Constitutive Expression of CCL22 Is Mediated by T Cell-Derived GM-CSF.

CCL22 is a key mediator of leukocyte trafficking in inflammatory immune responses, allergy, and cancer. It acts by attracting regulatory T cells and Th2 cells via their receptor CCR type 4 (CCR4). Beyond its role in inflammation, CCL22 is constitutively expressed at high levels in lymphoid organs during homeostasis, where it controls immunity by recruiting regulatory T cells to dendritic cells (DCs). In this study, we aimed to identify the mechanisms responsible for constitutive CCL22 expression. We confirmed that CD11c+ DCs are the exclusive producers of CCL22 in secondary lymphatic organs during homeostasis. We show that in vitro both murine splenocytes and human PBMCs secrete CCL22 spontaneously without any further stimulation. Interestingly, isolated DCs alone, however, are unable to produce CCL22, but instead require T cell help. In vitro, only the coculture of DCs with T cells or their supernatants resulted in CCL22 secretion, and we identified T cell-derived GM-CSF as the major inducer of DC-derived CCL22 expression. In vivo, Rag1 -/- mice, which lack functional T cells, have low CCL22 levels in lymphoid organs, and this can be restored by adoptive transfer of wild-type T cells or administration of GM-CSF. Taken together, we uncover T cell-derived GM-CSF as a key inducer of the chemokine CCL22 and thus, to our knowledge, identify a novel role for this cytokine as a central regulator of immunity in lymphatic organs. This knowledge could contribute to the development of new therapeutic interventions in cancer and autoimmunity.

CCL22 controls immunity by promoting regulatory T cell communication with dendritic cells in lymph nodes.

Chemokines have crucial roles in organ development and orchestration of leukocyte migration. The chemokine CCL22 is expressed constitutively at high levels in the lymph node, but the functional significance of this expression is so far unknown. Studying a newly established CCL22-deficient mouse, we demonstrate that CCL22 expression by dendritic cells (DCs) promotes the formation of cell-cell contacts and interaction with regulatory T cells (T reg) through their CCR4 receptor. Vaccination of CCL22-deficient mice led to excessive T cell responses that were also observed when wild-type mice were vaccinated using CCL22-deficient DCs. Tumor-bearing mice with CCL22 deficiency showed prolonged survival upon vaccination, and further, CCL22-deficient mice had increased susceptibility to inflammatory disease. In conclusion, we identify the CCL22-CCR4 axis as an immune checkpoint that is crucial for the control of T cell immunity.

Cancer cell-derived IL-1α induces CCL22 and the recruitment of regulatory T cells.

In cancer patients, immunosuppression through regulatory T cells (Treg) is a crucial component of tumor immune evasion and contributes to disease progression. Tumor-infiltrating Treg in particular suppress local effector T cell responses and are associated with poor prognosis in tumors such as human pancreatic cancer or hepatocellular carcinoma (HCC). The chemokine CCL22 is known to recruit Treg into the tumor tissue and many types of human tumors are known to express high levels of CCL22. The mechanisms leading to intratumoral secretion of CCL22 are so far unknown. We demonstrate here that intratumoral CCL22 is induced in tumor-infiltrating immune cells through cancer cell-derived interleukin-1 (IL-1α). In pancreatic cancer and HCC, CCL22 is produced by intratumoral dendritic cells, while the cancer cells themselves do not secrete CCL22 in vitro and in vivo. Incubation of human peripheral blood mononuclear cells (PBMC) or murine splenocytes with tumor cells or tumor cell supernatants strongly induced CCL22 secretion in vitro. Tumor cell supernatants contained IL-1 and CCL22 induction in PBMC could be specifically prevented by the IL-1 receptor antagonist anakinra or by transfection of tumor cell lines with IL-1 siRNA, leading to a suppression of Treg migration. In conclusion, we identify here tumor cell-derived IL-1α as a major inducer of the Treg attracting chemokine CCL22 in human cancer cells. Therapeutic blockade of the IL-1 pathway could represent a promising strategy to inhibit tumor-induced immunosuppression.

miRNA dynamics in tumor-infiltrating myeloid cells modulating tumor progression in pancreatic cancer.

Myeloid cells including tumor-associated macrophages (TAM) and myeloid-derived suppressor cells (MDSC) are known as important mediators of tumor progression in solid tumors such as pancreatic cancer. Infiltrating myeloid cells have been identified not only in invasive tumors, but also in early pre-invasive pancreatic intraepithelial precursor lesions (PanIN). The functional dynamics of myeloid cells during carcinogenesis is largely unknown. We aimed to systematically elucidate phenotypic and transcriptional changes in infiltrating myeloid cells during carcinogenesis and tumor progression in a genetic mouse model of pancreatic cancer. Using murine pancreatic myeloid cells isolated from the genetic mouse model at different time points during carcinogenesis, we examined both established markers of macrophage polarization using RT-PCR and FACS as well as transcriptional changes focusing on miRNA profiling. Myeloid cells isolated during carcinogenesis showed a simultaneous increase of established markers of M1 and M2 polarization during carcinogenesis, indicating that phenotypic changes of myeloid cells during carcinogenesis do not follow the established M1/M2 classification. MiRNA profiling revealed distinct regulations of several miRNAs already present in myeloid cells infiltrating pre-invasive PanIN lesions. Among them miRNA-21 was significantly increased in myeloid cells surrounding both PanIN lesions and invasive cancers. Functionally, miRNA-21-5p and -3p altered expression of the immune-modulating cytokines CXCL-10 and CCL-3 respectively. Our data indicate that miRNAs are dynamically regulated in infiltrating myeloid cells during carcinogenesis and mediate their functional phenotype by facilitating an immune-suppressive tumor-promoting micro-milieu.

Prevailing over T cell exhaustion: New developments in the immunotherapy of pancreatic cancer.

Pancreatic cancer is one of the most aggressive malignancies and has been considered poorly immunogenic for decades. However, this characterization might be over-simplistic. A more sophisticated approach is needed in order to develop new treatment strategies. In this review, we will focus on T cell exhaustion as a phenomenon of immune failure that is a useful paradigm to characterize immunosuppressive effects. Cancer creates an environment of constant antigen exposure and inflammation. In this setting, T cells transform into a differentiation state that has been termed T cell exhaustion, which is characterized by upregulation of inhibitory receptors, resulting in loss of effector function. The discovery of receptor-mediated immune checkpoints, which prevent uncontrolled T cell reactions, led to the development of a new class of antibodies termed checkpoint inhibitors. Unprecedented results in patients with metastatic melanoma and lung cancer have renewed interest in the immunotherapy of other solid tumor entities, including pancreatic cancer. Data on the efficacy of checkpoint inhibitors in pancreatic cancer are still sparse and indicate limited efficacy as single agents. Combination of checkpoint inhibitors with other immune-activating strategies or cytotoxic drugs might be a way to overcome therapy resistance in the treatment of pancreatic cancer.

NFATc1 Links EGFR Signaling to Induction of Sox9 Transcription and Acinar-Ductal Transdifferentiation in the Pancreas.

BACKGROUND & AIMS: Oncogenic mutations in KRAS contribute to the development of pancreatic ductal adenocarcinoma, but are not sufficient to initiate carcinogenesis. Secondary events, such as inflammation-induced signaling via the epidermal growth factor receptor (EGFR) and expression of the SOX9 gene, are required for tumor formation. Herein we sought to identify the mechanisms that link EGFR signaling with activation of SOX9 during acinar-ductal metaplasia, a transdifferentiation process that precedes pancreatic carcinogenesis. METHODS: We analyzed pancreatic tissues from Kras(G12D);pdx1-Cre and Kras(G12D);NFATc1(Δ/Δ);pdx1-Cre mice after intraperitoneal administration of caerulein, vs cyclosporin A or dimethyl sulfoxide (controls). Induction of EGFR signaling and its effects on the expression of Nuclear factor of activated T cells c1 (NFATc1) or SOX9 were investigated by quantitative reverse-transcription polymerase chain reaction, immunoblot, and immunohistochemical analyses of mouse and human tissues and acinar cell explants. Interactions between NFATc1 and partner proteins and effects on DNA binding or chromatin modifications were studied using co-immunoprecipitation and chromatin immunoprecipitation assays in acinar cell explants and mouse tissue. RESULTS: EGFR activation induced expression of NFATc1 in metaplastic areas from patients with chronic pancreatitis and in pancreatic tissue from Kras(G12D) mice. EGFR signaling also promoted formation of a complex between NFATc1 and C-JUN in dedifferentiating mouse acinar cells, leading to activation of Sox9 transcription and induction of acinar-ductal metaplasia. Pharmacologic inhibition of NFATc1 or disruption of the Nfatc1 gene inhibited EGFR-mediated induction of Sox9 transcription and blocked acinar-ductal transdifferentiation and pancreatic cancer initiation in mice. CONCLUSIONS: EGFR signaling induces expression of NFATc1 and Sox9, leading to acinar cell transdifferentiation and initiation of pancreatic cancer. Strategies designed to disrupt this pathway might be developed to prevent pancreatic cancer initiation in high-risk patients with chronic pancreatitis.

CUX1: a modulator of tumour aggressiveness in pancreatic neuroendocrine neoplasms.

Pancreatic neuroendocrine neoplasms (PNENs) constitute a rare tumour entity, and prognosis and treatment options depend on tumour-mediating hallmarks such as angiogenesis, proliferation rate and resistance to apoptosis. The molecular pathways that determine the malignant phenotype are still insufficiently understood and this has limited the use of effective combination therapies in the past. In this study, we aimed to characterise the effect of the oncogenic transcription factor Cut homeobox 1 (CUX1) on proliferation, resistance to apoptosis and angiogenesis in murine and human PNENs. The expression and function of CUX1 were analysed using knockdown and overexpression strategies in Ins-1 and Bon-1 cells, xenograft models and a genetically engineered mouse model of insulinoma (RIP1Tag2). Regulation of angiogenesis was assessed using RNA profiling and functional tube-formation assays in HMEC-1 cells. Finally, CUX1 expression was assessed in a tissue microarray of 59 human insulinomas and correlated with clinicopathological data. CUX1 expression was upregulated during tumour progression in a time- and stage-dependent manner in the RIP1Tag2 model, and associated with pro-invasive and metastatic features of human insulinomas. Endogenous and recombinant CUX1 expression increased tumour cell proliferation, tumour growth, resistance to apoptosis, and angiogenesis in vitro and in vivo. Mechanistically, the pro-angiogenic effect of CUX1 was mediated via upregulation of effectors such as HIF1α and MMP9. CUX1 mediates an invasive pro-angiogenic phenotype and is associated with malignant behaviour in human insulinomas.

The role of CUX1 in antagonizing NF-κB signaling in TAMs.

Macrophages represent a major component of the tumor microenvironment and contribute to neoplasia initiation and cancer progression. However, the molecular mechanisms underlying these phenomena are only partially understood. Manipulating the transcriptional activity of the macrophage functional specification factor NF-κB by virtue of a novel regulatory factor cut-like homeobox 1 (CUX1) may provide a potential target for therapeutic intervention.

Synthetic lethality screen identifies RPS6KA2 as modifier of epidermal growth factor receptor activity in pancreatic cancer.

Pancreatic cancer is characterized by a high degree of resistance to chemotherapy. Epidermal growth factor receptor (EGFR) inhibition using the small-molecule inhibitor erlotinib was shown to provide a small survival benefit in a subgroup of patients. To identify kinases whose inhibition acts synergistically with erlotinib, we employed a kinome-wide small-interfering RNA (siRNA)-based loss-of-function screen in the presence of erlotinib. Of 779 tested kinases, we identified several targets whose inhibition acted synergistically lethal with EGFR inhibition by erlotinib, among them the S6 kinase ribosomal protein S6 kinase 2 (RPS6KA2)/ribosomal S6 kinase 3. Activated RPS6KA2 was expressed in approximately 40% of 123 human pancreatic cancer tissues. RPS6KA2 was shown to act downstream of EGFR/RAS/mitogen-activated protein kinase kinase (MEK)/extracellular-signal regulated kinase (ERK) signaling and was activated by EGF independently of the presence of KRAS mutations. Knockdown of RPS6KA2 by siRNA led to increased apoptosis only in the presence of erlotinib, whereas RPS6KA2 activation or overexpression rescued from erlotinib- and gemcitabine-induced apoptosis. This effect was at least in part mediated by downstream activation of ribosomal protein S6. Genetic as well as pharmacological inhibition of RPS6KA2 by the inhibitor BI-D1870 acted synergistically with erlotinib. By applying this synergistic lethality screen using a kinome-wide RNA interference-library approach, we identified RPS6KA2 as potential drug target whose inhibition synergistically enhanced the effect of erlotinib on tumor cell survival. This kinase therefore represents a promising drug candidate suitable for the development of novel inhibitors for pancreatic cancer therapy.