Optogenetically engineered Ca2+ oscillation-mediated DRP1 activation promotes mitochondrial fission and cell death.

Mitochondrial dynamics regulate the quality and morphology of mitochondria. Calcium (Ca2+) plays an important role in regulating mitochondrial function. Here, we investigated the effects of optogenetically engineered Ca2+ signaling on mitochondrial dynamics. More specifically, customized illumination conditions could trigger unique Ca2+ oscillation waves to trigger specific signaling pathways. In this study, we found that modulating Ca2+ oscillations by increasing the light frequency, intensity and exposure time could drive mitochondria toward the fission state, mitochondrial dysfunction, autophagy and cell death. Moreover, illumination triggered phosphorylation at the Ser616 residue but not the Ser637 residue of the mitochondrial fission protein, dynamin-related protein 1 (DRP1, encoded by DNM1L), via the activation of Ca2+-dependent kinases CaMKII, ERK and CDK1. However, optogenetically engineered Ca2+ signaling did not activate calcineurin phosphatase to dephosphorylate DRP1 at Ser637. In addition, light illumination had no effect on the expression levels of the mitochondrial fusion proteins mitofusin 1 (MFN1) and 2 (MFN2). Overall, this study provides an effective and innovative approach to altering Ca2+ signaling for controlling mitochondrial fission with a more precise resolution than pharmacological approaches in the temporal dimension.

Interplay between HDAC3 and WDR5 is essential for hypoxia-induced epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is important for organ development, metastasis, cancer stemness, and organ fibrosis. Molecular mechanisms to coordinately regulate hypoxia-induced EMT remain elusive. Here, we show that HIF-1α-induced histone deacetylase 3 (hdac3) is essential for hypoxia-induced EMT and metastatic phenotypes. Change of specific chromatin states is associated with hypoxia-induced EMT. Under hypoxia, HDAC3 interacts with hypoxia-induced WDR5, recruits the histone methyltransferase (HMT) complex to increase histone H3 lysine 4 (H3K4)-specific HMT activity, and activates mesenchymal gene expression. HDAC3 also serves as an essential corepressor to repress epithelial gene expression. Knockdown of WDR5 abolishes mesenchymal gene activation but not epithelial gene repression during hypoxia. These results indicate that hypoxia induces different chromatin modifiers to coordinately regulate EMT through distinct mechanisms.

Amniotic membrane and adipose-derived stem cell co-culture system enhances bone regeneration in a rat periodontal defect model.

BACKGROUND/PURPOSE: Periodontal disease is a chronic inflammatory process that potentially leads to alveolar bone destruction and tooth loss. Tissue engineering combined with stem cell therapy is a potential effective treatment for periodontal bone loss. Amniotic membrane (AM) is a potential scaffold enriched with multiple growth factors. It has the effects of anti-inflammation, antiangiogenesis, and immunosuppression. Herein, we used adipose-derived stem cells (ADSCs) and an AM co-cultured system to study bone regeneration in a rat periodontal defect model in vivo. METHODS: Human ADSCs were isolated from the infrapatellar fat pad, and characterized by flow cytometry, reverse transcription-polymerase chain reaction, and multipotent differentiation assays. The co-culture system was applied in the periodontal two-wall osseous defect in a rat model, and computed tomography was used to measure the effect. RESULTS: Human ADSCs isolated from the infrapatellar fat pad showed spindle-like morphology. Flow cytometry results demonstrated that ADSCs expressed a high level of CD90 and CD105, but not CD31, CD34, and CD45. ADSCs strongly expressed stemness genes, including SOX2, OCT4, NANOG, and KLF4 on different passages. Furthermore, ADSCs were able to differentiate into osteogenic, chondrogenic, and adipogenic cells. In the periodontal osseous defect rat model, ADSCs and the AM co-culture system significantly increased bone regeneration. CONCLUSION: This study provides the basis for using ADSCs with an AM co-culture system as stem cell therapy and scaffold transplantation in clinical periodontology.

Prognostic Significance of CDCP1 Expression in Colorectal Cancer and Effect of Its Inhibition on Invasion and Migration.

BACKGROUND: To assess the correlations and functions of complement C1r/C1s, Uegf, Bmp1 domain-containing protein-1 (CDCP1) in identifying colorectal cancer (CRC) patients who are at high risk for metastasis. METHODS: Tumor specimens from 101 patients were analyzed by real-time polymerase chain reaction to detect CDCP1 expression. CDCP1 expression plasmids and shRNA were used to knock down CDCP1 expression in this study to investigate migratory and invasive abilities by Boyden chambers. The mRNA expression profiles in shCDCP1 transfectants were compared to those in control cells by conducting microarray analysis. Its downstream effectors were also invested in this study. RESULTS: CRC patients with a high CDCP1 expression had a statistically significant lower overall survival and disease-free survival compared to those exhibiting low CDCP1 expression. In vitro, knock-down CDCP1 expression significantly decreased migratory and invasive abilities in HCT116. Aberrant expression of CDCP1 increased cancer cell migration and invasion. By using integrated genomics, we identified ROCK1 (rho-associated, coiled-coil-containing protein kinase 1 pseudogene 1) as a downstream effector in CDCP1-mediated migration and as an invasion mediator. Clinically, ROCK1 and CDCP1 mRNA expression exhibited a strong positive correlation in CRC patient samples. CONCLUSIONS: Our results implicated CDCP1 as a key regulator of CRC migration and invasion, and suggest that it is a useful prognostic factor for patients with CRC. Improved identification of a high-risk subset of early metastatic patients may guide indications of individualized treatment in clinical practice.

Connective tissue growth factor inhibits gastric cancer peritoneal metastasis by blocking integrin α3ß1-dependent adhesion.

BACKGROUND: Connective tissue growth factor (CTGF) plays important roles in normal and pathological conditions. The aim of this study was to investigate the role of CTGF in peritoneal metastasis as well as the underlying mechanism in gastric cancer progression. METHODS: CTGF expression levels for wild-type and stable overexpression clones were determined by Western blotting and quantitative polymerase chain reaction (Q-PCR). Univariate and multivariate analyses, immunohistochemistry, and survival probability analyses were performed on gastric cancer patients. The extracellular matrix components involved in CTGF-regulated adhesion were determined. Recombinant CTGF was added to cells or coinoculated with gastric cancer cells into mice to evaluate its therapeutic potential. RESULTS: CTGF overexpression and treatment with the recombinant protein significantly inhibited cell adhesion. In vivo peritoneal metastasis demonstrated that CTGF-stable transfectants markedly decreased the number and size of tumor nodules in the mesentery. Statistical analysis of gastric cancer patient data showed that patients expressing higher CTGF levels had earlier TNM staging and a higher survival probability after the surgery. Integrin α3ß1 was the cell adhesion molecule mediating gastric cancer cell adhesion to laminin, and blocking of integrin α3ß1 prevented gastric cancer cell adhesion to recombinant CTGF. Coimmunoprecipitation results indicated that CTGF binds to integrin α3. Coinoculation of recombinant CTGF and gastric cancer cell lines in mice showed effective inhibition of peritoneal dissemination. CONCLUSIONS: Our results suggested that gastric cancer peritoneal metastasis is mediated through integrin α3ß1 binding to laminin, and CTGF effectively blocks the interaction by binding to integrin α3ß1, thus demonstrating the therapeutic potential of recombinant CTGF in gastric cancer patients.

Calreticulin activates ß1 integrin via fucosylation by fucosyltransferase 1 in J82 human bladder cancer cells.

Fucosylation regulates various pathological events in cells. We reported that different levels of CRT (calreticulin) affect the cell adhesion and metastasis of bladder cancer. However, the precise mechanism of tumour metastasis regulated by CRT remains unclear. Using a DNA array, we identified FUT1 (fucosyltransferase 1) as a gene regulated by CRT expression levels. CRT regulated cell adhesion through α1,2-linked fucosylation of ß1 integrin and this modification was catalysed by FUT1. To clarify the roles for FUT1 in bladder cancer, we transfected the human FUT1 gene into CRT-RNAi stable cell lines. FUT1 overexpression in CRT-RNAi cells resulted in increased levels of ß1 integrin fucosylation and rescued cell adhesion to type-I collagen. Treatment with UEA-1 (Ulex europaeus agglutinin-1), a lectin that recognizes FUT1-modified glycosylation structures, did not affect cell adhesion. In contrast, a FUT1-specific fucosidase diminished the activation of ß1 integrin. These results indicated that α1,2-fucosylation of ß1 integrin was not involved in integrin-collagen interaction, but promoted ß1 integrin activation. Moreover, we demonstrated that CRT regulated FUT1 mRNA degradation at the 3'-UTR. In conclusion, the results of the present study suggest that CRT stabilized FUT1 mRNA, thereby leading to an increase in fucosylation of ß1 integrin. Furthermore, increased fucosylation levels activate ß1 integrin, rather than directly modifying the integrin-binding sites.

Simvastatin inhibits cysteine-rich protein 61 expression in rheumatoid arthritis synovial fibroblasts through the regulation of sirtuin-1/FoxO3a signaling.

OBJECTIVE: To examine the role of sirtuin-1 (SIRT-1)/FoxO3a in the expression of cysteine-rich protein 61 (CYR-61) in rheumatoid arthritis synovial fibroblasts (RASFs) and the influence of simvastatin on this pathway, and to determine the relationship between disease progression and FoxO3a/CYR-61 signaling in synovial fibroblasts in vivo using a rat model of collagen-induced arthritis (CIA). METHODS: In RASFs, the expression of CYR-61 and SIRT-1, the localization of FoxO3a in the nucleus/cytoplasm, and the phosphorylation/acetylation of FoxO3a were examined by Western blotting. Secretion of CCL20 was assessed by enzyme-linked immunosorbent assay. Promoter activity of the Cyr61 gene was evaluated by luciferase assay, with or without forced expression of FoxO3a and SIRT-1 by lentiviral transduction. FoxO3a-Cyr61 promoter interaction was examined by chromatin immunoprecipitation. In rats with CIA, the expression of CYR-61 and phosphorylated FoxO3a in synovial fibroblasts was examined by immunohistochemistry. RESULTS: In RASFs, simvastatin suppressed the tumor necrosis factor α (TNFα)-induced production of CYR-61 and CCL20. Nuclear levels of FoxO3a were decreased after TNFα stimulation of RASFs, and forced expression of FoxO3a reversed the inductive effects of TNFα on CYR-61. Simvastatin inhibited the nuclear export, phosphorylation, and acetylation of FoxO3a and maintained its binding to the Cyr61 promoter. Forced expression of SIRT-1 in RASFs led to decreased levels of CYR-61 and deacetylation of FoxO3a. Following treatment with simvastatin, the expression of SIRT-1 was up-regulated and SIRT-1/FoxO3a binding was enhanced in RASFs. In rats with CIA, intraarticular injection of simvastatin alleviated arthritis and suppressed CYR-61 expression and FoxO3a phosphorylation in synovial fibroblasts. CONCLUSION: CYR-61 is important in the pathogenesis of RA, and SIRT-1/FoxO3a signaling is crucial to induction of CYR-61 in RASFs. Simvastatin plays a beneficial role in inflammatory arthritis through its up-regulation of SIRT-1/FoxO3a signaling in synovial fibroblasts. Continued study of the pathways linking sirtuins, FoxO proteins, and the inflammatory responses of RASFs may provide new insights into the pathophysiology of RA.

Changes in tumor growth and metastatic capacities of J82 human bladder cancer cells suppressed by down-regulation of calreticulin expression.

Bladder cancer is a common urothelial cancer. Through proteomic approaches, calreticulin (CRT) was identified and proposed as a urinary marker for bladder cancer. CRT is a multifunctional molecular chaperone that regulates various cellular functions such as Ca(2+) homeostasis and cell adhesion. CRT is overexpressed in various cancers, but its mechanism of action in the development of bladder tumors remains unclear. We generated J82 bladder cancer cells lines that either stably overexpressed or knocked down CRT to investigate the physiological effects of CRT on bladder tumors. Compared with the transfected control vector cells, the knockdown of CRT suppressed cell proliferation, migration, and attachment, whereas overexpression of CRT enhanced cell migration and attachment. We further demonstrated that the phosphorylation status of focal adhesion kinase and paxillin, important regulators of the focal adhesion complex, was also regulated in these cells. In contrast, phosphorylation of Src, a protein tyrosine kinase reported to be affected by CRT, was not significantly different between the control and CRT-RNAi groups. Most importantly, we observed that tumors derived from J82 CRT-RNAi cells were significantly smaller and had fewer metastatic sites in the lung and liver in vivo than did transfected control vector cells. In conclusion, our results suggest that alteration of CRT expression levels might affect bladder cancer progression in vitro and in vivo.

Diagnosis of neonatal intrahepatic cholestasis caused by citrin deficiency using high-resolution melting analysis and a clinical scoring system.

OBJECTIVE: To assess the diagnosis of neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) by using high-resolution melting (HRM) analysis and a clinical scoring system. STUDY DESIGN: Genetic variations in the 18 coding exons were prescreened using HRM analysis and then confirmed by direct sequencing. To establish a scoring system, clinical features of 20 patients with NICCD diagnosed in Taiwan between the years 2000 and 2008 were compared with those of 47 patients with biliary atresia and 35 with infantile cholestasis. RESULTS: Eight types of mutations/polymorphisms were identified in patients with NICCD, including 5 mutations in the coding region or splice site (c.851del4, c.1638ins23, R553Q, IVS6+5G > A, IVS11+1G > A), and 3 single-nucleotide polymorphisms (IVS11+17C > G, IVS4+6A > G/rs6957975, and c.1194A > G/rs2301629). The 3 hotspot mutations (c.851del4, c.1638ins23, and IVS6+5G > A) comprised 33/35 (94.3%) mutated alleles. The patients with NICCD had a higher frequency of the rs6957975 polymorphism compared with 103 healthy controls (P < .0001). A 6-point scoring system was proposed according to clinical parameters. The patients with NICCD tended to score ≥ 4 points, whereas biliary atresia and other infantile cholestasis tended to score <4 points (P < .0001). CONCLUSIONS: HRM analysis was efficient and effective in detecting mutations. Three common mutations comprised the majority of mutations found in our patients. The IVS4+6A > G polymorphism was associated with NICCD. A scoring system may help to differentiate patients with NICCD from those with biliary atresia.

Prognostic significance of TWEAK expression in colorectal cancer and effect of its inhibition on invasion.

BACKGROUND: Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) has been implicated in tumor development and progression. The aim of this study was to investigate the role of TWEAK in colorectal cancer (CRC) progression. METHODS: To investigate the involvement of TWEAK in the progression of human CRC, normal, and tumor specimens from 174 patients were analyzed immunohistochemically for the expression of TWEAK. TWEAK recombinant protein treatment, transfection of expression plasmids, and small interfering RNA to knockdown TWEAK expression were performed to test invasive ability with a Boyden chamber. The mRNA expression profile in recombinant TWEAK treatment was compared to a control group by microarray analysis. To identify downstream effectors, Raf kinase inhibitor (RKIP) and its correlation with TWEAK in vitro and in vivo were examined by quantitative real-time polymerase chain reaction and invasion assays. RESULTS: CRC patients whose tumors displayed high TWEAK expression had a statistically significantly higher overall survival and a disease-free advantage over those with a low TWEAK expression. In in vitro invasion assays, alterations in TWEAK expression in CRC cell lines inversely modulated their invasive ability. By means of integrated genomics, we identified RKIP as a downstream effector in TWEAK-mediated invasion inhibition. Knockout of RKIP expression in HCT116 cells by short hairpin RNA (shRKIP) resulted in increased invasiveness. Clinically, RKIP and TWEAK mRNA expression showed strong positive correlations in CRC patient samples. CONCLUSIONS: Our results implicate TWEAK as a key regulator of CRC invasion, and it appears to be a useful prognostic factor for patients with CRC.

Simvastatin inhibits cytokine-stimulated Cyr61 expression in osteoblastic cells: a therapeutic benefit for arthritis.

OBJECTIVE: To examine the effects of proinflammatory cytokines on Cyr61 expression in osteoblastic cells and the modulatory action of simvastatin, to assess the role of CREB in Cyr61 induction, and to investigate the relationship of osteoblastic expression of Cyr61 to disease progression in experimental arthritis. METHODS: Cyr61 expression and CREB phosphorylation at serine 133 were examined by Western blotting. Promoter activity of Cyr61 was assessed by luciferase assay with promoter deletion/mutagenesis and forced expression/gene silencing of CREB. Interaction between CREB and the Cyr61 promoter was evaluated by electrophoretic mobility shift assay and chromatin immunoprecipitation. CCL2 expression was examined by Northern blotting and enzyme-linked immunosorbent assay. In rats with collagen-induced arthritis (CIA), osteoblastic expression of Cyr61 was examined by immunohistochemistry, and disease progression was assessed by clinical, radiographic, and histologic examination. RESULTS: In primary human osteoblasts and U2OS cells, Cyr61 expression stimulated by tumor necrosis factor α, interleukin-1ß (IL-1ß), oncostatin M (OSM), and other IL-6-family cytokines was suppressed by simvastatin. In U2OS cells, simvastatin inhibited OSM-induced CREB phosphorylation and CREB-DNA binding. Knockdown of CREB by short hairpin RNA reduced Cyr61 synthesis. OSM-induced Cyr61 promoter activation was dependent on CRE-CREB interaction and inhibited by simvastatin. Cyr61 enhanced CCL2 expression by U2OS cells. Intraarticular injection of simvastatin inhibited CIA progression and diminished the number of Cyr61+ osteoblasts and infiltrating macrophages. CONCLUSION: Simvastatin inhibited cytokine-stimulated Cyr61 expression in osteoblastic cells and suppressed disease progression and osteoblastic expression of Cyr61 in inflammatory arthritis. This finding indicates that simvastatin may have potential as a therapeutic agent for inflammatory arthritis.

Insulin-like growth factor II mRNA-binding protein 3 expression predicts unfavorable prognosis in patients with neuroblastoma.

Insulin-like growth factor II mRNA-binding protein 3 (IMP3) has been reported to enhance proliferation and invasion in various cancers. The role of IMP3 on neuroblastoma (NB) is unknown. We aimed to clarify the prognostic significance of IMP3 expression in patients with NB. By microarray analysis, high IMP3 expression was found in patients with poor outcome. IMP3 expression in 90 NB samples was analyzed by immunohistochemical staining to correlate with clinical stages, histology, and patient outcome. Positive IMP3 expression was detected in 52 of 90 patients, and was significantly correlated with undifferentiated histology, advanced stages, MYCN amplification, and poor outcome. In subgroups, positive IMP3 expression could predict an even worse prognosis in patients with advanced disease, with normal MYCN status, or with MYCN amplification (P = 0.005, P = 0.001, and P = 0.033, respectively). The IMP3 expression decreased by induction of differentiation with retinoid acid treatment in SK-N-DZ and SK-N-SH cells in vitro. The invasion ability of NB cells also decreased as IMP3 knockdown by using RNA interference in vitro. In summary, high expression of IMP3 in NB might contribute to the undifferentiated phenotype and invasive behaviors, leading to a poor prognosis. Determining IMP3 expression in NB could help to improve a personalized therapy.

HDAC2 promotes cell migration/invasion abilities through HIF-1α stabilization in human oral squamous cell carcinoma.

BACKGROUND: Histone deacetylase 2 (HDAC2) expressions in oral squamous cell carcinoma (OSCC) had been implicated in advanced stage and poor prognosis. It suggests a possible link between the migration/invasion potential of oral cancer cells and the prevalent expression of HDAC2. METHODS: Five head and neck cancer (HNC) cell lines, including Ca9-22, Cal-27, HSC-3, SAS, and TW2.6, were used. Cells stably overexpressing HDAC2 and shRNA against HDAC2 were established to investigate migration/invasion ability in vitro and tumorigenesis and progression in vivo. RESULTS: We found that alterations in the HDAC2 level in OSCC cell lines modulated their invasive ability with a positive correlation. Animal model also showed that knockdown of HDAC2 expression in SAS cells, originally containing high endogenous HDAC2 expression, resulted in decrease in tumor initiation and progression. Using high-throughput transcriptome analysis, numerous genes involved in HIF-1α-associated pathways were found. At the mechanism levels, using agents to block de novo protein synthesis or prevent protein degradation by ubiquitination, we found the stability of hypoxia inducible factor 1α (HIF-1α) protein was maintained in OSCC cells with HDAC2 overexpression. In addition, co-immunoprecipitation assay also revealed that HDAC2-mediated HIF-1α protein stability is because of direct interaction of HIF-1α with von Hippel-Lindau (VHL) protein. CONCLUSIONS: Our work demonstrates that HDAC2 maintains HIF-1α stability, probably at the level of protein modification, which in turn leads to the increase in cell invasion/migration ability in oral cancer progression. These findings implicate the potential of HDAC inhibitors for oral cancer therapy.

Insulin-like growth factor II mRNA-binding protein 3 expression promotes tumor formation and invasion and predicts poor prognosis in oral squamous cell carcinoma.

BACKGROUND: Insulin-like growth factor II mRNA-binding protein 3 (IGF2BP3), an oncofetal RNA-binding protein, has been implicated in the enhancement of proliferation and invasion in various cancers. This study aimed to investigate the clinical significance and functional role of IGF2BP3 expression in oral squamous cell carcinoma (OSCC). METHODS: IGF2BP3 expression in 93 OSCC patients was investigated using immunohistochemical staining and correlated with clinical parameters and patients' survival. The effect of IGF2BP3 on cell invasion ability was evaluated by RNA interference in OSCC cell line. RESULTS: High expression of IGF2BP3 in OSCC was significantly correlated with large tumor size and lymph node metastasis. Kaplan-Meier analysis revealed that oral cancer patients with high IGF2BP3 expression had a significantly lower 5-year survival (P = 0.0017). Multivariate analysis of clinical samples demonstrated IGF2BP3 to be an independent prognosis factor (P = 0.003). Moreover, the IGF2BP3 shRNA significantly suppressed the invasion ability of OSCC in vitro, and the knockdown of endogenous IGF2BP3 expression also inhibited tumor formation in vivo. CONCLUSIONS: IGF2BP3 enhances cell invasion ability and tumorigenicity in human OSCC in vitro and in vivo. IGF2BP3 is an independent prognostic factor in patients with OSCC. Targeting of IGF2BP3 could potentially suppress the tumor growth and metastasis to improve the outcome of patients with OSCC.

Stromal cell-derived factor-1alpha (SDF-1alpha/CXCL12)-enhanced angiogenesis of human basal cell carcinoma cells involves ERK1/2-NF-kappaB/interleukin-6 pathway.

Stromal cell-derived factor 1alpha (SDF-1alpha) (CXCL12) has been observed to enhance tumor angiogenesis. However, the comprehensive role of SDF-1alpha (CXCL12)-CXCR4 interaction, exerted during angiogenesis, has not been well understood. We have previously demonstrated that human basal cell carcinoma (BCC) tissues and a BCC cell line (BCC-1/KMC) had significant expression of CXCR4, whose level was higher in invasive than in the non-invasive BCC types. Here, we observed that human BCC tissues with high expression levels of CXCR4 had higher vascularity. Further, among the 71 BCCs diagnosed between the years 2004-2005, BCCs with high CXCR4 expression had concomitantly higher microvessel density, as compared with those with low CXCR4 expression (P < 0.001). We found that SDF-1alpha induced angiogenic activity in human BCC cells, both in vitro and in vivo. SDF-1alpha significantly upregulated several angiogenesis-associated genes such as interferon-alpha-inducible protein 27, interleukin (IL)-6, bone morphogenetic protein (BMP)-6, SOCS2 and cyclooxygenase 2 (COX)-2 in human BCC cells. Among them, IL-6 was the earliest and highest upregulated gene whose induction was observed within 6 h of the commencement of SDF-1alpha-CXCR4 interaction. The mechanisms behind the SDF-1alpha-induced time and dose-dependent upregulation of messenger RNA expression and protein secretion of IL-6 were investigated. The transcriptional regulation of IL-6 by SDF-1alpha was mediated by phosphorylation of extracellular signal-related kinase 1/2 and activation of the nuclear factor-kappaB complex. The identification of the angiogenic profiles induced through SDF-1alpha-CXCR4 interactions in human BCC cells may contribute further insights into the mechanisms involved in the angiogenic potential of SDF-1alpha (CXCL12).

RNA-binding protein insulin-like growth factor II mRNA-binding protein 3 expression promotes tumor invasion and predicts early recurrence and poor prognosis in hepatocellular carcinoma.

UNLABELLED: Insulin-like growth factor II mRNA-binding protein 3 (IMP3) is an RNA-binding protein expressed in embryonic tissues and multiple cancers. To investigate the role of IMP3 in hepatocellular carcinoma (HCC), its protein expression in the surgically resected unifocal tumors of 377 HCC patients (296 men and 81 women) with ages ranging from 7 to 88 years (mean, 55.49 years) was analyzed by immunohistochemistry. IMP3 was expressed in 255 (67.6%) of 377 resected unifocal primary HCCs. IMP3 protein was predominantly expressed in tumor border and invasive front, and it was more abundant in the satellite nodules and tumor thrombi than in the main tumors. The expression correlated with high alpha-fetoprotein (>200 ng/mL, P < 1 x 10(-7)), larger tumor size (>5 cm, P = 0.006), high tumor grade (P < 1 x 10(-7)), and high tumor stage with vascular invasion and various degrees of intrahepatic metastasis (P < 1 x 10(-7)). IMP3 expression predicted early tumor recurrence (P < 1 x 10(-7)) and was a strong indicator of poor prognosis (P < 0.0001). Depletion of IMP3 with RNA interference in HCC cell line HA22T caused a decrease in cell motility, invasion, and transendothelial migration. Microarray analysis revealed that IMP3 depletion was associated with downregulation of multiple genes involved in tumor invasion. CONCLUSION: Our results indicate that IMP3 plays an important role in tumor invasion and metastasis and is a strong prognostic factor for patients with HCC.

Identification of calreticulin as a prognosis marker and angiogenic regulator in human gastric cancer.

The purpose of this study was to identify genes of interest for a subsequent functional and clinical cohort study using complementary (c)DNA microarrays. cDNA microarray hybridization was performed to identify differentially expressed genes between tumor and nontumor specimens in 30 gastric cancer patients. Subsequent functional studies of the selected gene were carried out, including cell cycle analysis, cell migration analysis, analyses of vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF), and oligo-microarray studies using two pairs of stable cell lines of the selected gene. Another independent cohort study of 79 gastric cancer patients was conducted to evaluate the clinical significance of the selected gene in human gastric cancer. Calreticulin (CRT) was selected for further investigation. Two pairs of stable cell lines of CRT overexpression and CRT knockdown were constructed to perform functional studies. CRT enhanced gastric cancer cell proliferation and migration. Overexpressed CRT upregulated the expression and secretion of PlGF and VEGF. CRT had a reciprocal effect on connective tissue growth factor (CTGF) expression. Positive immunohistochemical staining of calreticulin was significantly correlated with high microvessel density (MVD) (p = 0.014), positive serosal invasion (p = 0.013), lymph node metastasis (p = 0.002), perineural invasion (p = 0.008), and poor patient survival (p = 0.0014). Multivariate survival analysis showed that CRT, MVD, and serosal invasion were independent prognosticators. We conclude that CRT overexpression enhances angiogenesis, and facilitates proliferation and migration of gastric cancer cells, which is in line with the association of CRT with MVD, tumor invasion, lymph node metastasis, and survival in gastric cancer patients.

Lysophosphatidic acid upregulates vascular endothelial growth factor-C and tube formation in human endothelial cells through LPA(1/3), COX-2, and NF-kappaB activation- and EGFR transactivation-dependent mechanisms.

Lysophosphatidic acid (LPA) is a lipid bioactive mediator which binds to G-protein-coupled receptors and activates a variety of cellular functions. LPA modulates multiple behaviors in endothelial cells, including cell proliferation and migration, capillary-like tube formation in vitro, activation of proteases, interactions with leukocytes, and expressions of inflammation-related genes, thereby regulating vessel formation. LPA has been reported to modulate the angiogenesis process. However, the role of LPA in the lymphangiogenesis process has not been studied. In this study, we showed that LPA upregulated vascular endothelial growth factor-C (VEGF-C) mRNA expression in human umbilical vein endothelial cells (HUVECs) and subsequent endothelial cell tube formation in vitro and in vivo. These enhancement effects were LPA(1)- and LPA(3)-dependent and required cyclooxygenase-2 (COX-2), endothelial growth factor receptor (EGFR) transactivation and activation of nuclear factor kappaB (NF-kappaB). Moreover, LPA induced the protein expressions of the lymphatic markers, Prox-1, LYVE-1, and podoplanin, in HUVECs, and these enhancement effects were dependent on LPA(1) and LPA(3) activation and EGFR transactivation. Our results demonstrated that LPA might regulate VEGF-C and lymphatic marker expression in endothelial cells, which contributes to endothelial cell tube formation in vitro and in vivo, thus facilitating endothelial cell participation in the lymphangiogenesis process. This study clarifies the signaling mechanism of LPA-regulated VEGF-C expression and lymphatic marker expressions in endothelial cells, which suggest that LPA may be a suitable target for generating therapeutics against lymphangiogenesis and tumor metastasis.

CXCL12/CXCR4 promotes laryngeal and hypopharyngeal squamous cell carcinoma metastasis through MMP-13-dependent invasion via the ERK1/2/AP-1 pathway.

Laryngeal and hypopharyngeal squamous cell carcinomas (LHSCCs) are common head and neck cancers with a high propensity for lymph node (LN) and lung metastasis. Here, we report that LHSCCs express high levels of functional CXCR4 receptors, native for chemokine stromal cell-derived factor-1 (SDF-1/CXCL12). Primary tumor immunohistochemistry from LHSCC patients has revealed significant expression of CXCR4 and CXCL12. Greater expression of CXCR4 but not that of CXCL12 is correlated with LN and distant metastasis. Reverse transcription-polymerase chain reaction and western blots have demonstrated that CXCR4 messenger RNA (mRNA) and protein were expressed in LHSCC cell lines as well, but failed to detect CXCL12 mRNA expression. CXCL12 treatment enhanced extracellular signal-regulated kinase (ERK) pathway activation and the motility/invasiveness of LHSCC cell lines, which were blocked by treatment with a CXCR4 antagonist (AMD3100) and a specific MEK inhibitor (U0126). Results show that the mRNA and protein levels of matrix metalloproteinase (MMP)-13, but not MMP-2 or MMP-9, were elevated in HEp-2 cells in response to CXCL12. Again, U0126 almost inhibited the induction of MMP-13 in HEp-2 cells by stimulating CXCL12. The transcriptional factor, c-Jun, a downstream factor of ERK pathway, was found to be readily phosphorylated and translocated to the nucleus after 10 min of exposure to CXCL12. Blockage of c-Jun activity by transfection with c-jun antisense oligodeoxynucleotide significantly decreased CXCL12-induced MMP-13 expression and cell invasion. CXCL12 seems to enhance LHSCC cell invasion through paracrine-activated CXCR4, which triggers ERK/c-Jun-dependent MMP-13 upregulation.