Cancer cell-expressed BTNL2 facilitates tumour immune escape via engagement with IL-17A-producing γδ T cells.

Therapeutic blockade of the immune checkpoint proteins programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte antigen 4 (CTLA4) has transformed cancer treatment. However, the overall response rate to these treatments is low, suggesting that immune checkpoint activation is not the only mechanism leading to dysfunctional anti-tumour immunity. Here we show that butyrophilin-like protein 2 (BTNL2) is a potent suppressor of the anti-tumour immune response. Antibody-mediated blockade of BTNL2 attenuates tumour progression in multiple in vivo murine tumour models, resulting in prolonged survival of tumour-bearing mice. Mechanistically, BTNL2 interacts with local γδ T cell populations to promote IL-17A production in the tumour microenvironment. Inhibition of BTNL2 reduces the number of tumour-infiltrating IL-17A-producing γδ T cells and myeloid-derived suppressor cells, while facilitating cytotoxic CD8+ T cell accumulation. Furthermore, we find high BTNL2 expression in several human tumour samples from highly prevalent cancer types, which negatively correlates with overall patient survival. Thus, our results suggest that BTNL2 is a negative regulator of anti-tumour immunity and a potential target for cancer immunotherapy.

MHC Class I Deficiency in Solid Tumors and Therapeutic Strategies to Overcome It.

It is now well accepted that the immune system can control cancer growth. However, tumors escape immune-mediated control through multiple mechanisms and the downregulation or loss of major histocompatibility class (MHC)-I molecules is a common immune escape mechanism in many cancers. MHC-I molecules present antigenic peptides to cytotoxic T cells, and MHC-I loss can render tumor cells invisible to the immune system. In this review, we examine the dysregulation of MHC-I expression in cancer, explore the nature of MHC-I-bound antigenic peptides recognized by immune cells, and discuss therapeutic strategies that can be used to overcome MHC-I deficiency in solid tumors, with a focus on the role of natural killer (NK) cells and CD4 T cells.

PDIA6 promotes pancreatic cancer progression and immune escape through CSN5-mediated deubiquitination of ß-catenin and PD-L1.

Protein Disulfide Isomerase Family A Member 6 (PDIA6) is an endoplasmic reticulum protein that is capable of catalyzing protein folding and disulfide bond formation. Abnormally elevated expression of PDIA6 has been reported to predict poor outcomes in various cancers. Herein, gain-of- and loss-of-function experiments were performed to investigate how PDIA6 participated in the carcinogenesis of pancreatic cancer (PC). By analyzing the protein expression of PDIA6 in 28 paired PC and para carcinoma specimens, we first found that PDIA6 expression was higher in PC samples. Both the overall survival and disease-free survival rates of PC patients with higher PDIA6 expression were poorer than those with lower PDIA6 (n = 178). Furthermore, knockdown of PDIA6 impaired the malignancies of PC cells - suppressed cell proliferation, invasion, migration, cisplatin resistance, and xenografted tumor growth. PDIA6-silenced PC cells were more sensitive to cytotoxic natural killer (NK) cells. Overexpression of PDIA6 had opposite effects on PC cells. Interestingly, COP9 signalosome subunit 5 (CSN5), a regulator of E3 ubiquitin ligases known to promote deubiquitination of its downstream targets, was demonstrated to interact with PDIA6, and its expression was increased in PC cells overexpressing PDIA6. Additionally, PDIA6 overexpression promoted deubiquitination of ß-catenin and PD-L1 and subsequently upregulated their expression in PC cells. These alterations were partly reversed by CSN5 shRNA. Collectively, the above results demonstrate that PDIA6 contributes to PC progression, which may be associated with CSN5-regulated deubiquitination of ß-catenin and PD-L1. Our findings suggest PDIA6 as a potential target for the treatment of PC.

Specific N-cadherin-dependent pathways drive human breast cancer dormancy in bone marrow.

The challenge for treating breast cancer (BC) is partly due to long-term dormancy driven by cancer stem cells (CSCs) capable of evading immune response and resist chemotherapy. BC cells show preference for the BM, resulting in poor prognosis. CSCs use connexin 43 (Cx43) to form gap junctional intercellular communication with BM niche cells, fibroblasts, and mesenchymal stem cells (MSCs). However, Cx43 is an unlikely target to reverse BC dormancy because of its role as a hematopoietic regulator. We found N-cadherin (CDH2) and its associated pathways as potential drug targets. CDH2, highly expressed in CSCs, interacts intracellularly with Cx43, colocalizes with Cx43 in BC cells within BM biopsies of patients, and is required for Cx43-mediated gap junctional intercellular communication with BM niche cells. Notably, CDH2 and anti-apoptotic pathways maintained BC dormancy. We thereby propose these pathways as potential pharmacological targets to prevent dormancy and chemosensitize resistant CSCs.

Engineering T cells to enhance 3D migration through structurally and mechanically complex tumor microenvironments.

Defining the principles of T cell migration in structurally and mechanically complex tumor microenvironments is critical to understanding escape from antitumor immunity and optimizing T cell-related therapeutic strategies. Here, we engineered nanotextured elastic platforms to study and enhance T cell migration through complex microenvironments and define how the balance between contractility localization-dependent T cell phenotypes influences migration in response to tumor-mimetic structural and mechanical cues. Using these platforms, we characterize a mechanical optimum for migration that can be perturbed by manipulating an axis between microtubule stability and force generation. In 3D environments and live tumors, we demonstrate that microtubule instability, leading to increased Rho pathway-dependent cortical contractility, promotes migration whereas clinically used microtubule-stabilizing chemotherapies profoundly decrease effective migration. We show that rational manipulation of the microtubule-contractility axis, either pharmacologically or through genome engineering, results in engineered T cells that more effectively move through and interrogate 3D matrix and tumor volumes. Thus, engineering cells to better navigate through 3D microenvironments could be part of an effective strategy to enhance efficacy of immune therapeutics.

The binding of LDN193189 to CD133 C-terminus suppresses the tumorigenesis and immune escape of liver tumor-initiating cells.

The tumor-initiating cell (TIC) marker CD133 promotes TIC self-renewal and tumorigenesis through the tyrosine phosphorylation of its c-terminal domain. Therefore, finding compounds that target the phosphorylation of CD133 will provide an effective method for inhibiting TICs characteristics. Here, through small molecule microarray screening, compound LDN193189 was found to bind to the c-terminus of CD133 and influenced its tyrosine phosphorylation. LDN193189 inhibited the interaction between CD133 and p85, accompanied by a reduction in the self-renewal and tumorigenicity of liver TIC. In addition, LDN193189 inhibited the expression and transcription of Galectin-3 by reducing the tyrosine phosphorylation of CD133. Galectin-3 secreted by liver TICs inhibited the proliferation of activated CD8+ T cells by binding to PD-1. LDN193189 suppressed the immune escape ability of liver TICs by downregulating Galectin-3. Taken together, LDN193189 suppressed the tumorigenesis and immune escape of liver CSCs by targeting the CD133-Galectin-3 axis.

Double Insurance for OC: miRNA-Mediated Platinum Resistance and Immune Escape.

Ovarian cancer (OC) is still the leading cause of death among all gynecological malignancies, despite the recent progress in cancer therapy. Immune escape and drug resistance, especially platinum-based chemotherapy, are significant factors causing disease progression, recurrence and poor prognosis in OC patients. MicroRNAs(miRNAs) are small noncoding RNAs, regulating gene expression at the transcriptional level. Accumulating evidence have indicated their crucial roles in platinum resistance. Importantly, they also act as mediators of tumor immune escape/evasion. In this review, we summarize the recent study of miRNAs involved in platinum resistance of OC and systematically analyses miRNAs involved in the regulation of OC immune escape. Further understanding of miRNAs roles and their possible mechanisms in platinum resistance and tumor escape may open new avenues for improving OC therapy.

Anti-angiogenic agents - overcoming tumour endothelial cell anergy and improving immunotherapy outcomes.

Immune checkpoint inhibitors have revolutionized medical oncology, although currently only a subset of patients has a response to such treatment. A compelling body of evidence indicates that anti-angiogenic therapy has the capacity to ameliorate antitumour immunity owing to the inhibition of various immunosuppressive features of angiogenesis. Hence, combinations of anti-angiogenic agents and immunotherapy are currently being tested in >90 clinical trials and 5 such combinations have been approved by the FDA in the past few years. In this Perspective, we describe how the angiogenesis-induced endothelial immune cell barrier hampers antitumour immunity and the role of endothelial cell anergy as the vascular counterpart of immune checkpoints. We review the antitumour immunity-promoting effects of anti-angiogenic agents and provide an update on the current clinical successes achieved when these agents are combined with immune checkpoint inhibitors. Finally, we propose that anti-angiogenic agents are immunotherapies - and vice versa - and discuss future research priorities.

ARIH1 signaling promotes anti-tumor immunity by targeting PD-L1 for proteasomal degradation.

Cancer expression of PD-L1 suppresses anti-tumor immunity. PD-L1 has emerged as a remarkable therapeutic target. However, the regulation of PD-L1 degradation is not understood. Here, we identify several compounds as inducers of PD-L1 degradation using a high-throughput drug screen. We find EGFR inhibitors promote PD-L1 ubiquitination and proteasomal degradation following GSK3α-mediated phosphorylation of Ser279/Ser283. We identify ARIH1 as the E3 ubiquitin ligase responsible for targeting PD-L1 to degradation. Overexpression of ARIH1 suppresses tumor growth and promotes cytotoxic T cell activation in wild-type, but not in immunocompromised mice, highlighting the role of ARIH1 in anti-tumor immunity. Moreover, combining EGFR inhibitor ES-072 with anti-CTLA4 immunotherapy results in an additive effect on both tumor growth and cytotoxic T cell activation. Our results delineate a mechanism of PD-L1 degradation and cancer escape from immunity via EGFR-GSK3α-ARIH1 signaling and suggest GSK3α and ARIH1 might be potential drug targets to boost anti-tumor immunity and enhance immunotherapies.

Platelet-Mediated Protection of Cancer Cells From Immune Surveillance - Possible Implications for Cancer Immunotherapy.

The growing insights in the complex interactions between metastatic cancer-cells and platelets have revealed that platelet tumor cell interactions in the blood stream are an important factor supporting tumor metastasis. An increased coagulability of platelets facilitates the vascular evasion and establishment of solid tumor metastasis. Furthermore, platelets can support an immunosuppressive tumor microenvironment or shield tumor cells directly from engagement of cytotoxic lymphocytes as e.g., natural killer (NK) cells. Platelets are both in the tumor microenvironment and systemically the quantitatively most important source of TGF-ß, which is a key cytokine for immunosuppression in the tumor microenvironment. If similar platelet-tumor interactions are of physiological relevance in hematological malignancies remains less well-studied. This might be important, as T- and NK cell mediated graft vs. leukemia effects (GvL) are well-documented and malignant hematological cells have a high exposure to platelets compared to solid tumors. As NK cell-based immunotherapies gain increasing attention as a therapeutic option for patients suffering from hematological and other malignancies, we review the known interactions between platelets and NK cells in the solid tumor setting and discuss how these could also apply to hematological cancers. We furthermore explore the possible implications for NK cell therapy in patients with solid tumors and patients who depend on frequent platelet transfusions. As platelets have a protective and supportive effect on cancer cells, the impact of platelet transfusion on immunotherapy and the combination of immunotherapy with platelet inhibitors needs to be evaluated.

Evasion of Innate Immunity Contributes to Small Cell Lung Cancer Progression and Metastasis.

Small cell lung cancer (SCLC) is a pulmonary neuroendocrine cancer with very poor prognosis and limited effective therapeutic options. Most patients are diagnosed at advanced stages, and the exact reason for the aggressive and metastatic phenotype of SCLC is completely unknown. Despite a high tumor mutational burden, responses to immune checkpoint blockade are minimal in patients with SCLC. This may reflect defects in immune surveillance. Here we illustrate that evading natural killer (NK) surveillance contributes to SCLC aggressiveness and metastasis, primarily through loss of NK-cell recognition of these tumors by reduction of NK-activating ligands (NKG2DL). SCLC primary tumors expressed very low level of NKG2DL mRNA and SCLC lines express little to no surface NKG2DL at the protein level. Chromatin immunoprecipitation sequencing showed NKG2DL loci in SCLC are inaccessible compared with NSCLC, with few H3K27Ac signals. Restoring NKG2DL in preclinical models suppressed tumor growth and metastasis in an NK cell-dependent manner. Likewise, histone deacetylase inhibitor treatment induced NKG2DL expression and led to tumor suppression by inducing infiltration and activation of NK and T cells. Among all the common tumor types, SCLC and neuroblastoma were the lowest NKG2DL-expressing tumors, highlighting a lineage dependency of this phenotype. In conclusion, these data show that epigenetic silencing of NKG2DL results in a lack of stimulatory signals to engage and activate NK cells, highlighting the underlying immune avoidance of SCLC and neuroblastoma. SIGNIFICANCE: This study discovers in SCLC and neuroblastoma impairment of an inherent mechanism of recognition of tumor cells by innate immunity and proposes that this mechanism can be reactivated to promote immune surveillance.

Translational Regulation of Cancer Metastasis.

Deregulation of the mRNA translational process has been observed during tumorigenesis. However, recent findings have shown that deregulation of translation also contributes specifically to cancer cell spread. During metastasis, cancer cells undergo changes in cellular state, permitting the acquisition of features necessary for cell survival, dissemination, and outgrowth. In addition, metastatic cells respond to external cues, allowing for their persistence under significant cellular and microenvironmental stresses. Recent work has revealed the importance of mRNA translation to these dynamic changes, including regulation of cell states through epithelial-to-mesenchymal transition and tumor dormancy and as a response to external stresses such as hypoxia and immune surveillance. In this review, we focus on examples of altered translation underlying these phenotypic changes and responses to external cues and explore how they contribute to metastatic progression. We also highlight the therapeutic opportunities presented by aberrant mRNA translation, suggesting novel ways to target metastatic tumor cells.

Intermittent hypoxia induces tumor immune escape in murine S180 solid tumors via the upregulation of TGF-ß1 in mice.

PURPOSE: Studies have shown that intermittent hypoxia (IH) alters host immune functions and promotes tumor growth. However, the relevant mechanisms of these effects have not been completely elucidated. We hypothesized that IH promotes the growth of tumors by changing cytokine levels in the tumor microenvironment and inducing immune escape. METHODS: Sarcoma-180 (S180) solid tumor cells were injected into the right flank of Kunming mice. The mice were then randomly divided into the IH and room air (RA) groups. The mice were euthanized 2 weeks after IH exposure, and the weight of tumor tissues was measured. Next, IL-6, IL-17, IL-10, and TNF-α levels in tumor tissues were measured via enzyme linked immunosorbent assay (ELISA), and hypoxia inducible factor-1α (HIF-1α) and transforming growth factor ß1 (TGF-ß1) expressions were examined through Western blot analysis. RESULTS: Two weeks of IH exposure significantly accelerated the growth of S180 solid tumors. Western blot analysis results showed that the expression levels of HIF-1α and TGF-ß1 in S180 tumors in the IH group were significantly upregulated compared with those in the RA group. ELISA results showed that compared with the RA group, the IH group had significantly increased TNF-α and IL-10 (P < 0.05) and significantly decreased IL-17 (P < 0.05). CONCLUSION: IH might promote the growth of S180 solid tumors by inhibiting the antitumor immune response and inducing tumor immune escape via the upregulation of TGF-ß1.

Targeting Myeloid-Derived Suppressor Cells to Enhance the Antitumor Efficacy of Immune Checkpoint Blockade Therapy.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that are activated under pathological conditions, such as cancer, or mature myeloid cells that are converted immune-suppressive cells via tumor-derived exosomes, and potently support the tumor processes at different levels. Currently, multiple studies have demonstrated that MDSCs induce immune checkpoint blockade (ICB) therapy resistance through their contribution to the immunosuppressive network in the tumor microenvironment. In addition, non-immunosuppressive mechanisms of MDSCs such as promotion of angiogenesis and induction of cancer stem cells also exert a powerful role in tumor progression. Thus, MDSCs are potential therapeutic targets to enhance the antitumor efficacy of ICB therapy in cases of multiple cancers. This review focuses on the tumor-promoting mechanism of MDSCs and provides an overview of current strategies that target MDSCs with the objective of enhancing the antitumor efficacy of ICB therapy.

The Influence of Tumor Microenvironment on Immune Escape of Melanoma.

The low efficiency of currently-used anti-cancer therapies poses a serious challenge, especially in the case of malignant melanoma, a cancer characterized by elevated invasiveness and relatively high mortality rate. The role of the tumor microenvironment in the progression of melanoma and its acquisition of resistance to treatment seems to be the main focus of recent studies. One of the factors that, in normal conditions, aids the organism in its fight against the cancer and, following the malignant transformation, adapts to facilitate the development of the tumor is the immune system. A variety of cell types, i.e., T and B lymphocytes, macrophages, and dendritic and natural killer cells, as well as neutrophils, support the growth and invasiveness of melanoma cells, utilizing a plethora of mechanisms, including secretion of pro-inflammatory molecules, induction of inhibitory receptors expression, or depletion of essential nutrients. This review provides a comprehensive summary of the processes regulated by tumor-associated cells that promote the immune escape of melanoma cells. The described mechanisms offer potential new targets for anti-cancer treatment and should be further studied to improve currently-employed therapies.

Intratumoral Copper Modulates PD-L1 Expression and Influences Tumor Immune Evasion.

Therapeutic checkpoint antibodies blocking programmed death receptor 1/programmed death ligand 1 (PD-L1) signaling have radically improved clinical outcomes in cancer. However, the regulation of PD-L1 expression on tumor cells is still poorly understood. Here we show that intratumoral copper levels influence PD-L1 expression in cancer cells. Deep analysis of the The Cancer Genome Atlas database and tissue microarrays showed strong correlation between the major copper influx transporter copper transporter 1 (CTR-1) and PD-L1 expression across many cancers but not in corresponding normal tissues. Copper supplementation enhanced PD-L1 expression at mRNA and protein levels in cancer cells and RNA sequencing revealed that copper regulates key signaling pathways mediating PD-L1-driven cancer immune evasion. Conversely, copper chelators inhibited phosphorylation of STAT3 and EGFR and promoted ubiquitin-mediated degradation of PD-L1. Copper-chelating drugs also significantly increased the number of tumor-infiltrating CD8+ T and natural killer cells, slowed tumor growth, and improved mouse survival. Overall, this study reveals an important role for copper in regulating PD-L1 and suggests that anticancer immunotherapy might be enhanced by pharmacologically reducing intratumor copper levels. SIGNIFICANCE: These findings characterize the role of copper in modulating PD-L1 expression and contributing to cancer immune evasion, highlighting the potential for repurposing copper chelators as enhancers of antitumor immunity. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/19/4129/F1.large.jpg.

Palmitoylated Proteins on AML-Derived Extracellular Vesicles Promote Myeloid-Derived Suppressor Cell Differentiation via TLR2/Akt/mTOR Signaling.

Acute myeloid leukemia (AML) represents the most common acute leukemia among adults. Despite recent progress in diagnosis and treatment, long-term outcome remains unsatisfactory. The success of allogeneic stem cell transplantation underscores the immunoresponsive nature of AML, creating the basis for further exploiting immunotherapies. However, emerging evidence suggests that AML, similar to other malignant entities, employs a variety of mechanisms to evade immunosurveillance. In light of this, T-cell inhibitory myeloid-derived suppressor cells (MDSC) are gaining interest as key facilitators of immunoescape. Accumulation of CD14+HLA-DRlow monocytic MDSCs has been described in newly diagnosed AML patients, and deciphering the underlying mechanisms could help to improve anti-AML immunity. Here, we report that conventional monocytes readily take-up AML-derived extracellular vesicles (EV) and subsequently undergo MDSC differentiation. They acquired an CD14+HLA-DRlow phenotype, expressed the immunomodulatory indoleamine-2,3-dioxygenase, and upregulated expression of genes characteristic for MDSCs, such as S100A8/9 and cEBPß. The Akt/mTOR pathway played a critical role in the AML-EV-induced phenotypical and functional transition of monocytes. Generated MDSCs displayed a glycolytic switch, which rendered them more susceptible toward glycolytic inhibitors. Furthermore, palmitoylated proteins on the AML-EV surface activated Toll-like receptor 2 as the initiating event of Akt/mTOR-dependent induction of MDSC. Therefore, targeting protein palmitoylation in AML blasts could block MDSC accumulation to improve immune responses. SIGNIFICANCE: These findings indicate that targeting protein palmitoylation in AML could interfere with the leukemogenic potential and block MDSC accumulation to improve immunity.

Hepatoma cell-intrinsic TLR9 activation induces immune escape through PD-L1 upregulation in hepatocellular carcinoma.

A TLR9 agonist in combination with a PD-1 inhibitor produced powerful antitumor responses in a clinical trial despite TLR9 agonists as monotherapies failing to generate systemic antitumor immune responses due to immunosuppressive effects. However, the mechanism involved in the improved response induced by their combination remains unknown. Methods: Subcutaneous and orthotopic Hepa1-6 tumor model was used for single-drug and combined-drug treatment. We used TLR9 agonist stimulation or lentiviral vectors to overexpress TLR9 and activate TLR9 signaling. We next investigated the crosstalk between PARP1 autoPARylation and ubiquitination and between STAT3 PARylation and phosphorylation mediated by TLR9. Tissue chips were used to analyze the relationships among TLR9, PARP1, p-STAT3 and PD-L1 expression. Results: In this study, we found that the TLR9 agonist in combination with anti-PD-1 therapy or anti-PD-L1 therapy yielded an additive effect that inhibited HCC growth in mice. Mechanistically, we found that TLR9 promoted PD-L1 transcription by enhancing STAT3 Tyr705 phosphorylation. Then, we observed that TLR9 negatively regulated PARP1 expression, which mediated a decrease in STAT3 PARylation and an increase in STAT3 Tyr705 phosphorylation. Moreover, we found that TLR9 enhanced PARP1 autoPARylation by inhibiting PARG expression, which then promoted the RNF146-mediated ubiquitination and subsequent degradation of PARP1. Finally, we observed positive associations between TLR9 and p-STAT3 (Tyr705) or PD-L1 expression and negative associations between TLR9 and PARP1 in HCC patient samples. Conclusions: We showed that hepatoma cell-intrinsic TLR9 activation regulated the crosstalk between PARP1 autoPARylation and ubiquitination and between STAT3 PARylation and phosphorylation, which together upregulated PD-L1 expression and finally induces immune escape. Therefore, combination therapy with a TLR9 agonist and an anti-PD-1 antibody or anti-PD-L1 had much better antitumor efficacy than either monotherapy in HCC.