Deciphering the role of Enterococcus faecium cytidine deaminase in gemcitabine resistance of gallbladder cancer.

Gemcitabine-based chemotherapy is a cornerstone of standard care for gallbladder cancer (GBC) treatment. Still, drug resistance remains a significant challenge, influenced by factors such as tumor-associated microbiota impacting drug concentrations within tumors. Enterococcus faecium, a member of tumor-associated microbiota, was notably enriched in the GBC patient cluster. In this study, we investigated the biochemical characteristics, catalytic activity, and kinetics of the cytidine deaminase of E. faecium (EfCDA). EfCDA showed the ability to convert gemcitabine to its metabolite 2',2'-difluorodeoxyuridine. Both EfCDA and E. faecium can induce gemcitabine resistance in GBC cells. Moreover, we determined the crystal structure of EfCDA, in its apo form and in complex with 2', 2'-difluorodeoxyuridine at high resolution. Mutation of key residues abolished the catalytic activity of EfCDA and reduced the gemcitabine resistance in GBC cells. Our findings provide structural insights into the molecular basis for recognizing gemcitabine metabolite by a bacteria CDA protein and may provide potential strategies to combat cancer drug resistance and improve the efficacy of gemcitabine-based chemotherapy in GBC treatment.

MDSCs sneak CSCs out of (immuno)surveillance.

Cancer stem cells (CSCs) are known for their superior tumor-initiating and tumor-repopulating potential, partly reflecting their pronounced ability to evade immune recognition. Liu and colleagues recently identified a new aldehyde dehydrogenase (ALDH)-dependent mechanism whereby triple-negative breast CSCs evade immunosurveillance upon recruitment of myeloid-derived suppressor cells.

TRIM29 facilitates gemcitabine resistance via MEK/ERK pathway and is modulated by circRPS29/miR-770-5p axis in PDAC.

AIMS: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease. Chemotherapy based on gemcitabine (GEM) remains the first-line drug for patients with advanced PDAC. However, GEM resistance impairs its therapeutic effectiveness. Therefore, identifying effective therapeutic targets are urgently needed to overcome GEM resistance. METHODS: The clinical significance of Tripartite Motif Containing 29 (TRIM29) was identified by exploring GEO datasets and TCGA database and its potential biological functions were predicted by GSEA analysis. The regulatory axis was established by bioinformatics analysis and validated by mechanical experiments. Then, in vitro and in vivo assays were performed to validate the roles of TRIM29 in PDAC GEM resistance. RESULTS: High TRIM29 expression was associated with poor prognosis of PDAC and functional experiments demonstrated that TRIM29 promoted GEM resistance in PDAC GEM-resistant (GR) cells. Furthermore, we revealed that circRPS29 promoted TRIM29 expression via competitive interaction with miR-770-5p and then activated MEK/ERK signaling pathway. Additionally, both in vitro and in vivo functional experiments demonstrated that circRPS29/miR-770-5p/TRIM29 axis promoted PDAC GEM resistance via activating MEK/ERK signaling pathway. CONCLUSION: Our results identify the significance of the signaling axis, circRPS29/miR-770-5p/TRIM29-MEK/ERK, in PDAC GEM resistance, which will provide novel therapeutic targets for PDAC treatment.

LncRNA DYNLRB2-AS1 promotes gemcitabine resistance of nasopharyngeal carcinoma by inhibiting the ubiquitination degradation of DHX9 protein.

Gemcitabine (GEM) based induction chemotherapy is a standard treatment for locoregionally advanced nasopharyngeal carcinoma (NPC). However, approximately 15 % of patients are still resistant to GEM-containing chemotherapy, which leads to treatment failure. Nevertheless, the underlying mechanisms of GEM resistance remain poorly understood. Herein, based on a microarray analysis, we identified 221 dysregulated lncRNAs, of which, DYNLRB2-AS1 was one of the most upregulated lncRNAs in GEM-resistance NPC cell lines. DYNLRB2-AS1 was shown to function as contain an oncogenic lncRNA that promoted NPC GEM resistance, cell proliferation, but inhibited cell apoptosis. Mechanistically, DYNLRB2-AS1 could directly bind to the DHX9 protein and prevent its interaction with the E3 ubiquitin ligase PRPF19, and thus blocking PRPF19-mediated DHX9 degradation, which ultimately facilitated the repair of DNA damage in the presence of GEM. Clinically, higher DYNLRB2-AS1 expression indicated an unfavourable overall survival of NPC patients who received induction chemotherapy. Overall, this study identified the oncogenic lncRNA DYNLRB2-AS1 as an independent prognostic biomarker for patients with locally advanced NPC and as a potential therapeutic target for overcoming GEM chemoresistance in NPC.

Calcium channel blockers potentiate gemcitabine chemotherapy in pancreatic cancer.

There is currently no effective treatment for pancreatic ductal adenocarcinoma (PDAC). While palliative chemotherapy offers a survival benefit to most patients, nearly all will eventually progress on treatment and long-term survivability remains poor. Given the lack of subsequent line treatment options, in this study, we sought to identify novel strategies to prevent, delay, or overcome resistance to gemcitabine, one of the most widely used medications in PDAC. Using a combination of single-cell RNA sequencing and high-throughput proteomic analysis, we identified a subset of gemcitabine-resistant tumor cells enriched for calcium/calmodulin signaling. Pharmacologic inhibition of calcium-dependent calmodulin activation led to the rapid loss of drug-resistant phenotypes in vitro, which additional single-cell RNA sequencing identified was due to impaired activation of the RAS/ERK signaling pathway. Consistent with these observations, calcium chelation or depletion of calcium in the culture media also impaired ERK activation in gemcitabine-resistant cells, and restored therapeutic responses to gemcitabine in vitro. We observed similar results using calcium channel blockers (CCBs) such as amlodipine, which inhibited prosurvival ERK signaling in vitro and markedly enhanced therapeutic responses to gemcitabine in both orthotopic xenografts and transgenic models of PDAC. Combined, these results offer insight into a potential means of gemcitabine resistance and suggest that select CCBs may provide a clinical benefit to PDAC patients receiving gemcitabine-based chemotherapy.

Exploring the Molecular Therapeutic Mechanisms of Gemcitabine through Quantitative Proteomics.

Gemcitabine (GEM) is widely employed in the treatment of various cancers, including pancreatic cancer. Despite their clinical success, challenges related to GEM resistance and toxicity persist. Therefore, a deeper understanding of its intracellular mechanisms and potential targets is urgently needed. In this study, through mass spectrometry analysis in data-dependent acquisition mode, we carried out quantitative proteomics (three independent replications) and thermal proteome profiling (TPP, two independent replications) on MIA PaCa-2 cells to explore the effects of GEM. Our proteomic analysis revealed that GEM led to the upregulation of the cell cycle and DNA replication proteins. Notably, we observed the upregulation of S-phase kinase-associated protein 2 (SKP2), a cell cycle and chemoresistance regulator. Combining SKP2 inhibition with GEM showed synergistic effects, suggesting SKP2 as a potential target for enhancing the GEM sensitivity. Through TPP, we pinpointed four potential GEM binding targets implicated in tumor development, including in breast and liver cancers, underscoring GEM's broad-spectrum antitumor capabilities. These findings provide valuable insights into GEM's molecular mechanisms and offer potential targets for improving treatment efficacy.

Resistance to gemcitabine is mediated by the circ_0036627/miR-145/S100A16 axis in pancreatic cancer.

The development of gemcitabine (GEM) resistance severely limits the treatment efficacy in pancreatic cancer (PC) and increasing evidence highlights the vital roles of circular RNAs (circRNAs) in the tumorigenesis, progression and drug resistance of PC. However, the circRNAs underlying GEM resistance development of PC remains to be clarified. The current research aims to unveil the roles of circ_0036627 in dictating the aggressiveness and GEM sensitivity in PC. We reported the increased expression of circ_0036627 in PC tissues and PC cell lines. Elevated circ_0036627 expression level was correlated with advanced tumour grade and poor overall survival in PC patients. Functional assays and in vivo experiments demonstrated that circ_0036627 overexpression was required for the proliferation, migration invasion and GEM resistance in PC cells. circ_0036627 knockdown suppressed tumour development in vivo. The molecular analysis further showed that circ_0036627 increased S100A16 expression by sponging microRNA-145 (miR-145), a tumour-suppressive miRNA that could significantly attenuate PC cell proliferation, migration, invasion and GEM resistance. Furthermore, our findings suggested that S100A16 acted as an oncogenic factor to promote aggressiveness and GEM resistance in PC cells. In conclusion, the current findings provide new mechanistic insights into PC aggressiveness and GEM resistance, suggesting the critical role of circ_0036627/miR-145/S100A16 axis in PC progression and drug resistance development and offering novel therapeutic targets for PC therapy.

Removal of gemcitabine-induced senescent cancer cells by targeting glutaminase1 improves the therapeutic effect in pancreatic ductal adenocarcinoma.

Insufficient cancer treatment can induce senescent cancer cell formation and treatment resistance. The characteristics of induced senescent cancer (iSnCa) cells remain unclear. Pancreatic ductal adenocarcinoma (PDAC) has a low and nondurable response rate to current treatments. Our study aimed to analyze the properties of iSnCa cells and the relationship between cellular senescence and prognosis in PDAC. We evaluated the characteristics of gemcitabine-induced senescent cancer cells and the effect of senescence-associated secretory phenotype (SASP) factors released by iSnCa cells on surrounding PDAC cells. The relationship between cellular senescence and the prognosis was investigated in 50 patients with PDAC treated with gemcitabine-based neoadjuvant chemotherapy. Exposure to 5 ng/mL gemcitabine-induced senescence, decreased proliferation and increased senescence-associated ß-galactosidase-cell staining without cell death in PDAC cells; the expression of glutaminase1 (GLS1) and SASP factors also increased and caused epithelial-mesenchymal transition in surrounding PDAC cells. iSnCa cells were selectively removed by the GLS1 inhibitor bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) through apoptosis induction. Cellular senescence was induced in PDAC cells via insufficient gemcitabine in subcutaneous tumor model mice. GLS1 expression was an independent prognostic factor in patients with PDAC who received gemcitabine-based neoadjuvant chemotherapy. This is the first study to identify the relationship between senescence and GLS1 in PDAC. Low-dose gemcitabine-induced senescence and increased GLS1 expression were observed in PDAC cells. Cellular senescence may contribute to treatment resistance of PDAC, hence targeting GLS1 in iSnCa cells may improve the therapeutic effect.

Head-to-head comparison of treatment sequences in advanced pancreatic cancer-Real-world data from the prospective German TPK clinical cohort study.

There are no clear guidelines regarding the optimal treatment sequence for advanced pancreatic cancer, as head-to-head phase III randomised trials are missing. We assess real-world effectiveness of three common sequential treatment strategies by emulating a hypothetical randomised trial. This analysis included 1551 patients with advanced pancreatic cancer from the prospective, clinical cohort study Tumour Registry Pancreatic Cancer receiving FOLFIRINOX (n = 613) or gemcitabine/nab-paclitaxel (GEMNAB; n = 938) as palliative first-line treatment. We used marginal structural modelling to compare overall survival (OS) and time to deterioration (TTD) of health-related quality of life (HRQoL) between three common first- to second-line treatment sequences, adjusting for time-varying potential confounding. The sequences were: FOLFIRINOX→GEMNAB, GEMNAB→FOLFOX/OFF and GEMNAB→nanoliposomal irinotecan (NALIRI) + 5-fluorouracil. Outcome was also calculated stratified by patients' prognostic risk according to the Pancreatic Cancer Score. Median OS and TTD of HRQoL independent of risk were 10.7 [8.9, 11.9] and 6.4 [4.8, 7.7] months for FOLFIRINOX→GEMNAB, 8.4 [7.4, 9.7] and 5.8 [4.6, 7.1] months for GEMNAB→FOLFOX/OFF and 8.9 [7.8, 10.4] and 4.6 [4.1, 6.1] months for GEMNAB→NALIRI+5-fluorouracil. Compared to FOLFIRINOX→GEMNAB, OS and TTD were worse for poor-risk patients with GEMNAB→FOLFOX/OFF (OS: HR 2.09 [1.47, 2.98]; TTD: HR 1.97 [1.19, 3.27]) and those with GEMNAB→NALIRI+5-fluorouracil (OS: HR 1.35, [0.76, 2.39]; TTD: HR 2.62 [1.56, 4.42]). Brackets denote 95%-confidence intervals. The estimated real-world effectiveness of the three treatment sequences evaluated were largely comparable. Poor-risk patients might benefit from intensified treatment with FOLFIRINOX→GEMNAB in terms of clinical and patient-reported outcomes. Future randomised trials on sequential treatments in advanced pancreatic cancer are warranted.

Pharmacogenomic study of gemcitabine efficacy in patients with metastatic pancreatic cancer: A multicenter, prospective, observational cohort study (GENESECT study).

BACKGROUND: Genetic polymorphisms of molecules are known to cause individual differences in the therapeutic efficacy of anticancer drugs. However, to date, germline mutations (but not somatic mutations) for anticancer drugs have not been adequately studied. The objective of this study was to investigate the association between germline polymorphisms of gemcitabine metabolic and transporter genes with carbohydrate antigen 19-9 (CA 19-9) response (decrease ≥50% from the pretreatment level at 8 weeks) and overall survival (OS) in patients with metastatic pancreatic cancer who receive gemcitabine-based chemotherapy. METHODS: This multicenter, prospective, observational study enrolled patients with metastatic pancreatic cancer patients who were receiving gemcitabine monotherapy or gemcitabine plus nanoparticle albumin-bound paclitaxel combination chemotherapy. Thirteen polymorphisms that may be involved in gemcitabine responsiveness were genotyped, and univariate and multivariate logistic regression analyses were used to determine the association of these genotypes with CA 19-9 response and OS. The significance level was set at 5%. RESULTS: In total, 180 patients from 11 hospitals in Japan were registered, and 159 patients whose CA 19-9 response could be assessed were included in the final analysis. Patients who had a CA 19-9 response had significantly longer OS (372 vs. 241 days; p = .007). RRM1 2464A>G and RRM2 175T>G polymorphisms suggested a weak association with CA 19-9 response and OS, but it was not statistically significant. COX-2 -765G>C polymorphism did not significantly correlate with CA 19-9 response but was significantly associated with OS (hazard ratio, 2.031; p = .019). CONCLUSIONS: Genetic polymorphisms from the pharmacokinetics of gemcitabine did not indicate a significant association with efficacy, but COX-2 polymorphisms involved in tumor cell proliferation might affect OS.

Early Tumor Shrinkage and Depth of Response as Predictors of Survival for Advanced Biliary Tract Cancer: An Exploratory Analysis of JCOG1113.

BACKGROUND: Recent studies suggest that early tumor shrinkage (ETS) and depth of response (DpR) reflect outcomes of chemotherapy in various cancers. This study evaluated the association of ETS and DpR with clinical outcomes using data from JCOG1113, which demonstrated the non-inferiority of gemcitabine plus S-1 (GS) to gemcitabine plus cisplatin (GC) for chemotherapy-naïve advanced biliary tract cancer. MATERIAL AND METHODS: In total, 354 (289 with measurable target lesions) patients enrolled in JCOG1113 were divided into ETS-unachieved and ETS-achieved groups (≥20% tumor reduction at week 6) and DpR-low and DpR-high groups (≥40% maximum shrinkage) until 12 weeks after enrollment. The impact of ETS and DpR on survival outcome was evaluated using the multivariable Cox proportional hazard model. RESULTS: The proportions of patients in the ETS-achieved and DpR-high groups were similar between the 2 treatment arms. The hazard ratios (HRs) of progression-free survival (PFS) and overall survival (OS) for the ETS-achieved group were 0.70 (95% confidence interval (CI), 0.52-0.93) and 0.60 (95%CI, 0.44-0.81), respectively. The HRs of PFS and OS for the DpR-high group were 0.67 (95%CI, 0.48-0.94) and 0.64 (95%CI, 0.46-0.90), respectively. In the subpopulation treatment effect pattern plot analysis, most patients in the ETS-achieved group in the GC arm did not experience disease progression after 12 weeks from the landmark. CONCLUSION: As on-treatment markers, ETS and DpR were effective tools. ETS was clinically useful, because it can be used to evaluate the outcomes of treatment early at a specific time.

Nab-paclitaxel plus S-1 versus nab-paclitaxel plus gemcitabine in patients with advanced pancreatic cancer: a multicenter, randomized, phase II study.

BACKGROUND: Encouraging antitumor activity of nab-paclitaxel plus S-1 (AS) has been shown in several small-scale studies. This study compared the efficacy and safety of AS versus standard-of-care nab-paclitaxel plus gemcitabine (AG) as a first-line treatment for advanced pancreatic cancer (PC). METHODS: In this multicenter, randomized, phase II trial, eligible patients with unresectable, locally advanced, or metastatic PC were recruited and randomly assigned (1:1) to receive AS (nab-paclitaxel 125 mg/m2 on days 1 and 8; S-1 twice daily on days 1 through 14) or AG (nab-paclitaxel 125 mg/m2 on days 1 and 8; gemcitabine 1000 mg/m2 on days 1 and 8) for 6 cycles. The primary endpoint was progression-free survival (PFS). RESULTS: Between July 16, 2019, and September 9, 2022, 62 patients (AS, n = 32; AG, n = 30) were treated and evaluated. With a median follow-up of 8.36 months at preplanned interim analysis (data cutoff, March 24, 2023), the median PFS (8.48 vs 4.47 months; hazard ratio [HR], 0.402; P = .002) and overall survival (OS; 13.73 vs 9.59 months; HR, 0.226; P < .001) in the AS group were significantly longer compared to the AG group. More patients had objective response in the AS group than AG group (37.50% vs 6.67%; P = .005). The most common grade 3-4 adverse events were neutropenia and leucopenia in both groups, and gamma glutamyl transferase increase was observed only in the AG group. CONCLUSION: The first-line AS regimen significantly extended both PFS and OS of Chinese patients with advanced PC when compared with the AG regimen, with a comparable safety profile. (ClinicalTrials.gov Identifier: NCT03636308).

Targeting UBE2T Potentiates Gemcitabine Efficacy in Pancreatic Cancer by Regulating Pyrimidine Metabolism and Replication Stress.

BACKGROUND & AIMS: Although small patient subsets benefit from current targeted strategies or immunotherapy, gemcitabine remains the first-line drug for pancreatic cancer (PC) treatment. However, gemcitabine resistance is widespread and compromises long-term survival. Here, we identified ubiquitin-conjugating enzyme E2T (UBE2T) as a potential therapeutic target to combat gemcitabine resistance in PC. METHODS: Proteomics and metabolomics were combined to examine the effect of UBE2T on pyrimidine metabolism remodeling. Spontaneous PC mice (LSL-KrasG12D/+, LSL-Trp53R172H/+, Pdx1-Cre; KPC) with Ube2t-conditional knockout, organoids, and large-scale clinical samples were used to determine the effect of UBE2T on gemcitabine efficacy. Organoids, patient-derived xenografts (PDX), and KPC mice were used to examine the efficacy of the combination of a UBE2T inhibitor and gemcitabine. RESULTS: Spontaneous PC mice with Ube2t deletion had a marked survival advantage after gemcitabine treatment, and UBE2T levels were positively correlated with gemcitabine resistance in clinical patients. Mechanistically, UBE2T catalyzes ring finger protein 1 (RING1)-mediated ubiquitination of p53 and relieves the transcriptional repression of ribonucleotide reductase subunits M1 and M2, resulting in unrestrained pyrimidine biosynthesis and alleviation of replication stress. Additionally, high-throughput compound library screening using organoids identified pentagalloylglucose (PGG) as a potent UBE2T inhibitor and gemcitabine sensitizer. The combination of gemcitabine and PGG diminished tumor growth in PDX models and prolonged long-term survival in spontaneous PC mice. CONCLUSIONS: Collectively, UBE2T-mediated p53 degradation confers PC gemcitabine resistance by promoting pyrimidine biosynthesis and alleviating replication stress. This study offers an opportunity to improve PC survival by targeting UBE2T and develop a promising gemcitabine sensitizer in clinical translation setting.

Development and validation of AI-assisted transcriptomic signatures to personalize adjuvant chemotherapy in patients with pancreatic ductal adenocarcinoma.

BACKGROUND: After surgical resection of pancreatic ductal adenocarcinoma (PDAC), patients are predominantly treated with adjuvant chemotherapy, commonly consisting of gemcitabine-based regimens or the modified FOLFIRINOX regimen (mFFX). While mFFX has been shown to be more effective than gemcitabine-based regimens, it is also associated with higher toxicity. Current treatment decisions are based on patient performance status rather than on the molecular characteristics of the tumor. To address this gap, the goal of this study was to develop drug-specific transcriptomic signatures for personalized chemotherapy treatment. PATIENTS AND METHODS: We used PDAC datasets from preclinical models, encompassing chemotherapy response profiles for the mFFX-regimen components. From them we identified specific gene transcripts associated with chemotherapy response. Three transcriptomic AI-signatures were obtained by combining Independent Component Analysis, Least Absolute Shrinkage and the Selection Operator-Random Forest approach. We integrated a previously developed gemcitabine signature with three newly developed ones. The machine learning strategy employed to enhance these signatures incorporates transcriptomic features from the tumor microenvironment, leading to the development of the Pancreas-View tool ultimately clinically validated in a cohort of 343 patients from the PRODIGE-24/CCTG PA6 trial. RESULTS: Patients who were predicted to be sensitive to the administered drugs (n=164; 47.8%) had longer disease-free survival (DFS) than the other patients. The median DFS in the mFFX sensitive group treated with mFFX was 50.0 months (stratified HR: 0.31; 95% CI, 0.21-0.44; p<0.001) and 33.7 months (stratified HR: 0.40; 95% CI, 0.17-0.59; p<0.001) in the gemcitabine sensitive group when treated with gemcitabine. Comparatively patients with signature predictions unmatched with the treatments (n=86; 25.1%) or those resistant to all drugs (n=93; 27.1%) had shorter DFS (10.6 and 10.8 months, respectively). CONCLUSIONS: This study presents a transcriptome-based tool that was developed using preclinical models and machine learning to accurately predict sensitivity to mFFX and gemcitabine.

Camrelizumab plus gemcitabine and oxaliplatin for relapsed or refractory classical Hodgkin lymphoma: a phase II trial.

BACKGROUND: Classical Hodgkin lymphoma (cHL) is a highly curable disease, while novel therapy is needed for refractory or relapsed (R/R) patients. This phase II trial aimed to evaluate the role of camrelizumab plus gemcitabine and oxaliplatin (GEMOX) in R/R cHL patients. METHODS: Transplant-eligible patients with R/R cHL were enrolled and received two 14-day cycles of camrelizumab 200 mg intravenously (IV) and two 28-day cycles of camrelizumab 200 mg IV, gemcitabine 1000 mg/m2 IV, and oxaliplatin 100 mg/m2 IV on days 1 and 15. Patients with partial response (PR) or stable disease received an additional cycle of combination therapy. Those who achieved complete response (CR) or PR proceeded to autologous stem cell transplantation (ASCT). The primary endpoint was the CR rate at the end of protocol therapy before ASCT. RESULTS: Forty-two patients were enrolled. At the end of protocol therapy, the objective response rate and CR rate were 94.9% (37/39) and 69.2% (27/39) in the evaluable set, and 88.1% (37/42) and 64.3% (27/42) in the full analysis set, respectively. Twenty-nine patients (69.0%) proceeded to ASCT, and 4 of 5 patients with PR achieved CR after ASCT. After a median follow-up of 20.7 months, the 12-month progression-free survival rate was 96.6% and the 12-month overall survival rate was 100%. Grade 3 or higher treatment emergent adverse events occurred in 28.6% of patients (12/42), mainly hematological toxicity. CONCLUSIONS: Camrelizumab combined with GEMOX constitutes an effective salvage therapy for R/R cHL, proving to be relatively well-tolerated and facilitating ASCT in most patients, thus promoting sustained remission. TRIAL REGISTRATION: ClinicalTrials.gov NCT04239170. Registered on January 1, 2020.

ENO1 contributes to the gemcitabine resistance of pancreatic cancer through the YAP1 signaling pathway.

Pancreatic cancer (PC), a leading cause of cancer-related deaths, has a 5-year survival rate of approximately 10%. α-Enolase (ENO1) is a junction channel protein involved in tumor cell apoptosis and chemoresistance. However, the role of ENO1 in PC remains unclear. The expression and prognosis of ENO1 levels were determined in PC using public databases based on The Cancer Genome Atlas (TCGA) data sets. Cell viability, half maximal inhibitory concentration (IC50), autophagy, apoptosis, and autophagy markers were examined using cell counting kit-8 (CCK-8), transmission electron microscope, flow cytometry assays, and immunoblot, respectively. Using the Gene Expression Omnibus (GEO) and TCGA data sets, we found that ENO1 was significantly enriched in PC tumor tissues, and high expression levels of ENO1 were associated with an unfavorable prognosis. Whereas ENO1 silencing suppressed proliferation, autophagy, and induced cell apoptosis in PC cells, and inhibited tumor growth in vivo. Mechanistically, knockdown of ENO1 enhanced cellular cytotoxicity of gemcitabine (GEM), as well as reducing the expression of yes-associated protein 1 (YAP1), a major downstream effector of the Hippo pathway in vitro. YAP1 promoted autophagy and protected PC cells from GEM-induced apoptotic cell death. Furthermore, YAP1 overexpression attenuated the inhibition effects of ENO1 silencing. Our results suggest that ENO1 overexpression promotes cell growth and tumor progression by increasing the expression of YAP1 in PC. Further studies are required to understand the detailed mechanisms between ENO1 and YAP1 in PC.

Cancer-associated fibroblasts promote stemness maintenance and gemcitabine resistance via HIF-1α/miR-21 axis under hypoxic conditions in pancreatic cancer.

Gemcitabine (GEM) resistance affects chemotherapy efficacy of pancreatic cancer (PC). Cancer-associated fibroblasts (CAFs) possess the ability of regulating chemoresistance. This study probed the mechanism of hypoxia-treated CAFs regulating cell stemness and GEM resistance in PC. Miapaca-2/SW1990 were co-cultured with PC-derived CAFs under normoxic/hypoxic conditions. Cell viability/self-renewal ability was determined by MTT/sphere formation assays, respectively. Protein levels of CD44, CD133, Oct4, and Sox2 were determined by western blot. GEM tumoricidal assay was performed. PC cell GEM resistance was evaluated by MTT assay. CAFs were cultured at normoxia/hypoxia. HIF-1α and miR-21 expression levels were assessed by RT-qPCR and western blot, with their binding sites and binding relationship predicted and verified. CAF-extracellular vesicles (EVs) were incubated with Miapaca-2 cells. The RAS/AKT/ERK pathway activation was detected by western blot. PC xenograft models were established and treated with hypoxic CAF-EVs and GEM. CAFs and PC cell co-culture increased cell stemness maintenance, GEM resistance, cell viability, stem cell sphere number, and protein levels of CD44, CD133, Oct4, and Sox2, and weakened GEM tumoricidal ability to PC cells, with the effects further enhanced by hypoxia. Hypoxia induced HIF-1α and miR-21 overexpression in CAFs. Hypoxia promoted CAFs to secrete high-level miR-21 EVs via the HIF-1α/miR-21 axis, and activated the miR-21/RAS/AKT/ERK pathway. CAF-EVs promoted GEM resistance in PC via the miR-21/RAS/ATK/ERK pathway in vivo. Hypoxia promoted CAFs to secrete high-level miR-21 EVs through the HIF-1α/miR-21 axis, and activated the miR-21/RAS/AKT/ERK pathway via EVs to trigger stemness maintenance and GEM resistance in PC.

A Phase 2 Trial of Intravesical Gemcitabine and Docetaxel in the Treatment of Bacillus Calmette-Guérin‒Naïve Nonmuscle-Invasive Urothelial Carcinoma of the Bladder.

PURPOSE: Combination intravesical gemcitabine and docetaxel (GemDoce) has demonstrated efficacy as second-line therapy for patients with bacillus Calmette-Guérin (BCG)‒unresponsive nonmuscle-invasive urothelial carcinoma of the bladder (NMIBC). In the context of widespread BCG shortages, we performed a phase 2 prospective trial to assess GemDoce for BCG-naïve NMIBC. MATERIALS AND METHODS: This study is a prospective, single-arm, open-label phase 2 trial for patients with BCG-naïve high-risk NMIBC. Intravesical GemDoce was given weekly for 6 weeks as induction followed by monthly maintenance therapy for 2 years among responders. The primary end point was 3-month complete response, and key secondary end points included adverse events (AEs) and 12-month recurrence-free survival. RESULTS: Twenty-five patients were enrolled between August 2020 and August 2022 with median follow-up of 19.6 months. The pretreatment pathologic stages were high-grade (HG) T1 with carcinoma in situ (CIS; n = 7), HGT1 without CIS (n = 6), HGTa (n = 9), and CIS alone (n = 3). The 3-month complete response rate was 100% and recurrence-free survival at 12 months was 92%. Two patients with pretreatment HGT1 had HGT1 recurrences at 9 and 12 months. No patients progressed to T2 disease, underwent radical cystectomy, or had any radiographic evidence of progressive disease. Grade 1 AEs were common (23/25 patients) including hematuria, urinary frequency, urgency, and fatigue. Five patients (20%) experienced a grade 3 AE including hematuria and UTI. CONCLUSIONS: In this single-arm phase 2 trial, GemDoce was well tolerated with promising efficacy for patients with BCG-naïve high-risk NMIBC.

Skeletal Muscle-Derived Irisin Enhances Gemcitabine Sensitivity and Suppresses Migration Ability in Pancreatic Ductal Adenocarcinoma.

BACKGROUND: High skeletal muscle mass might be a prognostic factor for patients with pancreatic ductal adenocarcinoma (PDAC); however, the underlying reason is unclear. We hypothesized that myokines, which are cytokines secreted by the skeletal muscle, function as suppressors of PDAC. We specifically examined irisin, a myokine, which plays a critical role in the modulation of metabolism, to clarify the anticancer mechanisms. METHODS: First, the effect of the conditioned medium (CM) from skeletal muscle cells and from irisin-knockdown skeletal muscle cells on PDAC cell lines was evaluated. We then investigated the effects and anticancer mechanism of irisin in PDAC cells, and evaluated the anticancer effect of recombinant irisin in a PDAC xenograft mouse model. Finally, patients undergoing pancreatic resection for PDAC were divided into two groups based on their serum irisin level, and the long-term outcomes were evaluated. RESULTS: The CM enhanced gemcitabine sensitivity by inducing apoptosis and decreasing cell migration by inhibiting epithelial-mesenchymal transition (EMT) in PDAC cell lines. The CM derived from irisin-knockdown skeletal muscle cells did not affect the PDAC cell lines. The addition of recombinant irisin to PDAC cell lines facilitated sensitivity to gemcitabine by inhibiting the mitogen-activated protein kinase (MAPK) pathway, and decreased migration by inhibiting EMT via the transforming growth factor-ß/SMAD pathway. Xenografts injected with gemcitabine and recombinant irisin grew slower than the xenografts injected with gemcitabine alone. The overall survival was prolonged in the high-irisin group compared with that in the low-irisin group. CONCLUSIONS: Skeletal muscle-derived irisin may affect PDAC by enhancing its sensitivity to gemcitabine and suppressing EMT.

A fluorescent gemcitabine prodrug that follows a nucleoside transporter-independent internalization and bears enhanced therapeutic efficacy with respect to gemcitabine.

The multiplexity of cancer has rendered it the second leading cause of mortality worldwide and theragnostic prodrugs have gained popularity in recent years as a means of treatment. Theragnostic prodrugs enable the simultaneous diagnosis and therapy of tumors via high-precision real-time drug release monitoring. Herein, we report the development of the small theragnostic prodrug GF, based on the nucleoside anticancer agent gemcitabine and the fluorescent dye 5(6)-carboxyfluorescein. We have successfully demonstrated its efficient internalization in tumor cells, showing localization throughout both the early and late endocytic pathways. Its mechanism of cell internalization was evaluated, confirming its independence from nucleoside transporters. Its cellular localization via confocal microscopy revealed a clathrin-mediated endocytosis mechanism, distinguishing it from analogous compounds studied previously. Furthermore, GF exhibited stability across various pH values and in human blood plasma. Subsequently, its in vitro cytotoxicity was assessed in three human cancer cell lines (A549, U87 and T98). Additionally, its pharmacokinetic profile in mice was investigated and the consequent drug release was monitored. Finally, its in vivo visualization was accomplished in zebrafish xenotransplantation models and its in vivo efficacy was evaluated in A549 xenografts. The results unveiled an intriguing efficacy profile, positioning GF as a compelling candidate warranting further investigation.