Federica Liotti

Mast cells have a protumorigenic role in human thyroid cancer

In different human carcinoma types, mast cell infiltrate increases with respect to normal tissue and mast cell density correlates with a bad prognosis. To assess the role of mast cells in human thyroid cancer, we compared the density of tryptase-positive mast cells in 96 papillary thyroid carcinomas (PTCs) versus normal thyroid tissue from 14 healthy individuals. Mast cell density was higher in 95% of PTCs (n=91) than in control tissue. Mast cell infiltrate correlated with extrathyroidal extension (P=0.0005) of PTCs. We show that thyroid cancer cell-line-derived soluble factors induce mast cell activation and chemoattraction in vitro. Different mast cell lines (HMC-1 and LAD2) and primary human lung mast cells induced thyroid cancer cell invasive ability, survival and DNA synthesis in vitro. The latter effect was mainly mediated by three mast-cell-derived mediators: histamine, and chemokines CXCL1/GROα and CXCL10/IP10. We show that xenografts of thyroid carcinoma cells (8505-C) could recruit mast cells injected into the tail vein of mice. Co-injection of human mast cells accelerated the growth of thyroid cancer cell (8505-C) xenografts in athymic mice. This effect was mediated by increased tumor vascularization and proliferation, and was reverted by treating mice with sodium cromoglycate (Cromolyn), a specific mast cell inhibitor. In conclusion, our study data suggest that mast cells are recruited into thyroid carcinomas and promote proliferation, survival and invasive ability of cancer cells, thereby contributing to thyroid carcinoma growth and invasiveness.

Activation of TYRO3/AXL Tyrosine Kinase Receptors in Thyroid Cancer

Thyroid cancer is the most common endocrine cancer, but its key oncogenic drivers remain undefined. In this study we identified the TYRO3 and AXL receptor tyrosine kinases as transcriptional targets of the chemokine CXCL12/SDF-1 in CXCR4-expressing thyroid cancer cells. Both receptors were constitutively expressed in thyroid cancer cell lines but not normal thyroid cells. AXL displayed high levels of tyrosine phosphorylation in most cancer cell lines due to constitutive expression of its ligand GAS6. In human thyroid carcinoma specimens, but not in normal thyroid tissues, AXL and GAS6 were often coexpressed. In cell lines expressing both receptors and ligand, blocking each receptor or ligand dramatically affected cell viability and decreased resistance to apoptotic stimuli. Stimulation of GAS6-negative cancer cells with GAS6 increased their proliferation and survival. Similarly, siRNA-mediated silencing of AXL inhibited cancer cell viability, invasiveness, and growth of tumor xenografts in nude mice. Our findings suggest that a TYRO3/AXL-GAS6 autocrine circuit sustains the malignant features of thyroid cancer cells and that targeting the circuit could offer a novel therapeutic approach in this cancer.

Mast cells induce epithelial-to-mesenchymal transition and stem cell features in human thyroid cancer cells through an IL-8-Akt-Slug pathway.

There is increasing evidence that mast cells (MCs) and their mediators are involved in the remodeling of the tumor microenvironment and promote tumor growth, angiogenesis and metastasis. We have found that an increased density of MCs in thyroid cancer (TC) correlates with enhanced invasiveness. However, the MC-derived factors responsible for this activity and the mechanisms by which they enhance TC invasiveness remain unidentified. Here, we report that MCs, when activated by TC cells, produce soluble factors that induce epithelial-to-mesenchymal transition (EMT) and stemness features of TC cells. We identified CXCL8/interleukin (IL)-8 as the main mediator contained in activated MC conditioned media (CM) capable of inducing both EMT and stemness of TC cells. Mechanistically, MC CM or exogenous IL-8 stimulated Akt phosphorylation and Slug expression in TC cells. The inhibition of the Akt pathway or depletion of the Slug transcription factor by RNA interference, reverted EMT and stemness responses. TC cells stably transfected with exogenous IL-8 underwent EMT, displayed increased stemness and enhanced tumorigenicity with respect to control cells. The analysis of TC surgical specimens by immunohistochemical analysis demonstrated a positive correlation between MC density (Tryptase+ cells) and stemness features (OCT4 staining). Taken together, our data identify an MC-dependent IL-8–Akt–Slug pathway that sustains EMT/stemness of TC cells. The blockade of this circuit might be exploited for the therapy of advanced TC.

Formyl peptide receptors at the interface of inflammation, angiogenesis and tumor growth

N-formyl peptide receptors (FPRs) belong to the family of pattern recognition receptors (PRRs) that regulate innate immune responses. Three FPRs have been identified in humans: FPR1-FPR3. FPR expression was initially described in immune cells and subsequently in non-hematopoietic cells and certain tissues. Besides their involvement in inflammatory disorders, FPRs have been implicated in the regulation of tissue repair and angiogenesis. Angiogenesis is not only a key component of pathogen defence during acute infection and of chronic inflammatory disorders, but also plays a critical role in wound healing and tissue regeneration. Moreover, pathologic uncontrolled angiogenesis is central for tumour growth, progression, and the formation of metastases. In this review, we summarise the evidence for a central role of FPRs at the intersection between inflammation, physiologic angiogenesis and pathologic neovascularisation linked to cancer. These findings provide insights into the potential clinical relevance of new treatment regimens involving FPR modulation.

The formyl peptide receptor 1 exerts a tumor suppressor function in human gastric cancer by inhibiting angiogenesis

N-formyl peptide receptors (FPR1, FPR2 and FPR3) are involved in innate immunity, inflammation and cancer. FPR expression, initially described in immune cells, was later observed in non-hematopoietic cell populations and tissues. Several studies suggested a role for FPRs in the progression of various tumor histotypes, including gastric cancer (GC), for which a positive association with a specific FPR1 polymorphism has recently been described. We previously showed that FPRs are expressed on gastric epithelium and are required for wound repair and restitution of barrier integrity. Here we assess the role of FPRs in GC. We characterized the functions of FPRs in GC epithelial cells (MKN28, AGS and MKN45) cultured in vitro by assessing migration, proliferation, resistance to apoptosis and activation of the epithelial-to-mesenchymal transition. Activation of each FPR induced the epithelial-to-mesenchymal transition, proliferation, resistance to apoptosis and migration of GC cells in culture. Blocking compounds or RNA interference of each FPR reverted these effects. We also defined the in vivo tumorigenic potential of GC epithelial cells silenced for FPRs by xenograft experiments in immunocompromised mice. Interestingly, FPR1 silencing in GC cells (shFPR1) significantly enhanced xenograft growth with respect to shCTR, shFPR2 and shFPR3 xenografts, because of augmented vessel density and cell proliferation. Accordingly, HIF-1α and VEGF mRNA levels were higher in shFPR1 xenografts than in controls. Moreover, the in vitro production of proangiogenic factors in response to FPR2/3 agonists (WKYMVm, LL-37, uPA, uPAR84-95, AnxA1) or to other proinflammatory mediators (IL-1α) was higher in shFPR1 GC cells than in shCTR, shFPR2 and shFPR3 cells, suggesting that FPR1 functions as an inhibitor of CG angiogenesis. Thus, we propose that FPR1 stimulation may represent a novel therapeutic approach to counteract tumor angiogenesis.

Tumor-Associated Mast Cells in Thyroid Cancer

There is compelling evidence that the tumor microenvironment plays a major role in mediating aggressive features of cancer cells, including invasive capacity and resistance to conventional and novel therapies. Among the different cell populations that infiltrate cancer stroma, mast cells (MCs) can influence several aspects of tumor biology, including tumor development and progression, angiogenesis, lymphangiogenesis, and tissue remodelling. Thyroid cancer (TC), the most frequent neoplasia of the endocrine system, is characterized by a MC infiltrate, whose density correlates with extrathyroidal extension and invasiveness. Recent evidence suggests the occurrence of epithelial-to-mesenchymal transition (EMT) and stemness in human TC. The precise role of immune cells and their mediators responsible for these features in TC remains unknown. Here, we review the relevance of MC-derived mediators (e.g., the chemokines CXCL1/GRO-α, CXCL10/IP-10, and CXCL8/IL-8) in the context of TC. CXCL1/GRO-α and CXCL10/IP-10 appear to be involved in the stimulation of cell proliferation, while CXCL8/IL-8 participates in the acquisition of TC malignant traits through its ability to induce/enhance the EMT and stem-like features of TC cells. The inhibition of chemokine signaling may offer novel therapeutic approaches for the treatment of refractory forms of TC.

Formyl peptide receptor 1 suppresses gastric cancer angiogenesis and growth by exploiting inflammation resolution pathways

ABSTRACT Chronic inflammation can result from inadequate engagement of resolution mechanisms, mainly accomplished by specialized pro-resolving mediators (SPMs) arising from the metabolic activity of lipoxygenases (ALOX5/15) on ω-6 or ω-3 essential polyunsaturated fatty acids (PUFA). We previously demonstrated that formyl peptide receptor 1 (FPR1) suppresses gastric cancer (GC) by inhibiting its inflammatory/angiogenic potential. In this study, we asked whether FPR1 exploits inflammation resolution pathways to suppress GC angiogenesis and growth. Here, we demonstrate that genetic or pharmacologic modulation of FPR1 in GC cells regulated ALOX5/15 expression and production of the SPMs Resolvin D1 (RvD1) and Lipoxin B4 (LXB4). SPM treatment of GC cells abated their angiogenic potential. Genetic deletion of ALOX15 or of the RvD1 receptor GPR32 increased the angiogenic and tumorigenic activity of GC cells thereby mimicking FPR1 loss. Deletion/inhibition of ALOX5/15 or GPR32 blocked FPR1-mediated anti-angiogenic activities, indicating that ALOX5/15 and GPR32 are required for FPR1s pro-resolving action. An ω-3- or ω-6-enriched diet enforced SPM endogenous production in mice and inhibited growth of shFPR1 GC xenografts by suppressing their angiogenic activity. These data implicate that FPR1 and/or pro-resolving pathway components might be used as risk/prognostic markers for GC; ω-6/3-enriched diets, and targeting FPR1 or SPM machinery may be exploited for GC management.

Interleukin-8, but not the Related Chemokine CXCL1, Sustains an Autocrine Circuit Necessary for the Properties and Functions of Thyroid Cancer Stem Cells.

Interleukin‐8 (IL‐8/CXCL8) mediates its biological effects through two receptors, CXCR1 and CXCR2. While CXCR1 recognizes IL‐8 and granulocyte chemotactic protein‐2, CXCR2 binds to multiple chemokines including IL‐8, CXCL1, 2 and 3. Both IL‐8 and CXCL1 have been implicated in the neoplastic features of thyroid cancer (TC). Here, we assessed the role of the autocrine circuits sustained by IL‐8 and CXCL1 in determining TC stem cell (TC SC) features. Using immunohistochemistry, we found that thyroid epithelial cancerous, but not normal, cells stained positive for IL‐8, whose levels correlated with lymph‐nodal metastases. We assessed the expression of endogenous IL‐8 and CXCL1, by ELISA assays, and of their receptors CXCR1 and CXCR2, by flow cytometry, in a panel of TC cell lines. These molecules were expressed in TC cell lines grown in adherence, and at higher levels also in thyrospheres enriched in stem‐like cells. RNA interference demonstrated that IL‐8/CXCR1, but not CXCL1/CXCR2, is crucial for the sphere‐forming, self‐renewal and tumor‐initiating ability of TC cells. Accordingly, treatment of TC cells with IL‐8, but not with CXCL1, potentiated cell stemness. We identified CD34 as an IL‐8‐induced gene and as a TC SC marker, since it was overexpressed in thyrospheres compared to adherent cells. Moreover, CD34 is required for the efficient sphere‐forming ability and tumorigenicity of TC cells. Our data indicate that IL‐8, but not the CXCL1 circuit, is critical for the regulation of TC SCs, and unveils novel potential targets for the therapy of as yet untreatable forms of TC. Stem Cells 2017;35:135–146

New perspectives in cancer: Modulation of lipid metabolism and inflammation resolution

&NA; Inflammation is considered an enabling feature of cancer. Besides the persistence of inflammatory stimuli, also defective mechanisms of resolution can lead to chronic inflammation. Inflammation resolution is an active process controlled by lipidic specialized pro‐resolving mediators (SPMs), derived from &ohgr;‐3 or &ohgr;‐6 essential polyunsaturated fatty acids (PUFA) through the activity of lipoxygenases (ALOX5 and 15). Thus, a lack or defect in resolution mechanisms may affect cancer development and progression by prolonging inflammation. Components of pro‐resolving pathways (PUFA, enzymes, or SPMs) have been reported to modulate various cancer features by affecting both cancer cells and cancer‐associated stroma. Here, we will review the most important mechanisms by which SPMs, &ohgr;‐3/6 PUFA, and ALOXs affect cancer biology, paying particular attention to their role in the inhibition of inflammation and angiogenesis, two of the most important hallmarks of cancer. The collection of these results may suggest novel perspectives in cancer management based on the modulation of lipid metabolism and the production of SPMs. Graphical abstract Figure. No caption available.

Multiple anti-tumor effects of Reparixin on thyroid cancer

Background Expression of IL-8 and its receptors CXCR1 and CXCR2 is a common occurrence in human epithelial thyroid cancer (TC). In human TC samples, IL-8 expression is associated with tumor progression. IL-8 enhances proliferation, survival, motility, and leads to the maintenance of stemness features and tumor-initiating ability of TC cells. Here, we studied the effects of Reparixin (formerly Repertaxin), a small molecular weight CXCR1 and CXCR2 inhibitor, on the malignant phenotype of various TC cell lines. Results Reparixin impaired the viability of epithelial thyroid cancerous cells, but not that of the non-malignant counterpart. Reparixin treatment significantly decreased TC cell survival, proliferation, Epithelial-to-Mesenchymal Transition (EMT) and stemness. CXCR1 and CXCR2 silencing abolished these effects. Reparixin sensitized TC cells to Docetaxel and Doxorubicin in culture. Used as single agent, Reparixin significantly inhibited TC cell tumorigenicity in immunodeficient mice. Finally, Reparixin potentiated the effects of Docetaxel on TC cell xenotransplants in mice. Materials and Methods We assessed the effects of Reparixin on TC cell viability (by growth curves, BrdU incorporation, TUNEL assay), EMT (by RT-PCR, Flow Cytometry, Migration assays), stemness (by RT-PCR, Flow Cytometry, sphere-formation and self-renewal), and tumorigenicity (by xenotransplantation in nude mice). Conclusions The present study suggests that Reparixin, both alone and in combination with classic chemotherapics, represents a novel potential therapeutic strategy for aggressive forms of TC.