Characterization of HLA-G Regulation and HLA Expression in Breast Cancer and Malignant Melanoma Cell Lines upon IFN-γ Stimulation and Inhibition of DNA Methylation
IFN-γ 刺激和抑制 DNA 甲基化对乳腺癌和恶性黑色素瘤细胞系 HLA-G 调控和 HLA 表达的表征
- 作者列表："Nanna Jørgensen","Abid Sayed","Helene Bjerregaard Jeppesen","Gry Persson","Iben Weisdorf","Tina Funck","Thomas Vauvert Faurschou Hviid
The potential role of human leukocyte antigen (HLA)-G as a target for new cancer immunotherapy drugs has increased the interest in the analysis of mechanisms by which HLA-G expression is regulated, and how the expression can be manipulated. We characterized HLA expression in breast cancer and malignant melanoma cell lines and investigated the induction of HLA-G expression by two distinct mechanisms: stimulation with interferon (IFN)-γ or inhibition of methylation by treatment with 5-aza-2’-deoxycytidine (5-aza-dC). The effect of IFN-γ and 5-aza-dC on HLA expression was dependent on the cancer cell lines studied. However, in general, surface expression of HLA class Ia was induced on all cell lines. Surface expression of HLA-G was inconclusive but induction of HLA-G mRNA was prevalent upon treatment with 5-aza-dC and a combination of IFN-γ and 5-aza-dC. IFN-γ alone failed to induce HLA-G expression in the HLA-G-negative cell lines. The results support that HLA-G expression is regulated partly by DNA methylation. Furthermore, IFN-γ may play a role in the maintenance of HLA-G expression rather than inducing expression. The study demonstrates the feasibility of manipulating HLA expression and contributes to the exploration of mechanisms that can be potential targets for immunotherapy in breast cancer and malignant melanoma.
人类白细胞抗原 (HLA)-G作为新型癌症免疫治疗药物靶点的潜在作用增加了对HLA-G表达调控机制分析的兴趣，以及如何操纵表达式。我们表征了乳腺癌和恶性黑色素瘤细胞系中HLA的表达，并通过两种不同的机制研究了HLA-G表达的诱导: 干扰素 (IFN) 刺激-Γ 或通过 5-氮杂-2 '-脱氧胞苷 (5-aza-dC) 处理抑制甲基化。IFN-γ 和 5-aza-dC对HLA表达的影响依赖于所研究的癌细胞系。然而，一般来说，HLA Ia类的表面表达在所有细胞系上都被诱导。HLA-G的表面表达尚无定论，但在用 5-aza-dC和IFN-γ 和 5-aza-dC联合治疗时，HLA-G mRNA的诱导普遍存在。IFN-γ 单独诱导HLA-G阴性细胞系HLA-G表达失败。结果支持HLA-G表达部分受DNA甲基化调控。此外，IFN-γ 可能在HLA-G表达的维持中发挥作用，而不是诱导表达。该研究证明了操纵HLA表达的可行性，并有助于探索可作为乳腺癌和恶性黑色素瘤免疫治疗潜在靶点的机制。
METHODS::Macrophages (MΦ) play a critical role in tumor growth, immunosuppression and inhibition of adaptive immune responses in cancer. Hence, targeting signaling pathways in MΦs that promote tumor immunosuppression will provide therapeutic benefit. PI3Kγ has been recently established by our group and others as a novel immuno-oncology target. Herein, we report that a macrophage Syk-PI3K axis drives polarization of immunosuppressive MΦs which establish an immunosuppressive tumor microenvironment in in vivo syngeneic tumor models. Genetic or pharmacological inhibition of Syk and/or PI3Kγ in MΦs promotes a pro-inflammatory MΦphenotype, restores CD8+ T cell activity, destabilizes HIF under hypoxia, and stimulates an antitumor immune response. Assay for Transposase-accessible Chromatin using Sequencing (ATAC-seq) analyses on the bone marrow derived macrophages (BMDMs) show that inhibition of Syk kinase promotes activation and binding of NF-κB motif in SykMC-KO BMDMs, thus stimulating immunostimulatory transcriptional programming in MΦs to suppress tumor growth. Finally, we have developed in silico the "first in class" dual Syk/PI3K inhibitor, SRX3207, for the combinatorial inhibition of Syk and PI3K in one small molecule. This chemotype demonstrates efficacy in multiple tumor models and represents a novel combinatorial approach to activate antitumor immunity.
METHODS::Dysfunction of invariant natural killer T (iNKT) cells in tumor microenvironment hinders their anti-tumor efficacy, and the underlying mechanisms remain unclear. Here we report that iNKT cells increase lipid biosynthesis after activation, and that is promoted by PPARγ and PLZF synergically through enhancing transcription of Srebf1. Among those lipids, cholesterol is required for the optimal IFN-γ production from iNKT cells. Lactic acid in tumor microenvironment reduces expression of PPARγ in intratumoral iNKT cells and consequently diminishes their cholesterol synthesis and IFN-γ production. Importantly, PPARγ agonist pioglitazone, a thiazolidinedione drug for type 2 diabetes, successfully restores IFN-γ production in tumor-infiltrating iNKT cells from both human patients and mouse models. Combination of pioglitazone and alpha-galactosylceramide treatments significantly enhances iNKT cell-mediated anti-tumor immune responses and prolongs survival of tumor-bearing mice. Our studies provide a strategy to augment the anti-tumor efficacy of iNKT cell-based immunotherapies via promoting their lipid biosynthesis.
METHODS::Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed and selective cancer treatment that induces necrotic and immunogenic cell death and utilizes a monoclonal antibody conjugated to a photo-absorber dye, IR700DX, activated by NIR light. Although CD44 is surface cancer marker associated with drug resistance, anti-CD44-IR700 NIR-PIT results in inhibited cell growth and prolonged survival in multiple tumor types. Meanwhile, anti-CD25-IR700-targeted NIR-PIT has been reported to achieve selective and local depletion of FOXP3+CD25+CD4+ regulatory T cells (Tregs), which are primary immunosuppressive cells in the tumor microenvironment (TME), resulting in activation of local antitumor immunity. Combined NIR-PIT with CD44- and CD25-targeted agents has the potential to directly eliminate tumor cells and also amplify the immune response by removing FOXP3+CD25+CD4+ Tregs from the TME. We investigated the difference in therapeutic effects of CD44-targeted NIR-PIT alone, CD25-targeted NIR-PIT alone, and the combination of CD44- and CD25-targeted NIR-PIT in several syngeneic tumor models, including MC38-luc, LL/2, and MOC1. The combined NIR-PIT showed significant tumor growth inhibition and prolonged survival compared with CD44-targeted NIR-PIT alone in all tumor models and showed prolonged survival compared with CD25-targeted NIR-PIT alone in MC38-luc and LL/2 tumors. Combined CD44/CD25 NIR-PIT also resulted in some complete remissions, whereas this was not achieved with either type of NIR-PIT alone. Therefore, combined NIR-PIT simultaneously targeting cancer antigens and immunosuppressive cells in the TME may be more effective than either type of NIR-PIT alone and may have potential to induce prolonged immune responses in treated tumors.