Single-cell RNA-seq analysis reveals BHLHE40-driven pro-tumour neutrophils with hyperactivated glycolysis in pancreatic tumour microenvironment.

OBJECTIVE: Innate immunity plays important roles in pancreatic ductal adenocarcinoma (PDAC), as non-T-cell-enriched tumour. Neutrophils are major players in innate immune system. Here, we aimed to explore the heterogeneity and pro-tumour mechanisms of neutrophils in PDAC. DESIGN: We analysed single-cell transcriptomes of peripheral blood polymorphonuclear leucocytes (PMNs) and tumour-infiltrating immune cells from five patients with PDAC, and performed immunofluorescence/immunohistochemistry staining, multi-omics analysis and in vitro experiments to validate the discoveries of bioinformatics analysis. RESULTS: Exploration of the heterogeneity of tumour-associated neutrophils (TANs) revealed a terminally differentiated pro-tumour subpopulation (TAN-1) associated with poor prognosis, an inflammatory subpopulation (TAN-2), a population of transitional stage that have just migrated to tumour microenvironment (TAN-3) and a subpopulation preferentially expressing interferon-stimulated genes (TAN-4). Glycolysis signature was upregulated along neutrophil transition trajectory, and TAN-1 was featured with hyperactivated glycolytic activity. The glycolytic switch of TANs was validated by integrative multi-omics approach of transcriptomics, proteomics and metabolomics analysis. Activation of glycolytic activity by LDHA overexpression induced immunosuppression and pro-tumour functions in neutrophil-like differentiated HL-60 (dHL-60) cells. Mechanistic studies revealed BHLHE40, downstream to hypoxia and endoplasmic reticulum stress, was a key regulator in polarisation of neutrophils towards TAN-1 phenotype, and direct transcriptional regulation of BHLHE40 on TAN-1 marker genes was demonstrated by chromatin immunoprecipitation assay. Pro-tumour and immunosuppression functions were observed in dHL-60 cells overexpressing BHLHE40. Importantly, immunohistochemistry analysis of PDAC tissues revealed the unfavourable prognostic value of BHLHE40+ neutrophils. CONCLUSION: The dynamic properties of TANs revealed by this study will be helpful in advancing PDAC therapy targeting innate immunity.

Postoperative hyperprogression disease of pancreatic ductal adenocarcinoma after curative resection: a retrospective cohort study.

BACKGROUND: Prognosis for patients recurred rapidly after resection of pancreatic ductal adenocarcinoma (PDAC) was extremely poor. We proposed the concept of postoperative hyper-progression disease (PO-HPD) to define recurrence within 2 months after surgery, explored the role of surgery for postoperative HPD patients and determined the predictive preoperative risk factors and genomic features of PO-HPD. METHODS: 976 patients undergoing curative resection of PDAC were enrolled. Survival data of 1733 stage IV patients from the US Surveillance, Epidemiology and End Results database was also collected. Patients relapsed were grouped into 3 groups regarding of the recurrence time (within 2 months were PO-HPD, within 2 to 12 months were early recurrence (ER) and within > 12 months were late recurrence (LR)). Risk factors for PO-HPD were explored with logistic regression models. Genomic features of 113 patients were investigated using next-generation sequencing-based gene panel testing. RESULTS: 718 of 976 cases relapsed, 101were PO-HPD, 418 were ER and 199 were LR. Total survival of PO-HPD was 12.5 months, shorter than that of ER (16.7 months) and LR (35.1 months), and verged on that of stage IV patients (10.6 months). Preoperative risk factors for PO-HPD included red blood cell count < 3.94*10^12/L, CA19-9 ≥ 288.6 U/mL, CA125 ≥ 22.3 U/mL and tumor size≥3.45 cm. Mutations of CEBPA, ATR and JAK1 were only identified in PO-HPD and they owned lower level of CN gain compared to others. CONCLUSIONS: Prognosis of PO-HPD was extremely poor and the role of surgery for PO-HPD should be prudently assessed.

An allosteric PGAM1 inhibitor effectively suppresses pancreatic ductal adenocarcinoma.

Glycolytic enzyme phosphoglycerate mutase 1 (PGAM1) plays a critical role in cancer metabolism by coordinating glycolysis and biosynthesis. A well-validated PGAM1 inhibitor, however, has not been reported for treating pancreatic ductal adenocarcinoma (PDAC), which is one of the deadliest malignancies worldwide. By uncovering the elevated PGAM1 expressions were statistically related to worse prognosis of PDAC in a cohort of 50 patients, we developed a series of allosteric PGAM1 inhibitors by structure-guided optimization. The compound KH3 significantly suppressed proliferation of various PDAC cells by down-regulating the levels of glycolysis and mitochondrial respiration in correlation with PGAM1 expression. Similar to PGAM1 depletion, KH3 dramatically hampered the canonic pathways highly involved in cancer metabolism and development. Additionally, we observed the shared expression profiles of several signature pathways at 12 h after treatment in multiple PDAC primary cells of which the matched patient-derived xenograft (PDX) models responded similarly to KH3 in the 2 wk treatment. The better responses to KH3 in PDXs were associated with higher expression of PGAM1 and longer/stronger suppressions of cancer metabolic pathways. Taken together, our findings demonstrate a strategy of targeting cancer metabolism by PGAM1 inhibition in PDAC. Also, this work provided "proof of concept" for the potential application of metabolic treatment in clinical practice.

Long noncoding RNA NORAD, a novel competing endogenous RNA, enhances the hypoxia-induced epithelial-mesenchymal transition to promote metastasis in pancreatic cancer.

BACKGROUND: Pancreatic cancer, one of the top two most fatal cancers, is characterized by a desmoplastic reaction that creates a dense microenvironment, promoting hypoxia and inducing the epithelial-to-mesenchymal transition (EMT) to facilitate invasion and metastasis. Recent evidence indicates that the long noncoding RNA NORAD may be a potential oncogenic gene and that this lncRNA is significantly upregulated during hypoxia. However, the overall biological role and clinical significance of NORAD remains largely unknown. METHODS: NORAD expression was measured in 33 paired cancerous and noncancerous tissue samples by real-time PCR. The effects of NORAD on pancreatic cancer cells were studied by overexpression and knockdown in vitro. Insights into the mechanism of competitive endogenous RNAs (ceRNAs) were gained from bioinformatics analyses and luciferase assays. In vivo, metastatic potential was identified using an orthotopic model of PDAC and quantified using bioluminescent signals. Alterations in RhoA expression and EMT levels were identified and verified by immunohistochemistry and Western blotting. RESULTS: NORAD is highly expressed in pancreatic cancer tissues and upregulated in hypoxic conditions. NORAD upregulation is correlated with shorter overall survival in pancreatic cancer patients. Furthermore, NORAD overexpression promoted the migration and invasion of pancreatic carcinoma cells, while NORAD depletion inhibited EMT and metastasis in vitro and in vivo. In particular, NORAD may function as a ceRNA to regulate the expression of the small GTP binding protein RhoA through competition for hsa-miR-125a-3p, thereby promoting EMT. CONCLUSIONS: Elevated expression of NORAD in pancreatic cancer tissues is linked to poor prognosis and may confer a malignant phenotype upon tumor cells. NORAD may function as a ceRNA to regulate the expression of the small GTP binding protein RhoA through competition for hsa-miR-125a-3p. This finding may contribute to a better understanding of the role played by lncRNAs in hypoxia-induced EMT and provide a potential novel diagnostic and therapeutic target for pancreatic cancer.

MiR-216a decreases MALAT1 expression, induces G2/M arrest and apoptosis in pancreatic cancer cells.

How lncRNA MALAT1 is regulated by miRNAs at posttranscriptional level in pancreatic cancer and their regulative effects on the cancer cells remain largely unknown. By retrieving previous miRNA array data and performing primary qRT-PCR, we observed a significant negative correlation between miR-216a and MALAT1 in pancreatic ductal adenocarcinoma (PDAC) tissues and adjacent normal tissues. The following in vitro cell assay further confirmed a direct binding between miR-216a and MALAT1 and the suppressive effect of miR-216a on MALAT1 expression. MiR-216a overexpression had similar effects as MALAT1 siRNA on restoring p21 and p27 expression and inhibiting B-MYB, RAF1 and PCNA1 expression in both PANC-1 and BxPC3 cells. MiR-216a overexpression and MALAT1 knockdown induced cell cycle arrest at G2/M phase. MiR-216a overexpression not only significantly induced cell apoptosis, but also reduced cell viability and increased cell apoptosis in response to gemcitabine in the cancer cells. Based on these findings, we infer that miR-216a induces apoptosis both in the presence and absence of gemcitabine in pancreatic cancer cells by silencing MALAT1 expression.

Robot-Assisted Middle Pancreatectomy for Elderly Patients: Our Initial Experience.

BACKGROUND: The aim of this study was to evaluate the indications, safety, feasibility, and short- and long-term outcomes for elderly patients who underwent robot-assisted middle pancreatectomies (MPs). MATERIAL AND METHODS: Ten patients (≥60 years) underwent robot-assisted middle pancreatectomies from 2012 to 2015. The perioperative data, including tumor size, operating time, rate of postoperative pancreatic fistula (POPF), postoperative morbidity, and other parameters, were analyzed. We collected and analyzed the follow-up information. RESULTS: The mean age of patients was 64.30 years (range, 60-73 years). The average tumor size was 2.61 cm. The 10 cases were all benign or low-grade malignant lesions. The mean operating time was 175.00 min. The mean blood loss was 113.00 ml with no blood transfusion needed. Postoperative fistulas developed in 5 patients; there were 2 Grade A fistulas and 3 grade B fistulas. There were 3 patients who underwent postoperative complications, including 2 Grade 1 or 2 complications and 1 Grade 3 complication. No reoperation and postoperative mortality occurred. The mean hospital stay was 19.91 days. After a median follow-up of 23 months, new onset of diabetes mellitus developed in 1 patient and none suffered from deterioration of previously diagnosed diabetes or exocrine insufficiency, and no tumor recurrence happened. CONCLUSIONS: Robot-assisted middle pancreatectomy was safe and feasible for elderly people. It had low risk of exocrine or endocrine dysfunction and benefited patients' long-term outcomes. Incidence of POPF was relatively high but we could prevent it from resulting in bad outcomes by scientific perioperative care and systemic treatment.

Driver mutation subtypes involve with differentiated immunophenotypes influencing pancreatic cancer outcomes.

Despite many studies focusing on the prognostic biomarkers in pancreatic adenocarcinomas (PAADs), there is ill-informed about the relationships between their genomic features and immune characteristics. Herein, we deeply investigated the involvement of major driver mutation subtypes with immunophenotypes impacting PAAD outcomes. Based on public data analyses of RNA expression-based immune subtypes in PAAD, in contrast to KRAS G12D & TP53 co-mutant patients with poor outcomes, the best immune subtype C3 (inflammatory) characterized by high Th1/Th2 ratio was relatively enriched in KRASnon-G12DTP53wt patients with better survival, whereas the inferior subtype C2 (IFN-γ dominant) with low Th1/Th2 ratio was more common in the former than in the latter. Moreover, contrary to the highly immunosuppressive microenvironment (high Treg, high ratio of Treg to tumor-specific CD4+ T cell) in KRASG12DTP53mut patients, KRASG12VTP53wt individuals exhibited an inflamed context profiled by multiplex immunohistochemistry. It could be responsible for their outstanding survival advantage over others in postsurgical PAAD patients receiving adjuvant chemotherapy as shown by our cohort. Together, KRASG12VTP53wt may be a promising biomarker for prognostic evaluation and screening certain candidates with PAAD to get desirable survival benefit from adjuvant chemotherapy.

BHLHE40 Inhibits Ferroptosis in Pancreatic Cancer Cells via Upregulating SREBF1.

Pancreatic cancer (PCa) is one of the most fatal human malignancies. The enhanced infiltration of stromal tissue into the PCa tumor microenvironment limits the identification of key tumor-specific transcription factors and epigenomic abnormalities in malignant epithelial cells. Integrated transcriptome and epigenetic multiomics analyses of the paired PCa organoids indicate that the basic helix-loop-helix transcription factor 40 (BHLHE40) is significantly upregulated in tumor samples. Increased chromatin accessibility at the promoter region and enhanced mTOR pathway activity contribute to the elevated expression of BHLHE40. Integrated analysis of chromatin immunoprecipitation-seq, RNA-seq, and high-throughput chromosome conformation capture data, together with chromosome conformation capture assays, indicate that BHLHE40 not only regulates sterol regulatory element-binding factor 1 (SREBF1) transcription as a classic transcription factor but also links the enhancer and promoter regions of SREBF1. It is found that the BHLHE40-SREBF1-stearoyl-CoA desaturase axis protects PCa cells from ferroptosis, resulting in the reduced accumulation of lipid peroxidation. Moreover, fatostatin, an SREBF1 inhibitor, significantly suppresses the growth of PCa tumors with high expressions of BHLHE40. This study highlights the important roles of BHLHE40-mediated lipid peroxidation in inducing ferroptosis in PCa cells and provides a novel mechanism underlying SREBF1 overexpression in PCa.

CTC-derived pancreatic cancer models serve as research tools and are suitable for precision medicine approaches.

Pancreatic ductal adenocarcinoma (PDAC) poses significant clinical challenges, often presenting as unresectable with limited biopsy options. Here, we show that circulating tumor cells (CTCs) offer a promising alternative, serving as a "liquid biopsy" that enables the generation of in vitro 3D models and highly aggressive in vivo models for functional and molecular studies in advanced PDAC. Within the retrieved CTC pool (median 65 CTCs/5 mL), we identify a subset (median content 8.9%) of CXCR4+ CTCs displaying heightened stemness and metabolic traits, reminiscent of circulating cancer stem cells. Through comprehensive analysis, we elucidate the importance of CTC-derived models for identifying potential targets and guiding treatment strategies. Screening of stemness-targeting compounds identified stearoyl-coenzyme A desaturase (SCD1) as a promising target for advanced PDAC. These results underscore the pivotal role of CTC-derived models in uncovering therapeutic avenues and ultimately advancing personalized care in PDAC.

Comprehensive machine learning-generated classifier identifies pro-metastatic characteristics and predicts individual treatment in pancreatic cancer: A multicenter cohort study based on super-enhancer profiling.

Rationale: Accumulating evidence illustrated that the reprogramming of the super-enhancers (SEs) landscape could promote the acquisition of metastatic features in pancreatic cancer (PC). Given the anatomy-based TNM staging is limited by the heterogeneous clinical outcomes in treatment, it is of great clinical significance to tailor individual stratification and to develop alternative therapeutic strategies for metastatic PC patients based on SEs. Methods: In our study, ChIP-Seq analysis for H3K27ac was performed in primary pancreatic tumors (PTs) and hepatic metastases (HMs). Bootstrapping and univariate Cox analysis were implemented to screen prognostic HM-acquired, SE-associated genes (HM-SE genes). Then, based on 1705 PC patients from 14 multicenter cohorts, 188 machine-learning (ML) algorithm integrations were utilized to develop a comprehensive super-enhancer-related metastatic (SEMet) classifier. Results: We established a novel SEMet classifier based on 38 prognostic HM-SE genes. Compared to other clinical traits and 33 published signatures, the SEMet classifier possessed robust and powerful performance in predicting prognosis. In addition, patients in the SEMetlow subgroup owned dismal survival rates, more frequent genomic alterations, and more activated cancer immunity cycle as well as better benefits in immunotherapy. Remarkably, there existed a tight correlation between the SEMetlow subgroup and metastatic phenotypes of PC. Among 18 SEMet genes, we demonstrated that E2F7 may promote PC metastasis through the upregulation of TGM2 and DKK1. Finally, after in silico screening of potential compounds targeted SEMet classifier, results revealed that flumethasone could enhance the sensitivity of metastatic PC to routine gemcitabine chemotherapy. Conclusion: Overall, our study provided new insights into personalized treatment approaches in the clinical management of metastatic PC patients.

Targeting KRASG12V mutations with HLA class II-restricted TCR for the immunotherapy in solid tumors.

KRAS mutation is a significant driving factor of tumor, and KRASG12V mutation has the highest incidence in solid tumors such as pancreatic cancer and colorectal cancer. Thus, KRASG12V neoantigen-specific TCR-engineered T cells could be a promising cancer treatment approach for pancreatic cancer. Previous studies had reported that KRASG12V-reactive TCRs originated from patients' TILs could recognized KRASG12V neoantigen presented by specific HLA subtypes and remove tumor persistently in vitro and in vivo. However, TCR drugs are different from antibody drugs in that they are HLA-restricted. The different ethnic distribution of HLA greatly limits the applicability of TCR drugs in Chinese population. In this study, we have identified a KRASG12V-specific TCR which recognized classII MHC from a colorectal cancer patient. Interestingly, we observed that KRASG12V-specific TCR-engineered CD4+ T cells, not CD8+ T cells, demonstrated significant efficacy in vitro and in xenograft mouse model, exhibiting stable expression and targeting specificity of TCR when co-cultured with APCs presenting KRASG12V peptides. TCR-engineered CD4+ T cells were co-cultured with APCs loaded with neoantigen, and then HLA subtypes were identified by the secretion of IFN-γ. Collectively, our data suggest that TCR-engineered CD4+ T cells can be used to target KRASG12V mutation presented by HLA-DPB1*03:01 and DPB1*14:01, which provide a high population coverage and are more suitable for the clinical transformation for Chinese, and mediate tumor killing effect like CD8+ T cells. This TCR hold promise for precision therapy in immunotherapy of solid tumors as an attractive candidate.

Association between age at diabetes onset or diabetes duration and subsequent risk of pancreatic cancer: Results from a longitudinal cohort and mendelian randomization study.

Background: The aim of the study is to estimate the incidence of pancreatic cancer among individuals with new-onset type 2 Diabetes (T2DM) and evaluate the relationship of pancreatic cancer risk with age at diabetes onset and diabetes duration. Methods: This longitudinal cohort study included 428,362 new-onset T2DM patients in Shanghai and Mendelian randomization (MR) in the east-Asian population were used to investigate the association. Incidence rates of pancreatic cancer in all patients and by subgroups were calculated and compared to the general population. Findings: A total of 1056 incident pancreatic cancer cases were identified during eight consecutive years of follow-up. The overall pancreatic cancer annual incidence rate was 55·28/100,000 person years in T2DM patients, higher than that in the general population, with a standardized incidence ratio (SIR) of 1·54 (95% confidence interval [CI], 1·45-1·64). The incidence of pancreatic cancer increased with age and a significantly higher incidence was observed in the older groups with T2DM. However, the relative pancreatic cancer risk was inversely related to age of T2DM onset, and a higher SIR of 5·73 (95%CI, 4·49-7·22) was observed in the 20-54 years old group. The risk of pancreatic cancer was elevated at any diabetes duration. Fasting blood glucose ≥10·0 mmol/L was associated with increased risk of pancreatic cancer. MR analysis indicated a positive association between T2DM and pancreatic cancer risk. Interpretation: Efforts toward early and close follow-up programs, especially in individuals with young-onset T2DM, and the improvement of glucose control might represent effective strategies for improving the detection and results of treatment of pancreatic cancer. Funding: Chinese National Natural Science Foundation.

Specific human leukocyte antigen class I genotypes predict prognosis in resected pancreatic adenocarcinoma: a retrospective cohort study.

BACKGROUND: Patients with resected pancreatic adenocarcinoma (PAAD) often experience short-term relapse and dismal survival, suggesting an urgent need to develop predictive and/or prognostic biomarkers for these populations. Given the potential associations of the human leukocyte antigen class I ( HLA -I) genotype with oncogenic mutational profile and immunotherapy efficacy, we aimed to assess whether differential HLA -I genotype could predict the postoperative outcomes in resected PAAD patients. MATERIALS AND METHODS: HLA -I ( A , B , and C ) genotyping and somatic variants of 608 Chinese PAAD patients were determined by targeted next-generation sequencing of matched blood cells and tumor tissues. HLA - A / B alleles were classified with the available definition of 12 supertypes. The Kaplan-Meier curves of disease-free survival (DFS) and multivariable Cox proportional-hazards regression analyses were performed to determine the survival difference in 226 selected patients with radical resection. Early-stage (I-II) patients constituted the majority (82%, 185/226) and some stage I-II individuals with high-quality tumor samples were analyzed by RNA-sequencing to examine immunophenotypes. RESULTS: Patients with HLA-A02 + B62 + B44 - had significantly shorter DFS (median, 239 vs. 410 days; hazard ratio=1.65, P =0.0189) than patients without this genotype. Notably, stage I-II patients carrying HLA-A02 + B62 + B44 - had sharply shorter DFS than those without HLA-A02 + B62 + B44 - (median, 237 vs. 427 days; hazard ratio=1.85, P =0.007). Multivariate analysis revealed that HLA-A02 + B62 + B44 - was associated with significantly inferior DFS ( P =0.014) in stage I-II patients but not in stage III patients. Mechanistically, HLA-A02 + B62 + B44 - patients were associated with a high rate of KRAS G12D and TP53 mutations, lower HLA-A expression, and less inflamed T-cell infiltration. CONCLUSION: The current results suggest that a specific combination of germline HLA-A02/B62/B44 supertype, HLA-A02 + B62 + B44 - , was a potential predictor for DFS in early-stage PAAD patients after surgery.

LncRNA-PACERR induces pro-tumour macrophages via interacting with miR-671-3p and m6A-reader IGF2BP2 in pancreatic ductal adenocarcinoma.

BACKGROUND: LncRNA-PACERR plays critical role in the polarization of tissue-associated macrophages (TAMs). In this study, we found the function and molecular mechanism of PACERR in TAMs to regulate pancreatic ductal adenocarcinoma (PDAC) progression. METHODS: We used qPCR to analyse the expression of PACERR in TAMs and M1-tissue-resident macrophages (M1-NTRMs) which were isolated from 46 PDAC tissues. The function of PACERR on macrophages polarization and PDAC proliferation, migration and invasion were confirmed through in vivo and in vitro assays. The molecular mechanism of PACERR was discussed via fluorescence in situ hybridization (FISH), RNA pull-down, ChIP-qPCR, RIP-qPCR and luciferase assays. RESULTS: LncRNA-PACERR was high expression in TAMs and associated with poor prognosis in PDAC patients. Our finding validated that LncRNA-PACERR increased the number of M2-polarized cells and facilized cell proliferation, invasion and migration in vitro and in vivo. Mechanistically, LncRNA-PACERR activate KLF12/p-AKT/c-myc pathway by binding to miR-671-3p. And LncRNA-PACERR which bound to IGF2BP2 acts as an m6A-dependent manner to enhance the stability of KLF12 and c-myc in cytoplasm. In addition, the promoter of LncRNA-PACERR was a target of KLF12 and LncRNA-PACERR recruited EP300 to increase the acetylation of histone by interacting with KLF12 in nucleus. CONCLUSIONS: This study found that LncRNA-PACERR functions as key regulator of TAMs in PDAC microenvironment and revealed the novel mechanisms in cytoplasm and in nucleus.

A Novel c-MET-Targeting Antibody-Drug Conjugate for Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-associated death in the United States and has a 5-year survival rate of <4%. Although much effort has been invested in the research and development of pancreatic cancer drugs over the past 30 years, due to the lack of effective targetable carcinogenic drivers, no new targeted therapies that can improve patient prognosis have been approved for clinical use. SHR-A1403 is a new c-mesenchymal-epithelial transition factor (c-MET) antibody-drug conjugate that can be used for the targeted treatment of PDAC with high c-MET expression. This study reports for the first time the application prospects of SHR-A1403 in preclinical models of PDAC. SHR-A1403 significantly inhibited the proliferation, migration, and invasion of pancreatic cancer cells and induced cell cycle arrest and apoptosis. These changes were caused by inhibition of intracellular cholesterol biosynthesis by SHR-A1403. Therefore, targeting c-MET through SHR-A1403 showed strong preclinical anti-tumour efficacy in pancreatic cancer. Our work suggests the potential application of c-MET-targeted antibody-drug conjugate treatment for PDAC in clinical practise.

Identification of copy number variation-driven molecular subtypes informative for prognosis and treatment in pancreatic adenocarcinoma of a Chinese cohort.

BACKGROUND: Pancreatic adenocarcinoma (PAAD) is one of the most lethal carcinomas, and the current histopathological classifications are of limited use in clinical decision-making. There is an unmet need to identify new biomarkers for prognosis-informative molecular subtyping and ultimately for precision medicine. METHODS: We profiled genomic alterations for 608 PAAD patients in a Chinese cohort, including somatic mutations, pathogenic germline variants and copy number variations (CNV). Using the CNV information, we performed unsupervised consensus clustering of these patients, differential CNV analysis and functional/pathway enrichment analysis. Cox regression was conducted for progression-free survival analysis, the elastic net algorithm used for prognostic model construction, and rank-based gene set enrichment analysis for exploring tumor microenvironments. FINDINGS: Our data did not support prognostic value of point mutations in either highly mutated genes (such as KRAS, TP53, CDKN2A and SMAD4) or homologous recombination repair genes. Instead, associated with worse prognosis were amplified genes involved in DNA repair and receptor tyrosine kinase (RTK) related signalings. Motivated by this observation, we categorized patients into four molecular subtypes (namely repair-deficient, proliferation-active, repair-proficient and repair-enhanced) that differed in prognosis, and also constructed a prognostic model that can stratify patients with low or high risk of relapse. Finally, we analyzed publicly available datasets, not only reinforcing the prognostic value of our identified genes in DNA repair and RTK related signalings, but also identifying tumor microenvironment correlates with prognostic risks. INTERPRETATION: Together with the evidence from genomic footprint analysis, we suggest that repair-deficient and proliferation-active subtypes are better suited for DNA damage therapies, while immunotherapy is highly recommended for repair-proficient and repair-enhanced subtypes. Our results represent a significant step in molecular subtyping, diagnosis and management for PAAD patients. FUNDING: This work was supported by the National Natural Science Foundation of China (grant numbers 81470894, 81502695, 81672325, 81871906, 82073326, 82103482 and 32170663), the Shanghai Sailing Program (grant number 20YF1426900), and the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning (awarded to H.F.).

Tumor copy number instability is a significant predictor for late recurrence after radical surgery of pancreatic ductal adenocarcinoma.

OBJECTIVE: Our study examined the association between molecular features and clinical results of pancreatic ductal adenocarcinoma (PDAC) patients, aiming to explore the genomic determinants of the recurrence and prognosis of PDAC after surgical removal. METHODS: This retrospective study analyzed 181 PDAC patients who received pancreatectomy and adjuvant chemotherapy, with 67 patients in the training set. An internal validation set of 48 patients and an external validation set of 66 patients were used to validate the result. Comprehensive genomic profiling was performed on formalin-fixed paraffin-embedded (FFPE) tumor specimens to determine genomic features using the designed cancer-related gene panel based on next-generation sequencing (NGS). RESULTS: Significant differences were identified between the late recurrence (LR) group and early recurrence (ER) group in tumor copy number instability (CNI) levels. Next, the utility of low CNI (the middle and lowest tertile) with regard to predicting LR was confirmed in the training, internal, and external validation sets. Further univariate and multivariate analyses revealed that CNI was an independent predictive and prognostic biomarker, and had higher predictive accuracy for LR than CA19-9 level, pathological stage, tumor size, and age. In addition, CNI combined with lymph node (LN) metastasis status could provide a more accurate model for predicting LR of PDAC. CONCLUSION: We discovered and validated the association between CNI and clinical outcome in 181 patients with resectable PDAC, demonstrating the utility of lower tumor CNI levels as biomarkers of postoperative LR and favorable prognosis. Moreover, the combination of CNI and LN metastasis status elevated the predictive accuracy and illuminated strategies for patient stratification.

Melittin-induced long non-coding RNA NONHSAT105177 inhibits proliferation and migration of pancreatic ductal adenocarcinoma.

Long non-coding RNAs (lncRNAs) play crucial roles in the pathogenesis of pancreatic ductal adenocarcinoma (PDAC). Previously, we found that melittin treatment suppressed PDAC tumor growth. However, it is unclear whether lncRNAs have any role in the melittin-induced suppression of PDAC. In this study, we used microarray data to identify 844 lncRNAs that were significantly differentially expressed in response to melittin treatment. Of these lncRNAs, we focused on the lncRNA NONHSAT105177, which had about a 22-fold increase in expression with melittin treatment. We found that melittin treatment increased NONHSAT105177 expression in PDAC cell lines but not in normal pancreatic ductal epithelial cell line. NONHSAT105177 expression was significantly lower in PDAC cancer tissues than in adjacent noncancerous tissues. Additionally, overexpression of NONHSAT105177 inhibited PDAC cell proliferation, migration, and the epithelial-mesenchymal transition (EMT), both in vitro and in vivo. Consistent with the mechanism of action of melittin, NONHSAT105177 significantly downregulated cholesterol pathway genes, including Clusterin (CLU). Moreover, we found that NONHSAT105177 trafficking was mediated by exosomes. The combined findings of our current and previous studies suggest that NONHSAT105177 mediated the melittin-induced inhibition of PDAC cell growth and metastasis, which indicated a potential target for developing new strategies.

Melittin inhibits tumor growth and decreases resistance to gemcitabine by downregulating cholesterol pathway gene CLU in pancreatic ductal adenocarcinoma.

Melittin is a Chinese traditional medicine for treating chronic inflammation, immunological diseases and cancers, however, the efficacy of melittin and its mechanism for treating pancreatic ductal adenocarcinoma (PDAC) are still unknown. Here we investigated the anti-cancer activity of melittin and its regulated mechanism(s) in the PDAC models. Melittin was found to suppress tumor growth by promoting cell apoptosis and cell-cycle arrest. Interestingly, the microarray analyses demonstrated that melittin significantly regulated cholesterol biosynthesis pathway during treatment. For instance, the cholesterol pathway gene clusterin (CLU) was highly downregulated by melittin which also enhanced gemcitabine sensitivity in PDAC cells by inhibiting CLU expression. In contrast, overexpression of CLU significantly diminished melittin mediated tumor suppression and gemcitabine sensitization, suggesting that CLU is the target of melittin. Furthermore, in the xenograft mouse model, the combination therapy of melittin and gemcitabine is more efficacious for inhibiting PDAC tumor growth than either single regimen. Taken together, our study has indicated that melittin is capable of suppressing tumor growth and promoting gemcitabine sensitivity in PDAC by downregulating cholesterol pathway.