spread for most histologic types of RCC because no connective tissue separates thecortical columns of Bertin from the abundant lymphatics and vasculature within the sinus fat. Elf-3b will therefore be a good target for pharmacological response against CCRCC.

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