The insulin‐like growth factor system and sarcomas

Sarcomas are a diverse group of malignant mesenchymal tumours arising from bone and soft tissues. The identification of critical cellular signalling pathways in sarcomas is an important issue for the development of new targeted therapies. This review highlights the experimental and clinical evidence supporting the role of the insulin‐like growth factor (IGF) signalling system in the cellular transformation and progression of several types of sarcoma, including rhabdomyosarcoma, synovial sarcoma, leiomyosarcoma, Ewing's sarcoma and osteosarcoma. Preclinical data suggest that the IGF system could be a promising target for therapy in these sarcomas. Currently, therapies interrupting IGF signalling have been or are being developed. In recent phase 1 clinical studies with humanized monoclonal antibodies directed against IGF receptor type 1 (IGF‐1R), objective tumour responses were observed in several patients with Ewing's sarcoma, encouraging further clinical testing in Ewing's sarcoma and other sarcoma (sub)types. Moreover, the occasional occurrence of paraneoplastic hypoglycaemia as a result of the secretion of incompletely processed forms of pro‐IGF‐II by sarcomas is discussed. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Expression of Matrix Metalloproteinase‐9 mRNA and Vascular Endothelial Growth Factor Protein in Gastric Carcinoma and Its Relationship to Its Pathological Features and Prognosis

To investigate matrix metalloproteinase‐9 (MMP‐9) mRNA and vascular endothelial growth factor (VEGF) protein expression in gastric carcinoma and its correlation with microvascular density, growth‐pattern, invasion, metastasis, and prognosis. In situ hybridization of MMP‐9 mRNA and immunohistochemistry of VEGF and CD34 proteins were performed on surgical specimens of gastric cancers from 118 patients compared with 20 nonmalignant gastric mucosae. Their relationships to pathological parameters and survival times were determined by statistical analysis. The positive rate of MMP‐9 in noncancerous gastric mucosae was significantly lower than that of gastric cancer tissue (60.17%, P < 0.01). In patients with cancers of the infiltrating type, at stage T3‐T4, with vessel invasion, lymphatic metastasis, hepatic, or peritoneal metastasis, the positive expression rates of MMP‐9 mRNA, VEGF protein, and CD34 were significantly higher than those for patients with tumors of the expanding type (P < 0.01), at stage T1–T2 (P < 0.01), with nonvessel invasion (P < 0.05), without lymphatic metastasis (P < 0.05), and without hepatic (P < 0.001) or peritoneal metastasis (P < 0.001), respectively. Expression of MMP‐9 mRNA was positively related to that of VEGF protein (P < 0.001) and microvascular density (P < 0.001). Patients with higher MMP‐9 mRNA and VEGF expression demonstrated vivid tumor angiogenesis and poor 5‐year survival rate. MMP‐9 and VEGF expression is associated with enhanced tumor angiogenesis and may play crucial roles in the invasion and metastasis of gastric carcinoma. Therefore, MMP‐9 and VEGF may represent prognostic biomarkers and promising targets for therapeutic intervention. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

β ‐Adrenoceptor agonist sensitizes the dopamine‐1 receptor in renal tubular cells

The renal effects of dopamine are mainly mediated via the dopamine‐1 receptor (D1 receptor). This receptor is recruited from intracellular compartments to the plasma membrane by dopamine and atrial natriuretic peptide (ANP), via adenylyl cyclase activation. We have studied whether isoproterenol, a β‐adrenoceptor (β‐AR) agonist that may interact with dopamine in the regulation of rat renal Na+, K⁺‐adenosine triphosphatase (ATPase) activity, can recruit D1 receptors to the plasma membrane. The spatial regulation of D1 receptors was examined using confocal microscopy techniques in LLCPK cells and the functional interaction between dopamine and isoproterenol was examined by studying their effects on Na+, K⁺‐ATPase activity in microdissected single proximal tubular segments from rat. Isoproterenol was found to translocate the D1 receptors from the interior of the cell towards the plasma membrane. The recruitment of dopamine 1 receptors was found to be cyclic adenosine phosphate (cAMP) dependent, while protein kinase C (PKC) activation was not involved. The functional studies on Na+, K⁺‐ATPase activity showed that the effect of isoproterenol was abolished by a D1‐like receptor antagonist (SCH 23390), and mediated via protein kinase A (PKA) and PKC dependent pathways. The results provide an explanation for the interaction between G protein‐coupled receptors. The effects of isoproterenol on Na+, K⁺‐ATPase activity can be explained by a heterologous recruitment of D1 receptors to the plasma membrane.     

Immunohistochemical expression of matrix metalloproteinase‐1, matrix metalloproteinase‐2 and matrix metalloproteinase‐9, myofibroblasts and Ki‐67 in actinic cheilitis and lip squamous cell carcinoma

Matrix metalloproteinases (MMPs), myofibroblasts (MFs) and epithelial proliferation have key roles in neoplastic progression. In this study immunoexpression of MMP‐1, MMP‐2 and MMP‐9, presence of MFs and the epithelial proliferation index were investigated in actinic cheilitis (AC), lip squamous cell carcinoma (LSCC) and mucocele (MUC). Thirty cases of AC, thirty cases of LSCC and twenty cases of MUC were selected for immunohistochemical investigation of the proteins MMP‐1, MMP‐2, MMP‐9, α‐smooth muscle actin (α‐SMA) and Ki‐67. The MMP‐1 expression in the epithelial component was higher in the AC than the MUC and LSCC. In the connective tissue, the expression was higher in the LSCC. MMP‐2 showed lower epithelial and stromal immunostaining in the LSCC when compared to the AC and MUC. The epithelial staining for MMP‐9 was higher in the AC when compared to the LSCC. However, in the connective tissue, the expression was lower in the AC compared to other lesions. The cell proliferation rate was increased in proportion to the severity of dysplasia in the AC, while in the LSCC it was higher in well‐differentiated lesions compared to moderately differentiated. There were no statistically significant differences in number of MFs present in the lesions studied. The results suggest that MMPs could affect the biological behaviour of ACs and LSCCs inasmuch as they could participate in the development and progression from premalignant lesions to malignant lesions.     

Methylthioadenosine phosphorylase and activated insulin‐like growth factor‐1 receptor/insulin receptor: potential therapeutic targets in chordoma

Currently there is no effective chemotherapy for chordoma. Recent studies report co‐expression of insulin‐like growth factor‐1 receptor (IGF1R) and its cognate ligand in chordoma, but it is unknown whether this receptor tyrosine kinase is activated in these tumours. Additionally, genetic studies have confirmed frequent deletions of chromosome 9p in chordomas, which encompasses the cyclin‐dependent kinase inhibitor 2A (CDKN2A) locus. Another gene in this region, methylthioadenosine phosphorylase (MTAP), is an essential enzyme of the purine salvage pathway and has therapeutic relevance because MTAP‐deficient cells are particularly sensitive to inhibitors of de novo purine synthesis. We investigated whether these pathways might be potential therapeutic targets for chordoma. Paraffin‐embedded tissue samples from 30 chordomas were analysed by immunohistochemistry for expression of the phosphorylated isoforms of IGF1R or the insulin receptor (pIGF1R/pIR) and selected downstream signalling molecules, including BCL2‐associated agonist of cell death protein (BAD). Expression of CDKN2A and MTAP proteins was also assessed. Skeletal chondrosarcomas, benign notochordal cell tumours, and fetal notochord were studied for comparison. Phosphorylated IGF1R/IR was detected in 41% of chordomas, together with activated downstream signalling molecules, and pIGF1R/pIR was absent in benign notochordal cell tumours and fetal notochord. Thirty‐nine per cent of chordomas were negative for MTAP immunoreactivity. Patients with pIGF1R/pIR‐positive tumours showed significantly decreased median disease‐free survival in multivariate survival analysis (p = 0.036), whereas phosphorylation of BAD at serine‐99 was found to be associated with a favourable prognosis (p = 0.002). Approximately 40% of chordomas demonstrate evidence of activation of the IGF1R/IR signalling pathway or loss of a key enzyme in the purine salvage pathway. Aberrant signalling cascades and disrupted metabolic pathways such as these may represent opportunities for novel targeted therapeutic approaches for the treatment of chordoma. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Neural tube defects and impaired neural progenitor cell proliferation in Gβ1‐deficient mice

Heterotrimeric G proteins are well known for their roles in signal transduction downstream of G protein–coupled receptors (GPCRs), and both Gα subunits and tightly associated Gβγ subunits regulate downstream effector molecules. Compared to Gα subunits, the physiological roles of individual Gβ and Gγ subunits are poorly understood. In this study, we generated mice deficient in the Gβ1 gene and found that Gβ1 is required for neural tube closure, neural progenitor cell proliferation, and neonatal development. About 40% Gβ1^?/? embryos developed neural tube defects (NTDs) and abnormal actin organization was observed in the basal side of neuroepithelium. In addition, Gβ1^?/? embryos without NTDs showed microencephaly and died within 2 days after birth. GPCR agonist‐induced ERK phosphorylation, cell proliferation, and cell spreading, which were all found to be regulated by Gαi and Gβγ signaling, were abnormal in Gβ1^?/? neural progenitor cells. These data indicate that Gβ1 is required for normal embryonic neurogenesis. Developmental Dynamics 239:1089–1101, 2010. © 2010 Wiley‐Liss, Inc.     

Transforming growth factor-beta type 1 receptor (ALK5) and Smad proteins mediate TIMP-1 and collagen synthesis in experimental intestinal fibrosis

Transforming growth factor β (TGF-β) is known to play a key role in intestinal fibrosis; however, the underlying mechanisms are not well understood. TGF-β signal transduction is through TGF-β receptors, including the TGF-β type 1 receptor. Most cell types contain a TGF-β type 1 receptor form known as activin receptor-like kinase 5 (ALK5), which propagates the signal to the nucleus through the phosphorylation of Smad2 and Smad3 proteins. Therefore, we assessed the effect of the disruption of TGF-β/ALK5/Smad signalling by an ALK5 inhibitor (SD-208) in two experimental animal models of intestinal fibrosis: anaerobic bacteria- and trinitrobenzensulphonic acid-induced colitis. In addition, isolated myofibroblasts were pretreated with SD-208 and exposed to recombinant TGF-β1. Finally, myofibroblasts were transfected with ALK5, Smad2, and Smad3-specific siRNA. Up-regulation of ALK5 and TIMP-1, phosphorylation of Smad2 and Smad3 proteins, and increased intestinal wall collagen deposition were found in both experimental animal models. These effects were decreased by SD-208. TGF-β1 treatment also induced phosphorylation of Smad2 and Smad3 and up-regulation of ALK5 protein, TIMP-1, and α2 type 1 collagen gene expression in isolated myofibroblasts. Again these effects were inhibited by SD-208. Also, ALK5, Smad2, and Smad3 siRNA abolished the induction of TIMP-1 and α2 type 1 collagen. Our findings provide evidence that the TGF-β/ALK5/Smad pathway participates in the pathogenesis of experimental intestinal fibrosis. These data show promise for the development of an effective therapeutic intervention in this condition.     

Oncogenic S1P signalling in EBV‐associated nasopharyngeal carcinoma activates AKT and promotes cell migration through S1P receptor 3

Undifferentiated nasopharyngeal carcinoma (NPC) is a cancer with high metastatic potential that is consistently associated with Epstein–Barr virus (EBV) infection. In this study, we have investigated the functional contribution of sphingosine‐1‐phosphate (S1P) signalling to the pathogenesis of NPC. We show that EBV infection or ectopic expression of the EBV‐encoded latent genes (EBNA1, LMP1, and LMP2A) can up‐regulate sphingosine kinase 1 (SPHK1), the key enzyme that produces S1P, in NPC cell lines. Exogenous addition of S1P promotes the migration of NPC cells through the activation of AKT; shRNA knockdown of SPHK1 resulted in a reduction in the levels of activated AKT and inhibition of cell migration. We also show that S1P receptor 3 (S1PR3) mRNA is overexpressed in EBV‐positive NPC patient‐derived xenografts and a subset of primary NPC tissues, and that knockdown of S1PR3 suppressed the activation of AKT and the S1P‐induced migration of NPC cells. Taken together, our data point to a central role for EBV in mediating the oncogenic effects of S1P in NPC and identify S1P signalling as a potential therapeutic target in this disease. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Induction of interleukin-10 in the T helper type 17 effector population by the G protein coupled estrogen receptor (GPER) agonist G-1

Interleukin-10 (IL-10) is a potent suppressor of the immune system, commonly produced by CD4(+) T cells to limit ongoing inflammatory responses minimizing host damage. Many autoimmune diseases are marked by large populations of activated CD4(+) T cells within the setting of chronic inflammation; therefore, drugs capable of inducing IL-10 production in CD4(+) T cells would be of great therapeutic value. Previous reports have shown that the small molecule G-1, an agonist of the membrane-bound G-protein-coupled estrogen receptor GPER, attenuates disease in an animal model of autoimmune encephalomyelitis. However, the direct effects of G-1 on CD4(+) T-cell populations remain unknown. Using ex vivo cultures of purified CD4(+) T cells, we show that G-1 elicits IL-10 expression in T helper type 17 (Th17) -polarized cells, increasing the number of IL-10(+) and IL-10(+) IL-17A(+) cells via de novo induction of IL-10. T-cell cultures differentiated in the presence of G-1 secreted threefold more IL-10, with no change in IL-17A, tumour necrosis factor-α, or interferon-γ. Moreover, inhibition of extracellular signal-regulated kinase (but not p38 or Jun N-terminal kinase) signalling blocked the response, while analysis of Foxp3 and RORγt expression demonstrated increased numbers of IL-10(+) cells in both the Th17 (RORγt(+)) and Foxp3(+) RORγt(+) hybrid T-cell compartments. Our findings translated in vivo as systemic treatment of male mice with G-1 led to increased IL-10 secretion from splenocytes following T-cell receptor cross-linking. These results demonstrate that G-1 acts directly on CD4(+) T cells, and to our knowledge provide the first example of a synthetic small molecule capable of eliciting IL-10 expression in Th17 or hybrid T-cell populations.     

From the black widow spider to human behavior: Latrophilins,a relatively unknown class of G protein‐coupled receptors,are implicated in psychiatric disorders

The findings of a recent study associate LPHN3, a member of the latrophilin family, with an increased risk of developing attention deficit/hyperactivity disorder (ADHD), the most common psychiatric disorder in childhood and adolescence. Latrophilins comprise a new family of G protein‐coupled receptors of unknown native physiological function that mediate the neurotoxic effects of α‐latrotoxin, a potent toxin found in black widow spider venom. This receptor–toxin interaction has helped to elucidate the mechanistic aspects of neurotransmitter and hormone release in vertebrates. Such unprecedented discovery points to a new direction in the assessment of ADHD and suggest that further study of this receptor family may provide novel insights into the etiology and treatment of ADHD and other related psychiatric conditions. © 2010 Wiley‐Liss, Inc.     

Intimal sarcoma of the abdominal aorta with platelet‐derived growth factor receptor α overexpression and amplification in mural invasive cells and pulmonary metastatic cells but not in intimal spreading cells

Intimal sarcoma (IS) is the most common sarcoma of the aorta. The platelet‐derived growth factor receptor α (PDGFRA), murine double minute 2 (MDM2), and cyclin‐dependent kinase 4 (CDK4) genes are often simultaneously amplified in IS. While immunohistochemical analysis of IS tissue has demonstrated frequent overexpression of the MDM2 and CDK4 proteins, the expression pattern of PDGFRA has not been well characterized, particularly in terms of intratumoral heterogeneity. Here, we present the case of a 46‐year‐old man who presented with a backache and was subsequently diagnosed with IS. Intratumoral heterogeneity of PDGFRA gene amplification was observed using fluorescence in situ hybridization and was positively correlated with PDGFRA protein expression using immunohistochemistry (IHC). The expression of PDGFRA was also correlated with cytological atypia: PDGFRA was not overexpressed in intimal spreading cells that displayed the lowest degree of atypia while PDGFRA overexpression and amplification were observed in invasive cells of progressive areas such as the aortic wall and a pulmonary metastatic site, which showed increased cytological atypia. Although PDGFRA has not been well examined on IHC, IHC of PDGFRA could be useful to diagnose IS. However, the areas within the tumor from which specimens are derived are important given potential intratumoral heterogeneity.     

Caveolin-1 promotes an invasive phenotype and predicts poor prognosis in large cell lung carcinoma

Purpose

This study investigated the relationships of caveolin-1 expression with clinical pathologic parameters and the prognosis of patients with large cell lung carcinoma. This study also explored the roles of caveolin-1 in cell invasiveness, matrix metalloproteinase (MMP) expression, and non-small cell lung carcinoma activity in vitro.

Methods

A total of 120 tissue samples were immunohistochemically analyzed for caveolin-1 expression. Cell invasion ability was measured by a Transwell invasion assay. Protein expression was assessed by Western blotting. MMP activity was detected by gelatin zymography.

Results

Caveolin-1 was expressed in 54 of 120 (45.0%) cases of large cell lung carcinoma. Caveolin-1 expression was significantly correlated with node status (N0 vs. N1, N2, and N3; P = 0.005) and advanced pTNM stage (Stages I and II vs. Stage III, P < 0.001). Patients with caveolin-1-positive expression had a poorer prognosis than did those with caveolin-1-negative expression (P < 0.001). The knockdown of caveolin-1 in H460 and 95D cells reduced the invasive ability of the cells and the expression of phosphorylated epidermal growth factor receptor (EGFR), phospho-extracellular signal-regulated kinases 1 and 2, MMP2, and MMP9; the protein level and activity of MMP2 and MMP9 were also decreased by the inhibition of EGFR activity in H460 and 95D cells.

Conclusions

The expression of caveolin-1 was positively correlated with an advanced pathologic stage. Thus, caveolin-1 could act as a predictor of a poor prognosis in patients with large cell lung carcinoma. In addition, the downregulation of caveolin-1 reduced both the invasive ability of tumor cells and the protein and activity levels of MMP2 and MMP9 in vitro, suggesting the involvement of EGFR/MMP signaling in this process.     

Transforming Growth Factor‐β Suppressed Id‐1 Expression in a smad3‐Dependent Manner in LoVo Cells

TGF‐β plays an important role in regulating cell differentiation and proliferation in human cancers such as colorectal cancer. Id‐1 has been identified as a marker in colorectal cancer progression. The aim of this study was to investigate the role of TGF‐β in regulating Id‐1 in LoVo cells. siRNA was used to silence smad2, smad3, and p38 MAPK gene expression in Lovo cells. Interference efficiency and the role of TGF‐β on Id‐1 expression were analyzed using a luciferase reporter assay, RT‐PCR, and Western blotting. Cell viability was determined using the MTT assay. In this study, we demonstrated that TGF‐β1 downregulated Id‐1 protein expression in LoVo cells. Smad2 and smad3 siRNA inhibited TGF‐β1‐induced 4×SBE luciferase reporter activity. p38 MAPK siRNA inhibited TGF‐β1‐induced 3×AP‐1 luciferase reporter activity. However, the suppression of Id‐1 by TGF‐β1 was recovered by smad3 siRNA but not smad2 or p38 MAPK siRNA. Moreover, TGF‐β1 stimulated cellular proliferation and p21^Waf1 protein expression, which might be mediated by suppressing Id‐1 expression. In conclusion, this study demonstrated that TGF‐β1 suppressed Id‐1 expression in a smad3‐dependent manner in LoVo cells using RNAi technology. These results provide new insight into the mechanisms of TGF‐β function in colorectal cancer cells. Anat Rec, 2010. © 2009 Wiley‐Liss, Inc.     

Innate immune receptor NOD2 promotes vascular inflammation and formation of lipid‐rich necrotic cores in hypercholesterolemic mice

Atherosclerosis is an inflammatory disease associated with the activation of innate immune TLRs and nucleotide‐binding oligomerization domain‐containing protein (NOD)‐like receptor pathways. However, the function of most innate immune receptors in atherosclerosis remains unclear. Here, we show that NOD2 is a crucial innate immune receptor influencing vascular inflammation and atherosclerosis severity. 10‐week stimulation with muramyl dipeptide (MDP), the NOD2 cognate ligand, aggravated atherosclerosis, as indicated by the augmented lesion burden, increased vascular inflammation and enlarged lipid‐rich necrotic cores in Ldlr^?/? mice. Myeloid‐specific ablation of NOD2, but not its downstream kinase, receptor‐interacting serine/threonine‐protein kinase 2, restrained the expansion of the lipid‐rich necrotic core in Ldlr^?/? chimeric mice. In vitro stimulation of macrophages with MDP enhanced the uptake of oxidized low‐density lipoprotein and impaired cholesterol efflux in concordance with upregulation of scavenger receptor A1/2 and downregulation of ATP‐binding cassette transporter A1. Ex vivo stimulation of human carotid plaques with MDP led to increased activation of inflammatory signaling pathways p38 MAPK and NF‐κB‐mediated release of proinflammatory cytokines. Altogether, this study suggests that NOD2 contributes to the expansion of the lipid‐rich necrotic core and promotes vascular inflammation in atherosclerosis.