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Background Desmoplastic stroma, a feature of pancreatic ductal adenocarcinoma (PDAC), contains abundant activated pancreatic stellate cells (PSCs). How PSCs promote PDAC progression remains incompletely understood. Methods Effect of epithelium-specific E-twenty six factor 3 (ESE3)-positive PSCs on PDAC fibrosis and chemoresistance was examined by western blot, RT-PCR, immunofluorescence, flow cytometry assay, chromatin immunoprecipitation, luciferase assay, immunohistochemistry and subcutaneous pancreatic cancer mouse model. Results ESE3 expression increased in PSCs in PDAC tissues compared with those in normal PSCs. Clinical data showed that ESE3 upregulation in PSCs was positively correlated with tumour size, pTNM stage, CA19-9, carcinoembryonic antigen and serum CA242 level. ESE3 overexpression in PSCs was an independent negative prognostic factor for disease-free survival and overall survival amongst patients with PDAC. Mechanistically, the conditional medium from the loss and gain of ESE3-expressing PSCs influenced PDAC chemoresistance and tumour growth. ESE3 directly induced the transcription of α-SMA, collagen-I and IL-1β by binding to ESE3-binding sites on their promoters to activate PSCs. IL-1β upregulated ESE3 in PSCs through NF-κB activation, and ESE3 was required for PSC activation by tumour cell-derived IL-1β. Conclusion Inhibiting the IL-1β/ESE3 (PSCs)/IL-1β-positive feedback loop is a promising therapeutic strategy to reduce tumour fibrosis and increase chemotherapeutic efficacy in PDAC.

The ETS family transcription factor ESE3 is a crucial element in differentiation and development programs for many epithelial tissues. Here we report its role as a tumor suppressor in pancreatic cancer. We observed drastically lower ESE3 expression in pancreatic ductal adenocarcinomas (PDAC) compared with adjacent normal pancreatic tissue. Reduced expression of ESE3 in PDAC correlated closely with an increase in lymph node metastasis and vessel invasion and a decrease in relapse-free and overall survival in patients. In functional experiments, downregulating the expression of ESE3 promoted PDAC cell motility and invasiveness along with metastasis in an orthotopic mouse model. Mechanistic studies in PDAC cell lines, the orthotopic mouse model, and human PDAC specimens demonstrated that ESE3 inhibited PDAC metastasis by directly upregulating E-cadherin expression at the level of its transcription. Collectively, our results establish ESE3 as a negative regulator of PDAC progression and metastasis by enforcing E-cadherin upregulation. Cancer Res; 77(4); 874–85. ©2016 AACR.

Figure S1. Effects of Methylation-reducing agent and ESE3 on pancreatic cancer growth and migration in vitro. Figure S2. The basal expression of ESE3 in four PDAC cell lines by Western blotting experiment. Figure S3. Regulation of EMT marker expression by ESE3. Figure S4. Impact of altered expression of ESE3 on pancreatic tumor growth in nude mice.

Purpose:Oxygen and glucose deprivation is a common feature of the solid tumor. Regulatory network underlying the adaptation of cancer cells to the harsh microenvironment remains unclear. We determined the mechanistic role of LIM and senescent cell antigen-like–containing domain protein 1 (LIMS1) in cancer cell survival under oxygen–glucose deprivation conditions.Experimental Design:The expression level of LIMS1 was determined by IHC staining and analyzing the mRNA expression profiles from The Cancer Genome Atlas of three human solid tumors. Roles of LIMS1 in cancer cell metabolism and growth were determined by molecular and cell biology methods. A jetPEI nanocarrier was used as the vehicle for anti-LIMS1 siRNAs in mouse models of cancer therapeutics.Results:LIMS1 expression was drastically elevated in pancreatic ductal adenocarcinoma (PDAC). High LIMS1 level was associated with advanced TNM stage and poor prognosis of patients with tumor. Increased LIMS1 expression was pivotal for tumor cells to survive in the oxygen–glucose deprivation conditions. Mechanistically, LIMS1 enhanced GLUT1 expression and membrane translocation, which facilitated tumor cell adaptation to the glucose deprivation stress. Furthermore, LIMS1 promoted HIF1A protein translation by activating AKT/mTOR signaling, while hypoxia-inducible factor 1 (HIF1) transactivated LIMS1 transcription, thus forming a positive feedback loop in PDAC cell adaptation to oxygen deprivation stress. Inhibition of LIMS1 with jetPEI nanocarrier–delivered anti-LIMS1 siRNAs significantly increased cell death and suppressed tumor growth.Conclusions:LIMS1 promotes pancreatic cancer cell survival under oxygen–glucose deprivation conditions by activating AKT/mTOR signaling and enhancing HIF1A protein translation. LIMS1 is crucial for tumor adaptation to oxygen–glucose deprivation conditions and is a promising therapeutic target for cancer treatment.

Pancreatic cancer is a lethal disease owing to its intrinsic and acquired resistance to therapeutic modalities. The altered balance between pro- and antiapoptosis signals within cancer cells is critical to therapeutic resistance. However, the molecular mechanisms underlying increased antiapoptosis signals remain poorly understood. In this study, we report that PRMT1 expression is increased in pancreatic cancer tissues and is associated with higher tumor grade, increased aggressiveness, and worse prognosis. PRMT1 overexpression increased arginine methylation of HSPs of 70 kDa (HSP70); this methylation enhanced HSP70 binding and stabilization of BCL2 mRNA through AU-rich elements in 3′-untranslated region and consequentially increased BCL2 protein expression and protected cancer cells from apoptosis induced by cellular stresses and therapeutics. RNA binding and regulation function of HSP70 was involved in pancreatic cancer drug resistance and was dependent on protein arginine methylation. These findings not only reveal a novel PRMT1–HSP70–BCL2 signaling axis that is crucial to pancreatic cancer cell survival and therapeutic resistance, but they also provide a proof of concept that targeted inhibition of this axis may represent a new therapeutic strategy.Significance:This study demonstrates that a PRMT1-mediated stabilization of BCL2 mRNA contributes to therapeutic resistance in pancreatic cancer and that targeting this pathway could overcome said resistance.

Supplementary Figures and Tables and Methods 8 Supplemental Figures Figure S1. PRMT1 is overexpressed in multiple gastrointestinal tumors and is associated with patient overall survival. Figure S2. Genetic alterations and inflammatory factors contribute to increased PRMT1 expression during pancreatic cancer (PDA) progression. Figure S3. Altered expression and activity of PRMT1 impacts pancreatic cancer (PDA) cell migration, invasion, and growth. Figure S4. PRMT1 and HSP70 proteins exhibit subcellular co-localization and binding motifs of PRMT1-HSP70 interaction are evident. Figure S5. Arginine methylation sites of HSP70 are identified. Figure S6. Arginine methylation of HSP70 protects cells against various stresses. Figure S7. Arginine methylation of HSP70 promotes its mRNA binding and stabilization activity. Figure S8. Arginine methylation of heat shock proteins mediates therapeutic resistance in pancreatic cancer. 3 Supplemental Tables Table S1. Clinicopathologic parameters and PRMT1 expression in the pancreatic cancer patients Table S2. HA-PRMT1 and HA-ctrl binding proteins identified in mass spectrometry Table S3. Primers and peptides used in the study. Detailed Supplemental Methods for Cell Lines and Cell Culture; Plasmids and Cloning Procedures; Antibodies, siRNAs, Primers and Compounds; shRNA construct and transfection; Western blot Analysis; Alcian Blue Staining; Cell Scratch-Wound Healing Assay; Cell Invasion Assay; In Vitro Cytotoxicity and Cell Viability Assay; Immunoprecipitation Assay; Protein Purification from Bacterial or Mammalian Cells; Immunofluorescence Assay; In Vitro Methylation Assay; Protein Sequence Alignment; Mass Spectrometry Analysis; Generation of Gene Knockout Cell Lines; Annexin V-Propidium Iodide Assay; Quantitative Real-time PCR Assay; Ribonucleoprotein Immunoprecipitation; mRNA Decay Assay; Protein-Nucleic Acid Binding Assay; and Luciferase Assay.

15 Supplementary Figures: Supplementary Figure S1. The expression and subcellular localization of LIMS1 in PDAC cell lines. Supplementary Figure S2. LIMS1 is overexpressed in esophageal carcinoma and negatively correlated with survival. Supplementary Figure S3. LIMS1 is overexpressed in lung adenocarcinoma and negatively correlated with survival. Supplementary Figure S4. LIMS1 had no effect on cell viability under normal culture condition. Supplementary Figure S5. LIMS1 facilitates glucose uptaking and surviving of esophageal carcinoma. Supplementary Figure S6. LIMS1 facilitates glucose uptaking and surviving of lung adenocarcinoma. Supplementary Figure S7. LIMS1 expression does not alter the half-life of HIF1A. Supplementary Figure S8. LIMS1 plays important roles in maintain the activation of AKT signaling. Supplementary Figure S9. Impact of altered expression of LIMS1 on tumorigenicity and therapeutic efficacy. Supplementary Figure S10. The effect of altered LIMS1 expression on extracellular acidification. Supplementary Figure S11. LIMS1 expression level positively correlates with glucose uptaking in PETCT scanning of patients with PDAC, esophageal carcinoma and lung adenocarcinoma. Supplementary Figure S12. The LIMS1-HIF1 positive feedback loop is pivotal to oxygen-glucose deprivation stress resistance. Supplementary Figure S13. Impact of altered expression on LIMS1 on cell survival under hypoxia. Supplementary Figure S14. Impact of altered expression of LIMS1 and HIF1 on tumorigenicity. Supplementary Figure S15. The effect of LIMS1 on IRES-dependent translation of HIF1A.

Figure S1. Effects of Methylation-reducing agent and ESE3 on pancreatic cancer growth and migration in vitro. Figure S2. The basal expression of ESE3 in four PDAC cell lines by Western blotting experiment. Figure S3. Regulation of EMT marker expression by ESE3. Figure S4. Impact of altered expression of ESE3 on pancreatic tumor growth in nude mice.

Supplementary Table 1. Antibodies and primers Supplementary Table 2. Clinicopathologic characteristics of TMA

7 Supplementary Tables: Supplementary Table S1. Association between LIMS1 expression and clinicopathological features of patients with PDAC. Supplementary Table S2. Association between LIMS1 mRNA expression and clinicopathological features of PDAC patients from TCGA. Supplementary Table S3. Cox's proportional hazards model analysis of prognostic factors in patients with pancreatic ductal adenocarcinoma (PDAC) Supplementary Table S4. Association between LIMS1 expression and clinicopathological features of patients with esophageal cancer. Supplementary Table S5. Association between LIMS1 expression and clinicopathological features of patients with lung adenocarcinoma. Supplementary Table S6. Main antibodies and reagents. Supplementary Table S7. Primers and oligonucleotides sequences.

Supplementary Table 1. Antibodies and primers.

BxPC-3 and Panc-1 cells were transfected with pcDNA3.1 and pcDNA3.1-LASP1 plasmids (2 μg) for 48 h and assessed by RT-PCR and Western blot analyses.

(A) The morphology of Panc-1/pLV vector and Panc-1/pLV LASP-1 cells. (B) LASP-1 expression determined by Western blot analysis in Panc-1/pLV vector andPanc-1/pLV LASP-1 cells.

HIF-1α protein expression in CFPAC-1 cell transfected with negative control siRNA (siNC) and HIF-1α siRNA (si HIF-1α#1-3) (50 nM) for 48 hours determined by Western blot. β-actin was used as a loading control.

Preoperative serum CA19-9 and histology grade could show the biological characteristics of pancreatic ductal adenocarcinoma (PDAC). This study aims to explore the combined effect of preoperative CA19-9 and histology grade on the prognosis of patients with PDAC. A total of 612 patients with PDAC undergoing curative pancreatectomy were retrospectively enrolled. 360 (58.8%) patients had preoperative CA19-9 > 112 U/ml and 348 (56.9%) patients had high histology grade. A biological risk model was established based on preoperative CA19-9 and histology grade. Prognostic analysis showed that biological risk based on preoperative CA19-9 and histology grade was independently associated with survival of PDAC patients. Then the biological risk was incorporated into the eighth edition of the TNM staging system and a modified TNM (mTNM) staging system was developed. The ROC curves showed that the area under curve(AUC) of the mTNM staging system was significantly greater than that of the TNM staging system. Biological risk based on preoperative CA19-9 and histology grade was an independent prognostic factor for patients with PDAC. Incorporating the biological risk into the TNM staging system could improve the the accuracy of the TNM staging system in predicting prognosis of patients with PDAC.

Background Pancreatic cancer has a high malignancy and rapid progression. It is very easy to metastasize in clinical cases. In recent years, molecular targeted therapy has made some progress in the treatment of some tumors. In this study, we explored the application of miR-25-3p in targeted therapy of pancreatic cancer to improve the survival time of patients with this treatment in the future. Methods qRT‒PCR, scratch healing experiments and transwell experiments were applied in this study. iRGD peptide-modified exosomes carrying anti-miR-25-3p and in situ model mice were constructed. The HEK-293T cell line overexpressing the CD63-iRGD-3×FLAG fusion protein and overexpressing the miR-25-3p antisense sequence was obtained by fluorescence sorting using flow cytometry, and exosomes in the culture supernatant of this cell line were obtained by ultracentrifugation. Treatment of pancreatic cancer was simulated by regular tail vein injections of BALB/c nu mice overexpressing CD63-iRGD-3×FLAG fusion protein and loaded with miR-25-3p antisense sequence. The liver and spleen of the mice were stained with HE after a certain course of treatment, and the effect of treatment with exosomes overexpressing CD63-iRGD-3×FLAG fusion protein and loaded with miR-25-3p antisense sequence on liver metastasis of pancreatic cancer was observed. Results There was a positive correlation between miR-25-3p and the invasive migration ability of pancreatic cancer cell lines in the Panc-1 and Panc-3 cell lines, and the invasive migration ability of pancreatic cancer was enhanced by overexpression of miR-25-3p (P

Background Malnutrition and muscle wasting are common features frequently observed in pancreatic ductal adenocarcinoma (PDAC) patients with cancer cachexia. They are associated with reduced survival and quality of life. Nutrition therapy is an important part of multimodal cancer care in PDAC. However, due to the complexity of nutrition assessment, only 30–60% of patients with nutritional risks receive nutritional treatment at present. It is important to identify biomarkers that may be used to improve management of PDAC‐associated malnutrition. Serum insulin‐like growth factor binding protein 2 (IGFBP2) has emerged as a potential serum biomarker in a variety of tumours. However, its association with malnutrition and muscle wasting in PDAC is unclear. Methods We evaluated the tumour IGFBP2 expression and serum IGFBP2 level in 98 PDAC patients using immunohistochemistry and enzyme‐linked immunosorbent assay and analysed the correlation between them. Furthermore, we explored the relationship between IGFBP2 of both tumour and serum and nutritional status (Patient‐Generated Subjective Global Assessment and skeletal muscle index). Pan02 IGFBP2 stable transfection cell lines, Pan02 PLV‐IGFBP2 cells, and PLKO‐IGFBP2 cells were injected subcutaneously into the flank of C57BL/6 mouse. Serum IGFBP2 levels, food intake, and body weight of these mice were measured. The degree of muscle atrophy is characterized by haematoxylin and eosin, Oil Red O, and Masson's trichrome staining. The mRNA and protein expression of several essential muscle‐related signal proteins such as atrogin‐1 and muscle RING finger 1 was measured. Results Among 98 patients, we found that tumour IGFBP2 expression is related to plasma IGFBP2 levels (r s = 0.562, P

Human interleukin (IL)-37 is a member of the IL-1 family with potent anti-inflammatory and immunosuppressive properties. Previously, it has been reported that IL-37 suppresses tumor growth and progression. However, the roles of IL-37 in pancreatic cancer development and chemo-resistance remain unknown. Methods: Immunohistochemistry was used to analyze the correlation between IL-37 expression and clinicopathological features of pancreatic ductal adenocarcinoma (PDAC). Western-blot and RT-PCR was used to verify the correlation between IL-37 and hypoxia-inducible factor (HIF)-1α. We performed chromatin immunoprecipitation and luciferase assays to validate HIF-1α suppression of IL-37 expression. Moreover, gain- and loss-of-function studies in vitro and in vivo were used to demonstrate the biological function of IL-37 on PDAC development and chemo-resistance. Results: Our results showed that IL-37 expression was remarkably decreased in PDAC tissues when compared to adjacent normal pancreatic tissues. Reduced IL-37 expression in PDACs was associated with increased PDAC histological grade, tumor size, lymph node metastasis and vessel invasion. IL-37 low patients also have remarkably shorter relapse-free and overall survival. Importantly, IL-37 expression was positively correlated with Gemcitabine efficacy. Mechanistically, HIF-1α attenuated IL-37 transcription by binding to the hypoxia response elements (HREs) in IL-37 promoter. Conversely, IL-37 suppressed HIF-1α expression through STAT3 inhibition. Functionally, downregulation of IL-37 in PDAC cells promoted chemo-resistance, migration and progression in vivo and in vitro. Conclusions: Collectively, our data uncovered IL-37/ STAT3/ HIF-1α negative feedback signaling drives Gemcitabine resistance in PDAC.

Patients with resectable pancreatic ductal adenocarcinoma (PDAC) show differential prognosis after radical resection. Currently, cancer grading and surgical criteria depend heavily on imaging and anatomical diagnosis. It's essential to set up a model with reliable prognostic factors during the perioperative period to assess prognosis in PDAC patients. In this study, 103 patients diagnosed with PDAC who underwent radical resection were recruited. The predictive value of preoperative carcinoembryonic antigen (CEA), postoperative CA24-2 and the combination of two for overall survival (OS) were evaluated. Both pre-CEA and post-CA24-2 were found to be independent prognostic factors for OS according to multivariate analyses. Kaplan-Meier analysis revealed that CEA and CA24-2 as well as the combination of two were correlated with poor OS. In addition, patients with both markers elevated have worse prognosis than patients with either pre-CEA or post-CA24-2 elevated. Thus, we concluded that the combination of CEA and CA24-2 can be used as a prognostic factor for stage I and II resectable PDAC patients.

744 Background: Pancreatic cancer (PC) is one of the leading causes of cancer death. Identifying molecular residual disease (MRD) with tailored tumor-informed ctDNA-based next-generation sequencing (NGS) assays after curative surgery could facilitate the individualized management of patients with resected pancreatic cancer (PC). Here, we prospectively evaluated the clinical performance of tumor-informed ctDNA mutation analysis using a novel Burning Rock Patient Specific Prognostic and Potential Therapeutic Marker Tracking (brPROPHET) approach for assessing MRD in patients with resected PC. Methods: The prospective cohort study recruited patients (n = 20) diagnosed with resectable stage I-III PC. Plasma samples (n = 63) were collected before surgery (baseline), 7-days after surgery (landmark), 30-days after surgery (before any adjuvant therapy), during the adjuvant therapy and follow-up. Individual tumors and matched white blood cells (WBCs) were whole-exome sequenced and somatic mutations were identified. Serial plasma samples were analyzed by a personalized, tumor-informed ctDNA-based NGS assay (brPROPHET) designed to target up to 50 variants per patient. Results: A total of 20 patients (stage I/II 10 [50%]/9[ 45%]) were analyzed. Patients were followed for a median of 190 days (range: 158 days to 396 days). Preoperative ctDNA was detected in 100% (20/20) of the patients. MRD status was analyzed postoperatively on day 7 and day 30 with a positivity rate of 11% (2/18) and 17% (2/12), respectively. Of the 4 patients with known recurrences, 2 had detectable ctDNA (50%, 2/4) at 7 days after surgery, whereas all disease-free patients were ctDNA-negative (100%, 13/13) at this time point. Detection at the landmark timepoint 7 days after surgery was associated with shorter recurrence-free survival (hazard ratio [HR]: 16.87, p=0.00363). The landmark negative predictive value (NPV) and positive predictive value (PPV) were 87.5% and 100%, respectively. When integrating all timepoints, 100% (4/4) of relapse patients were ctDNA positive before relapse, with the median leading time by brPROPHET assay to radiological recurrence was 126 days, whereas all of the patients with constant ctDNA negative status were disease-free (longitudinal PPV 80%, NPV 100%). In one relapsed patient who had elevated preoperative carbohydrate antigen (CA) 19-9 and constant normal postoperative CA19-9 (

Desmoplastic stroma, a feature of pancreatic ductal adenocarcinoma (PDAC), contains abundant activated pancreatic stellate cells (PSCs). How PSCs promote PDAC progression remains incompletely understood.Effect of epithelium-specific E-twenty six factor 3 (ESE3)-positive PSCs on PDAC fibrosis and chemoresistance was examined by western blot, RT-PCR, immunofluorescence, flow cytometry assay, chromatin immunoprecipitation, luciferase assay, immunohistochemistry and subcutaneous pancreatic cancer mouse model.ESE3 expression increased in PSCs in PDAC tissues compared with those in normal PSCs. Clinical data showed that ESE3 upregulation in PSCs was positively correlated with tumour size, pTNM stage, CA19-9, carcinoembryonic antigen and serum CA242 level. ESE3 overexpression in PSCs was an independent negative prognostic factor for disease-free survival and overall survival amongst patients with PDAC. Mechanistically, the conditional medium from the loss and gain of ESE3-expressing PSCs influenced PDAC chemoresistance and tumour growth. ESE3 directly induced the transcription of α-SMA, collagen-I and IL-1β by binding to ESE3-binding sites on their promoters to activate PSCs. IL-1β upregulated ESE3 in PSCs through NF-κB activation, and ESE3 was required for PSC activation by tumour cell-derived IL-1β.Inhibiting the IL-1β/ESE3 (PSCs)/IL-1β-positive feedback loop is a promising therapeutic strategy to reduce tumour fibrosis and increase chemotherapeutic efficacy in PDAC.

Soy isoflavones are natural tyrosine kinase inhibitors closely associated with decreased morbidity and mortality of various tumors. The activation of tyrosine kinases such as ERBB2 is the mechanism by which cholecystitis transforms into gallbladder cancer (GBC), therefore, it is important to investigate the relationship between long-term exposure to soy isoflavones and the occurrence and progression of GBC. This case-control study (n = 85 pairs) found that the high level of plasma soy isoflavone-genistein (GEN) was associated with a lower risk of gallbladder cancer (≥326.00 ng/mL compared to ≤19.30 ng/mL, crude odds ratio 0.15, 95% CI 0.04-0.59; P for trend = 0.016), and that the level of GEN exposure negatively correlated with Ki67 expression in GBC tissue (n = 85). Consistent with these results, the proliferation of GBC cells was inhibited in the long-term exposure models of GEN in vitro and in vivo. The long-term exposure to GEN reduced the tyrosine kinase activity of ERBB2 and impaired the function of the PTK6-AKT-GSK3β axis, leading to downregulation of the MCM complex in GBC cells. In summary, long-term exposure to GEN associated with soy products intake might play a certain role in preventing GBC and even inhibiting the proliferation of GBC cells.

Gemcitabine (GEM) resistance is one of the major causes of treatment failure in pancreatic ductal adenocarcinoma (PDAC) in clinic. Here, through CRISPR/Cas9 activation library screen, we found that MTA3 mediates the GEM resistance of PDAC and thus might be a potential therapeutic target for combination chemotherapy. The CRISPR library screening showed that MTA3 is the most enriched gene in the surviving GEM-treated cells, and bioinformatic and histology analysis implied its high correlation with GEM resistance. MTA3 promoted GEM resistance of PDAC cells in in vitro and in vivo experiments. Mechanistically, as a component of the Mi-2/nucleosome remodeling and deacetylase transcriptional repression complex, MTA3 transcriptionally represses CRIP2, a transcriptional repressor of NF-κB/p65, activating NF-κB signaling and consequently leading to GEM resistance. Furthermore, the treatment of GEM increases MTA3 expression in PDAC cells via activating STAT3 signaling, thereby inducing the acquired chemoresistance of PDAC to GEM. In patients derived xenografts (PDX) mouse model, Colchicine suppresses the expression of MTA3 and increases the sensitivity of tumor cells to GEM. Based on these findings, MTA3 plays a key role in GEM resistance in pancreatic cancer and is a promising therapeutic target for reversing GEM chemotherapy resistance.

Perineural invasion (PNI) is a typical pathological feature of pancreatic ductal adenocarcinoma (PDAC). PNI is associated with poor prognosis of PDAC patients, however, the underlying molecular mechanisms in the development of PNI have not been fully revealed. In this study, we found that the expression of GM-CSF and HIF-1α were dramatically increased in PDAC cells. The overexpression of HIF-1α and GM-CSF closely correlated with increased occurrence of PNI and cancer-related pain and shortened overall survival in PDAC patients. GM-CSF expression in PDAC cells was mediated by HIF-1α through direct binding to the hypoxia response element in the GM-CSF promoter. The activated HIF-1α can up-regulate GM-CSF expression and secretion, which promoted the migration of Schwann cells in tumor microenvironment. Furthermore, GM-CSF overexpression could rescue the inhibition of Schwann cells migration by HIF-1α knockdown. Moreover, HIF-1α inhibition with PX478 drastically reduced the expression level of GM-CSF and decreased the PNI in a PDAC xenograft mouse model. Overall, our results demonstrated that the HIF-1α/GM-CSF pathway is involved in the tumor-nerve interactions and promotes the occurrence of PNI. The blockade of this signal might help to inhibit PDAC progression.

Purpose: Oxygen and glucose deprivation is a common feature of the solid tumor. Regulatory network underlying the adaptation of cancer cells to the harsh microenvironment remains unclear. We determined the mechanistic role of LIM and senescent cell antigen-like–containing domain protein 1 (LIMS1) in cancer cell survival under oxygen–glucose deprivation conditions. Experimental Design: The expression level of LIMS1 was determined by IHC staining and analyzing the mRNA expression profiles from The Cancer Genome Atlas of three human solid tumors. Roles of LIMS1 in cancer cell metabolism and growth were determined by molecular and cell biology methods. A jetPEI nanocarrier was used as the vehicle for anti-LIMS1 siRNAs in mouse models of cancer therapeutics. Results: LIMS1 expression was drastically elevated in pancreatic ductal adenocarcinoma (PDAC). High LIMS1 level was associated with advanced TNM stage and poor prognosis of patients with tumor. Increased LIMS1 expression was pivotal for tumor cells to survive in the oxygen–glucose deprivation conditions. Mechanistically, LIMS1 enhanced GLUT1 expression and membrane translocation, which facilitated tumor cell adaptation to the glucose deprivation stress. Furthermore, LIMS1 promoted HIF1A protein translation by activating AKT/mTOR signaling, while hypoxia-inducible factor 1 (HIF1) transactivated LIMS1 transcription, thus forming a positive feedback loop in PDAC cell adaptation to oxygen deprivation stress. Inhibition of LIMS1 with jetPEI nanocarrier–delivered anti-LIMS1 siRNAs significantly increased cell death and suppressed tumor growth. Conclusions: LIMS1 promotes pancreatic cancer cell survival under oxygen–glucose deprivation conditions by activating AKT/mTOR signaling and enhancing HIF1A protein translation. LIMS1 is crucial for tumor adaptation to oxygen–glucose deprivation conditions and is a promising therapeutic target for cancer treatment.

EHF transcriptionally inhibits the expressions of TGFβ1 and GM-CSF to decrease T reg cell and MDSC accumulation, making it a promising biomarker to evaluate the immune microenvironment in PDAC. EHF overexpression may improve the efficacy of checkpoint immunotherapy in PDAC., Pancreatic ductal adenocarcinoma (PDAC) is a highly immune-suppressive tumor with a low response rate to single checkpoint blockade therapy. ETS homologous factor (EHF) is a tumor suppressor in PDAC. Here, we report a novel function of EHF in pancreatic cancer immune microenvironment editing and efficacy prediction for anti-PD1 therapy. Our findings support that the deficiency of tumoral EHF induced the accumulation of regulatory T (T reg) cells and myeloid-derived suppressor cells (MDSCs) and a decrease in the number of tumor-infiltrating CD8+ T cells. Mechanistically, EHF deficiency induced the conversion and expansion of T reg cells and MDSCs through inhibiting tumor TGFβ1 and GM-CSF secretion. EHF suppressed the transcription of TGFB1 and CSF2 by directly binding to their promoters. Mice bearing EHF overexpression tumors exhibited significantly better response to anti-PD1 therapy than those with control tumors. Our findings delineate the immunosuppressive mechanism of EHF deficiency in PDAC and highlight that EHF overexpression may improve PDAC checkpoint immunotherapy.

CD73, a cell surface-localized ecto-5'-nucleotidase, is the major enzymatic source of extracellular adenosine. Canonically, it plays multiple roles in cancer-related processes via its metabolite. As a druggable target, clinical trials targeting CD73 in various malignant diseases are currently ongoing. Here, we report the ecto-5'-nucleotidase-independent functions of CD73 in pancreatic ductal adenocarcinoma (PDAC). Our findings support that the elevated expression of CD73 in PDAC cells promotes gemcitabine (GEM) resistance by activating AKT. We discovered that a large amount of intracellular CD73 are localized in the endoplasmic reticulum membrane. Intracellular CD73 physically interacts with major vault protein to activate the SRC-AKT circuit. Troglitazone (TGZ) is a peroxisome proliferator-activated receptor gamma agonist that could inhibit the expression of CD73. The administration of TGZ markedly enhances sensitivity to GEM via downregulating CD73 in PDAC. Our findings support that CD73 could be targeted to overcome chemoresistance in PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by remarkable desmoplasia, usually driven by cancer-associated fibroblasts (CAFs), influencing patient prognosis. CAFs are a group of plastic cells responsible for tumor growth and metastasis. Fibroblasts have been reported to directly contribute to angiogenesis by undergoing mesenchymal-endothelial transition (MEndoT) after ischemic injury in the heart, brain, and hindlimbs. However, whether CAFs can undergo similar transdifferentiation in the hostile tumor microenvironment and directly contribute to tumor angiogenesis remains unclear. Herein, we provide evidence that CAFs can adopt an endothelial cell-like phenotype and directly contribute to tumor angiogenesis in vitro and in vivo. Furthermore, this program is regulated by the PERK-eIF2α-ERK1/2 axis. Pharmacological inhibition of PERK with GSK2606414 limited the phenotypic transition of CAFs. In conclusion, our results suggest that CAFs contribute to tumor angiogenesis by undergoing the MEndoT, thus representing therapeutic targets for improving PDAC prognosis.

Pancreatic stellate cells (PSCs) play a pivotal role in pancreatic fibrosis and pancreatic ductal adenocarcinoma (PDAC) progression. The mechanisms controlling PSC activation is not completely understood. Here we investigated the role of ESE3 (Epithelium-Specific ETS factor 3) in PSC activation. We discovered that in PDAC patients ESE3 expression was increased in PSC while decreased in tumor cells. ESE3 overexpression in PSC promoted PSC activation. Condition medium from ESE3-overexprssing PSC promotes PDAC cell migration, chemoresistance, tumor growth and fibrosis. ESE3 directly induced the transcription of α-SMA, Collagen 1 and IL-1β by binding to ESE3 binding sites on their promoters to activate PSC. On the other hand, IL-1β upregulates ESE3 in PSC through NFκB activation and ESE3 is required for PSC activation by tumor cell derived IL-1β. Clinical data showed ESE3 upregulation in PSC was positively correlated with tumor size, pTNM stage, CA19-9, CEA and CA242 level in serum. ESE3 overexpression in PSC was an independent negative prognostic factor for disease-free survival and overall survival among PDAC patients. Inhibition of the IL-1β/ ESE3 (PSC)/ IL-1β positive feedback loop represents a promising therapeutic strategy to reduce tumor fibrosis and increase chemotherapeutic efficacy in PDAC.

Background and aimsThe crosstalk between cancer stem cells (CSCs) and their niche is required for the maintenance of stem cell-like phenotypes of CSCs. Here, we identified E26 transformation-specific homologous factor (EHF) as a key molecule in decreasing the sensitivity of pancreatic cancer (PC) cells to CSCs’ niche stimulus. We also explored a therapeutic strategy to restore the expression of EHF.DesignWe used a LSL-KrasG12D/+mice, LSL-Trp53R172H/+ and Pdx1-Cre (KPC) mouse model and samples from patients with PC. Immunostaining, flow cytometry, sphere formation assays, anchorage-independent growth assay, in vivo tumourigenicity, reverse transcription PCR, chromatin immunoprecipitation (ChIP) and luciferase analyses were conducted in this study.ResultsCXCL12 derived from pancreatic stellate cells (PSCs) mediates the crosstalk between PC cells and PSCs to promote PC stemness. Tumorous EHF suppressed CSC stemness by decreasing the sensitivity of PC to CXCL12 stimulus and inhibiting the crosstalk between PC and CSC-supportive niches. Mechanically, EHF suppressed the transcription of the CXCL12 receptor CXCR4. EHF had a cell autonomous role in suppressing cancer stemness by inhibiting the transcription of Sox9, Sox2, Oct4 and Nanog. Rosiglitazone suppressed PC stemness and inhibited the crosstalk between PC and PSCs by upregulating EHF. Preclinical KPC mouse cohorts demonstrated that rosiglitazone sensitised PDAC to gemcitabine therapy.ConclusionsEHF decreased the sensitivity of PC to the stimulus from PSC-derived CSC-supportive niche by negatively regulating tumorous CXCR4. Rosiglitazone could be used to target PC stem cells and the crosstalk between CSCs and their niche by upregulating EHF.

Interleukin 35 (IL-35) is a novel member of the IL-12 family, consisting of an EBV-induced gene 3 (EBI3) subunit and a P35 subunit. IL-35 is an immune-suppressive cytokine mainly produced by regulatory T cells. However, the role of IL-35 in cancer metastasis and progression is not well understood. Here we demonstrate that IL-35 is overexpressed in human pancreatic ductal adenocarcinoma (PDAC) tissues, and that IL-35 overexpression is associated with poor prognosis in PDAC patients. IL-35 has critical roles in PDAC cell extravasation and metastasis by facilitating the adhesion to endothelial cells and transendothelial extravasation. Mechanistically, IL-35 promotes ICAM1 overexpression through a GP130-STAT1 signalling pathway, which facilitates endothelial adhesion and transendothelial migration via an ICAM1–fibrinogen–ICAM1 bridge. In an orthotopic xenograft model, IL-35 promotes spontaneous pancreatic cancer metastasis in an ICAM1-dependent manner. Together, our results indicate additional functions of IL-35 in promoting PDAC metastasis through mediating ICAM1 expression., Interleukin-35 (IL-35) is an immune suppressive cytokine produced by T-cells. Here, the authors show that IL-35 is overexpressed by human pancreatic cancer cells and show, in a mouse model, that IL-35 promotes metastasis in an autocrine/paracrine manner via induction of ICAM-1 expression.

Forkheadbox protein 3 (FOXP3), initially identified as a key transcription factor for regulatory T cells (Treg cells), was also expressed in many tumors including pancreatic ductal adenocarcinoma (PDAC). However, its role in PDAC progression remains elusive. In this study, we utilized 120 PDAC tissues after radical resection to detect cancer-FOXP3 and Treg cells by immunohistochemistry and evaluated clinical and pathological features of these patients. Cancer-FOXP3 was positively correlated with Treg cells accumulation in tumor tissues derived from PDAC patients. In addition, high cancer-FOXP3 expression was associated with increased tumor volumes and poor prognosis in PDAC especially combined with high levels of Treg cells. Overexpression of cancer-FOXP3 promoted the tumor growth in immunocompetent syngeneic mice but not in immunocompromised or Treg cell-depleted mice. Furthermore, CCL5 was directly trans-activated by cancer-FOXP3 and promoted the recruitment of Treg cells from peripheral blood to the tumor site in vitro and in vivo. This finding has been further reinforced by the evidence that Treg cells recruitment by cancer-FOXP3 was impaired by neutralization of CCL5, thereby inhibiting the growth of PDAC. In conclusion, cancer-FOXP3 serves as a prognostic biomarker and a crucial determinant of immunosuppressive microenvironment via recruiting Treg cells by directly trans-activating CCL5. Therefore, cancer-FOXP3 could be used to select patients with better response to CCL5/CCR5 blockade immunotherapy.