YBX1 as a therapeutic target to suppress the LRP1-ß-catenin-RRM1 axis and overcome gemcitabine resistance in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is highly malignant and has a poor prognosis, without effective therapeutic targets in common gene mutations. Gemcitabine, a first-line chemotherapeutic for PDAC, confers <10 % 5-year survival rate because of drug resistance. Y-box binding protein 1 (YBX1), associated with multidrug-resistance gene activation, remains unelucidated in PDAC gemcitabine resistance. In vivo and in vitro, we verified YBX1's promotional effects, especially gemcitabine resistance, in pancreatic cancer cells. YBX1-induced LRP1 transcription by binding to the LRP1 promoter region significantly altered the concentration and distribution of ß-catenin in pancreatic cancer cells. Through TCF3, ß-catenin bound to the promoter region of RRM1, a key gene for gemcitabine resistance, that promotes RRM1 expression. Combination therapy with the YBX1 inhibitor SU056 and gemcitabine effectively reduced gemcitabine resistance in in vivo and in vitro experiments. High YBX1 expression promoted pathogenesis and gemcitabine resistance in pancreatic cancer through the YBX1-LRP1-ß-catenin-RRM1 axis. Combining YBX1 inhibitors with gemcitabine may provide a new direction for combination chemotherapy to overcome gemcitabine resistance, which frequently occurs during chemotherapy for pancreatic cancer.

A distinct subset of urothelial cells with enhanced EMT features promotes chemotherapy resistance and cancer recurrence by increasing COL4A1-ITGB1 mediated angiogenesis.

Drug resistance and tumor recurrence remain clinical challenges in the treatment of urothelial carcinoma (UC). However, the underlying mechanism is not fully understood. Here, we performed single-cell RNA sequencing and identified a subset of urothelial cells with epithelial-mesenchymal transition (EMT) features (EMT-UC), which is significantly correlated with chemotherapy resistance and cancer recurrence. To validate the clinical significance of EMT-UC, we constructed EMT-UC like cells by introducing overexpression of two markers, Zinc Finger E-Box Binding Homeobox 1 (ZEB1) and Desmin (DES), and examined their histological distribution characteristics and malignant phenotypes. EMT-UC like cells were mainly enriched in UC tissues from patients with adverse prognosis and exhibited significantly elevated EMT, migration and gemcitabine tolerance in vitro. However, EMT-UC was not specifically identified from tumorous tissues, certain proportion of them were also identified in adjacent normal tissues. Tumorous EMT-UC highly expressed genes involved in malignant behaviors and exhibited adverse prognosis. Additionally, tumorous EMT-UC was associated with remodeled tumor microenvironment (TME), which exhibited high angiogenic and immunosuppressive potentials compared with the normal counterparts. Furthermore, a specific interaction of COL4A1 and ITGB1 was identified to be highly enriched in tumorous EMT-UC, and in the endothelial component. Targeting the interaction of COL4A1 and ITGB1 with specific antibodies significantly suppressed tumorous angiogenesis and alleviated gemcitabine resistance of UC. Overall, our findings demonstrated that the driven force of chemotherapy resistance and recurrence of UC was EMT-UC mediated COL4A1-ITGB1 interaction, providing a potential target for future UC treatment.

E3 ubiquitin ligase UBR5 promotes gemcitabine resistance in pancreatic cancer by inducing O-GlcNAcylation-mediated EMT via destabilization of OGA.

Pancreatic cancer (PC) is among the deadliest malignancies, with an extremely poor diagnosis and prognosis. Gemcitabine (GEM) remains the first-line drug for treating PC; however, only a small percentage of patients benefit from current immunotherapies or targeted therapies. Resistance to GEM is prevalent and affects long-term survival. We found that ubiquitin-protein ligase E3 module N-recognition 5 (UBR5) is a therapeutic target against GEM resistance. UBR5 was markedly upregulated in clinical GEM-resistant PC samples and GEM-resistant PC cells. UBR5 knockdown markedly increased GEM sensitivity in GEM-resistant PC cell lines. UBR5-mediated GEM resistance was accompanied by activation of epithelial-mesenchymal transition (EMT) and could be mitigated by inhibiting EMT. Further analysis revealed that UBR5 promoted GEM resistance in PC cells by enhancing O-GlcNAcylation-mediated EMT. In addition, UBR5 knockdown resulted in increased O-GlcNAase (OGA) levels, an essential negatively regulated enzyme in the O-GlcNAcylation process. We identified a negative association between OGA and UBR5 levels, which further supported the hypothesis that O-GlcNAcylation-mediated GEM resistance induced by UBR5 is OGA-dependent in PC cells. Mechanistic studies revealed that UBR5 acts as an E3 ubiquitin ligase of OGA and regulates O-GlcNAcylation by binding and modulating OGA, facilitating its degradation and ubiquitination. Additionally, high-throughput compound library screening using three-dimensional protein structure analysis and drug screening identified a Food and Drug Administration drug, Y-39983 dihydrochloride, as a potent GEM sensitiser and UBR5 inhibitor. The combination of Y-39983 dihydrochloride and GEM attenuated tumour growth in a mouse xenograft tumour model. Collectively, these data demonstrated that UBR5 plays a pivotal role in the sensitisation of PC to GEM and provides a potential therapeutic strategy to overcome GEM resistance.

The double life of a chemotherapy drug: Immunomodulatory functions of gemcitabine in cancer.

Although the development of immunotherapies has been revolutionary in the treatment of several cancers, many cancer types remain unresponsive to immune-based treatment and are largely managed by chemotherapy drugs. However, chemotherapeutics are not infallible and are frequently rendered ineffective as resistance develops from prolonged exposure. Recent investigations have indicated that some chemotherapy drugs have additional functions beyond their normative cytotoxic capacity and are in fact immune-modifying agents. Of the pharmaceuticals with identified immune-editing properties, gemcitabine is well-studied and of interest to clinicians and scientists alike. Gemcitabine is a chemotherapy drug approved for the treatment of multiple cancers, including breast, lung, pancreatic, and ovarian. Because of its broad applications, relatively low toxicity profile, and history as a favorable combinatory partner, there is promise in the recharacterization of gemcitabine in the context of the immune system. Such efforts may allow the identification of suitable immunotherapeutic combinations, wherein gemcitabine can be used as a priming agent to improve immunotherapy efficacy in traditionally insensitive cancers. This review looks to highlight documented immunomodulatory abilities of one of the most well-known chemotherapy agents, gemcitabine, relating to its influence on cells and proteins of the immune system.

ECM1 and KRT6A are involved in tumor progression and chemoresistance in the effect of dexamethasone on pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) has a very poor prognosis. Neoadjuvant chemotherapy is an effective PDAC treatment option, but chemotherapy causes unfavorable side effects. Glucocorticoids (e.g., dexamethasone [DEX]) are administered to reduce side effects of chemotherapy for solid tumors, including pancreatic cancer. Glucocorticoids have both beneficial and detrimental effects, however. We investigated the functional changes and gene-expression profile alterations induced by DEX in PDAC cells. PDAC cells were treated with DEX, and the cell proliferation, migration, invasion, and chemosensitivity to gemcitabine (GEM) were evaluated. The results demonstrated decreased cell proliferative capacity, increased cell migration and invasion, and decreased sensitivity to GEM. A comprehensive genetic analysis revealed marked increases in ECM1 and KRT6A in DEX-treated PDAC cells. We evaluated the effects of ECM1 and KRT6A expression by using PDAC cells transfected with those genes. Neither ECM1 nor KRT6A changed the cells' proliferation, but each enhanced cell migration and invasion. ECM1 decreased sensitivity to GEM. We also assessed the clinicopathological significance of the expressions of ECM1 and KRT6A in 130 cases of PDAC. An immunohistochemical analysis showed that KRT6A expression dominated the poorly differentiated areas. High expressions of these two proteins in PDAC were associated with a poorer prognosis. Our results thus demonstrated that DEX treatment changed PDAC cells' functions, resulting in decreased cell proliferation, increased cell migration and invasion, and decreased sensitivity to GEM. The molecular mechanisms of these changes involve ECM1 and KRT6A, whose expressions are induced by DEX.

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.

Enfortumab Vedotin and Pembrolizumab in Untreated Advanced Urothelial Cancer.

BACKGROUND: No treatment has surpassed platinum-based chemotherapy in improving overall survival in patients with previously untreated locally advanced or metastatic urothelial carcinoma. METHODS: We conducted a phase 3, global, open-label, randomized trial to compare the efficacy and safety of enfortumab vedotin and pembrolizumab with the efficacy and safety of platinum-based chemotherapy in patients with previously untreated locally advanced or metastatic urothelial carcinoma. Patients were randomly assigned in a 1:1 ratio to receive 3-week cycles of enfortumab vedotin (at a dose of 1.25 mg per kilogram of body weight intravenously on days 1 and 8) and pembrolizumab (at a dose of 200 mg intravenously on day 1) (enfortumab vedotin-pembrolizumab group) or gemcitabine and either cisplatin or carboplatin (determined on the basis of eligibility to receive cisplatin) (chemotherapy group). The primary end points were progression-free survival as assessed by blinded independent central review and overall survival. RESULTS: A total of 886 patients underwent randomization: 442 to the enfortumab vedotin-pembrolizumab group and 444 to the chemotherapy group. As of August 8, 2023, the median duration of follow-up for survival was 17.2 months. Progression-free survival was longer in the enfortumab vedotin-pembrolizumab group than in the chemotherapy group (median, 12.5 months vs. 6.3 months; hazard ratio for disease progression or death, 0.45; 95% confidence interval [CI], 0.38 to 0.54; P<0.001), as was overall survival (median, 31.5 months vs. 16.1 months; hazard ratio for death, 0.47; 95% CI, 0.38 to 0.58; P<0.001). The median number of cycles was 12 (range, 1 to 46) in the enfortumab vedotin-pembrolizumab group and 6 (range, 1 to 6) in the chemotherapy group. Treatment-related adverse events of grade 3 or higher occurred in 55.9% of the patients in the enfortumab vedotin-pembrolizumab group and in 69.5% of those in the chemotherapy group. CONCLUSIONS: Treatment with enfortumab vedotin and pembrolizumab resulted in significantly better outcomes than chemotherapy in patients with untreated locally advanced or metastatic urothelial carcinoma, with a safety profile consistent with that in previous reports. (Funded by Astellas Pharma US and others; EV-302 ClinicalTrials.gov number, NCT04223856.).

Deoxycytidine kinase inactivation enhances gemcitabine resistance and sensitizes mitochondrial metabolism interference in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is considered one of the most lethal forms of cancer. Although in the last decade, an increase in 5-year patient survival has been observed, the mortality rate remains high. As a first-line treatment for PDAC, gemcitabine alone or in combination (gemcitabine plus paclitaxel) has been used; however, drug resistance to this regimen is a growing issue. In our previous study, we reported MYC/glutamine dependency as a therapeutic target in gemcitabine-resistant PDAC secondary to deoxycytidine kinase (DCK) inactivation. Moreover, enrichment of oxidative phosphorylation (OXPHOS)-associated genes was a common property shared by PDAC cell lines, and patient clinical samples coupled with low DCK expression was also demonstrated, which implicates DCK in cancer metabolism. In this article, we reveal that the expression of most genes encoding mitochondrial complexes is remarkably upregulated in PDAC patients with low DCK expression. The DCK-knockout (DCK KO) CFPAC-1 PDAC cell line model reiterated this observation. Particularly, OXPHOS was functionally enhanced in DCK KO cells as shown by a higher oxygen consumption rate and mitochondrial ATP production. Electron microscopic observations revealed abnormal mitochondrial morphology in DCK KO cells. Furthermore, DCK inactivation exhibited reactive oxygen species (ROS) reduction accompanied with ROS-scavenging gene activation, such as SOD1 and SOD2. SOD2 inhibition in DCK KO cells clearly induced cell growth suppression. In combination with increased anti-apoptotic gene BCL2 expression in DCK KO cells, we finally reveal that venetoclax and a mitochondrial complex I inhibitor are therapeutically efficacious for DCK-inactivated CFPAC-1 cells in in vitro and xenograft models. Hence, our work provides insight into inhibition of mitochondrial metabolism as a novel therapeutic approach to overcome DCK inactivation-mediated gemcitabine resistance in PDAC patient treatment.

Reprogramming the Intrahepatic Cholangiocarcinoma Immune Microenvironment by Chemotherapy and CTLA-4 Blockade Enhances Anti-PD-1 Therapy.

Intrahepatic cholangiocarcinoma (ICC) has limited therapeutic options and a dismal prognosis. Adding blockade of the anti-programmed cell death protein (PD)-1 pathway to gemcitabine/cisplatin chemotherapy has recently shown efficacy in biliary tract cancers but with low response rates. Here, we studied the effects of anti-cytotoxic T lymphocyte antigen (CTLA)-4 when combined with anti-PD-1 and gemcitabine/cisplatin in orthotopic murine models of ICC. This combination therapy led to substantial survival benefits and reduction of morbidity in two aggressive ICC models that were resistant to immunotherapy alone. Gemcitabine/cisplatin treatment increased tumor-infiltrating lymphocytes and normalized the ICC vessels and, when combined with dual CTLA-4/PD-1 blockade, increased the number of activated CD8+Cxcr3+IFNγ+ T cells. CD8+ T cells were necessary for the therapeutic benefit because the efficacy was compromised when CD8+ T cells were depleted. Expression of Cxcr3 on CD8+ T cells is necessary and sufficient because CD8+ T cells from Cxcr3+/+ but not Cxcr3-/- mice rescued efficacy in T cell‒deficient mice. Finally, rational scheduling of anti-CTLA-4 "priming" with chemotherapy followed by anti-PD-1 therapy achieved equivalent efficacy with reduced overall drug exposure. These data suggest that this combination approach should be clinically tested to overcome resistance to current therapies in ICC patients.

Is switching intravesical chemotherapeutic agents beneficial in short-term recurrent high-risk non-muscle-invasive bladder tumors? A 5-year retrospective study.

OBJECTIVE: To explore if switching intravesical chemotherapeutic agents is beneficial in short-term recurrences of high-risk non-muscle-invasive bladder cancer (NMIBC) following the failure of preceding intravesical therapy. MATERIALS AND METHODS: From June 2010 to October 2015, 205 patients with NMIBC who experienced tumor recurrence within a year after receiving first-line intravesical chemotherapy (IVC) were classified into two groups. After a second complete transurethral resection (TUR) process, we immediately altered the intravesical instillation agent for 107 patients (group A). In contrast, the remaining 98 patients (group B) continued using their original intravesical instillation agent. After transurethral resection of the bladder tumor (TURBT), all patients received either an immediate instillation of epirubicin (EPI), gemcitabine (GEM), or hydroxycamptothecin (HCPT), followed by regular induction and maintenance instillations. Recurrence and progression rates were evaluated using the Chi-square test, and recurrence-free survival (RFS) and progression-free survival (PFS) were calculated using the Kaplan-Meier method. RESULTS: In this study, there was no significant difference in either the 5-year tumor recurrence or progression rates between the two groups (p > 0.05) The Kaplan-Meier plot showed no difference in progression-free or recurrence-free survival between the two groups. CONCLUSION: Switching IVC agents does not improve RFS and PFS for patients with short-term recurrent high-risk NMIBC.

Toripalimab Plus Chemotherapy for Recurrent or Metastatic Nasopharyngeal Carcinoma: The JUPITER-02 Randomized Clinical Trial.

Importance: There are currently no therapies approved by the US Food and Drug Administration for nasopharyngeal carcinoma (NPC). Gemcitabine-cisplatin is the current standard of care for the first-line treatment of recurrent or metastatic NPC (RM-NPC). Objective: To determine whether toripalimab in combination with gemcitabine-cisplatin will significantly improve progression-free survival and overall survival as first-line treatment for RM-NPC, compared with gemcitabine-cisplatin alone. Design, Setting, and Participants: JUPITER-02 is an international, multicenter, randomized, double-blind phase 3 study conducted in NPC-endemic regions, including mainland China, Taiwan, and Singapore. From November 10, 2018, to October 20, 2019, 289 patients with RM-NPC with no prior systemic chemotherapy in the RM setting were enrolled from 35 participating centers. Interventions: Patients were randomized (1:1) to receive toripalimab (240 mg [n = 146]) or placebo (n = 143) in combination with gemcitabine-cisplatin for up to 6 cycles, followed by maintenance with toripalimab or placebo until disease progression, intolerable toxicity, or completion of 2 years of treatment. Main Outcome: Progression-free survival as assessed by a blinded independent central review. Secondary end points included objective response rate, overall survival, progression-free survival assessed by investigator, duration of response, and safety. Results: Among the 289 patients enrolled (median age, 46 [IQR, 38-53 years; 17% female), at the final progression-free survival analysis, toripalimab treatment had a significantly longer progression-free survival than placebo (median, 21.4 vs 8.2 months; HR, 0.52 [95% CI, 0.37-0.73]). With a median survival follow-up of 36.0 months, a significant improvement in overall survival was identified with toripalimab over placebo (hazard ratio [HR], 0.63 [95% CI, 0.45-0.89]; 2-sided P = .008). The median overall survival was not reached in the toripalimab group, while it was 33.7 months in the placebo group. A consistent effect on overall survival, favoring toripalimab, was found in subgroups with high and low PD-L1 (programmed death-ligand 1) expression. The incidence of all adverse events, grade 3 or greater adverse events, and fatal adverse events were similar between the 2 groups. However, adverse events leading to discontinuation of toripalimab or placebo (11.6% vs 4.9%), immune-related adverse events (54.1% vs 21.7%), and grade 3 or greater immune-related adverse events (9.6% vs 1.4%) were more frequent in the toripalimab group. Conclusions and Relevance: The addition of toripalimab to chemotherapy as first-line treatment for RM-NPC provided statistically significant and clinically meaningful progression-free survival and overall survival benefits compared with chemotherapy alone, with a manageable safety profile. These findings support the use of toripalimab plus gemcitabine-cisplatin as the new standard of care for this patient population. Trial Registration: ClinicalTrials.gov Identifier: NCT03581786.

A Practical Guide for the Systemic Treatment of Biliary Tract Cancer in Canada.

Biliary tract cancers (BTC) are rare and aggressive tumors with poor prognosis. Radical surgery offers the best chance for cure; however, most patients present with unresectable disease, and among those receiving curative-intent surgery, recurrence rates remain high. While other locoregional therapies for unresectable disease may be considered, only select patients may be eligible. Consequently, systemic therapy plays a significant role in the treatment of BTC. In the adjuvant setting, capecitabine is recommended following curative-intent resection. In the neoadjuvant setting, systemic therapy has mostly been explored for downstaging in borderline resectable tumours, although evidence for its routine use is lacking. For advanced unresectable or metastatic disease, gemcitabine-cisplatin plus durvalumab has become the standard of care, while the addition of pembrolizumab to gemcitabine-cisplatin has also recently demonstrated improved survival compared to chemotherapy alone. Following progression on gemcitabine-cisplatin, several chemotherapy combinations and biomarker-driven targeted agents have been explored. However, the optimum regimen remains unclear, and access to targeted agents remains challenging in Canada. Overall, this article serves as a practical guide for the systemic treatment of BTC in Canada, providing valuable insights into the current and future treatment landscape for this challenging disease.

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

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

N 6-methyladenosine Modification of FZR1 mRNA Promotes Gemcitabine Resistance in Pancreatic Cancer.

The therapeutic options for treating pancreatic ductal adenocarcinoma (PDAC) are limited, and resistance to gemcitabine, a cornerstone of PDAC chemotherapy regimens, remains a major challenge. N6-methyladenosine (m6A) is a prevalent modification in mRNA that has been linked to diverse biological processes in human diseases. Herein, by characterizing the global m6A profile in a panel of gemcitabine-sensitive and gemcitabine-insensitive PDAC cells, we identified a key role for elevated m6A modification of the master G0-G1 regulator FZR1 in regulating gemcitabine sensitivity. Targeting FZR1 m6A modification augmented the response to gemcitabine treatment in gemcitabine-resistant PDAC cells both in vitro and in vivo. Mechanistically, GEMIN5 was identified as a novel m6A mediator that specifically bound to m6A-modified FZR1 and recruited the eIF3 translation initiation complex to accelerate FZR1 translation. FZR1 upregulation maintained the G0-G1 quiescent state and suppressed gemcitabine sensitivity in PDAC cells. Clinical analysis further demonstrated that both high levels of FZR1 m6A modification and FZR1 protein corresponded to poor response to gemcitabine. These findings reveal the critical function of m6A modification in regulating gemcitabine sensitivity in PDAC and identify the FZR1-GEMIN5 axis as a potential target to enhance gemcitabine response. SIGNIFICANCE: Increased FZR1 translation induced by m6A modification engenders a gemcitabine-resistant phenotype by inducing a quiescent state and confers a targetable vulnerability to improve treatment response in PDAC.

Combination of Tissue Microarray Profiling and Multiplexed IHC Approaches to Investigate Transport Mechanism of Nucleoside Analog Drug Resistance.

Nucleoside analogs (NAs) are an established class of anticancer agents being used clinically for the treatment of diverse cancers, either as monotherapy or in combination with other established anticancer or pharmacological agents. To date, nearly a dozen anticancer NAs are approved by the FDA, and several novel NAs are being tested in preclinical and clinical trials for future applications. However, improper delivery of NAs into tumor cells because of alterations in expression of one or more drug carrier proteins (e.g., solute carrier (SLC) transporters) within tumor cells or cells surrounding the tumor microenvironment stands as one of the primary reasons for therapeutic drug resistance. The combination of tissue microarray (TMA) and multiplexed immunohistochemistry (IHC) is an advanced, high-throughput approach over conventional IHC that enables researchers to effectively investigate alterations to numerous such chemosensitivity determinants simultaneously in hundreds of tumor tissues derived from patients. In this chapter, taking an example of a TMA from pancreatic cancer patients treated with gemcitabine (a NA chemotherapeutic agent), we describe the step-by-step procedure of performing multiplexed IHC, imaging of TMA slides, and quantification of expression of some relevant markers in these tissue sections as optimized in our laboratory and discuss considerations while designing and carrying out this experiment.

Nab-paclitaxel and gemcitabine plus camrelizumab and radiotherapy versus nab-paclitaxel and gemcitabine alone for locally advanced pancreatic adenocarcinoma: a prospective cohort study.

Treatment options specifically for patients with locally advanced pancreatic adenocarcinoma (LAPC) are scare and chemotherapy alone delivers limited efficacy. Immunotherapy and radiotherapy are potential effective treatments for LAPC, and both of them may synergize with chemotherapy. Therefore, in this prospective cohort study, we compared the efficacy and safety of nab-paclitaxel plus gemcitabine combined with anti-programmed cell death (PD-1) immunotherapy and radiotherapy (hereafter, combination treatment) versus nab-paclitaxel plus gemcitabine (chemotherapy alone) in the treatment of LAPC. In the combination group, participants received conventional fractionated radiotherapy with doses ranging from 54 to 63 Gy in 28 fractions, intravenous camrelizumab 200 mg once every 3 weeks, and intravenous nab-paclitaxel plus gemcitabine on day 1 and 8 of a 21-day cycle for eight cycles until disease progression, death or unacceptable toxicity. In the chemotherapy group, participants received intravenous nab-paclitaxel plus gemcitabine on day 1 and 8 of a 21-day cycle for eight cycles. From April, 2020 to December, 2021, 96 participants with LAPC were prospectively enrolled with 32 received combination treatment and 64 received chemotherapy alone at a single center. The combination treatment yielded significantly longer median overall-survival (22.3 months vs. 18.6 months, P = 0.031) and progression-free survival (12.0 months vs. 10.5 months, P = 0.043) than chemotherapy alone did. And the incidence of severe adverse events was not significantly different between the combination group and chemotherapy group (P = 0.856). In conclusion, nab-paclitaxel plus gemcitabine combined with anti-PD-1 immunotherapy and radiotherapy was effective and safe for LAPC patients, and it warrants further investigation in larger randomized trials.

Effect of a MUC5AC Antibody (NPC-1C) Administered With Second-Line Gemcitabine and Nab-Paclitaxel on the Survival of Patients With Advanced Pancreatic Ductal Adenocarcinoma: A Randomized Clinical Trial.

Importance: Treatment options are limited for patients with advanced pancreatic ductal adenocarcinoma (PDAC) beyond first-line 5-fluorouracil, leucovorin, irinotecan, and oxaliplatin (FOLFIRINOX), with such individuals commonly being treated with gemcitabine and nab-paclitaxel. Objective: To determine whether NPC-1C, an antibody directed against MUC5AC, might increase the efficacy of second-line gemcitabine and nab-paclitaxel in patients with advanced PDAC. Design, Setting, and Participants: This multicenter, randomized phase II clinical trial enrolled patients with advanced PDAC between April 2014 and March 2017 whose disease had progressed on first-line FOLFIRINOX. Eligible patients had tumors with at least 20 MUC5AC staining by centralized immunohistochemistry review. Statistical analysis was performed from April to May 2022. Interventions: Patients were randomly assigned to receive gemcitabine (1000 mg/m2) and nab-paclitaxel (125 mg/m2) administered intravenously on days 1, 8, and 15 of every 4-week cycle, with or without intravenous NPC-1C 1.5 mg/kg every 2 weeks. Main Outcomes and Measures: The primary end point was overall survival (OS). Secondary end points were progression-free survival (PFS), objective response rate (ORR), and safety. Pretreatment clinical variables were explored with Cox proportional hazards analysis. Results: A total of 78 patients (median [range] age, 62 [36-78] years; 32 [41%] women; 9 [12%] Black; 66 [85%] White) received second-line treatment with gemcitabine plus nab-paclitaxel (n = 40) or gemcitabine plus nab-paclitaxel and NPC-1C (n = 38). Median OS was 6.6 months (95% CI, 4.7-8.4 months) with gemcitabine plus nab-paclitaxel vs 5.0 months (95% CI, 3.3-6.5 months; P = .22) with gemcitabine plus nab-paclitaxel and NPC-1C. Median PFS was 2.7 months (95% CI, 1.9-4.1 months) with gemcitabine plus nab-paclitaxel vs 3.4 months (95% CI, 1.9-5.3 months; P = .80) with gemcitabine plus nab-paclitaxel and NPC-1C. The ORR was 3.1% (95% CI, 0.4%-19.7%) in the gemcitabine plus nab-paclitaxel and NPC-1C group and 2.9% (95% CI, 0.4%-18.7%) in the gemcitabine plus nab-paclitaxel group. No differences in toxicity were observed between groups, except that grade 3 or greater anemia occurred more frequently in patients treated with gemcitabine plus nab-paclitaxel and NPC-1C than gemcitabine plus nab-paclitaxel (39% [15 of 38] vs 10% [4 of 40]; P = .003). The frequency of chemotherapy dose reductions was similar in both groups (65% vs 74%; P = .47). Lower performance status, hypoalbuminemia, PDAC diagnosis less than or equal to 18 months before trial enrollment, lymphocyte-to-monocyte ratio less than 2.8, and CA19-9 greater than 2000 IU/mL were independently associated with poorer survival. Conclusions and Relevance: In this randomized clinical trial of advanced PDAC, NPC-1C did not enhance the efficacy of gemcitabine/nab-paclitaxel. These data provide a benchmark for future trials investigating second-line treatment of PDAC. Trial Registration: ClinicalTrials.gov Identifier: NCT01834235.

Elevated PAF1-RAD52 axis confers chemoresistance to human cancers.

Cisplatin- and gemcitabine-based chemotherapeutics represent a mainstay of cancer therapy for most solid tumors; however, resistance limits their curative potential. Here, we identify RNA polymerase II-associated factor 1 (PAF1) as a common driver of cisplatin and gemcitabine resistance in human cancers (ovarian, lung, and pancreas). Mechanistically, cisplatin- and gemcitabine-resistant cells show enhanced DNA repair, which is inhibited by PAF1 silencing. We demonstrate an increased interaction of PAF1 with RAD52 in resistant cells. Targeting the PAF1 and RAD52 axis combined with cisplatin or gemcitabine strongly diminishes the survival potential of resistant cells. Overall, this study shows clinical evidence that the expression of PAF1 contributes to chemotherapy resistance and worse clinical outcome for lethal cancers.

ADAM12 promotes gemcitabine resistance by activating EGFR signaling pathway and induces EMT in bladder cancer.

BACKGROUND: Gemcitabine (GEM)-based chemotherapy regimens is widely used in bladder cancer (BC) patients. However, GEM resistance may occur and result in treatment failure and disease progression. A disintegrin and metalloprotease 12 (ADAM12) plays a critical role in many cancers. However, the role of ADAM12 in GEM resistance of BC remains unclear. METHODS: We analyzed the relationship between ADAM12 expression and tumor characteristics using the data downloaded from The Cancer Genome Atlas (TCGA) database and Gene Expression Omnibus (GEO) database. Then, we established GEM resistant BC cell lines and used quantitative real-time PCR, western blot, cell counting kit-8, immunohistochemistry, and xenograft mouse model to investigate the role of ADAM12 in GEM resistance. RESULTS: In general, ADAM12 was found to be upregulated in GEM resistant BC cells. ADAM12 knockdown increased the chemosensitivity of BC cells. We further proved that ADAM12 could promote GEM resistance by activating the epidermal growth factor receptor (EGFR) signaling pathway in BC. Furthermore, the epithelial-mesenchymal transition (EMT) phenotype was observed in GEM resistant BC cells. ADAM12 induced EMT process and promotes tumor progression in BC. CONCLUSION: Our findings suggested that ADAM12 was a key gene for GEM resistance and positively correlated with malignancy of BC. It might serve as a novel and valuable therapeutic target for BC.