Reprogramming Of Pancreatic Cancer Cells Research Articles

Abstract 392: MYB regulates a specific subset of hypoxia-induced genes and exhibits altered genomic occupancy under hypoxia

Abstract Hypoxia, an unavoidable microenvironmental stress, greatly influences the tumor cell behavior and therapeutic outcomes. Under low oxygen condition, tumor cells undergo transcriptional reprogramming, largely mediated through hypoxia-inducible factors (HIFs), triggering adaptive response pathways. We recently demonstrated a significant role of MYB in hypoxic survival of pancreatic cancer cells by facilitating adaptive changes in cellular metabolism in collaboration with HIF1α. Here, we employed genome-wide deep sequencing approaches to study the impact of hypoxia on transcriptional reprogramming of pancreatic cancer cells and investigate the role of MYB in the hypoxia-dependent processes. A large number of differentially expressed genes (DEGs) were identified in pancreatic cancer cells subjected to hypoxia (1% O2) and associated with multiple adaptive response pathways. Specifically, we found that pathways related to autophagy, cell migration, epithelial-to-mesenchymal transition, endocytosis, cytoskeleton reorganization were induced, while those associated with RNA processing, ribosome biogenesis, DNA replication and cell cycle were suppressed. To analyze the role of MYB in these adaptive responses, we compared the relative abundance of transcripts in MYB-overexpressing control and MYB knockout (KO) pancreatic cancer cells when cultured under normoxic or hypoxic conditions. We observed that the transcriptional output of MYB was drastically changed under hypoxia with a vast majority of genes being distinctly altered in response to the loss of MYB expression under normoxia and hypoxia. To examine if it resulted from altered genomic occupancy of MYB under normoxia and hypoxia, we compared MYB ChIP-seq peaks under these conditions. We observed a decrease in overall MYB-bound genomic regions under hypoxia despite the fact that we did not observe a decrease in nuclear MYB expression. More interestingly, the genomic occupancy of MYB shifted substantially from regions proximal to transcription start site to distal intergenic or intronic regions under hypoxia. Studies are currently underway to examine the mechanisms underlying the altered MYB genomic occupancy and its impact on adaptive physiological response of pancreatic cancer cells under hypoxia. Together, our findings bring unprecedented insight into the mechanistic role of MYB in hypoxia adaptation and pancreatic cancer pathobiology. Citation Format: Shashi Anand, Mohammad Aslam Khan, Kunwar Somesh Vikramdeo, Seema Singh, Ajay Pratap Singh. MYB regulates a specific subset of hypoxia-induced genes and exhibits altered genomic occupancy under hypoxia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 392.

Altered histone acetylation patterns in pancreatic cancer cell lines induce subtype‑specific transcriptomic and phenotypical changes.

Pancreatic ductal adenocarcinoma (PDAC) is often diagnosed at advanced tumor stages with chemotherapy as the only treatment option. Transcriptomic analysis has defined a classical and basal‑like PDAC subtype, which are regulated by epigenetic modification. The present study aimed to determine if drug‑induced epigenetic reprogramming of pancreatic cancer cells affects PDAC subtype identity and chemosensitivity. Classical and basal‑like PDAC cell lines PaTu‑S, Capan‑1, Capan‑2, Colo357, PaTu‑T, PANC‑1 and MIAPaCa‑2, were treated for a short (up to 96h) and long (up to 30weeks) period with histone acetyltransferase (HAT) and histone deacetylase (HDAC) inhibitors. The cells were analyzed using gene expression approaches, immunoblot analysis, and various cell assays to assess cell characteristics, such as proliferation, colony formation, cell migration and sensitivity to chemotherapeutic drugs. Classical and basal‑like PDAC cell lines showed pronounced epigenetic regulation of subtype‑specific genes through acetylation of lysine 27 on Histone H3 (H3K27ac). Moreover, classical cell lines revealed a significantly decreased expression of HDAC2 and increased total levels of H3K27ac in comparison with the basal‑like cell lines. Following HAT inhibitor treatment, classical cell lines exhibited a loss of epithelial marker gene expression, decreased chemotherapy response gene score and increased cell migration invitro, indicating a tumor‑promoting phenotype. HDAC inhibitor treatment, however, exerted minimal reprogramming effects in both subtypes. Epigenetic reprogramming of classical and basal‑like tumor cells did not have a major impact on gemcitabine response, although the gemcitabine transporter gene SLC29A1 (solute carrier family 29 member 1) was epigenetically regulated.

Metabolomic Investigations into Hypoxia-Mediated Metabolic Reprogramming of Pancreatic Cancer Cells.

Hypoxia is a crucial microenvironmental factor that defines tumor cell growth and aggressiveness. Cancer cells adapt to hypoxia by altering their metabolism. These alterations impact various cellular and physiological functions, including energy metabolism, vascularization, invasion and metastasis, genetic instability, cell immortalization, stem cell maintenance, and resistance to chemotherapy (Li et al. Technol Cancer Res Treat 20:15330338211036304, 2021). Hypoxia-inducible factor-1α (HIF-1α) is known to be a critical regulator of glycolysis that directly regulates the transcription of multiple key enzymes of the glycolysis pathway. Moreover, HIF-1α stabilization can be directly modulated by TCA-derived metabolites, including 2-ketoglutarate and succinate (Infantino et al, Int J Mol Sci 22(22), https://doi.org/10.3390/ijms22115703 , 2021). Overall, the molecular mechanisms underlying the adaptation of cellular metabolism to hypoxia impact the metabolic phenotype of cancer cells. Such adaptations include increased glucose uptake, increased lactate production, and increased levels of other metabolites that stabilize HIF-1α, leading to a vicious circle of hypoxia-induced tumor growth.

Novel strategy for oncogenic alteration-induced lipid metabolism reprogramming in pancreatic cancer.

The pathogenesis of pancreatic cancer involves substantial metabolic reprogramming, resulting in abnormal proliferation of tumor cells. This tumorigenic reprogramming is often driven by genetic mutations, such as activating mutations of the KRAS oncogene and inactivating or deletions of the tumor suppressor genes SMAD4, CDKN2A, and TP53, which play a critical role in the initiation and development of pancreatic cancer. As a normal cell gradually develops into a cancer cell, a series of signature characteristics are acquired: activation of signaling pathways that sustain proliferation; an ability to resist growth inhibitory signals and evade apoptosis; and an ability to generate new blood vessels and invade and metastasize. In addition to these features, recent research has revealed that metabolic reprogramming and immune escape are two other novel characteristics of tumor cells. The effect of the interactions between tumor and immune cells on metabolic reprogramming is a key factor determining the antitumor immunotherapy response. Lipid metabolism reprogramming, a feature of many malignancies, not only plays a role in maintaining tumor cell proliferation but also alters the tumor microenvironment by inducing the release of metabolites that in turn affect the metabolism of normal immune cells, ultimately leading to the attenuation of the antitumor immune response and resistance to immunotherapy. Pancreatic cancer has been found to have substantial lipid metabolism reprogramming, but the mechanisms remain elusive. Therefore, this review focuses on the mechanisms regulating lipid metabolism reprogramming in pancreatic cancer cells to provide new therapeutic targets and aid the development of new therapeutic strategies for pancreatic cancer.

KRAS, A Prime Mediator in Pancreatic Lipid Synthesis through Extra Mitochondrial Glutamine and Citrate Metabolism.

Kirsten rat sarcoma viral oncogene homolog (KRAS)-driven pancreatic cancer is very lethal, with a five-year survival rate of <9%, irrespective of therapeutic advances. Different treatment modalities including chemotherapy, radiotherapy, and immunotherapy demonstrated only marginal efficacies because of pancreatic tumor specificities. Surgery at the early stage of the disease remains the only curative option, although only in 20% of patients with early stage disease. Clinical trials targeting the main oncogenic driver, KRAS, have largely been unsuccessful. Recently, global metabolic reprogramming has been identified in patients with pancreatic cancer and oncogenic KRAS mouse models. The newly reprogrammed metabolic pathways and oncometabolites affect the tumorigenic environment. The development of methods modulating metabolic reprogramming in pancreatic cancer cells might constitute a new approach to its therapy. In this review, we describe the major metabolic pathways providing acetyl-CoA and NADPH essential to sustain lipid synthesis and cell proliferation in pancreatic cancer cells.

Undermining Glutaminolysis Bolsters Chemotherapy While NRF2 Promotes Chemoresistance in KRAS-Driven Pancreatic Cancers.

Pancreatic cancer is a disease with limited therapeutic options. Resistance to chemotherapies poses a significant clinical challenge for patients with pancreatic cancer and contributes to a high rate of recurrence. Oncogenic KRAS, a critical driver of pancreatic cancer, promotes metabolic reprogramming and upregulates NRF2, a master regulator of the antioxidant network. Here, we show that NRF2 contributed to chemoresistance and was associated with a poor prognosis in patients with pancreatic cancer. NRF2 activation metabolically rewired and elevated pathways involved in glutamine metabolism. This curbed chemoresistance in KRAS-mutant pancreatic cancers. In addition, manipulating glutamine metabolism restrained the assembly of stress granules, an indicator of chemoresistance. Glutaminase inhibitors sensitized chemoresistant pancreatic cancer cells to gemcitabine, thereby improving the effectiveness of chemotherapy. This therapeutic approach holds promise as a novel therapy for patients with pancreatic cancer harboring KRAS mutation. SIGNIFICANCE: These findings illuminate the mechanistic features of KRAS-mediated chemoresistance and provide a rationale for exploiting metabolic reprogramming in pancreatic cancer cells to confer therapeutic opportunities that could be translated into clinical trials. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/8/1630/F1.large.jpg.

P53-dependent transcriptional suppression of BAG3 protects cells against metabolic stress via facilitation of p53 accumulation.

Solid tumour frequently undergoes metabolic stress during tumour development because of inadequate blood supply and the high nutrient expenditure. p53 is activated by glucose limitation and maintains cell survival via triggering metabolic checkpoint. However, the exact downstream contributors are not completely identified. BAG3 is a cochaperone with multiple cellular functions and is implicated in metabolic reprogramming of pancreatic cancer cells. The current study demonstrated that glucose limitation transcriptionally suppressed BAG3 expression in a p53‐dependent manner. Importantly, hinderance of its down‐regulation compromised cellular adaptation to metabolic stress triggered by glucose insufficiency, supporting that BAG3 might be one of p53 downstream contributors for cellular adaptation to metabolic stress. Our data showed that ectopic BAG3 expression suppressed p53 accumulation via direct interaction under metabolic stress. Thereby, the current study highlights the significance of p53‐mediated BAG3 suppression in cellular adaptation to metabolic stress via facilitating p53 accumulation.

Metabolic reprogramming of pancreatic cancer cells by protein arginine methyltransferase 3

Metabolic reprogramming of pancreatic cancer cells by protein arginine methyltransferase 3

Metabolic Mechanisms and a Rational Combinational Application of Carboxyamidotriazole in Fighting Pancreatic Cancer Progression after Chemotherapy.

The anticancer and anti-inflammatory effects of carboxyamidotriazole (CAI) have been demonstrated in several studies, but the underlying mechanisms remain to be elucidated. This study showed that CAI caused metabolic reprogramming of pancreatic cancer cells. The inhibition of mitochondrial oxidative metabolism by CAI led to increased glutamine-dependent reductive carboxylation and enhanced glycolytic metabolism. The presence of environmental substances that affect cellular metabolism, such as glutamine and pyruvate, attenuated the anticancer efficacy of CAI. Based on the action of CAI: 1) when glutamine was removed, the NAD+/NADH ratio was decreased, the synthesis of cellular aspartate was reduced, and autophagy flux was blocked; and 2) when glycolysis was pharmacologically inhibited, the ATP level was significantly decreased, the cell viability was greatly inhibited, and the compensatory rescue effect of glutamine was eliminated. When combined with chemotherapy, cotreatment with CAI and the glycolysis inhibitor 2-deoxyglucose (2-DG) inhibited the pancreatic cancer progression after chemotherapy. As the inhibition of mitochondrial oxidative metabolism can explain several anticancer activities of CAI reported previously, including inhibition of calcium entry and induction of reactive oxygen species, we demonstrate that inhibition of mitochondrial oxidative phosphorylation may be the fundamental mechanism of CAI. The combination of CAI and 2-DG causes energy depletion in cancer cells, eliminating the rescue effect of the metabolic environment. Inhibiting pancreatic cancer progression after chemotherapy is a rational application of this metabolism-disturbing combination strategy.

Unique cellular interactions between pancreatic cancer cells and the omentum.

Pancreatic cancer is a common cause of cancer-related mortality. Omental spread is frequent and usually represents an ominous event, leading to patient death. Omental metastasis has been studied in ovarian cancer, but data on its role in pancreatic cancer are relatively scarce and the molecular biology of this process has yet to be explored. We prepared tissue explants from human omental fat, and used conditioned medium from the explants for various in vitro and in vivo experiments designed to evaluate pancreatic cancer development, growth, and survival. Mass spectrometry identified the fat secretome, and mRNA array identified specific fat-induced molecular alternations in tumor cells. Omental fat increased pancreatic cancer cellular growth, migration, invasion, and chemoresistance. We identified diverse potential molecules secreted by the omentum, which are associated with various pro-tumorigenic biological processes. Our mRNA array identified specific omental-induced molecular alternations that are associated with cancer progression and metastasis. Omental fat increased the expression of transcription factors, mRNA of extracellular matrix proteins, and adhesion molecules. In support with our in vitro data, in vivo experiments demonstrated an increased pancreatic cancer tumor growth rate of PANC-1 cells co-cultured for 24 hours with human omental fat conditioned medium. Our results provide novel data on the role of omental tissue in omental metastases of pancreatic cancer. They imply that omental fat secreted factors induce cellular reprogramming of pancreatic cancer cells, resulting in increased tumor aggressiveness. Understanding the mechanisms of omental metastases may enable us to discover new potential targets for therapy.

Alterations of Epigenetic Regulators in Pancreatic Cancer and Their Clinical Implications.

Pancreatic cancer is one of the most aggressive human cancer types with a five-year survival less than 7%. Emerging evidence revealed that many genetic alterations in pancreatic cancer target epigenetic regulators. Some of these mutations are driver mutations in cancer development. Several most important mechanisms of epigenetic regulations include DNA methylation, histone modifications (methylation, acetylation, and ubiquitination), chromatin remodeling, and non-coding ribonucleic acids (RNAs). These modifications can alter chromatin structure and promoter accessibility, and thus lead to aberrant gene expression. However, exactly how these alterations affect epigenetic reprogramming in pancreatic cancer cells and in different stages of tumor development is still not clear. This mini-review summarizes the current knowledge of epigenetic alterations in pancreatic cancer development and progression, and discusses the clinical applications of epigenetic regulators as diagnostic biomarkers and therapeutic targets in pancreatic cancer.

Abstract 10: Silibinin exhibits anti-cachectic and anti-cancerous property by modulating metabolic properties of pancreatic cancer cells

Abstract Cancer cachexia is a systemic syndrome characterized by progressive weight loss of the patient due to muscle wasting and fat depletion with or without anorexia. About 80% of pancreatic ductal adenocarcinoma (PDAC) patients exhibits cachectic phenotype and it significantly contributes in mortality and morbidity of the disease. In present study we have evaluated the effect of bioactive molecule silibinin on pancreatic cancer progression and cachectic properties by utilizing in vitro as well as in vivo models of PDAC. We observed that silibinin inhibits growth and induces apoptosis in multiple pancreatic cancer cell lines in a dose-dependent manner. We also observed silibinin-mediated reduction in the expression of key glycolytic genes and inhibition of glucose uptake and lactate secretion. By performing LC-MS/MS based metabolomics, we observed that silibinin treatment leads to global metabolic alterations in pancreatic cancer cells. Pancreatic cancer cells treated with silibinin exhibited reduced expression of c-MYC level, a key metabolic regulator. Furthermore, we observed that silibinin-mediated STAT3 inhibition leads to reduced c-MYC expression. Ectopic expression of constitutively active STAT3 significantly attenuated the effect of silibinin on c-MYC expression and metabolic phenotype of pancreatic cancer cells. Silibinin treatment also inhibited tumor growth and progression of cachexia. Silibinin treatment to tumor-bearing mice also lead to increased food intake, increased grip strength and body coordination. Overall, our results demonstrate that silibinin exhibits anti-cachectic and anti-cancerous properties by inducing metabolic reprogramming in pancreatic cancer cells. Citation Format: Surendra K. Shukla, Aneesha Dasgupta, Kamiya Mehla, Venugopal Gunda, Enza Vernucci, Joshua Soucheck, Gennifer Goode, Ryan King, Anusha Mishra, Ibha Rai, Sangeetha Natrajan, Nina Chaika, Fang Yu, Pankaj K. Singh. Silibinin exhibits anti-cachectic and anti-cancerous property by modulating metabolic properties of pancreatic cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 10.

Silibinin-mediated metabolic reprogramming attenuates pancreatic cancer-induced cachexia and tumor growth

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths in the US. Cancer-associated cachexia is present in up to 80% of PDAC patients and is associated with aggressive disease and poor prognosis. In the present studies we evaluated an anti-cancer natural product silibinin for its effectiveness in targeting pancreatic cancer aggressiveness and the cachectic properties of pancreatic cancer cells and tumors. Our results demonstrate that silibinin inhibits pancreatic cancer cell growth in a dose-dependent manner and reduces glycolytic activity of cancer cells. Our LC-MS/MS based metabolomics data demonstrates that silibinin treatment induces global metabolic reprogramming in pancreatic cancer cells. Silibinin treatment diminishes c-MYC expression, a key regulator of cancer metabolism. Furthermore, we observed reduced STAT3 signaling in silibinin-treated cancer cells. Overexpression of constitutively active STAT3 was sufficient to substantially revert the silibinin-induced downregulation of c-MYC and the metabolic phenotype. Our in vivo investigations demonstrate that silibinin reduces tumor growth and proliferation in an orthotopic mouse model of pancreatic cancer and prevents the loss of body weight and muscle. It also improves physical activity including grip strength and latency to fall in tumor-bearing mice. In conclusion, silibinin-induced metabolic reprogramming diminishes cell growth and cachectic properties of pancreatic cancer cells and animal models.

Abstract 2154: ERK/MAPK pathway inhibits tumorigenesis and cellular reprogramming of pancreatic cancer cells

Abstract Reprogrammation of cancer cells into stem-like state is one the most important mechanisms implicated in tumor initiation, metastasis and resistance to chemotherapies. Acquisition of stem-like cell property is directly correlated with the level of activation of the ERK/MAPK pathway. Our study focuses on the implication of the downregulation of this pathway during the transition of pancreatic benign neoplasms to pancreatic ductal adenocarcinoma. This transformation is directly correlated with the acquisition of stem-like cells properties. Conversely, hyperactivation of the ERK pathway using phosphatase inhibitors abrogates the stem-like cell phenotype. We analyzed the variation of genes expression by microarrays analysis between cells obtained from pancreatic intraepithelial neoplasias and pancreatic cancer. These analyses implicate the expression of DUSPs and genes related to stem cell biology in tumor progression during pancreatic cancer. Citation Format: Benjamin le Calvé, Xavier Deschenes-Ximard, Filippos Kottakis, Véronique Bourdeau, Frédéric Lessard, Karine Moineau-Vallée, Emmanuelle Saint-Germain, Julien Fitamant, Rushika Miriam Perera, Nabeel Bardeesy, Gerardo Ferbeyre. ERK/MAPK pathway inhibits tumorigenesis and cellular reprogramming of pancreatic cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2154. doi:10.1158/1538-7445.AM2015-2154

MUC16-mediated activation of mTOR and c-Myc reprograms pancreatic cancer metabolism.

MUC16, a transmembrane mucin, facilitates pancreatic adenocarcinoma progression and metastasis. In the current studies, we observed that MUC16 knockdown pancreatic cancer cells exhibit reduced glucose uptake and lactate secretion along with reduced migration and invasion potential, which can be restored by supplementing the culture media with lactate, an end product of aerobic glycolysis. MUC16 knockdown leads to inhibition of mTOR activity and reduced expression of its downstream target c-MYC, a key player in cellular growth, proliferation and metabolism. Ectopic expression of c-MYC in MUC16 knockdown pancreatic cancer cells restores the altered cellular physiology. Our LC-MS/MS based metabolomics studies indicate global metabolic alterations in MUC16 knockdown pancreatic cancer cells, as compared to the controls. Specifically, glycolytic and nucleotide metabolite pools were significantly decreased. We observed similar metabolic alterations that correlated with MUC16 expression in primary tumor tissue specimens from human pancreatic adenocarcinoma cancer patients. Overall, our results demonstrate that MUC16 plays an important role in metabolic reprogramming of pancreatic cancer cells by increasing glycolysis and enhancing motility and invasiveness.

Metabolic reprogramming induced by ketone bodies diminishes pancreatic cancer cachexia.

BackgroundAberrant energy metabolism is a hallmark of cancer. To fulfill the increased energy requirements, tumor cells secrete cytokines/factors inducing muscle and fat degradation in cancer patients, a condition known as cancer cachexia. It accounts for nearly 20% of all cancer-related deaths. However, the mechanistic basis of cancer cachexia and therapies targeting cancer cachexia thus far remain elusive. A ketogenic diet, a high-fat and low-carbohydrate diet that elevates circulating levels of ketone bodies (i.e., acetoacetate, β-hydroxybutyrate, and acetone), serves as an alternative energy source. It has also been proposed that a ketogenic diet leads to systemic metabolic changes. Keeping in view the significant role of metabolic alterations in cancer, we hypothesized that a ketogenic diet may diminish glycolytic flux in tumor cells to alleviate cachexia syndrome and, hence, may provide an efficient therapeutic strategy.ResultsWe observed reduced glycolytic flux in tumor cells upon treatment with ketone bodies. Ketone bodies also diminished glutamine uptake, overall ATP content, and survival in multiple pancreatic cancer cell lines, while inducing apoptosis. A decrease in levels of c-Myc, a metabolic master regulator, and its recruitment on glycolytic gene promoters, was in part responsible for the metabolic phenotype in tumor cells. Ketone body-induced intracellular metabolomic reprogramming in pancreatic cancer cells also leads to a significantly diminished cachexia in cell line models. Our mouse orthotopic xenograft models further confirmed the effect of a ketogenic diet in diminishing tumor growth and cachexia.ConclusionsThus, our studies demonstrate that the cachectic phenotype is in part due to metabolic alterations in tumor cells, which can be reverted by a ketogenic diet, causing reduced tumor growth and inhibition of muscle and body weight loss.