Quang-Dé Nguyen

Tenascin-C and SF/HGF produced by myofibroblasts in vitro provide convergent pro-invasive signals to human colon cancer cells through RhoA and Rac

Myofibroblasts are present at the invasion front in colon cancer. In an attempt to understand their putative proinvasive activity, we have developed an in vitro model. Myofibroblasts isolated from colon cancer tissue or obtained through transdifferentiation of colon fibroblasts by transforming growth factor (TGF)‐β stimulate invasion of colon cancer cells into collagen type I and Matrigel. We identified two convergent proinvasive agents secreted by myofibroblasts: namely scatter factor/hepatocyte growth factor (SF/HGF) and the TGF‐β‐upregulated extracellular matrix glycoprotein tenascin‐C (TNC), each of which is necessary though not sufficient for invasion. Myofibroblast‐stimulated invasion into collagen type I is characterized by a change from a round, nonmigratory morphotype with high RhoA and low Rac activity to an elongated, migratory morphotype with low RhoA and high Rac activity. RhoA inactivation is determined by the epidermal growth factor (EGF)‐like repeats of TNC through EGF‐receptor signaling that confers a permissive and priming signal for the proinvasive activity of SF/HGF that activates Rac via c‐Met. We confirmed the validity of this mechanism by using pharmacological modulators and dominant negative or constitutive active mutants that interfere with RhoA‐Rho kinase and Rac signaling. Our in vitro results point to a new putative proinvasive signal for colon cancer cells provided by myofibroblasts in the tumor stroma.

Positron emission tomography imaging of drug-induced tumor apoptosis with a caspase-3/7 specific [18F]-labeled isatin sulfonamide

Of the molecular biochemical alterations that occur during apoptosis, activation of caspases, notably caspase-3, is probably the most attractive for developing specific in vivo molecular imaging probes. We recently designed a library of isatin-5 sulfonamides and selected [18F]ICMT-11 for further evaluation on the basis of subnanomolar affinity for activated capsase-3, high metabolic stability, and facile radiolabeling. In this present study, we have demonstrated that [18F]ICMT-11 binds to a range of drug-induced apoptotic cancer cells in vitro and to 38C13 murine lymphoma xenografts in vivo by up to 2-fold at 24 h posttreatment compared to vehicle treatment. We further demonstrated that the increased signal intensity in tumors after drug treatment, detected by whole body in vivo microPET imaging, was associated with increased apoptosis. In summary, we have characterized [18F]ICMT-11 as a caspase-3/7 specific PET imaging radiotracer for the assessment of tumor apoptosis that could find utility in anticancer drug development and the monitoring of early responses to therapy.

Trefoil peptides as proangiogenic factors in vivo and in vitro: implication of cyclooxygenase-2 and EGF receptor signaling

We previously established that the trefoil peptides (TFFs) pS2, spasmolytic polypeptide, and intestinal trefoil factor are involved in cellular scattering and invasion in kidney and colonic cancer cells. Using the chorioallantoic membrane (CAM) assay and the formation of tube‐like structures by human umbilical vein endothelial cells (HUVEC) plated on the Matrigel matrix substratum, we report here that TFFs are proangiogenic factors. Angiogenic activity of TFFs is comparable to that induced by vascular endothelial growth factor, leptin, and transforming growth factor‐a. Stimulation of angiogenesis by pS2 in the CAM assay is blocked by pharmacological inhibitors of cyclooxygenase COX‐2 (NS‐398) and epidermal growth factor receptor (EGF‐R) tyrosine kinase (ZD1839), but is independent of KDR/Flk‐1 and thromboxane A2 receptors. In contrast, the morphogenic switch induced by pS2 in HUVEC cells could be inhibited by the specific KDR heptapeptide antagonist ATWLPPR and by inhibitors of COX‐2 and EGF‐R signaling. These results implicate TFFs in the formation of new blood vessels during normal and pathophysiological processes linked to wound healing, inflammation, and cancer progression in the digestive mucosa and other human solid tumors associated with aberrant expression of TFFs.—Rodrigues, S., Van Aken, E., Van Bocxlaer, S., Attoub, S., Nguyen, Q.‐D., Bruyneel, E., Westley, B. R., May, F. E. B., Thim, L., Mareel, M., Gespach, C., Emami, S. Trefoil peptides as proangiogenic factors in vivo and in vitro: implication of cyclooxygenase −2 and EGF receptor signaling. FASEB J. 17, 7–16 (2003)

Activation of cellular invasion by trefoil peptides and src is mediated by cyclooxygenase- and thromboxane A2 receptor-dependent signaling pathways

We have investigated the possible functional relationships between cellular invasion pathways induced by trefoil factors (TFFs), src, and the cyclooxygenases COX‐1 and COX‐2. Pharmacological inhibitors of the Rho small GTPase (C3 exoenzyme), phospholipase C (U‐73122), cyclooxygenases (SC‐560, NS‐398), and the thromboxane A2 receptor (TXA2‐R) antagonist SQ‐295 completely abolished invasion induced by intestinal trefoil factor, pS2, and src in kidney and colonic epithelial cells MDCKts.src and PCmsrc. In contrast, invasion was induced by the TXA2‐R mimetic U‐46619, constitutively activated forms of the heterotrimeric G‐proteins Gαq (AGαq), Gα12, Gα13 (AGα12/13), which are signaling elements downstream of TXA2‐R. Ectopic overexpression of pS2 cDNA and protein in MDCKts.src‐pS2 cells and human colorectal cancer cells HCT8/S11‐pS2 initiate distinct invasion signals that are Rho independent and COX and TXA2‐R dependent. We detected a marked induction of COX‐2 protein and accumulation of the stable PGH2/TXA2 metabolite TXB2 in the conditioned medium from cells transformed by src. This led to activation of the TXA2‐R‐dependent invasion pathway, which is monitored via a Rho‐ and Gα12/Gα13‐independent mechanism using the Gαq/PKC signaling cascade. These findings identify a new intracrine/paracrine loop that can be monitored by TFFs and src in inflammatory diseases and progression of colorectal cancers.—Rodrigues, S., Nguyen, Q.‐ D., Faivre, S., Bruyneel, E., Thim, L., Westley, B., May, F., Flatau, G., Mareel, M., Gespach, C., Emami, S. Activation of cellular invasion by trefoil peptides and src is mediated by cyclooxygenase‐ and thromboxane A2 receptor‐dependent signaling pathways. FASEB J. 15, 1517–1528 (2001)

CXCR4-Targeted and MMP-Responsive Iron Oxide Nanoparticles for Enhanced Magnetic Resonance Imaging†

MRI offers high spatial resolution with excellent tissue penetration but it has limited sensitivity and the commonly administered contrast agents lack specificity. In this study, two sets of iron oxide nanoparticles (IONPs) were synthesized that were designed to selectively undergo copper-free click conjugation upon sensing of matrix metalloproteinase (MMP) enzymes, thereby leading to a self-assembled superparamagnetic nanocluster network with T2 signal enhancement properties. For this purpose, IONPs with bioorthogonal azide and alkyne surfaces masked by polyethylene glycol (PEG) layers tethered to CXCR4-targeted peptide ligands were synthesized and characterized. The IONPs were tested in vitro and T2 signal enhancements of around 160 % were measured when the IONPs were incubated with cells expressing MMP2/9 and CXCR4. Simultaneous systemic administration of the bioorthogonal IONPs in tumor-bearing mice demonstrated the signal-enhancing ability of these ‘smart’ self-assembling nanomaterials.

Reactivating HIF prolyl hydroxylases under hypoxia results in metabolic catastrophe and cell death.

Cells exposed to low-oxygen conditions (hypoxia) alter their metabolism to survive. This response, although vital during development and high-altitude survival, is now known to be a major factor in the selection of cells with a transformed metabolic phenotype during tumorigenesis. It is thought that hypoxia-selected cells have increased invasive capacity and resistance to both chemo- and radiotherapies, and therefore represent an attractive target for antitumor therapy. Hypoxia inducible factors (HIFs) are responsible for the majority of gene expression changes under hypoxia, and are themselves controlled by the oxygen-sensing HIF prolyl hydroxylases (PHDs). It was previously shown that mutations in succinate dehydrogenase lead to the inactivation PHDs under normoxic conditions, which can be overcome by treatment with α-ketoglutarate derivatives. Given that solid tumors contain large regions of hypoxia, the reactivation of PHDs in these conditions could induce metabolic catastrophe and therefore prove an effective antitumor therapy. In this report we demonstrate that derivatized α-ketoglutarate can be used as a strategy for maintaining PHD activity under hypoxia. By increasing intracellular α-ketoglutarate and activating PHDs we trigger PHD-dependent reversal of HIF1 activation, and PHD-dependent hypoxic cell death. We also show that derivatized α-ketoglutarate can permeate multiple layers of cells, reducing HIF1α levels and its target genes in vivo.

RhoA- and RhoD-dependent regulatory switch of Gα subunit signaling by PAR-1 receptors in cellular invasion

Thrombin and proteinase‐activated receptors (PAR) specifically regulate several functions that markedly enhance the transformation phenotype such as inflammation, cell proliferation, tumor growth, and metastasis. We recently reported that thrombin inhibits cellular invasion induced by src, hepatocyte growth factor (HGF), and leptin in kidney and colonic epithelial cells via predominant activation of the pertussis toxin (PTx) ‐sensitive G‐proteins Gαo/Gαi. We provide pharmacological and biochemical evidence that in the presence of PTx, PAR‐1 induced cellular invasion through Gα12/Gα13‐ and RhoA/Rho kinase (ROCK) ‐dependent signaling. However, inhibition of the endogenous small GTPase RhoA by the C3 exoenzyme, dominant‐negative N19‐RhoA, activated G26VRhoD, and activators of the nitric oxide/cGMP pathways conferred invasive activity to PAR‐1 via a signaling cascade using Gαq, phospholipase C (PLC), Ca2+/ calmodulin myosin light chain kinase (CaM‐MLCK), and phosphorylation of MLC. We found that cellular invasion induced by the src oncogene is abrogated by inhibitors of the RhoA/ROCK pathway and is independent of PLC/CaM‐MLCK signaling. Our data demonstrate that the RhoA and RhoD small GTPases are acting as a molecular switch of cellular invasion and reveal a novel critical mechanism by which PAR‐1 bypass Gαo/i and RhoA inhibition via differential coupling to heterotrimeric G‐proteins linked to divergent or convergent biological responses. Our data also indicate that Rho GTPases and ROCK mediate a src‐dependent invasion signal in kidney and colonic cancer cells. We conclude that dynamic regulation of Rho GTPases activation and inactivation by oncogenes, growth factors, cGMP‐inducing agents, and adhesion molecules can initiate convergent invasion signals controlled by the thrombin PAR‐1 in cancer cells.—Nguyen, Q.‐D., Faivre, S., Bruyneel, E., Rivat, C., Seto, M., Endo, T., Mareel, M., Emami, S., Gespach, C. RhoA‐ and RhoD‐dependent regulatory switch of Gα subunit signaling by PAR‐1 receptors in cellular invasion. FASEB J. 16, 565–576 (2002)

Selective abrogation of the proinvasive activity of the trefoil peptides pS2 and spasmolytic polypeptide by disruption of the EGF receptor signaling pathways in kidney and colonic cancer cells

Trefoil peptides (TFFs) are now considered as scatter factors, proinvasive and angiogenic agents acting through cyclooxygenase-2 (COX-2)- and thromboxane A2 receptor (TXA2-R)-dependent signaling pathways. As expression and activation levels of the epidermal growth factor receptor (EGFR) predict the metastatic potential of human colorectal cancers, the purpose of this study was to establish whether the EGF receptor tyrosine kinase (EGFR-TK) contributes to cellular invasion induced by TFFs in kidney and colonic cancer cells. Both the dominant negative form of the EGFR (HER-CD533) and the EGFR-TK inhibitor ZD1839 (Iressa) abrogated cellular invasion induced by pS2, spasmolytic polypeptide (SP) and the src oncogene, but not by ITF and the TXA2-R. Similarly, EGFR-TK inhibition by ZD1839 reversed the invasive phenotype promoted by the constitutively activated form of the EGFR (EGFRvIII) and the EGFR agonists transforming growth factor alpha (TGFα), amphiregulin and EGF. We also provide evidence that TFFs, EGFRvIII, and TGFα trigger common proinvasive pathways using the PI3′-kinase and Rho/Rho- kinase cascades. These findings identify the EGFR-TK as a key signaling element for pS2- and SP-mediated cellular invasion. It is concluded that although pS2, SP and ITF belong to the same family of inflammation- and cancer-associated regulatory peptides, they do not control identical signaling networks.

Heterogeneity in Lung 18 FDG Uptake in Pulmonary Arterial Hypertension Potential of Dynamic 18 FDG Positron Emission Tomography With Kinetic Analysis as a Bridging Biomarker for Pulmonary Vascular Remodeling Targeted Treatments

Background— Pulmonary arterial hypertension (PAH) is a disease of progressive vascular remodeling, characterized by dysregulated growth of pulmonary vascular cells and inflammation. A prevailing view is that abnormal cellular metabolism, notably aerobic glycolysis that increases glucose demand, underlies the pathogenesis of PAH. Increased lung glucose uptake has been reported in animal models. Few data exist from patients with PAH. Methods and Results— Dynamic positron emission tomography imaging with fluorine-18–labeled 2-fluoro-2-deoxyglucose (18FDG) ligand with kinetic analysis demonstrated increased mean lung parenchymal uptake in 20 patients with PAH, 18 with idiopathic PAH (IPAH) (FDG score: 3.27±1.22), and 2 patients with connective tissue disease (5.07 and 7.11) compared with controls (2.02±0.71; P<0.05). Further compartment analysis confirmed increased lung glucose metabolism in IPAH. Lung 18FDG uptake and metabolism varied within the IPAH population and within the lungs of individual patients, consistent with the recognized heterogeneity of vascular pathology in this disease. The monocrotaline rat PAH model also showed increased lung 18FDG uptake, which was reduced along with improvements in vascular pathology after treatment with dicholoroacetate and 2 tyrosine kinase inhibitors, imatinib and sunitinib. Hyperproliferative pulmonary vascular fibroblasts isolated from IPAH patients exhibited upregulated glycolytic gene expression, along with increased cellular 18FDG uptake; both were reduced by dicholoroacetate and imatinib. Conclusions— Some patients with IPAH exhibit increased lung 18FDG uptake. 18FDG positron emission tomography imaging is a tool to investigate the molecular pathology of PAH and its response to treatment.

Mechanism of action of the Aurora kinase inhibitor CCT129202 and in vivo quantification of biological activity

The Aurora family of serine/threonine kinases is important for the regulation of centrosome maturation, chromosome segregation, and cytokinesis during mitosis. Overexpression of Aurora kinases in mammalian cells leads to genetic instability and transformation. Increased levels of Aurora kinases have also been linked to a broad range of human tumors. Here, we describe the properties of CCT129202, a representative of a structurally novel series of imidazopyridine small-molecule inhibitors of Aurora kinase activity. This compound showed high selectivity for the Aurora kinases over a panel of other kinases tested and inhibits proliferation in multiple cultured human tumor cell lines. CCT129202 causes the accumulation of human tumor cells with ≥4N DNA content, leading to apoptosis. CCT120202-treated human tumor cells showed a delay in mitosis, abrogation of nocodazole-induced mitotic arrest, and spindle defects. Growth of HCT116 xenografts in nude mice was inhibited after i.p. administration of CCT129202. We show that p21, the cyclin-dependent kinase inhibitor, is induced by CCT129202. Up-regulation of p21 by CCT129202 in HCT116 cells led to Rb hypophosphorylation and E2F inhibition, contributing to a decrease in thymidine kinase 1 transcription. This has facilitated the use of 3′-deoxy-3′[18F]fluorothymidine-positron emission tomography to measure noninvasively the biological activity of the Aurora kinase inhibitor CCT129202 in vivo. [Mol Cancer Ther 2007;6(12):3147–57]