Sensitivity Of Pancreatic Cancer Cells Research Articles

Exposure to endocrine disruptor DEHP promotes the progression and radiotherapy resistance of pancreatic cancer cells by increasing BMI1 expression and properties of cancer stem cells

Most patients diagnosed with pancreatic cancer are initially at an advanced stage, and radiotherapy resistance impact the effectiveness of treatment. This study aims to investigate the effects of endocrine disruptor Di-(2-ethylhexyl) phthalate (DEHP) on various biological behaviors and the radiotherapy sensitivity of pancreatic cancer cells, as well as its potential mechanisms. Our findings indicate that exposure to DEHP promotes the proliferation of various cancer cells, including those from the lung, breast, pancreas, and liver, in a time- and concentration-dependent manner. Furthermore, DEHP exposure could influence several biological behaviors of pancreatic cancer cells in vivo and vitro. These effects include reducing cell apoptosis, causing G0/G1 phase arrest, increasing migration capacity, enhancing tumorigenicity, elevating the proportion of cancer stem cells (CSCs), and upregulating expression levels of CSCs markers such as CD133 and BMI1. DEHP exposure can also increase radiation resistance, which can be reversed by downregulating BMI1 expression. In summary our research suggests that DEHP exposure can lead to pancreatic cancer progression and radiotherapy resistance, and the mechanism may be related to the upregulation of BMI1 expression, which leads to the increase of CSCs properties.

EPIGALLOCATECHIN GALLATE SENSITIZES PANCREATIC CANCER CELLS TO GEMCITABINE BY MODULATING MICRORNA EXPRESSION PROFILE

Pancreatic cancer is a leading cause of cancer-related
 deaths in developed countries, with a 5-year average
 survival rate of less than 5%, making it a malignant
 disease. Gemcitabine (GEM), an FDA-approved
 pyrimidine antimetabolite, is widely used in pancreatic
 cancer treatment. However, due to its targeting of
 all dividing cells, severe side effects are frequently
 observed in patients undergoing GEM treatment for
 pancreatic cancer. Consequently, meta-analyses have
 shown that the combination of GEM with other active
 compounds significantly improves the 1-year survival
 rate of pancreatic cancer patients. Epigallocatechin-
 3-gallate (EGCG), an active compound found in
 green tea (Camellia sinensis), has proven anticancer
 activity in pancreatic cancer. Subsequent studies have
 demonstrated that EGCG enhances the sensitivity of
 pancreatic cancer cells to GEM. However, among
 the studies conducted to date, the impact of the
 combination of EGCG and GEM on the expression
 of critical microRNAs, which act as key epigenetic
 regulators in pancreatic cancer pathology, has not
 been investigated. This study aims to determine the
 cytotoxic and apoptotic effects of the combination of
 GEM and EGCG on PANC1 cells and to examine its
 effectiveness on the expression levels of microRNAs
 involved in cancer progression.
 Material and Method
 Cytotoxicity of GEM and EGCG in PANC1 cells was
 assessed using the WST-1 assay, and combination
 effects were analyzed using isobologram analysis.
 Apoptosis analysis was performed using the Annexin
 V method. miRNA isolation was conducted with the
 miRNeasy Kit, followed by cDNA synthesis using
 the miScript II Reverse Transcription Kit. Changes
 in the expression of miRNAs involved in cancer
 cell proliferation, apoptosis, and metastasis were
 examined using real-time qRT-PCR analysis.
 Results
 The IC50 values for GEM at 24, 48, and 72 hours were
 determined as 72.85 μM, 26.55 μM, and 9.38 μM,
 respectively. EGCG's IC50 values at 24, 48, and 72
 hours were determined as 64.36 μM, 48.34 μM, and
 19.73 μM, respectively. When combined at a 2:3 ratio
 (GEM: EGCG) at 24 and 72 hours, a synergistic effect
 was observed, while at 48 hours, a strong synergistic
 drug interaction was observed. At a concentration of
 only 26.55 μM, the group treated with GEM showed
 a 4.2-fold increase in apoptosis compared to the
 control group receiving fresh medium. In contrast,
 the combination treatment (EGCG: 4.71 μM, GEM:
 3.14 μM) resulted in a remarkable 12.04-fold increase
 in apoptosis. After combination treatment, the
 expression of tumor suppressor miRNAs, miR-137,
 and miR-130a-3p, increased, while the expression of
 oncogenic miRNAs, including miR-27a-3p, miR-425-
 5p, miR-183-5p, miR-187-3p, miR-21-5p, miR-324-5p,
 and miR-486-5p, decreased.
 Conclusion
 EGCG can sensitize pancreatic cancer to GEM
 through epigenetic mechanisms, shedding light on
 novel therapeutic approaches.

Abstract LB_C03: Protein translation inhibition enforces histone deacetylase inhibitor activity resulting in synergistic pancreatic cancer cell death

Abstract Management of pancreatic cancer presents significant challenges due to late-stage diagnosis, aggressive tumor biology, and limited efficacy of existing treatment options. Gemcitabine and FOLFIRINOX (folinic acid, 5-FU, irinotecan hydrochloride, oxaliplatin) are standard treatments for advanced pancreatic cancer. Despite these therapies the overall 5-year survival rate for pancreatic cancer patients is at 12%, largely due to drug resistance. Thus, there is a critical need to explore new therapeutic strategies. Our study explores a small molecule combination that induces epigenetic reprogramming as a potential novel therapeutic strategy for pancreatic cancer. We have identified marked sensitivity of pancreatic cancer cells to the combination of a novel inhibitor of RNA helicase eIF4A, DMPatA (MZ735), and a histone deacetylase (HDAC) inhibitor, romidepsin. A short-term, low dose romidepsin treatment, when combined with MZ735, triggers persistent global histone acetylation, transcriptional upregulation, replication inhibition, and augmented DNA damage. This combined effect induces a synergistic anti-proliferative response in pancreatic cancer cells. We have identified a tolerable protocol for this combination regimen in animals, demonstrating its efficacy against tumor growth in a MIA PaCa-2 xenograft mouse model of pancreatic cancer. Treatment with the combination resulted in inhibition and then complete suppression of tumor growth in animals for 39 days. Our in vitro studies aimed at understanding the mechanism of this combination treatment showed that at low doses of romidepsin monotherapy, histone acetylation rapidly faded, while robust acetylation persisted to 18 and 24 hours with the combination. Additionally, R-loop accumulation was observed using confocal microscopy and DRIP-qPCR, along with a reduction in replication fork speed, which might account for the identified DNA damage and genome instability. The combination treatment significantly reduces acetyl-CoA and c-MYC protein levels and disrupts glycolysis and oxidative phosphorylation. We show that a structurally different HDAC inhibitor exhibits the same synergistic effects when co-administered with MZ735, confirming a unique method of eliciting synthetic lethality in PDAC cells. In conclusion, this combination shows significant antitumor activity in both in vitro and in vivo models of pancreatic cancer and warrant translation to the clinic. Citation Format: Maryam Safari, Luigi Scotto, Agnes Basseville, Thomas Litman, Haoran Xue, Luba Petrukhin, Ping Zhou, Christopher Damoci, Minhzhao Zhu, Kenneth Hull, Kenneth P Olive, Antonio T Fojo, Daniel Romo, Susan E Bates. Protein translation inhibition enforces histone deacetylase inhibitor activity resulting in synergistic pancreatic cancer cell death [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_C03.

Overexpression of ZNF488 supports pancreatic cancer cell proliferation and tumorigenesis through inhibition of ferroptosis via regulating SCD1-mediated unsaturated fatty acid metabolism

BackgroundPancreatic cancer is a malignancy with high mortality. Once diagnosed, effective treatment strategies are limited and the five-year survival is extremely poor. Recent studies have shown that zinc finger proteins play important roles in tumorigenesis, including pancreatic cancer. However, it remains unknown on the clinical significance, function and underlying mechanisms of zinc finger protein 488 (ZNF488) during the development of pancreatic cancer.MethodsThe clinical relevance of ZNF488 and stearoyl-CoA desaturase 1 (SCD1) was examined by analyzing the data from The Cancer Genome Atlas (TCGA) and immunohistochemical staining of the tissue microarray. Gain-of-function and loss-of-function experiments were performed by transfecting the cells with overexpressing lentivirus and siRNAs or shRNA lentivirus, respectively. The function of ZNF488 in pancreatic cancer was assessed by CCK8, colony formation, EdU staining, PI/Annexin V staining and xenografted tumorigenesis. Chip-qPCR assay was conducted to examine the interaction between ZNF488 and the promoter sequence of SCD1. Transcription activity was measured by dual luciferase reporter assay. mRNA and protein expression was detected by qRT-PCR and immunoblotting experiment, respectively. Fatty acid was quantified by gas chromatography mass spectrometry.ResultsZNF488 was overexpressed in pancreatic cancer samples compared with normal tissues. High expression of ZNF488 predicted the poor prognosis of the patients. In vitro, ZNF488 upregulation contributed to the EuU cooperation, proliferation and colony formation of MIAPaCa-2 and PANC-1 cells. Based on PI/Annexin V and trypan blue staining results, we showed that ZNF488 suppressed the ferroptosis and apoptosis of pancreatic cancer cells. Mechanistically, ZNF488 directly interacted with the promoter sequence of SCD1 gene and promoted its transcription activity, which resulted in enhanced palmitoleic and oleic acid production, as well as the peroxidation of fatty acid. In vivo, ZNF488 overexpression promoted the xenograted tumorigenesis of PANC-1, which was reversed by SCD1 knockdown. Importantly, combination of erastin and SCD1 inhibitors A939572 completely blunted the growth of ZNF488 overexpressed MIAPaCa-2 and PANC-1 cells. Usage of A939572 or erastin recovered the sensitivity of pancreatic cancer cells to the treatment of gemcitabine. Lastly, we found a positive correlation between ZNF488 and SCD1 in pancreatic cancer patients based on TCGA and immunohistochemical staining results.ConclusionOverexpression of ZNF488 suppresses the ferroptosis and apoptosis to support the growth and tumorigenesis of pancreatic cancer through augmentation of SCD1-mediated unsaturated fatty acid metabolism. Combination of SCD1 inhibitors, ferroptosis inducers or gemcitabine could be applied for the treatment of pancreatic cancer with overexpression of ZNF488.

Sonodynamic Therapy of NRP2 Monoclonal Antibody-Guided MOFs@COF Targeted Disruption of Mitochondrial and Endoplasmic Reticulum Homeostasis to Induce Autophagy-Dependent Ferroptosis.

The lethality and chemotherapy resistance of pancreatic cancer necessitates the urgent development of innovative strategies to improve patient outcomes. To address this issue, we designed a novel drug delivery system named GDMCN2,which uses iron-based metal organic framework (Fe-MOF) nanocages encased in a covalent organic framework (COF) and modified with the pancreatic cancer-specific antibody, NRP2. After being targeted into tumor cells, GDMCN2gradually release the sonosensitizer sinoporphyrin sodium (DVDMS) and chemotherapeutic gemcitabine (GEM) and simultaneously generated reactive oxygen species (ROS) under ultrasound (US) irradiation. This system can overcome gemcitabine resistance in pancreatic cancer and reduce its toxicity to non-targeted cells and tissues. In a mechanistic cascade, the release of ROS activates the mitochondrial transition pore (MPTP), leading to the release of Ca2+ and induction of endoplasmic reticulum (ER) stress. Therefore, microtubule-associated protein 1A/1B-light chain 3 (LC3) is activated, promoting lysosomal autophagy. This process also induces autophagy-dependent ferroptosis, aided by the upregulation of Nuclear Receptor Coactivator 4 (NCOA4). This mechanism increases the sensitivity of pancreatic cancer cells to chemotherapeutic drugs and increases mitochondrial and DNA damage. The findings demonstrate the potential of GDMCN2 nanocages as a new avenue for the development of cancer therapeutics.

Targeting AHR Increases Pancreatic Cancer Cell Sensitivity to Gemcitabine through the ELAVL1-DCK Pathway.

The aryl hydrocarbon receptor (AHR) is a transcription factor that is commonly upregulated in pancreatic ductal adenocarcinoma (PDAC). AHR hinders the shuttling of human antigen R (ELAVL1) from the nucleus to the cytoplasm, where it stabilises its target messenger RNAs (mRNAs) and enhances protein expression. Among these target mRNAs are those induced by gemcitabine. Increased AHR expression leads to the sequestration of ELAVL1 in the nucleus, resulting in chemoresistance. This study aimed to investigate the interaction between AHR and ELAVL1 in the pathogenesis of PDAC in vitro. AHR and ELAVL1 genes were silenced by siRNA transfection. The RNA and protein were extracted for quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot (WB) analysis. Direct binding between the ELAVL1 protein and AHR mRNA was examined through immunoprecipitation (IP) assay. Cell viability, clonogenicity, and migration assays were performed. Our study revealed that both AHR and ELAVL1 inter-regulate each other, while also having a role in cell proliferation, migration, and chemoresistance in PDAC cell lines. Notably, both proteins function through distinct mechanisms. The silencing of ELAVL1 disrupts the stability of its target mRNAs, resulting in the decreased expression of numerous cytoprotective proteins. In contrast, the silencing of AHR diminishes cell migration and proliferation and enhances cell sensitivity to gemcitabine through the AHR-ELAVL1-deoxycytidine kinase (DCK) molecular pathway. In conclusion, AHR and ELAVL1 interaction can form a negative feedback loop. By inhibiting AHR expression, PDAC cells become more susceptible to gemcitabine through the ELAVL1-DCK pathway.

ROS-Sp1 axis is involved in thermochemotherapy-enhanced sensitivity of pancreatic cancer cells to gemcitabine.

Gemcitabine (GEM)-based chemotherapy represents the first choice for locally unresectable advanced pancreatic cancer, while the benefit is limited due to acquired chemoresistance or drug delivery insufficiency. Hyperthermia treatment potentially improves the clinical efficacy of GEM. However, the underlying mechanism is incompletely revealed. Our study aims to investigate the effect and involved mechanism of thermochemotherapy on cell survival. Pancreatic cancer cells PANC-1 and ASPC-1 were either treated with GEM or thermochemotherapy, then cell viability, apoptosis, migration, invasion, reactive oxygen species (ROS) production, and Sp1 expression were evaluated. The results showed that GEM dose and time-dependently affected cell viability, and 30 μM GEM achieved favorable effect in suppressing cancer cell growth. Meanwhile, hyperthermia preconditioning (43°C for 60 min) 24 h before GEM treatment yielded superior effect than other treatment sequence. GEM caused significant cell apoptosis and proapoptotic genes of both cancer cells, and thermochemotherapy further promoted apoptosis and genes transcription, accompanied by excessive ROS production. Thermochemotherapy was superior to GEM in suppressing cell migration and invasion of pancreatic cancer cells. Meanwhile, GEM significantly reduced Sp1 mRNA and protein and its downstream gene Cox-2, and thermochemotherapy further suppressed their expressions. ROS elimination with N-acetyl-l-cysteine notably neutralizes the suppressive effect of GEM and thermochemotherapy on cell growth and expressions of Sp1 and Cox-2. In conclusion, thermochemotherapy inhibited pancreatic cell viability, migration and invasion, and promoted cell apoptosis by inducing excessive ROS production, which subsequently suppressed Sp1 expression and the downstream Cox-2.

Autophagy Inhibition Increased Sensitivity of Pancreatic Cancer Cells to Carbon Ion Radiotherapy.

Pancreatic cancer has the poorest survival rate among all cancer types. Therefore, it is essential to develop an effective treatment strategy for this cancer. We performed carbon ion radiotherapy (CIRT) in human pancreatic cancer cell lines and analyzed their survival, apoptosis, necrosis, and autophagy. To investigate the role of CIRT-induced autophagy, autophagy inhibitors were added to cells prior to CIRT. To evaluate tumor formation, we inoculated CIRT-treated murine pancreatic cancer cells on the flank of syngeneic mice and measured tumor weight. We immunohistochemically measured autophagy levels in surgical sections from patients with pancreatic cancer who received neoadjuvant chemotherapy (NAC) plus CIRT or NAC alone. CIRT reduced the survival fraction of pancreatic cancer cells and induced apoptotic and necrotic alterations, along with autophagy. Preincubation with an autophagy inhibitor accelerated cell death. Mice inoculated with control pancreatic cancer cells developed tumors, while those inoculated with CIRT/autophagy inhibitor-treated cells showed significant evasion. Surgical specimens of NAC-treated patients expressed autophagy comparable to control patients, while those in the NAC plus CIRT group expressed little autophagy and nuclear staining. CIRT effectively killed the pancreatic cancer cells by inhibiting their autophagy-inducing abilities.

Thymoquinone affects the gemcitabine sensitivity of pancreatic cancer by regulating collagen via hypoxia inducible factor-1α.

Objective: To clarify the potential therapeutic effects of thymoquinone (TQ) on pancreatic cancer and its gemcitabine (GEM) sensitivity. Methods: The expression levels of hypoxia inducible factor-1α (HIF-1α), collagens (COL1A1, COL3A1, and COL5A1), and transforming growth factor-β1 (TGFβ1) in pancreatic cancer and para-carcinoma tissues were compared using immunohistochemical methods, and their relationships with TNM staging were analyzed. The effects of TQ on apoptosis, migration, invasion, and GEM sensitivity of pancreatic cancer cells were assessed using in vitro and in vivo experiments. Western blot and immunohistochemistry were used to detect the expression levels of HIF-1α, extracellular matrix (ECM) production pathway-related proteins, and TGFβ/Smad signaling pathway-related proteins. Results: The expression levels of HIF-1α, COL1A1, COL3A1, COL5A1, and TGFβ1 in pancreatic cancer tissues were significantly higher than those in para-carcinoma tissues and correlated with TNM staging (p < 0.05). TQ and GEM administration inhibited the migration and invasion of the human pancreatic cancer cell line PANC-1 and promoted the apoptosis of PANC-1 cells. The combination of TQ and GEM was more effective than GEM alone. Western blot analysis showed that the expression levels of HIF-1α, ECM production pathway-related proteins, and TGFβ/Smad signaling pathway-related proteins were significantly decreased when TQ was used to treat PANC-1 cells (p < 0.05), and the expression levels of these proteins in the TQ + GEM group were significantly more decreased than those in the GEM group. Overexpression or knockdown of HIF-1α in PANC-1 cells showed the same effects as those induced by TQ administration. In vivo experiments showed that in PANC-1 tumor-bearing mice, tumor volume and tumor weight in mice treated with GEM and TQ were significantly lower than those in control or GEM-treated mice, whereas cell apoptosis was significantly increased (p < 0.05). Western blot and immunohistochemistry results showed that the levels of HIF-1α, ECM production pathway-related proteins, and TGFβ/Smad signaling pathway-related proteins in the GEM + TQ treatment group were further decreased compared to the control group or the GEM treatment group (p < 0.05). Conclusion: In pancreatic cancer cells, TQ can promote apoptosis, inhibit migration, invasion, and metastasis, and enhance the sensitivity to GEM. The underlying mechanism may involve the regulation of ECM production through the TGFβ/Smad pathway, in which HIF-1α plays a key role.

Matrix stiffness triggers chemoresistance through elevated autophagy in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) has a signature of extremely high matrix stiffness caused by a special desmoplastic reaction, which dynamically stiffens along with the pathological process. The poor prognosis and low five-year survival rate of PDAC are partly owing to chemoresistance triggered by substrate stiffness. Understanding the potential mechanisms of matrix stiffness causing PDAC chemoresistance is of great significance. In this study, methacrylated gelatin hydrogel was used as platform for PANC-1 and MIA-PaCa2 cell culture. The results indicated that compared to soft substrate, stiff substrate distinctively reduced the gemcitabine sensitivity of pancreatic cancer. Intriguingly, transmission electron microscopy, immunofluorescence staining, western blot and qRT-PCR assay showcased that the number of autophagosomes and the expression of LC3 were elevated. The observations indicate that matrix stiffness may regulate the autophagy level, which plays a vital role during chemoresistance. In brief, soft substrate exhibited low autophagy level, while the counterpart displayed elevated autophagy level. In order to elucidate the underlying interaction between matrix stiffness-mediated cell autophagy and chemoresistance, rescue experiments with rapamycin and chloroquine were conducted. We found that inhibiting cell autophagy dramatically increased the sensitivity of pancreatic cancer cells to gemcitabine in the stiff group, while promoting autophagy-driven chemoresistance in the soft group, demonstrating that matrix stiffness modulated chemoresistance via autophagy. Furthermore, RNA-seq results showed that miR-1972 may regulate autophagy level in response to matrix stiffness. Overall, our research shed light on the synergistic therapy of PDAC combined with gemcitabine and chloroquine, which is conducive to promoting a therapeutic effect.

Effects of chloroquine and hydroxychloroquine on the sensitivity of pancreatic cancer cells to targeted therapies.

Approaches to improve pancreatic cancer therapy are essential as this disease has a very bleak outcome. Approximately 80% of pancreatic cancers are pancreatic ductal adenocarcinomas (PDAC). PDAC is a cancer which is difficult to effectively treat as it is often detected late in the disease process. Almost all PDACs (over 90%) have activating mutations in the GTPase gene KRAS. These mutations result in constitutive KRas activation and the mobilization of downstream pathways such as the Raf/MEK/ERK pathway. Small molecule inhibitors of key components of the KRas/Raf/MEK/ERK pathways as well as monoclonal antibodies (MoAbs) specific for upstream growth factor receptors such insulin like growth factor-1 receptor (IGF1-R) and epidermal growth factor receptors (EGFRs) have been developed and have been evaluated in clinical trials. An additional key regulatory gene frequently mutated (∼75%) in PDAC is the TP53 tumor suppressor gene which controls the transcription of multiple genes involved in cell cycle progression, apoptosis, metabolism, cancer progression and other growth regulatory processes. Small molecule mutant TP53 reactivators have been developed which alter the structure of mutant TP53 protein and restore some of its antiproliferative activities. Some mutant TP53 reactivators have been examined in clinical trials with patients with mutant TP53 genes. Inhibitors to the TP53 negative regulator Mouse Double Minute 2 (MDM2) have been developed and analyzed in clinical trials. Chloroquine and hydroxychloroquine are established anti-malarial and anti-inflammatory drugs that also prevent the induction of autophagy which can have effects on cancer survival. Chloroquine and hydroxychloroquine have also been examined in various clinical trials. Recent studies are suggesting effective treatment of PDAC patients may require chemotherapy as well as targeting multiple pathways and biochemical processes.

Hypoxia activated HGF expression in pancreatic stellate cells confers resistance of pancreatic cancer cells to EGFR inhibition.

Epidermal growth factor receptor (EGFR) is an essential target for cancer treatment. However, EGFR inhibitor erlotinib showed limited clinical benefit in pancreatic cancer therapy. Here, we showed the underlying mechanism of tumor microenvironment suppressing the sensitivity of EGFR inhibitor through the pancreatic stellate cell (PSC). The expression of alpha-smooth muscle actin (α-SMA) and hypoxia marker in human pancreatic cancer tissues were detected by immunohistochemistry, and their correlation with overall survival was evaluated. Human immortalized PSC was constructed and used to investigate the potential effect on pancreatic cancer cell lines in hypoxia and normoxia. Luciferase reporter assay and Chromatin immunoprecipitation were performed to explore the potential mechanisms invitro. The combined inhibition of EGFR and Met was evaluated in an orthotopic xenograft mouse model of pancreatic cancer. We found that high expression levels of α-SMA and hypoxia markers are associated with poor prognosis of pancreatic cancer patients. Mechanistically, we demonstrated that hypoxia induced the expression and secretion of HGF in PSC via transcription factor HIF-1α. PSC-derived HGF activates Met, the HGF receptor, suppressing the sensitivity of pancreatic cancer cells to EGFR inhibitor in a KRAS-independent manner by activating the PI3K-AKT pathway. Furthermore, we found that the combination of EGFR inhibitor and Met inhibitor significantly suppressed tumor growth in an orthotopic xenograft mouse model. Our study revealed a previously uncharacterized HIF1α-HGF-Met-PI3K-AKT signaling axis between PSC and cancer cells and indicated that EGFR inhibition plus Met inhibition might be a promising strategy for pancreatic cancer treatment. This study was supported by The National Natural Science Foundation of China.

Dysregulation of HNF1B/Clusterin axis enhances disease progression in a highly aggressive subset of pancreatic cancer patients.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy and is largely refractory to available treatments. Identifying key pathways associated with disease aggressiveness and therapeutic resistance may characterize candidate targets to improve patient outcomes. We used a strategy of examining the tumors from a subset of PDAC patient cohorts with the worst survival to understand the underlying mechanisms of aggressive disease progression and to identify candidate molecular targets with potential therapeutic significance. Non-negative matrix factorization (NMF) clustering, using gene expression profile, revealed three patient subsets. A 142-gene signature specific to the subset with the worst patient survival, predicted prognosis and stratified patients with significantly different survival in the test and validation cohorts. Gene-network and pathway analysis of the 142-gene signature revealed dysregulation of Clusterin (CLU) in the most aggressive patient subset in our patient cohort. Hepatocyte nuclear factor 1 b (HNF1B) positively regulated CLU, and a lower expression of HNF1B and CLU was associated with poor patient survival. Mechanistic and functional analyses revealed that CLU inhibits proliferation, 3D spheroid growth, invasiveness and epithelial-to-mesenchymal transition (EMT) in pancreatic cancer cell lines. Mechanistically, CLU enhanced proteasomal degradation of EMT-regulator, ZEB1. In addition, orthotopic transplant of CLU-expressing pancreatic cancer cells reduced tumor growth in mice. Furthermore, CLU enhanced sensitivity of pancreatic cancer cells representing aggressive patient subset, to the chemotherapeutic drug gemcitabine. Taken together, HNF1B/CLU axis negatively regulates pancreatic cancer progression and may potentially be useful in designing novel strategies to attenuate disease progression in PDAC patients.

Pyroptosis-related genes regulate proliferation and invasion of pancreatic cancer and serve as the prognostic signature for modeling patient survival

ObjectivePancreatic ductal adenocarcinoma (PDAC) has high mortality and poor prognosis. Pyroptosis can influence the prognosis of patients by regulating the proliferation, invasion, and metastasis of cancer cells. However, the role of pyroptosis-related genes (PRGs) in PDAC remains unclear.MethodsIn this study, based on the Cancer Genome Atlas (TCGA) cohort of PDAC samples, univariate Cox analysis and LASSO regression analysis were used to screen the prognostic PRGs and establish the gene signature. To further evaluate the functional significance of CASP4 and NLRP1 in PDAC, we also conducted an in vitro study to explore the mechanism of CASP4 and NLRP1 regulating the occurrence and development of PDAC. Finally, we investigated the relationship between CASP4 and NLRP1 expression levels and drug sensitivity in pancreatic cancer cells.ResultsA risk prediction model based on CASP4 and NLRP1 was established, which can distinguish high-risk patients from low-risk patients (P < 0.001). Both internal validation and external GEO data sets validation demonstrate good predictive capability of the model (AUC = 0.732, AUC = 0.802, AUC = 0.632, P < 0.05). In vitro, CCK8 and Transwell assay suggested that CASP4 may accelerate the progression of PDAC by promoting proliferation and migration of pancreatic cancer cells, while NLRP1 has been found to have tumor suppressive effect. It should be noted that knockdown of CASP4 reduced the level of coke death, the expression levels of acetyl-CoA carboxylase, FASN, SREBP-1 and SREBP-2 were decreased, and the number of lipid droplets was also significantly reduced. Moreover, the enrichment of signaling pathways showed that NLRP1 was significantly correlated with MAPK and RAS/ERK signaling pathways, and knocking down NLRP1 could indeed up-regulate p-ERK expression. Finally, high expression of CASP4 and low expression of NLRP1 increased the sensitivity of pancreatic cancer cells to ERK inhibitors.ConclusionsIn especial, CASP4 can promote tumor progression by promoting the synthesis and accumulation of fatty acids, while NLRP1 acts on RAS/ERK signaling pathway. Both of genes play an important role in the diagnosis and treatment of PDAC, which may also affect the inhibitors of MAPK/ERK efficiency.

Starvation mediates pancreatic cancer cell sensitivity to ferroptosis via ERK1/2, JNK and changes in the cell mesenchymal state.

Pancreatic cancer is a highly metastatic and therapy-resistant disease. In the present study, the prospects of a novel approach to kill pancreatic cancer cells were examined: Starvation combined with ferroptosis induction. Established pancreatic cancer cell lines (Miapaca2, Panc-1, Su.86.86 and T3M4), as well as a unique cell line, Capan-26, which was originally derived in the authors' laboratory, were used. Cells were deprived from growth factors, amino acids and pseudo-starved using treatment with mTOR inhibitors; erastin was used to induce ferroptosis. Cell viability and lipid peroxidation measurements using flow cytometry revealed that the starved pancreatic cancer cells reacted differently to ferroptosis induction: The Panc-1, Su.86.86 and T3M4 cells gained sensitivity, while the Miapaca2 cells acquired resistance. Fluorescence microscopy revealed that ERK1/2 translocated to the nucleus of the starved pancreatic cancer cells. Moreover, ERK1/2 pharmacological inhibition with SCH772984 prevented erastin-induced ferroptosis in the starved Panc-1, Su.86.86 and T3M4 cells. Confocal microscopy also indicated JNK activation. However, the inhibition of this kinase revealed its unexpected role in oxidative stress management: Treatment with the JNK inhibitor, SP600125, increased the viability of pseudo-starved cells following erastin treatment. In addition, the FBS-starved Miapaca2 and Capan-26 cells transitioned between epithelial and mesenchymal cell states. The results were further confirmed using wound healing assays, western blot analysis and microscopic analysis of epithelial-to-mesenchymal transition (EMT) markers. Mesenchymal properties were associated with a higher sensitivity to erastin, whereas epithelial-like cells were more resistant. Finally, it was demonstrated that compounds targeting EMT-related signaling pathways increased cell sensitivity to erastin. On the whole, these results confirm that in starved pancreatic cancer cells, ERK1/2 and JNK signaling, as well as switching between epithelial and mesenchymal states mediates sensitivity to erastin and reveal novel therapeutic prospects of the combination of starvation with ferroptosis induction.

Wild type and gain of function mutant TP53 can regulate the sensitivity of pancreatic cancer cells to chemotherapeutic drugs, EGFR/Ras/Raf/MEK, and PI3K/mTORC1/GSK-3 pathway inhibitors, nutraceuticals and alter metabolic properties.

TP53 is a master regulator of many signaling and apoptotic pathways involved in: aging, cell cycle progression, gene regulation, growth, apoptosis, cellular senescence, DNA repair, drug resistance, malignant transformation, metastasis, and metabolism. Most pancreatic cancers are classified as pancreatic ductal adenocarcinomas (PDAC). The tumor suppressor gene TP53 is mutated frequently (50–75%) in PDAC. Different types of TP53 mutations have been observed including gain of function (GOF) point mutations and various deletions of the TP53 gene resulting in lack of the protein expression. Most PDACs have point mutations at the KRAS gene which result in constitutive activation of KRas and multiple downstream signaling pathways. It has been difficult to develop specific KRas inhibitors and/or methods that result in recovery of functional TP53 activity. To further elucidate the roles of TP53 in drug-resistance of pancreatic cancer cells, we introduced wild-type (WT) TP53 or a control vector into two different PDAC cell lines. Introduction of WT-TP53 increased the sensitivity of the cells to multiple chemotherapeutic drugs, signal transduction inhibitors, drugs and nutraceuticals and influenced key metabolic properties of the cells. Therefore, TP53 is a key molecule which is critical in drug sensitivity and metabolism of PDAC.

Fructose-1,6-bisphosphatase 1 (FBP1) is an independent biomarker associated with a favorable prognosis in esophageal adenocarcinoma

IntroductionDespite modern multimodal therapeutic regimens, the prognosis of esophageal adenocarcinoma (EAC) is still poor and there is a lack of biological markers estimating the patients’ prognosis. Fructose-1,6-biphosphatase (FBP1) is a key enzyme in gluconeogenesis and is associated with tumor initiation in several cancers. Therefore, this study aims to characterize its implication for EAC patients.Methods and materialsA total of 571 EAC patients who underwent multimodal treatment between 1999 and 2017 were analyzed for FBP1 expression using immunohistochemistry.Results82.5% of the EACs show FBP1 expression in the tumor albeit with different intensities categorizing specimens accordingly into score 0 (no expression), score 1 (weak expression), score 2 (moderate expression) and score 3 (strong expression) (score 1 = 25.0%, score 2 = 35.9%, score 3 = 21.5%). Intratumoral FBP1 expression was significantly associated with a better prognosis (p = 0.024). This observation was particularly relevant among patients who received primary surgery without neoadjuvant treatment (p = 0.004). In multivariate analysis, elevated FBP1 expression was an independent biomarker associated with a favorable prognosis.DiscussionDespite being associated with a favorable prognosis, the majority of patients with high FBP1 expression also require individualized therapy options to ensure long-term survival. Recently, it has been shown that the presence of the FBP1 protein increases the sensitivity of pancreatic cancer cells to the bromodomain and extraterminal domain (BET) inhibitor JQ1.ConclusionWe described for the first time the prognostic and possibly therapeutic relevance of FBP1 in EAC. The efficiency of the BET inhibitor in EAC should be verified in clinical studies and special attention should be paid to the effects of neoadjuvant therapy on FBP1 expression.

Expression, Prognostic Value, and Functional Mechanism of the KDM5 Family in Pancreatic Cancer.

Background: The histone lysine demethylase KDM5 family is an important epigenetic state-modifying enzyme family. Increasing evidence supports that epigenetic abnormalities in the KDM5 family are related to multiple cancers in humans. However, the role of the KDM5 family in pancreatic cancer is not clear, and related research is very scarce. Methods: R software, Kaplan–Meier Plotter, cBioPortal, TIMER, LinkedOmics, STRING, Metascape, TISIDB, and the GSCA Lite online tool were utilized for bioinformatics analysis. Results: KDM5A/B/C was significantly overexpressed in many kinds of tumor tissues, including pancreatic adenocarcinoma (PAAD), while the expression of KDM5D was significantly downregulated. The high expression of KDM5A/B/C was related to poor clinical features, such as worse treatment efficacy, higher tumor grade, and more advanced clinical stage. Patients with a family history of breast cancer and melanoma, history of drinking or history chronic pancreatitis were more likely to have KDM5A/B/C gene abnormalities, which were related to a variety of adverse clinical features. The results of gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) pathway analyses of the KDM5 family and its 800 co-expressed genes showed that many gene terms related to cell proliferation, migration and many carcinogenic pathways. Notably, we found that the expression level of KDM5A/B/C was positively correlated with the expression of multiple key driver genes such as KRAS, BRCA1, and BRCA2 etc. In addition, PPI network analysis showed KDM5 family proteins have strong interactions with histone deacetylase family 1 (HDAC1), which could modify the lysines of histone H3, and co-act on many pathways, including the “longevity-regulating pathway” and “Notch signaling pathway”. Moreover, the upregulation of KDM5A/B/C expression was associated with an increase in the infiltration of B cells, CD8+ T cells and other infiltrating immune lymphocytes and the expression levels of immune molecules such as NT5E and CD274. Interestingly, the overexpression of KDM5A/C was also corelated with reduced sensitivity of pancreatic cancer cells to many kinds of pancreatic cancer-targeting or chemotherapeutic drugs, including axitinib and gemcitabine. Conclusion: KDM5 family members may be prognostic markers and new therapeutic targets for patients with pancreatic cancer.

STING agonist cGAMP enhances anti-tumor activity of CAR-NK cells against pancreatic cancer

ABSTRACT Activation of the stimulator of interferon gene (STING)-mediated innate immune response has been suggested as a promising therapeutic strategy for cancers. However, the effects of STING agonist on natural killer (NK) cell-mediated anti-tumor responses in pancreatic cancer remains unknown. Herein, we evaluated the effects of a classical STING agonist cyclic GMP-AMP (cGAMP) on NK cells in pancreatic cancer. We found that cGAMP could directly activate NK cells and enhance the sensitivity of pancreatic cancer cells to NK cell cytotoxicity, suggesting that cGAMP may become a potential adjuvant for NK cell therapy. In addition, combination of CAR-NK-92 cells targeting mesothelin and cGAMP displayed greater antitumor efficacy by inhibiting tumor growth and prolonging survival of the mouse model of pancreatic cancer. These results suggest that the combination of a STING agonist and NK cells may become a novel immunotherapy strategy for pancreatic cancer.

Down-regulation of USP22 reduces cell stemness and enhances the sensitivity of pancreatic cancer cells to cisplatin by inactivating the Wnt/β-catenin pathway

ObjectivePancreatic cancer (PC) has the worst prognosis of all common cancers worldwide. This study was intended to investigate the role of ubiquitin specific peptidase 22 (USP22) in cisplatin sensitivity of PC cells and its regulatory mechanism. MethodsThe expression of USP22 and the toxicity of cisplatin to PC cells were detected. The two cell lines AsPC-1 and CAPAN-1 with the most differential drug resistance were selected. By down-expressing USP22 in CAPAN-1 cells and over-expressing USP22 in AsPC-1 cells, the survival rate of PC cells treated with cisplatin was detected. The mRNA expressions of stem cell markers, cell stemness, migration ability and apoptosis of PC cells were detected. The expression of Wnt/β-catenin pathway related proteins was detected. The role of the Wnt/β-catenin pathway in PC cell stemness and cisplatin sensitivity was explored after adding the inhibitor HLY78 and activator DKK1. ResultsUSP22 was highly-expressed in PC cells, and the sensitivity of PC cells to cisplatin was negatively-correlated with USP22 expression. Downregulation of USP22 raised the sensitivity of PC cells to cisplatin, reduced the levels of stem cell markers, reduced the tumor sphere formation and migration, and promoted apoptosis. Silencing USP22 inhibited the Wnt/β-catenin pathway. Inhibition of USP22 reduced the cell stemness and augmented the sensitivity of PC cells to cisplatin by inhibiting the Wnt/β-catenin pathway. ConclusionSilencing USP22 can inhibit the Wnt/β-catenin pathway to reduce cell stemness and enhance the sensitivity of PC cells to cisplatin.