Long-chain polyunsaturated lipids associated with responsiveness to anti-PD-1 therapy are colocalized with immune infiltrates in the tumor microenvironment.

The programmed cell death protein-1 (PD-1) is highly expressed on the surface of antigen-specific exhausted T cells and, upon interaction with its ligand PD-L1, can result in inhibition of the immune response. Anti-PD-1 treatment has been shown to extend survival and result in durable responses in several cancers, yet only a subset of patients benefit from this therapy. Despite the implication of metabolic alteration following cancer immunotherapy, mechanistic associations between antitumor responses and metabolic changes remain unclear. Here, we used desorption electrospray ionization mass spectrometry imaging to examine the lipid profiles of tumor tissue from three syngeneic murine models with varying treatment sensitivity at the baseline and at three time points post-anti-PD-1 therapy. These imaging experiments revealed specific alterations in the lipid profiles associated with the degree of response to treatment and allowed us to identify a significant increase of long-chain polyunsaturated lipids within responsive tumors following anti-PD-1 therapy. Immunofluorescence imaging of tumor tissues also demonstrated that the altered lipid profile associated with treatment response is localized to dense regions of tumor immune infiltrates. Overall, these results indicate that effective anti-PD-1 therapy modulates lipid metabolism in tumor immune infiltrates, and we thereby propose that further investigation of the related immune-metabolic pathways may be useful for better understanding success and failure of anti-PD-1 therapy.

Oncolytic virus V937 in combination with PD-1 blockade therapy to target immunologically quiescent liver and colorectal cancer.

V937 is an investigational, genetically unmodified Kuykendall strain of coxsackievirus A21, which has been evaluated in the clinic for advanced solid tumor malignancies. V937 specifically infects and lyses tumor cells that overexpress intercellular adhesion molecule-1 (ICAM-1). Intratumoral V937 as a monotherapy and in combination with anti-PD-1 antibody pembrolizumab has shown clinical response in patients with metastatic melanoma, which overexpresses ICAM-1. Here, we investigate in preclinical studies the potential bidirectional cross-talk between hepatocellular carcinomas (HCC) or colorectal carcinomas (CRC) and immune cells when treated with V937 alone or in combination with pembrolizumab. We show that while V937 treatment of tumor cell lines or organoids or peripheral blood mononuclear cells (PBMCs) alone induced a minimal immunological response, V937 treatment of non-contact co-cultures of tumor cell lines or CRC organoids with PBMCs led to robust production of proinflammatory cytokines and immune cell activation. In addition, both recombinant interferon-gamma and pembrolizumab increased ICAM-1 on tumor cell lines or organoids and, in turn, amplified V937-mediated oncolysis and immunogenicity. These findings provide critical mechanistic insights on the cross-talk between V937-mediated oncolysis and immune responses, demonstrating the therapeutic potential of V937 in combination with PD-1 blockade to treat immunologically quiescent cancers.

Cyclic GMP/protein kinase G type-Iα (PKG-Iα) signaling pathway promotes CREB phosphorylation and maintains higher c-IAP1, livin, survivin, and Mcl-1 expression and the inhibition of PKG-Iα kinase activity synergizes with cisplatin in non-small cell lung cancer cells.

Previously, our laboratory showed that nitric oxide (NO)/cyclic GMP (cGMP)/protein kinase G type-Iα (PKG-Iα) signaling pathway plays an important role in preventing spontaneous apoptosis and promoting cell proliferation in both normal cells (bone marrow stromal cells and vascular smooth muscle cells) and certain cancer cells (ovarian cancer cells). In the present study, we investigated the novel role of the cGMP/PKG-Iα pathway in preventing spontaneous apoptosis, promoting colony formation and regulating phosphorylation of cAMP response element binding (CREB) protein and protein expression of inhibitor of apoptosis proteins (IAPs) and anti-apoptotic Bcl-2-related proteins in NCI-H460 and A549 non-small cell lung cancer (NSCLC) cells. 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), which blocks endogenous NO-induced activation of cGMP/PKG-Iα, induced apoptosis and decreased colony formation. ODQ also decreased CREB ser133 phosphorylation and protein expression of c-IAP1, livin, and survivin. DT-2 (inhibitor of PKG-Iα kinase activity) increased apoptosis by twofold and decreased CREB ser133 phosphorylation and c-IAP1, livin, and survivin expression. Gene knockdown of PKG-Iα expression using small-interfering RNA increased apoptosis and decreased CREB ser133 phosphorylation, and c-IAP1, livin, survivin, and Mcl-1 expression. Inhibition of PKG-Iα kinase activity with DT-2 dramatically enhanced pro-apoptotic effects of the chemotherapeutic agent cisplatin. Combined treatment of DT-2 and cisplatin increased apoptosis compared with cisplatin or DT-2 alone, showing a synergistic effect. The data suggest that the PKG-Iα kinase activity is necessary for maintaining higher levels of CREB phosphorylation at ser133 and protein expression of c-IAP1, livin, survivin, and Mcl-1, preventing spontaneous apoptosis and promoting colony formation in NSCLC cells, which may limit the effectiveness of chemotherapeutic agents like cisplatin.

Essential roles of the nitric oxide (no)/cGMP/protein kinase G type-Iα (PKG-Iα) signaling pathway and the atrial natriuretic peptide (ANP)/cGMP/PKG-Iα autocrine loop in promoting proliferation and cell survival of OP9 bone marrow stromal cells.

Inappropriate signaling conditions within bone marrow stromal cells (BMSCs) can lead to loss of BMSC survival, contributing to the loss of a proper micro-environmental niche for hematopoietic stem cells (HSCs), ultimately causing bone marrow failure. In the present study, we investigated the novel role of endogenous atrial natriuretic peptide (ANP) and the nitric oxide (NO)/cGMP/protein kinase G type-Iα (PKG-Iα) signaling pathway in regulating BMSC survival and proliferation, using the OP9 BMSC cell line commonly used for facilitating the differentiation of HSCs. Using an ANP-receptor blocker, endogenously produced ANP was found to promote cell proliferation and prevent apoptosis. NO donor SNAP (S-nitroso-N-acetylpenicillamine) at low concentrations (10 and 50 µM), which would moderately stimulate PKG activity, protected these BMSCs against spontaneous apoptosis. YC-1, a soluble guanylyl cyclase (sGC) activator, decreased the levels of apoptosis, similar to the cytoprotective effects of low-level NO. ODQ (1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one), which blocks endogenous NO-induced activation of sGC and thus lowers endogenous cGMP/PKG activity, significantly elevated apoptotic levels by 2.5- and three-fold. Pre-incubation with 8-Bromo-cGMP or ANP, which bypass the ODQ block, almost completely prevented the ODQ-induced apoptosis. A highly-specific PKG inhibitor, DT-3, at 20, and 30 µM, caused 1.5- and two-fold increases in apoptosis, respectively. ODQ and DT-3 also decreased BMSCs proliferation and colony formation. Small Interfering RNA gene knockdown of PKG-Iα increased apoptosis and decreased proliferation in BMSCs. The data suggest that basal NO/cGMP/PKG-Iα activity and autocrine ANP/cGMP/PKG-Iα are necessary for preserving OP9 cell survival and promoting cell proliferation and migration.

Adamts1 responds to systemic cues and gates adipogenesis.

Intuitively, excess caloric intake causes adipose tissue expansion. However, the signals and mechanisms by which this systemic trigger directs a local response in the adipose tissue are incompletely understood. Both hypertrophy of existing adipocytes and the generation of new adipocytes through differentiation of adipocyte precursor cells (APCs), contribute to adipose tissue expansion in response to changes in the diet. Ex vivo studies of this process elucidated an elegant network of mostly transcription factors that drive APCs through the differentiation (adipogenesis) process. Here we discuss our study that identified an Adamts1 signal as a glucocorticoid and diet responsive regulator of an extracellular relay system that modulates the initiation of this intracellular adipogenesis program in APCs. Furthermore, we describe how we applied sensitive tools that enable monitoring of endogenous APC activity to study the early response to high-fat diet in vivo.

Pancreatic-ß-cell survival and proliferation are promoted by protein kinase G type Iα and downstream regulation of AKT/FOXO1.

Early studies showed nitric oxide as a pro-inflammatory-cytokine-induced toxin involved in pancreatic ß-cell destruction during pathogenesis of type-1 diabetes. However, nitric oxide has both cytotoxic and cytoprotective effects on mammalian cells, depending on concentration and micro-environmental surroundings. Our studies have shown that low/physiological-level nitric oxide selectively activates protein kinase G type Iα isoform, promoting cytoprotective/pro-cell-survival effects in many cell types. In bone marrow-derived stromal/mesenchymal stem cells, protein kinase G type Iα mediates autocrine effects of nitric oxide and atrial natriuretic peptide, promoting DNA-synthesis/proliferation and cell survival. In this study, endothelial nitric oxide synthase/neuronal nitric oxide synthase inhibitor L-NIO (L-N(5)-(1-iminoethyl)ornithine), soluble guanylyl cyclase inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3,-a] quinoxalin-1-one), atrial natriuretic peptide-receptor inhibitor A71915 and protein kinase G type Iα kinase activity inhibitor DT-2 all increased apoptosis and decreased insulin secretion in RINm5F pancreatic ß-cells, suggesting autocrine regulatory role for endogenous nitric oxide- and atrial natriuretic peptide-induced activation of protein kinase G type Iα. In four pancreatic ß-cell lines, Beta-TC-6, RINm5F, INS-1 and 1.1B4, protein kinase G type Iα small-interfering RNA decreased phospho-serine-239-VASP (indicator of endogenous protein kinase G type Iα kinase activity), increased apoptosis and decreased proliferation. In protein kinase G type Iα-knockdown ß-cell lines, expressions of phospho-protein kinase B (PKB/AKT) (AKT), phospho-Forkhead box protein O1 (FOXO1) (transcriptional repressor of pancreas duodenum homobox-1) and pancreas duodenum homobox-1 were decreased, suppressing proliferation and survival in pancreatic ß-cells. The data suggest autocrine nitric oxide/atrial natriuretic peptide-induced activation of protein kinase G type Iα/p-AKT/p-FOXO1 promotes survival and proliferation in pancreatic ß-cells, providing therapeutic implications for development of new therapeutic agents for diabetes.

A glucocorticoid- and diet-responsive pathway toggles adipocyte precursor cell activity in vivo.

Obesity is driven by excess caloric intake, which leads to the expansion of adipose tissue by hypertrophy and hyperplasia. Adipose tissue hyperplasia results from the differentiation of adipocyte precursor cells (APCs) that reside in adipose depots. Investigation into this process has elucidated a network of mostly transcription factors that drive APCs through the differentiation process. Using in vitro and in vivo approaches, our study revealed a signaling pathway that inhibited the initiation of the adipocyte differentiation program. Mouse adipocytes secreted the extracellular protease ADAMTS1, which triggered the production of the cytokine pleiotrophin (PTN) through the Wnt/ß-catenin pathway, and promoted proliferation rather than differentiation of APCs. Glucocorticoid exposure in vitro or in vivo reduced ADAMTS1 abundance in adipocytes. In addition, mice fed a high-fat diet showed decreased Adamts1 expression in the visceral perigonadal adipose depot, which expanded by adipogenesis in response to the diet, and increased Adamts1 expression in the subcutaneous inguinal adipose depot, which did not induce adipogenesis. Similar to what occurred in mouse subcutaneous adipose tissue, diet-induced weight gain increased the expression of ADAMTS1, PTN, and certain Wnt target genes in the subcutaneous adipose depot of human volunteers, suggesting the relevance of this pathway to physiological adipose tissue homeostasis and the pathogenesis of obesity. Thus, this pathway functions as a toggle on APCs, regulating a decision between differentiation and proliferation and coordinating the response of adipose tissue to systemic cues.

Resveratrol at anti-angiogenesis/anticancer concentrations suppresses protein kinase G signaling and decreases IAPs expression in HUVECs.

BACKGROUND: Resveratrol increases nitric oxide (NO) production via increased expression and activation of endothelial-form-NO-synthase (eNOS) in endothelial cells. However, the role of downstream cGMP/protein kinase G (PKG) signaling, a pathway activated by NO/eNOS, in pro- and anti-angiogenic effects of resveratrol is still unclear. MATERIALS AND METHODS: Endogenous NO/cGMP/PKG pathway and downstream cell-survival proteins (Inhibitor of Apoptosis Proteins, IAPs) were studied in relation to pro- and anti-angiogenic effects of resveratrol in human umbilical vein endothelial cells (HUVECs). RESULTS: Resveratrol at higher/anti-angiogenic concentrations inhibits HUVEC tube formation and cell migration/invasion (indices of angiogenesis). Resveratrol at lower concentrations stimulates proliferation and protects HUVECs against spontaneous apoptosis. 8-Br-cGMP, a direct activator of PKG, protects against pro-apoptotic effects of high-concentration resveratrol. Western blot analyses showed that anti-angiogenic concentrations of resveratrol suppress endogenous PKG kinase activity and decrease the expression of four cell-survival proteins, c-IAP1, c-IAP2, livin and XIAP. CONCLUSION: Resveratrol-induced anti-angiogenesis/pro-apoptosis induced suppression of PKG signaling and decreased expression of the cell-survival proteins c-IAP1, c-IAP2, livin and XIAP.

Protein kinase G activity prevents pathological-level nitric oxide-induced apoptosis and promotes DNA synthesis/cell proliferation in vascular smooth muscle cells.

BACKGROUND: Protein kinase G (PKG), a recognized downstream mediator of nitric oxide, is a key regulator of cardiovascular physiology and pathology. High-level stimulation of cyclic guanosine monophosphate/PKG signaling using high concentrations of nitric oxide donors, mimicking pathological conditions, induces apoptosis in vascular smooth muscle cells. In contrast, we have found that PKG at basal and moderately elevated activity prevents both spontaneous and toxin-induced apoptosis in many other cells. We hypothesized that PKG's apoptosis-regulatory role in vascular smooth muscle cells depends on PKG activation levels [low/basal-level activation prevents apoptosis, whereas high-level activation (hyperactivation) causes apoptosis]. Furthermore, we hypothesized that, although PKG hyperactivation inhibits vascular smooth muscle cell proliferation (potentially causing anti-atherogenic effects), basal PKG activity may promote vascular smooth muscle cell proliferation/atherogenesis. METHODS: Involvement of PKG in apoptosis and proliferation was determined in unpassaged vascular smooth muscle cells from mouse aorta. Western blot analysis was used to determine PKG expression, and activators/inhibitors of PKG activity were used to determine involvement in apoptosis (Hoechst staining and DNA-fragmentation ELISAs) and proliferation (cell count, MTT assay, and BrdU incorporation). RESULTS: Both PKG-Iα and PKG-Iß isoforms were expressed. Lower-level stimulation of PKG using the nitric oxide donor S-nitroso-acetylpenacillamine (10, 50 µM) significantly (P<.05) lowered spontaneous apoptosis, whereas S-nitroso-acetylpenacillamine at higher concentrations (500, 1000 µM) elevated apoptosis. Twenty-four-hour pretreatment with atrial natriuretic peptide, a PKG activator, completely prevented high-concentration, nitric oxide-induced apoptosis. Inhibition of basal PKG activity using highly selective PKG inhibitors, DT-2 and DT-3, significantly (P<.001) increased apoptosis and inhibited DNA synthesis/proliferation. CONCLUSION: The data suggest that basal/moderately elevated PKG activity protects against high/pathological-level nitric oxide-induced apoptosis and promotes DNA synthesis/proliferation in vascular smooth muscle cells, potentially important for atherogenesis.

Protein kinase G type Ialpha activity in human ovarian cancer cells significantly contributes to enhanced Src activation and DNA synthesis/cell proliferation.

Previously, we showed that basal activity of nitric oxide (NO)/cyclic GMP (cGMP)/protein kinase G (PKG) signaling pathway protects against spontaneous apoptosis and confers resistance to cisplatin-induced apoptosis in human ovarian cancer cells. The present study determines whether basal PKG kinase activity regulates Src family kinase (SFK) activity and proliferation in these cells. PKG-Ialpha was identified as predominant isoform in both OV2008 (cisplatin-sensitive, wild-type p53) and A2780cp (cisplatin-resistant, mutated p53) ovarian cancer cells. In both cell lines, ODQ (inhibitor of endogenous NO-induced cGMP biosynthesis), DT-2 (highly specific inhibitor of PKG-Ialpha kinase activity), and PKG-Ialpha knockdown (using small interfering RNA) caused concentration-dependent inhibition of DNA synthesis (assessed by bromodeoxyuridine incorporation), indicating an important role of basal cGMP/PKG-Ialpha kinase activity in promoting cell proliferation. DNA synthesis in OV2008 cells was dependent on SFK activity, determined using highly selective SFK inhibitor, 4-(4'-phenoxyanilino)-6,7-dimethoxyquinazoline (SKI-1). Studies using DT-2 and PKG-Ialpha small interfering RNA revealed that SFK activity was dependent on PKG-Ialpha kinase activity. Furthermore, SFK activity contributed to endogenous tyrosine phosphorylation of PKG-Ialpha in OV2008 and A2780cp cells. In vitro coincubation of recombinant human c-Src and PKG-Ialpha resulted in c-Src-mediated tyrosine phosphorylation of PKG-Ialpha and enhanced c-Src autophosphorylation/activation, suggesting that human c-Src directly tyrosine phosphorylates PKG-Ialpha and the c-Src/PKG-Ialpha interaction enhances Src kinase activity. Epidermal growth factor-induced stimulation of SFK activity in OV2008 cells increased PKG-Ialpha kinase activity (indicated by Ser(239) phosphorylation of the PKG substrate vasodilator-stimulated phosphoprotein), which was blocked by both SKI-1 and SU6656. The data suggest an important role of Src/PKG-Ialpha interaction in promoting DNA synthesis/cell proliferation in human ovarian cancer cells. The NO/cGMP/PKG-Ialpha signaling pathway may provide a novel therapeutic target for disrupting ovarian cancer cell proliferation.