eIF4A1 was highly expressed in pancreatic ductal adenocarcinoma and predicted a poor prognosis
Firstly, we analyzed the expression of eIF4A1 in pan-cancer using data from Gene Expression Profiling Interactive Analysis (GEPIA, http://gepia.cancer-pku.cn/). eIF4A1 was highly expressed in multiple cancer types, including pancreatic adenocarcinoma, thymoma, glioblastoma multiforme, diffuse large B cell lymphoma, and testicular germ cell tumors (Fig. 1a). To evaluate the whole eIF family expression in pancreatic adenocarcinoma, we analyze the RNA-seq data from TCGA and GTEx. Two samples were deleted after data quality control, and finally, 179 cases of pancreatic tumor tissues and 169 cases of normal pancreatic tissues were obtained. We ranked differential expression of all the eIF (Fig. 1b) and the result showed that the expression level of eIF4A1 in pancreatic tumor tissues was significantly higher than in normal tissues (Fig. 1c).
Subsequently, we analyze the prognostic role of eIF4A1 in PDAC patients. eIF4A1 was mainly localized in the cytosol (Fig. 1d), which was consistent with the function of assisting translation initiation. We detected the eIF4A1 expression in a tissue microarray using immunohistochemistry from 53 PDAC patients confirmed by surgeries and pathologists from 2009 (Fig. 1e). The results indicated that patients with high eIF4A1 expression suggest a poor prognosis, the median OS of high eIF4A1 expression patients was significantly shortened compared with patients with low eIF4A1 (6.0 months VS 9.0 months, HR = 2.10, 95% CI: 1.44–5.24, P = 0.0061) (Fig. 1f). Furtherly, we examined the correlations between eIF4A1 expression and multiple clinical features (Table 1, * P < 0.05; χ2 test or Fisher’s exact test). Importantly, we found that a high level of eIF4A1 expression was significantly correlated with tumor size and lymph node metastasis. In summary, these results showed that eIF4A1 was highly expressed in pancreatic adenocarcinoma tissues, and high expression of eIF4A1 suggested a poor prognosis. The expression of eIF4A1 was positively correlated with lymph node metastasis which is a major way for cancer cell metastasis.
Table 1
Correlation between eIF4A1 expression and clinical characteristics of PDAC patients
Clinical Characteristics | High - eIF4A1 | Low - eIF4A1 | P-value |
Age, mean ± SD, years | 66.04 ± 9.69 | 64.10 ± 10.07 | 0.482 |
Male, n (%) | 13 (56.53%) | 21 (70.00%) | 0.311 |
Overall survival, median, months | 6.0 (4.0, 9.0) | 9.0 (8.0, 15.5) | 0.006* |
Differentiation status | | 0.523 |
Well differentiated | 11 (20.75%) | 17 (32.08%) | |
Moderately to poorly differentiated | 12 (22.64%) | 13 (24.53%) |
Tumor size, mean ± SD, cm | 4.57 ± 1.40 | 3.75 ± 1.28 | 0.032* |
Recurrences, n (%) | 17 (73.91%) | 20 (66.67%) | 0.569 |
Location | | 0.267 |
Head, n (%) | 15 (28.30%) | 18 (33.96%) | |
Body/tail, n (%) | 7 (13.21%) | 20 (37.74%) |
Diffusion involvement, n(%) | 1 (1.89%) | 2 (3.77%) |
TNM stage# | | 0.077 |
I – II stage, n (%) | 14 | 24 | |
III – IV stage, n (%) | 9 | 5 |
Lymph node metastasis, n (%) | 14 (63.64%) | 10 (35.71%) | 0.0498* |
Vascular Infiltration, n (%) | 10 (43.48%) | 3 (10.00%) | 0.272 |
# NCCN Version 3.2019 Pancreatic Adenocarcinoma |
* P < 0.05, significant difference |
eIF4A1 targeted c-MYC to regulate the metastasis in pancreatic cancer cells
To elucidate the mechanism underlying eIF4A1 regulating the biological behavior of tumor cells, we searched the GEO database for all the translational profiling by ribosome profiling that included pancreatic cancer cells, and ultimately, datasets GSE120159 was selected for further analysis. The data included 3 Panc-1 cell samples treated by rocaglate CR31B, a small-molecule inhibitor of the eIF4A helicase, and 3 Panc-1 cell samples treated by DMSO. A differential expression analysis using R package DEseq2 identified 179 differentially expressed proteins (DEP) between the CR31B-treated group and the DMSO-treated group, with a p. adjusted (FDR) value < 0.05 and |log2FC (fold change) |> 1 as the cut-offs. Among these proteins, 128 were downregulated and 51 were upregulated. The heatmap displayed the top overexpressed and suppressed molecules (Fig. 2a). The expression of c-MYC was significantly downregulated (log2FC = -1.05, FDR = 0.00192) in the CR31B-treated group compared with the control group, ranked the top 0.6% in all the regulated gene lists.
To furtherly screen the key target of eIF4A to promote metastasis, we repressed the eIF4A1 expression (eIF4A1 siRNA) and analyzed the alternation of protein profile expression compared to the control group. Notably, the protein abundance at 49kDa, which is the molecular weight of c-MYC remarkably decreased after the deletion of eIF4A1 (additional file 1: Figure S1).
Gene Ontology (GO) analysis revealed that the DEPs enriched in the GO terms for 497 biological processes (BPs), 56 cellular components (CCs), 48 molecular functions (MFs) with statistical significance (Fig. 2b). Through the GO analysis, we can conclude that the DEPs enriched in metastasis relevant functions including signal transduction, cytoskeleton, lymph-angiogenesis, cell junction. Gene set enrichment analysis (GSEA) performed with ribosomal profiling showed that the enrichment of EMT-related gene sets reduced significantly after CR31B treatment (Fig. 2c). These results indicated that eIF4A1 could target c-MYC to regulate the biological behaviors of pancreatic cancer cells.
eIF4A1 promoted EMT and metastasis through c-MYC/miR-9 signaling
Recent studies have demonstrated that c-MYC promoted EMT through upregulating the expression of miR-9, and miR-9 could competitively bind with E-cadherin encoding sequence which led to EMT occurrence (20, 28). Before exploring the role of eIF4A1 and c-MYC in the regulation effect in pancreatic cells, we analyzed the expression of eIF4A1 in pancreatic cell lines Panc-1, Capan-2, AsPC-1, MiaPaca-2, and normal pancreatic ductal epithelial cell line HPDE. Western blot showed that eIF4A1 was notably higher expressed in the aggressive pancreatic cancer cell line AsPC-1 and relatively lower expressed in the indolent Capan-2 cell line and normal HPDE cell line (Fig. 3a). Therefore, we selected AsPC-1 and Capan-1 in the follow-up studies. We repressed the eIF4A1 or c-MYC expression in aggressive AsPC-1 cells (eIF4A1 siRNA, c-MYC siRNA) and overexpressed the eIF4A1 or c-MYC expression in indolent Capan-2 cells (pcDNA3.1-eIF4A1, pcDNA3.2-myc). Western blot and qRT-PCR showed that down-regulation of eIF4A1 in aggressive AsPC-1 decreased the expression of EMT-related gene (c-MYC, snail, and miR-9) and increased the expression of E-cadherin (Fig. 3b). Downregulation of c-MYC resulted in the same expression alternation (Figure, 3c), however, repressing the c-MYC expression did not influence the expression of eIF4A1. Accordingly, the results of eIF4A1 and c-MYC upregulation in Capan-2 were consistent with the trends of downregulation experiments (Fig. 3d, 3e). Transwell migration and invasion assays showed that eIF4A1/c-MYC-downregulated AsPC-1 cells displayed significantly lower migratory and invasive abilities (Fig. 3f), and the abilities of eIF4A1/c-MYC upregulated Capan-2 cells increased significantly than control (Fig. 3g). Based on the changes of EMT-related molecule expression level and the changes of migratory and invasive capabilities, these results indicated that eIF4A1 could promote EMT through targeting c-MYC/miR-9 signaling.
Overexpression of eIF4A1 increased the MYC-downregulated AsPC-1 cells’ invasive, migratory, and metastatic capabilities in vitro and in vivo
The relation between eIF4A1 and c-MYC is not simply direct upstream and downstream, recent studies revealed that overexpressed c-MYC could increase the expression level of eIF4A1 reversely (15, 29). To further explore the regulatory relations between eIF4A1 and c-MYC, lentivirus was used to regulate the eIFA1 and c-MYC expression. The setting groups were as follow: 1) e-U-M-D sequential regulation group: overexpressed eIF4A1 expression (Lv- eIF4A1) of AsPC-1 followed by repressing c-MYC expression (Lv-sh-c-MYC); 2) e-U group: singly overexpressed eIF4A1 (Lv- eIF4A1) expression; 3) M-D group: singly repressed c-MYC (Lv-sh-c-MYC) expression; 4): Vector group (Lv-sh-control).
Western blots showed that the expression level of E-cadherin significantly decreased in e-U AsPC-1 cells, and significantly increased in the M-D AsPC-1 cells. The E-cadherin expression level of e-U- M-D AsPC-1 cells was between e-U group and M-D group, however, still higher than the level of vector group cells (Fig. 4a). Consistent with the trends of western blot results, Transwell migration and invasion assays showed that the migratory and invasive abilities of e-U- M-D sequential regulation group cells were superior to the M-D group cells, but inferior to the vector group and e-U group (Fig. 4b). In vivo, the metastatic potentials of the above group cells were examined using a mouse metastasis model via caudal vein injection. The results (Fig. 4c) also showed that the luminescence intensity of e-U-M-D group cells was significantly higher than M-D group cells (1.008e + 10 vs 5.387e + 9, P = 0.0349), accordingly weaker to the e-U group cells (1.008e+ 10 vs 2.410e+ 10, P = 0.2369). Collectively, both in vitro and in vitro results showed that the EMT level and metastatic capabilities of e-U- M-D sequential group cells were between e-U group and M-D group. These results indicated that overexpression of eIF4A1 expression could attenuate the inhibition of MYC-downregulated pancreatic cancer cells’ capabilities of EMT and metastasis.
RocA alone was not inferior to RocA plus Mycro3 joint intervention to inhibit EMT and metastasis in vitro and in vivo
Our previous studies demonstrated that c-MYC was not the only target of eIF4A1 to promote EMT and metastasis. When c-MYC was repressed, eIF4A1 might expert pro-EMT effect through other pathways to compensate for the inhibition. Considering the complex loop relation between eIF4A1 and c-MYC, we adopted joint intervention of RocA (eIF4A1 inhibitor) plus Mycro3 (c-MYC inhibitor) to explore whether joint intervention is superior to the two inhibitors intervention alone.
To select the optimal drug concentration, we conducted series of drug concentration gradient experiments. The western blot results showed that the expression level of eIF4A1 decreased significantly at 100nM RocA and the expression level of c-MYC decreased significantly at 5000nM Mycro3 (Fig. 5a). There was crystal precipitation when concentration increasing. Thus, we selected 100nM RocA and 5000nM Mycro3 in the follow-up studies. To compare the safety and efficiency of different intervention methods, we set 4 groups: 1) RocA + Mycro3 group; 2) RocA group; 3) Myro3 group; 4) DMSO control group. Western blots and qRT-PCR showed that all 3 intervention methods significantly increased the E-cadherin expression level and decreased the c-MYC expression level of AsPC-1 cells (Fig. 5b). Compared with the control group, all 3 intervention methods also remarkably decreased the migratory and invasive abilities. All 3 intervention methods could decrease the EMT level of pancreatic cancer cells (Fig. 5c). However, there was no statistical significance among the 3 groups.
To examine the efficiency and safety of different intervention methods in vivo, we used a mouse metastasis model via caudal vein injection. We found that the mouse mortality rates of RocA group (40.0%) and joint intervention group (62.5%) were relatively high. The autopsy showed that there were multiple cases present with nonocclusive intestine dilation. Considering the possible high-dose and frequent RocA use induced intolerance, we decreased the dose intensity from 5mg/kg/d qd to 2.5mg/kg/d once on alternate day by intraperitoneal injection, and the dosage of mycro3 remains intragastric 100mg/kg/d qd. The mice were well-tolerant to the modified regimen without more death cases. The luminescence intensity from RocA single-dose group was significantly weaker than the control group (1.393e + 9 vs 2.707e + 9, P = 0.0474). However, there were no statistical differences in luminescence intensity between the control group and the myro3 single-dose group/ joint intervention group (Fig. 5d). Taken together, these data demonstrated that the modified regimen of RocA single-dose presented an obvious anti-metastasis effect, even superior to mycro3 or joint-intervention.
To further testify the efficacy of RocA, we used a subcutaneous xenograft nude mice model. The 2-way ANOVA analysis indicated that the tumor volumes of RocA group were significantly smaller than those of the control group (P < 0.0001) (Figure. 5e) which indicated that RocA notably suppressed the growth of tumor. And western blot showed that RocA markedly decreased the expression of eIF4a1, c-MYC, and snail whereas increased the expression of E-cadherin (Fig. 5f) in vivo.