Dataset Information

Hypoxia-Induced Intracellular and Extracellular Heat Shock Protein gp96 Increases Paclitaxel-Resistance and Facilitates Immune Evasion in Breast Cancer.

ABSTRACT:

Backgrounds

Hypoxia contributes to cancer progression, drug resistance and immune evasion in various cancers, including breast cancer (BC), but the molecular mechanisms have not been fully studied. Thus, the present study aimed to investigate this issue.

Methods

The paclitaxel-sensitive BC (PS-BC) cells were administered with continuous low-dose paclitaxel treatment to establish paclitaxel-resistant BC (PR-BC) cells. Exosomes were isolated/purified by using the commercial kit, which were observed by Transmission electron microscopy (TEM). Cell viability was measured by MTT assay, cell apoptosis was determined by flow cytometer (FCM). Gene expressions were respectively measured by Real-Time qPCR, Western Blot and immunofluorescence staining assay. The peripheral mononuclear cells (PBMCs) derived CD8⁺ T cells were obtained and co-cultured with gp96-containing exosomes, and cell proliferation was evaluated by EdU assay. ELISA was employed to measure cytokine secretion in CD8⁺ T cells' supernatants.

Results

HSP gp96 was significantly upregulated in the cancer tissues and plasma exosomes collected from BC patients with paclitaxel-resistant properties. Also, continuous low-dose paclitaxel treatment increased gp96 levels in the descendent PR-BC cells and their exosomes, in contrast with the parental PS-BC cells. Upregulation of gp96 increased paclitaxel-resistance in PS-BC cells via degrading p53, while gp96 silence sensitized PR-BC cells to paclitaxel treatments. Moreover, PR-BC derived gp96 exosomes promoted paclitaxel-resistance in PS-BC cells and induced pyroptotic cell death in the CD8⁺ T cells isolated from human peripheral blood mononuclear cells (pPBMCs). Furthermore, we noticed that hypoxia promoted gp96 generation and secretion through upregulating hypoxia-inducible factor 1 (HIF-1), and hypoxia increased paclitaxel-resistance and accelerated epithelial-mesenchymal transition (EMT) in PS-BC cells.

Conclusions

Hypoxia induced upregulation of intracellular and extracellular gp96, which further degraded p53 to increase paclitaxel-sensitivity in BC cells and activated cell pyroptosis in CD8⁺ T cells to impair immune surveillance.

SUBMITTER: Tian T

PROVIDER: S-EPMC8722103 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The extracellular form of the abundant heat-shock protein, gp96, is involved in human autoimmune pathologies. In patients with type 1 diabetes, circulating gp96 is found to be elevated, and is bound to the acute-phase protein, α1-antitrypsin (AAT). The two molecules also engage intracellularly during the physiological folding of AAT. AAT therapy promotes pancreatic islet survival in both transplantation and autoimmune diabetes models, and several clinical trials are currently examining AAT therapy for individuals with type 1 diabetes. However, its mechanism of action is yet unknown. Here, we examine whether the protective activity of AAT is related to binding of extracellular gp96. Primary mouse islets, macrophages, and dendritic cells were added recombinant gp96 in the presence of clinical-grade human AAT (hAAT, Glassia™, Kamada Ltd., Israel). Islet function was evaluated by insulin release. The effect of hAAT on IL-1β/IFNγ-induced gp96 cell-surface levels was also evaluated. In vivo, skin transplantation was performed for examination of robust immune responses, and systemic inflammation was induced by cecal puncture. Endogenous gp96 was inhibited by gp96-inhibitory peptide (gp96i, Compugen Ltd., Israel) in an allogeneic islet transplantation model. Our findings indicate that hAAT binds to gp96 and diminishes gp96-induced inflammatory responses; e.g., hAAT-treated gp96-stimulated islets released less pro-inflammatory cytokines (IL-1β by 6.16-fold and TNFα by 2.69-fold) and regained gp96-disrupted insulin release. hAAT reduced cell activation during both skin transplantation and systemic inflammation, as well as lowered inducible surface levels of gp96 on immune cells. Finally, inhibition of gp96 significantly improved immediate islet graft function. These results suggest that hAAT is a regulator of gp96-mediated inflammatory responses, an increasingly appreciated endogenous damage response with relevance to human pathologies that are exacerbated by tissue injury.