Fibroblast growth factor-2/platelet-derived growth factor enhances atherosclerotic plaque stability.

Increased immature neovessels contribute to plaque growth and instability. Here, we investigated a method to establish functional and stable neovessel networks to increase plaque stability. Rabbits underwent aortic balloon injury and were divided into six groups: sham, vector and lentiviral transfection with vascular endothelial growth factor-A (VEGF)-A, fibroblast growth factor (FGF)-2, platelet-derived growth factor (PDGF)-BB and FGF-2 + PDGF-BB. Lentivirus was percutaneously injected into the media-adventitia of the abdominal aorta by intravascular ultrasound guidance, and plaque-rupture rate, plaque-vulnerability index and plaque neovessel density at the injection site were evaluated. Confocal microscopy, Prussian Blue assay, Evans Blue, immunofluorescence and transmission electron microscopy were used to assess neovessel function and pericyte coverage. To evaluate the effect of FGF-2/PDGF-BB on pericyte migration, we used the mesenchymal progenitor cell line 10T1/2 as an in vitro model. VEGF-A- and FGF-2-overexpression increased the number of immature neovessels, which caused intraplaque haemorrhage and inflammatory cell infiltration, eventually resulting in the plaque vulnerability; however, FGF-2/PDGF-BB induced mature and functional neovessels, through increased neovessel pericyte coverage. Additionally, in vitro analysis of 10T1/2 cells revealed that FGF-2/PDGF-BB induced epsin-2 expression and enhanced the VEGF receptor-2 degradation, which negatively regulated pericyte function consistent with the in vivo data. These results showed that the combination of FGF-2 and PDGF-BB promoted the function and maturation of plaque neovessels, thereby representing a novel potential treatment strategy for vulnerable plaques.

The Role of Psychological Stress on Heart Autophagy in Mice With Heart Failure.

OBJECTIVE: Psychological stress in chronic heart failure (CHF) is associated with systemic neurohormonal and immune system responses and increased mortality. Autophagy refers to the biological process of degradation and recycling of dysfunctional cellular components. We investigated the role of psychological stress on autophagy function in CHF mice. METHODS: C57BL/6 mice underwent transverse aortic constriction, with or without combined acoustic and restraint stress, and cardiac function was assessed by echocardiography analysis. Serum corticosterone and angiotensin II (Ang II) were determined using enzyme-linked immunosorbent assay (ELISA). Autophagy and oxidative stress were measured with immunohistochemistry and quantitative polymerase chain reaction, and chloroquine and rapamycin were used to detect autophagy flux. In vivo, cardiomyocytes were cultured with or without Ang II or N-acetylcysteine, and autophagy and oxidative stress were also detected. RESULTS: A 1-week stress exposure significantly increased serum levels of corticosterone and Ang II (p = .000), increased levels of oxidative stress, induced overt heart failure, and increased mortality (p = .002). Furthermore, stress exposure unregulated messenger RNA expression of Bcl-2-interacting coiled-coil protein 1 (10.891 [3.029] versus 4.754 [1.713], p = .001), cysteine-rich domain containing beclin-1 interacting (6.403 [1.813] versus 3.653 [0.441], p = .006), and autophagy 7 (111.696 [4.049] versus 6.189 [1.931], p = .017), increased expression of autophagosomal, and decreased clearance of autophagosomes. In vitro, Ang II significantly increased autophagy flux in cultured cardiomyocytes, which could be partly inhibited by N-acetylcysteine. CONCLUSIONS: Psychological stress may contribute to the development of CHF by enhancing heart oxidative stress and impairing autophagy flux.

Anti-hepatitis B virus effect of matrine-type alkaloid and involvement of p38 mitogen-activated protein kinase and tumor necrosis factor receptor-associated factor 6.

The matrine-type alkaloid, oxymatrine inhibits hepatitis B virus (HBV) replication but very little is known about these effects in other matrine-type alkaloids, including sophoridine and sophocarpine. Therefore, we compared the in vitro anti-HBV effects of matrine, oxymatrine, sophocarpine, and sophoridine by treating an HBV-transfected cell line (HepG2.2.15) with 0.4-1.6mM of the compounds for 24 or 72h. The levels of the HBV surface antigen (HBsAg) and e antigen (HBeAg) in the culture medium, as well as the intracellular and extracellular HBV DNA levels, were determined. Metabolomic analysis and detection of the mRNA level of p38 mitogen-activated protein kinase (MAPK), tumor necrosis factor receptor-associated factor (TRAF) 6, extracellular signal-regulated kinase (ERK) 1, NOD-like receptor family pyrin domain containing 10 (NLRP10), and caspase-1 were conducted in sophoridine-treated HepG2.2.15 cells. HepG2.2.15 cell exposure to 0.4-1.6mM sophocarpine or sophoridine for 24h reduced the HBsAg level of the medium more effectively than exposure to matrine and oxymatrine did, and reduced the HBeAg levels more effectively than these compounds did at 1.6mM. Sophoridine (0.4-1.6mM) reduced the cell medium HBV DNA levels more than the same concentrations of matrine, oxymatrine, or sophocarpine did. After 72h, 0.4 and 0.8mM sophoridine reduced HBsAg and intracellular HBV DNA levels more potently than matrine, oxymatrine, or sophocarpine did. Furthermore, sophoridine (0.8mM) potently reduced the cell medium HBeAg levels while the metabolomic analyses revealed that HepG2.2.15 cells exposed to 0.8mM sophoridine for 72h exhibited reduced cycloleucine and phytosphingosine levels. In addition, the mRNA expression analyses revealed that HepG2.2.15 cells exposed to 0.8mM sophoridine showed reduced levels of p38 MAPK, TRAF6, ERK1, NLRP10, and caspase-1. Sophoridine produced more potent anti-HBV effects than matrine, oxymatrine, and sophocarpine did. These effects may be related to the sophoridine-mediated reduction of p38 MAPK and TRAF6 levels.

[Thymopolypeptides combined with matrine type alkaloids suppress HBV replication].

To investigate the antiviral effect of thymopolypeptides combined with 4 kinds of matrine type alkaloids on HepG2.2.15 cells, oxymatrine, sophocarpidine, sophocarpine, and sophoridine (at concentration of 0.2 mmol•L⁻¹ respectively) were respectively combined with thymopolypeptides (0.025, 0.1 g•L⁻¹), and after 48 h and 72 h treatment on HepG2.2.15 cells, the cells and supernatants were collected. The cells activity in various groups was determined by CCK-8 method to evaluate the toxic effects of the drugs on HepG2.2.15 cells. Enzyme linked immunosorbent assay (ELISA) was used to determine HBeAg and HBsAg levels in cellular supernatants. HBV DNA levels in cellular supernatants andcells were quantified with fluorogenic quantitative PCR method; and the expression level of IFN-α in supernatants was detected with CBA method. The results indicated that single thymopolypeptides at 0.025-0.4 g•L⁻¹ had no toxicity to cells. Thymopolypeptides in this concentration range combined with 0.2 mmol•L⁻¹ matrine type alkaloids also had no toxicity to cells. Anti-HBV activity of drug combination was better than that of alkali or thymopolypeptides alone. Thymopolypeptides at 0.025 g•L⁻¹ had better inhibitory effect than thymopolypeptides at 0.1 g•L⁻¹ on intracellular HBV DNA expression, but the inhibitory effect on supernatant HBeAg level was on the contrary. Anti-HBV activity was similar between alkaloids combined with 0.1 g•L⁻¹ and alkaloids combined with 0.025 g•L⁻¹. There was no statistical difference in anti-HBV effect between various combined groups (P<0.05). In general, 72 h anti-HBV effect was better than 48 h anti-HBV effect (P<0.05). The expression of IFN-α was increased after drug combination, with positive correlation to the changes of other four indicators (P<0.05). In conclusion, oxymatrine, sophocarpidine, sophocarpine and sophoridine combined with thymopolypeptides could inhibit HBsAg and HBeAg secretion in HepG2.2.15 cells and HBV DNA replication, and further promote the antiviral effect by promoting the expression of IFN-α.

Papillary glioneuronal tumors: histological and molecular characteristics and diagnostic value of SLC44A1-PRKCA fusion.

INTRODUCTION: Papillary Glioneuronal Tumor (PGNT) is a grade I tumor which was classified as a separate entity in the World Health Organization Classification of the Central Nervous System 2007 in the group of mixed glioneuronal tumors. This tumor is rare and subclassifying PGNT represents a challenge. Recently, a fusion between SLC44A1 and PRKCA which encodes a protein kinase C involved in MAPK signaling pathway has been described in two studies (five cases). The current study aimed at raising the cytogenetic, histological and molecular profiles of PGNT and to determine if SLC44A1-PRKCA fusion represented a specific diagnostic marker to distinguish it from other glioneuronal tumors. RESULTS: We report on four pediatric cases of PGNT, along with clinico-radiologic and immunohistological features for which SLC44A1-PRKCA fusion assessment by fluorescence in situ hybridization, BRAF V600E and FGFR1 mutation by immunohistochemistry and direct DNA sequencing and KIAA1549-BRAF fusion by RT-PCR were performed. MAPK signaling pathway activation was investigated using phospho-ERK immunohistochemistry and western blot. We analyzed fifteen cases of tumors with challenging histological or clinical differential diagnoses showing respectively a papillary architecture or periventricular location (PGNT mimics). fluorescence in situ hybridization analysis revealed a constant SLC44A1-PRKCA fusion signal in all PGNTs. None of PGNT mimics showed the SLC44A1-PRKCA fusion signal pattern. All PGNTs were negative for BRAF V600E and FGFR1 mutation, and KIAA1549-BRAF fusion. Phospho-ERK analysis provides arguments for the activation of the MAPK signaling pathway in these tumors. CONCLUSIONS: Here we confirmed and extended the molecular data on PGNT. These results suggest that PGNT belong to low grade glioma with MAPK signaling pathway deregulation. SLC44A1-PRKCA fusion seems to be a specific characteristic of PGNT with a high diagnostic value and detectable by FISH.

Tongxinluo Protects against Hypertensive Kidney Injury in Spontaneously-Hypertensive Rats by Inhibiting Oxidative Stress and Activating Forkhead Box O1 Signaling.

Hypertension is an independent risk factor for the progression of chronic renal failure, and oxidative stress plays a critical role in hypertensive renal damage. Forkbox O1(FoxO1) signaling protects cells against oxidative stress and may be a useful target for treating oxidative stress-induced hypertension. Tongxinluo is a traditional Chinese medicine with cardioprotective and renoprotective functions. Therefore, this study aimed to determine the effects of Tongxinluo in hypertensive renal damage in spontaneously hypertensive rats(SHRs)and elucidate the possible involvement of oxidative stress and FoxO1 signaling in its molecular mechanisms. SHRs treated with Tongxinluo for 12 weeks showed a reduction in systolic blood pressure. In addition to increasing creatinine clearance, Tongxinluo decreased urinary albumin excretion, oxidative stress injury markers including malondialdehyde and protein carbonyls, and expression of nicotinamide adenine dinucleotide phosphate oxidase subunits and its activity in SHR kidneys. While decreasing phosphorylation of FoxO1, Tongxinluo also inhibited the phosphorylation of extracellular signal-regulated kinase1/2 and p38 and enhanced manganese superoxide dismutase and catalase activities in SHR kidneys. Furthermore, histology revealed attenuation of glomerulosclerosis and renal podocyte injury, while Tongxinluo decreased the expression of α-smooth muscle actin, extracellular matrixprotein, transforming growth factor ß1 and small mothers against decapentaplegic homolog 3,and improved tubulointerstitial fibrosis in SHR kidneys. Finally, Tongxinluo inhibited inflammatory cell infiltration as well as expression of tumor necrosis factor-α and interleukin-6. In conclusion, Tongxinluo protected SHRs against hypertension-induced renal injury by exerting antioxidant, antifibrotic, and anti-inflammatory activities. Moreover, the underlying mechanisms of these effects may involve inhibition of oxidative stress and functional activation of FoxO1 signaling.

Tumoral TP53 and/or CDKN2A alterations are not reliable prognostic biomarkers in patients with localized Ewing sarcoma: a report from the Children's Oncology Group.

BACKGROUND: A growing collection of retrospective studies have suggested that TP53 mutations and/or CDKN2A deletions have prognostic significance in Ewing sarcoma. We sought to evaluate these variables in patients with localized disease treated prospectively on a single Children's Oncology Group protocol. PROCEDURE: Of the 568 patients enrolled on Children's Oncology Group protocol AEWS0031 (NCT00006734), 112 had tumor specimens of sufficient quality and quantity to allow for analysis of TP53 mutations status by DNA sequencing, and CDKN2A deletion by dual color fluorescent in situ hybridization. RESULTS: Eight of 93 cases (8.6%) were found to have TP53 point mutations and 12 of 107 cases (11.2%) demonstrated homozygous CDKN2A deletion. Two cases were found to have an alteration in both genes. There was no significant difference in event-free survival of patients with TP53 mutations and/or CDKN2A deletions compared to patients with normal TP53/CDKN2A gene status, as demonstrated by log rank test (p = 0.58). CONCLUSIONS: Although previous retrospective studies suggest their significance, TP53 mutation and/or CDKN2A deletion are not reliable prognostic biomarkers in localized Ewing sarcoma.

Specific matrix metalloproteinases play different roles in intraplaque angiogenesis and plaque instability in rabbits.

BACKGROUND: Ectopic angiogenesis within the intima and media is considered to be a hallmark of advanced vulnerable atherosclerotic lesions. Some studies have shown that specific matrix metalloproteinases (MMPs) might play different roles in angiogenesis. Therefore, we investigated the predominant effects of specific MMPs in intraplaque angiogenesis and plaque instability in a rabbit model of atherosclerosis. METHODS AND RESULTS: New Zealand rabbits underwent balloon injury of the abdominal artery and ingestion of a high-cholesterol (1%) diet to establish an atherosclerotic animal model. At weeks 4, 6, 8, 10, and 12 after balloon injury, five rabbits were euthanized and the abdominal aorta was harvested. Blood lipid analysis, intravascular ultrasound imaging, pathologic and immunohistochemical expression studies, and western blotting were performed. From weeks 4 to 12, the expression of MMP-1, -2, -3, and -9 and vascular endothelial growth factor A (VEGF-A) increased with atherosclerotic plaque development in the abdominal aorta, while the expression of MMP-14 substantially decreased. The vulnerability index (VI) gradually increased over time. Intraplaque neovessels appeared at week 8. The microvessel density (MVD) was greater at week 12 than at week 8. The VI, MVD, and VEGF-A level were positively correlated with the MMP-1, -2,-3, and -9 levels within plaques. Negative correlations were noted between the MMP-14 level and the VI, MVD, and VEGF-A level. CONCLUSION: Upregulation of MMP-1, -2, -3, and -9 and downregulation of MMP-14 may contribute to intraplaque angiogenesis and plaque instability at the advanced stage of atherosclerosis in rabbits.

Resveratrol-enhanced autophagic flux ameliorates myocardial oxidative stress injury in diabetic mice.

Autophagic dysfunction is observed in diabetes mellitus. Resveratrol has a beneficial effect on diabetic cardiomyopathy. Whether the resveratrol-induced improvement in cardiac function in diabetes is via regulating autophagy remains unclear. We investigated the mechanisms underlying resveratrol-mediated protection against heart failure in diabetic mice, with a focus on the role of sirtuin 1 (SIRT1) in regulating autophagic flux. Diabetic cardiomyopathy in mice was induced by streptozotocin (STZ). Long-term resveratrol treatment improved cardiac function, ameliorated oxidative injury and reduced apoptosis in the diabetic mouse heart. Western blot analysis revealed that resveratrol decreased p62 protein expression and promoted SIRT1 activity and Rab7 expression. Inhibiting autophagic flux with bafilomycin A1 increased diabetic mouse mortality and attenuated resveratrol-induced down-regulation of p62, but not SIRT1 activity or Rab7 expression in diabetic mouse hearts. In cultured H9C2 cells, redundant or overactive H2O2 increased p62 and cleaved caspase 3 expression as well as acetylated forkhead box protein O1 (FOXO1) and inhibited SIRT1 expression. Sirtinol, SIRT1 and Rab7 siRNA impaired the resveratrol amelioration of dysfunctional autophagic flux and reduced apoptosis under oxidative conditions. Furthermore, resveratrol enhanced FOXO1 DNA binding at the Rab7 promoter region through a SIRT1-dependent pathway. These results highlight the role of the SIRT1/FOXO1/Rab7 axis in the effect of resveratrol on autophagic flux in vivo and in vitro, which suggests a therapeutic strategy for diabetic cardiomyopathy.

Period 2 is essential to maintain early endothelial progenitor cell function in vitro and angiogenesis after myocardial infarction in mice.

Cellular therapeutic neovascularization has been successfully performed in clinical trials for patients with ischaemia diseases. Despite the vast knowledge of cardiovascular disease and circadian biology, the role of the circadian clock in regulating angiogenesis in myocardial infarction (MI) remains poorly understood. In this study, we aimed to investigate the role and underlying mechanisms of Period 2 (Per2) in endothelial progenitor cell (EPC) function. Flow cytometry revealed lower circulating EPC proportion in per2(-/-) than in wild-type (WT) mice. PER2 was abundantly expressed in early EPCs in mice. In vitro, EPCs from per2(-/-) mice showed impaired proliferation, migration, tube formation and adhesion. Western blot analysis demonstrated inhibited PI3k/Akt/FoxO signalling and reduced C-X-C chemokine receptor type 4 (CXCR4) protein level in EPCs of per2(-/-) mice. The impaired proliferation was blocked by activated PI3K/Akt/FoxO signalling. Direct interaction of CXCR4 and PER2 was detected in WT EPCs. To further study the effect of per2 on in vivo EPC survival and angiogenesis, we injected saline or DiI-labelled WT or per2(-/-) EPC intramyocardially into mice with induced MI. Per2(-/-) reduced the retention of transplanted EPCs in the myocardium, which was associated with significantly reduced DiI expression in the myocardium of MI mice. Decreased angiogenesis in the myocardium of per2(-/-) EPC-treated mice coincided with decreased LV function and increased infarct size in the myocardium. Per2 may be a key factor in maintaining EPC function in vitro and in therapeutic angiogenesis in vivo.

Tongxinluo Improves Cardiac Function and Ameliorates Ventricular Remodeling in Mice Model of Myocardial Infarction through Enhancing Angiogenesis.

Background. Myocardial infarction (MI) is a major cause of morbidity and mortality in the world. Tongxinluo (TXL) is a traditional Chinese compound prescription which has cardioprotective functions. The present study was aimed to determine the effect of TXL on postischemic cardiac dysfunction and cardiac remodeling and to elucidate the underlying mechanisms. Methods and Results. MI was performed by ligation of left anterior descending coronary artery (LAD) in male adult mice. Mice were randomly divided into four groups: (1) sham group (Sham); (2) MI-control group (Control); (3) MI-low dose TXL group (TXL-L); and (4) MI-high dose TXL (TXL-H) group. Compared with the control group, TXL treatment restored cardiac function, increased revascularization, attenuated cardiomyocyte apoptosis, and reduced interstitial fibrosis. TXL treatment increased the phosphorylation of Akt, extracellular signal regulated kinase (ERK), and endothelial nitric oxide synthase (eNOS); the expression of phosphatidylinositol3-kinase (PI3K), hypoxia-inducible factors 1 α (HIF-1 α ), and vascular endothelial growth factor (VEGF); and the DNA binding activity of HIF-1 α after MI. Conclusion. TXL may improve cardiac function and ameliorate cardiac remodeling by increasing neovascularization through enhancing the phosphorylation of Akt and ERK, the expression and activity of HIF-1 α , and the protein level of VEGF and p-eNOS.

Identification of a novel, recurrent SLC44A1-PRKCA fusion in papillary glioneuronal tumor.

Mixed neuronal-glial tumors are rare and challenging to subclassify. One recently recognized variant, papillary glioneuronal tumor (PGNT), is characterized by prominent pseudopapillary structures and glioneuronal elements. We identified a novel translocation, t(9;17)(q31;q24), as the sole karyotypic anomaly in two PGNTs. A fluorescence in situ hybridization (FISH)-based positional cloning strategy revealed SLC44A1, a member of the choline transporter-like protein family, and PRKCA, a protein kinase C family member of serine/threonine-specific protein kinases, as the 9q31 and 17q24 breakpoint candidate genes, respectively. Reverse transcription-polymerase chain reaction (RT-PCR) analysis using a forward primer from SLC44A1 exon 5 and a reverse primer from PRKCA exon 10 confirmed the presence of a SLC44A1-PRKCA fusion product in both tumors. Sequencing of each chimeric transcript uncovered an identical fusion cDNA junction occurring between SLC44A1 exon 15 and PRKCA exon 9. A dual-color breakpoint-spanning probe set custom-designed for interphase cell recognition of the translocation event identified the fusion in a third PGNT. These results suggest that the t(9;17)(q31;q24) with the resultant novel fusion oncogene SLC44A1-PRKCA is the defining molecular feature of PGNT that may be responsible for its pathogenesis. The FISH and RT-PCR assays developed in this study can serve as valuable diagnostic adjuncts for this rare disease entity.

Unpredictable chronic mild stress promotes atherosclerosis in high cholesterol-fed rabbits.

OBJECTIVES: Chronic psychological stress is associated with an increased risk of atherosclerosis in humans. Experimental studies using various stress models have yielded controversial results. This study investigated the effects of unpredictable chronic mild stress (UCMS) on atherogenesis in New Zealand white rabbits. METHODS: Rabbits were fed with a cholesterol-enriched (1%) diet for 4 to 16 weeks, with or without concomitant UCMS treatment. Atherosclerosis was assessed in the abdominal aorta by serial sectioning and morphological analysis. Expressions of inflammatory factors were measured with immunohistochemistry and quantitative polymerase chain reaction. Serum nitrate/nitrite levels were determined with Griess assay, and corticosterone and inflammatory markers were determined using enzyme-linked immunosorbent assay. RESULTS: High-cholesterol feeding resulted in hypercholesterolemia and formation of atherosclerotic plaques in the aorta. UCMS exposure significantly increased the plaque size (p = .003) and decreased the plaque stability (decreased the contents of collagen and smooth muscle and increased the amount of macrophage and matrix metalloproteinases). The proatherogenic effects of UCMS were unrelated to changes in serum cholesterol level but accompanied by increased blood pressure (p < .001) and vascular inflammation (up-regulation of tumor necrosis factor α, C-reactive protein, and monocyte chemoattractant protein 1, all p values < .01). Serum concentrations of nitrate/nitrite were lower in UCMS-treated animals (p = .01). Vessels from UCMS-treated animals exhibited augmented phosphorylation of p38 and c-Jun N-terminal kinase and activation of nuclear factor κB. CONCLUSIONS: Chronic psychological stress may contribute to the development of atherosclerosis by enhancing vascular inflammation and decreasing endothelial nitric oxide bioavailability.

WITHDRAWN: Restoration of transforming growth factor-beta receptor II expression in colon cancer cells with microsatellite instability increases metastatic potential in vivo.

Microsatellite instability (MSI), which occurs in 15% of colorectal cancer, has been shown to have a lower incidence of metastasis and better patient survival rates compared with microsatellite stable colorectal cancer. However, a mechanistic understanding of the basis for this difference is very limited. Here, we show that restoration of TGFß signaling by re-expression of TGFß receptor II in MSI colon cancer cells increased PI3K/AKT activation, conferred resistance to growth factor deprivation stress-induced apoptosis, and promoted cell motility in vitro. Treatment with a potent PI3K inhibitor (LY294002) blocked the prosurvival and promotility effects of TGFß, indicating that TGFß-mediated promotion of cell survival and motility is dependent upon activation of the PI3K/AKT pathway. Analysis of apoptotic effectors that are affected by TGFß signaling indicated that Bim is an effector of TGFß-mediated survival. In addition, TGFß-induced down-regulation of E-cadherin contributed to the prosurvival effect of TGFß, and restoration of TGFß signaling in MSI colon cancer cells increased liver metastasis in an orthotopic model in vivo. Taken together, our results demonstrate that restoration of TGFß signaling promotes cell survival, motility, and metastatic progression in MSI colon cancer cells and indicate that TGFß receptor II mutations contribute to the favorable outcomes in colon cancer patients with MSI.

Phosphatase PRL-3 is a direct regulatory target of TGFbeta in colon cancer metastasis.

Metastasis causes most deaths from cancer yet mechanistic understanding and therapeutic options remain limited. Overexpression of the phosphatase PRL-3 (phosphatase of regenerating liver) is associated with metastasis of colon cancer. Here, we show that PRL-3 is a direct target of signaling by TGFß, which is broadly implicated in progression and metastasis. We found that suppression of PRL-3 expression by TGFß was mediated by Smad-dependent inhibition of PRL-3 transcription at the level of promoter activity. PRL-3 activation stimulated PI3K/AKT signaling that caused resistance to stress-induced apoptosis. PRL-3 overexpression promoted metastatic colonization in an orthotopic mouse model of colon cancer, whereas PRL-3 knockdown reduced metastatic potential. Altered metastatic phenotypes were not derivative of primary tumor development or local invasion but could be attributed to PRL-3-mediated cell survival. Our findings suggest that inhibiting PRL-3 expression might be an important mechanism through which TGFß suppresses metastasis in colon cancer. In addition, our findings suggest that loss of TGFß signaling, which occurs commonly during colon cancer progression, is sufficient to activate a PRL-3-mediated cell survival pathway that can selectively promote metastasis. Therefore, a major implication of our findings is that PRL-3 antagonists may offer significant value for antimetastatic therapy in patients with colon cancer.

Knockdown of Ron kinase inhibits mutant phosphatidylinositol 3-kinase and reduces metastasis in human colon carcinoma.

Abnormal accumulation and activation of receptor tyrosine kinase Ron (recepteur d'origine nantais) has been demonstrated in a variety of primary human cancers. We show that RNA interference-mediated knockdown of Ron kinase in a highly tumorigenic colon cancer cell line led to reduced proliferation as compared with the control cells. Decreased Ron expression sensitized HCT116 cells to growth factor deprivation stress-induced apoptosis as reflected by increased DNA fragmentation and caspase 3 activation. In addition, cell motility was decreased in Ron knockdown cells as measured by wound healing assays and transwell assays. HCT116 cells are heterozygous for gain of function mutant PIK3CA H1047R. Analysis of signaling proteins that are affected by Ron knockdown revealed that phosphatidylinositol 3-kinase (PI3K) activity of the mutant PI3K as well as AKT phosphorylation was substantially reduced in the Ron knockdown cells compared with the control cells. Moreover, we demonstrated in vivo that knockdown of Ron expression significantly reduced lung metastasis as compared with the control cells in the orthotopic models. In summary, our results demonstrate that Ron plays an essential role in maintaining malignant phenotypes of colon cancer cells through regulating mutant PI3K activity. Therefore, targeting Ron kinase could be a potential strategy for colon cancer treatment, especially in patients bearing gain of function mutant PI3K activity.

Transforming growth factor beta induces apoptosis through repressing the phosphoinositide 3-kinase/AKT/survivin pathway in colon cancer cells.

FET cells, derived from an early-stage colon carcinoma, are nontumorigenic in athymic mice. Stable transfection of a dominant-negative transforming growth factor beta (TGFbeta) type II receptor (DNRII) into FET cells that express autocrine TGFbeta shows loss of TGFbeta signaling and increased tumorigenicity in vivo indicating tumor suppressor activity of TGFbeta signaling in this model. The ability of tumorigenic cells to withstand growth factor and nutrient deprivation stress (GFDS) is widely regarded as a key attribute for tumor formation and progression. We hypothesized that increased tumorigenicity of FET/DNRII cells was due to loss of participation of autocrine TGFbeta in a "fail-safe" mechanism to generate cell death in response to this stress. Here, we document that loss of autocrine TGFbeta in FET/DNRII cells resulted in greater endogenous cell survival in response to GFDS due to activation of the phosphoinositide 3-kinase (PI3K)/Akt/survivin pathway. Treatment of FET DNRII cells with a PI3K inhibitor (LY294002) inhibited Akt phosphorylation and reduced survivin expression resulting in increased apoptosis in FET/DNRII cells. We also show that exogenous TGFbeta increased apoptosis in FET cells through repression of the PI3K/Akt/survivin pathway during GFDS. These results indicate that the PI3K/Akt/survivin pathway is blocked by TGFbeta signaling and that loss of autocrine TGFbeta leads to increased cell survival during GFDS through the novel linkage of TGFbeta-mediated repression of survivin expression. Inhibition of survivin function by dominant-negative approaches showed that this inhibitor of apoptosis family member is critical to cell survival in the FET/DNRII cells, thus indicating the importance of this target for TGFbeta-mediated apoptosis.

High frequency somatic mutations in RASSF1A in nasopharyngeal carcinoma.

High frequency loss of 3p21.3 region is a common event in various kinds of tumors including nasopharyngeal carcinoma (NPC). RASSF1A has been identified as a putative tumor suppressor gene residing in this region. Chromosome alterations and epigenetic changes are commonly observed as mechanisms for inactivation of RASSF1A function. In this study, we applied the PCR-cloning-sequencing strategy to examine somatic mutations in RASSF1A in NPC tissues as compared with the sequences detected in the matched peripheral blood lymphocytes. Our results revealed a high incidence of RASSF1A mutation in primary tumor tissues of NPC. There are totally 35 mutations identified in 74% (17/23) of these NPC cases, including 30 transitions, three transversions and two deletions. Most of these mutations result in amino acid changes: three nonsense (stop codon) mutations, two-1 bp deletion (frameshift), 26 missense and the remaining four are synonymous (silent). No obvious 'hot-spot' mutations were observed in this study. A similarly high rate (74%) of promoter methylation of RASSF1A was also detected in the same group of NPC tissues, but no significant correlation between mutation and methylation was detected. Our results suggest various mechanisms involved in inactivation of RASSF1A function and indicate a critical role of RASSF1A in NPC development.

Transcriptional expression of RPMS1 in nasopharyngeal carcinoma and its oncogenic potential.

The Epstein-Barr virus (EBV) BamHI A rightward transcripts (BARTs) were originally identified in C15 xenograft of nasopharyngeal carcinoma (NPC) and easily detected in a wide variety of EBV latent infection and EBV-associated tumors. It had been reported that p31 cosmid containing BARTs immortalized monkey epithelial cells, but which particular gene among BARTs family participates in the transformation procedure remains to be identified. RPMS1 is the only full-length cDNA confirmed so far and one of the most abundant spliced forms in BARTs family. To investigate the involvement of RPMS1 gene in NPC, we examined the expression of RPMS1 transcripts in NPC biopsies from Guangdong and its oncogenic potential. Our results revealed that RPMS1 mRNA preferentially expressed in primary NPC to non-carcinoma tissue of nasopharynx and peripheral blood lymphocytes (PBLs) of NPC patients. Furthermore, by introducing RPMS1 ORF into HEK293 cells, these transfectants enhanced the anchorage-independent growth and produced tumors in nude mice. These data imply that RPMS1 gene might play an important role in the development of NPC.