The interaction of mast cells with membranes from lung cancer cells induces the release of extracellular vesicles with a unique miRNA signature.

Mast cells (MCs) are immune cells that play roles in both normal and abnormal processes. They have been linked to tumor progression in several types of cancer, including non-small cell lung cancer (NSCLC). However, the exact role of MCs in NSCLC is still unclear. Some studies have shown that the presence of a large number of MCs is associated with poor prognosis, while others have suggested that MCs have protective effects. To better understand the role of MCs in NSCLC, we aimed to identify the initial mechanisms underlying the communication between MCs and lung cancer cells. Here, we recapitulated cell-to-cell contact by exposing MCs to membranes derived from lung cancer cells and confirming their activation, as evidenced by increased phosphorylation of the ERK and AKT kinases. Profiling of the microRNAs that were selectively enriched in the extracellular vesicles (EVs) released by the lung cancer-activated MCs revealed that they contained significantly increased amounts of miR-100-5p and miR-125b, two protumorigenic miRNAs. We explored the pathways regulated by these miRNAs via enrichment analysis using the KEGG database, demonstrating that these two miRNAs regulate p53 signaling, cancer pathways, and pathways associated with apoptosis and the cell cycle.

Mast Cells Are Directly Activated by Cancer Cell-Derived Extracellular Vesicles by a CD73- and Adenosine-Dependent Mechanism.

We have recently shown that mast cells (MCs), which constitute an important part of the tumor microenvironment (TME), can be directly activated by cancer cells under conditions that recapitulate cell to cell contact. However, MCs are often detected in the tumor periphery rather than intratumorally. Therefore, we investigated the possibility of MC activation by cancer cell-derived extracellular vesicles (EVs). Here we show that exposure of MCs to EVs derived from pancreatic cancer cells or non-small cell lung carcinoma results in MC activation, evident by the increased phosphorylation of the ERK1/2 MAP kinases. Further, we show that, similarly to activation by cancer cell contact, activation by EVs is dependent on the ecto enzyme CD73 that mediates extracellular formation of adenosine and on signaling by the A3 adenosine receptor. Finally, we show that activation by either cell contact or EVs upregulates expression of angiogenic and tissue remodeling genes, including IL8, IL6, VEGF, and amphiregulin. Collectively, our findings indicate that both intratumorally localized MCs and peripheral MCs are activated and reprogrammed in the TME either by contact with the cancer cells or by their released EVs.

Mammalian diaphanous-related formin 1 (mDia1) coordinates mast cell migration and secretion through its actin-nucleating activity.

BACKGROUND: Actin remodeling is a key regulator of mast cell (MC) migration and secretion. However, the precise mechanism underlying the coordination of these processes has remained obscure. OBJECTIVE: We sought to characterize the actin rearrangements that occur during MC secretion or chemotactic migration and identify the underlying mechanism of their coordination. METHODS: Using high-resolution microscopy, we analyzed the dynamics of actin rearrangements in MCs triggered to migration by IL-8 or prostaglandin E2 or to FcεRI-stimulated secretion. RESULTS: We show that a major feature of the actin skeleton in MCs stimulated to migration is the buildup of pericentral actin clusters that prevent cell flattening and converge the secretory granules (SGs) in the cell center. This migratory phenotype is replaced on encounter of an IgE cross-linking antigen that stimulates secretion through a secretory phenotype characterized by cell flattening, reduction of actin mesh density, ruffling of cortical actin, and mobilization of SGs. Furthermore, we show that knockdown of mammalian diaphanous-related formin 1 (mDia1) inhibits chemotactic migration and its typical actin rearrangements, whereas expression of an active mDia1 mutant recapitulates the migratory actin phenotype and enhances cell migration while inhibiting FcεRI-triggered secretion. However, mice deficient in mDia1 appear to have normal numbers of MCs in various organs at baseline. CONCLUSION: Our results demonstrate a unique role of actin rearrangements in clustering the SGs and inhibiting their secretion during MC migration. We identify mDia1 as a novel regulator of MC response that coordinates MC chemotaxis and secretion through its actin-nucleating activity.

Role of Mast Cell-Derived Adenosine in Cancer.

Accumulating evidence has highlighted the accumulation of mast cells (MCs) in tumors. However, their impact on tumor development remained controversial. Indeed, cumulative data indicate an enigmatic role for MCs in cancer, whereby depending on the circumstances, which still need to be resolved, MCs function to promote or restrict tumor growth. By responding to multiple stimuli MCs release multiple inflammatory mediators, that contribute to the resolution of infection and resistance to envenomation, but also have the potency to promote or inhibit malignancy. Thus, MCs seem to possess the power to define tumor projections. Given this remarkable plasticity of MC responsiveness, there is an urgent need of understanding how MCs are activated in the tumor microenvironment (TME). We have recently reported on the direct activation of MCs upon contact with cancer cells by a mechanism involving an autocrine formation of adenosine and signaling by the A3 adenosine receptor. Here we summarized the evidence on the role of adenosine signaling in cancer, in MC mediated inflammation and in the MC-cancer crosstalk.

Mast cells are directly activated by contact with cancer cells by a mechanism involving autocrine formation of adenosine and autocrine/paracrine signaling of the adenosine A3 receptor.

Mast cells (MCs) constitute an important part of the tumor microenvironment (TME). However, their underlying mechanisms of activation within the TME remain poorly understood. Here we show that recapitulating cell-to-cell contact interactions by exposing MCs to membranes derived from a number of cancer cell types, results in MC activation, evident by the increased phosphorylation of the ERK1/2 MAP kinases and Akt, in a phosphatidylinositol 3-kinase dependent fashion. Activation is unidirectional since MC derived membranes do not activate cancer cells. Stimulated ERK1/2 phosphorylation is strictly dependent on the ecto enzyme CD73 that mediates autocrine formation of adenosine, and is inhibited by knockdown of the A3 adenosine receptor (A3R) as well as by an A3R antagonist or by agonist-stimulated down-regulation of the A3R. We also show that cancer cell mediated triggering upregulates expression and stimulates secretion of interleukin 8 from the activated MCs. These findings provide evidence for a novel mode of unidirectional crosstalk between MCs and cancer cells implicating direct activation by cancer cells in MC reprogramming into a pro tumorigenic profile.

Combining an EGFR directed tyrosine kinase inhibitor with autophagy-inducing drugs: a beneficial strategy to combat non-small cell lung cancer.

The potential therapeutic value of combinatorial regimens based on an EGF receptor tyrosine kinase inhibitor (TKI) and autophagy inducing drugs was evaluated by comparing their molecular impacts on H1299 and A549 non-small cell lung cancer (NSCLC) cells, which overexpress wild type EGF receptor, but are either deficient or have wild type p53 alleles, respectively. We show that H1299 cells display a considerably lower sensitivity to erlotinib treatment, which can be restored by combining erlotinib with rapamycin or with imatinib, though to a lesser extent. Cytotoxicity was associated with increased autophagy and hyperpolarization of the mitochondrial membrane potential. Therefore, combining an EGF receptor directed TKI with an autophagy-inducing drug, preferably, rapamycin, might be beneficial in treating poor responding NSCLC patients.

Molecular impacts of rapamycin-based drug combinations: combining rapamycin with gemcitabine or imatinib mesylate (Gleevec) in a human leiomyosarcoma model.

Drug combinations may provide a therapeutic benefit in treating cancer patients. However when considering a drug combination, it is important to assess how the molecular impact of the combination relates to the effects manifested by each drug alone and whether or not it varies depending on the tumor type. In this study, we have analyzed the molecular impact on a human leiomyosarcoma cell line (SK-LMS-1) of a combination consisting of the mTOR inhibitor rapamycin and either the anti-metabolite drug gemcitabine (Gemzar) or the protein tyrosine kinase inhibitor imatinib mesylate (Gleevec, STI571). We show that imatinib mesylate depolarizes the mitochondrial membrane potential (DeltaPhim) and inhibits protein tyrosine phosphorylation, but displays only minor effects on cell proliferation when added alone or in combin-ation with rapamycin. Gemcitabine or rapamycin, when added alone, inhibit protein tyrosine phosphorylation as well as phosphorylation of the MAP kinases ERK1/2. Both drugs also affect the cell cycle, arresting the cells at the S or G1 phase respectively. Rapamycin elevates significantly DeltaPhim but produces only a moderate effect on cell growth. Gemcitabine inhibits considerably cell growth but exerts no effect on DeltaPhim. Combining gemcitabine and rapamycin produces a major effect on the cell cycle, elevates the DeltaPhim even further and maintains the molecular impacts exerted by each single drug. Therefore, consistent with our clinical observation, these results suggest that combining gemcitabine and rapamycin may be beneficial in treating leiomyosarcoma patients.

Synaptotagmin (Syt) IX is an essential determinant for protein sorting to secretory granules in mast cells.

The secretory granules (SGs) of secretory cells of the hematopoietic lineage, such as the mast cells, are lysosome-related organelles whose membrane proteins travel through the plasma membrane and the endocytic system. Therefore, a mechanism must exist to prevent proteins destined to recycling or to the trans-Golgi network (TGN) from reaching the SGs. We now show that synaptotagmin (Syt) IX, a Syt homologue that is required for recycling from the endocytic recycling compartment (ERC) in rat basophilic leukemia (RBL-2H3) cultured mast cells, is involved in segregating recycling proteins from the SGs. By using as a marker the recycling protein TGN38, which cycles between the TGN, plasma membrane, and the ERC, we show that knock-down of Syt IX results in mistargeting of HA-tagged TGN38 to the SGs. We further demonstrate that Syt IX binds directly the small GTPase ARF1 and associates with the clathrin adaptor complex AP-1. These results therefore implicate Syt IX as an essential factor for the correct sorting of SGs proteins. Moreover, they place Syt IX as part of the machinery that is involved in the formation of transport carriers that mediate SGs protein sorting.

Classical protein kinase C(s) regulates targeting of synaptotagmin IX to the endocytic recycling compartment.

Neuronal and non-neuronal tissues show distinctly different intracellular localization of synaptotagmin (Syt) homologues. Therefore, cell type-specific mechanisms are likely to direct Syt homologues to their final cellular destinations. Syt IX localizes to dense core vesicles in PC12 cells. However, in the rat basophilic leukemia (RBL-2H3) mast cell line, as well as in CHO cells, Syt IX is localized at the endocytic recycling compartment (ERC). We show that targeting of Syt IX to the ERC involves constitutive trafficking to the plasma membrane followed by internalization and transport to the ERC. We further show that internalization from the plasma membrane and delivery to the ERC are dependent on phosphorylation by Ca(2+)-dependent protein kinase Calpha or beta. As such, correct targeting of Syt IX is facilitated by the phorbol ester TPA but prevented by the cPKC inhibitor Go 6976.