MicroRNA-211 Modulates the DUSP6-ERK5 Signaling Axis to Promote BRAFV600E-Driven Melanoma Growth In Vivo and BRAF/MEK Inhibitor Resistance.

MicroRNAs (miRs) are important posttranscriptional regulators of cell fate in both normal and disease states. miR-211 has previously been shown to be a direct regulator of metabolism in BRAFV600E-mutant melanoma cells in vitro. Here, we report that miR-211 expression promotes the aggressive growth of BRAFV600E-mutant melanoma xenografts in vivo. miR-211 promoted proliferation through the posttranscriptional activation of extracellular signal-regulated kinase (ERK) 5 signaling, which has recently been implicated in the resistance to BRAF and MAPK/ERK kinase inhibitors. We therefore examined whether miR-211 similarly modulated melanoma resistance to the BRAF inhibitor vemurafenib and the MAPK/ERK kinase inhibitor cobimetinib. Consistent with this model, miR-211 expression increased melanoma cell resistance to both the inhibitors, and this resistance was associated with an increased ERK5 phosphorylation. miR-211 mediates these effects by directly inhibiting the expression of DUSP6, an ERK5 pathway-specific phosphatase and now shown to be an miR-211 target gene. These results dissect the role of the miR-211-DUSP6-ERK5 axis in melanoma tumor growth and suggest a mechanism for the development of drug-resistant tumors and a target for overcoming resistance.

Absence of HIF1A Leads to Glycogen Accumulation and an Inflammatory Response That Enables Pancreatic Tumor Growth.

Cancer cells respond to hypoxia by upregulating the hypoxia-inducible factor 1α (HIF1A) transcription factor, which drives survival mechanisms that include metabolic adaptation and induction of angiogenesis by VEGF. Pancreatic tumors are poorly vascularized and severely hypoxic. To study the angiogenic role of HIF1A, and specifically probe whether tumors are able to use alternative pathways in its absence, we created a xenograft mouse tumor model of pancreatic cancer lacking HIF1A. After an initial delay of about 30 days, the HIF1A-deficient tumors grew as rapidly as the wild-type tumors and had similar vascularization. These changes were maintained in subsequent passages of tumor xenografts in vivo and in cell lines ex vivo. There were many cancer cells with a "clear-cell" phenotype in the HIF1A-deficient tumors; this was the result of accumulation of glycogen. Single-cell RNA sequencing (scRNA-seq) of the tumors identified hypoxic cancer cells with inhibited glycogen breakdown, which promoted glycogen accumulation and the secretion of inflammatory cytokines, including interleukins 1ß (IL1B) and 8 (IL8). scRNA-seq of the mouse tumor stroma showed enrichment of two subsets of myeloid dendritic cells (cDC), cDC1 and cDC2, that secreted proangiogenic cytokines. These results suggest that glycogen accumulation associated with a clear-cell phenotype in hypoxic cancer cells lacking HIF1A can initiate an alternate pathway of cytokine and DC-driven angiogenesis. Inhibiting glycogen accumulation may provide a treatment for cancers with the clear-cell phenotype. SIGNIFICANCE: These findings establish a novel mechanism by which tumors support angiogenesis in an HIF1α-independent manner.

YAP-IL-6ST autoregulatory loop activated on APC loss controls colonic tumorigenesis.

Loss of tumor suppressor adenomatous polyposis coli (APC) activates ß-catenin to initiate colorectal tumorigenesis. However, ß-catenin (CTNNB1) activating mutations rarely occur in human colorectal cancer (CRC). We found that APC loss also results in up-regulation of IL-6 signal transducer (IL-6ST/gp130), thereby activating Src family kinases (SFKs), YAP, and STAT3, which are simultaneously up-regulated in the majority of human CRC. Although, initial YAP activation, which stimulates IL6ST gene transcription, may be caused by reduced serine phosphorylation, sustained YAP activation depends on tyrosine phosphorylation by SFKs, whose inhibition, along with STAT3-activating JAK kinases, causes regression of established colorectal tumors. These results explain why APC loss is a more potent initiating event than the mere activation of CTNNB1.

The TRPA1 ion channel is expressed in CD4+ T cells and restrains T-cell-mediated colitis through inhibition of TRPV1.

OBJECTIVE: Transient receptor potential ankyrin-1 (TRPA1) and transient receptor potential vanilloid-1 (TRPV1) are calcium (Ca2+)-permeable ion channels mostly known as pain receptors in sensory neurons. However, growing evidence suggests their crucial involvement in the pathogenesis of IBD. We explored the possible contribution of TRPA1 and TRPV1 to T-cell-mediated colitis. DESIGN: We evaluated the role of Trpa1 gene deletion in two models of experimental colitis (ie, interleukin-10 knockout and T-cell-adoptive transfer models). We performed electrophysiological and Ca2+ imaging studies to analyse TRPA1 and TRPV1 functions in CD4+ T cells. We used genetic and pharmacological approaches to evaluate TRPV1 contribution to the phenotype of Trpa1-/- CD4+ T cells. We also analysed TRPA1 and TRPV1 gene expression and TRPA1+TRPV1+ T cell infiltration in colonic biopsies from patients with IBD. RESULTS: We identified a protective role for TRPA1 in T-cell-mediated colitis. We demonstrated the functional expression of TRPA1 on the plasma membrane of CD4+ T cells and identified that Trpa1-/- CD4+ T cells have increased T-cell receptor-induced Ca2+ influx, activation profile and differentiation into Th1-effector cells. This phenotype was abrogated upon genetic deletion or pharmacological inhibition of the TRPV1 channel in mouse and human CD4+ T cells. Finally, we found differential regulation of TRPA1 and TRPV1 gene expression as well as increased infiltration of TRPA1+TRPV1+ T cells in the colon of patients with IBD. CONCLUSIONS: Our study indicates that TRPA1 inhibits TRPV1 channel activity in CD4+ T cells, and consequently restrains CD4+ T-cell activation and colitogenic responses. These findings may therefore have therapeutic implications for human IBD.

The digestive tract as the origin of systemic inflammation.

Failure of gut homeostasis is an important factor in the pathogenesis and progression of systemic inflammation, which can culminate in multiple organ failure and fatality. Pathogenic events in critically ill patients include mesenteric hypoperfusion, dysregulation of gut motility, and failure of the gut barrier with resultant translocation of luminal substrates. This is followed by the exacerbation of local and systemic immune responses. All these events can contribute to pathogenic crosstalk between the gut, circulating cells, and other organs like the liver, pancreas, and lungs. Here we review recent insights into the identity of the cellular and biochemical players from the gut that have key roles in the pathogenic turn of events in these organ systems that derange the systemic inflammatory homeostasis. In particular, we discuss the dangers from within the gastrointestinal tract, including metabolic products from the liver (bile acids), digestive enzymes produced by the pancreas, and inflammatory components of the mesenteric lymph.

Neuronal TRPV1 activation regulates alveolar bone resorption by suppressing osteoclastogenesis via CGRP.

The transient receptor potential vanilloid 1 (TRPV1) channel is abundantly expressed in peripheral sensory neurons where it acts as an important polymodal cellular sensor for heat, acidic pH, capsaicin, and other noxious stimuli. The oral cavity is densely innervated by afferent sensory neurons and is a highly specialized organ that protects against infections as well as physical, chemical, and thermal stresses in its capacity as the first part of the digestive system. While the function of TRPV1 in sensory neurons has been intensively studied in other organs, its physiological role in periodontal tissues is unclear. In this study we found that Trpv1(-/-) mice developed severe bone loss in an experimental model of periodontitis. Chemical ablation of TRPV1-expressing sensory neurons recapitulated the phenotype of Trpv1(-/-) mice, suggesting a functional link between neuronal TRPV1 signaling and periodontal bone loss. TRPV1 activation in gingival nerves induced production of the neuropeptide, calcitonin gene-related peptide (CGRP), and CGRP treatment inhibited osteoclastogenesis in vitro. Oral administration of the TRPV1 agonist, capsaicin, suppressed ligature-induced bone loss in mice with fewer tartrate-resistant acid phosphatase (TRAP)-positive cells in alveolar bone. These results suggest that neuronal TRPV1 signaling in periodontal tissue is crucial for the regulation of osteoclastogenesis via the neuropeptide CGRP.

Definition of a Novel Feed-Forward Mechanism for Glycolysis-HIF1α Signaling in Hypoxic Tumors Highlights Aldolase A as a Therapeutic Target.

The hypoxia-inducible transcription factor HIF1α drives expression of many glycolytic enzymes. Here, we show that hypoxic glycolysis, in turn, increases HIF1α transcriptional activity and stimulates tumor growth, revealing a novel feed-forward mechanism of glycolysis-HIF1α signaling. Negative regulation of HIF1α by AMPK1 is bypassed in hypoxic cells, due to ATP elevation by increased glycolysis, thereby preventing phosphorylation and inactivation of the HIF1α transcriptional coactivator p300. Notably, of the HIF1α-activated glycolytic enzymes we evaluated by gene silencing, aldolase A (ALDOA) blockade produced the most robust decrease in glycolysis, HIF-1 activity, and cancer cell proliferation. Furthermore, either RNAi-mediated silencing of ALDOA or systemic treatment with a specific small-molecule inhibitor of aldolase A was sufficient to increase overall survival in a xenograft model of metastatic breast cancer. In establishing a novel glycolysis-HIF-1α feed-forward mechanism in hypoxic tumor cells, our results also provide a preclinical rationale to develop aldolase A inhibitors as a generalized strategy to treat intractable hypoxic cancer cells found widely in most solid tumors. Cancer Res; 76(14); 4259-69. ©2016 AACR.

ERK5 signalling rescues intestinal epithelial turnover and tumour cell proliferation upon ERK1/2 abrogation.

The ERK1/2 MAPK signalling module integrates extracellular cues that induce proliferation and differentiation of epithelial lineages, and is an established oncogenic driver, particularly in the intestine. However, the interrelation of the ERK1/2 module relative to other signalling pathways in intestinal epithelial cells and colorectal cancer (CRC) is unclear. Here we show that loss of Erk1/2 in intestinal epithelial cells results in defects in nutrient absorption, epithelial cell migration and secretory cell differentiation. However, intestinal epithelial cell proliferation is not impeded, implying compensatory mechanisms. Genetic deletion of Erk1/2 or pharmacological targeting of MEK1/2 results in supraphysiological activity of the ERK5 pathway. Furthermore, targeting both pathways causes a more effective suppression of cell proliferation in murine intestinal organoids and human CRC lines. These results suggest that ERK5 provides a common bypass route in intestinal epithelial cells, which rescues cell proliferation upon abrogation of ERK1/2 signalling, with therapeutic implications in CRC.

A gp130-Src-YAP module links inflammation to epithelial regeneration.

Inflammation promotes regeneration of injured tissues through poorly understood mechanisms, some of which involve interleukin (IL)-6 family members, the expression of which is elevated in many diseases including inflammatory bowel diseases and colorectal cancer. Here we show in mice and human cells that gp130, a co-receptor for IL-6 cytokines, triggers activation of YAP and Notch, transcriptional regulators that control tissue growth and regeneration, independently of the gp130 effector STAT3. Through YAP and Notch, intestinal gp130 signalling stimulates epithelial cell proliferation, causes aberrant differentiation and confers resistance to mucosal erosion. gp130 associates with the related tyrosine kinases Src and Yes, which are activated on receptor engagement to phosphorylate YAP and induce its stabilization and nuclear translocation. This signalling module is strongly activated upon mucosal injury to promote healing and maintain barrier function.

TRPV1: Turning up the heat on intestinal tumorigenesis.

TRP channels are associated with the development and progression of cancer but their precise molecular roles in these processes are unclear. Recently, we showed that the transient receptor potential cation channel, subfamily V, member 1 (TRPV1) ion channel is part of a negative feedback loop downstream of epidermal growth factor receptor signaling that suppresses intestinal tumorigenesis.

Ion channel TRPV1-dependent activation of PTP1B suppresses EGFR-associated intestinal tumorigenesis.

The intestinal epithelium has a high rate of turnover, and dysregulation of pathways that regulate regeneration can lead to tumor development; however, the negative regulators of oncogenic events in the intestinal epithelium are not fully understood. Here we identified a feedback loop between the epidermal growth factor receptor (EGFR), a known mediator of proliferation, and the transient receptor potential cation channel, subfamily V, member 1 (TRPV1), in intestinal epithelial cells (IECs). We found that TRPV1 was expressed by IECs and was intrinsically activated upon EGFR stimulation. Subsequently, TRPV1 activation inhibited EGFR-induced epithelial cell proliferation via activation of Ca2+/calpain and resulting activation of protein tyrosine phosphatase 1B (PTP1B). In a murine model of multiple intestinal neoplasia (Apc(Min/+) mice), TRPV1 deficiency increased adenoma formation, and treatment of these animals with an EGFR kinase inhibitor reversed protumorigenic phenotypes, supporting a functional association between TRPV1 and EGFR signaling in IECs. Administration of a TRPV1 agonist suppressed intestinal tumorigenesis in Apc(Min/+) mice, similar to--as well as in conjunction with--a cyclooxygenase-2 (COX-2) inhibitor, which suggests that targeting both TRPV1 and COX-2 has potential as a therapeutic approach for tumor prevention. Our findings implicate TRPV1 as a regulator of growth factor signaling in the intestinal epithelium through activation of PTP1B and subsequent suppression of intestinal tumorigenesis.

STAT3 regulates monocyte TNF-alpha production in systemic inflammation caused by cardiac surgery with cardiopulmonary bypass.

BACKGROUND: Cardiopulmonary bypass (CPB) surgery initiates a controlled systemic inflammatory response characterized by a cytokine storm, monocytosis and transient monocyte activation. However, the responsiveness of monocytes to Toll-like receptor (TLR)-mediated activation decreases throughout the postoperative course. The purpose of this study was to identify the major signaling pathway involved in plasma-mediated inhibition of LPS-induced tumor necrosis factor (TNF)-α production by monocytes. METHODOLOGY/PRINCIPAL FINDINGS: Pediatric patients that underwent CPB-assisted surgical correction of simple congenital heart defects were enrolled (n = 38). Peripheral blood mononuclear cells (PBMC) and plasma samples were isolated at consecutive time points. Patient plasma samples were added back to monocytes obtained pre-operatively for ex vivo LPS stimulations and TNF-α and IL-6 production was measured by flow cytometry. LPS-induced p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB activation by patient plasma was assessed by Western blotting. A cell-permeable peptide inhibitor was used to block STAT3 signaling. We found that plasma samples obtained 4 h after surgery, regardless of pre-operative dexamethasone treatment, potently inhibited LPS-induced TNF-α but not IL-6 synthesis by monocytes. This was not associated with attenuation of p38 MAPK activation or IκB-α degradation. However, abrogation of the IL-10/STAT3 pathway restored LPS-induced TNF-α production in the presence of suppressive patient plasma. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that STAT3 signaling plays a crucial role in the downregulation of TNF-α synthesis by human monocytes in the course of systemic inflammation in vivo. Thus, STAT3 might be a potential molecular target for pharmacological intervention in clinical syndromes characterized by systemic inflammation.

Hsp70 and cardiac surgery: molecular chaperone and inflammatory regulator with compartmentalized effects.

Open heart surgery is a unique model to study the interplay between cellular injury, regulation of inflammatory responses and tissue repair. Stress-inducible heat shock protein 70-kDa (Hsp70) provides a molecular link between these events. In addition to molecular chaperoning, Hsp70 exerts modulatory effects on endothelial cells and leukocytes involved in inflammatory networks. Hsp70 residing in the intracellular compartment is part of an inhibitory feedback loop that acts on nuclear factor kappaB (NF-kappaB). In contrast, extracellular Hsp70 is recognized by multiple germline-encoded immune receptors, e.g., Toll-like receptor (TLR) 2, TLR4, LOX-1, CD91, CD94, CCR5 and CD40. Hsp70 is thereby able to enhance chemotaxis, phagocytosis and cytolytic activity of innate immune cells and stimulate antigen-specific responses. These apparent contradictory pro- and anti-inflammatory effects of endogenous Hsp70 in the context of cardiac surgery are still not fully understood. An all-embracing model of the compartmentalized effects of endogenous Hsp70 in the orchestration of inflammatory responses in cardiac surgery is proposed.