ELK3 is highly expressed in PDAC
To clarify the role of ELK3 in PDAC, we first analyzed the expression of ELK3 using Oncomine database. The mRNA level of ELK3 was significantly upregulated in pancreatic cancer tissues in two datasets (Badea Pancreas Statistics, P = 6.36E-9, Segara Pancreas Statistics, P = 8.46E-5) (Fig. 1A). Furthermore, overexpression of ELK3 in PDAC was confirmed by analyzing two NCBI Gene Expression Omnibus (GEO) datasets (GSE15471, P = 4.78E-10, GSE71987, P = 1.08E-7) (Fig. 1B) and The Cancer Genome Atlas (TCGA) dataset (P < 0.05) (Fig. 1C). The Kaplan-Meier curve analyses revealed that elevated ELK3 level indicated poorer overall survival (OS) and relapse free survival (RFS) (P < 0.05) (Fig. 1D). To further determine the expression level of ELK3, immunohistochemical (IHC) analysis of the tissue microarray (TMA) containing 70 cases of PDAC tissues with corresponding normal tissues were conducted. As shown in Fig.1E and F, the expression level of ELK3 was higher in PDAC tissues than that in adjacent normal tissues. Overall, we conclude that ELK3 is frequently elevated in PDAC, and the roles of ELK3 remain to be explored.
ELK3 promotes PDAC cells proliferation, migration and invasion in vitro
To investigate the functional roles of ELK3 in PDAC, ELK3 was knocked down and overexpressed in PANC-1 and MIA PaCa-2 cells, which have been used in our previous experiments [27]. We constructed three shRNAs (sh-1, sh-2, sh-3) and a lentiviral overexpression vector targeting ELK3. The qRT-PCR results showed that ELK3 level was significantly down-regulated or up-regulated in PDAC cells transfected with the indicated shRNAs or overexpression vector respectively (Fig. S1A). Among the three shRNAs, sh-1 (sh-ELK3) was selected for further study because of its highest inhibitory efficiency. In addition, the successful knockdown and overexpression of ELK3 were confirmed at protein levels (Fig. S1B). Colony formation and EdU assays revealed that knockdown of ELK3 suppressed the proliferation of both PANC-1 and MIA PaCa-2 cells (Fig. 2A-B). Conversely, overexpression of ELK3 had the opposite effects on cell proliferation (Fig. 2A-B). The wound-healing assay demonstrated that ELK3 depletion inhibited the mobility of PANC-1 cells, while forced expression of ELK3 increased the migration speed of them (Fig. 2C). Correspondingly, the effect was confirmed by transwell migration and matrigel invasion assays (Fig.2D). Additionally, similar results were obtained in MIA PaCa-2 cells (Fig. 2C-D). To conclude, our findings indicate that ELK3 may be involved in cell proliferation, migration and invasion, acting as a positive regular.
ELK3 promotes pancreatic tumor growth and metastasis in vivo
To verify the function of ELK3 in pancreatic tumor growth and metastasis in vivo, we injected pancreatic cancer cells with stable knockdown or overexpression of ELK3 into the armpit or tail vein of nude mice. The results showed that tumors in sh-ELK3/MIA PaCa-2 group grown more slowly than those in sh-NC/ MIA PaCa-2 group, and this phenomenon was also reflected by tumor volume and final tumor weight (Fig. 3A-C). Additionally, the volume and weight of xenograft tumors in ELK3/PANC-1 group were significantly higher than the control tumors of PANC-1 cells (Fig. 3D-F). In the lung metastasis model, we discovered that the metastatic nodules in mice injected with sh-ELK3/ MIA PaCa-2 cells were less than in mice injected with sh-NC/ MIA PaCa-2 cells (Fig. 3G-H), while a higher number of metastatic nodules was observed in mice injected with ELK3/PANC-1 cells than in these injected with Ctrl/PANC-1 cells (Fig. 3I-J). Taken together, these in vivo results suggest that ELK3 plays an important role in pancreatic tumor growth and metastasis.
ELK3 is required in TGFβ-induced EMT
As we all know, EMT process plays important roles in cancer cell invasion and tumor metastasis [16]. TGFβ is a potent inducer of EMT, and TGFβ stimulation could irritate changes in cell morphology and biological behavior [28, 29]. Our results have showed that ELK3 was associated with malignant progression of pancreatic cancer. This prompted us to explore the underlying effects of ELK3 on TGFβ-induced EMT process. Western blot and confocal immunofluorescence analysis indicated that TGFβ treatment markedly decreased E-cadherin and increased N-cadherin and Vimentin expression (Fig. 4A-B). However, in sh-ELK3/PANC-1 and sh-ELK3/MIA PaCa-2 cells simultaneous treatment with TGFβ, these molecular events induced by TGFβ were completely abolished by ELK3 depletion (Fig. 4A-B). Additionally, ELK3 knockdown also effectively quenched the wound healing, cell migration and invasion abilities induced by TGFβ in PANC-1 and MIA PaCa-2 cells (Fig. 4C-D). These results demonstrate that ELK3 is crucial for TGFβ-induced EMT in PDAC.
ELK3 promotes the progression of pancreatic cancer cells through Wnt/β-catenin signaling pathway
Considering the importance of β-catenin signaling in the development of cancer and EMT process [30, 31], we were inspired to explore whether ELK3 could regulate Wnt/β-catenin signaling pathway in pancreatic cancer. To verify the effects of ELK3 on Wnt/β-catenin, we performed western blot assay. The results showed that neither knockdown nor overexpression of ELK3 significantly affected the total β-catenin level (Fig. 5A). However, ELK3 depletion decreased the level of nuclear β-catenin and increased the cytosolic β-catenin levels, whereas ELK3 overexpression had the opposite effects on the subcellular location of β-catenin (Fig. 5A). TOP-Flash and FOP-Flash luciferase reporters were used to further test the activity of the Wnt/β-catenin signaling pathway. As shown in Fig. S2, the TOP/FOP luciferase activities in PDAC cells transfected with sh-ELK3 groups were much lower than in the control group, and were significantly higher in ELK3 overexpressed cells. Furthermore, to determine whether β-catenin is essential for the functions of ELK3, ELK3/PANC-1 and ELK3/MIA PaCa-2 cells were transfected with siβ-catenin. We observed that β-catenin suppression dampened ELK3-mediated cell wound healing (Fig. 5B), migration and invasion (Fig. 5C). These data confirmed Wnt/β-catenin signaling pathway plays a vital role in ELK3-mediated pancreatic cancer progression.
ZEB1 transcriptionally activates ELK3 expression
To dissect the molecular mechanism of ELK3 overexpression in PDAC, we first explored the genetic or epigenetic dysregulation of ELK3 in pancreatic adenocarcinoma (TCGA, Firehose Legacy) from the cBioPortal database. However, we discovered no evidence regarding the dysregulation of ELK3 at the genetic or methylation levels (Fig. S3A), suggesting that genetic alterations (mutation, amplification and deletion) and methylation modification may not be the main causes of the overexpression of ELK3 in PDAC. Then, we would explore its overexpression at transcriptional level. As one of the most important EMT-inducing transcription factors, ZEB1 not only transcriptionally represses but also activates some EMT-related genes, and its overexpression promotes tumorigenesis and metastasis in human carcinomas [32-34]. A recent study showed that ZEB1 could collaborate with ELK3 to regulate gene expression [35]. Thus, we are inspired to explore whether ZEB1 could transcriptionally activate ELK3 expression in PDAC. Analyzing from the JASPAR database, we found the binding motifs of ZEB1 and five potential ZEB1 binding sites on the ELK3 promoter (Fig. 6A, B and Fig. S3B). QRT-PCR and western blotting analysis demonstrated that forced expression of ZEB1 significantly increased ELK3 mRNA and protein levels, while ZEB1 deletion exhibited an opposite effect (Fig. 6C, D and Fig. S3C, D). ChIP-qPCR results indicated that ZEB1 could interact with ELK3 promoter within the -641 to -631bp region (Fig. 6E-F). In addition, we found a significantly decreased ZEB1 enrichement in the ELK3 promoter following ZEB1 silencing, while ZEB1 overexpression increased occupancy of ZEB1 in the ELK3 promoter (Fig. 6G). To further investigate the regulatory role of ZEB1 on ELK3 transcription, we constructed wild type (WT) and mutant (Mut) reporter plasmids. For mutant plasmid, several bases were replaced in the binding site#2, and wild type reporter contained intact binding site#2 (Fig. 6H). Luciferase reporter assays showed that overexpression of ZEB1 could activate the luciferase activity of WT plasmids, but failed to activate Mut reporters (Fig. 6I). To sum up, we concluded that ZEB1 binds to the region between -641 to -631 bp of the ELK3 promoter to activate its’ transcriptional activity in pancreatic cancer.
ELK3 is critical for the function of ZEB1 on PDAC cell proliferation and migration
ZEB1 has been shown to promote the proliferation and metastasis of PDAC cells [34]. Since ZEB1 could increase ELK3 level in PDAC, we investigated whether ELK3 was necessary for mediating the effect of ZEB1 on the cellular proliferation and metastasis of PDAC. As shown in Fig. 7A and Fig. S4A, ZEB1-enhanced cell proliferation was inhibited by ELK3 knockdown. Moreover, ZEB1-enhanced cell migration and invasion ability was decreased when ELK3 was knockdown (Fig. 7B, C and Fig. S4B, C). As an EMT-activator, western blot and immunofluorescence results shown that ZEB1 could promote the EMT process of PDAC cells, while this effect was reversed by ELK3 knockdown (Fig. 7D, E, and Fig. S4D, E). In summary, these results demonstrated that ELK3 was important for the oncogenic effect of ZEB1 on PDAC progression.
Clinical pathological features of ZEB1 and ELK3 in PDAC patients
First, we found that ZEB1 expression was also upregulated in GSE15471 (P = 1.75E-07) and GSE71987 (P = 6.79E-06) datasets (Fig. S5A). Scatter plot analysis showed a positive correlation between the mRNA levels of ZEB1 and ELK3 (GSE15471, R = 0.6792, P<0.0001, GSE71987, R = 0.8890, P < 0.0001) (Fig. S5B). In addition, the protein level of ZEB1 was examined in above 70 paired pancreatic cancer tissues and paracancerous tissues, and the results of IHC analysis showed that ZEB1 was highly expressed in pancreatic cancer tissues compared with matched normal tissues (Fig. 8A-B). Moreover, nearly 64.3% of pancreatic cancer samples where ZEB1 was more highly expressed presented stronger ELK3 staining, while approximately 71.2% of those with lower ZEB1 expression exhibited weaker ELK3 staining (Fig. 8C). Pearson correlation analysis confirmed the positive correlation between ZEB1 and ELK3 proteins in TMAs (R = 0.848, P < 0.0001) (Fig. S5C). Based on the median expression of ELK3 or ZEB1 in TMAs, the samples were divided into ELK3 high expression group and ELK3 low expression group or ZEB1 high expression group and ZEB1 low expression group. As shown in Table1 and Fig. S5D, ELK3 expression was significantly higher in pancreatic cancer tissues of T3 stage, N1stage, distant metastasis M1 stage and AJCC stage IIB-IV than in these of T1-T2 stage, N0 stage, M1 stage and AJCC-IIA stage respectively (P < 0.05 for all). ZEB1 expression was positive with pathological grade, N stage and AJCC stage (P < 0.05 for all, Table2, Fig. S5E). Kaplan-Meier survival analysis showed that patients with higher ELK3 and ZEB1 expression level were both associated with worse overall survival (OS) (Fig. 8D,E). Moreover, the combination of these two elements demonstrated that pancreatic cancer individuals with the expression of ZEB1highELK3high had an even worse OS rate than any other groups (P = 0.0014) (Fig. 8F). Taken together, ZEB1 and ELK3 predicated poor survival in clinical samples and might be indicators of efficient prognostic factors in PDAC patients.