TNBC-derived Gal3BP/Gal3 complex induces immunosuppression through CD45 receptor

ABSTRACTA preliminary study investigating immunotherapy strategies for aggressive triple negative breast cancer (TNBC) revealed an overexpression of genes involved in the release of extracellular vesicles (EVs). Proteins expressed by EVs play a role in reprogramming the tumor microenvironment and impeding effective responses to immunotherapy. Galectin 3 (Gal3), found in the extracellular space of breast cancer cells, downregulates T-cell receptor expression. Gal3 binds to several receptors, including CD45, which is required for T-cell receptor activation. Previously, we reported a novel tumor escape mechanism, whereby TNBC cells suppress immune cells through CD45 intracellular signals. The objective of this study was to determine the potential association of Gal3 with TNBC-secreted EVs induction of immunosuppression via the CD45 signaling pathway. EVs were isolated from MDA-MB-231 cells and the plasma of patients with TNBC. Mass spectrometry revealed the presence of Gal3 binding protein (Gal3BP) in the isolated small EVs, which interacted with TNBC secreted Gal3. Gal3BP and Gal3 form a complex that induces a significant increase in T-regulatory cells in peripheral blood mononuclear cells (PBMCs). This increase correlates with a significant increase in suppressive interleukins 10 and 35. Blocking the CD45 receptor in PBMCs cultured with tumor-derived EVs impeded the immunosuppression exerted by the Gal3BP/Gal3 complex. This led to an increase in IFN-γ and the activation of CD4, CD8 and CD56 effector cells. This study suggests a tumor escape mechanism that may contribute to the development of a different immunotherapy strategy that complements current therapies used for TNBC

Oxidative stress-induced DNA damage and repair in human peripheral blood mononuclear cells: protective role of hemoglobin.

BACKGROUND: DNA repair is a cellular defence mechanism responding to DNA damage caused in large part by oxidative stress. There is a controversy with regard to the effect of red blood cells on DNA damage and cellular response. AIM: To investigate the effect of red blood cells on H2O2-induced DNA damage and repair in human peripheral blood mononuclear cells. METHODS: DNA breaks were induced in peripheral blood mononuclear cells by H2O2 in the absence or presence of red blood cells, red blood cells hemolysate or hemoglobin. DNA repair was measured by (3)H-thymidine uptake, % double-stranded DNA was measured by fluorometric assay of DNA unwinding. DNA damage was measured by the comet assay and by the detection of histone H2AX phosphorylation. RESULTS: Red blood cells and red blood cells hemolysate reduced DNA repair in a dose-dependent manner. Red blood cells hemolysate reduced % double-stranded DNA, DNA damage and phosphorylation of histone H2AX. Hemoglobin had the same effect as red blood cells hemolysate on % double-stranded DNA. CONCLUSION: Red blood cells, via red blood cells hemolysate and hemoglobin, reduced the effect of oxidative stress on peripheral blood mononuclear cell DNA damage and phosphorylation of histone H2AX. Consequently, recruitment of DNA repair proteins diminished with reduction of DNA repair. This suggests that anemia predisposes to increased oxidative stress induced DNA damage, while a higher hemoglobin level provides protection against oxidative-stress-induced DNA damage

The Therapeutic Potential of AN-7, a Novel Histone Deacetylase Inhibitor, for Treatment of Mycosis Fungoides/Sezary Syndrome Alone or with Doxorubicin.

The 2 histone deacetylase inhibitors (HDACIs) approved for the treatment of cutaneous T-cell lymphoma (CTCL) including mycosis fungoides/sezary syndrome (MF/SS), suberoylanilide hydroxamic acid (SAHA) and romidepsin, are associated with low rates of overall response and high rates of adverse effects. Data regarding combination treatments with HDACIs is sparse. Butyroyloxymethyl diethylphosphate (AN-7) is a novel HDACI, which was found to have selective anticancer activity in several cell lines and animal models. The aim of this study was to compare the anticancer effects of AN-7 and SAHA, either alone or combined with doxorubicin, on MF/SS cell lines and peripheral blood lymphocytes (PBL) from patients with Sezary syndrome (SPBL). MyLa cells, Hut78 cells, SPBL, and PBL from healthy normal individuals (NPBL) were exposed to the test drugs, and the findings were analyzed by a viability assay, an apoptosis assay, and Western blot. AN-7 was more selectively toxic to MyLa cells, Hut78 cells, and SPBL (relative to NPBL) than SAHA and also acted more rapidly. Both drugs induced apoptosis in MF/SS cell lines, SAHA had a greater effect on MyLa cell line, while AN-7 induced greater apoptosis in SPBL; both caused an accumulation of acetylated histone H3, but AN-7 was associated with earlier kinetics; and both caused a downregulation of the HDAC1 protein in MF/SS cell lines. AN-7 acted synergistically with doxorubicin in both MF/SS cell lines and SPBL, and antagonistically with doxorubicin in NPBL. By contrast, SAHA acted antagonistically with doxorubicin on MF/SS cell lines, SPBL, and NPBL, leaving <50% viable cells. In conclusion, AN-7 holds promise as a therapeutic agent in MF/SS and has several advantages over SAHA. Our data provide a rationale for combining AN-7, but not SAHA, with doxorubicin to induce the cell death in MF/SS

Dose-response curve of the effect of RBC hemolysate (% ^v/_v in medium) on H₂O₂-induced DNA repair (% of control) in PBMC.

<p>For the effects of 0.3, 0.6, 1 and 2% ^v/_v hemolysate, p = NS, p = NS, p<0.005 and p<0.005, respectively.</p

Dose-response curve of the effect of RBC hemolysate (% ^v/_v in medium) on ds-DNA ratio in H₂O₂-stimulated PBMC.

<p>p<0.001 for the effects of the 0.3, 0.6, 1 and 2% ^v/_v RBC hemolysate concentrations.</p

Dose response curve of the effect of H₂O₂ on ds DNA.

<p>Effect of 10, 20, 50, 100 and 200 µmol/L H₂O₂ on PBMC was tested: 100 and 200 µmol/L reduced %ds DNA significantly (p<0.02, p<0.01, respectively).</p

ds-DNA ratio in H₂O₂-stimulated PBMC: effect of purchased human hemoglobin (1.8 mg/ml) and RBC hemolysate (2% ^v/_v, mean hemoglobin content = 2.00±0.07 mg/ml).

<p>b vs a, p<0.001; c vs a, p<0.001; N = 8.</p

Comet assay – a representative experiment (N = 3).

<p>The left image depicts the control. The middle image depicts the effect of H₂O₂. The right image depicts the effect of H₂O₂ and RBC hemolysate.</p

Correlation between ds-DNA ratio (control = 1.00) and DNA repair (% of control) in H₂O₂-stimulated PBMC, at concentrations of 0, 0.3, 0.6, 1 and 2% ^v/_v RBC hemolysate.

<p>For each point, N = 8. Coefficient r = −0.925, p<0.025. Regression line: y = −0.131x +2.432.</p