Inhibition of MNKs promotes macrophage immunosuppressive phenotype to limit CD8+ T cell antitumor immunity.

To elicit effective antitumor responses, CD8+ T cells need to infiltrate tumors and sustain their effector function within the immunosuppressive tumor microenvironment (TME). Here, we evaluate the role of MNK activity in regulating CD8+ T cell infiltration and antitumor activity in pancreatic and thyroid tumors. We first show that human pancreatic and thyroid tumors with increased MNK activity are associated with decreased infiltration by CD8+ T cells. We then show that, while MNK inhibitors increase CD8+ T cells in these tumors, they induce a T cell exhaustion phenotype in the tumor microenvironment. Mechanistically, we show that the exhaustion phenotype is not caused by upregulation of programmed cell death ligand 1 (PD-L1) but is caused by tumor-associated macrophages (TAMs) becoming more immunosuppressive following MNK inhibitor treatment. Reversal of CD8+ T cell exhaustion by an anti-PD-1 antibody or TAM depletion synergizes with MNK inhibitors to control tumor growth and prolong animal survival. Importantly, we show in ex vivo human pancreatic tumor slice cultures that MNK inhibitors increase the expression of markers associated with immunosuppressive TAMs. Together, these findings demonstrate a role of MNKs modulating a protumoral phenotype in macrophages and identify combination regimens involving MNK inhibitors to enhance antitumor immune responses.

P2Y purinergic signaling in prostate cancer: Emerging insights into pathophysiology and therapy.

Despite recent advances in the treatment landscape for prostate cancer, many challenges still remain. A more profound understanding of prostate cancer pathogenesis and the underlying mechanisms is critical to developing novel therapeutics strategies. Extracellular nucleotides play a central role in the growth and progression of a variety of cancer types - almost all tumor cells and immune cells express purinergic membrane receptors for extracellular nucleotides (ATP, ADP, UTP, UDP, UDP-sugar) and their metabolic nucleoside products (e.g., adenosine). Herein we review the pathological and immunomodulatory roles of P2Y purinergic nucleotide receptors in prostate cancer and their potential as therapeutic targets to address some of the clinical limitations in prostate cancer treatment.

Gα13 loss in Kras/Tp53 mouse model of pancreatic tumorigenesis promotes tumors susceptible to rapamycin.

Gα13 transduces signals from G-protein-coupled receptors. While Gα13 functions as a tumor suppressor in lymphomas, it is not known whether Gα13 is pro-tumorigenic or tumor suppressive in genetically engineered mouse (GEM) models of epithelial cancers. Here, we show that loss of Gα13 in the Kras/Tp53 (KPC) GEM model promotes well-differentiated tumors and reduces survival. Mechanistically, tumors developing in KPC mice with Gα13 loss exhibit increased E-cadherin expression and mTOR signaling. Importantly, human pancreatic ductal adenocarcinoma (PDAC) tumors with low Gα13 expression also exhibit increased E-cadherin expression and mTOR signaling. Treatment with the mTOR inhibitor rapamycin decreases the growth of syngeneic KPC tumors with Gα13 loss by promoting cell death. This work establishes a tumor-suppressive role of Gα13 in pancreatic tumorigenesis in the KPC GEM model and suggests targeting mTOR in human PDAC tumors with Gα13 loss.

P2 Receptors as Therapeutic Targets in the Salivary Gland: From Physiology to Dysfunction.

Although often overlooked in our daily lives, saliva performs a host of necessary physiological functions, including lubricating and protecting the oral cavity, facilitating taste sensation and digestion and maintaining tooth enamel. Therefore, salivary gland dysfunction and hyposalivation, often resulting from pathogenesis of the autoimmune disease Sjögren's syndrome or from radiotherapy of the head and neck region during cancer treatment, severely reduce the quality of life of afflicted patients and can lead to dental caries, periodontitis, digestive disorders, loss of taste and difficulty speaking. Since their initial discovery in the 1970s, P2 purinergic receptors for extracellular nucleotides, including ATP-gated ion channel P2X and G protein-coupled P2Y receptors, have been shown to mediate physiological processes in numerous tissues, including the salivary glands where P2 receptors represent a link between canonical and non-canonical saliva secretion. Additionally, extracellular nucleotides released during periods of cellular stress and inflammation act as a tissue alarmin to coordinate immunological and tissue repair responses through P2 receptor activation. Accordingly, P2 receptors have gained widespread clinical interest with agonists and antagonists either currently undergoing clinical trials or already approved for human use. Here, we review the contributions of P2 receptors to salivary gland function and describe their role in salivary gland dysfunction. We further consider their potential as therapeutic targets to promote physiological saliva flow, prevent salivary gland inflammation and enhance tissue regeneration.

Purinergic signaling in Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is characterized by three major histopathological markers: amyloid-ß (Aß) plaques, neurofibrillary tangles and gliosis in the central nervous system (CNS). It is now accepted that neuroinflammatory events in the CNS play a crucial role in the development of AD. This review focuses on neuroinflammatory signaling mediated by purinergic receptors (P1 adenosine receptors, P2X ATP-gated ion channels and G protein-coupled P2Y nucleotide receptors) and how therapeutic modulation of purinergic signaling influences disease progression in AD patients and animal models of AD.

P2X7 receptor antagonism prevents IL-1ß release from salivary epithelial cells and reduces inflammation in a mouse model of autoimmune exocrinopathy.

Salivary gland inflammation is a hallmark of Sjögren's syndrome (SS), a common autoimmune disease characterized by lymphocytic infiltration of the salivary gland and loss of saliva secretion, predominantly in women. The P2X7 receptor (P2X7R) is an ATP-gated nonselective cation channel that induces inflammatory responses in cells and tissues, including salivary gland epithelium. In immune cells, P2X7R activation induces the production of proinflammatory cytokines, including IL-1ß and IL-18, by inducing the oligomerization of the multiprotein complex NLRP3-type inflammasome. Here, our results show that in primary mouse submandibular gland (SMG) epithelial cells, P2X7R activation also induces the assembly of the NLRP3 inflammasome and the maturation and release of IL-1ß, a response that is absent in SMG cells isolated from mice deficient in P2X7Rs (P2X7R-/-). P2X7R-mediated IL-1ß release in SMG epithelial cells is dependent on transmembrane Na+ and/or K+ flux and the activation of heat shock protein 90 (HSP90), a protein required for the activation and stabilization of the NLRP3 inflammasome. Also, using the reactive oxygen species (ROS) scavengers N-acetyl cysteine and Mito-TEMPO, we determined that mitochondrial reactive oxygen species are required for P2X7R-mediated IL-1ß release. Lastly, in vivo administration of the P2X7R antagonist A438079 in the CD28-/-, IFNγ-/-, NOD.H-2h4 mouse model of salivary gland exocrinopathy ameliorated salivary gland inflammation and enhanced carbachol-induced saliva secretion. These findings demonstrate that P2X7R antagonism in vivo represents a promising therapeutic strategy to limit salivary gland inflammation and improve secretory function.

Increased Expression of TGF-ß Signaling Components in a Mouse Model of Fibrosis Induced by Submandibular Gland Duct Ligation.

Transforming growth factor-ß (TGF-ß) is a multi-functional cytokine with a well-described role in the regulation of tissue fibrosis and regeneration in the liver, kidney and lung. Submandibular gland (SMG) duct ligation and subsequent deligation in rodents is a classical model for studying salivary gland damage and regeneration. While previous studies suggest that TGF-ß may contribute to salivary gland fibrosis, the expression of TGF-ß signaling components has not been investigated in relation to mouse SMG duct ligation-induced fibrosis and regeneration following ductal deligation. Following a 7 day SMG duct ligation, TGF-ß1 and TGF-ß3 were significantly upregulated in the SMG, as were TGF-ß receptor 1 and downstream Smad family transcription factors in salivary acinar cells, but not in ductal cells. In acinar cells, duct ligation also led to upregulation of snail, a Smad-activated E-cadherin repressor and regulator of epithelial-mesenchymal transition, whereas in ductal cells upregulation of E-cadherin was observed while snail expression was unchanged. Upregulation of these TGF-ß signaling components correlated with upregulation of fibrosis markers collagen 1 and fibronectin, responses that were inhibited by administration of the TGF-ß receptor 1 inhibitors SB431542 or GW788388. After SMG regeneration following a 28 day duct deligation, TGF-ß signaling components and epithelial-mesenchymal transition markers returned to levels similar to non-ligated controls. The results from this study indicate that increased TGF-ß signaling contributes to duct ligation-induced changes in salivary epithelium that correlate with glandular fibrosis. Furthermore, the reversibility of enhanced TGF-ß signaling in acinar cells of duct-ligated mouse SMG after deligation indicates that this is an ideal model for studying TGF-ß signaling mechanisms in salivary epithelium as well as mechanisms of fibrosis initiation and their resolution.

P2Y2 nucleotide receptor activation enhances the aggregation and self-organization of dispersed salivary epithelial cells.

Hyposalivation resulting from salivary gland dysfunction leads to poor oral health and greatly reduces the quality of life of patients. Current treatments for hyposalivation are limited. However, regenerative medicine to replace dysfunctional salivary glands represents a revolutionary approach. The ability of dispersed salivary epithelial cells or salivary gland-derived progenitor cells to self-organize into acinar-like spheres or branching structures that mimic the native tissue holds promise for cell-based reconstitution of a functional salivary gland. However, the mechanisms involved in salivary epithelial cell aggregation and tissue reconstitution are not fully understood. This study investigated the role of the P2Y2 nucleotide receptor (P2Y2R), a G protein-coupled receptor that is upregulated following salivary gland damage and disease, in salivary gland reconstitution. In vitro results with the rat parotid acinar Par-C10 cell line indicate that P2Y2R activation with the selective agonist UTP enhances the self-organization of dispersed salivary epithelial cells into acinar-like spheres. Other results indicate that the P2Y2R-mediated response is dependent on epidermal growth factor receptor activation via the metalloproteases ADAM10/ADAM17 or the α5ß1 integrin/Cdc42 signaling pathway, which leads to activation of the MAPKs JNK and ERK1/2. Ex vivo data using primary submandibular gland cells from wild-type and P2Y2R(-/-) mice confirmed that UTP-induced migratory responses required for acinar cell self-organization are mediated by the P2Y2R. Overall, this study suggests that the P2Y2R is a promising target for salivary gland reconstitution and identifies the involvement of two novel components of the P2Y2R signaling cascade in salivary epithelial cells, the α5ß1 integrin and the Rho GTPase Cdc42.