Cell Invasion Activity Research Articles

Nijmegen breakage syndrome protein 1 (NBS1) modulates hypoxia inducible factor-1α (HIF-1α) stability and promotes in vitro migration and invasion under ionizing radiation

Hypoxia-inducible factor (HIF) is a heterodimer transcription factor complex that monitors the cellular response to the oxygen levels in cells. Hypoxia-inducible factor-1α (HIF-1α) has been shown to be stabilized by ionizing radiation (IR) and its stabilization promotes tumor progression and metastasis. Nijmegen breakage syndrome protein 1 (NBS1), a component of the MRE11–RAD50–NBS1 complex, plays an important role in the cellular response to DNA damage but its overexpression contributes to transformation and has been found to correlate with metastasis. However, whether NBS1 participates in IR-induced metastasis needs to be further determined. The aim of this study is to investigate whether radiation-induced HIF-1α stabilization is regulated by NBS1 and thereby promotes tumor cell migration/invasion. Here, we show that both NBS1 and HIF-1α expression are up-regulated after exposure to IR, and NBS1 increases HIF-1α expression at the protein level. In addition, IR treatment promotes the epithelial–mesenchymal transition (EMT) and in vitro cell migration and invasion activity, which could be abolished by suppression of NBS1. Furthermore, NBS1 directly interacts with HIF-1α and reduces the ubiquitination of HIF-1α⋅ Co-expression of HIF-1α and NBS1 in primary tumors of patients with lung adenocarcinoma correlates with a worse prognosis. These results provide a new function of NBS1 in stabilizing HIF-1α under IR, which leads to enhanced cancer cell migration and invasion.

High expression of protein phosphatase 4 is associated with the aggressive malignant behavior of colorectal carcinoma.

BackgroundRecent evidence suggests an important role of protein phosphatase 4 (PP4C) in the progression of several cancers, including breast cancer, lung cancer and pancreatic ductal adenocarcinoma. However, the contribution of PP4C to colorectal carcinoma (CRC) remains elusive.MethodsThe expression of PP4C in CRC tissues compared with matched non-tumor tissues and CRC cells was detected using quantitative RT-PCR, immunohistochemistry and western blotting assays. Through univariate and Kaplan-Meier analysis, we correlated the PP4C expression with clinicopathological features and patient survival. A series of experiments, including cell proliferation, lentiviral infection, cell invasion and MMP gelatinase activity assays, were performed to investigate the underlying mechanisms. Through further experiments, tumor growth and metastasis were evaluated in vivo using a xenogenous subcutaneously implant model and a tail vein metastasis model.ResultsIn the present study, we found that PP4C expression is frequently increased in human CRC and that the upregulation of PP4C correlates with a more invasive tumor phenotype and poor prognosis. The ectopic expression of PP4C promoted CRC cell proliferation, migration and invasion in vitro and tumor growth and lung metastasis in vivo. Silencing the expression of PP4C resulted in the inhibition of cell proliferation and invasion. Further investigations showed that phosphorylated Akt (p-AKT) is required for the PP4C-mediated upregulation of MMP-2 and MMP-9, which promotes cell invasion.ConclusionsOur data suggested a potential role of PP4C in tumor progression and provided novel insights into the mechanism of how this factor positively regulated cell proliferation and invasion in CRC cells.

Effect of adenovirus- mediated interleukin- 6 gene on biological behavior of spinal cord astrocytes

Objective To investigate the effect of interleukin- 6(IL- 6)gene on proliferation, migration and invasion activity of spinal cord astrocytes. Methods IL-6 gene was cloned into adenovirus vector, then the astrocytes particles expressing IL-6 were infected into astrocytes. Real- time quantitative polymerase chain reaction(Real- time PCR)and Western blotting were performed to examine the expression of IL-6 in infected astrocytes. The growth ability was detected by methyl thiazol tetrazolium(MTT) assay.The cell cycle was analyzed by flow cytometry. The adhension and migration were examined by matrix(MTX)and multi tetrazolium compounds(MTS)assays. Results Comparing to controls group and Ad- IL- 6- green fluorescent protein(GFP)group, the expression levels of IL- 6 mRNA and protein were both significantly up- regulated after 4 days of IL- 6 adenovirus infection. Comparing with the Ad- GFP group, MTT assay showed that the A490 of Ad- IL- 6- GFP group was up- regulated after adenovirus infection(P<0.05). Comparing with the Ad- GFP group, cell cycle test revealed IL- 6 gene could accelerate cells in G1[(5.40±0.90)%], and the proportion of KF cells was increased in S phase [(7.26±0.40)%]and G2 phase[(83.52±0.50)%]after cells were infected(P<0.01). Transfection of IL- 6 adenovirus stimulated the migration and invasion activity of astrocytes. Conclusion Adenovirus- mediated IL- 6 gene efficiently stimulate the proliferation, cell cycle progression, migration and invasion activity of astrocytes. Key words: Interleukin-6; Spinal cord; Astrocyte; Proliferation; Migration; Adhension

MMP-9 is increased in the pathogenesis of gastric cancer by the mediation of HER2.

Human epidermal growth factor receptor 2 (HER2) overexpression is not only closely associated with the tumor growth, but is also related to tumor invasion. We here aimed to investigate the mechanism of HER2 mediation in the pathogenesis of gastric cancer. The human gastric cancer cell lines SGC-7901, MKN-45, AGS, the immortalized cell line GES-1 derived from normal gastric mucosa. Cell transfection and selection of stable cell lines and the gene and protein levels of HER2 and Matrix metalloproteinase-9 (MMP-9) were examined to determine the molecular relationship between them in the pathogenesis of gastric cancer. The human gastric cancer cell lines SGC-7901, MKN-45, AGS, the immortalized cell line GES-1 derived from normal gastric mucosa. Cell transfection and selection of stable cell lines and the gene and protein levels of HER2 and MMP-9 were examined to determine the molecular relationship between them in the pathogenesis of gastric cancer. We demonstrated that vector-based shRNA significantly knocked down the expression of HER2 and considerably inhibited both the migration and invasion of gastric cancer cells. HER2 knockdown resulted in the downregulation of the expression of MMP-9, whereas HER2 overexpression improved the transcription of MMP-9 through the activation of an MMP-9 promoter. The promoter region of MMP-9 between -2500 and -2000 bp was found to be crucial for the upregulation of HER2-mediated transcription. Furthermore, a truncated promoter (-70 to +63) did not display any transcriptional activity. Cell invasion activity was almost completely inhibited when MMP-9 was knocked down. Conversely, the overexpression of MMP-9 partly rescued the invasion ability of cell strains with knockdown HER2. These findings help further understanding of the molecular mechanisms through which HER2 promotes malignancy, and suggest that targeting both HER2 and MMP-9 may be required to effectively block HER2 signaling in gastric cancer therapy.

Monocyte-Induced Prostate Cancer Cell Invasion is Mediated by Chemokine ligand 2 and Nuclear Factor-κB Activity.

Study BackgroundThe tumor microenvironment contains inflammatory cells which can influence cancer growth and progression; however the mediators of these effects vary with different cancer types. The mechanisms by which prostate cancer cells communicate with monocytes to promote cancer progression are incompletely understood. This study tested prostate cancer cell and monocyte interactions that lead to increased prostate cancer cell invasion.MethodsWe analyzed the prostate cancer cell invasion and NF-κB activity and cytokine expression during interaction with monocyte-lineage cells in co-cultures. The roles of monocyte chemotactic factor (MCP-1/CCL2) and NF-κB activity for co-culture induced prostate cancer invasion were tested. Clinical prostate cancer NF-κB expression was analyzed by immunohistochemistry.ResultsIn co-cultures of prostate cancer cell lines with monocyte-lineage cells, (C-C motif) ligand 2 (CCL2) levels were significantly increased when compared with monocytes or cancer cells cultured alone. Prostate cancer cell invasion was induced by recombinant CCL2 in a dose dependent manner, similar to co-cultures with monocytes. The monocyte-induced prostate cancer cell invasion was inhibited by CCL2 neutralizing antibodies and by the CCR2 inhibitor, RS102895. Prostate cancer cell invasion and CCL2 expression induced in the co-cultures was inhibited by Lactacystin and Bay11-7082 NF-κB inhibitors. Prostate cancer cell NF-κB DNA binding activity depended on CCL2 dose and was inhibited by CCL2 neutralizing antibodies. Clinical prostate cancer NF-κB expression correlated with tumor grade.ConclusionsCo-cultures with monocyte-lineage cell lines stimulated increased prostate cancer cell invasion through increased CCL2 expression and increased prostate cancer cell NF-κB activity. CCL2 and NF-κB may be useful therapeutic targets to interfere with inflammation-induced prostate cancer invasion.

Neurotrophic Factor Artemin Promotes Invasiveness and Neurotrophic Function of Pancreatic Adenocarcinoma In Vivo and In Vitro

ObjectivesThe aim of this study was to investigate the effect of the neurotrophic factor Artemin on neuroplasticity and perineural invasion of pancreatic adenocarcinoma.MethodsArtemin expressions were detected in human pancreatic adenocarcinoma tissues by Western blot and immunohistochemistry. Artemin overexpression and RNA interference in the pancreatic cancer cell lines were performed to evaluate the effects of Artemin on cell proliferation, invasion, and neurotrophic activity in vitro and in nude orthotopic transplantation tumor models.ResultsArtemin expression in pancreatic cancer tissues was related to the incidence of lymphatic metastasis and perineural invasion as well as the mean density and total area of nerve fibers. Overexpression of Artemin in pancreatic cancer cell lines improved colony formation, cell migration, matrigel invasion, and neurotrophic activity in vitro. This overexpression also increased the volume of nude orthotopic transplantation tumors; promoted cancer cell invasion of the peripheral organs, nerves, vessels, and lymph nodes; and stimulated the proliferation of peritumoral nerve fibers. Artemin depletion by RNA interference had an inhibitory effect mentioned previously.ConclusionsArtemin could promote invasiveness and neurotrophic function of pancreatic adenocarcinoma in vivo and in vitro. Therefore, Artemin could be used as a new therapeutic target of pancreatic carcinoma.

Hsp27 regulates EGF/β-catenin mediated epithelial to mesenchymal transition in prostate cancer.

Increased expression of the molecular chaperone Hsp27 is associated with the progression of prostate cancer (PCa) to castration-resistant disease, which is lethal due to metastatic spread of the prostate tumor. Metastasis requires epithelial to mesenchymal transition (EMT), which endows cancer cells with the ability to disseminate from the primary tumor and colonize new tissue sites. A wide variety of secreted factors promote EMT, and while overexpression and constitutive activation of epidermal growth factor (EGF) signaling is associated with poor prognosis of PCa, a precise role of EGF in PCa progression to metastasis remains unclear. Here, we show that Hsp27 is required for EGF-induced cell migration, invasion and MMPs activity as well as the expression of EMT markers including Fibronectin, Vimentin and Slug with concomitant decrease of E-cadherin. Mechanistically, we found that Hsp27 is required for EGF-induced AKT and GSK3β phosphorylation and β-catenin nuclear translocation. Moreover, silencing Hsp27 decreases EGF dependent phosphorylation of β-catenin on tyrosine 142 and 654, enhances β-catenin ubiquitination and degradation, prevents β-catenin nuclear translocation and binding to the Slug promoter. These data suggest that Hsp27 is required for EGF-mediated EMT via modulation of the β-catenin/Slug signaling pathway. Together, our findings underscore the importance of Hsp27 in EGF induced EMT in PCa and highlight the use of Hsp27 knockdown as a useful strategy for patients with advanced disease.

MiR-203 inhibits melanoma invasive and proliferative abilities by targeting the polycomb group gene BMI1

Metastasis is the major problem in malignant melanoma, posing a therapeutic challenge to clinicians. The investigation of the underlying mechanism driving this progress remains a large unmet need. In this study, we revealed a miR-203-BMI1 axis that regulated melanoma metastasis. We found significantly deregulation of miR-203 and up-regulation of BMI1 in melanoma, particularly in metastatic melanoma. An inverse correlation between the levels of miR-203 and BMI1 was further observed in melanoma tissues and cell lines. We also identified BMI1 as a downstream target gene of miR-203, which bound to the 3′UTR of BMI1. Overexpression of miR-203 was associated with decreased BMI1 expression and impaired cell invasion and tumor sphere formation activities. Re-expression of BMI1 markedly rescued miR-203-mediated suppression of these events. Taken together, our results demonstrated that miR-203 regulated melanoma invasive and proliferative abilities in part by targeting BMI1, providing new insights into potential mechanisms of melanoma metastasis.

Cotylenin A and arsenic trioxide cooperatively suppress cell proliferation and cell invasion activity in human breast cancer cells.

Arsenic trioxide (ATO) is an approved treatment for acute promyelocytic leukemia (APL). It has also shown potential for treatment of multiple myeloma and various solid tumors including breast cancer. The requirement of high, toxic concentrations for the induction of apoptosis in non-APL and solid tumor cells is a major limitation for its use in other hematological malignancies and solid tumors. We have examined whether inducers of differentiation of leukemia cells can control the growth of solid tumor cells. In the present study, we found that cotylenin A, a plant growth regulator and a potent inducer of differentiation in myeloid leukemia cells, significantly potentiated both ATO-induced inhibition of cell growth in a liquid culture, and ATO-induced inhibition of anchorage-independent growth in a semi-solid culture in human breast cancer MCF-7 and MDA-MB-231 cells. ISIR-005 (a synthetic cotylenin A-derivative) was also able to enhance ATO-induced growth inhibition. The combined treatment with cotylenin A and ATO induced cleaved caspase-7 in MCF-7 cells at the concentrations which ATO alone scarcely induced and cotylenin A alone only weakly induced. Expression of survivin in MCF-7 cells was markedly decreased with the presence of both cotylenin A and ATO, although the expression of survivin was only slightly decreased by cotylenin A or ATO alone. The pretreatment with N-acetylcysteine significantly reduced the combination treatment-induced cell growth inhibition. These data suggest that induction of cleaved caspase-7, inhibition of survivin and oxidative responses are important events in the corporative inhibition in the growth of MCF-7 cells induced by both cotylenin A and ATO. Furthermore, we found that the combined treatment with cotylenin A and ATO also could be effective in suppressing the invasive capacity of MDA-MB-231 cells determined with the impedance-based xCELLigence Real-Time Cell Analysis technology. These results suggest that cotylenin A is an attractive enhancer for the ATO-induced anticancer activities in human breast cancer.

Inhibition of MDA-MB-231 breast cancer cell migration and invasion activity by andrographolide via suppression of nuclear factor-κB-dependent matrix metalloproteinase-9 expression.

Breast cancer is one of the most common types of cancer worldwide. The majority of patients with cancer succumb to the disease as a result of distant metastases (for example, in the bones), which cause severe complications. Despite advancements in breast cancer treatment, chemotherapeutic outcomes remain far from satisfactory, prompting a search for effective natural agents with few side-effects. Andrographolide (AP), a natural diterpenoid lactone isolated from Andrographis paniculata, inhibits cancer cell growth. The current study aimed to examine the effect of AP on breast cancer cell proliferation, survival and progression in vitro and also its inhibitory activity on breast cancer bone metastasis in vivo. To achieve this, CCK8, flow cytometry, migration, invasion, western blot, PCR and luciferase reporter assay analyses were performed in vitro as well as establishing intratibial xenograft model of breast cancer bone metastasis in vivo. The results demonstrated that AP inhibits the migration and invasion of the MBA-MD-231 aggressive breast cancer cell line at non-lethal concentrations, in addition to suppressing proliferation and inducing apoptosis at high concentrations in vitro. In vivo, AP significantly inhibited the growth of tumors planted in bone and attenuated cancer-induced osteolysis. Tartrate-resistant acid phosphatase staining revealed osteoclast activation in tumor-bearing mice and AP was observed to attenuate this activation. The anti-tumor activity of AP in vitro and in vivo correlates with the downregulation of the nuclear factor κB signaling pathway and the inhibition of matrix metalloproteinase-9 expression levels. These results indicate that AP may be an effective anti-tumor agent for the treatment of breast cancer bone metastasis.

Phosphatidylinositol 3-Kinase Mediates the Ability of Retinol to Decrease Colorectal Cancer Cell Invasion

Previously, we showed that retinol (vitamin A) decreased both colorectal cancer cell invasion and phosphatidylinositol 3-kinase (PI3K) activity through a retinoic acid receptor–independent mechanism. Here, we determined if these phenomena were related by using parental HCT-116 cells that harbor 1 allele of wild-type PI3K and 1 allele of constitutively active (ca) PI3K and 2 mutant HCT-116 cell lines homozygous for caPI3K. In vitro, treatment of parental HCT-116 cells with 10 μM retinol reduced cell invasion whereas treatment of mutant HCT-116 cell lines with retinol did not. Treatment with 10 μM retinol also decreased the activity of matrixmetalloproteinase-9 and increased tissue inhibitor of matrixmetalloproteinase-I levels in parental, but not mutant, HCT-116 cells. Finally, parental or mutant cells were intrasplenically injected into athymic mice consuming diets with or without supplemental vitamin A. As expected, vitamin A supplementation tended (P = 0.18) to reduce the incidence of metastases in mice injected with the parental cell line and consuming the supplemented diet. In contrast, metastatic incidence was not affected (P = 1.00) by vitamin A supplementation in mice injected with mutant cells. These data indicate that the capacity of retinol to inhibit PI3K activity confers its ability to decrease colorectal cancer metastasis.

WAVE3-NFκB interplay is essential for the survival and invasion of cancer cells.

The WAVE3 cytoskeletal protein promotes cancer invasion and metastasis. We have shown that the WAVE3-mediated activation of cancer cell invasion is due, in part, to its regulation of expression and activity of key metalloproteinases (MMPs), including MMP9, which is centrally involved in invadopodia-mediated degradation of the extracellular matrix (ECM). MMP9 is also a major NFκB target gene, suggesting a potential linkage of WAVE3 to this pathway, which we sought to investigate. Mechanistically, we found that loss of WAVE3 in cancer cells leads to inhibition of NFκB signaling as a result of a decrease in the nuclear translocation of NFκB and therefore loss of activation of NFκB target genes. Conversely, overexpression of WAVE3 was sufficient to enhance NFκB activity. Both pharmacologic and genetic manipulations of NFκB effector molecules show that the biological consequence of loss of WAVE3 function in the NFκB pathway result the inhibition of invadopodia formation and ECM degradation by cancer cells, and these changes are a consequence of decreased MMP9 expression and activity. Loss of WAVE3 also sensitized cancer cells to apoptosis and cell death driven by TNFα, through the inhibition of the AKT pro-survival pathway. Our results identify a novel function of WAVE3 in NFκB signaling, where its activity is essential for the regulation of invadopodia and ECM degradation. Therefore, targeted therapeutic inhibition of WAVE3 will sensitize cancer cells to apoptosis and cell death, and suppress cancer invasion and metastasis.

Progeny from irradiated colorectal cancer cells acquire an EMT-like phenotype and activate Wnt/β-catenin pathway.

Radiotherapy remains a major approach to adjuvant therapy for patients with advanced colorectal cancer, however, the fractionation schedules frequently allow for the repopulation of surviving tumors cells, neoplastic progression, and subsequent metastasis. The aim of the present study was to analyze the transgenerational effects induced by radiation and evaluate whether it could increase the malignant features on the progeny derived from irradiated parental colorectal cancer cells, Caco-2, HT-29, and HCT-116. The progeny of these cells displayed a differential radioresistance as seen by clonogenic and caspase activation assay and had a direct correlation with survivin expression as observed by immunoblotting. Immunofluorescence showed that the most radioresistant progenies had an aberrant morphology, disturbance of the cell-cell adhesion contacts, disorganization of the actin cytoskeleton, and vimentin filaments. Only the progeny derived from intermediary radioresistant cells, HT-29, reduced the E-cadherin expression and overexpressed β-catenin and vimentin with increased cell migration, invasion, and metalloprotease activation as seen by immunoblotting, wound healing, invasion, and metalloprotease activity assay. We also observed that this most aggressive progeny increased the Wnt/β-catenin-dependent TCF/LEF activity and underwent an upregulation of mesenchymal markers and downregulation of E-cadherin, as determined by qRT-PCR. Our results showed that the intermediate radioresistant cells can generate more aggressive cellular progeny with the EMT-like phenotype. The Wnt/β-catenin pathway may constitute an important target for new adjuvant treatment schedules with radiotherapy, with the goal of reducing the migratory and invasive potential of the remaining cells after treatment.

Abstract 1354: Relevance of Sp1 and survivin expression in epithelial ovarian cancer patients and their usefulness as potential therapeutic targets

Abstract Objectives: Specificity protein 1 (Sp1) is a transcription factor that is over-expressed in several cancers and is inversely associated with survival. Survivin is known to cause resistance to chemo- and radiation therapy. Herein, we evaluated the correlation of Sp1 and survivin expression in epithelial ovarian cancer (EOC) patients and tested a pre-clinical strategy to target these candidates using tolfenamic acid (TA) for enhancing the efficacy of a chemotherapeutic agent, Cisplatin (Cis). Methods: Expression of Sp1 and survivin in clinical specimens was determined by qPCR and Western blot analysis. Anti-proliferative response of EOC cell lines (ES2 and OVCAR-3) was assessed in the presence of Cis, TA or Cis+TA using CellTiter-Glo. Apoptosis was determined by flow cytometry using annexin-V staining, measuring the effector caspases activity using caspase3/7 Glo kit. Cell cycle analysis was carried out using flow cytometry. Matrigel coated ranswell chambers were used to assess cell migration/invasion. Proteomics nalysis was performed at Agilent Technologies, Inc. Results: qPCR showed an increase in the expression of urvivin (∼5-fold) and Sp1 (∼2-fold) in clinical specimens, which was corroborated by Western blot analysis [Sp1 (&amp;gt;2.6-fold) and survivin &amp;gt;100-fold)]. TA and Cis showed a dose- and time-dependent inhibition of cell viability in EOC cell lines [50 µM TA caused 50% (ES2) and 40% (OVCAR-3) cell growth inhibition; Cis (5 µM) caused 60% (ES2) and 40% (OVCAR-3) inhibition at 48 h post-treatment]. Combination of TA (50 µM) and Cis (5 µM) resulted in a synergistic response causing higher growth inhibition (ES2: ∼80%, p&amp;lt;0.001; OVCAR-3: 60%, p&amp;lt;0.001) compared to single-agent. This is accompanied by cell-cycle arrest (G2/M phase), an increase in apoptosis, as determined by Caspase 3/7 activity, annexin-V staining, PARP cleavage, and inhibition of ES2 cell invasion and migration. Global proteomic profiling of ES2 cells indicated that the combination treatment up-regulated proteins associated with oxidative phosphorylation, apoptotic execution, and electron transport chain and down-regulated ribosomal proteins, translational actors, DNA damage and cell cycle proteins. Conclusions: Elevated expression of Sp1 and survivin suggests a strong association of these two agents in EOC. Our pre-clinical results confirmed that targeting these candidates with small molecule (TA) potentially sensitizes EOC cells to chemotherapy. Citation Format: Umesh T. Sankpal, Susan B. Ingersoll, Vadiraja B. Bhat, Sarfraz Ahmad, Robert W. Holloway, Riyaz M. Basha. Relevance of Sp1 and survivin expression in epithelial ovarian cancer patients and their usefulness as potential therapeutic targets. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1354. doi:10.1158/1538-7445.AM2014-1354

Abstract 1126: Tristetraprolin inhibits Twist1-induced cancer cell migration

Abstract Epithelial-Mesenchymal Transition (EMT) enhances tumor cell migration. However, detail mechanisms for the regulation of EMT are not well known. Tristetraprolin (TTP) is an AU-rich element (ARE) binding protein that can promote the decay of the transcripts. This study demonstrates that TTP plays a critical role in EMT process. Overexpression of TTP in ovarian cancer cells decreased the expression of mesenchymal markers (N-cadherin and Vimentin), increased the expression of epithelial marker (E-cadherin), and inhibited cell migration and invasion activities. On the contrary, inhibition of TTP by siRNA decreased the expression of E-cadherin, increased the expression of N-cadherin and Vimentin, and promoted cell migration and invasion. The cDNA microarray analysis revealed that Twist1 was significantly down-regulated in TTP-overexpressed cells. Twist1 mRNA contains ARE within its 3′-UTR and TTP enhances the decay of Twist1 mRNA through binding to its 3′-UTR. In addition, TTP directly bound to the ARE of Twist1 mRNA by EMSA assay. Ectopic expression of Twist1 gene without 3′UTR blocked the inhibitory of TTP on the EMT. We also observed enhanced levels of Twist1 and reduced TTP levels in human ovarian adenocarcinoma. Collectively, our data indicate that TTP conducts anti-metastatic activity by down-regulating Twist1, presenting a novel mechanism of TTP-mediated regulation of tumor metastasis. Citation Format: Wha Ja Cho, Nal Ae Yoon, Mai-Tram Vo, Young Joo Min, Jeong Woo Park. Tristetraprolin inhibits Twist1-induced cancer cell migration. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1126. doi:10.1158/1538-7445.AM2014-1126

Abstract 3145: eIF4E, an adverse prognostic marker of melanoma patient survival, increases melanoma cell invasion

Abstract Human cutaneous melanoma is a life-threatening skin cancer due to its invasive nature and high metastatic potential, leading to poor prognosis for melanoma patients. However, the mechanisms for melanoma invasion and metastasis are poorly understood. Human eukaryotic translation initiation factor 4E (eIF4E) has been shown to be associated with tumor progression in multiple cancer types. However, the role of eIF4E in melanoma progression is not very well known. We examined eIF4E expression in 448 melanocytic lesions at different stages using tissue microarray and found that positive eIF4E staining was significantly increased in primary melanomas compared to dysplastic nevi (P&amp;lt;0.001), and further increased in metastatic melanomas compared to primary melanomas (P=0.008). EIF4E expression was correlated with melanoma thickness (P&amp;lt;0.001) and was inversely correlated with overall and disease-specific 5-year survival of all and primary melanoma patients especially those with tumor ≥4mm thick combined with metastatic melanoma patients. Furthermore, positive eIF4E expression was significantly higher in AJCC stage I-II melanomas compared to stage III-IV melanomas. Multivariate Cox regression analysis also revealed that eIF4E is an independent prognostic marker. FACS analysis and sulforhodamine B (SRB) cell proliferation assay showed that eIF4E knockdown in melanoma cells resulted in a significant increase and decrease in apoptosis and cell proliferation, respectively. EIF4E knockdown in melanoma cells also resulted in a down regulation of mesenchymal markers such as vimentin, N-cadherin, α-smooth muscle actin (α-SMA) and the EMT inducing transcription factor Twist. Additionally eIF4E knockdown in melanoma cells led to a decrease in the expression of c-Myc and Bcl2 and an increase in the expression of cleaved PARP and cleaved Caspase3. Moreover, down regulation of eIF4E resulted in a decrease in both melanoma cell invasion and MMP-2 activity. Taken together our data suggests the eIF4E may promote melanoma cell invasion and metastasis by inducing EMT and preventing apoptosis and also by increasing MMP-2 activity. EIF4E may also serve as a promising prognostic marker and a potential therapeutic target for melanoma. Citation Format: Shahram Khosravi, Gholamreza Safaee Ardekani, Magdalena Martinka, Christopher John Ong. eIF4E, an adverse prognostic marker of melanoma patient survival, increases melanoma cell invasion. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3145. doi:10.1158/1538-7445.AM2014-3145

Abstract 4986: APG101 inhibits CD95-Ligand induced invasion of glioblastoma in 3D-models in vitro and ex vivo

Abstract CD95 (APO-1/Fas) is a receptor that belongs to the Tumor Necrosis Factor Receptor Super Family (TNFRSF). Originally discovered as a death receptor inducing apoptosis upon binding to its ligand CD178 (CD95L/FasL), recent research indicates that CD95 also mediates non-apoptotic functions (e.g. liver regeneration, neuronal development, inflammation and cellular migration/invasion of tumor cells) depending on the cellular context. The potential impact on multiple cellular processes classifies the CD95 pathway as an attractive target for pharmacological interference. Apogenix developed APG101, a recombinant human fusion protein consisting of the extracellular domain of CD95 and the Fc-domain of an IgG antibody for the treatment of glioblastoma, a malignancy characterized by aggressive and invasive growth. Preclinical development activities to elucidate the mode of action of APG101 included the comparison of 3-dimensional in vitro and ex vivo models employing U87-MG and U373-MG cells on murine organotypic brain tissue cultures. Expression of CD95L and CD95 in these cells was reduced by semi-stable transfection of shRNA vectors to assess contribution of the CD95/CD95L system to invasion and proliferation of glioma cell lines and the anti-invasive effect of APG101 treatment. While knockdown of CD95 leads to death of U373-MG cells, we observed significant reduction of proliferation rates in U87-MG cells. The effect of CD95L knockdown on proliferation was only minor in U87-MG and was absent in U373-MG cells. Consistently, we found that treatment of glioma cells with APG101 had no effect on proliferation rates or cell cycle regulation of either cell line. The main effect of CD95L knockdown was a strongly reduced invasiveness of both cell lines in vitro and ex vivo. Invasiveness could be restored by exogenous addition of recombinant CD95L, and this induced invasion was inhibited in the presence of APG101. In contrast, a CD95L binding defective CD95-Fc mutant APG122 did not have an anti-invasive effect in our experimental models. We conclude that: Results obtained from the in vitro and ex vivo assay models reflect each other and are suitable to study the role of CD95 signaling in tumor cell invasion activation of CD95 on glioma cells by CD95L contributes to invasive spread of tumor cells to the surrounding brain tissue, and Apogenix drug APG101 is able to block the pro-invasive activity of CD95L. Based on these observations, an important mode of action for APG101 is inhibition of CD95 ligand induced invasion of glioma cells. Citation Format: Christian Merz, Alexander Strecker, Jaromir Sykora, Oliver Hill, Gieffers Christian, Harald Fricke, Peter Angel, Heike Peterziel. APG101 inhibits CD95-Ligand induced invasion of glioblastoma in 3D-models in vitro and ex vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4986. doi:10.1158/1538-7445.AM2014-4986

Elastin-derived peptides stimulate trophoblast migration and invasion: a positive feedback loop to enhance spiral artery remodelling.

Elastin breakdown in the walls of uterine spiral arteries during early pregnancy facilitates their transformation into dilated, high-flow, low-resistance channels. Elastin-derived peptides (EDP) can influence cell migration, invasion and protease activity, and so we hypothesized that EDP released during elastolysis promote extravillous trophoblast (EVT) invasion and further elastin breakdown. Treatment of the trophoblast cell line SGHPL4 with the elastin-derived matrikine VGVAPG (1 μg/ml) significantly increased total elastase activity, promoted migration in a wound healing assay and increased invasion through Matrigel-coated transwells compared with vehicle control (0.1% DMSO) or the scrambled sequence VVGPGA. Furthermore, treatment of first-trimester placental villous explants with this EDP significantly increased both the area of trophoblast outgrowth and distance of migration away from the villous tips. Primary first-trimester cytotrophoblast exposed to VGVAPG (1 μg/ml) for 30 min showed increased phosphorylation of endothelial nitric oxide synthase and activation of the mitogen activated protein kinase pathway, events also associated with tumour cell migration and invasion. These in vitro observations suggest liberation of bioactive EDP during induction of elastolysis in the uterine spiral arteries may orchestrate a positive feedback loop that promotes EVT invasion and further elastin breakdown, contributing to the process of vascular remodelling.

Thymoquinone inhibits proliferation and invasion of human nonsmall-cell lung cancer cells via ERK pathway.

Thymoquinone (TQ) is the primary bioactive component of Nigella sativa Linn seed oil and used as anti-inflammatory, anti-oxidant, and anti-neoplastic agent. Previous studies have shown that TQ exhibits inhibitory effects on multiple cancers. However, the detailed antineoplastic effects and its molecular mechanisms of TQ on lung cancer are not entirely elucidated yet. In the present study, we aimed to investigate the effects of TQ on cell proliferation, migration, and invasion as well as its underlying anti-metastatic mechanisms in A549 cells. Lung cancer cell line A549 cells were treated with different concentration of TQ for different period of time, and the growth-inhibitory effects of TQ was measured by MTT and cell count assays; cell cycle was determined by flow cytometry; wound healing and transwell assays were used to assess the cell migration and invasion activities; Western blot and real-time quantitative RT-PCR were used to determine the expression of proliferation and invasion associated genes as well as MAPKs pathway molecules; gelatinase activity was estimated using gelatin zymography assay. The results show that TQ played a role in inhibiting the proliferation, migration, and invasion of A549 lung cancer cells, it also inhibited the expression level of PCNA, cyclin D1, MMP2, and MMP9 mRNA and protein in a dose- and time-dependent manner especially at 10, 20, 40μmol/L concentrations. The cell cycle inhibitor P16 expression and the gelatinase activities of MMP2 and MMP9 were also inhibited by TQ dramatically. TQ reduced phosphorylation of ERK1/2; however, the proliferation and invasion inhibitory effects of TQ on A549 cells were neutralized by ERK1/2 inhibitor PD98059. In conclusion, our study confirmed that TQ could inhibit A549 cell proliferation, migration, and invasion through ERK1/2 pathway, as proposed the therapeutic potential of TQ as an anti-metastatic agent in human lung cancer treatment.

Targeting filamin B induces tumor growth and metastasis via enhanced activity of matrix metalloproteinase-9 and secretion of VEGF-A.

Filamins regulate cell locomotion and associate with diverse signaling molecules. We have recently found that targeting filamin A (FLNA) reduces RAS-induced lung adenocarcinomas. In this study, we explored the role of another major filamin isoform, filamin B (FLNB), in tumor development. In contrast to FLNA, we report that targeting FLNB enhances RAS-induced tumor growth and metastasis which is associated with higher matrix metallopeptidase-9 (MMP-9) and extracellular signal-regulated kinase (ERK) activity. Flnb deficiency in mouse embryonic fibroblasts results in increased proteolytic activity of MMP-9 and cell invasion mediated by the RAS/ERK pathway. Similarly, silencing FLNB in multiple human cancer cells increases the proteolytic activity of MMP-9 and tumor cell invasion. Furthermore, we observed that Flnb-deficient RAS-induced tumors display more capillary structures that is correlated with increased vascular endothelial growth factor-A (VEGF-A) secretion. Inhibition of ERK activation blocks phorbol myristate acetate-induced MMP-9 activity and VEGF-A secretion in vitro. In addition, silencing FLNB in human ovarian cancer cells increases secretion of VEGF-A that induces endothelial cells to form more vascular structures in vitro. We conclude that FLNB suppresses tumor growth and metastasis by regulating the activity of MMP-9 and secretion of VEGF-A which is mediated by the RAS/ERK pathway.