Research Report on the Mechanism of BAG6 Restricting Pancreatic Cancer Progression

Research Background

Pancreatic Ductal Adenocarcinoma (PDAC) has an extremely poor prognosis, with a median survival of only 6 months, urgently requiring new treatment methods. In recent years, studies have found that extracellular vesicles (EVs) released by tumor cells play an important role in pancreatic cancer progression. EVs are phospholipid bilayer nanoparticles secreted by cells, carrying biological molecules such as proteins and RNA from secreting cells, influencing cancer cell and tumor microenvironment (TME) cell signaling pathways through interaction with surface receptors/ligands or by being internalized by receptor cells. However, the driving genes guiding EV function, EV receptor cells, and their cellular responses to EV uptake remain unclear. This paper mainly studies the role of BCL-2-associated antigen 6 (BAG6), a gene regulating EV generation, in cancer progression.

Paper Source

This paper was co-authored by Bilal Alashkar Alhamwe and several other scholars, mainly from renowned institutions such as Philipps-University, Harvard Medical School, and the University of Cologne. The paper was published in the journal “Cellular & Molecular Immunology” in June 2024.

Research Process

The research team used a Cre recombinase/LoxP-based reporting system combined with single-cell RNA sequencing technology to monitor EV uptake and changes in the tumor microenvironment in a pancreatic cancer mouse model with BAG6 deficiency. The research steps are as follows:

1. Model Establishment: BAG6 gene knockout (KO) and wild-type (WT) Pan02 tumor cells were transplanted into immunocompetent mice to observe subcutaneous and orthotopic tumor growth.
2. Immunohistochemical Analysis: Infiltration of CD4+ T cells, CD8+ T cells, and CD56+ natural killer (NK) cells was detected in tumor tissues, and gene expression analysis confirmed these cellular changes.
3. EV Inhibition Experiment: GW4869 was used to inhibit EV generation and release, observing its effect on tumor growth.
4. Experimental Transgenic System: A Cre-LoxP reporting system was used to track the transfer of EVs released by tumor cells in receptor cells.
5. Single-cell RNA Sequencing: Single-cell RNA sequencing was performed on orthotopic tumors, annotating different cell clusters and analyzing EV uptake.
6. Mast Cell (MC) Related Experiments: Immunohistochemistry and RT-qPCR methods were used to detect MC activation and gene expression. Imatinib was used for MC depletion experiments, and its effect on tumor growth was analyzed.
7. IL33-IL33R Signaling Pathway Analysis: ELISA and flow cytometry were used to detect the expression of IL33 and its receptor (IL1RL1) in EVs, and in vitro experiments verified the role of IL33 in MC activation.

Main Research Findings

1. BAG6 Deficiency Accelerates Tumor Growth and Alters TME: In BAG6-deficient mouse tumors, infiltration of CD4+ T cells and CD8+ T cells decreased, while inflammatory cancer-associated fibroblasts (iCAFs) increased. Spearman correlation analysis showed that BAG6 deficiency positively correlated with the accumulation of MCs and iCAFs.
2. EV Release Associated with Tumor Progression: BAG6 KO cells released slightly higher numbers of EVs than WT cells. After injection of the EV inhibitor GW4869, the growth of BAG6 KO tumors significantly decreased, indicating that EVs play a key role in tumor progression caused by BAG6 deficiency.
3. MCs are the Main EV Receptor Cells: Single-cell RNA sequencing showed that almost 100% of MCs in BAG6 KO tumors received EV signals, and IL33 receptor (IL1RL1) expression in MCs significantly increased. Immunohistochemistry further verified this finding.
4. Role of IL33 in MC Activation: Experiments indicated that IL33 was significantly elevated in EVs released by BAG6 KO cells, activating MCs through the IL33-IL1RL1 pathway, promoting the expression of inflammatory factors (such as IL6, LIF, TNFα, etc.). Flow cytometry and ELISA proved that IL33 is mainly mediated through the neutral sphingomyelinase 2 (nSMase) pathway for its release.

Conclusions and Significance

1. New Mechanism of BAG6 Inhibiting Tumor Development: BAG6 inhibits the activation of MCs by regulating the degradation and secretion of IL33 and inhibiting the release of EVs, thereby weakening the pro-cancer effects of the tumor microenvironment. BAG6 expression levels positively correlate with patient survival, while MC infiltration negatively correlates. This suggests that targeting MCs, especially using imatinib for MC depletion, may provide effective treatment for pancreatic cancer patients with low BAG6 expression and high MC infiltration.
2. Research Highlights: This study reveals for the first time the EV biogenesis mechanism centered on BAG6 in PDAC and its role in TME, proposes the key role of MCs in pancreatic cancer progression, and provides scientific basis for adopting precision medicine strategies to treat specific pancreatic cancer patients.

This research not only expands our understanding of BAG6 in pancreatic cancer but also provides possible targets for future treatments. By deepening our understanding of EVs and their role in the cancer microenvironment, we may be able to develop more effective treatment methods to improve the survival prognosis of pancreatic cancer patients.