JAK-STAT-mediated chronic inflammation impairs cytotoxic T lymphocyte activation to decrease anti-PD-1 immunotherapy efficacy in pancreatic cancer.
ABSTRACT: Human pancreatic cancer does not respond to immune check point blockade immunotherapy. One key feature of pancreatic cancer is the association between its progression and chronic inflammation. Emerging evidence supports a key role for the JAK-STAT pathway in pancreatic cancer inflammation. We aimed at testing the hypothesis that sustained JAK-STAT signaling suppresses cytotoxic T lymphocyte (CTL) activation to counteract anti-PD-1 immunotherapy-induced CTL activity in pancreatic cancer. We show that human pancreatic carcinomas express high level of PD-L1 and exhibit low level of CTL infiltration. JAK-STAT inhibitor Ruxolitinib selectively inhibits STAT1 and STAT3 activation and increases CTL infiltration to induce a Tc1/Th1 immune response in the tumor microenvironment in an orthotopic pancreatic cancer mouse model. Ruxilitinib-mediated tumor suppressive efficacy diminishes in T-cell-deficient mice. Pancreatic tumor grows significantly faster in IFN?-deficient mice. However, neutralizing IFN? does not alter tumor growth but diminishes Ruxolitinib-induced tumor suppression in vivo, indicating that lymphocytes and IFN? are essential for Ruxolitinib-induced host antitumor immune response. Both type I and type II interferons upregulate PD-L1 expression through the JAK-STAT signaling pathway in mouse pancreatic tumor cells. Tumor cells respond to activated T cells by activating STAT3. The inhibition of STAT3 downregulates immune suppressive cytokines production by tumor cells, resulting in increased T cell activation and effector function. Consequently, Ruxolitinib significantly improves the efficacy of anti-PD-1 immunotherapy. Our data demonstrate that Ruxolitinib is effective in the inhibition of systemic inflammation in the tumor microenvironment and therefore upregulates CTL infiltration and activation to overcome pancreatic cancer resistance to anti-PD-1 immunotherapy.
Project description:Tumor-associated macrophages contribute to tumor progression and therapeutic resistance in breast cancer. Within the tumor microenvironment, tumor-derived factors activate pathways that modulate macrophage function. Using in vitro and in vivo models, we find that tumor-derived factors induce activation of the Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) pathway in macrophages. We also demonstrate that loss of STAT3 in myeloid cells leads to enhanced mammary tumorigenesis. Further studies show that macrophages contribute to resistance of mammary tumors to the JAK/STAT inhibitor ruxolitinib in vivo and that ruxolitinib-treated macrophages produce soluble factors that promote resistance of tumor cells to JAK inhibition in vitro. Finally, we demonstrate that STAT3 deletion and JAK/STAT inhibition in macrophages increases expression of the pro-tumorigenic factor cyclooxygenase-2 (COX-2) and that COX-2 inhibition enhances responsiveness of tumors to ruxolitinib. These findings define a novel mechanism through which macrophages promote therapeutic resistance and highlight the importance of understanding the impact of targeted therapies on the tumor microenvironment. Overall design: Primary human macrophages were treated with breast tumor cell (MCF7, MDA-MB-231, or control) conditioned medium, in combination with ruxolitinib (or control), and processed for gene expression analysis in triplicate. Bone marrow derived macrophages from STAT3 wild type or STAT3 conditional knockout mice were extracted and were processed for gene expression analysis in triplicate.
Project description:Myxoid liposarcoma (MLS) shows extensive intratumoural heterogeneity with distinct subpopulations of tumour cells. Despite improved survival of MLS patients, existing therapies have shortcomings as they fail to target all tumour cells. The nature of chemotherapy-resistant cells in MLS remains unknown. Here, we show that MLS cell lines contained subpopulations of cells that can form spheres, efflux Hoechst dye and resist doxorubicin, all properties attributed to cancer stem cells (CSCs). By single-cell gene expression, western blot, phospho-kinase array, immunoprecipitation, immunohistochemistry, flow cytometry and microarray analysis we showed that a subset of MLS cells expressed JAK-STAT genes with active signalling. JAK1/2 inhibition via ruxolitinib decreased, while stimulation with LIF increased, phosphorylation of STAT3 and the number of cells with CSC properties indicating that JAK-STAT signalling controlled the number of cells with CSC features. We also show that phosphorylated STAT3 interacted with the SWI/SNF complex. We conclude that MLS contains JAK-STAT-regulated subpopulations of cells with CSC features. Combined doxorubicin and ruxolitinib treatment targeted both proliferating cells as well as cells with CSC features, providing new means to circumvent chemotherapy resistance in treatment of MLS patients.
Project description:Numerous prior studies on fighting cancer have been based on using inhibitors of JAK-STAT pathway (signal transducer and activator of transcription 3 (STAT3) inhibitor in particular), a signaling pathway responsible for progression of many types of cancer cells. However, recent studies have shown that STAT3 activation leads to upregulation of program death receptor-ligand 1 (PD-L1, an immune checkpoint protein that plays a major role behind evasion of immune systems by growing tumors) expression levels in tumor cells, leading to enhanced immune suppression. This is why global efforts are being witnessed in combating cancer through use of immune checkpoint inhibitors. Herein, we report on the design, synthesis, physicochemical characterizations, and bioactivity evaluation of novel tumor- and tumor-vasculature-targeting noncytotoxic Au-CGKRK nanoconjugates (17-80 nm) for combating tumor. Using a syngeneic mouse tumor model, we show that intraperitoneal (i.p.) administration of the Au-CGKRK nanoparticles (NPs) complexed with both PD-L1siRNA (the immune checkpoint inhibitor) and STAT3siRNA (the JAK-STAT pathway inhibitor) results in significant (>70%) enhancement in overall survivability (OS) in melanoma-bearing mice (n = 5) when compared to the OS in the untreated mice group. The expression levels of CD8 and CD4 proteins in the tumor lysates of differently treated mice groups (by Western blotting) are consistent with the observed OS enhancement being a T-cell-driven process. Biodistribution study using near-infrared dye-loaded Au-CGKRK nanoconjugates revealed selective accumulation of the dye in mouse tumor. Notably, the overall survival benefits were significantly less (?35%) when melanoma-bearing mice were treated (i.p.) with Au-CGKRK NPs complexed with only PD-L1siRNA or with STAT3siRNA alone. The presently described Au-CGKRK nanoconjugates are expected to find future use in therapeutic RNA-interference-based cancer immunotherapy.
Project description:Myeloproliferative neoplasms (MPN) are a group of clonal disorders that affect hematopoietic stem/progenitor cells. These disorders are often caused by oncogenic driver mutations associated with persistent Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling. While JAK inhibitors, such as ruxolitinib, reduce MPN-related symptoms in myelofibrosis, they do not influence the underlying cause of the disease and are not curative. Due to these limitations, there is a need for alternative therapeutic strategies and targets. Heat shock proteins (HSPs) are cytoprotective stress-response chaperones involved in protein homeostasis and in many critical pathways, including inflammation. Over the last decade, several research teams have unraveled the mechanistic connection between STAT signaling and several HSPs, showing that HSPs are potential therapeutic targets for MPN. These HSPs include HSP70, HSP90 (chaperoning JAK2) and both HSP110 and HSP27, which are key factors modulating STAT3 phosphorylation status. Like the HSPs, the PD-1/PD-L1 signaling pathway has been widely studied in cancer, but the importance of PD-L1-mediated immune escape in MPN was only recently reported. In this review, we summarize the role of HSPs and PD-1/PD-L1 signaling, the modalities of their experimental blockade, and the effect in MPN. Finally, we discuss the potential of these emerging targeted approaches in MPN therapy.
Project description:The JAK-STAT signalling pathway regulates cellular processes like cell division, cell death and immune regulation. Dysregulation has been identified in solid tumours and STAT3 activation is a marker for poor outcome. The aim of this study was to explore potential therapeutic strategies by targeting this pathway in bladder cancer (BC). High STAT3 expression was detected in 51.3% from 149 patient specimens with invasive bladder cancer by immunohistochemistry. Protein expression of JAK, STAT and downstream targets were confirmed in 10 cell lines. Effects of the JAK inhibitors Ruxolitinib and BSK-805, and STAT3/5 inhibitors Stattic, Nifuroxazide and SH-4-54 were analysed by cell viability assays, immunoblotting, apoptosis and cell cycle progression. Treatment with STAT3/5 but not JAK1/2 inhibitors reduced survival, levels of phosphorylated STAT3 and Cyclin-D1 and increased apoptosis. Tumour xenografts, using the chicken chorioallantoic membrane (CAM) model responded to Stattic monotherapy. Combination of Stattic with Cisplatin, Docetaxel, Gemcitabine, Paclitaxel and CDK4/6 inhibitors showed additive effects. The combination of Stattic with the oncolytic adenovirus XVir-N-31 increased viral replication and cell lysis. Our results provide evidence that inhibitors against STAT3/5 are promising as novel mono- and combination therapy in bladder cancer.
Project description:Ovarian carcinoma-associated mesenchymal stem cells (CA-MSC) produce not only high levels of interleukin-6 (IL6) but also the related cytokine leukemia inhibitory factor (LIF). IL6-mediated activation of STAT3 is implicated as a critical therapeutic target for cancer therapy. Less is known about the role of LIF, which can similarly activate STAT3, in ovarian cancer. We therefore sought to evaluate the tumorigenic effects of CA-MSC paracrine LIF signaling and the redundancy of IL6 and LIF in activating ovarian cancer STAT3 mediated cancer growth. As expected, we found that both IL6 and LIF induce STAT3 phosphorylation in tumor cells. In addition, both IL6 and LIF increased the percentage of ALDH+?ovarian cancer stem-like cells (CSC). Supporting redundancy of function by the two cytokines, CA-MSC induced STAT3 phosphorylation and increased cancer cell "stemness". This effect was not inhibited by LIF or IL6 blocking antibodies alone, but was prevented by dual IL6/LIF blockade or JAK2 inhibition. Similarly, small hairpin RNA (shRNA)-mediated reduction of IL6 or LIF in CA-MSC partially decreased but could not completely abrograte the ability of CA-MSC to induce STAT3 phosphorylation and stemness. Importantly, the in vivo pro-tumorigenic effect of CA-MSC is abrogated by dual blockade with the JAK2 inhibitor ruxolitinib to a much greater extent than treatment with anti-IL6 or anti-LIF antibody alone. Ruxolitinib treatment also improves survival in the immunocompetent ovarian cancer mouse model system with ID8 tumor cells plus MSC. Ruxolitinib-treated tumors in both the immunocompromised and immunocompetent animal models demonstrate decreased phospho-STAT3, indicating on-target activity. In conclusion, CA-MSC activate ovarian cancer cell STAT3 signaling via IL6 and LIF and increase tumor cell stemness. This functional redundancy suggests that therapeutic targeting of a single cytokine may be less effective than strategies such as dual inhibitor therapy or targeting shared downstream factors of the JAK/STAT pathway.
Project description:Ovarian granulosa cells are fundamental for oocyte maintenance and maturation. Recent studies have demonstrated the importance of members of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signalling pathway in the granulosa cell population of mouse and horse ovaries, with perturbation of JAK1 signalling in the mouse shown to impair oocyte maintenance and accelerate primordial follicle activation. The presence and role of the JAK/STAT pathway in human granulosa cells has yet to be elucidated. In this study, expression of JAK1, STAT1 and STAT3 was detected in oocytes and granulosa cells of human ovarian sections from fetal (40 weeks gestation) and premenopausal ovaries (34-41 years of age; n=3). To determine the effects of JAK1 signalling in granulosa cells, the human granulosa-like cell line COV434 was used, with JAK1 inhibition using ruxolitinib. Chemical inhibition of JAK1 in COV434 cells with 100nM ruxolitinib for 72h resulted in significant increases in STAT3 mRNA (P=0.034) and p-Y701-STAT1 protein (P=0.0117), demonstrating a role for JAK1 in modulating STAT in granulosa cells. This study implicates a conserved role for JAK/STAT signalling in human ovary development, warranting further investigation of this pathway in human granulosa cell function.
Project description:Although squamous cell carcinomas (SqCCs) of the lungs, head and neck, oesophagus, and cervix account for up to 30% of cancer deaths, the mechanisms that regulate disease progression remain incompletely understood. Here, we use gene transduction and human tumor xenograft assays to establish that the tumour suppressor Cell adhesion molecule 1 (CADM1) inhibits SqCC proliferation and invasion, processes fundamental to disease progression. We determine that the extracellular domain of CADM1 mediates these effects by forming a complex with HER2 and integrin ?6?4 at the cell surface that disrupts downstream STAT3 activity. We subsequently show that treating CADM1 null tumours with the JAK/STAT inhibitor ruxolitinib mimics CADM1 gene restoration in preventing SqCC growth and metastases. Overall, this study identifies a novel mechanism by which CADM1 prevents SqCC progression and suggests that screening tumours for loss of CADM1 expression will help identify those patients most likely to benefit from JAK/STAT targeted chemotherapies.
Project description:The programmed death protein 1 (PD-1) and its ligand (PD-L1) represent a well-characterized immune checkpoint in cancer, effectively targeted by monoclonal antibodies that are approved for routine clinical use. The regulation of PD-L1 expression is complex, varies between different tumor types and occurs at the genetic, transcriptional and post-transcriptional levels. Copy number alterations of PD-L1 locus have been reported with varying frequency in several tumor types. At the transcriptional level, a number of transcriptional factors seem to regulate PD-L1 expression including HIF-1, STAT3, NF-??, and AP-1. Activation of common oncogenic pathways such as JAK/STAT, RAS/ERK, or PI3K/AKT/MTOR, as well as treatment with cytotoxic agents have also been shown to affect tumoral PD-L1 expression. Correlative studies of clinical trials with PD-1/PD-L1 inhibitors have so far shown markedly discordant results regarding the value of PD-L1 expression as a marker of response to treatment. As the indications for immune checkpoint inhibition broaden, understanding the regulation of PD-L1 in cancer will be of utmost importance for defining its role as predictive marker but also for optimizing strategies for cancer immunotherapy. Here, we review the current knowledge of PD-L1 regulation, and its use as biomarker and as therapeutic target in cancer.
Project description:Activating Janus kinase (JAK) and signal transducer and activator of transcription (STAT) mutations have been discovered in many T-cell malignancies, including anaplastic lymphoma kinase (ALK)- anaplastic large cell lymphomas (ALCLs). However, such mutations occur in a minority of patients. To investigate the clinical application of targeting JAK for ALK- ALCL, we treated ALK- cell lines of various histological origins with JAK inhibitors. Interestingly, most exogenous cytokine-independent cell lines responded to JAK inhibition regardless of JAK mutation status. JAK inhibitor sensitivity correlated with the STAT3 phosphorylation status of tumor cells. Using retroviral shRNA knockdown, we have demonstrated that these JAK inhibitor-sensitive cells are dependent on both JAK1 and STAT3 for survival. JAK1 and STAT3 gain-of-function mutations were found in some, but not all, JAK inhibitor-sensitive cells. Moreover, the mutations alone cannot explain the JAK1/STAT3 dependency, given that wild-type JAK1 or STAT3 was sufficient to promote cell survival in the cells that had either JAK1or STAT3 mutations. To investigate whether other mechanisms were involved, we knocked down upstream receptors GP130 or IL-2R?. Knockdown of GP130 or IL-2R? induced cell death in selected JAK inhibitor-sensitive cells. High expression levels of cytokines, including IL-6, were demonstrated in cell lines as well as in primary ALK- ALCL tumors. Finally, ruxolitinib, a JAK1/2 inhibitor, was effective in vivo in a xenograft ALK- ALCL model. Our data suggest that cytokine receptor signaling is required for tumor cell survival in diverse forms of ALK- ALCL, even in the presence of JAK1/STAT3 mutations. Therefore, JAK inhibitor therapy might benefit patients with ALK- ALCL who are phosphorylated STAT3<sup/>.