Gallbladder cancer (GBC) is an extremely malignant tumor with a high mortality rate. Little is known about its invasion and metastasis mechanism so far. To identify the driver genes in GBC metastasis, we performed a mRNA microarray of metastatic GBC and paired non-tumor samples, and found PLEK2 was markedly upregulated in GBC tissues. Next, the expression of PLEK2 in GBC were examined in a larger cohort of patients by qRT-PCR, western blot and IHC staining. The clinicopathologic correlation of PLEK2 was determined by statistical analyses. The biological involvement of PLEK2 in GBC metastasis and the underlying mechanisms were investigated. In this study, we found that PLEK2 had higher expression in GBC tumor tissues compared to non-cancerous adjacent tissues and cholecystolithiasis tissues. The clinicopathologic analyses showed PLEK2 expression was positively correlated with tumor TNM stage, distant metastasis and PLEK2 was an independent predictor of overall survival (OS) in GBC patients. The cellular function assays showed PLEK2 promoted GBC cells migration, invasion and liver metastasis in mouse model via the regulation of epithelial-mesenchymal transition (EMT) process. Our mass spectrum and co-immunoprecipitation (co-IP) assays demonstrated that PLEK2 could interact with the kinase domain of EGFR and suppress EGFR ubiquitination mediated by c-CBL, leading to constitutive activation of EGFR signaling. Furthermore, RNA-sequencing and qRT-PCR results demonstrated chemokine (C-C motif) ligand 2 (CCL2), a target gene downstream of PLEK2/EGFR signaling, mediated the motility-promoting function of PLEK2. On the basis of these collective data, we propose that PLEK2 promotes the invasion and metastasis of GBC by EGFR/CCL2 pathway and PLEK2 can serve as a potential therapeutic target for GBC treatment.

Background: Gallbladder carcinoma is highly aggressive and resistant to chemotherapy, with no consistent strategy to guide first line chemotherapy. However, patient‐derived xenograft (PDX) model has been increasingly used as an effective model for in preclinical study of chemosensitivity. Methods: Mini‐PDX model was established using freshly resected primary lesions from 12 patients with gallbladder to examine the sensitivity with five of the most commonly used chemotherapeutic agents, namely gemcitabine, oxaliplatin, 5‐fluorouracil, nanoparticle albumin‐bound (nab)‐paclitaxel, and irinotecan. The results were used to guide the selection of chemotherapeutic agents for adjunctive treatment after the surgery. Kaplan–Meier method was used to compare overall survival (OS) and disease free survival (DFS) with 45 patients who received conventional chemotherapy with gemcitabine and oxaliplatin. Results: Cell viability assays based on mini‐PDX model revealed significant heterogeneities in drug responsiveness. Kaplan–Meier analysis showed that patients in the PDX‐guided chemotherapy group had significantly longer median OS (18.6 months; 95% CI 15.9–21.3 months) than patients in the conventional chemotherapy group (13.9 months; 95% CI 11.7–16.2 months) ( P = 0.030; HR 3.18; 95% CI 1.47–6.91). Patients in the PDX‐guided chemotherapy group also had significantly longer median DFS (17.6 months; 95% CI 14.5–20.6 months) than patients in the conventional chemotherapy group (12.0 months; 95% CI 9.7–14.4 months) ( P = 0.014; HR 3.37; 95% CI 1.67–6.79). Conclusion: The use of mini‐PDX model to guide selection of chemotherapeutic regimens could improve the outcome in patients with gallbladder carcinoma.

Adolescents and young adults (AYAs) diagnosed with cancer between ages 15~45 years may exhibit unique biologic and genomic characteristics as well as clinical features, resulting in differences in clinical characters and drug resistance. However, compared to other solid cancers, relatively few studies have been conducted in this age group in cholangiocarcinoma (CCA). This study is to investigate the clinical and molecular features of AYAs with CCA. Three cohorts, including the external dataset (TCGA and MSKCC) and the perihilar CCA databank of Chinese tertiary hospitals, were contained in this study. Pathway and process enrichment analysis had been carried out with the following ontology sources: KEGG Pathway, GO Biological Processes, Reactome Gene Sets, Canonical Pathways, and CORUM. Metascape and GEPIA datasets were used for bioinformatic analysis. P< 0.05 was considered statistically significant. All statistical analyses were performed with GraphPad Prism (version 7.0; GraphPad Software, La Jolla, California) and R studio (version 3.6.1; R studio, Boston, Massachusetts). Compared to older adults, AYAs with CCA presented with worse overall survival, though the difference was not significant. Specific to patients with stage IV CCAs who underwent chemotherapy, AYAs were associated with significantly poorer OS (Fig.1B. p=0.03, HR 3.01, 95%CI 1.14~4.91). From the anatomical perspective, more eCCA was detected in the AYA group. MSI occurred in 3% of older patients in the present study. Nevertheless, none of the AYAs had MSI status. Here in this study, AYAs gained an enhanced frequency of ASXL1(p=0.02) and KMT2C(p=0.02) mutation than their older counterparts. Besides ASXL and KMT2C, the genes enriched in AYAs with CCA were analyzed by pathway and process enrichment analysis. And those genes were found to be associated with poorer differentiation, deubiquitination, and WNT signal pathway. Moreover, AYAs were relevant to poor differentiation and advanced tumor stage. This study offered a preliminary landscape of the clinical and molecular features of early onset biliary cancers. Further studies including more samples are essential to investigate whether ASXL1 and KMT2C could be considered as potentially targetable genomic signatures for young patients.

Background: Recent studies have reported that IGF2BP3 is linked to the pathogenesis of various malignancies. Since IGF2BP3 is associated with poor outcomes of gallbladder carcinoma (GBC), we aimed to explore the association between its N⁶-methyladenosine (m6A) RNA methylation and GBC progression.Methods: Bioinformatic analysis of GSE136982, GSE104165, and RNA-seq was performed. In vitro and in vivo gain- and loss-of-function assays were done. qPCR, Western blotting, and IHC were conducted in cells or in collected clinical tissue samples. RNA immunoprecipitation, RNA stability measurement, methylated RNA immunoprecipitation, and dual-luciferase reporter assays were performed in this study.Results: The expression of IGF2BP3 was higher in GBC tissues than in peritumoral tissues. Functions such as cell proliferation and migration, both in vitro and in vivo, were inhibited by downregulation of IGF2BP3. The analysis of RNA-seq indicated that KLK5 was a downstream target of IGF2BP3. The expression of KLK5 was measured in GBC cells and tumor samples. It was found to be positively correlated with IGF2BP3 level. Upon IGF2BP3 depletion, ectopic expression of KLK5 could rescue cell function in part. Mechanistically, we found that IGF2BP3 directly binds to KLK5 mRNA and regulates its stability in an m6A-dependent manner. As a result, inhibition of KLK5 decreased the expression of PAR2, and deregulated phospho-Akt. Using bioinformatic prediction combined with miRNA microarray analysis, we identified that let-7g-5p is an inhibitor of IGF2BP3, and let-7g-5p expression was negatively correlated with IGF2BP3. Overexpression of let-7g-5p affected the aggressive phenotype of GBC cells by deregulating IGF2BP3, and inhibiting the KLK5/PAR2/AKT axis.Conclusions: Our data showed that IGF2BP3 is associated with the aggressive phenotype of GBC. Mechanistically, IGF2BP3 activated the PAR2/AKT axis by stabilizing KLK5 mRNA in an m6A-dependent manner. The loss of let-7g-5p enhanced the expression of IGF2BP3 and improved GBC progression. Thus, IGF2BP3 plays a crucial role in GBC, and the let-7g-5p/IGF2BP3/KLK5/PAR2 axis may be a therapeutic target for GBC.

The prognostic benefits and safety of extended lymphadenectomy for hilar cholangiocarcinoma remain uncertain. The available evidence is still insufficient concerning its retrospective aspect. The aim of this study is to explore the clinical effect and safety of extended lymphadenectomy compared to regional lymphadenectomy in patients with hilar cholangiocarcinoma. The Relay-HC trial is a prospective, multicenter, and randomized controlled trial. Seven hundred and thirty-four eligible patients with resectable perihilar cholangiocarcinoma across 15 tertiary hospitals in China will be randomly assigned (1:1) to receive either regional lymphadenectomy or extended lymphadenectomy. The primary objective is to determine the overall survival after the two approaches. Secondary objectives of the study include the evaluation of perioperative mortality, postoperative complication, and disease-free survival. This study has been approved by the ethics committee of each participating hospital. The Relay-HC trial is designed to investigate the prognostic benefits and safety of expanded lymphadenectomy for hilar cholangiocarcinoma. Currently, it has never been investigated in a prospective randomized controlled clinical trial. Chinese Clinical Trial Registry (ChiCTR), ChiCTR1800015688 . Registered on 15 April 2018.

Pyroptosis, a novel form of programmed cell death, has demonstrated significant antitumor effects. However, enhancing the precision of pyroptosis and improving the treatment efficacy for malignant tumors still remain formidable challenges. In this work, we propose a novel synergistic chemo-photodynamic therapy strategy that targets endogenous esterase in the tumor microenvironment (TME), aiming to achieve precise pyroptosis and facilitate the release of chemotherapeutic drugs to promote pyroptosis. An esterase-activated diketopyrrolopyrrole-based theranostic prodrug DPP-QS was synthesized by the conjugation of a chemotherapeutic drug chlorambucil, with DPP-QE characteristic of aggregation-induced emission (AIE) through a one-step nucleophilic reaction. Besides, the detection limit of DPP-QS toward esterase in vitro was 5.13 × 10⁻⁵ U mL⁻¹. Upon cleavage by esterase endogenously expressed in pancreatic cancer cells, the released DPP-QE moiety selectively accumulated in lipid droplets (LDs) with a significantly enhanced fluorescence due to AIE, and upon subsequent exposure to light, reactive oxygen species (ROS) are generated to induce pyroptosis. Simultaneously, chlorambucil entered the nucleus of cancer cells, causing DNA damage, which further enhanced the effect of pyroptosis. In vivo experiments demonstrated that the prodrug displayed significant TME-activatable features and excellent tumor therapeutic effects through synergistic chemo-photodynamic therapy.