Sensitivity Of Pancreatic Cancer Research Articles

Combined Autophagy Inhibition and Dendritic Cell Recruitment Induces Antitumor Immunity and Enhances Immune Checkpoint Blockade Sensitivity in Pancreatic Cancer.

The effect of immune checkpoint inhibitors is extremely limited in patients with pancreatic ductal adenocarcinoma (PDAC) due to the suppressive tumor immune microenvironment (TIME). Autophagy, which has been shown to play a role in anti-tumor immunity, has been proposed as a therapeutic target for PDAC. Here, single-cell RNA-sequencing of autophagy-deficient murine PDAC tumors revealed that autophagy inhibition in cancer cells induced dendritic cell (DC) activation. Analysis of human PDAC tumors substantiated a negative correlation between autophagy and DC activation signatures. Mechanistically, autophagy inhibition increased intracellular accumulation of tumor antigens, which could activate DCs. Administration of chloroquine (CQ), an autophagy inhibitor, in combination with Flt3 ligand (Flt3L)-induced DC infiltration inhibited tumor growth and increased tumor-infiltrating T lymphocytes. However, autophagy inhibition in cancer cells also induced CD8+ T cell exhaustion with high expression of immune checkpoint LAG3. A triple therapy comprising CQ, Flt3L, and an anti-LAG3 antibody markedly reduced tumor growth in orthotopic syngeneic PDAC mouse models. Thus, targeting autophagy in cancer cells and activating DCs sensitizes PDAC tumors to immune checkpoint inhibitor therapy, warranting further development of this treatment approach to overcome immunosuppression in pancreatic cancer.

Sphingosine-1-Phosphate Inhibition Increases Endoplasmic Reticulum Stress to Enhance Oxaliplatin Sensitivity in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer resistant to current therapies, including oxaliplatin (Oxa). Growing evidence supports the ability of cancers to harness sphingolipid metabolism for survival. Sphingosine-1-phosphate (S1P) is an anti-apoptotic, pro-survival mediator that can influence cellular functions such as endoplasmic reticulum (ER) stress. We hypothesize that PDAC drives dysregulated sphingolipid metabolism and that S1P inhibition can enhance ER stress to improve therapeutic response to Oxa in PDAC. RNA sequencing data of sphingolipid mediators from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression Project (GTEx) datasets were analyzed. Murine and human PDAC cell lines were treated with small interfering RNA (siRNA) against sphingosine kinase-2 (SPHK2) or ABC294640 (ABC) and incubated with combinations of vehicle control or Oxa. In an orthotopic syngeneic KPC PDAC model, tumors were treated with either vehicle control, Oxa, ABC, or combination therapy. RNA sequencing analysis revealed multiple significantly differentially expressed sphingolipid mediators (P < 0.05). In vitro, both siRNA knockdown of SPHK2 and ABC sensitized cells to Oxa therapy (P < 0.05), and induced eukaryotic initiation factor 2α (eIF2α) and protein kinase RNA-like endoplasmic reticulum kinase (PERK) phosphorylation, hallmarks of ER stress. In vitro therapy also increased extracellular high mobility group box 1 (HMGB1) release (P < 0.05), necessary for immunogenic cell death (ICD). In vivo combination therapy increased apoptotic markers as well as the intensity of HMGB1 staining compared to control (P < 0.05). Our evidence suggests that sphingolipid metabolism is dysregulated in PDAC. Furthermore, S1P inhibition can sensitize PDAC to Oxa therapy through increasing ER stress and can potentiate ICD induction. This highlights a potential therapeutic target for chemosensitizing PDAC as well as an adjunct for future chemoimmunotherapy strategies.

Abstract 4658: Genome wide CRISPR/Cas9 library screening identifies aurora kinase A as a regulator of elraglusib sensitivity in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy with a dismal 5-year survival rate, highlighting the urgent need to improve drug response and overcome primary or acquired (secondary) resistance to conventional chemotherapy. Glycogen synthase kinase 3β (GSK-3β) is a target in PDAC and the GSK-3 inhibitor, elraglusib, has been shown to improve chemosensitivity of PDAC and survival in preclinical cancer models. In addition, data from phase II clinical trials in patients with previously untreated metastatic pancreatic cancer is demonstrating preliminary evidence of clinical benefit in patients receiving a combination of gemcitabine/nab-paclitaxel. While this early evidence is highly encouraging, the biological determinants of elraglusib response are unknown. Using genome wide CRISPR/Cas9 library screening, we identify aurora kinase A (AURKA), an oncogenic kinase with major roles in mitosis, as a critical regulator for elraglusib sensitivity. Elraglusib treatment activates AURKA by inducing a G2/M cell cycle arrest. We show that knocking out AURKA reduces the phosphorylation of polo like kinase 1 (PLK1), arresting cells in mitosis, and leading to mitotic catastrophe and significant apoptosis after elraglusib treatment. Strikingly, the AURKA inhibitor, alisertib, works synergistically with elraglusib to inhibit PDAC cell line growth and induce apoptosis. In summary, our results demonstrate that targeting AURKA is an effective approach to sensitize PDAC tumors to elraglusib. Citation Format: Li Ding, Easton Maeder, Cheng Zhang, Taylor Weiskittel, Daniel Schmitt, Andrew Mazar, Hu Li, Daniel Billadeau. Genome wide CRISPR/Cas9 library screening identifies aurora kinase A as a regulator of elraglusib sensitivity in pancreatic cancer [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 4658.

Abstract 1269: Large-scale validaton studies of a blood-based effective and affordable test for multicancer early detection

Abstract Cancer early detection aims at reducing cancer deaths. Unfortunately, many established cancer screening methods are not suitable for use in low- and middle-income countries (LMICs) due to cost, complexity, and dependency on extensive medical infrastructure. Nearly 10,000 participants (2003 cancer cases and 7888 non-cancer cases) were divided into one training and five independent validation cohorts across different races, sample types and platforms. One tube of peripheral blood was collected from each participant and quantified using a panel of seven protein tumor markers (PTMs) consisting of AFP, CA125, CA15-3, CA19-9, CA72-4, CEA and CYFRA 21-1 by common clinical immunoassay analyzers. An algorithm named OncoSeek was established using artificial intelligence (AI) to distinguish cancer cases from non-cancer cases by calculating the probability of cancer (POC) index based on the quantification of the seven PTMs and clinical information including sex and age, and to predict the possible affected tissue of origin (TOO). The conventional clinical method that relied only on a single threshold for each PTM would make a big problem when combining the results of those markers as the false positive rate would accumulate as the number of markers increased. Nevertheless, OncoSeek was empowered by AI to significantly reduce the false positive rate, increasing the specificity from 54.0% to 93.0%. The overall sensitivity of OncoSeek was 51.7%, resulting in 84.6% accuracy. The performance was consistent in the training and the five validation cohorts from three countries (Brazil, China and United States) including two sample types (plasma and serum) and three different platforms (Roche, Luminex and ELISA). The sensitivities ranged from 39.0% to 77.6% for the detection of the nine common cancer types (breast, colorectum, liver, lung, lymphoma, oesophagus, ovary, pancreas and stomach), which account for 59.2% of global cancer deaths annually. Furthermore, it has shown excellent sensitivity in several high-mortality cancer types for which there are lacking routine screening tests in the clinic, such as the sensitivity of pancreatic cancer was 77.6%. The overall accuracy of TOO prediction in the true positives was 65.4%, which could assist the clinical diagnostic workup. OncoSeek significantly outperforms the conventional clinical method, representing a novel blood-based test for multicancer early detection (MCED) that is non-invasive, easy, efficient and robust. Moreover, the accuracy of TOO facilitates the follow-up diagnostic workup. OncoSeek is affordable (~$20) and accessible requiring nothing more than a blood draw at the screening sites, which makes it adoptable in LMICs. Citation Format: Mao Mao, Bing Wei, Qingxia Xu, Yong Shen, Raphael Brandão, Shiyong Li, Wei Wu, Pingping Xing, Yinyin Chang, Dandan Zhu. Large-scale validaton studies of a blood-based effective and affordable test for multicancer early detection [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 1269.

Myoglobin improves doxycycline sensitivity in pancreatic cancer through promoting heme oxygenase-1-mediated ferroptosis.

Ferroptosis is expected to be a therapeutic target for cancers including pancreatic cancer. We aimed to screen genes that regulate ferroptosis and doxycycline resistance in pancreatic cancer and to explore the underlying mechanisms. Bioinformatics analysis was performed to identify genes that respond to ferroptosis in two human pancreatic cancer cells with GOT1 knocked down or not. 325 and 842 genes were upregulated in MiaPaCa and Tu8902 cells in response to GOT1 knockdown, with 43 genes shared. Among the 43 genes, 14 genes were identified to interact with ferroptosis key genes. MB and HMOX1 were the genes most sensitive to Erastin and doxycycline. Moreover, MB and HMOX1 expression was higher in human normal pancreatic duct epithelial cells than in pancreatic cancer cells. MB and HMOX1 proteins physically bound and promoted each other's expression. By interacting with HMOX1, MB suppressed pancreatic cancer cell proliferation, colony formation and invasion, and promoted cell ferroptosis and sensitivity to erastin and doxycycline. Silencing HMOX1 reversed the promoting effect of MB on cell ferroptosis and sensitivity to doxycycline. A pancreatic cancer xenograft model was established by subcutaneous injection of Panc-1 cells transfected with or without Ad-MB, and doxycycline was administered intraperitoneally. Overexpression of MB enhanced the inhibitory effect of doxycycline on xenograft growth. In conclusion, MB facilitated doxycycline sensitivity in pancreatic cancer cells through promoting HMOX1-mediated ferroptosis.

Deciphering the Pathways to PARP Sensitivity in Pancreatic Cancer.

A recent article analyzed paired cell-free DNA among patients with platinum-sensitive BRCA- or PALB2-mutated pancreatic cancer who received maintenance olaparib. Reversion mutations were linked with worse outcomes. These types of paired correlative studies are needed to improve our understanding of drug resistance and guide future clinical trials. See related article by Brown et al., p. 5207.

A panel of seven protein tumour markers for effective and affordable multi-cancer early detection by artificial intelligence.

155 Background: Cancer early detection aims at reducing cancer deaths. Unfortunately, many established cancer screening technologies are not suitable for use in low- and middle-income countries (LMICs) due to cost, complexity, and dependency on extensive medical infrastructure. Methods: Nearly 10,000 participants [1959 cancer cases (containing more than nine common cancer types) and 7423 non-cancer cases] were divided into one training and two independent validation cohorts. One tube of peripheral blood samples was collected from each participant and quantified using a panel of seven selected protein tumour markers (PTMs) by a common clinical electrochemiluminescence immunoassay analyser. An algorithm named OncoSeek was established using artificial intelligence (AI) to distinguish cancer from non-cancer cases by calculating the probability of cancer (POC) index based on the quantification results of the seven PTMs and clinical information including sex and age of the subjects, and to predict the possible affected tissue of origin (TOO) for those who have been detected with cancer signal. Results: The conventional clinical method relied only on a single threshold for each PTM which would make a big problem when combining the results of those markers, the false positive rate would accumulate as the number of markers increased. Nevertheless, OncoSeek was empowered by AI technology to significantly reduce the false positive rate, increasing the specificity from 56·9% [95% confidence interval (CI): 55·8% to 58·0%] to 92·9% (95% CI: 92·3% to 93·5%). In all cancer types, the overall sensitivity of the OncoSeek test was 51·7% (95% CI: 49·4% to 53·9%). And the performance was generally consistent in the training and the two validation cohorts. The sensitivities ranged from 37·1% to 77·6% for the detection of the nine common cancer types (breast, colorectum, liver, lung, lymphoma, oesophagus, ovary, pancreas, and stomach), which account for ~59·2% of global cancer deaths annually. Furthermore, it has shown excellent sensitivity in several high-mortality cancer types for which there are lacking routine screening tests in the clinic, such as the sensitivity of pancreatic cancer was 77·6% (95% CI: 69·3% to 84·6%). The overall accuracy of TOO prediction in the true positives was 66·8%, which could assist the clinical diagnostic workup. Conclusions: OncoSeek significantly outperforms the conventional clinical method, representing a novel blood-based test for multi-cancer early detection (MCED) which is a non-invasive, easy, efficient, and robust approach. Moreover, the accuracy of TOO facilitates the follow-up diagnostic workup and this method is affordable (less than $25) and accessible requiring nothing more than a blood draw at the screening sites, which makes it acceptable and sustainable in LMICs.

STAT3 inhibition enhances gemcitabine sensitivity in pancreatic cancer by suppressing EMT, immune escape and inducing oxidative stress damage

Pancreatic cancer (PC) is a highly-malignant tumor of the digestive system with a very poor prognosis and high mortality. Chemotherapy and PD-1/PD-L1 immune checkpoint blockade are important treatment strategies for advanced PC. However, chemotherapy resistance and poor therapeutic effect of immune checkpoint inhibitors is are the main clinical problems to be solved urgently at present. The effects of combined application of gemcitabine and STAT3 inhibition on the proliferation, apoptosis, migration, and invasion of PC cells (PCCs) were investigated. In addition, oxidative stress (OS), ferroptosis, immune escape, and the epithelial-mesenchymal transition (EMT) were evaluated. STAT3 inhibition with Stattic enhanced the inhibitory activity of gemcitabine on PCC proliferation by regulating the cell cycle. STAT3 inhibition enhanced mitochondrial-dependent apoptosis in gemcitabine-treated PCCs, but did not induce autophagy and ferroptosis. Further study showed that the anti-proliferative and pro-apoptotic effects may be associated with increased OS damage by inactivating Nrf2-HO-1 signaling, as well as DNA damage by inducing the imbalance between ATM andATR-Chk1 pathway. In addition, STAT3 inhibition strengthened gemcitabine-mediated suppression in PCC invasion and migration by antagonizing Smad2/3-dependent EMT. Moreover, the anti-tumorimmuneresponse of gemcitabine was upregulated by Stattic through reducing the expression of PD-L1 and CD47. Mechanistically, combined application of gemcitabine and Stattic suppressed the phosphorylation and nuclear expression of STAT3. Interestingly, the activities of AKT and β-catenin signaling were also regulated, suggesting that drug combination has a broad-spectrum signal regulation effect. STAT3 inhibition enhanced the sensitivity of PCCs to the chemotherapy drug gemcitabine by suppressing EMT and immune escape and inducing OS damage.

A panel of seven protein tumour markers for effective and affordable multi-cancer early detection by artificial intelligence: a large-scale and multicentre case–control study

A panel of seven protein tumour markers for effective and affordable multi-cancer early detection by artificial intelligence: a large-scale and multicentre case–control study

Single-cell RNA sequencing revealed subclonal heterogeneity and gene signatures of gemcitabine sensitivity in pancreatic cancer.

Introduction: Resistance to gemcitabine is common and critically limits its therapeutic efficacy in pancreatic ductal adenocarcinoma (PDAC). Methods: We constructed 17 patient-derived xenograft (PDX) models from PDAC patient samples and identified the most notable responder to gemcitabine by screening the PDX sets in vivo. To analyze tumor evolution and microenvironmental changes pre- and post-chemotherapy, single-cell RNA sequencing (scRNA-seq) was performed. Results: ScRNA-seq revealed that gemcitabine promoted the expansion of subclones associated with drug resistance and recruited macrophages related to tumor progression and metastasis. We further investigated the particular drug-resistant subclone and established a gemcitabine sensitivity gene panel (GSGP) (SLC46A1, PCSK1N, KRT7, CAV2, and LDHA), dividing PDAC patients into two groups to predict the overall survival (OS) in The Cancer Genome Atlas (TCGA) training dataset. The signature was successfully validated in three independent datasets. We also found that 5-GSGP predicted the sensitivity to gemcitabine in PDAC patients in the TCGA training dataset who were treated with gemcitabine. Discussion and conclusion: Our study provides new insight into the natural selection of tumor cell subclones and remodeling of tumor microenvironment (TME) cells induced by gemcitabine. We revealed a specific drug resistance subclone, and based on the characteristics of this subclone, we constructed a GSGP that can robustly predict gemcitabine sensitivity and prognosis in pancreatic cancer, which provides a theoretical basis for individualized clinical treatment.

A panel of seven protein tumor markers for multi-cancer early detection by artificial intelligence.

3067 Background: Early cancer detection is key to reducing cancer deaths. Unfortunately, many established cancer screening technologies are not suitable for use in low- and middle-income countries due to expenses, complexity, and dependency on extensive medical infrastructure. Therefore, a simple, affordable, efficient multi-cancer early detection test is a major unmet need. Methods: This study enrolled nearly 10,000 participants (1959 cancer cases and 7423 non-cancer cases) containing more than nine common cancer types. One tube of peripheral blood samples was collected from each participant and quantified a panel of seven selected protein tumor markers (PTMs) by a clinical common electrochemiluminescence immunoassay analyzer. A protein assay named OncoSeek was established using artificial intelligence to distinguish cancer from non-cancer cases by calculating the probability of cancer (POC) index based on the quantification results of 7 PTMs and clinical information including gender and age of the subjects. Then using another model to predict the possible affected tissue of origin (TOO) who has been detected with cancer signal. Results: In this study, we found that the conventional clinical method relied only on a single threshold for each protein tumor marker which would make a big problem that was when combining the result of those markers, the false positive rate would accumulate as the number of tests increased. Nevertheless, OncoSeek was empowered by artificial intelligence technology to significantly reduce the false positive rate, increasing the specificity from 56.9% (95% confidence interval (CI): 55.8% to 58.0%) to 92.9% (95% CI: 92.3% to 93.5%). In all cancer types sensitivity of the OncoSeek test was 51.7% (95% CI: 49.4% to 53.9%). The sensitivities ranged from 37.1% to 77.6% for the detection of the nine common cancer types (breast, colorectum, esophagus, liver, lung, lymphoma, ovary, pancreas, stomach), which account for ~59.2% of global cancer deaths annually. Furthermore, it has shown excellent sensitivity in several high-mortality cancer types for which there are a lack of routine screening tests in the clinic, such as the sensitivity of pancreatic cancer was 77.6% (95% CI: 69.3% to 84.6%). The overall accuracy of tissue of origin (TOO) prediction in true positives was 66.8%, which reduced the clinical diagnostic workup. Conclusions: In summary, this study supported that OncoSeek significantly outperforms the conventional clinical method, representing a novel blood-based test for multi-cancer early detection which is a non-invasive, easier, and efficient approach. Moreover, the accuracy of TOO of it which facilitates the follow-up diagnostic workup. As well as this method is affordable (the cost of the test is less than $25) and requires nothing more than a blood draw at the screening sites, which makes it more practical in low- and middle-income countries.

Wogonin increases gemcitabine sensitivity in pancreatic cancer by inhibiting Akt pathway.

Pancreatic cancer has a high degree of malignancy and a low 5-year survival rate, and drug resistance is one of the main factors leading to poor prognosis of pancreatic cancer. Wogonin is a flavonoid drug isolated from Scutellaria baicalensis, which has certain antitumor activity. Hence the purpose of this study was to investigate whether wogonin can be used to enhance the sensitivity of pancreatic cancer to gemcitabine chemotherapy, and investigate its possible sensitization mechanism. In vitro, MTT assay showed that wogonin increased gemcitabine cytotoxicity in gemcitabine-resistant pancreatic cancer cells. In vivo, Wogonin combined with gemcitabine was found to inhibit tumor growth in orthotopic pancreatic cancer mouse model. In order to explore the sensitization mechanism, the differentially expressed genes (DEGs) of the gemcitabine-resistant cell line Panc-1 and the gemcitabine-sensitive cell line Bxpc-3 were screened through the GEO database, and 15 differentially expressed genes were obtained by intersecting with the potential targets of wogonin. Gene Ontology and KEGG enrichment analysis was performed. Bioinformatics results predicted that wogonin promoted pancreatic cancer cell apoptosis by inhibiting protein kinase B (Akt) signaling, thereby enhancing the sensitivity of gemcitabine to Pancreatic cancer. The above results were also verified by flow cytometry and Western blotting experiments. In conclusion, wogonin may enhance the sensitivity of gemcitabine by inhibiting Akt pathway.

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.

Turning towards nonimmunoreactive tumors: Evaluation of cancer-associated fibroblasts enables prediction of the immune microenvironment and treatment sensitivity in pancreatic cancer

Increasing evidence has confirmed that cancer-associated fibroblasts (CAFs) recruit and induce regulatory T cells (Tregs) and macrophages but inhibit cytotoxic T lymphocyte infiltration to a certain extent, indicating that CAFs have a significant influence on the immunosuppressive microenvironment. However, the effect of CAFs on the immune microenvironment and immunotherapy response in pancreatic cancer remains unclear. Our research identified remarkable variation in CAF-associated molecules in multiple cancer types at the genetic and transcriptome levels. Two phenotypes were identified for 476 pancreatic cancer samples, and the different phenotypes exhibited significant variation in immune and inflammatory characteristics. Phenotype 1 exhibited higher levels of immune infiltration and lower expression of tumor-associated gene signatures than phenotype 2. We used a multipart approach to assess the prognostic value of CAF-associated molecules and constructed a CAF score model that could accurately predict patient prognosis. The CAF score accurately predicted infiltrating immune cell abundance, chemosensitivity, and the response to immunotherapy. Additionally, we found that the CAF-associated molecule FGFR4 may promote the proliferation and migration and inhibit the apoptosis of pancreatic cancer cells and is correlated with immune infiltration, suggesting its potential role as an oncogene. CAFs may promote the malignant biological behavior of pancreatic cancer through FGFR4. In summary, our research highlights potential relationships of the dysregulation of CAF-associated molecules with genome alterations and carcinogenesis in multiple malignancies. Our CAF-associated phenotypes and scoring system may enhance the understanding of pancreatic cancer chemotherapy sensitivity and immunotherapy response, providing new insights for personalized chemotherapy and immunotherapy.

PVT1 Knockdown Inhibits Autophagy and Improves Gemcitabine Sensitivity by Regulating the MiR-143/HIF-1α/VMP1 Axis in Pancreatic Cancer.

Elucidation of the regulatory mechanisms of gemcitabine sensitivity is needed to improve the therapeutic effects of this drug in pancreatic cancer. PANC-1 cells were transfected with small hairpin RNA against PVT1 or microRNA (miR)-143 mimics or inhibitor. The gemcitabine sensitivity of pancreatic cancer was evaluated. Autophagosomes were analyzed with an immunofluorescence assay. Cell viability and proliferation were examined with MTT assays. Quantitative reverse transcription-polymerase chain reaction and Western blotting were used to analyze the expression of PVT1, miR-143, HIF-1α, VMP1, LC3I/II, p62, and Beclin-1. The interactions of PVT1/miR-143 and miR-143/HIF-1α were assessed by dual-luciferase reporter assays. PVT1 was upregulated while miR-143 was downregulated in pancreatic cancer. Both PVT1 knockdown and miR-143 overexpression suppressed autophagy and improved gemcitabine sensitivity in pancreatic cancer. PVT1 directly sponged miR-143 to regulate HIF-1α expression. MiR-143 inhibitor reversed the effect of PVT1 knockdown on autophagy and gemcitabine sensitivity. PVT1 knockdown inhibited autophagy and improved gemcitabine sensitivity via the miR-143/HIF-1α/VMP1 axis in pancreatic cancer. Our investigation elucidated a novel regulatory mechanism of gemcitabine sensitivity and may contribute to improve the therapeutic effects of chemotherapy drugs on pancreatic cancer.

Synergistic Interactions between GW8510 and Gemcitabine in an In Vitro Model of Pancreatic Cancer.

One of the main reasons for the poor survival rates of pancreatic cancer patients is the development of gemcitabine resistance, indicating that novel treatment strategies that have the ability to improve gemcitabine sensitivity are in need to combat this devastating disease. TCGA PAAD data was used to determine the clinicopathological significance of high RRM2 (Ribonucleotide reductase subunit M2) expression for Pancreatic Ductal Adenocarcinoma (PDAC). The effects of GW8510 and gemcitabine on PANC-1 cell viability were determined using WST-8 assay. The potential synergistic interaction between GW8510 and gemcitabine was evaluated by the Combination Index (CI) analysis. The effects of GW8510 treatment on apoptosis, cell cycle, and cell migration, either in combination with gemcitabine or alone, were investigated. The effect of GW8510 on RRM2 protein levels was evaluated using ELISA assay. RRM2 is significantly over-expressed in PDAC compared to healthy pancreatic tissues (p <0.0001). RRM2 mRNA expression was found to be significantly correlated with the overall survival rate of patients (HR=2.17 [1.44-3.27], p=0.00016) and the pathological stages of the disease (p=0.0054). GW8510 significantly decreased the RRM2 protein levels compared to the control. Cell viability analysis showed that GW8510 has a similar effect to gemcitabine in inhibiting PANC-1 cell viability. GW8510 was found to synergize with gemcitabine to inhibit PANC-1 cell viability and migration. However, the effects of GW8510 on PANC-1 cells could not be explained by induction of apoptosis or cell cycle arrest. Targeting RRM2 using GW8510 may have the potential to increase gemcitabine sensitivity in pancreatic cancer.

Abstract CT119: Organoid sensitivity in pancreatic cancer correlates with clinical response to treatment and reveals utility for reducing toxicity: Preliminary results from the HOPE trial

Abstract The HOPE trial (Harnessing Organoids for PErsonalized Therapy) was a pilot study to test the feasibility of generating patient derived organoids (PDOs) from patients with pancreatic cancer under real world conditions, test drug sensitivity against these PDOs, and correlate these findings with clinical outcomes. Biopsies were obtained primarily during routine clinical care from surgical specimens, ascites, fine needle biopsies (FNB) of primary tumors, and IR guided core biopsies of liver and lymph node metastases. PDOs were grown in WNT free media according to our previously published methods. PDO drug sensitivity testing was performed on a panel of drugs, AUC calculated, and sensitivity ranked. Patients were followed clinically and assessed for disease control. At data cutoff (January 2020), we enrolled a total of 76 subjects representing all stages of disease. Drug testing was performed successfully on PDOs generated from 12 of these subjects (16%). Factors contributing to success obtaining sufficient cells for PDO generation included modality, body part, and tumor cellularity. H&amp;E and IHC corresponded in matched PDOs and donor tumors, as did DNA alterations. Transcriptomes of PDOs were classified as both ‘basal' and ‘classical' subtypes. When AUC values were annotated with clinical data, the Jenks break of 1.69 segregated matched PDO/AUC values into disease control and progressive disease. We estimated that a PDO AUC value &amp;lt;1.66 yields a &amp;gt; 99% probability of disease control from a regimen that contains this drug, whereas if all drugs in a regimen had an AUC &amp;gt; 2.75 there is a &amp;gt; 80% probability of accurately predicting resistance. To illustrate the potential of PDO testing to tailor treatment for an individual patient, we described a case of a subject with stage IV PDAC with a KRAS mutation and ERBB2 amplification. The subject had disease control with FOLFIRINOX, which was held for toxicity. The PDO showed resistance to oxaliplatin and the patient subsequently had an extended period of disease control with regimens which did not include oxaliplatin, highlighting the potential of PDO drug sensitivity testing to exclude ineffective treatments from combination chemotherapy and limit toxicity. In conclusion, we have shown the feasibility of collecting material via real-world clinical practice sufficient to develop PDOs suitable for rapidly screening multiple drugs, and have shown a high degree of correlation between clinical outcomes in patients with PDAC and matched PDO drug sensitivity. We determined preliminary criteria based on the AUC of individual drugs in PDOs to predict drug sensitivity in subjects. These results highlight the potential of PDOs to personalize therapy and allow for the exclusion of ineffective drugs from combination regimens thereby reducing toxicity. We anticipate this approach will be used in future trials to prospectively inform treatment selection for patients with PDAC. Citation Format: Joseph Elan Grossman, Ling Huang, Lakshmi Muthuswamy, Sofia Perea, Dipikaa Akshinthala, Raul Gonzalez, Leo Tsai, Jonah Cohen, Mandeep Sawhney, Douglas Pleskow, Tyler M. Berzin, Bruno Bockorny, Andrea Bullock, Benjamin Schlechter, Mary Linton B. Peters, Catherine Conahan, Supraja Narasimhan, Christine Lim, Roger Davis, Robert Besaw, Martin Smith, Tara Kent, Mark Callery, Senthil K. Muthuswamy, Manuel Hidalgo. Organoid sensitivity in pancreatic cancer correlates with clinical response to treatment and reveals utility for reducing toxicity: Preliminary results from the HOPE trial [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT119.

TLE2 is associated with favorable prognosis and regulates cell growth and gemcitabine sensitivity in pancreatic cancer.

BackgroundThe transducin-like enhancer of split (TLE) proteins are a group of transcriptional corepressors. They play a crucial role in cellular homeostasis and are involved in various cancers. Compared with other TLE family members, little is known about the role and the underlying mechanism of TLE2 in human cancers. This study aimed to investigate the role of TLE2 in pancreatic ductal adenocarcinoma (PDAC) using in silico analysis and in vitro experiments.MethodsData were obtained from the Cancer Genome Atlas (TCGA) database to evaluate the prognostic value of TLE2 in PDAC. The MiaPaCa-2 cell line was transfected with siRNA to inhibit endogenous TLE2 expression, and a PANC-1 cell line with stable TLE2 overexpression was constructed using lentiviral transfection, which were confirmed by real-time polymerase chain reaction and western blotting. MTT assay, transwell invasion assays, and flow cytometry were carried out to assess cell viability, invasion, and apoptosis, respectively. TLE2 expression in PDAC cells was altered to evaluate their sensitivity to gemcitabine. Gene set enrichment analysis (GSEA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted to predict the biological role of TLE2.ResultsHigh expression of TLEs was significantly associated with increased overall survival (OS) and disease-free survival (DFS) in patients with PDAC. Among the PDAC cell lines, TLE2 expression was lowest and highest in PANC-1 cells and MiaPaCa-2 cells, respectively. TLE2 overexpression impaired the proliferation ability of PANC-1 cells and downregulation of TLE2 promoted the proliferation of MiaPaCa-2 cells. Upregulation of TLE2 in PANC-1 cells induced S-phase accumulation and sensitivity to gemcitabine. In contrast, the downregulation of TLE2 in MiaPaCa-2 cells promoted resistance to gemcitabine. Moreover, bioinformatics analysis also revealed the potential tumor suppressor role of TLE2 and uncovered a close relationship between TLE2 expression and cell cycle regulation.ConclusionsOur results suggest that TLE2 expression is correlated with prognosis in patients with PDAC and show that TLE2 plays a central role in the regulation of cell proliferation, the cell cycle, and gemcitabine sensitivity. This study provides new insights and evidence that TLE2 functions as a tumor suppressor gene and prognostic marker in PDAC.

STAT1-mediated inhibition of FOXM1 enhances gemcitabine sensitivity in pancreatic cancer.

Forkhead box protein M1 (FOXM1) was identified as an oncogenic transcription factor and master regulator of tumor progression and metastasis. FOXM1 expression often correlates with poor prognosis and chemotherapy resistance. In the present study, we investigated the association of FOXM1 expression and chemoresistance in pancreatic cancer. Elevated FOXM1 protein levels were associated with gemcitabine chemoresistance in patients with pancreatic cancer. In gemcitabine resistance cell line models of pancreatic cancer, FOXM1 expression increased, which induced gemcitabine chemoresistance in vitro. In pancreatic cancer cells treated with gemcitabine, FOXM1 affected nuclear factor κB (NF-κB) signaling activity. Immunohistochemical analysis demonstrated a negative association of FOXM1 expression and the level of phosphorylated signal transducer and activator of transcription 1 (pSTAT1) in human pancreatic cancer tissues. Dual-luciferase reporter assays and chromatin-immunoprecipitation assays demonstrated that pSTAT1 directly binds to the FOXM1 promoter to down-regulate its transcription. Interferon γ (IFNγ) promoted gemcitabine-induced cell apoptosis and inhibited cell proliferation in vitro and in vivo by FOXM1 inhibition. These data suggested that FOXM1 enhances chemoresistance to gemcitabine in pancreatic cancer. IFNγ could be used to down-regulate the expression of FOXM1 through STAT1 phosphorylation, thereby increasing the sensitivity of pancreatic cancer cells to gemcitabine. These studies suggested the sensitization by IFNγ in pancreatic ductal adenocarcinoma (PDAC) chemotherapy, which requires further clinical studies.

LAT2 regulates glutamine-dependent mTOR activation to promote glycolysis and chemoresistance in pancreatic cancer

BackgroundReprogrammed energy metabolism has become an emerging hallmark of cancer in recent years. Transporters have been reported to be amino acid sensors involved in controlling mTOR recruitment and activation, which is crucial for the growth of both normal and tumor cells. L-type amino acid transporter 2 (LAT2), encoded by the SLC7A8 gene, is a Na+-independent neutral amino acid transporter and is responsible for transporting neutral amino acids, including glutamine, which can activate mTOR. Previous studies have shown that LAT2 was overexpressed in gemcitabine-resistant pancreatic cancer cells. However, the role of LAT2 in chemoresistance in pancreatic cancer remains uncertain and elusive.MethodsThe effects of LAT2 on biological behaviors were analyzed. LAT2 and LDHB levels in tissues were detected, and the clinical value was evaluated.ResultsWe demonstrated that LAT2 emerged as an oncogenic protein and could decrease the gemcitabine sensitivity of pancreatic cancer cells in vitro and in vivo. The results of a survival analysis indicated that high expression levels of both LAT2 and LDHB predicted a poor prognosis in patients with pancreatic cancer. Furthermore, we found that LAT2 could promote proliferation, inhibit apoptosis, activate glycolysis and alter glutamine metabolism to activate mTOR in vitro and in vivo. Next, we found that gemcitabine combined with an mTOR inhibitor (RAD001) could reverse the decrease in chemosensitivity caused by LAT2 overexpression in pancreatic cancer cells. Mechanistically, we demonstrated that LAT2 could regulate two glutamine-dependent positive feedback loops (the LAT2/p-mTORSer2448 loop and the glutamine/p-mTORSer2448/glutamine synthetase loop) to promote glycolysis and decrease gemcitabine (GEM) sensitivity in pancreatic cancer.ConclusionTaken together, our data reveal that LAT2 functions as an oncogenic protein and could regulate glutamine-dependent mTOR activation to promote glycolysis and decrease GEM sensitivity in pancreatic cancer. The LAT2-mTOR-LDHB pathway might be a promising therapeutic target in pancreatic cancer.