Regulatory Role of ELAVL1 in EMT-Driven Pancreatic Cancer Progression

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths, with a five-year survival rate below 10%. Its aggressiveness and chemotherapy resistance are linked to epithelial-mesenchymal transition (EMT), a process enabling epithelial cells to gain mesenchymal properties, enhancing migration and metastasis. EMT is driven by transcription factors such as SNAIL, SLUG, ZEB1/2, and TWIST. SNAI1 plays a key role in metastasis, while high ZEB1 expression correlates with poor survival. This process is tightly regulated by signalling networks, including ELAVL1, which encodes HuR, a protein that stabilizes mRNAs by binding to AU-rich elements (AREs) in their 3′ untranslated regions (3′UTRs), enhancing translation. Tumor tissues from 65 PDAC patients undergoing surgical resection were analyzed. Total RNA was extracted and converted into cDNA for qRT-PCR. BxPC-3, MiaPaCa-2, and Su.86.86 cell lines were cultured in RPMI medium with 10% fetal bovine serum and 1% antibiotics at 37°C in a 5% CO2 humidified environment. For immunoprecipitation, 1–2 × 107 cells were lysed using a manufacturer-provided protocol. Mouse monoclonal anti-HuR (ELAVL1) antibody was used for protein binding, with normal mouse IgG as a control. Samples were analyzed via qRT-PCR. Binding sites were annotated using the CISBP-RNA database, considering only RNAdirect-confirmed 3′UTR motifs. Target gene 3′UTR sequences were retrieved from GENCODE V47 (GRCh38). Statistical analysis was performed using GraphPad Prism, applying the Kruskal-Wallis test with Dunn’s multiple comparisons and Spearman’s correlation. Data were presented as median with ± interquartile range, with significance at p<0.05. A strong positive correlation was identified between ELAVL1 and the transcription factors ZEB1, SNAI1, and SNAI2 (r = 0.74, 0.76, and 0.76, respectively). Expression levels of EMT-TFs and ELAVL1 were classified as high and low. Elevated ELAVL1 expression was associated with a 71.65-fold increase in ZEB1, whereas high ZEB1 levels corresponded to a 96.1% reduction in ELAVL1 expression. Similarly, high ELAVL1 levels resulted in a substantial upregulation of SNAI1 (312.35-fold), while even low ELAVL1 expression maintained elevated SNAI1 levels (8.45-fold). However, when SNAI1 expression was high, ELAVL1 levels declined by 90%, whereas simultaneous high expression of both factors led to a moderate 3.71-fold increase in ELAVL1. High SNAI2 expression was associated with a 93% reduction in ELAVL1, whereas low SNAI2 levels corresponded to a striking 198.11-fold increase in ELAVL1. Structural analysis identified multiple ELAVL1 binding sites: 34 in ZEB1, 1 in SNAI1, and 9 in SNAI2. Immunoprecipitation analysis confirmed that ELAVL1 can potentially bind to ZEB1, SNAI1, and SNAI2 in MiaPaCa-2 and Su.86.86 cell lines. In conclusion, our findings highlight a significant regulatory interplay between ELAVL1 and key EMT transcription factors in PDAC. The strong positive correlation between ELAVL1 and these genes, along with their observed expression patterns, suggests that ELAVL1 plays a crucial role in EMT regulation. Furthermore, structural mapping identified multiple ELAVL1 binding sites within these genes, reinforcing its post-transcriptional regulatory function. These insights lay the groundwork for further investigations into ELAVL1 as a potential therapeutic target in EMT-driven pancreatic cancer progression.