Ginsenoside Rh2 enhances immune surveillance of natural killer (NK) cells via inhibition of ERp5 in breast cancer.

BACKGROUND: One critical component of the immune system that prevents breast cancer cells from forming distant metastasis is natural killer (NK) cells participating in immune responses to tumors. Ginsenoside Rh2 (GRh2) as one of the major active ingredients of ginseng has been employed in treatment of cancers, but the function of GRh2 in modulating the development of breast cancer remains elusive. PURPOSE: This study was to dissect the effect of GRh2 against breast cancer and its potential mechanisms associated with NK cells, both in vitro and in vivo. METHODS: MDA-MB-231 and 4T1 cells were used to establish in situ and hematogenous mouse models. MDA-MB-231 and MCF-7 were respectively co-cultured with NK92MI cells or primary NK cells in vitro. Anti-tumor efficacy of GRh2 was verified by immunohistochemistry (IHC), Cell Counting Kit-8 (CCK8), high resolution micro-computed tomography (micro-CT) scanning of lungs and hematoxylin and eosin (H&E) staining. Lactate dehydrogenase (LDH) cytotoxicity assay, flow cytometry, in vivo depletion of NK cells, enzyme-linked immunosorbent assay (ELISA), western blot, quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunofluorescence and cell transfection were performed for investigating the anti-tumor mechanisms of GRh2. Molecular docking, microscale thermophoresis (MST) and cellular thermal shift assay (CETSA) were employed to determine the binding between endoplasmic reticulum protein 5 (ERp5) and GRh2. RESULTS: We demonstrated that GRh2 exerted prominent impacts on retarding the growth and metastasis of breast cancer through boosting the cytotoxic function of NK cells, as validated by the elevated release of perforin, granzyme B and interferon-γ (IFN-γ). Mechanistical studies revealed that GRh2 was capable of diminishing the expression of ERp5 and GRh2 directly bound to ERp5 in MDA-MB-231 cells as well as on a recombinant protein level. GRh2 prevented the formation of soluble MICA (sMICA) and upregulated the expression level of MICA in vivo and in vitro. Importantly, the reduced lung metastasis of breast cancer by GRh2 was almost abolished upon the depletion of NK cells. Moreover, GRh2 was able to insert into the binding pocket of ERp5 directly. CONCLUSION: We firstly demonstrated that GRh2 played a pivotal role in augmenting NK cell activity by virtue of modulating the NKG2D-MICA signaling axis via directly binding to ERp5, and may be further optimized to a therapeutic agent for the treatment of breast cancer.

PDIA6 and Maspin in Prostate Cancer.

BACKGROUND/AIM: PDIA6 is a disulphide isomerase of the PDI family, known to mediate disulphide bond formation in the endoplasmic reticulum. However, PDI-related proteins also function in other parts of the cell and PDIA6 has been shown to be involved in many types of cancers. We previously identified PDIA6 as a putative Maspin interactor. Maspin has itself been implicated in prostate cancer progression. Our aim was to further explore the roles of Maspin in prostate cancer and establish whether PDIA6 is also involved in prostate cancer. MATERIALS AND METHODS: RNA levels of PDIA6 and Maspin in prostate cell lines were measured using RT-PCR. Bioinformatics analysis of the TCGA database was used to find RNA levels of PDIA6 and Maspin in prostate cancer. siRNAs were used to knock-down PDIA6, and proliferation and migration assays were conducted on those cells. RESULTS: PDIA6 and Maspin RNA were shown to be expressed at varying levels in prostate cell lines. RNAseq data showed that PDIA6 expression was significantly increased in prostate adenocarcinoma samples, while Maspin RNA expression was decreased. When PDIA6 expression was knocked-down using siRNA in prostate cell lines, proliferation was decreased substantially in the two prostate cancer cell lines (DU145 and PC3) and also decreased in the normal prostate cell line (PNT1a), though less strongly. CONCLUSION: PDIA6 expression is higher in prostate cancer cells compared to normal prostate cells. Decreasing PDIA6 expression decreases proliferation. Thus, PDIA6 is a promising target for prostate cancer therapeutics.

Targeting thiol isomerase activity with zafirlukast to treat ovarian cancer from the bench to clinic.

Thiol isomerases, including PDI, ERp57, ERp5, and ERp72, play important and distinct roles in cancer progression, cancer cell signaling, and metastasis. We recently discovered that zafirlukast, an FDA-approved medication for asthma, is a pan-thiol isomerase inhibitor. Zafirlukast inhibited the growth of multiple cancer cell lines with an IC50 in the low micromolar range, while also inhibiting cellular thiol isomerase activity, EGFR activation, and downstream phosphorylation of Gab1. Zafirlukast also blocked the procoagulant activity of OVCAR8 cells by inhibiting tissue factor-dependent Factor Xa generation. In an ovarian cancer xenograft model, statistically significant differences in tumor size between control vs treated groups were observed by Day 18. Zafirlukast also significantly reduced the number and size of metastatic tumors found within the lungs of the mock-treated controls. When added to a chemotherapeutic regimen, zafirlukast significantly reduced growth, by 38% compared with the mice receiving only the chemotherapeutic treatment, and by 83% over untreated controls. Finally, we conducted a pilot clinical trial in women with tumor marker-only (CA-125) relapsed ovarian cancer, where the rate of rise of CA-125 was significantly reduced following treatment with zafirlukast, while no severe adverse events were reported. Thiol isomerase inhibition with zafirlukast represents a novel, well-tolerated therapeutic in the treatment of ovarian cancer.

Mechano-redox control of integrin de-adhesion.

How proteins harness mechanical force to control function is a significant biological question. Here we describe a human cell surface receptor that couples ligand binding and force to trigger a chemical event which controls the adhesive properties of the receptor. Our studies of the secreted platelet oxidoreductase, ERp5, have revealed that it mediates release of fibrinogen from activated platelet αIIbß3 integrin. Protein chemical studies show that ligand binding to extended αIIbß3 integrin renders the ßI-domain Cys177-Cys184 disulfide bond cleavable by ERp5. Fluid shear and force spectroscopy assays indicate that disulfide cleavage is enhanced by mechanical force. Cell adhesion assays and molecular dynamics simulations demonstrate that cleavage of the disulfide induces long-range allosteric effects within the ßI-domain, mainly affecting the metal-binding sites, that results in release of fibrinogen. This coupling of ligand binding, force and redox events to control cell adhesion may be employed to regulate other protein-protein interactions.

A genome-wide screen for sporulation-defective mutants in Schizosaccharomyces pombe.

Yeast sporulation is a highly regulated developmental program by which diploid cells generate haploid gametes, termed spores. To better define the genetic pathways regulating sporulation, a systematic screen of the set of ~3300 nonessential Schizosaccharomyces pombe gene deletion mutants was performed to identify genes required for spore formation. A high-throughput genetic method was used to introduce each mutant into an h(90) background, and iodine staining was used to identify sporulation-defective mutants. The screen identified 34 genes whose deletion reduces sporulation, including 15 that are defective in forespore membrane morphogenesis. In S. pombe, the total number of sporulation-defective mutants is a significantly smaller fraction of coding genes than in S. cerevisiae, which reflects the different evolutionary histories and biology of the two yeasts.

How to exploit stress-related immunity against Hodgkin's lymphoma: Targeting ERp5 and ADAM sheddases.

Stress-related immunity can be activated in the course of lymphoproliferative disorders, including Hodgkin's lymphoma, upon the interaction between killer cell lectin-like receptor subfamily K, member 1 (KLRK1, best known as NKG2D) on effector lymphocytes and NKG2D ligands (NKG2DL), such as MHC class I polypeptide-related sequence A (MICA), MICB and various UL16-binding proteins (ULBPs), on lymphoma cells. However, NKG2DLs can also bind NKG2D upon shedding, thus affecting the recognition of lymphoma cells by the immune system. The proteolytic cleavage of MICA depends on protein disulfide isomerase family A, member 6 (PDIA6, a thiol isomerase best known as ERp5) as well as on the disintegrins and metalloproteinases ADAM metallopeptidase domain 10 (ADAM10) and ADAM17, which also cleave ULPBs. These enzymes can be targeted in novel therapeutic schemes to avoid the escape of malignant cells from stress-evoked immune responses.