Stachnevičiūtė, Gabija

Background/Objectives.
Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal malignancies due to its aggressive nature and resistance to therapy. The epithelial-mesenchymal transition (EMT) drives cancer progression, regulated by transcription factors (TFs) such as SNAI1, SNAI2, ZEB1, ZEB2, and TWIST. This study evaluates EMT-TF expression in PDAC and its clinical relevance.
Methods.
PDAC tissues from 45 patients were analyzed using qRT-PCR and Western blot. Clinical features and survival outcomes were statistically examined for correlations with EMT-TF levels.
Results.
mRNA levels of SNAI1 (16.4-fold, p = 0.02), SNAI2 (21.8-fold, p = 0.028), ZEB1 (17.2-fold, p = 0.037) were significantly elevated in PDAC tissues compared to healthy controls. TWIST showed 3.75-fold increase in PDAC tissue; however, this elevation was not significant (p = 0.124). Corresponding protein-level increases were observed for Snail1/Slug and Zeb1. High SNAI1 expression correlated with peripancreatic invasion (p = 0.026) while ZEB1 overexpression was significantly associated with shorter survival (15.2 vs. 33.3 months, p = 0.037) and remained an independent prognostic factor in multivariate analysis. ZEB2 mRNA was reduced, however, protein levels were elevated. TWIST mRNA overexpression was not reflected at the protein level. Conclusions.
Overexpression of EMT transcription factors SNAI1 and ZEB1 reflect more aggressive histopathological patterns of PDAC. The strong correlation of SNAI1 expression with the expression of other EMT-TFs highlights its’ role in peripancreatic invasion, as well as impact on overall survival and may serve as an argument defining the leading role of SNAI1 in this context.

Pancreatic ductal adenocarcinoma (PDAC) remains largely unresponsive to immunotherapy because of its highly immunosuppressive tumor microenvironment. Aryl hydrocarbon receptor (AHR), a ligand-dependent transcription factor, has emerged as a key regulator of immune homeostasis and inflammation. However, its systemic immunomodulatory role in PDAC, particularly outside the tumor microenvironment, remains poorly understood.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers, partly because the cancer cells can evade the immune system, also the pancreatic cancer cells are highly heterogeneous, and this tumor has a late onset of symptoms, which become visible only in advanced stages. For this study, we wanted to investigate the role of the aryl hydrocarbon receptor (AhR), a potential regulator of immune response and inflammatory pathways in PDAC. Using patient-derived peripheral blood mononuclear cells (PBMCs), we sought to determine how modulation of AhR activity affects the expression and production of immune checkpoint molecules and inflammatory mediators. To reflect the subtle effects of AhR activity on immunity, we divided PBMCs from 22 PDAC patients into four groups: unmodulated control, AhR stimulated by carbidopa, AhR stimulated by tapinarof, and AhR suppressed by bay. After 24 hours of modulation, the PBMCs were frozen for gene expression analysis using RT-PCR. The culture supernatants were also frozen for target protein analysis using ELISA. After experimental analysis all patient results were separated into two parts based on the AhR expression level of the unmodulated group: low AhR and high AhR. The Mann-Whitney test was used to determine the statistical significance in GraphPad software. Our study demonstrated different patterns of gene regulation in response to AhR modulation. In the high AhR expression group, carbidopa significantly downregulated AhR expression (p < 0.05), while in the low AhR expression group, tapinarof induced a significant upregulation of AhR (p < 0.05). Regardless of baseline AhR levels, tapinarof stimulation consistently increased CYP1A1 transcription (p < 0.05) across both groups. Conversely, AhR inhibition with bay resulted in a decreasing trend of CYP1A1 expression in both AhR groups. Interestingly, PTGS2 (encoding COX-2) remained downregulated in AhR-stimulated groups, but no reliable changes were observed. AhR modulation also affected immune checkpoint gene expression. Tapinarof treatment showed an upregulating trend in CD274 (PD-L1) transcription, independent of the initial AhR levels, while CD279 (PD-1) expression remained relatively stable across all conditions. At the protein level, carbidopa treatment was associated with a decreasing trend in soluble PD-L1 concentrations across both AhR expression groups. In contrast, soluble PD-1 levels remained elevated following AhR stimulation with carbidopa and tapinarof. Notably, soluble PGE2 levels showed a decreasing trend in the low AhR group after suppression with bay. These findings suggest the unique immunological role of AhR primary expression, functional activity, and different modulators in PDAC patients' blood. AhR activation enhances CYP1A1 expression, slightly reduces PD-L1 protein production with carbidopa, and affects inflammation-related genes in a context-dependent manner. AhR inhibition attenuates these responses, particularly in patients with low baseline AhR expression. However, these results suggest that AhR may be one of the main targets as a key regulator of immune responses in PDAC, and present new strategies for improving the efficacy of immunotherapeutic interventions.

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.

Aryl hydrocarbon receptor (AHR) is a transcription factor that’s commonly upregulated in pancreatic ductal adenocarcinoma (PDAC). There are evidence that upregulated AHR increases PDAC aggressiveness, invasiveness, migration and decreases survival rates. This can be linked to epithelial-mesenchymal transition, when cells acquire a migratory mesenchymal phenotype. AHR expression levels have been shown to influence epithelial or mesenchymal phenotype in some cancer types, however little is known how targeting AHR would affect cell migration and invasiveness in pancreatic cancer. The aim of this study was to investigate how targeting AHR would affect cell migration and invasiveness in pancreatic cancer cells. Two PDAC cell were used for the study (BxPC-3 and Su.86.86). AHR was silenced by lipofectamine mediated siRNA transfection, inhibited by AHR inhibitor (BAY compound) or knocked-out by using CRISPR-CAS system. After silencing, inhibiting or knocking-out AHR, scratch assay was performed to asses the effect on cell migration in 2D system. AHR protein inhibition influence on cell invasiveness and migration was also assessed in 3D system by growing them in low-adherence plates until they formed spheroids. The spheroids were then moved into adherence allowing plates to asses migration. Alternatively the spheroids were put into an extracellular matrix to asses invasion. The results showed that targeting AHR decreases PDAC cells migration and invasiveness. 2D Scratch assay showed that the migration was decreased by silencing AHR (BxPC-3 by 73 %; Su.86.86 by 54 %), inhibiting (BxPC-3 by 36 %; Su.86.86 – no change) and knocking-out (BxPC-3 by 51 %; Su.86.86 – KO not tested). 3D spheroid assays revealed similar trends of reducing cell migration (by 33-41 % depending on cell line) and invasiveness (by 20-43 % depending on cell line) after inhibiting AHR with BAY compound. Targeting AHR could prove to be a viable strategy in slowing PDAC progression by reduce cell invasiveness and migratory capabilities, however differences between cell lines suggest that such strategy should be pursued as a personalized treatment with other molecular mechanisms in mind.

Introduction: Aryl hydrocarbon receptor (AHR) is a transcription fac -tor that’s commonly upregulated in pancreatic ductal adenocarcinoma(PDAC). There are evidence that upregulated AHR increases cancer aggressiveness, invasiveness, migration and decreases survival rates. This can be linked to epithelial-mesenchymal transition, when cells acquire a migratory mesenchymal phenotype. AHR expression levels have been shown to influence epithelial or mesenchymal phenotype, however the exact mechanism is not yet know. EMT transcription factors (EMT-TF)(ZEB1, ZEB2, SNAI1, SNAI2, TWIST) can regulate EMT, but their relation-ship with AHR or the possibility to decrease PDAC migration and invasion by targeting AHR has not been widely studied. Aims & Methods: The aim of this study was to investigate correlations of AHR and EMT-TF RNA expression in PDAC tissue, patient outcomes and the effects of AHR modulation on PDAC cell migration and invasion. Pancreatic carcinoma tissue and next-to-cancer tissue samples were obtained from 86 patients who underwent partial duodenopancreatectomy (Whip-pleresection) for pancreatic carcinoma. The presence of cancerous tissue in the samples was confirmed histologically. Cancerous and next-to-cancer tissues were taken from the same patients. Next-to-cancer tissues were used as reference controls. Total RNA was extracted after which it was converted to cDNA and RNA expression was analyzed using real time polymerase chain reaction using AHR, GAPDH, ZEB1, ZEB2, SNAI1, SNAI2and TWIST primers. Expression changes were calculated using 2(-Delta Delta CT) method, correlations were calculated using Spearman method, Survival analysis was done using Log-Rank method. PDAC cells (BxPC-3;Su.86.86) were grown in RPMI medium with additives and treated for 24hours with AHR inhibitor (BAY) after which they were seeded for cell migration analysis using scratch assay and 3D spheroid assays and invasive-ness analysis using 3D spheroid method utilizing a basement membranematrix. Results: The results showed that AHR expression change in cancer com-pared to next-to-cancer tissue positively correlated with ZEB1 (r=0.801),SNAI1 (r=0.687) and SNAI2 (r=0.802) expression changes but did not correlate with ZEB2 or TWIST expression changes. Grouping the patient into AHR low (1/2 lowest AHR expression patients) and AHR high (1/2 highest AHR expression patients) showed similar results, with most EMT-TF relative expression being decreased compared to next-to-cancer tissue (AHR0.19-fold, ZEB1 0.15-fold, ZEB2 0.62-fold, SNAI1 0.12-fold, SNAI2 0.21-fold,TWIST – no change). In AHR high group most of EMT-TF relative expression was increased (AHR 11.7-fold, ZEB1 6.6-fold, ZEB2 – no change, SNAI112.4-fold, SNAI2, 67.2-fold, TWIST – no change). Survival analysis of AHR low and AHR high groups revealed a significantly higher survival rates in AHR low group (54 months median) compared to AHR high group (19months median). The results of PDAC cell line migration showed a statistically significant decrease (by 30-49 % depending on cell line and method)as well as decreased invasiveness (20-45 % depending on cell line).Conclusion: AHR plays a role in PDAC patient survival possibly through its involvement with a number of EMT-TF. AHR inhibition could prove a viable strategy in reducing PDAC aggressiveness through EMT process modulation.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths, with a five-year survival rate un-der 10% [1]. Its lethality stems from its aggressive progression and strong chemotherapy resistance, both closely linked to epithelial-mesenchymal transition (EMT). EMT enables epithelial cells to gain migratory mesenchymal traits and is regulated by transcription factors such as SNAIL, SLUG,ZEB1/2, and TWIST [2].Among these, SNAIL plays a major role in metastasis [3], while ZEB1 is as-sociated with poor prognosis [4]. EMT is tightly modulated by networks including ELAVL1, which encodes the RNA-binding protein HuR. HuR stabilizes mRNAs by binding AU-rich elements (AREs) in their 3′ untranslated regions (3′UTRs), promoting their expression [5].Aims & Methods: This study aimed to identify post-transcriptional interactions between ELAVL1 and EMT-related genes and assess their prognostic value in PDAC. mRNA was extracted from cancerous and adjacent normal tissues (n=65), converted to cDNA, and analyzed by RT-PCR.BxPC-3, MiaPaCa-2, and Su.86.86 cell lines were cultured under standard conditions. Immunoprecipitation was performed on 1–2×10⁷ cells using a mouse monoclonal anti-HuR antibody, with IgG as control. qRT-PCR assessed mRNA enrichment. Binding sites were annotated using CISBP-RNA, considering only RNA direct-confirmed 3′UTR motifs. Target 3′UTRsequences were retrieved from GENCODE V47. Statistical analysis was performed using GraphPad Prism, applying the Wilcoxon signed-rank test, Kruskal-Wallis test with Dunn’s correction, and Spearman correlation. Data were presented as median ± interquartile range, with p<0.05considered significant. Results: Expression levels of EMT-related genes and ELAVL1 varied according to survival outcomes. In short-term survivors (1–12 months), SNAIL and SLUG were significantly upregulated (2.19- and 1.92-fold), while ZEB2was downregulated (0.65-fold). Medium-term survivors (13–35 months)showed reduced ZEB1 (0.53), SNAIL (0.30), SLUG (0.35), and TWIST (0.68).Long-term survivors (36–125 months) had the lowest levels of ZEB1 (0.24),ZEB2 (0.50), SLUG (0.24), TWIST (0.35), and ELAVL1 (0.72). ELAVL1 showed strong positive correlations with ZEB1, SNAIL, and SLUG (r = 0.74–0.76),whereas overall survival was weakly negatively correlated with the ex -pression of ZEB1, SNAIL, SLUG, and TWIST (r = -0.25 to -0.32).Although ZEB1, SNAIL, and TWIST did not show significant survival differences individually, low SLUG predicted better early survival (36 vs. 15months, p = 0.0278). ELAVL1 expression was stratified as high and low. High ELAVL1 corresponded to a 71.65-fold increase in ZEB1, whereas highZEB1 reduced ELAVL1 by 96.1%. High ELAVL1 also led to a 312.35-fold in-crease in SNAIL, while low ELAVL1 still supported elevated SNAIL expression (8.45-fold). High SNAIL expression reduced ELAVL1 by 90%, though simultaneous high expression of both resulted in a 3.71-fold ELAVL1 in-crease. High SLUG reduced ELAVL1 by 93%, whereas low SLUG led to a198.11-fold increase. Binding site analysis revealed ELAVL1 targets within3′UTRs: 34 in ZEB1, 1 in SNAIL, and 9 in SLUG. Conclusion: ELAVL1 (HuR) likely plays a central role in regulating EMT in PDAC through stabilization of key transcription factor mRNAs. High levels of ELAVL1, SNAIL, SLUG, and ZEB1 were associated with worse outcomes, particularly in short-term survivors. Conversely, low expression of SLUG was linked to improved survival. The strong correlations and validated binding sites suggest ELAVL1 as a promising prognostic biomarker and potential therapeutic target in PDAC.

Objective It has been suggested that Epithelial-mesenchymal transition (EMT) program might be associated with carcinogenesis of pancreatic adenocarcinoma. Furthermore it has been described the association between ZEB1 and worse survival of PDAC patients. However the post-transcriptional regulation pathways remain unclear as well as the role of other EMT-TFs. The purpose of our study to determine the association of EMT-TFs expression with clinicopathological parameters and prognosis of PDAC. Methods Data of 45 patients following pancreatoduodenectomy for PDAC between 2009- 2017 were analyzed. Patient’s mRNA expression levels of Snail1, Snail2, ZEB1, ZEB2 and TWIST in PDAC were compared with normal pancreatic tissue. The correlations among EMT-TFs and HuR as well as clinicopathological parameters were analyzed. The Kaplan-Meier method and log-rank tests were used for univariate analysis. Results Snail1, Snail2, ZEB1 and Twist expression were accordingly 1643.05 fold, 2186.02- fold, 1723.33-fold and 3.72-fold higher, while ZEB2 expression were 0.53-fold lower when compared to normal pancreatic tissue. Univariate analysis revealed that high expression of all EMT-TFs was associated with worse overall survival (0S) of PDAC patients. High expression levels of ZEB1 were associated with peripancreatic invasion. Conclusions Hyperexpression of EMT-TFs is associated with more aggressive clinicopathological features of pancreatic cancer and worse overall survival.

Purpose: It has been suggested that HuR is involved in the post-transcriptional regulation of the Epithelial-mesenchymal transition (EMT) program through major EMT-activating transcription factors (EMT-TFs) as Snail1, Snail2, ZEB1, ZEB2, TWIST. However, the impact of HuR on post-transcriptional regulation of EMT-TFs and their impact on carcinogenesis in the pancreatic adenocarcinoma (PDAC) still remains unclear. The purpose of our study is to assess the role of transcription factor HuR on the EMT-TFs and to determine the association with clinicopathological parameters and prognosis of PDAC. Methods: Data of 45 patients after pancreatodeduodenectomy for PDAC between 2009- 2017 were analyzed. Patient’s mRNA expression levels of Snail, Snail2, ZEB1, ZEB2 and TWIST in PDAC were compared with normal pancreatic tissue. The correlations among EMT-TFs with clinicopathological parameters were analyzed. The Kaplan-Meier method and log-rank tests were used for univariate analysis. Pancreatic cancer cell line BxPC-3 was transfected with anti-HuR siRNR and RT-PCR analysis was carried out for EMT-TFs expression. Results: Snail1, Snail2, ZEB1 and Twist expression were accordingly 1643.05 fold, 2186.02-fold, 1723.33-fold and 3.72-fold higher, while ZEB2 expression were 0.53-fold lower when compared to normal pancreatic tissue. Univariate analysis revealed that high expression of all EMT-TFs was associated with worse overall survival (0S) of PDAC patients. High expression levels of ZEB1 were associated with peripancreatic invasion. Following siHuR transfection, the upregulation of EMT-TFs expression was observed. Conclusion: Our results suggests that upregulation of HuR significantly affects EMT program in the pancreatic cancer cell lines and might be related with poor outcomes in PDAC patients.

Pancreatic cancer is the fourth highest cause of cancer fatalities, with a 5-year survival rate of less than 10% [1]. The ability of tumor cells to resist treatment and invade surrounding tissues is influenced by a reversible developmental process known as epithelial-mesenchymal transition (EMT). EMT is triggered by transcription factors including SNAIL, SLUG, ZEB1/2 and TWIST [2]. ZEB1 is regarded as the most important EMT-TF, as it promotes pancreatic cancer stemness, invasion, and metastasis [3]. HuR, an RNA-binding protein, is a member of the ELAV family and is found in nearly all carcinomas, including pancreatic cancer. This protein is considered to be a key factor in cancer-related gene expression and EMT regulation, although its specific mechanisms in EMT are not yet fully understood [4]. To develop a new treatment method for pancreatic cancer it is necessary to determine the expression changes of EMT-TFs and HuR in pancreatic cancer pathogenesis. The aim of this study was to evaluate the expression of mRNA molecules responsible for post- transcriptional regulation and EMT in pancreatic cancer tissues. Postoperative pancreatic cancerous and precancerous tissues (n=30) were frozen at -80oC and used for this research. The RNA from the tissues was isolated using TRIzol as instructed by “Abexa” manufacturer. cDNAs were synthesized using a Reverse Transcription Kit and converted to 1 µg. The gene expressions of the tissues were measured using TaqMan® Fast Universal PCR Master Mix and TaqMan probes. The data was standardized using GAPDH and the relative change in expression was computed using the 2-ΔΔCq (Livak) method. The statistical analysis was conducted using GraphPad Prism and the Wilcoxon test with a p-value of <0.05 considered significant. All data was presented as median +/- interquartile range. Our results showed that all genes were down-regulated in comparison between the patient's cancerous and precancerous tissue. ZEB1 was found to have a median expression level of 0.2 with a p-valueof <0.001. ZEB2 had a median of 0.3 with a p-value of 0.00. SNAIL1 had a median of 0.1 with a p-value of <0.005, while TWIST had a median expression level of 0.4 with a p-value of <0.008. The post-transcriptional markerHuR and another marker that plays a role in EMT, known as SLUG, did not show any significant statistical difference as their p-value was greater than 0.05. Although the median was lower, 7 out of 30 patients showed higher ZEB1 expression and 5 of same patients has higher HuR expression. This enables us to categorize patients based on their ZEB1 and HuR expression and analyze clinical features within each group separately. In conclusion, our results suggest that the expression of EMT-TFs has been decreased, indicating EMT has already occurred in cancerous tissues. However, expression of ZEB1 along with HuR were increased in 5 pancreatic cancer patients. This would allow us to conclude that ZEB1 could be regulated by HuR. Grouping patients according to ZEB1 and HuR expression will let us evaluate clinical aspects in one or the other study group for future analysis.