Promoted Cell Mobility Research Articles

CXCR3 isoform A promotes head and neck cancer progression by enhancing stem-like property and chemoresistance.

The chemokine network orchestrates the cancer stem-like property and consequently participates in cancer progression. CXCR3 contributes cancer progressive property and immunomodulation in the tumor microenvironment. The two major isoforms of CXCR3 are scrutinized and the divergence is showed that CXCR3A promotes cancer cell growth and motility while CXCR3B functions contrarily in many studies. However, rare studies illustrate the role of CXCR3 isoforms in cancer stem-like property and chemoresistance, especially in head and neck cancer (HNC). Levels of CXCR3, CXCR3B, and Sox2 were determined in HNC tissue microarray by immunohistochemistry staining to explore potential clinical relevance. Lentivirus-mediated CXCR3-isoform overexpression with MTS assay, clonogenic assay, transwell migration, sphere formation, and chemo-drug susceptibility were implemented to investigate the role of CXCR3-isofoms in HNC. High levels of CXCR3 were significantly associated with advanced stage (p < 0.01), regional lymph node metastasis (p < 0.05), and poor differentiation (p < 0.005) and further correlated with worse survival rate in oral cancer patients (p=0.036). Higher levels of CXCR3B were found in regional lymphatic invasion of HNC and progressive stage of squamous cell carcinoma. Elevated Sox2 expression was significantly associated with the advanced stage of HNC in the oral cavity, and demonstrated a co-expression pattern with CXCR3B. Furthermore, lentivirus-mediated overexpression of CXCR3A and CXCR3B in SAS human oral cancer cells promoted cell mobility. CXCR3A overexpression enhanced sphere-forming ability and chemoresistance of CSCs by upregulating stemness-related genes. This study first provides a novel insight of CXCR3 isoform A in HNC cancer progression via regulating cancer stem-like properties and chemoresistance, suggesting that CXCR3A may be a prognostic marker and novel target for HNC therapy.

Placenta-specific 8 (PLAC8) mediates inflammation and mobility of the hPDLCs via MEK/ERK signaling pathway

BackgroundPlacenta-specific 8 (PLAC8) is reported to regulate cellular functions in the progression of various diseases. However, its role in periodontitis is still unclear. MethodsHuman periodontal ligament cells (hPDLCs) were treated with lipopolysaccharide of Porphyromonas Gingivalis (LPS-PG) to mimic periodontitis in vitro. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to measure the mRNA expression levels and western blot was for protein levels. Wound healing and transwell migration assays were performed to assess the cell mobility of hPDLCs. Both mRNA and protein levels of inflammatory cytokines including IFN-γ, IL-17, TNF-α, IL-4, IL-10 and IL-13 were accessed to evaluated process of periodontitis in vitro. Furthermore, the protein expressions of mitogen-activated protein kinase kinase (MEK), extracellular regulated protein kinase (ERK) and their phosphorylated products quantified by western blotting assay were determined to confirm the activation of the MEK/ERK signaling pathway. ResultsThe microarray analysis results showed that PLAC8 was most significantly downregulated in periodontium samples of patients with periodontitis, which participates in blood coagulation and integrin-mediated signaling pathway. PLAC8 was also markedly downregulated in the LPS-PG-treated hPDLCs. Moreover, overexpression of PLAC8 ameliorated inflammation and promoted cell mobility of LPS-PG-treated hPDLCs, while inhibition of PLAC8 exhibited the opposite effects. MEK/ERK was selected based on analyses of the protein-protein interaction (PPI) network as the potential signaling pathway interacted with PLAC8, and PLAC8 showed regulatory function on activation of the MEK/ERK pathway. Additionally, U0126, the inhibitor of MEK, abrogated the effects of PLAC8 on inflammation and cell mobility of LPS-PG-treated hPDLCs. ConclusionOverexpression of PLAC8 protected hPDLCs from dysfunction of inflammation and cell mobility via activating MEK/ERK pathway, indicating a novel therapeutic target for periodontitis.

Roughness and dynamics of proliferating cell fronts as a probe of cell\u2013cell interactions

Juxtacellular interactions play an essential but still not fully understood role in both normal tissue development and tumour invasion. Using proliferating cell fronts as a model system, we explore the effects of cell–cell interactions on the geometry and dynamics of these one-dimensional biological interfaces. We observe two distinct scaling regimes of the steady state roughness of in-vitro propagating Rat1 fibroblast cell fronts, suggesting different hierarchies of interactions at sub-cell lengthscales and at a lengthscale of 2–10 cells. Pharmacological modulation significantly affects the proliferation speed of the cell fronts, and those modulators that promote cell mobility or division also lead to the most rapid evolution of cell front roughness. By comparing our experimental observations to numerical simulations of elastic cell fronts with purely short-range interactions, we demonstrate that the interactions at few-cell lengthscales play a key role. Our methodology provides a simple framework to measure and characterise the biological effects of such interactions, and could be useful in tumour phenotyping.

Inhibition of the Notch signaling pathway attenuates progression of cell motility, metastasis, and epithelial-to-mesenchymal transition-like phenomena induced by low concentrations of cisplatin in osteosarcoma

Although advances in osteosarcoma treatment have been made in recent decades, the survival rate for patients suffering from metastatic disease, especially lung metastasis, remains disappointing. Previous studies have confirmed that epithelial-to-mesenchymal transition (EMT) is associated with tumor metastasis, and several studies have suggested that osteosarcoma cells also exhibit EMT-like characteristics. In addition, Notch signaling is known to be related to the development and progression of human malignancies, including osteosarcoma. However, whether chemotherapy affects the EMT-like events and whether these events are medicated by Notch signaling remain to be elucidated. To address these issues, in the current work, osteosarcoma 143B cells were exposed to sublethal concentrations of the first-line chemotherapeutic agent cisplatin (DDP), which promoted cell migration, in vitro invasion, and in vivo lung metastasis. Furthermore, low concentrations of DDP upregulated mesenchymal phenotype-related genes and proteins and promoted EMT-like properties in osteosarcoma cells. In addition, low concentrations of DDP could activate the Notch receptor and its target genes. Finally, combined treatment of DDP with the Notch signaling pathway inhibitor DAPT, which can effectively downregulate mesenchymal phenotype-related genes and proteins, inhibited cell migration and invasion in vitro, and it decreased pulmonary metastatic nodules in vivo. The results of the current study supported the idea that low concentrations of DDP could induce EMT-like characteristics in osteosarcoma cells and could promote cell mobility in vitro, as well as pulmonary metastasis in vivo. Importantly, however, these biological processes are mediated by the Notch signaling pathway. Blocking the Notch signaling pathway can effectively attenuate the osteosarcoma EMT-like phenotype and its associated migration, invasion, and metastasis.

CD142 plays an important role in the mobility of colorectal cancer cells

CD142 promotes cell mobility, which contributes to carcinogenesis. However, the role of CD142 on colorectal cancer (CRC) mobility is unclear. This study showed that CD142 expression increased in CRC tissues, especially in those with invasion or metastasis. The positive sorting or overexpression of CD142 promoted the invasion and migration of CRC cells. Overall, CD142 may be responsible for CRC mobility.

T cell and chemokine receptors differentially control CD8 T cell motility behavior in the infected airways immediately before and after virus clearance in a primary infection.

In mice, experimental influenza virus infection stimulates CD8 T cell infiltration of the airways. Virus is cleared by day 9, and between days 8 and 9 there is an abrupt change in CD8 T cell motility behavior transitioning from low velocity and high confinement on day 8, to high velocity with continued high confinement on day 9. We hypothesized that loss of virus and/or antigen signals in the context of high chemokine levels drives the T cells into a rapid surveillance mode. Virus infection induces chemokine production, which may change when the virus is cleared. We therefore sought to examine this period of rapid changes to the T cell environment in the tissue and seek evidence on the roles of peptide-MHC and chemokine receptor interactions. Experiments were performed to block G protein coupled receptor (GPCR) signaling with Pertussis toxin (Ptx). Ptx treatment generally reduced cell velocities and mildly increased confinement suggesting chemokine mediated arrest (velocity <2 μm/min) (Friedman RS, 2005), except on day 8 when velocity increased and confinement was relieved. Blocking specific peptide-MHC with monoclonal antibody unexpectedly decreased velocities on days 7 through 9, suggesting TCR/peptide-MHC interactions promote cell mobility in the tissue. Together, these results suggest the T cells are engaged with antigen bearing and chemokine producing cells that affect motility in ways that vary with the day after infection. The increase in velocities on day 9 were reversed by addition of specific peptide, consistent with the idea that antigen signals become limiting on day 9 compared to earlier time points. Thus, antigen and chemokine signals act to alternately promote and restrict CD8 T cell motility until the point of virus clearance, suggesting the switch in motility behavior on day 9 may be due to a combination of limiting antigen in the presence of high chemokine signals as the virus is cleared.

Circular RNA circTMEM45A Acts as the Sponge of MicroRNA-665 to Promote Hepatocellular Carcinoma Progression

Mounting evidences indicate that circular RNAs (circRNAs) play vital roles in the development and progression of various cancers. However, the detailed functions and underlying mechanisms of circRNAs in hepatocellular carcinoma (HCC) remain largely unknown. The expression profile of circRNAs was screened by circRNA microarrays. Quantitative real-time PCR was used to determine the level-10 circRNAs selected from the top five upregulated (hsa_circ_0001955, hsa_circ_0001535, hsa_circ_0061395, hsa_circ_0000502, and hsa_circ_0066659) and top five downregulated circRNAs (hsa_circ_0046366, hsa_circ_0003418, hsa_circ_0026134, hsa_circ_0005692, and hsa_circ_0014130). The effects of circTMEM45A in HCC cells were studied both in vitro (in a Cell Counting Kit-8 assay, apoptosis analysis, and cell cycle assays) and in vivo (by means of tumor xenografts in nude mice). Luciferase reporter, RNA immunoprecipitation (RIP), and rescued assays were used to confirm the interactions between circTMEM45A, miR-665, and insulin growth factor 2 (IGF2). We found that the level of circTMEM45A was significantly upregulated in HCC and was positively correlated with clinicopathological features and poor prognosis of patients with HCC. Functionally, circTMEM45A promoted cell mobility in vitro, as well as in vivo tumorigenesis. Mechanistically, circTMEM45A acted as a miR-65 sponge to relieve the repressive effect of miR-665 on its target IGF2. Moreover, circTMEM45A was upregulated in serum exosomes from HCC patients. circTMEM45A promotes HCC progression through the miR-665/IGF2 axis and may serve as a novel diagnostic marker and target for treatment of HCC patients.

Abstract 4155: ARID1A loss leads to chemotherapy resistance and is associated with a poor clinic outcome for patients with non-small cell lung cancer

Abstract Purpose: ARID1A (AT-rich interactive domain 1A) is a member of the switch/sucrose nonfermentable (SWI/SNF) chromatin remodeling complex, and plays important roles in the regulation of gene transcription and DNA damage responses. ARID1A mutations occur frequently in non-small cell lung cancer (NSCLC), but the functions of ARID1A loss in the initiation, progression and maintenance of NSCLC remains largely unknown. This study aimed to investigate the clinical, pathological, and functional significance of ARID1A in NSCLC. Experimental design: We first analyzed the prognostic value of ARID1A using a gene-expression microarray from the Director's Challenge Lung Study. Kaplan-Meier method and Log-rank tests were used to estimate the correlation between ARID1A expression and overall survival (OS). Univariate and multivariate Cox regression models were used to investigate the relationship between ARID1A and other clinical covariates. We further established stable ARID1A knockdown H1299 and H358 cell lines, and ARID1A exogenously expressing H460 and A549 cell lines. We performed functional assays to study the effects of ARID1A on proliferation, migration, invasion, cell cycle progression, sensitivity to chemotherapy, and in vivo tumor growth. Results: In the Director's Challenge Study, decreased expression of ARID1A was associated with lymph node dissemination, higher T-stage (T3/T4), and worse survival compared with high expression in patients in the whole group (n=440) and subgroup who received surgery alone and no adjuvant therapy (n=328). ARID1A ablation by shRNA suppressed cell proliferation, promoted cell mobility, increased G2/M phase cell cycle distribution, and protected NSCLC cells from chemotherapy; whereas overexpression of ARID1A showed the opposite biological effects. Consistent with in vitro findings, in vivo tumor xenografts demonstrated ARID1A depletion led to reductions in cell growth but resistance of NSCLC to chemotherapy. Conclusions: Our results suggest that loss of ARID1A suppresses NSCLC cell growth perhaps by inducing G2/M arrest, but promotes cell migration/invasion, and protect cells from chemotherapy. Further study of ARID1A is needed to better define its roles in the initiation and progression of NSCLC and response to chemotherapy. Citation Format: Linlin Yang, Changxian Shen, Xiaokui Mo, Terence M. Williams. ARID1A loss leads to chemotherapy resistance and is associated with a poor clinic outcome for patients with non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4155.

Circular RNA circNHSL1 promotes gastric cancer progression through the miR-1306-3p/SIX1/vimentin axis

BackgroundMounting evidences indicate that circular RNAs (circRNAs) play vital roles in the development and progression of various cancers. However, the detail functions and underlying mechanisms of circRNAs in gastric cancer remain largely unknown.MethodsThe expression profile of metastasis-related circRNAs was screened by RNA-seq analysis. qRT-PCR was used to determine the level and prognostic values of circNHSL1 in gastric cancer tissues. In vitro cell wound healing and transwell (migration and invasion) and in vivo tumorigenesis and metastasis assays were performed to evaluate the functions of circNHSL1. Luciferase reporter, RNA immunoprecipitation (RIP) and rescued assays were employed to confirm the interactions between circNHSL1, miR-1306-3p and SIX1. It’s widely accepted that as a mesenchymal marker, Vimentin promotes invasion and metastasis in various cancers. Luciferase reporter assay was used to determine the regulation of SIX1 on Vimentin. In addition, In situ hybridization (ISH) was performed to detect the level and prognostic values of miR-1306-3p.ResultsWe found that the level of circNHSL1 was significantly up-regulated in gastric cancer, and positively correlated with clinicopathological features and poor prognosis of patients with gastric cancer. Functionally, circNHSL1 promoted cell mobility and invasion, as well as in vivo tumorgenesis and metastasis. Mechanistically, circNHSL1 acted as a miR-1306-3p sponge to relieve the repressive effect of miR-1306-3p on its target SIX1. Moreover, SIX1 enhanced Vimentin expression in the transcriptional level through directly binding to the promoter domain of Vimentin, thereby promoting cell migration and invasion. In addition, miR-1306-3p was down-regulated and negatively correlated with pathological features and poor prognosis in gastric cancer.ConclusionsCircNHSL1 promotes gastric cancer progression through miR-1306-3p/SIX1/Vimentin axis, and may serve as a novel diagnostic marker and target for treatment of gastric cancer patients.

Nanoscale and Macroscale Scaffolds with Controlled-Release Polymeric Systems for Dental Craniomaxillofacial Tissue Engineering.

Tremendous progress in stem cell biology has resulted in a major current focus on effective modalities to promote directed cellular behavior for clinical therapy. The fundamental principles of tissue engineering are aimed at providing soluble and insoluble biological cues to promote these directed biological responses. Better understanding of extracellular matrix functions is ensuring optimal adhesive substrates to promote cell mobility and a suitable physical niche to direct stem cell responses. Further, appreciation of the roles of matrix constituents as morphogen cues, termed matrikines or matricryptins, are also now being directly exploited in biomaterial design. These insoluble topological cues can be presented at both micro- and nanoscales with specific fabrication techniques. Progress in development and molecular biology has described key roles for a range of biological molecules, such as proteins, lipids, and nucleic acids, to serve as morphogens promoting directed behavior in stem cells. Controlled-release systems involving encapsulation of bioactive agents within polymeric carriers are enabling utilization of soluble cues. Using our efforts at dental craniofacial tissue engineering, this narrative review focuses on outlining specific biomaterial fabrication techniques, such as electrospinning, gas foaming, and 3D printing used in combination with polymeric nano- or microspheres. These avenues are providing unprecedented therapeutic opportunities for precision bioengineering for regenerative applications.

MEG3/miR‑21 axis affects cell mobility by suppressing epithelial‑mesenchymal transition in gastric cancer.

The prognosis of patients with gastric cancer remains poor mainly due to distant metastasis. Maternally expressed gene 3 (MEG3), a long non-coding RNA (lncRNA), is downregulated in various tumor tissues and suppresses tumor progression. miR-21 is a microRNA which is expressed highly in tumor tissues. In the present study, we investigated the relationship between MEG3 and miR-21 in regards to the cell mobility of gastric cancer. Our data demonstrated that MEG3 was downregulated while miR-21 was upregulated in gastric cancer tissues and cell lines by qRT-PCR. Overexpression of MEG3 suppressed cell mobility of gastric cancer cells (AGS) by downregulating the expression of MMP-3, MMP-9 and VEGF. As shown by western blot analysis, overexpression of MEG3 also suppressed epithelial-mesenchymal transition (EMT) by increasing the expression of an epithelial marker (E-cadherin) and downregulating the expression of mesenchymal markers (N-cadherin, Snail and β-catenin), indicating that MEG3 suppressed cell mobility through the inhibition of EMT in gastric cancer. The expression of miR-21 was negatively regulated by MEG3 and overexpression of miR-21 promoted cell mobility of AGS through activation of EMT. Co-transfection of lncRNA-MEG3 and miR-21 mimic counteracted the inhibitory effect on the cell mobility attributed to MEG3, suggesting that the MEG3/miR-21 axis affects cell mobility by suppressing EMT in gastric cancer. Using a mouse xenograft tumor model, we found that the overexpression of MEG3 suppressed tumor growth and metastasis while overexpression of miR-21 had the opposite effects. The MEG3/miR-21 axis affected gastric cancer growth and metastasis through inhibition of EMT in vivo. In conclusion, we demonstrated that the MEG3/miR-21 axis participates in the tumor progression and metastasis of gastric cancer through the regulation of EMT.

P53-R273H upregulates neuropilin-2 to promote cell mobility and tumor metastasis

Mounting evidence indicates that hotspot p53 mutant proteins often possess gain-of-function property in promoting cell mobility and tumor metastasis. However, the molecular mechanisms are not totally understood. In this study, we demonstrate that the hotspot mutation, p53-R273H, promotes cell migration, invasion in vitro and tumor metastasis in vivo. p53-R273H significantly represses expression of DLX2, a homeobox protein involved in cell proliferation and pattern formation. We show that p53-R273H-mediated DLX2 repression leads to upregulation of Neuropilin-2 (NRP2), a multifunctional co-receptor involved in tumor initiation, growth, survival and metastasis. p53-R273H-induced cell mobility is effectively suppressed by DLX2 expression. Furthermore, knockdown of NRP2 significantly inhibits p53-R273H-induced tumor metastasis in xenograft mouse model. Together, these results reveal an important role for DLX2-NRP2 in p53-R273H-induced cell mobility and tumor metastasis.

Upregulation of Wnt signaling under hypoxia promotes lung cancer progression.

The hypoxic tumor microenvironment induces epithelial-mesenchymal transition (EMT) in tumor cells and increases tumor cell malignancy. Previous studies indicated that malfunction of Wnt signaling is observed in some lung cancer patients. Athough crosstalk between hypoxia and Wnt signaling in tumor cells has recently been revealed, the detailed underlying mechanisms have not been well defined. In the present study, we demonstrated that hypoxia in lung adenocarcinoma cells can enhance Wnt signaling activity by stabilizing β-catenin and altering its localization into the nucleus. Overexpression of HIF-2α increased β-catenin expression, promoted cell mobility, and induced morphological changes to a greater degree than HIF-1α overexpression. Knockdown of HIF-2α decreased β-catenin expression and inhibited hypoxia-induced cell mobility. Moreover, we identified that phosphorylational activation of AKT1 by hypoxia and HIF-2α was required for Wnt activation upon hypoxia treatment. Downregulation of HIF-2α and β-catenin reduced colony formation when cells were exposed to long-term hypoxia treatment. Taken together, our data support that hypoxia activates PI3K/AKT as well as Wnt signaling in a HIF-2α-dependent manner, thus elevating the resistance of lung cancer cells to chronic hypoxia-induced stress.

LGR5 Promotes Breast Cancer Progression and Maintains Stem-Like Cells Through Activation of Wnt/β-Catenin Signaling.

The cancer stem cell (CSC) hypothesis suggests that a subset of cancer cells possesses stem cell properties and is crucial in tumor initiation, metastasis, and drug resistance. To determine the mechanism of CSCs in breast cancer, we focused on LGR5, a marker of adult stem cells that potentially serves as a functional factor in CSCs. LGR5 overexpression was detected in breast cancer and significantly associated with breast cancer recurrence and poor outcome. LGR5 promoted cell mobility, tumor formation, and epithelial-mesenchymal transition in breast cancer cells by activating Wnt/β-catenin signaling. In addition, LGR5 was more highly expressed in tumorspheres and increased the stemness of breast cancer cells. Compared with LGR5 low-expression (LGR5(low) ) cells, LGR5(high) cells exhibited CSC/tumor-initiating cell-like properties, including the formation of self-renewing spheres and high tumorigenicity. Importantly, our studies indicate that LGR5 activation of Wnt/β-catenin signaling is a possible mechanism to regulate breast CSC/tumor-initiating cell renewal. These findings indicate that LGR5 not only participates in carcinogenesis but also maintained stemness by activating Wnt/β-catenin signaling in breast cancer.

Piezo1 is as a novel trefoil factor family 1 binding protein that promotes gastric cancer cell mobility in vitro.

Trefoil factor family 1 (TFF1) is a member of the TFF-domain peptide family involved in epithelial restitution and cell motility. Recently, we screened Piezo1 as a candidate TFF1-binding protein. We aimed to confirm Piezo1 as a novel TFF1 binding protein and to assess the role of this interaction in mediating gastric cancer cell mobility. This interaction was confirmed by co-immunoprecipitation and co-localisation of TFF1 and Piezo1 in GES-1 cells. We used stable RNA interference to knockdown Piezo1 protein expression and restored the expression of TFF1 in the gastric cancer cell lines SGC-7901 and BGC-823. Cell motility was evaluated using invasion assay and migration assay in vitro. The expression levels of the integrin subunits β1, β5, α1 as well as the expression of β-catenin and E-cadherin were detected by Western blot. We demonstrate that TFF1, but not TFF2 or TFF3, bind to and co-localize with Piezo1 in the cytoplasm in vitro. TFF1 interacts with the C-terminal portion of the Piezo1 protein. Wound healing and trans-well assays demonstrated that the restored expression of TFF1 promoted cell mobility in gastric cancer cells, and this effect was attenuated by the knockdown of Piezo1. Western blots demonstrated the decreased expression of integrin β1 in Piezo1-knockdown cells. Our data demonstrate that Piezo1 is a novel TFF1 binding protein that is important for TFF1-mediated cell migration and suggest that this interaction may be a therapeutic target in the invasion and metastasis of gastric cancer.

Identification of LASEP3 as a new serological and prognostic biomarker for lung and various type of cancer.

e13565 Background: Identification of early cancer detection and prognostic biomarkers is urgently required to improve clinical outcome. Methods: We have screened cancer biomarkers using a strategy as follows; i)To identify up-regulated genes in non-small cell lung cancers (NSCLCs) using the cDNA microarray representing 27,648 genes and 120 lung cancers, ii) To verify the candidate genes for their no or low expression in 23 normal tissues by northern-blot analysis, iii)To validate clinicopathological significance of their protein expression by tissue microarray consisting of 259 lung cancers, iv) To verify whether they are essential for the growth of cancer cells by siRNA assay, and v) To measure their serum protein levels by ELISA in patients with various type of cancers. Results: We identified LASEP3 (Lung cancer Associated Serum Protein 3) encoding secreted protein as a candidate. Immunohistochemical analysis showed that LASEP3 was frequently over-expressed in various histological types of lung cancers; positive staining of LASEP3 was observed in 198 (54.8%) of 361 NSCLCs. In addition, a high level of LASEP3 expression was associated with poor prognosis of patients with NSCLC (p=0.0183 by log-rank test). Serum LASEP3 levels were higher in NSCLC patients than in healthy volunteers. The proportion of serum LASEP3-positive cases was 160 (61.8%) of 259 NSCLCs; 131 (63.6%) of 206 ADCs and 29 (54.7%) of 53 SCCs, while 6 (5.5%) of 109 healthy volunteers were falsely diagnosed. Serum LASEP3 levels were significantly higher in breast and colon cancer patients than in healthy volunteers, and tissue LASEP3 protein was overexpressed in breast and colon cancer tissues. Furthermore, treatment of lung cancer cells with siRNAs against LASEP3 suppressed its expression and resulted in growth suppression of cancer cells. Induction of exogenous expression of LASEP3 promoted cell mobility in vitro. We found a 50-kDa receptor (LASEPR) which interacts with LASEP3 on cancer cell surface, suggesting that LASEP3 is an autocrine/paracrine growth factor. Conclusions: LASEP3 is a potential target for the development of diagnostic and prognostic biomarkers for lung and various types of cancers

Epithelial to Mesenchymal Transition Is Activated in Metastatic Pheochromocytomas and Paragangliomas Caused by SDHB Gene Mutations

Pheochromocytoma and paraganglioma are rare neural-crest-derived tumors. They are metastatic in 15% of cases, and the identification of a germline mutation in the SDHB gene is a predictive risk factor for malignancy and poor prognosis. To date, the link between SDHB mutations and malignancy is still missing. Epithelial to mesenchymal transition (EMT) is a developmental event, reactivated in cancer cells to promote cell mobility and invasiveness. The aim of this study was to address the participation of EMT in the metastatic evolution of pheochromocytoma/paraganglioma. Transcriptomic profiling of EMT was performed on 188 tumor samples, using a set of 94 genes implicated in this pathway. Activation of EMT was further confirmed at protein level by immunohistochemistry in a second set of 93 tumors. Hierarchical unsupervised classification showed that most SDHB-metastatic samples clustered together, indicating that EMT is differently regulated in these tumors. Major actors of EMT, metalloproteases and components of cellular junctions, were either up-regulated (LOXL2, TWIST, TCF3, MMP2, and MMP1) or down-regulated (KRT19 and CDH2) in SDHB-metastatic tumors compared with nonmetastatic ones. Interestingly, within metastatic tumors, most of these genes (LOXL2, TWIST, TCF3, MMP2, and KRT19) also allowed us to discriminate SDHB-mutated from non-SDHB-related tumors. In the second set of tumors, we studied Snail1/2 expression by immunohistochemistry and observed its specific nuclear translocation in all SDHB-metastatic tumors. We have identified the first pathway that distinguishes SDHB-metastatic from all other types of pheochromocytomas/paragangliomas and suggest that activation of the EMT process might play a critical role in the particularly invasive phenotype of this group of tumors.

Transactivation of EGFR/PI3K/Akt involved in ATP‐induced inflammatory protein expression and cell motility

Phenotype transition of vascular smooth muscle cells (VSMCs) is important in vascular diseases, such as atherosclerosis and restenosis. Once released, ATP may promote activation of VSMCs by stimulating cyclooxygenase-2 (COX-2), cytosolic phospholipase A(2) (cPLA(2)) expression and prostaglandin (PG)E(2) synthesis via activation of MAPKs and NF-κB. However, whether alternative signaling pathways participated in regulating COX-2 and cPLA(2) expression associated with cell migration were investigated in rat VSMCs. Western blot analysis, RT-PCR, promoter assay and PGE(2) ELISA were used to determine expression of COX-2, cPLA(2) and PGE(2). Specific inhibitors and siRNAs against various protein kinases or transcription factors were used to investigate the related signaling components in inflammatory protein induction by ATPγS. We found that ATPγS-induced COX-2 and cPLA(2) expression and PGE(2) release was attenuated by the pharmacological inhibitors or transfection with siRNA against PKCδ, c-Src, EGFR, PI3-K, Akt, p44/p42 MAPK or Elk-1. Moreover, ATPγS-stimulated phosphorylation of PKCδ, c-Src, EGFR, Akt, p42/p44 MAPK and Elk-1, suggesting the participation of PKCδ/c-Src/EGFR/PI3-K/Akt/p42/p44 MAPK cascade in mediating Elk-1 activities in VSMCs. In addition, migration assay revealed that ATPγS promoted cell mobility through up-regulation of COX-2 and cPLA(2) expression and PGE(2) release, which was attenuated by pretreatment with PGE(2) receptor antagonists. Taken together, these data showed that ATPγS up-regulated the expression of COX-2 and cPLA(2) through transactivation of PKCδ/c-Src/EGFR/PI3K/Akt/Elk-1 pathway. Newly synthesized PGE(2) acted on its receptors to promote cell motility of ATPγS-stimulated VSMCs.

ADH1, an N-cadherin inhibitor, evaluated in preclinical models of angiogenesis and androgen-independent prostate cancer

The conversion from E-cadherin to N-cadherin has been observed in several human cancer types, including prostate cancer, with more homogenous expression of N-cadherin detected in high-grade prostate tumors. N-cadherin, in vitro, has been shown to promote cell mobility, migration and invasion of several cancer cell lines, indicating the possibility of N-cadherin as a molecular target of cancer therapy. Herein, we examined the potential of an N-cadherin inhibitor, ADH1, in reducing tumor angiogenesis ex vivo and delaying tumor progression in vivo. Our data demonstrate that ADH1, at the dosages evaluated, does not display either antiangiogenic activity in a rat aortic ring assay or antitumor potential in a PC3 subcutaneous xenograft tumor model. We detected cytotoxic activity in human umbilical vein endothelial cells, PC3, and Tsu-Pr1 cells, when ADH1 exposure was evaluated at 500 micromol/l or above.

An Interaction between Insulin-like Growth Factor-binding Protein 2 (IGFBP2) and Integrin α5 Is Essential for IGFBP2-induced Cell Mobility

In the study we report here, we tested the hypothesis that insulin-like growth factor-binding protein 2 (IGFBP2) promotes cell mobility through its interaction with integrin alpha5. Our previous microarray studies showed that IGFBP2 activates the expression of integrin alpha5. In addition, IGFBP2 has an Arg-Gly-Asp (RGD) domain, which is a known integrin binding motif. We first confirmed our microarray results by showing that the expression of integrin alpha5 is indeed up-regulated at the protein level in IGFBP2-overexpressing SNB19 glioma cells. Using co-immunoprecipitation, we confirmed that IGFBP2 does interact with integrin alpha5. To confirm that IGFBP2 interacts directly with integrin alpha5 through the RGD domain, we created an RGD --> RGE mutant (D306E) IGFBP2 and stably overexpressed the mutant IGFBP2 in the same cell line. Co-immunoprecipitation then showed that D306E-IGFBP2 had no detectable binding with integrin alpha5. We further observed that IGFBP2-overexpressing cells have extensive cell surface lamellipodia, whereas D306E-IGFBP2-overexpressing cells show abundant cell surface focal adhesions. Consistent with this, phenotype analysis then showed that IGFBP2-overexpressing cells have elevated migration rates compared with vector control; in contrast, the migration rates of the D306E-IGFBP2-overexpressing cells were not elevated and were comparable with that of vector control. Decreased expression of integrin alpha5 by small interference RNA in IGFBP2-overexpressing cells also reduced cell mobility. Therefore, we have concluded that one mechanism by which IGFBP2 activates IGFBP2-induced cell mobility is through its interaction with integrin alpha5 and this interaction is specifically mediated through the RGD domain on IGFBP2.