Targeting epigenetic modifications and gene expression in pancreatic cancer: antitumoral effect of combined therapies
- Autores: Immacolata Maietta
- Directores de la Tesis: Rosana Simón Vázquez (dir. tes.), África González Fernández (dir. tes.)
- Lectura: En la Universidade de Vigo ( España ) en 2025
- Idioma: inglés
- Tribunal Calificador de la Tesis: Monica Neagu (presid.), Susana Magadán Mompó (secret.), Jorge Barbazán García (voc.)
- Programa de doctorado: Programa de Doctorado en Metodología y Aplicaciones en Ciencias de la Vida por la Universidad de Vigo
- Materias:
- Enlaces
- Tesis en acceso abierto en: Investigo
- Resumen
Pancreatic cancers can originate from either exocrine or endocrine cells, being pancreatic ductal adenocarcinoma (PDAC) the most common and aggressive one, accounting for around 85% of the pancreatic tumors. PDAC has a notoriously poor prognosis, with a 5-year survival rate of only 10% of patients. Other pancreatic cancers include neuroendocrine tumors (PNETs), which may be functional (secreting hormones) or non-functional (often asymptomatic until they grow large enough to cause complications). PDAC is characterized by multifactorial etiopathogenesis, involving genetic factors (such as mutations in KRAS, TP53, and BRCA genes), environmental factors (smoking, alcohol), and conditions like chronic pancreatitis and diabetes.
The development of PDAC involves four key stages: initiation (mutations in pancreatic cells), promotion (rapid cell division, forming precancerous lesions called PanIN), progression (genetic mutations lead to invasive cancer), and metastasis (cancer spreads to other organs). Mutations in genes like KRAS (found in 90% of PDAC patients) and TP53 are critical in driving the disease. KRAS mutations, for instance, lock the protein in an active state, continuously signalling cell growth. This pathway, known as Ras/MAPK, is essential in regulating cell growth, differentiation, and survival. Other genetic changes, such as inactivation of tumor suppressor genes like CDKN2A and SMAD4, contribute to the progression of the disease.
Epigenetic modifications, such as DNA methylation and histone changes, also play a significant role in the progression of pancreatic cancer. These modifications can silence tumor suppressor genes or activate oncogenes, contributing to the cancer's aggressive nature. DNA methylation, for example, represses genes when cytosine residues in CpG islands are methylated. Enzymes like DNMTs (DNA methyltransferases) facilitate this process, which can lead to gene silencing and tumor progression. Histone modifications, such as acetylation and methylation, further regulate gene expression. In PDAC, overexpression of EZH2, a histone methyltransferase, promotes drug resistance and immune evasion.
The tumor microenvironment in PDAC is another major factor contributing to the disease's aggressiveness and therapy resistance. This environment includes cancer stem cells, cancer-associated fibroblasts (CAFs), immune cells, and a dense extracellular matrix (ECM) that forms a fibrotic barrier around the tumor. This stromal barrier not only hinders drug penetration but also promotes tumor survival by creating a protective niche for cancer cells. Immune cells, including tumor-infiltrating lymphocytes (TILs), macrophages, and myeloid-derived suppressor cells (MDSCs), play complex roles in the PDAC microenvironment, either supporting or suppressing tumor growth.
Therapeutically, PDAC is notoriously resistant to conventional treatments like chemotherapy, radiation, and immunotherapy. Surgical resection is the only potentially curative treatment but is often only feasible in early-stage disease. Chemotherapy regimens, such as FOLFIRINOX and gemcitabine combined with nab-paclitaxel, are commonly used to treat advanced PDAC, but their effectiveness is very limited. The tumor's dense stroma, hypoxic environment, and immunosuppressive microenvironment contribute to this resistance. Emerging new treatments, including targeted therapies, immunotherapies, and the use of drugs like epigenetic inhibitors, offer promise in overcoming some of these barriers.
The PDAC involves dysregulated signalling pathways, notably in the KRAS/MAPK and Hippo routes, leading to uncontrolled cell growth and survival. Therea are two proteins, YAP-1 (Yes-associated protein 1) and FOSL1 (Fos-like antigen 1), which play critical roles in PDAC progression. Normally, the Hippo pathway suppresses YAP, but when dysregulated, YAP enters the nucleus and activates genes that promote proliferation, survival, and stemness. YAP drives cell cycle regulators like Cyclin D1 and prevents apoptosis by upregulating proteins such as Bcl-xL. It also maintains the cancer stem cell population, contributing to therapy resistance and recurrence.
YAP-1 influences the TME by activating cancer-associated fibroblasts (CAFs), leading to the production of extracellular matrix components like collagen and fibronectin, which provide a physical barrier and pro-survival signals. YAP-1 is also involved in epithelial-to-mesenchymal transition (EMT), enhancing metastasis by activating transcription factors like ZEB1. It drives cell cycle regulators like Cyclin D1 and prevents apoptosis by upregulating proteins such as Bcl-xL.
FOSL-1, part of the AP-1 transcription factor complex, works synergistically with YAP-1. Activated by the MAPK pathway downstream of KRAS mutations, FOSL1 amplifies tumor progression by regulating genes involved in EMT and drug resistance. The YAP1-FOSL1 axis promotes aggressive PDAC behaviour and chemoresistance, making these proteins key therapeutic targets. Their interaction also facilitates immune evasion by recruiting immunosuppressive cells, further limiting immunotherapy efficacy.
The YAP1-FOSL1 axis is a critical driver of PDACs aggressive behaviour, contributing to cell proliferation, survival, immune evasion, and metastasis. This makes it a promising but challenging target for therapies, particularly in combination with other treatments that address the tumor`s dense stroma, hypoxic environment, and immune-suppressive microenvironment.
Innovative research into PDAC is being supported by advances in experimental models, ranging from traditional 2D cell cultures to more advanced 3D systems like spheroids, organoids / tumoroids. These models, particularly organoids derived from patient biopsies, offer valuable insights into the disease's biology and allow personalized medicine approaches. They better replicate the tumor's microenvironment and heterogeneity compared to 2D cultures. However, challenges remain in replicating the full complexity of PDAC, including immune interactions and vascular systems.
Looking ahead, gene therapy and nanotechnology-based drug delivery systems are among the most promising approaches for treating PDAC. For instance, small interfering RNA (siRNA) targeting KRAS mutations has shown potential in preclinical models. These gene-silencing strategies, combined with nanotechnology to improve drug delivery, could overcome some of the challenges posed by the tumor's dense stroma and drug resistance mechanisms.
In conclusion, pancreatic ductal adenocarcinoma is a highly aggressive and lethal cancer with complex genetic, epigenetic, and environmental factors contributing to its development and progression. Despite advances in understanding the molecular and cellular mechanisms driving PDAC, effective treatments remain elusive due to the tumor's heterogeneity, dense stroma, and resistance to conventional therapies. However, emerging approaches, including targeted therapies, immunotherapies, and novel experimental models, are strongly needed.
Taking this in mind, we decided to study the efficacy of a novel combined therapy that targets both the molecular and epigenetic mechanisms involved in the aggressive phenotype of PDAC, with the aim of converting PDAC into a treatable tumor. The work of this doctoral thesis has been structured in 3 different chapters:
Chapter I The chapter focuses on the resistance of PDAC to traditional chemotherapies, and the exploration of novel therapeutic strategies involving epigenetic inhibitors. The chapter outlines the frequent genetic mutations in PDAC and the standard treatments for patients, such as surgery and chemotherapy, depending on whether the tumors are resectable or metastatic. Treatments like gemcitabine, FOLFIRINOX, and nab-paclitaxel target DNA replication, but their antitumoral efficacy is limited due to the development of chemoresistance, particularly to gemcitabine. To address this, the study explores epigenetic inhibitors that target key pathways in the tumoral cell survival. These drugs were developed by the Organic Chemistry group (ORCHID), led by Dr. Angel de Lera at CINBIO, University of Vigo. The inhibitors tested include UVI5008, MS275 (commercial name: Entinostat), Psammaplin A, BIX01294. The drugs target enzymes involved in histone modification and DNA methylation, which are critical for tumor growth and resistance. For instance, UVI5008 inhibits the histone deacetylases HDAC1, HDAC4, and DNA DNA (cytosine-5)-methyltransferase 3A (DNMT3a), and sirtuin 1 (SIRT1). Entinostat is also an HDAC inhibitor, while BIX01294 targets Euchromatic histone-lysine N-methyltransferase 2 (EHMT2). Psammaplin A is a marine metabolite, potent inhibitor of HDAC and DNA methyltransferases and aminopeptidase N. The study also evaluated P53R3, which restores the function of the protein called p53, a tumor suppressor factor.
The study in this chapter aimed to compare the effectiveness of these drugs on three human PDAC cell lines (SKPC-1, MIA PaCa-2, and BxPC-3), both alone and in combination with gemcitabine, trying to find synergistic effects. In vitro results showed that these inhibitors significantly reduced cell viability in a dose-dependent manner across the three cell lines, with UVI5008 and BIX01294 showing the highest efficacy. BxPC-3 was the most sensitive cell line, while MIA PaCa-2, known for its gemcitabine resistance, exhibited some resistance to these drugs. UVI5008 demonstrated broad anti-tumoral activity, reactivating tumor suppressor genes and inducing apoptosis. Entinostat (MS275) showed synergy with gemcitabine, increasing apoptosis and inhibiting proliferation in gemcitabine- resistant pancreatic cells.
Further analysis included real-time cell viability assays using the xCelligence RTCA system, revealing time-dependent effects of the drugs. For example, Entinostat was more effective after 48 hours, while UVI5008 and BIX01294 worked more rapidly. The chapter also introduced three-dimensional (3D) cell cultures to better mimic the tumor microenvironment. These 3D assays confirmed the effectiveness of the drugs, with UVI5008, Entinostat and BIX01294 outperforming gemcitabine, especially in gemcitabine-resistant cell lines like MIA PaCa-2. The 3D models emphasized the importance of the extracellular matrix (ECM) and cell-cell interactions in evaluating drug efficacy.
The combination therapy highlighted the potential for Entinostat and UVI5008 to work synergistically with gemcitabine. This combination achieved higher anti-tumoral activity at lower doses of each drug, suggesting a way to overcome resistance while reducing chemotherapy toxicity. The synergistic effect was most pronounced in MIA PaCa-2 cells, a model for gemcitabine-resistant PDAC, where combinations of Entinostat or UVI5008 plus gemcitabine significantly reduced cell viability, compared to individual treatments.
RNA sequencing (RNA-seq) was performed on cells treated with drug combinations to identify differentially expressed (DE) genes that could explain the observed synergy. This analysis revealed significant transcriptional changes, including the downregulation of genes involved in tumor progression and survival, such as those in the KRAS signalling pathway and epithelial mesenchymal transition (EMT). The combination therapies also impacted estrogenic-related pathways, suggesting a novel mechanism for reducing tumor proliferation in PDAC.
The study identified several synergy candidate (SC) genes that were uniquely mobilized by the drug combinations. In BxPC-3 cells treated with MS275 plus gemcitabine, notable genes like GABRQ and DCLK2 were significantly downregulated, potentially impairing tumor cell viability. In MIA PaCa-2 cells treated with UVI5008 plus gemcitabine, upregulated genes included those linked to anti-tumor activity, such as HPSE2, while genes involved in cancer progression, like SPRY1, were downregulated.
To assess the clinical relevance of these findings, the study correlated the expression of SC genes with patient survival data. Several SC genes, including GRIA1 and KRT19, were associated with improved survival outcomes in PDAC patients, suggesting their potential as biomarkers for therapy response. Additionally, the combination therapies downregulated pro-tumorigenic genes like P-cadherin (CDH3), further supporting their efficacy.
The chapter concludes that either UVI5008 or Entinostat, both in combination with gemcitabine, represent promising therapeutic strategies for gemcitabine-resistant PDAC. These findings underscore the need for personalized therapies based on the genetic and epigenetic profiles of individual tumors. The study also emphasizes the importance of further preclinical testing, including in vivo models and patient-derived organoids, to validate the efficacy of these drug combinations in a clinical setting. Moreover, the development of nanocarriers for these hydrophobic epigenetic inhibitors could enhance their bioavailability and reduce systemic toxicity, potentially translating these results into more effective treatments for PDAC patients.
Chapter II This chapter investigates a novel combined therapy for pancreatic ductal adenocarcinoma (PDAC). The study focuses on improving treatment efficacy by combining three therapeutic approaches: epigenetic inhibition using Entinostat, chemotherapy with gemcitabine, and gene silencing via small interfering RNA (siRNA) targeting key pro-tumoral agents: YAP-1 and FOSL-1. This strategy aims to address both the tumor cells and the surrounding stroma, which plays a crucial role in the development of drug resistance.
As we indicated in chapter I, standard treatments like gemcitabine often fail due to PDAC s dense, fibrotic stroma, which acts as a physical barrier to effective drug delivery. We demonstrated that combination of some epigenetic inhibitors (like Entinostat) plus gemcitabine, were much more effective that the individual drugs, especially in those gemcitabine resistant tumoral cells.
Now we want to go one step further by silencing genes involved in tumor progression. In this study, we focus on YAP-1 and FOSL-1, two transcription factors that play significant roles in cancer cell proliferation, epithelial-mesenchymal transition (EMT), and resistance to therapy. YAP-1 interacts with the KRAS pathway and contributes to the desmoplastic stroma typical of PDAC.
Small interfering RNA (siRNA) can induce efficient gene silencing but it needs a vector to enter inside the tumoral cells. The experimental design included the use of lipoplexes (liposome-formulated containing several siRNAs) to target YAP-1 and FOSL-1 in MIA PaCa-2 cells and stromal fibroblasts. Cell viability and proliferation assays indicated that siRNA-mediated gene silencing has minimal impact on cell viability, but it does reduce cell proliferation, suggesting that while gene silencing alone may not be sufficient to eliminate cancer cells, it can slow down their growth.
Our study explored the combined effect of the siRNA therapy with gemcitabine plus Entinostat. MIA PaCa-2 cells and fibroblasts were treated with gemcitabine + Entinostat after siRNA transfection. The combination therapy results in a significant reduction in cell viability and an increase in cell death compared to treatments with individual therapies using gemcitabine or Entinostat alone. This suggests a synergistic effect between the siRNA-mediated silencing of YAP-1 and FOSL-1 and the chemotherapy.
We also used xenograft mouse models to test the in vivo efficacy of this combination therapy. Immunocompromised nude mice were injected subcutaneously with human MIA PaCa-2 cells to develop tumor, being further treated with liposomes-siRNA targeting YAP-1 and FOSL-1 (peritumoral injections), followed by gemcitabine + Entinostat. Mouse weight, survival and tumor volume were measured over time. The results showed a significant reduction in tumor size compared to untreated controls or to those receiving only individual treatments.
Histological analysis of tumor tissues reveals that the combined therapy induces significant apoptosis (cell death) in the tumoral cells, as evidenced by increased staining of apoptotic markers. Moreover, the fibrotic stroma surrounding the tumor was notably reduced in the treated groups, as shown by Masson s trichrome staining, which measures collagen deposition. This reduction in the stromal content is important because the dense stroma in PDAC often acts as a barrier for an effective drug delivery. By reducing the stroma, the combined therapy may enhance the penetration of chemotherapeutic drugs into the tumor.
The study also examined the presence of innate immune cells in the histological samples. Because we used nude mice (carrying a mutation in the Foxn1 gene, Foxn1nu), they do not have mature T cells, allowing not only allografts but also xenografts (like we performed using human cells).
Immunofluorescence staining shows increased infiltration of immune cells, including natural killer (NK) cells and M1 macrophages into the tumor tissue of treated mice with the combined therapy. M1 macrophages (identified as CD11b+ CD80+) are associated with pro-inflammatory, anti-tumoral responses, while NK cells (CD56+) are known for their ability to kill cancer cells. The presence of these immune cells suggests that the combination therapy not only affects to the tumoral cells, but also stimulates an immune response that contributes to modify the tumor microenvironment. Conversely, the number of M2 macrophages (identified as CD11b+ CD206+), which are associated with tissue repair and immunosuppression, was reduced in the treated groups. This shift from an immunosuppressive to an immune-activating environment may further enhance the anti-tumoral effects of this therapy.
Overall, our study demonstrates the potential of combining epigenetic inhibition, chemotherapy, and RNAi-based gene silencing to overcome the challenges of treating PDAC. The results indicate that the combination of siRNA targeting YAP-1 and FOSL-1 with gemcitabine plus Entinostat effectively reduces tumor size, disrupts the fibrotic stroma, and stimulates a robust anti-tumoral innate immune response. These findings underscore the importance of addressing both the cancer cells and the tumor microenvironment in PDAC therapy.
While the results are very promising, the study highlights the need for further research to fully understand the mechanisms behind the observed synergistic effects and to optimize the treatment regimen. Future investigations should explore the potential of this combination therapy in other models of PDAC, before initiating clinical trials. For example, mice with a humanized immune system, to analyse the therapy in environments including human T cells. Additionally, the development of improved delivery systems for siRNA, such as nanoparticle-based carriers, could enhance the stability and targeting of RNA molecules, further improving therapeutic outcomes. This research opens new avenues for the treatment of PDAC and represents a significant step towards developing more effective therapies for this challenging cancer.
Chapter III This chapter investigates the role of organoid bioengineering in modern medical research, particularly its applications in cancer treatment and drug development. Organoids are three-dimensional models created from patient-derived cells, which replicate the architecture and function of human tissues, including tumors, more accurately than traditional two-dimensional cell cultures. This technology allows personalized disease modelling and a powerful platform for developing targeted therapies. By utilizing organoids derived from patient s cells, we can better understand disease mechanisms and tailor treatments, significantly advancing precision medicine.
Organoids were first conceptualized in the 1940s, and their development has since then been driven by key advances in stem cell biology and tissue engineering. These models are typically generated from embryonic stem cells (ESCs), adult stem cells (ASCs), or induced pluripotent stem cells (iPSCs). Each type of stem cell brings unique capabilities: ESCs can differentiate into any cell type, ASCs replicate the regenerative properties of specific tissues, and iPSCs are reprogrammed to replicate early developmental stages, useful for studying congenital diseases. Organoids derived from these sources faithfully replicate the structure and function of various human organs, including the lungs, liver, pancreas, and brain. Their self-organizing capacity within a three-dimensional matrix allows them to mimic the complexity of human tissues, including cellular diversity, functionality, and response to biochemical signals.
The chapter III emphasizes the growing role of patient-derived tumor organoids (PDOs) in cancer research, particularly in modelling tumor heterogeneity a major barrier to effective cancer treatment. Tumor heterogeneity, both inter- and intra-patient, poses significant challenges in eradicating cancer cells, as various populations of cells within a tumor may respond differently to treatment. PDOs offer a way to address this complexity by preserving the genetic and phenotypic diversity of the original tumor, making them ideal for testing drug responses and developing personalized therapies. These models provide a more physiologically relevant platform for drug screening and for studying cancer biology compared to traditional cell lines and animal models.
PDOs are particularly useful in studying cancers like PDAC. This chapter presents an innovative approach using hybrid organoids (human PDAC cells and murine tumor environment) to perform the characterization of the mechanism involved in the efficacy of the combined therapy tested in Chapter II (epigenetic inhibitors + gemcitabine + siRNA targeting YAP-1 and FOSL-1). By simultaneously targeting both tumor cells and the surrounding stroma, this therapy aims to enhance drug delivery and efficacy. The organoids provide an excellent source of tumor sample, reducing the need for animal testing and facilitating a better characterization of this novel therapy.
A key component of this research was conducted during a three-month stay at Dr. Christine Chio s laboratory at Columbia University in New York. The Chio lab specializes in understanding the biology of PDAC and developing therapeutic interventions. Using genetically modified mouse models, ex vivo co-culture systems, and patient-derived organoid transplant models, the lab investigates PDAC at multiple levels. During this period, valuable experience was gained in 3D culture techniques, molecular biology, and pharmacological treatments applied to PDAC organoids. At the beginning, organoid development from both normal and tumoral cells was the primary focus, followed by mastering techniques such as organoid dissociation, flow cytometry, Western blotting, PCR, and viability assays. The study advanced to treat organoids with pharmacological agents, providing critical insights for testing epigenetic therapies and novel drug combinations in realistic PDAC models. This collaboration has also been extended to the biobank at Alvaro Cunqueiro Hospital in Vigo, focusing on organoids derived from pancreatic, lung, and gastric tumors, further enhancing the clinical relevance of this 3D culture.
As initial step, pancreatic tumor organoids were developed from untreated mouse tumor tissues to evaluate the combined efficacy of siRNA, gemcitabine, and the histone deacetylase inhibitor Entinostat. Organoids were cultured in a three-dimensional matrix, closely mimicking the in vivo conditions. Once established, these organoids were treated with liposomal siRNA targeting YAP-1 and FOSL-1, followed by gemcitabine and entinostat, both alone and in combination.
The results showed that the combination therapy significantly reduced organoid viability compared to individual treatments. Mouse organoids treated with the combination of siRNA, gemcitabine, and entinostat showed the most pronounced reduction in cell viability, demonstrating a synergistic effect, in agreement with the in vivo results (Chapter II).
Morphologically, the treated organoids exhibited substantial structural changes. While untreated organoids retained their rounded, homogeneous shape, those exposed to the combination therapy displayed irregular, fragmented structures, indicating the therapy s disruptive effects on tumor architecture. These observations were further supported by immunofluorescence analysis, which showed a marked reduction in the expression of fibroblast and tumoral markers (-SMA and CK-19, respectively) in treated organoids. This reduction highlights the therapy s ability to target both tumor and stromal cells, disrupting the tumor microenvironment a key factor in PDAC s resistance to conventional treatments.
Our findings suggest that targeting multiple components of the tumor microenvironment could be an effective strategy for overcoming therapeutic resistance in PDAC. By disrupting the interactions between tumor cells and the fibrotic stroma, the combination of liposomal siRNA, gemcitabine, and entinostat may improve drug delivery and enhance therapeutic efficacy. The use of a 3D organoid model was crucial in revealing these interactions, as traditional 2D cultures fail to replicate the complexity of the tumor microenvironment, thereby limiting the relevance of experimental outcomes.
Besides showing the potential of combination therapies, this study also highlights the broader implications of using organoid models in cancer research. Organoids provide a unique platform for studying not only tumor biology but also drug responses and resistance mechanisms in a patient-specific context. As personalized medicine continues to evolve, organoids are expected to play a key role in developing tailored treatments based on an individual s tumor characteristics. Furthermore, their ability to mimic tumor heterogeneity and the tumor microenvironment makes them invaluable for preclinical testing of new drugs and therapeutic combinations.
