Aberrant function of pathogenic STAT3 mutant proteins is linked to altered stability of monomers and homodimers.

STAT3 mutations, predominantly in the DNA-binding domain (DBD) and Src-homology 2 domain (SH2D), cause rare cases of immunodeficiency, malignancy, and autoimmunity. The exact mechanisms by which these mutations abrogate or enhance STAT3 function are not completely understood. Here, we examined how loss-of-function (LOF) and gain-of-function (GOF) STAT3 mutations within the DBD and SH2D affect monomer and homodimer protein stability as well as their effect on key STAT3 activation events, including recruitment to phosphotyrosine (pY) sites within peptide hormone receptors, tyrosine phosphorylation at Y705, dimerization, nuclear translocation, and DNA binding. The DBD LOF mutants showed reduced DNA binding when homodimerized, whereas the DBD GOF mutants showed increased DNA binding. DBD LOF and GOF mutants showed minimal changes in other STAT3 functions or in monomer or homodimer protein stability. However, SH2D LOF mutants demonstrated reduced conformational stability as either monomers or homodimers, leading to decreased pY-peptide recruitment, tyrosine phosphorylation, dimerization, nuclear localization, and DNA binding. In contrast, cancer-causing SH2D GOF mutants showed increased STAT3 homodimer stability, which increased their DNA binding. Of note, a small-molecule inhibitor of STAT3 that targets the tyrosine phosphopeptide-binding pocket within the STAT3 SH2D potently inhibited cell proliferation driven by STAT3 SH2D GOF mutants. These findings indicate that the stability of STAT3 protein monomer and homodimer is critical for the pathogenesis of diseases caused by SH2D LOF and GOF mutations and suggest that agents that modulate STAT3 monomer and/or homodimer protein stability may have therapeutic value in diseases caused by these mutations.

Targeting Janus Kinases and Signal Transducer and Activator of Transcription 3 to Treat Inflammation, Fibrosis, and Cancer: Rationale, Progress, and Caution.

Before it was molecularly cloned in 1994, acute-phase response factor or signal transducer and activator of transcription (STAT)3 was the focus of intense research into understanding the mammalian response to injury, particularly the acute-phase response. Although known to be essential for liver production of acute-phase reactant proteins, many of which augment innate immune responses, molecular cloning of acute-phase response factor or STAT3 and the research this enabled helped establish the central function of Janus kinase (JAK) family members in cytokine signaling and identified a multitude of cytokines and peptide hormones, beyond interleukin-6 and its family members, that activate JAKs and STAT3, as well as numerous new programs that their activation drives. Many, like the acute-phase response, are adaptive, whereas several are maladaptive and lead to chronic inflammation and adverse consequences, such as cachexia, fibrosis, organ dysfunction, and cancer. Molecular cloning of STAT3 also enabled the identification of other noncanonical roles for STAT3 in normal physiology, including its contribution to the function of the electron transport chain and oxidative phosphorylation, its basal and stress-related adaptive functions in mitochondria, its function as a scaffold in inflammation-enhanced platelet activation, and its contributions to endothelial permeability and calcium efflux from endoplasmic reticulum. In this review, we will summarize the molecular and cellular biology of JAK/STAT3 signaling and its functions under basal and stress conditions, which are adaptive, and then review maladaptive JAK/STAT3 signaling in animals and humans that lead to disease, as well as recent attempts to modulate them to treat these diseases. In addition, we will discuss how consideration of the noncanonical and stress-related functions of STAT3 cannot be ignored in efforts to target the canonical functions of STAT3, if the goal is to develop drugs that are not only effective but safe. SIGNIFICANCE STATEMENT: Key biological functions of Janus kinase (JAK)/signal transducer and activator of transcription (STAT)3 signaling can be delineated into two broad categories: those essential for normal cell and organ development and those activated in response to stress that are adaptive. Persistent or dysregulated JAK/STAT3 signaling, however, is maladaptive and contributes to many diseases, including diseases characterized by chronic inflammation and fibrosis, and cancer. A comprehensive understanding of JAK/STAT3 signaling in normal development, and in adaptive and maladaptive responses to stress, is essential for the continued development of safe and effective therapies that target this signaling pathway.

Novel STAT3 small-molecule inhibitors identified by structure-based virtual ligand screening incorporating SH2 domain flexibility.

Efforts to develop STAT3 inhibitors have focused on its SH2 domain starting with short phosphotyrosylated peptides based on STAT3 binding motifs, e.g. pY905LPQTV within gp130. Despite binding to STAT3 with high affinity, issues regarding stability, bioavailability, and membrane permeability of these peptides, as well as peptidomimetics such as CJ-887, have limited their further clinical development and led to interest in small-molecule inhibitors. Some small molecule STAT3 inhibitors, identified using structure-based virtual ligand screening (SB-VLS); while having favorable drug-like properties, suffer from weak binding affinities, possibly due to the high flexibility of the target domain. We conducted molecular dynamic (MD) simulations of the SH2 domain in complex with CJ-887, and used an averaged structure from this MD trajectory as an "induced-active site" receptor model for SB-VLS of 110,000 compounds within the SPEC database. Screening was followed by re-docking and re-scoring of the top 30% of hits, selection for hit compounds that directly interact with pY + 0 binding pocket residues R609 and S613, and testing for STAT3 targeting in vitro, which identified two lead hits with good activity and favorable drug-like properties. Unlike most small-molecule STAT3 inhibitors previously identified, which contain negatively-charged moieties that mediate binding to the pY + 0 binding pocket, these compounds are uncharged and likely will serve as better candidates for anti-STAT3 drug development. IMPLICATIONS: SB-VLS, using an averaged structure from molecular dynamics (MD) simulations of STAT3 SH2 domain in a complex with CJ-887, a known peptidomimetic binder, identify two highly potent, neutral, low-molecular weight STAT3-inhibitors with favorable drug-like properties.

Impact of chronic hepatitis C virus infection on the survival of patients with oropharyngeal cancer.

BACKGROUND: Although an association between hepatitis C virus (HCV) infection and oropharyngeal cancers (OPCs) has been reported, to the authors' knowledge the clinical significance of this epidemiological finding remains unknown. Therefore, the authors analyzed the oncologic outcomes of HCV-infected patients with OPCs. METHODS: In this retrospective cohort study, all patients with OPCs who were seen at The University of Texas MD Anderson Cancer Center between January 2004 and December 2015 were reviewed. HCV infection was defined as detectable HCV RNA in the serum. Five-year overall survival and progression-free survival rates were compared between patients infected with HCV and those not infected. RESULTS: A total of 161 patients were examined. The majority of the patients were white (141 patients; 88%) and male (132 patients; 82%) and had TNM stage III or IV disease (147 patients; 91%). The OPC involved the tonsils (83 patients; 52%), base of the tongue (67patients; 42%), or the soft palate (11 patients; 7%). The median follow-up after an OPC diagnosis was 3 years (range, 1-13 years). HCV-infected patients (25 patients) and HCV-uninfected patients (136 patients) were comparable with regard to smoking and alcohol status. In multivariate analysis, HCV was associated with increased cancer-specific mortality (hazard ratio, 2.15; 95% confidence interval, 1.08-6.85 [P = .02]) and risk of OPC progression (hazard ratio, 5.42; 95% confidence interval, 2.64-11.14 [P = .0008]) independent of age and cirrhosis status. Antivirals were administered after the diagnosis of OPC in 8 of the 25 HCV-infected patients (32%). HCV-infected patients who received antivirals were found to have better 5-year overall survival (70% vs 12%; P = .005) and progression-free survival (72% vs 19%; P = .005) compared with patients who did not. CONCLUSIONS: The early detection of HCV is important in patients with OPC because this infection may affect their oncologic outcomes. Cancer 2018;124:960-5. © 2017 American Cancer Society.

Contribution of STAT3 to Inflammatory and Fibrotic Diseases and Prospects for its Targeting for Treatment.

Signal transducer and activator of transcription (STAT) 3 plays a central role in the host response to injury. It is activated rapidly within cells by many cytokines, most notably those in the IL-6 family, leading to pro-proliferative and pro-survival programs that assist the host in regaining homeostasis. With persistent activation, however, chronic inflammation and fibrosis ensue, leading to a number of debilitating diseases. This review summarizes advances in our understanding of the role of STAT3 and its targeting in diseases marked by chronic inflammation and/or fibrosis with a focus on those with the largest unmet medical need.

A novel role of microRNA146b in promoting mammary alveolar progenitor cell maintenance.

In this report, we have shown that miR146b promotes the maintenance of pregnancy-derived mammary luminal alveolar progenitors. MiR146b expression was significantly higher in the mammary glands of pregnant and lactating mice than in virgin mice. Furthermore, miR146b levels were significantly higher in mouse mammary glands exposed to the sex hormones, estrogen and progesterone, compared with those of untreated control animals. Pregnancy-derived primary mouse mammary epithelial cells in which miR146b was knocked down showed a significant reduction in the number of hollow acinar organoid structures formed on three-dimensional Matrigel and in ß-casein expression. This demonstrates that miR146b promotes the maintenance of pregnancy-derived mammary luminal alveolar progenitors. It has been shown that mouse mammary luminal progenitors give rise to hollow organoid structures, whereas solid organoid structures are derived from stem cells. Among several miR146b targets, miR146b knockdown resulted in preferential STAT3ß overexpression. In the primary mouse mammary epithelial cells, overexpression of STAT3ß isoform caused mammary epithelial cell death and a significant reduction in ß-casein mRNA expression. Therefore, we conclude that during pregnancy miR146b is involved in luminal alveolar progenitor cell maintenance, at least partially, by regulating STAT3ß.

A novel lentiviral vector-based approach to generate chimeric antigen receptor T cells targeting Aspergillus fumigatus.

Invasive aspergillosis (IA) is a common and deadly mold infection in immunocompromised patients. As morbidity and mortality of IA are primarily driven by poor immune defense, adjunct immunotherapies, such as chimeric antigen receptor (CAR) T cells, are direly needed. Here, we propose a novel approach to generate Aspergillus fumigatus (AF)-CAR T cells using the single-chain variable fragment domain of monoclonal antibody AF-269-5 and a lentiviral vector system. These cells successfully targeted mature hyphal filaments of representative clinical and reference AF isolates and elicited a potent release of cytotoxic effectors and type 1 T cell cytokines. Furthermore, AF-CAR T cells generated from peripheral blood mononuclear cells of four healthy human donors and expanded with either of three cytokine stimulation regimens (IL-2, IL-2 + IL-21, or IL-7 + IL-15) significantly suppressed mycelial growth of AF-293 after 18 hours of co-culture and synergized with the immunomodulatory antifungal agent caspofungin to control hyphal growth for 36 hours. Moreover, cyclophosphamide-immunosuppressed NSG mice with invasive pulmonary aspergillosis that received two doses of 5 million AF-CAR T cells (6 and 48 hours after AF infection) showed significantly reduced morbidity on day 4 post-infection (P < 0.001) and significantly improved 7-day survival (P = 0.049) compared with mice receiving non-targeting control T cells, even without concomitant antifungal chemotherapy. In conclusion, we developed a novel lentiviral strategy to obtain AF-CAR T cells with high targeting efficacy, yielding significant anti-AF activity in vitro and short-term protection in vivo. Our approach could serve as an important steppingstone for future clinical translation of antifungal CAR T-cell therapy after further refinement and thorough preclinical evaluation.IMPORTANCEInvasive aspergillosis (IA) remains a formidable cause of morbidity and mortality in patients with hematologic malignancies and those undergoing hematopoietic stem cell transplantation. Despite the introduction of several new Aspergillus-active antifungals over the last 30 years, the persisting high mortality of IA in the setting of continuous and profound immunosuppression is a painful reminder of the major unmet need of effective antifungal immune enhancement therapies. The success of chimeric antigen receptor (CAR) T-cell therapy in cancer medicine has inspired researchers to translate this approach to opportunistic infections, including IA. Aiming to refine anti-Aspergillus CAR T-cell therapy and improve its feasibility for future clinical translation, we herein developed and validated a novel antibody-based CAR construct and lentiviral transduction method to accelerate the production of CAR T cells with high targeting efficacy against Aspergillus fumigatus. Our unique approach could provide a promising platform for future clinical translation of CAR T-cell-based antifungal immunotherapy.

Therapeutic Potential of a Small-Molecule STAT3 Inhibitor in a Mouse Model of Colitis.

BACKGROUND AND AIMS: Inflammatory bowel disease (IBD) predisposes to colorectal cancer (CRC). In the current studies, we used the dextran sodium sulfate (DSS) murine model of colitis, which is widely used in preclinical studies, to determine the contribution of STAT3 to IBD. STAT3 has two isoforms: (STAT3 α; which has pro-inflammatory and anti-apoptotic functions, and STAT3ß; which attenuates the effects of STAT3α). In the current study, we determined the contribution of STAT3 to IBD across all tissues by examining DSS-induced colitis in mice that express only STAT3α and in mice treated with TTI-101, a direct small-molecule inhibitor of both isoforms of STAT3. METHODS: We examined mortality, weight loss, rectal bleeding, diarrhea, colon shortening, apoptosis of colonic CD4+ T-cells, and colon infiltration with IL-17-producing cells following 7-day administration of DSS (5%) to transgenic STAT3α knock-in (STAT3ß-deficient; ΔßΔß) mice and wild-type (WT) littermate cage control mice. We also examined the effect of TTI-101 on these endpoints in DSS-induced colitis in WT mice. RESULTS: Each of the clinical manifestations of DSS-induced colitis examined was exacerbated in ΔßΔß transgenic versus cage-control WT mice. Importantly, TTI-101 treatment of DSS-administered WT mice led to complete attenuation of each of the clinical manifestations and also led to increased apoptosis of colonic CD4+ T cells, reduced colon infiltration with IL-17-producing cells, and down-modulation of colon mRNA levels of STAT3-upregulated genes involved in inflammation, apoptosis resistance, and colorectal cancer metastases. CONCLUSIONS: Thus, small-molecule targeting of STAT3 may be of benefit in treating IBD and preventing IBD-associated colorectal cancer.

Blockade of the PD-1/PD-L1 Immune Checkpoint Pathway Improves Infection Outcomes and Enhances Fungicidal Host Defense in a Murine Model of Invasive Pulmonary Mucormycosis.

Anecdotal clinical reports suggested a benefit of adjunct immune checkpoint inhibitors (ICIs) to treat invasive mucormycosis. However, proof-of-concept data in animal models and mechanistic insights into the effects of ICIs on host defense against Mucorales are lacking. Therefore, we studied the effects of PD-1 and PD-L1 inhibitors (4 doses of 250 µg/kg) on outcomes and immunopathology of invasive pulmonary mucormycosis (IPM) in cyclophosphamide- and cortisone acetate-immunosuppressed mice. Rhizopus arrhizus-infected mice receiving either of the ICI treatments had significantly improved survival, less morbidity, and lower fungal burden compared to isotype-treated infected mice. While early improvement of morbidity/mortality was comparable between the ICI treatments, anti-PD-L1 provided more consistent sustained protection through day 7 post-infection than anti-PD-1. Both ICIs enhanced the fungicidal activity of ex-vivo splenocytes and effectively counteracted T-cell exhaustion; however, macrophages of ICI-treated mice showed compensatory upregulation of other checkpoint markers. Anti-PD-1 elicited stronger pulmonary release of proinflammatory cytokines and chemokines than anti-PD-L1, but also induced cytokines associated with potentially unfavorable type 2 T-helper-cell and regulatory T-cell responses. Although no signs of hyperinflammatory toxicity were observed, mice with IPM receiving ICIs, particularly anti-PD-1, had elevated serum levels of IL-6, a cytokine linked to ICI toxicities. Altogether, inhibition of the PD-1/PD-L1 pathway improved clinical outcomes of IPM in immunosuppressed mice, even without concomitant antifungals. PD-L1 inhibition yielded more favorable immune responses and more consistent protection from IPM-associated morbidity and mortality than PD-1 blockade. Future dose-effect studies are needed to define the "sweet spot" between ICI-induced augmentation of antifungal immunity and potential immunotoxicities.

Mesothelin overexpression promotes autocrine IL-6/sIL-6R trans-signaling to stimulate pancreatic cancer cell proliferation.

Mesothelin (MSLN) overexpression in pancreatic cancer (PC) leads to enhanced cell survival/proliferation and tumor progression. After screening for a number of growth factors/cytokines, we found that the MSLN expression correlated closely with interleukin (IL)-6 in human PC specimens and cell lines. Stably overexpressing MSLN in different PC cell lines (MIA-MSLN and Panc1-MSLN) led to higher IL-6 production. Silencing MSLN by small interfering RNA (siRNA) significantly reduced IL-6 levels. Blocking the observed constitutive activation of nuclear factor-kappaB (NF-κB) with IKK inhibitor wedelolactone in MIA-MSLN cells also reduced IL-6. Silencing IL-6 by siRNA reduced cell proliferation, cell cycle progression and induced apoptosis with significant decrease of c-myc/bcl-2. Interestingly, recombinant IL-6-induced proliferation of MIA-MSLN cells but not MIA-V cells. Although messenger RNA/protein levels of IL-6R did not vary, soluble IL-6R (sIL-6R) was significantly elevated in MIA-MSLN and was reduced by treatment with the TACE/ADAM17 inhibitor TAPI-1, indicating intramembrane IL-6R cleavage and IL-6 trans-signaling may be operative in MIA-MSLN cells. Blocking the IL-6/sIL-6R axis using sIL-6R antibody abrogated basal proliferation/survival as well as recombinant human IL-6-induced cell proliferation. Our data suggest that MSLN-activated NF-κB induces elevated IL-6 expression, which acts as a growth factor to support PC cell survival/proliferation through a novel auto/paracrine IL-6/sIL-6R trans-signaling. In addition, using a panel of PC cells with varying MSLN/IL-6 expressions, we showed that MSLN/IL-6 axis is a major survival axis in PC supporting tumor cell growth under anchorage-dependent and independent conditions. The close correlation between MSLN and IL-6 provides a new rationale for combination therapy for effective control of MSLN-overexpressing PCs.

Mesothelin confers pancreatic cancer cell resistance to TNF-α-induced apoptosis through Akt/PI3K/NF-κB activation and IL-6/Mcl-1 overexpression.

BACKGROUND: Previous studies showed that mesothelin (MSLN) plays important roles in survival of pancreatic cancer (PC) cells under anchorage dependent/independent conditions as well as resistance to chemotherapy. The recent success of intratumorally-injected adeno-encoded, chemo/radiation-inducible-promoter driven hTNF-α, (TNFerade) + gemcitabine in pre-clinical models of PC have renewed interest in use of TNF-α as a therapeutic component. To help find additional factors which might affect the therapy, we examined the resistance of MSLN-overexpressing pancreatic cancer cell lines to TNF-α-induced growth inhibition/apoptosis. METHODS: Stable MSLN overexpressing MIA PaCa-2 cells (MIA-MSLN), stable MSLN-silenced AsPC-1 cells (AsPC-shMSLN) and other pancreatic cells (MIA-PaCa2, Panc 28, Capan-1, BxPC3, PL 45, Hs 766T, AsPC-1, Capan-2, Panc 48) were used. NF-κB activation was examined by western blots and luciferase reporter assay. TNF-α induced growth inhibition/apoptosis was measured by MTT, TUNEL assay and caspase activation. IL-6 was measured using luminex based assay. RESULTS: Compared to low endogenous MSLN-expressing MIA PaCa-2 and Panc 28 cells, high endogenous MSLN-expressing Capan-1, BxPC3, PL 45, Hs 766T, AsPC-1, Capan-2, Panc 48 cells were resistant to TNF-α induced growth inhibition. Stable MSLN overexpressing MIA-PaCa2 cells (MIA-MSLN) were resistant to TNF-α-induced apoptosis while stable MSLN-silenced AsPC1 cells (AsPC-shMSLN) were sensitive. Interestingly, TNF-α-treated MIA-MSLN cells showed increased cell cycle progression and cyclin A induction, both of which were reversed by caspase inhibition. We further found that MIA-MSLN cells showed increased expression of anti-apoptotic Bcl-XL and Mcl-1; deactivated (p-Ser75) BAD, and activated (p-Ser70) Bcl-2. Constitutively activated NF-κB and Akt were evident in MIA-MSLN cells that could be suppressed by MSLN siRNA with a resultant increase in sensitivity of TNF-α induced apoptosis. Blocking NF-κB using IKK inhibitor wedelolactone also increased sensitivity to TNF-α-mediated cytotoxicity with concomitant decrease in Mcl-1. Blocking Akt using PI3K inhibitor also had a likewise effect presumably affecting cell cycle. MIA-MSLN cells produced increased IL-6 and were increased furthermore by TNF-α treatment. SiRNA-silencing of IL-6 increased TNF-α sensitivity of MIA-MSLN cells. CONCLUSIONS: Our study delineates a MSLN-Akt-NF-κB-IL-6-Mcl-1 survival axis that may be operative in PC cells, and might help cancer cells' survival in the highly inflammatory milieu evident in PC. Further, for the success of TNFerade + gemcitabine to be successful, we feel the simultaneous inhibition of components of this axis is also essential.

Soluble CD40 ligand induces endothelial dysfunction in human and porcine coronary artery endothelial cells.

The purpose of this study was to determine the effects and mechanisms of sCD40L on endothelial dysfunction in both human coronary artery endothelial cells (HCAECs) and porcine coronary artery rings. HCAECs treated with sCD40L showed significant reductions of endothelial nitric oxide synthase (eNOS) mRNA and protein levels, eNOS mRNA stability, eNOS enzyme activity, and cellular NO levels, whereas superoxide anion (O(2)(-)) production was significantly increased. sCD40L enhanced eNOS mRNA 3'UTR binding to cytoplasmic molecules and induced a unique expression pattern of 95 microRNAs. sCD40L significantly decreased mitochondrial membrane potential, and catalase and SOD activities, whereas it increased NADPH oxidase (NOX) activity. sCD40L increased phosphorylation of MAPKs p38 and ERK1/2 as well as IkappaBalpha and enhanced NF-kappaB nuclear translocation. In porcine coronary arteries, sCD40L significantly decreased endothelium-dependent vasorelaxation and eNOS mRNA levels, whereas it increased O(2)(-) levels. Antioxidant seleno-l-methionine; chemical inhibitors of p38, ERK1/2, and mitochondrial complex II; as well as dominant negative mutant forms of IkappaBalpha and NOX4 effectively blocked sCD40L-induced eNOS down-regulation in HCAECs. Thus, sCD40L reduces eNOS levels, whereas it increases oxidative stress through the unique molecular mechanisms involving eNOS mRNA stability, 3'UTR-binding molecules, microRNAs, mitochondrial function, ROS-related enzymes, p38, ERK1/2, and NF-kappaB signal pathways in endothelial cells.

Aberrant expression of zinc transporter ZIP4 (SLC39A4) significantly contributes to human pancreatic cancer pathogenesis and progression.

Zinc is an essential trace element and catalytic/structural component used by many metalloenzymes and transcription factors. Recent studies indicate a possible correlation of zinc levels with the cancer risk; however, the exact role of zinc and zinc transporters in cancer progression is unknown. We have observed that a zinc transporter, ZIP4 (SLC39A4), was substantially overexpressed in 16 of 17 (94%) clinical pancreatic adenocarcinoma specimens compared with the surrounding normal tissues, and ZIP4 mRNA expression was significantly higher in human pancreatic cancer cells than human pancreatic ductal epithelium (HPDE) cells. This indicates that aberrant ZIP4 up-regulation may contribute to the pancreatic cancer pathogenesis and progression. We studied the effects of ZIP4 overexpression in pancreatic cancer cell proliferation in vitro and pancreatic cancer progression in vivo. We found that forced expression of ZIP4 increased intracellular zinc levels, increased cell proliferation by 2-fold in vitro, and significantly increased tumor volume by 13-fold in the nude mice model with s.c. xenograft compared with the control cells. In the orthotopic nude mice model, overexpression of ZIP4 not only increased the primary tumor weight (7.2-fold), it also increased the peritoneal dissemination and ascites incidence. Moreover, increased cell proliferation and higher zinc content were also observed in the tumor tissues that overexpressed ZIP4. These data reveal an important outcome of aberrant ZIP4 expression in contributing to pancreatic cancer pathogenesis and progression. It may suggest a therapeutic strategy whereby ZIP4 is targeted to control pancreatic cancer growth.

Bempegaldesleukin selectively depletes intratumoral Tregs and potentiates T cell-mediated cancer therapy.

High dose interleukin-2 (IL-2) is active against metastatic melanoma and renal cell carcinoma, but treatment-associated toxicity and expansion of suppressive regulatory T cells (Tregs) limit its use in patients with cancer. Bempegaldesleukin (NKTR-214) is an engineered IL-2 cytokine prodrug that provides sustained activation of the IL-2 pathway with a bias to the IL-2 receptor CD122 (IL-2Rß). Here we assess the therapeutic impact and mechanism of action of NKTR-214 in combination with anti-PD-1 and anti-CTLA-4 checkpoint blockade therapy or peptide-based vaccination in mice. NKTR-214 shows superior anti-tumor activity over native IL-2 and systemically expands anti-tumor CD8+ T cells while inducing Treg depletion in tumor tissue but not in the periphery. Similar trends of intratumoral Treg dynamics are observed in a small cohort of patients treated with NKTR-214. Mechanistically, intratumoral Treg depletion is mediated by CD8+ Teff-associated cytokines IFN-γ and TNF-α. These findings demonstrate that NKTR-214 synergizes with T cell-mediated anti-cancer therapies.

Mesothelin-induced pancreatic cancer cell proliferation involves alteration of cyclin E via activation of signal transducer and activator of transcription protein 3.

Mesothelin (MSLN) is a cell surface glycoprotein that is overexpressed in human pancreatic cancer. Although its value as a tumor marker for diagnosis and prognosis and as a preferred target of immunointervention has been evaluated, there is little information on the growth advantage of MSLN on tumor cells. In this study, we examined the effect of MSLN on pancreatic cancer cell proliferation, cell cycle progression, expression of cell cycle regulatory proteins, and signal transduction pathways in two pancreatic cancer cell lines, MIA-MSLN (overexpressing MSLN in MIA PaCa-2 cells) and BxPC-siMSLN (silencing MSLN in BxPC-3 cells). Increased cyclin E and cyclin-dependent kinase 2 expression found in MIA-MSLN cells correlated with significantly increased cell proliferation and faster cell cycle progression compared with control cells. BxPC-siMSLN cells showed slower proliferation and slower entry into the S phase than control cells. Signal transducer and activator of transcription protein 3 (Stat3) was constitutively activated in MIA-MSLN cells, but not in control cells. Inhibition of Stat3 activation in MIA-MSLN cells by the Janus-activated kinase-selective inhibitor tyrphostin AG490 was followed by a marked decrease in proliferation of the cells. Small interfering RNA against Stat3 significantly reduced the MIA-MSLN cell cycle progression with a concomitant decrease in cyclin E expression. Our data indicate that overexpression of MSLN in pancreatic cancer cells leads to constitutive activation of the transcription factor Stat3, which results in enhanced expression of cyclin E and cyclin E/cyclin-dependent kinase 2 complex formation as well as increased G(1)-S transition.

C-reactive protein inhibits cholesterol efflux from human macrophage-derived foam cells.

OBJECTIVE: The objective of this study was to determine the effects and potential mechanisms of C-reactive protein (CRP) on cholesterol efflux from human macrophage foam cells, which may play a critical role in atherogenesis. METHODS AND RESULTS: Human THP-1 monocytes and peripheral blood mononuclear cells (PBMCs) were preincubated with acetylated LDL and [3H]-cholesterol to form foam cells, which were then treated with apolipoprotein A-I (apoA-I) or HDL for cholesterol efflux assay. Clinically relevant concentrations of CRP significantly reduced cholesterol efflux from THP-1 and PBMCs to apoA-I or HDL. CRP significantly decreased the expression of ATP-binding membrane cassette transporter A-1 (ABCA1) and ABCG1, whereas it increased superoxide anion production. Furthermore, CRP substantially activated ERK1/2 in THP-1-derived foam-like cells. Reducing superoxide anion by antioxidant seleno-L-methionine or SOD mimetic (MnTBAP) effectively abolished the CRP-induced decrease in cholesterol efflux and the expression of ABCA1 and ABCG1. Inhibiting ERK1/2 activation by its specific inhibitor PD98059 or by a dominant negative mutant of ERK2 could also block CRPs action on THP-1 cells. CONCLUSIONS: CRP inhibits cholesterol efflux from human foam cells derived from THP-1 and PBMCs in vitro though oxidative stress, ERK1/2 activation, and downregulation of intracellular cholesterol transport molecules ABCA1 and ABCG1.

Elevated interleukin-6 and G-CSF in human pancreatic cancer cell conditioned medium suppress dendritic cell differentiation and activation.

Although dendritic cell (DC) function is impaired in pancreatic cancer patients, the underlying mechanisms are unknown. This study analyzed the soluble factors released by pancreatic cancer cells responsible for inhibiting DC differentiation and activation. Medium conditioned by a highly metastatic human pancreatic cancer cell line BxPC-3 [BxPC-3 conditioned medium (BxCM)] was mainly used for the study. Both CD34+ hematopoietic progenitor cell-derived and CD14+ monocyte-derived immature DCs and mature DCs (mDCs) were inhibited by BxCM. Allostimulation of CD4+ and CD8+ T cells by BxCM-treated mDCs was inefficient and resulted in production of lower levels of Th1 and Th2 cytokines. Antigen-specific T-cell activation capability was also reduced in BxCM-treated mDCs. Addition of exogenous interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF), which were present in high amounts in BxCM, mimicked the inhibitory effect of BxCM on DC differentiation and maturation. IL-6 was able to suppress DC differentiation and G-CSF mainly acted on the suppressing allostimulatory capacity of DCs. In addition, pancreatic cancer patient sera were able to inhibit DC differentiation of CD14+ monocytes obtained from healthy donors. Depleting IL-6 or G-CSF from BxCM could reverse the DC-inhibitory properties of BxCM. Furthermore, BxCM, IL-6, or G-CSF led to the activation of signal transducer and activator of transcription 3 (STAT3) in CD14+ monocytes to different degrees. Blocking BxCM-induced STAT3 activation also reversed the inhibitory effect of BxCM on DC differentiation. Therefore, IL-6 and G-CSF in BxCM represent two main factors responsible for suppression of DC differentiation, maturation, and antigen presentation, and this suppression of DC functions may be due to the aberrant activation of STAT3 by BxCM.

Mesothelin is a malignant factor and therapeutic vaccine target for pancreatic cancer.

Given the high fatality rate of pancreatic cancer, an effective treatment for this devastating disease is urgently needed. We have shown that mesothelin expression was higher in human pancreatic cancer cells than in human pancreatic duct epithelial cells, and mesothelin mRNA was substantially overexpressed in 18 of 21 (86%) clinical pancreatic adenocarcinoma specimens when compared with the surrounding normal tissues. However, the biological functions of mesothelin in tumor progression are not clearly understood. Here we studied the effects of mesothelin overexpression in pancreatic cancer cell proliferation and migration in vitro and pancreatic cancer progression in vivo. We found that forced expression of mesothelin significantly increased tumor cell proliferation and migration by 90% and 300%, respectively, and increased tumor volume by 4-fold in the nude mice xenograft model when compared with the vector control cell line. Silencing of mesothelin inhibited cell proliferation and migration in pancreatic cancer cells and ablated tumor progression in vivo. Vaccination with chimeric virus-like particles that contain human mesothelin substantially inhibited tumor progression in C57BL/6J mice. The increases in mesothelin-specific antibodies and CTL activity and the decrease in regulatory T cells correlated with reduced tumor progression and prolonged survival. This study revealed novel functions of mesothelin and suggested a new therapeutic vaccine strategy whereby mesothelin is targeted to control pancreatic cancer progression.

Correction: Small-molecule inhibition of STAT3 in radioresistant head and neck squamous cell carcinoma.

[This corrects the article DOI: 10.18632/oncotarget.8368.].

IL-6 stimulates Th2 type cytokine secretion and upregulates VEGF and NRP-1 expression in pancreatic cancer cells.

BACKGROUND: Although upregulation of interleukin-6 (IL-6) is associated with many solid tumors, its role in pancreatic cancer has not been well elucidated. In this study, we examined the expression of IL-6 in pancreatic cancer cells, and determined the effect of exogenous IL-6 on cytokine secretion, gene expression and signaling in human pancreatic cancer cells. METHODS: The mRNA levels of IL-6, VEGF165, neuropilin-1 (NRP-1) and neuropilin-2 (NRP-2) were determined by real-time RT PCR. Phosphorylation of ERK2 in pancreatic cancer cells was determined by using Bio-Plex phosphoprotein assay. The expression of IL-6 and other cytokines in human pancreatic cancer cell lines was determined with Bio-Plex cytokine assay. RESULTS: Pancreatic cancer cell lines expressed higher levels of IL-6 than normal human pancreatic ductal epithelium (HPDE) cells. Exogenous IL-6 increased the secretion of multiple Th2 type of cytokines in Panc-1, MIA PaCa-2 and BxPC-3 cells. IL-6 also upregulated the expression of VEGF165, and NRP-1, and both IL-6 and VEGF165 were inducible by hypoxia. In addition, IL-6 activated ERK2 signaling pathways in pancreatic cancer cells. CONCLUSIONS: IL-6 may be involved in promoting human pancreatic cancer develop ment by furnishing Th2 type of cytokine environment and upregulating cell proliferation and angiogenesis related genes. Targeting IL-6 might be an effective treatment for pancreatic cancer.