Karvelytė, Gabrielė

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.

Pancreatic cancer (PC) presents a significant challenge in treatment efficacy due to late-stage diagnosis and chemoresistance. The effects of the combination of a selective small-molecule AHR inhibitor and gemcitabine treatmenteffectiveness in PC cells has been a focus of research. This study utilized the PC cell lines BxPC-3 and Su.86.86 to investigate the impact of AHR activity modulation on gene and protein expression related to the gemcitabine response. Assays including viability measurement, combinational index calculation, qRT-PCR, Western blot analysis, immunocytofluorescence, and clonogenic assays, were employed. Additionally, patient tissue samples were analysed for AHR, ELAVL1, and DCK levels. The results show that AHR activity modulation influenced ELAVL1 localization, DCK expression, and gemcitabine response. Inhibition of AHR activity caused synergistic effects with gemcitabine, whereas activation had an antagonistic effect. Regarding colony formation, inhibition of AHR increased gemcitabine effectiveness by 30-41%, whereas activation decreased the response by 11-28%. Patient tissue analysis revealed correlations between AHR, ELAVL1, and DCK mRNA levels and showed increased levels of AHR protein (2.2-fold) and decreased DCK protein levels (36% decrease) in tumor tissue compared to next-to-cancer tissue. These findings demonstrate the potential of AHR modulation to improve gemcitabine treatment outcomes. This study highlights the significance of AHR modulation in influencing the gemcitabine response in PC cells. By inhibiting AHR activity, cells exhibited improved gemcitabine response, offering a promising avenue for enhancing treatment efficacy. These findings suggest that AHR could serve as a target for optimizing gemcitabine treatment and potentially reducing cancer aggressiveness.

Įvadas Viena iš mirtiniausių vėžio formų, kuriai būdinga vos 10 % 5 metų išgyvenamumas, yra kasos vėžys [1]. Dideliam mirtingumo rodikliui turi įtakos agresyvi ligos eiga. Ankstyvosiose stadijose, liga pasireiškia nespecifiniais simptomais, todėl dažnai pacientai yra vėlai diagnozuojami, kuomet vėžys yra pažengęs. Tokiais atvejais intervencijos ir gydymo pasirinkimai yra riboti, o taikomi terapijos metodai nėra pakankamai efektyvūs, dėl dažnai stebimo auglio įgimto, arba įgyto atsparumo gydymui [2]. Norint pagerinti pacientų išgyvenimo galimybes, būtina tirti potencialiai onkogenezėje dalyvajaunčių faktorių įtaką kasos vėžio progresijai, siekiant pritaikyti šias žinias gydymo strategijų vystymui. Šiam tikslui patrauklus tyrimų taikinys yra su onkogenetiniais reiškiniais asocijuojamas baltymas AhR (angl. Aryl Hydrocarbon Receptor). AhR yra nuo ligandų priklausomas transkripcijos faktorius, kuris, aktyvuotas, branduolyje kartu su papildomais koaktyvatoriais, inicijuoja specifinių genų transkripciją. Daugelyje vėžio formų, įskaitant kasos vėžį, stebimas reikšmingas AhR iRNR, pačio transkripcijos faktoriaus raiškos, bei aktyvacijos suintensyvėjimas, lyginant su sveikais audiniais [3]. Toks ryškus baltymo reguliacinis pokytis leidžia daryti prielaidą, dėl galimo jo vaidmens onkogeneziniuose procesuose. Tyrimai su skirtingo tipo vėžiais atskleidė daugialypį AhR vaidmenį navikų progresijoje. Vėžinėse ląstelėse stebima nuolatinė AhR aktyvacija tam tikruose navikuose skatina migraciją, invaziją, angiogenezę ir kitus pyktybėjimo procesus [3, 4]. Tai įrodančių duomenų gausa sudaro įtikinantį argumentą AhR veiklos slopinimo naudai, tačiau, dėl šio baltymo funkcijų įvairovės, toks sprendimas nėra toks paprastas. Pastebėta, jog AhR slopinimas, priklausomai nuo audinio ir ligando prigimties, gali sukelti nepageidautiną poveikį [4]. Todėl, norint pritaikyti AhR baltymą kasos vėžio terapijos kurime, būtini išsamūs baltymo moduliacijos tyrimai. Taigi, šio tyrimo tikslas buvo įvertinti AhR aktyvacijos ir slopinimo įtaką kasos vėžio ląstelių išgyvenamumui ir migracijai. [...].

Introduction Pancreatic cancer (PC) is a notoriously difficult to treat disease with an overwhelmingly high mortality rate worldwide [1]. A major determining factor is the aggressive nature of its progression. In the early stages of the disease, most patients present with nonspecific symptoms, often delaying diagnosis. By the time of diagnosis, the initial tumor has metastasized, making treatment significantly more complicated [2]. Treatment options are very limited therefore exploring different therapeutic approaches is critical. PTGS2 is an enzyme which has been noted to be up-regulated in several gastrointestinal cancers, including PC, and is known to be involved in tumorigenic processes [3]. For this reason, it is a promising target for therapy development and is already applied in the case of colorectal cancer [4]. AhR is a transcription factor which is a point of interest due its many supposed roles in the progression of different types of cancer [5]. Interaction between AhR and PTGS2 has been observed in the event of TCDD induced AhR activation and the inflammatory processes that follow it [6]. This means in normal circumstances there is a relationship between them and investigating this interaction in PC cells could potentially provide a perspective for new research. [...].

Introduction An RNA binding protein, human antigen R (ELAVL1), is a key regulator of a molecular mechanism that is responsible for posttranscriptional gene regulation which is altered in pancreatic ductal adenocarcinoma (PDAC) cells. This, in turn, supports the pro-survival phenotype intrinsic to PDAC cells (1). Therefore, ELAVL1 inhibition could be considered a viable direction for cancer therapeutics (2), however, it is still not clear how different inhibitors affect the cell. [...].