Translation Initiation Factor eIF3b Expression in Human Cancer and Its Role in Tumor Growth and Lung Colonization

Deregulated protein synthesis and degradation contribute to cancer genesis and progression. Control of protein synthesis directs both global and selective translation of specific mRNAs, and the protein products then promote tumor cell survival, angiogenesis, invasion, and metastasis (1). Protein synthesis in eukaryotes is primarily regulated at initiation, the rate-limiting step, and one that involves a large number of eukaryotic initiation factors (eIF). To date, well over a dozen eIFs have been identified, several operating within large multiprotein complexes (2, 3). The components of several of these complexes are aberrantly expressed or activated in different types of human cancer (1, 4). Hence, targeting such factors offers promise for the development of a new class of cancer therapeutics. For example, overexpression of eIF4E is associated with poor prognosis in several human cancers (5), yet despite this factor being previously considered rate limiting in cap-dependent protein synthesis, its depletion with siRNA did not cause host toxicity in mice (6). This finding led to clinical trials in patients with cancer using eIF4E siRNA (6–8).

Recent transcriptomic analyses carried out in the context of biomarker discovery (9–11) suggested that eukaryotic initiation factor 3b (eIF3b) may be elevated in human bladder and prostate cancer. eIF3b is a subunit of the eIF3 translation initiation factor complex. This complex has a molecular weight of 550 to 700 kDa and consists of 13 putative subunits in humans (12). By virtue of increased expression of several of its subunits (13), the eIF3 complex has been implicated in cancer. For example, eIF3a (p170) is overexpressed in several human cancers (13), and its depletion by antisense cDNA reversed the malignant phenotype in human cancer cells (14). Overexpression of a truncated eIF3e mutant caused transformation of mammary epithelial cells both in vitro and in vivo (15). eIF3h, located on chromosome region 8q, is frequently amplified in breast cancer, prostate cancer, and non–small cell lung cancer alone with the adjacent MYC proto-oncogene, and its overexpression promotes cancer cell growth (16, 17). The eIF3b (hPrt1) subunit is particularly interesting because it serves a critical scaffolding function for the entire eIF3 complex (12, 18, 19). Indeed, ectopic overexpression of eIF3b transforms NIH3T3 cells (20), and overexpression of this protein is observed in breast cancer (21). However, these data fall short of implicating eIF3b as a prognostic factor or requirement for the maintenance or progression of human cancer.

Here, we show that eIF3b is overexpressed in human bladder and prostate cancer and that higher expression is associated with advanced grade, stage, and poor prognosis. Using complementary molecular approaches in multiple cellular models in vitro and in vivo, we show that eIF3b expression is important to the malignant phenotype and consequently, as with eIF4E, eIF3b may be a viable therapeutic target in cancer.

Two publicly available microarray datasets were used for the analysis of eIF3b expression in bladder cancer. The preprocessed data from Kim and colleagues (22) were downloaded from Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/, accession GSE13507). The raw data from Stransky and colleagues (23) were downloaded from ArrayExpress (http://www.ebi.ac.uk/arrayexpress/, accession E-TABM-147) and normalized using the Robust Multichip Average algorithm (24). Patient demographics and clinicopathologic data are shown in Supplementary Table S1.

If there were multiple probe sets for eIF3b in a dataset, the probe set with the highest mean expression across samples was selected to represent eIF3b expression. The 2 datasets were mean centered so that samples have mean expression of zero in each dataset. Dotplots of eIF3b expression comparing different groups of samples were plotted, and differences in distributions were tested by Mann–Whitney U tests. As a means to check whether eIF3b expression stratifies disease-specific patient survival, patients were divided into 2 groups using the 70th percentile of eIF3b expression and compared using Cox proportional hazards models and log-rank tests.

Bladder cancer tissue microarrays were constructed at the Spanish National Cancer Center (details in Supplementary Materials and Methods), including a total of 143 bladder tumors (70 nonmuscle invasive T1G3 and 73 muscle invasive T2-4 tumors). Protein expression patterns of eIF3b were assessed using standard avidin–biotin immunoperoxidase procedures (25). The primary antibody for immunohistochemistry (IHC) was eIF3b (LifeSpan Biosciences LS-C138931), rabbit monoclonal at 1:50. The secondary antibody was a biotinylated horse anti-rabbit antibody (Vector Laboratories, 1:1,000 dilution).

eIF3b expression was evaluated as continuous variables based on the number of cells expressing the protein in the cytoplasm. The intensity of the staining was categorized from negative (−) to low (+), intermediate (++), and high (+++). The associations of these proteins with disease-specific overall survival were also evaluated using the log-rank test in those cases for which follow-up information was available. Disease-specific overall survival time was defined as the months elapsed between transurethral resection or cystectomy and death as a result of disease (or the last follow-up date). Patients who were alive at the last follow-up or lost to follow-up were censored. Survival curves were plotted using the standard Kaplan–Meier methodology (25). Statistical analyses were conducted using the SPSS statistical package (version 18.0).

UMUC3, Lul2 human bladder cancer cells were cultured in minimum essential medium plus 10% FBS, 1 mmol/L sodium pyruvate. siRNA transfection was carried out using Oligofectamine (Invitrogen) according to the manufacturer's instructions. Predesigned siRNA duplexes were purchased from Dharmacon as follows: eIF3b-3: 5′-GAGTATGAACGGTGCCTTATT-3′; eIF3b-4: 5′-AGAGATCAGTACAGTGTGATT-3′; Luciferase GL2: 5′-GTACGCGGAATACTTCGA-3′. Integrin α5 siRNA, a pool of 3 target-specific siRNAs, was purchased from Santa Cruz. For Western blot analysis, transfected cells were harvested after 72 hours and equal amounts of total proteins were subjected to SDS-PAGE and analyzed using specific antibody. Antibodies used for Western blot analysis are listed in the Supplementary Materials and Methods.

Quantitative reverse transcription PCR (qRT-PCR) was carried out on iCycler Optical Module (Bio-Rad) with IQ SYBR Green fluorescent dye (Bio-Rad) included in the PCR to determine the amount of mRNA. Total RNA was purified from cells using RNeasy Mini kit (Qiagen). Human 12p primers developed by our laboratory were used for tumor quantization in the lungs (26). DNA from mouse lungs was purified using MasterPure DNA Purification Kit (Epicentre Biotech).

Monolayer growth was evaluated by AlamarBlue assay and anchorage-independent growth by soft agar assay (27). The cell cycle was evaluated by propidium iodide (PI) staining as described (27). A cell migration assay was carried out by Boyden Chamber assay as described (28).

Cells were plated on cover slips and transfected with eIF3b or GL2 control siRNA the following day. Seventy-two hours later, cells were fixed with 4% formaldehyde and stained with phalloidin-AlexaFluo 594 (Molecular Probes) or anti-myosinIIA (Abcam) to visualize actin filaments. Anti-P-FAK (Invitrogen) or Paxillin (BD transduction) staining was used to identify focal adhesions and 4′, 6-diamidino-2-phenylindole (DAPI) as a nuclear marker. A Zeiss LSM 510-UX or Olympus FV1000 Laser Scanning confocal microscope was used for capturing immunofluorescence images.

New protein synthesis was measured using Click-iT Metabolic Labeling Reagents [L-azidohomoalanine (L-AHA)] and the Click-iT protein reaction buffer kit from Invitrogen according to the manufacturer's instructions. Newly synthesized proteins were detected by anti-TAMRA antibody as per manufacturer's instructions (Thermo Scientific). Total protein loading on the gel was detected by Ponceau S staining.

Female 6-week-old athymic mice (Ncr nu/nu) were obtained from the National Cancer Institute. Twenty-four hours after siRNA transfection, 10⁶ of UMUC3 cells were injected subcutaneously; tumors were measured by calipers weekly, and volume was calculated (29). For lung colonization, mice were injected via tail vein with 10⁶ of Lul2 cells transfected with siRNA 24 hours later. The images of lung metastases were evaluated by Xenogen Bioluminescent imaging system (Caliper Life Sciences) as described (30) and quantified using IGOR Pro 4.09A image analysis software. Quantification of tumor in the lungs was conducted by qRT-PCR using human 12p primers as described (26).

Discovery transcriptomic analysis of human cancers using previously published and newly profiled tumor and cell line microarray data (9–11) showed that probes associated with eIF3b were increased in bladder and prostate cancer. To evaluate the clinical relevance of this finding, we compared eIF3b expression in normal bladder urothelium versus tumors, low-grade versus high-grade, and non–muscle invasive (NMI; pTa and pT1) versus muscle invasive (MI; pT2 and above) samples. We observed that eIF3b mRNA expression was higher in cancer than normal samples in high-grade than low-grade samples (Supplementary Fig. S1A and S1B) and in muscle invasive than NMI samples (Fig. 1A). Higher eIF3b expression was also associated with worse patient outcome (Fig. 1B).

Next, we developed immunohistochemical staining for formalin-fixed paraffin-embedded (FFPE) materials and carried it out on human bladder tumor samples. Western blot analysis and staining of eIF3b overexpressing cell lysates or FFPE cell pellets showed specificity of the eIF3b antibody used for IHC (Supplementary Fig. S2A and S2B). Representative IHC results on human tumors are shown in Fig. 1C. The next set of analyses searched for associations with clinicopathologic variables of patients with bladder cancer. It was observed that both the number of tumor cells displaying cytoplasmic expression and the intensity of the cytoplasmic expression of eIF3b were significantly associated with tumor stage (Kruskall–Wallis, P = 0.004, and P = 0.036 respectively). Furthermore, tumors expressing cytoplasmic eIF3b in at least 80% of the cancer cells were significantly associated with poor disease-specific survival (Fig. 1D). These observations indicated that increased cytoplasmic eIF3b is associated with higher tumor stage and shorter survival, consistent with the transcriptomic analysis. We also did a similar analysis on prostate cancer and found that eIF3b mRNA expression was higher in prostate cancer compared with normal prostate tissue and had a positive correlation with Gleason score. Higher eIF3b expression was also associated with androgen independence and worse disease-specific survival in patients with prostate cancer (Supplementary Fig. S3A–S3D).

To determine the importance of eIF3b expression for cancer cell growth, we depleted eIF3b protein via transfer of siRNAs into human bladder (UMUC3, Lul2) and prostate (PC3, DU145) cancer cells. A time-course of eIF3b protein levels showed significant reduction at 48 to 96 hours after transfection that lasted for 6 days (Supplementary Fig. S4). eIF3b depletion significantly reduced the proliferation of UMUC3 and Lul2 cells (Fig. 2A), and these findings were recapitulated with PC3 and DU145 cells (Supplementary Fig. S5). We next evaluated the ability of eIF3b-depleted cancer cells to form colonies in soft agar. Compared with the control siRNA–transfected cells, UMUC3 and Lul2 cells with reduced eIF3b had much lower numbers of colonies (Fig. 2B).

To begin identifying the mechanisms underlying these effects, we evaluated whether eIF3b depletion had any effect on the cell cycle. At 96 hours after transfection, the percentage of cells in S-phase was lower in samples treated with eIF3b siRNAs, with a concomitant increase in the G₁ phase in both UMUC3 (Fig. 2C) and Lul2 cells (Supplementary Fig. S6A), compared with samples treated with nonrelevant GL2 siRNA. Because this suggested inhibition of the G₁–S transition, we sought to determine whether any of the regulators involved in this process are affected by eIF3b depletion. Interestingly, cyclin A, cyclin E, Rb protein expression, and Rb phosphorylation were significantly decreased with eIF3b knockdown in UMUC3 cells (Fig. 2D). Knockdown of eIF3b also led to an increase in cyclin-dependent kinase inhibitor p27Kip1 in UMUC3, Lul2, and PC3 cells (Fig. 2D and Supplementary Fig. S6B). In contrast, the protein levels of RhoGDP dissociation inhibitor 1 and 2, known suppressors of tumor growth (RhoGDI1) and colonization (RhoGDI2) in human bladder cancer (31–33), were not changed (Fig. 2D). Importantly, although cells transfected with eIF3b siRNA showed significantly decreased eIF3b mRNA, control GUSB and mRNA levels of the above-mentioned genes were not altered by eIF3b depletion (Supplementary Fig. S6C). These data suggest that changes of protein levels of cell-cycle regulators seen with depletion of eIF3b are not due to the changes of mRNA transcription and also that this effect is not general to all proteins.

Because eIF3b depletion led to the reduced cell numbers in vitro (Fig. 2A), we sought to determine whether apoptosis played a role in this process. We did not observe dramatic changes by Annexin V-fluorescein isothiocyanate (FITC)/PI staining apoptosis assay in response to eIF3b depletion (Supplementary Fig. S7A). Furthermore, we also analyzed the cell lysates by Western blotting and did not observe cleaved PARP or activated caspases (Supplementary Fig. S7B). These results indicate that no apoptosis was induced by eIF3b depletion.

Cell migration is an important component of metastasis (34). Furthermore, the ability of cancer cells to attach successfully via focal adhesions to the extracellular matrix has been shown to be critical to both migration and growth at metastatic sites (35, 36). UMUC3 cells with eIF3b knockdown showed approximately 50% reduced Transwell cell migration compared with control cells (Fig. 3A). We also observed dramatic cell morphology changes after eIF3b depletion in UMUC3 and PC3, with eIF3b-depleted cells being smaller and rounded, suggesting impaired spreading compared with control cells (Fig. 3B; Supplementary Fig. S8A). Next, we evaluated the intracellular distribution of proteins related to cell migration by immunofluorescence microscopy of UMUC3 cells. In control cells stained with phalloidin and myosin IIA for actin cytoskeleton evaluation, actin was present at the plasma membrane and formed stress fibers. In contrast, cells depleted of eIF3b revealed disorganization of the actin cytoskeleton, reduction of stress fibers, and an increase of cortical actin (Fig. 3C). Similarly, we observed decreased vinculin (actin-binding protein) staining in eIF3b-depleted PC3 cells (Supplementary Fig. S8A). Paxillin is an established marker of focal adhesions. Reduction of eIF3b levels led to a loss of paxillin distribution, suggesting loss of focal adhesions in these cells (Fig. 3C). Focal adhesion kinase (FAK), an important signaling molecule in the integrin pathway, localizes to and is activated (by autophosphorylation at Y397 site) within focal adhesions (37, 38). Consistent with the notion that eIF3b depletion induces focal adhesion disruption, this also led to decreased phosphorylation of FAK in the focal adhesion (Fig. 3C). This finding was confirmed by Western blot analysis with anti-phospho-FAK antibody (Fig. 3D). In contrast, overall levels of these proteins seemed unaltered by eIF3b depletion (Fig. 3D and Supplementary Fig. S8B). These results suggest that depletion of eIF3b decreases human bladder cancer cell migration in part by interrupting actin cytoskeleton organization and focal adhesion formation. Because FAK mediates several cell survival pathways, we evaluated phosphorylation of Akt and found this was reduced in parallel to depletion of eIF3b and reduction of FAK phosphorylation (Fig. 3D).

Because integrins are upstream regulators of FAK, we sought to evaluate their expression as a function of eIF3b depletion. Western blot analysis revealed that integrin α5 protein level was dramatically decreased upon eIF3b knockdown in both UMUC3 and PC3 cells (Fig. 4A and Supplementary Fig. S8B), whereas other integrin proteins were not changed. Quantitation of integrin mRNA levels showed no statistically significant changes (Fig. 4B). Furthermore, we knocked down integrin α5 by siRNA in UMUC3 cells and observed that the cells were smaller and rounded and had decreased spreading compared with control cells treated with GL2 siRNA (Fig. 4C), a phenocopy of the knockdown of eIF3b. Knockdown of integrin α5 also showed decreased phosphorylation of FAK and Akt, whereas the total protein levels stayed the same (Fig. 4D), an effect similar to that observed upon knockdown of eIF3b. Taken together, these data suggest that the decreased cell migration and the change of cell morphology and adhesion induced by eIF3b knockdown are mediated in part through integrin α5.

Given that depletion of eIF3b leads to many cell changes, we wondered whether this is caused by the decreasing of global protein synthesis and/or by a specific pool of proteins. To evaluate global protein synthesis, we used the Click-iT metabolic labeling assay. As shown in Fig. 5A, untreated UMUC3 cells exhibited high levels of newly synthesized proteins, but as expected, protein synthesis was almost completely blocked by cycloheximide treatment. Reduction of eIF3b inhibited global protein synthesis by about 50% (Fig. 5A). Despite this, we found that upon eIF3b depletion, the total levels of some proteins were maintained compared with the untreated control, whereas others decreased. Specifically, actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and FAK levels remained high, but the level of integrin α5 was decreased dramatically (Fig. 5B) in the samples with eIF3b depletion (Fig. 5C). This is consistent with the results of the total lysate Western blot analysis (Fig. 3D and 4D; Supplementary Fig. S8B) and suggests that although global protein synthesis is inhibited by depletion of eIF3b, the effect on total protein levels may be specific to individual proteins.

To determine whether the effect on global translation is eIF3b specific or more likely due to disruption of the eIF3 complex, we examined the expression levels of all 13 eIF3-subunit mRNAs in human bladder cancer. In addition to eIF3b, both eIF3c and eIF3i were higher in cancer than in normal samples in both datasets (Supplementary Table S3). We chose eIF3c to deplete based on the global architecture of eIF3, which places eIF3c at the center of the complex (12) and thus its depletion is more likely to affect the entire complex. In addition, eIF3c is increased in testicular seminomas (39) and ectopic overexpression of eIF3c transforms NIH3T3 (20), both findings supporting a role for this protein in cancer. Depletion of eIF3c significantly inhibited UMUC3 cell growth and new protein synthesis (Supplementary Fig. S9A and S9C), decreased integrin α5 expression, and disrupted actin cytoskeleton organization and focal adhesion formation (Supplementary Fig. S9B and S9D). Thus, changes induced by eIF3c depletion match those observed with eIF3b depletion, suggesting that the observed effects of depletion of either protein are due to an overall disruption of eIF3 complex stoichiometry or abundance that leads to the inhibition of translation.

To determine the in vivo role of eIF3b expression in cancer, we injected UMUC3 cells transfected with control or eIF3b siRNA subcutaneously into mice and evaluated the kinetics of tumor formation and the growth of established tumors. eIF3b-depleted cells had delayed tumor formation and overall reduced tumor take (Fig. 6A). The tumors that did form grew more slowly compared with the group injected with control siRNA (Fig. 6B). We then investigated whether eIF3b was critical for bladder tumor colonization of the lung. For this evaluation, we selected Lul2 cells, a highly metastatic human bladder cell line (40). eIF3b-depleted Lul2 cells were inoculated in the tail vein of mice and bioluminescent in vivo imaging of metastases was carried out weekly. Five weeks after injection, imaging showed much stronger signals in the GL2 siRNA group than those in the eIF3b siRNA group (Fig. 6C). To confirm that these lung signals were indeed Lul2 cells, all mice were necropsied at 5 weeks, and the tumor burden was quantified by quantitative PCR (qPCR) with human-specific primers on 12p (26). Compared with the GL2 siRNA group, the mice injected with Lul2 cells transfected with eIF3b siRNA showed dramatically less human genomic DNA (Fig. 6D). Importantly, the results of the tumor burden quantitated by qPCR were consistent with the quantitation of the radiance of luciferase (Fig. 6D), suggesting the latter was a true marker of lung colonization by human cancer cells.

Although overexpression of eIF3b in mouse fibroblast cells causes transformation (20), until our work here, the role of eIF3b in human cancer was unclear. Data presented support a model in which eIF3b is required for maintenance of the malignant state. Knockdown of eIF3b inhibits cancer cell growth by inhibition of the G₁–S cell-cycle transition, but this did not seem to induce apoptosis of these cells. We investigated the molecular mechanism involved in cell-cycle regulation by eIF3b. Given that eIF3b knockdown inhibits global translation and cyclins are unstable proteins (Supplementary Table S4) actively degraded in G₁ phase, a reduction in protein synthesis would be expected to lead to the reduced cyclin levels. This is consistent with our data (Fig. 2D). The observed increase in p27Kip levels along with decreased cyclins would also be expected to lead to hypophosphorylated Rb, which leads to G₁ arrest (41). Also, G₁ arrest is the major size regulatory “R or restriction point” checkpoint; if cells are too small, they cannot pass this checkpoint (42). Because reduction in protein synthesis leads to smaller cells (Fig. 4C), we expected and indeed observed, eIF3b knockdown-induced translation depression to lead to arrest in G₁ as a result of this checkpoint, consistent with recent findings (43, 44).

In addition to the cell-cycle effects, we found that lowering eIF3b levels in UMUC3 cells inhibited cell migration. We also observed cell morphology changes and decreased spreading. Cytoskeleton organization and adhesion formation are critical for cell motility and structural support (45); our data indicate that eIF3b knockdown disrupts both, raising the question of how eIF3b regulates these processes. Integrins link the extracellular matrix to intracellular cytoskeleton and focal adhesions and thus control a variety of signal transduction pathways, including cell proliferation and survival (46). Because depletion of eIF3b markedly inhibited integrin α5 expression, integrin α5 might be one of eIF3b targets responsible for the disrupted actin cytoskeleton and focal adhesion. This is supported by our observation that knockdown of integrin α5 phenocopied the morphologic changes seen with depletion of eIF3b. Knockdown of eIF3b and integrin α5 also had similar effects on the phosphorylation of FAK and Akt, which is significant because Akt plays a key role in cell proliferation and survival. This suggests that dramatically decreased bladder cancer cell growth induced by the depletion of eIF3b is not only due to effects on the cell cycle but also to effects on integrin/FAK/Akt signaling that are induced by a decrease in protein synthesis. Finally, because integrin α5 promotes cancer cell invasion and metastasis formation (47–49), our data support the idea that the decreased integrin α5 induced by the knockdown of eIF3b contributes to the inhibition of lung colonization in bladder cancer.

Consistent with its role in translation initiation, depletion of eIF3b inhibited global protein synthesis (Fig. 5A), but interestingly not all protein levels were decreased equally (Fig. 2–4D and 5B), probably because not all mRNAs had an equal decrease in translation (7, 50). In fact, because mRNAs vary widely in their inherent “translatability,” they are preferentially and disproportionately affected when global translation is inhibited. For example, the mTOR inhibitor Torin 1 caused a severe defect in global protein synthesis, but a subset of mRNAs, so called top or top-like mRNAs, are specifically regulated by mTOR inhibition (50). Also, another subset of mRNAs, which have lengthy, high G+C, highly structured 5′-UTRs, so called “weak” mRNAs, are particularly sensitive to alterations in eIF4E levels and eIF4F complex formation (7). In our study, depletion of eIF3b led to decreased global translation, and more profoundly to a subset of mRNAs including cyclins and integrin α5. Interestingly, depletion of eIF3c induced similar changes on targeted proteins and cell morphology, suggesting that the eIF3 complex is disrupted by depletion of either eIF3 subunit. Comparative studies using polysome RNA microarray or other techniques will be needed to show whether any RNAs are regulated in a subunit-specific way.

In summary, our results indicate that eIF3b expression is necessary for multiple cellular processes, including cell-cycle progression, focal adhesion maintenance, tumorigenesis, and progression in human cancer cells through global changes in translation and potentially through alteration in the rates of synthesis of specific proteins. Because we did not see depression of all proteins when depleting eIF3b levels in human cancer cells, we speculate that, like eIF4E (7), eIF3b might be another tractable pharmaceutical target for cancer treatment.

No potential conflicts of interest were disclosed.

Conception and design: H. Wang, X. Wang, D. Theodorescu

Development of methodology: H. Wang, X. Wang

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): H. Wang, M. Sanchez-Carbayo, X. Wang

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): H. Wang, Y. Ru, M. Sanchez-Carbayo, X. Wang, J.S. Kieft

Writing, review, and/or revision of the manuscript: H. Wang, Y. Ru, M. Sanchez-Carbayo, X. Wang, J.S. Kieft, D. Theodorescu

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): D. Theodorescu

Study supervision: D. Theodorescu

This work is supported by NIH grants CA075115 and CA104106 (to D. Theodorescu).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.