Facilitate Tumor Cell Invasion Research Articles

Knockdown of hTERT by siRNA inhibits cervical cancer cell growth in vitro and in vivo.

Human telomerase reverse transcriptase (hTERT) is the catalytic component of telomerase that facilitates tumor cell invasion and proliferation. It has been reported that telomerase and hTERT are significantly upregulated in majority of cancers including cervical cancer, thus, downregulation of hTERT is a promising target in malignant tumor treatment. We established a short interfering RNA (siRNA) targeting hTERT, and transfected it into HeLa cells (a cervical cancer cell line) to investi-gate the effect of cell proliferation, apoptosis, migration and invasion in cervical cancer cells. The results showed that siRNA targeting hTERT could effectively knock down hTERT expression, remarkably suppress telomerase activity, cell proliferation, migration and invasion, and induced cell apoptosis of cervical cancers cells in vitro. In addition, we evaluated whether siRNA targeting hTERT affects tumor growth in nude mice, and found that it dramatically inhibited tumorigenesis and growth of mice injected with siRNA targeting hTERT. Furthermore, we also found that knockdown of hTERT was able to significantly suppress constitutive phosphorylation of Akt, PI3K, which might imply that reduction of hTERT inhibited tumor growth via the PI3K/Akt signaling pathway to some extent. These results suggest that the suppression of hTERT expression by siRNA inhibits cervical cancer cell growth in vitro and in vivo, and may provide a novel target for anticancer gene therapy.

Matrix metalloproteinase-10 promotes tumor progression through regulation of angiogenic and apoptotic pathways in cervical tumors.

BackgroundCancer invasion and metastasis develops through a series of steps that involve the loss of cell to cell and cell to matrix adhesion, degradation of extracellular matrix and induction of angiogenesis. Different protease systems (e.g., matrix metalloproteinases, MMPs) are involved in these steps. MMP-10, one of the lesser studied MMPs, is limited to epithelial cells and can facilitate tumor cell invasion by targeting collagen, elastin and laminin. Enhanced MMP-10 expression has been linked to poor clinical prognosis in some cancers, however, mechanisms underlying a role for MMP-10 in tumorigenesis and progression remain largely unknown. Here, we report that MMP-10 expression is positively correlated with the invasiveness of human cervical and bladder cancers.MethodsUsing commercial tissue microarray (TMA) of cervical and bladder tissues, MMP-10 immunohistochemical staining was performed. Furthermore using a panel of human cells (HeLa and UROtsa), in vitro and in vivo experiments were performed in which MMP-10 was overexpressed or silenced and we noted phenotypic and genotypic changes.ResultsExperimentally, we showed that MMP-10 can regulate tumor cell migration and invasion, and endothelial cell tube formation, and that MMP-10 effects are associated with a resistance to apoptosis. Further investigation revealed that increasing MMP-10 expression stimulates the expression of HIF-1α and MMP-2 (pro-angiogenic factors) and PAI-1 and CXCR2 (pro-metastatic factors), and accordingly, targeting MMP-10 with siRNA in vivo resulted in diminution of xenograft tumor growth with a concomitant reduction of angiogenesis and a stimulation of apoptosis.ConclusionTaken together, our findings show that MMP-10 can play a significant role in tumor growth and progression, and that MMP-10 perturbation may represent a rational strategy for cancer treatment.

RhoC Interacts with Integrin α5β1 and Enhances Its Trafficking in Migrating Pancreatic Carcinoma Cells

Human pancreatic ductal adenocarcinoma (PDAC) is characterized by early systemic dissemination. Although RhoC has been implicated in cancer cell migration, the relevant underlying molecular mechanisms remain unknown. RhoC has been implicated in the enhancement of cancer cell migration and invasion, with actions which are distinct from RhoA (84% homology), and are possibly attributed to the divergent C-terminus domain. Here, we confirm that RhoC significantly enhances the migratory and invasive properties of pancreatic carcinoma cells. In addition, we show that RhoC over-expression decreases cancer cell adhesion and, in turn, accelerates cellular body movement and focal adhesion turnover, especially, on fibronectin-coated surfaces. Whilst RhoC over-expression did not alter integrin expression patterns, we show that it enhanced integrin α5β1 internalization and re-cycling (trafficking), an effect that was dependent specifically on the C-terminus (180-193 amino acids) of RhoC protein. We also report that RhoC and integrin α5β1 co-localize within the peri-nuclear region of pancreatic tumor cells, and by masking the CAAX motif at the C-terminal of RhoC protein, we were able to abolish this interaction in vitro and in vivo. Co-localization of integrin α5β1 and RhoC was demonstrable in invading cancer cells in 3D-organotypic cultures, and further mimicked in vivo analyses of, spontaneous human, (two distinct sources: operated patients and rapid autopsy programme) and transgenic murine (LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre), pancreatic cancers. In both cases, co-localization of integrin α5β1 and RhoC correlated with poor differentiation status and metastatic potential. We propose that RhoC facilitates tumor cell invasion and promotes subsequent metastasis, in part, by enhancing integrin α5β1 trafficking. Thus, RhoC may serve as a biomarker and a therapeutic target.

Vitronectin–αvβ3 Integrin Engagement Directs Hypoxia-Resistant mTOR Activity and Sustained Protein Synthesis Linked to Invasion by Breast Cancer Cells

The tumor microenvironment is a crucial player in the ability of cancer cells to acquire the ability to survive under the hypoxic environment and promote migration and invasion. Translational regulation is an essential part of cancer development and progression. Protein synthesis consumes considerable cellular metabolic energy and is therefore highly regulated, in turn controlling tumor cell proliferation and survival in extreme tumor-host conditions. Protein synthesis is typically downregulated by hypoxia, impairing cell proliferation and migration. Here, we show that breast cancer cells expressing integrin αvβ3, when engaging the extracellular matrix (ECM) protein vitronectin, strongly upregulate both mTOR activity and cap-dependent mRNA translation, which overrides their inhibition by hypoxia and facilitates tumor cell invasion. Interaction of vitronectin with integrin αvβ3 results in the continued activation of the kinase mTOR despite hypoxia through a mechanism that is dependent on integrin-linked kinase but is independent of focal adhesion kinase. Continuous activation of mTOR despite hypoxia involves release of translation initiation factor eIF4E from its repressor protein 4E-BP1, which is required for vitronectin-mediated tumor cell invasion. As integrin αvβ3 is associated with breast cancer cell invasion and metastasis to bone, we propose that the interaction with specific ECM proteins can influence cancer cell invasion, in part, by hyperactivation of mTOR, thereby promoting and sustaining protein synthesis under hypoxic conditions.

Plasma Fibronectin Promotes Tumor Cell Survival and Invasion through Regulation of Tie2

Our previous research has shown that plasma fibronectin promotes lung metastasis by facilitating tumor cell invasion in clotted plasma. To evaluate the role of clotted plasma for tumor cell survival, we treated B16F1 cells embedded in a 3-dimensional matrix of fibrin with tumor necrosis factor α (TNFα), a cytokine with anti-tumor activity. Under these conditions, TNFα caused significant cytotoxicity, which was prevented when we added plasma fibronectin to the fibrin clot. Fibronectin-mediated TNFα resistance was dependent on PI3-kinase, which also mediated the pro-adhesive and pro-invasive effects of plasma fibronectin on tumor cells. To further investigate the role of plasma fibronectin in tumor cell signaling, we performed a gene array that showed specific upregulation of Tie2 in B16F1 cells embedded in fibrin-fibronectin compared to fibrin. Importantly, inhibition of Tie2 resulted in decreased tumor cell invasion, reduced colony formation and increased tumor cell death in response to TNFα. Together, our findings indicate that plasma fibronectin induces tumor cell invasion and protects tumor cells from the cytotoxic effects of inflammatory mediators through up-regulation of Tie2.

Cystatins in Immune System

Cystatins comprise a large superfamily of related proteins with diverse biological activities. They were initially characterised as inhibitors of lysosomal cysteine proteases, however, in recent years some alternative functions for cystatins have been proposed. Cystatins possessing inhibitory function are members of three families, family I (stefins), family II (cystatins) and family III (kininogens). Stefin A is often linked to neoplastic changes in epithelium while another family I cystatin, stefin B is supposed to have a specific role in neuredegenerative diseases. Cystatin C, a typical type II cystatin, is expressed in a variety of human tissues and cells. On the other hand, expression of other type II cystatins is more specific. Cystatin F is an endo/lysosome targeted protease inhibitor, selectively expressed in immune cells, suggesting its role in processes related to immune response. Our recent work points on its role in regulation of dendritic cell maturation and in natural killer cells functional inactivation that may enhance tumor survival. Cystatin E/M expression is mainly restricted to the epithelia of the skin which emphasizes its prominent role in cutaneous biology. Here, we review the current knowledge on type I (stefins A and B) and type II cystatins (cystatins C, F and E/M) in pathologies, with particular emphasis on their suppressive vs. promotional function in the tumorigenesis and metastasis. We proposed that an imbalance between cathepsins and cystatins may attenuate immune cell functions and facilitate tumor cell invasion.

Glioma-related edema: new insight into molecular mechanisms and their clinical implications

Glioma-related edema (GRE) is a significant contributor to morbidity and mortality from glioma. GRE is a complicated process involving not only peritumoral edema but also the water content of the tumor body. In terms of etiology, this condition derives from both GRE in the untreated state and GRE secondary to clinical intervention, and different cell types contribute to distinct components of GRE. Peritumoral edema was previously believed to loosen glioma tissue, facilitating tumor-cell invasion; however, the nutrition hypothesis of the tumor microecosystem suggests that tumor cells invade for the sake of nutrition. Edema is the pathologic consequence of the reconstructed trophic linkage within the tumor microecosystem. Glioma cells induce peritumoral brain edema via an active process that supplies a suitable niche for peritumoral invasive cells, suggesting that glioma-related peritumoral brain edema is determined by the invasive property of tumor cells. There are differences between pivotal molecular events and reactive molecular events in the development of GRE. Molecular therapy should target the former, as targeting reactive molecular events will produce undesired or even adverse results. At present, brain glioma angiogenesis models have not been translated into a new understanding of the features of brain images. The effect of these models on peritumoral brain edema is unclear. Clinical approaches should be transformed on the basis of new knowledge of the molecular mechanism underlying GRE. Exploring clinical assessment methods, optimizing the existing control strategy of GRE, and simultaneously developing new treatments are essential.

Special Issue on Immune Responses in Tumors and Non-Transformed Inflammatory Microenvironments

Special Issue on Immune Responses in Tumors and Non-Transformed Inflammatory Microenvironments

Tissue Factor Pathway Inhibitor 2 (TFPI2) Is Commonly Methylated in Multiple Myeloma

Abstract 4617 Background:Tissue Factor Pathway Inhibitor 2 (TFPI2) is a member of the Kunitz-type serine proteinase inhibitor family that regulates extracellular tissue homeostasis by inhibiting matrix metalloproteinases. Transcriptional silencing of TFPI2 due to DNA methylation of the CpG island of its promoter has been reported in several tumors and has been suggested to facilitate tumor cell invasion and angiogenesis. We investigated the DNA methylation status of TFPI2 in multiple myeloma (MM) patients as part of a program of epigenetic profiling of multiple myeloma. Methods:Genomic DNA extracted from two human MM cell lines (U-266 and RPMI-8226) and bone marrow aspirate samples from a well chracterized series of MM patients was modified by sodium bisulphite using the EZ DNA methylation kit, ZymoResearch. Bisulfite modified DNA was used as a template for Methylation Specific PCR with primers specific for unmethylated and methylated alleles. Control methylated (CpG Genome™ Universal Methylated, Chemicon International) and unmethylated genomic DNA was included in each experiment. Chi-square test, logistic regression analysis and unpaired t-test were used to investigate associations between gene methylation and ISS stage, presence of extramedullary disease, bone disease, anemia (hemoglobin ≤10 mg/dL), renal failure, serum albumin and beta (2)-microglobulin level. Kaplan-Meier curves were used to estimate the probabilities of survival and the Log-rank test to assess the statistical significance of differences in event rates. Results:Forty six patients with MM (26 male, 20 female; mean age 64) and two human MM cell lines were studied. According to the International Staging System (ISS) 21 patients were classified as stage I, 10 stage II and 15 as stage III disease. Methylation in the CpG island of the promoter of TFPI2 was detected in 30 patients (65%, 95%CI= 52,6–78,7%) and in both cell lines (100%). Patients with methylated TFPI2 had significantly elevated beta 2 microglobulin levels (5553 vs 3787 mg/L mean values, p=0.0002) but we did not detect a statistically significant difference in overall survival among patients with methylated versus non methylated TFPI2. No other relevant correlations were detected. Conclusion:TFPI2 is commonly methylated in MM patients. Transcriptional silencing of TFPI2 may play a role in the mechanism of myelomatogenesis and its methylation status warrants further evaluation as a diagnostic co-marker for this disease. Disclosures:No relevant conflicts of interest to declare.

Pokemon Silencing Leads to Bim-Mediated Anoikis of Human Hepatoma Cell QGY7703

Pokemon is an important proto-oncogene that plays a critical role in cellular oncogenic transformation and tumorigenesis. Anoikis, which is regulated by Bim-mediated apoptosis, is critical to cancer cell invasion and metastasis. We investigated the role of Pokemon in anoikis, and our results show that Pokemon renders liver cells resistant to anoikis via suppression of Bim transcription. We knocked-down Pokemon in human hepatoma cells QGY7703 with small interfering RNAs (siRNA). Knockdown of Pokemon alone did not significantly affect the growth and survival of QGY7703 cells but notably enhanced their sensitivity to apoptotic stress due to the presence of chemical agents or cell detachment, thereby inducing anoikis, as evidenced by flow cytometry and caspase-3 activity assays. In contrast, ectopic expression of Pokemon in HL7702 cells led to resistance to anoikis. Dual-luciferase reporter and ChIP assays illustrated that Pokemon suppressed Bim transcription via direct binding to its promoter. Our results suggest that Pokemon prevents anoikis through the suppression of Bim expression, which facilitates tumor cell invasion and metastasis. This Pokemon-Bim pathway may be an effective target for therapeutic intervention for cancer.

Abstract 5250: Optimization of an orthotopic mouse model for in vivo fluorescent uPA imaging in breast cancer

Abstract The urokinase plasminogen activator (uPA) system is one of the serine protease systems involved in extracellular matrix degradation, which will facilitate tumor cell invasion and intravasation. Recently, the Medicinal Chemistry group of the University of Antwerp (UAMC) developed some very selective covalently binding uPA inhibitors. Besides its function as possible therapeutic target, uPA and its endogenous inhibitor PAI-1 are described as prognostic biomarkers for breast cancer with the highest level of evidence. Commercial ELISA and PCR kits are available for the screening of uPA in breast tumors. These ex vivo assays measure or predict the total uPA content (active and inactive). We present here a more advanced technique, which will be able to monitor the actual enzymatic uPA activity in a non-invasive manner. The technology is based on the modification of the UAMC inhibitors towards activity based probes containing a fluorescent label. Currently, we are evaluating these probes as imaging tools for uPA monitoring. The developed imaging probes will be profoundly tested in an orthotopic model of human MDA-MB-231 and MCF7 breast cancer cells since these cell lines show a high and low uPA expression respectively. We will use these models to study the potential beneficial characteristics of activity based probes in the field of bio-imaging applications targeted to uPA as a validated biomarker. The current protocol, which is still under evaluation, can be described as followed: Cell suspensions (2.106 cells/100μl) of each cell line were implanted in the mammary fat pads at both flanks of 7 mice, alternatively with and without Matrigel. Tumor size was monitored using bioluminescent imaging (BLI) and caliper measurements. When the tumor volume reached 200 - 400mm3, the rhodamine labeled uPA-inhibitor (uPA-probe) was administered (0.3 mg / kg i.v.). Fluorescent images were taken at 1, 2, 4, 6, 8, 12, 24 and 48 hours after injection. At 48 hours, the mice were sacrificed and the tumor, liver and lung were excised for ex vivo bioluminescent or fluorescent imaging and stored for histological examination. In this presentation, we describe the evaluation of the model and the first promising results. Considering the role of uPA in migration and invasion, the uPA-probe will also be tested in the future for the detection of micrometastases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5250. doi:1538-7445.AM2012-5250

The Process of Macrophage Migration Promotes Matrix Metalloproteinase-Independent Invasion by Tumor Cells

Tumor-associated macrophages are known to amplify the malignant potential of tumors by secreting a variety of cytokines and proteases involved in tumor cell invasion and metastasis, but how these macrophages infiltrate tumors and whether the macrophage migration process facilitates tumor cell invasion remain poorly documented. To address these questions, we used cell spheroids of breast carcinoma SUM159PT cells as an in vitro model of solid tumors. We found that macrophages used both the mesenchymal mode requiring matrix metalloproteinases (MMPs) and the amoeboid migration mode to infiltrate tumor cell spheroids. Whereas individual SUM159PT cells invaded Matrigel using an MMP-dependent mesenchymal mode, when they were grown as spheroids, tumor cells were unable to invade the Matrigel surrounding spheroids. When spheroids were infiltrated or in contact with macrophages, tumor cell invasiveness was restored. It was dependent on the capacity of macrophages to remodel the matrix and migrate in an MMP-independent mesenchymal mode. This effect of macrophages was much reduced when spheroids were infiltrated by Matrigel migration-defective Hck(-/-) macrophages. In the presence of macrophages, SUM159PT migrated into Matrigel in the proximity of macrophages and switched from an MMP-dependent mesenchymal migration to an amoeboid mode resistant to protease inhibitors.Thus, in addition to the well-described paracrine loop between macrophages and tumor cells, macrophages can also contribute to the invasiveness of tumor cells by remodeling the extracellular matrix and by opening the way to exit the tumor and colonize the surrounding tissues in an MMP-dispensable manner.

Epithelial-Mesenchymal Transition Induced by Senescent Fibroblasts

Depending on the cell type and tissue environment, epithelial and mesenchymal cell phenotypes are not static and can be highly dynamic. Epithelial-mesenchymal transitions (EMTs) and reverse EMTs provide flexibility during embryogenesis. While EMTs are a critical normal process during development and wound healing, properties of the EMT have been implicated in human pathology, particularly cancer metastasis. A normal undamaged epithelium does not typically exhibit features of an EMT. However, particularly under the influence of the surrounding microenvironment, cancer cells may reactivate developmental phenotypes out of context in the adult. This reactivation, such as the EMT, can facilitate tumor cell invasion and metastasis, and therefore is a major mechanism of tumor progression. Conversely, cellular senescence, which is associated with aging, is a process by which cells enter a state of permanent cell cycle arrest, thereby constituting a potent tumor suppressive mechanism. However, accumulating evidence shows that senescent cells can have deleterious effects on the tissue microenvironment. The most significant of these effects is the acquisition of a senescence-associated secretory phenotype (SASP) that turns senescent fibroblasts into pro-inflammatory cells having the ability to promote tumor progression, in part by inducing an EMT in nearby epithelial cells. Here, we summarize the potential impacts of SASP factors, particularly interleukins, on tissue microenvironments and their ability to stimulate tumor progression through induction of an EMT.

Role of Hyaluronan-Mediated CD44 Signaling in Head and Neck Squamous Cell Carcinoma Progression and Chemoresistance

Head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy that may involve the oral cavity, pharynx, larynx, and paranasal sinuses. The mechanisms of tumor progression underlying the clinical behavior of HNSCC remain unclear. CD44 comprises a family of transmembrane receptors that can give rise to multiple CD44 variant isoforms. Hyaluronan (HA), a major extracellular matrix component is the primary ligand for CD44 receptors. HA and CD44 signaling play an important role in HNSCC progression. Several CD44 variant isoforms (including v3-, v6-, and v10-containing isoforms) are associated with advanced disease, possibly through unique growth factor interactions with binding domains in the inserted variant regions of the cytoplasmic domain of CD44. In HNSCC, HA mediates the formation of a complex including CD44 and the epidermal growth factor receptor (EGFR) which is overexpressed in a large proportion of HNSCCs. Downstream effectors under EGFR regulation are activated, promoting promote cell growth and tumor survival. The leukemia-associated Rho-guanine nucleotide exchange factor (LARG) also associates with CD44 and EGFR to promote several Ras and RhoA pathway effectors, leading to cell migration, growth, and tumor survival. The secretion of matrix metalloproteinases, necessary for tumor cell invasion, is also regulated by these HA/CD44-mediated pathways. Finally, EGFR-mediated pathways play major roles in the HA/CD44 promotion of chemoresistance in HNSCC. Understanding HA/CD44-mediated signaling pathways may lead to improved treatment of HNSCC.

614 Mechanisms of cell signalling in metastasis

Aberrant TGFβ signaling is common in human cancers and contributes to tumor metastasis. Here, we demonstrate that Gr-1+CD11b+ myeloid cells are recruited into mammary carcinomas with type II TGFβ receptor gene (Tgfbr2) deletion and directly promote tumor metastasis. Gr-1+CD11b+ cells infiltrate into the invasive front of tumor tissues and facilitate tumor cell invasion and metastasis through a process involving metalloproteinase activity. This infiltration of Gr-1+CD11b+ cells also results in increased abundance of TGFβ1 in tumors with Tgfbr2 deletion. The recruitment of Gr-1+CD11b+ cells into tumors with Tgfbr2 deletion involves two chemokine receptor axes, the SDF-1/CXCR4 and CXCL5/CXCR2 axes. Together, these data indicate that Gr-1+CD11b+ cells contribute to TGFβ-mediated metastasis through enhancing tumor cell invasion and metastasis.

Abstract 2237: Expression of the trefoil protein TFF3 in human breast cancers

Abstract Expression of the trefoil protein TFF3 in human breast cancers The purpose of this study is to investigate expression of the trefoil factor family protein, TFF3 in a large series of human breast cancers. Trefoil factors are small secreted proteins that are expressed in mucus secreting epithelia. They are involved in mucosal protection in part by stimulation of cell motility. It has been proposed that the motogenic actions of trefoil proteins may facilitate tumor cell invasion and metastasis. Expression of TFF3 mRNA is regulated by estrogen in human breast cancer cell lines and strong association between estrogen receptor and TFF3 mRNA expression has been demonstrated in breast tumors by expression microarray analyses. A tissue microarray was constructed from 300 primary breast tumors and 76 metastatic deposits of breast tumors from patients who presented in the northern region of England between 2002 and 2003. TFF3 expression was evaluated by immunohistochemistry with an antibody raised against correctly-folded human TFF3 protein. Each sample was analyzed in duplicate. The intensity of the immunoreactions in 1,000 tumor cells was evaluated as: absent, weak, moderate or strong. The association of TFF3 expression with various clinico-pathological features and with estrogen receptor protein expression was tested statistically with SPSS software (p<0.01). Clear TFF3 expression is demonstrated in normal and malignant breast epithelial cells. There is no expression in stromal, endothelial or immune cells. Expression of TFF3 varied enormously between tumor samples. TFF3 expression is highest in mucinous and tubular breast adenocarcinomas. TFF3 is expressed at higher levels in lobular than in ductal breast cancers. TFF3 expression is associated strongly with estrogen receptor expression. There is a negative association between TFF3 expression and tumor grade. There is however a positive association between TFF3 expression and presence of vascular invasion and TFF3 expression is higher in metastatic breast tumor cells than in primary breast tumor cells. This is the first report of TFF3 protein expression in a large series of human breast tumors. The strong association between TFF3 and estrogen receptor suggests that TFF3 expression is dependent on estrogen in a significant proportion of breast tumors. The high expression of TFF3 in breast tumors with vascular invasion and in metastatic breast tumor cells supports the hypothesis that TFF3 predisposes towards breast cancer cell invasion. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2237.

Knockdown of hTERT and concurrent treatment with interferon-gamma inhibited proliferation and invasion of human glioblastoma cell lines

Human telomerase reverse transcriptase (hTERT) is the catalytic component of telomerase that facilitates tumor cell invasion and proliferation. Telomerase and hTERT are remarkably upregulated in majority of cancers including glioblastoma. Interferon-gamma (IFN-γ) modulates several cellular activities including cell cycle and multiplication through transcriptional regulation. The present investigation was designed to unravel the molecular mechanisms of the inhibition of cell proliferation, migration, and invasion of human glioblastoma SNB-19 and LN-18 cell lines after knockdown of hTERT using a plasmid vector based siRNA and concurrent treatment with IFN-γ. We observed more than 80% inhibition of cell proliferation, migration, and invasion of both cell lines after the treatment with combination of hTERT siRNA and IFN-γ. Our studies also showed accumulation of apoptotic cells in subG1 phase and an increase in cell population in G0/G1 with a reduction in G2/M phase indicating cell cycle arrest in G0/G1 phase for apoptosis. Semiquantitative and real-time RT-PCR analyses demonstrated significant downregulation of c-Myc and upregulation of p21 Waf1 and p27 Kip1. Western blotting confirmed the downregulation of the molecules involved in cell proliferation, migration, and invasion and also showed upregulation of cell cycle inhibitors. In conclusion, our study demonstrated that knockdown of hTERT and concurrent treatment with IFN-γ effectively inhibited cell proliferation, migration, and invasion in glioblastoma cells through downregulation of the molecules involved in these processes and cell cycle inhibition. Therefore, the combination of hTERT siRNA and IFN-γ offers a potential therapeutic strategy for controlling growth of human glioblastoma cells.

Inhibition of Urokinase Activity Reduces Primary Tumor Growth and Metastasis Formation in a Murine Lung Carcinoma Model

Lung cancer is the most common malignancy in humans. Urokinase (uPA) plays a crucial role in carcinogenesis by facilitating tumor cell invasion and metastasis. We investigated the effect of the highly specific urokinase inhibitor CJ-463 (benzylsulfonyl-D-Ser-Ser-4-amidinobenzylamide) on tumor growth, metastasis formation, and tumor vascularization in the murine Lewis lung carcinoma (LLC) and a human small lung cancer model. A quantity of 3 x 10(6) LLC cells were subcutaneously injected into the right flank of C57Bl6/N mice, uPA knock out, and uPA receptor knockout mice. Seven days later mice were randomized to receive intraperitoneally either saline (control group), CJ-463 (10 and 100 mg/kg, twice a day), or its ineffective stereoisomer (10 mg/kg, twice a day). Tumor volume was measured every second day and metastasis formation was monitored by volumetric-computed tomography. Twelve days after onset of treatment mice were killed and tumors were prepared for histologic examination. Treatment with CJ-463 resulted in a significant inhibition of primary tumor growth, with the highest efficacy seen in the 100 mg/kg group. In addition, histological analysis of the lung revealed a significant reduction in lung micrometastasis in the 100 mg/kg group. Similarly, a reduced seeding of tumor cells into the lung after intravenous injection of LLC cells was observed in inhibitor-treated mice. In these mice, treatment with CJ-463 appeared not to significantly alter the relative extent of tumor vascularization. In vitro, proliferation of LLC cells remained unchanged upon inhibitor treatment. CJ-463 was found to similarly reduce tumor growth in uPA receptor knockout mice, but was ineffective in uPA knockout mice. Our results suggest that synthetic low-molecular-weight uPA-inhibitors offer as novel agents for treatment of lung cancer.

Cadmium and Breast Cancer: Exposure Associated with Basal-Like Phenotype

Cadmium has been linked with several human diseases including chronic kidney disease and cancer. As a carcinogen, cadmium targets several sites that are considered endocrine-sensitive, and some data suggest the breast may be among them. Although cadmium has been hypothesized to act as a metalloestrogen—a metal that triggers an estrogen-like reaction—research to date has not confirmed this as a mechanism linking cadmium and breast cancer. Additionally, although many breast cancers are estrogen-dependent, some of the most difficult-to-treat cases are not. A new study finds that cadmium can induce malignant transformation in breast cells in vitro regardless of the absence of estrogen receptors, strengthening evidence that cadmium exposure may be a factor in breast cancer, a leading cause of cancer deaths among women [EHP 117:1847–1852; Benbrahim-Tallaa et al.]. MCF-10A cells, which are derived from normal human breast epithelium, were grown with either no cadmium exposure or continuous cadmium exposure (2.5 μM) for up to 40 weeks. Positive controls included MCF-7 human breast cancer cells (which express the estrogen receptors ER-α and ER-β) and SKBR3 breast cancer cells (which express HER2, a receptor that can be overexpressed in certain malignant breast cancer cells). In contrast, MCF-10A cells do not express ER-α, ER-β, or HER2 proteins, although expression can be acquired in carcinogenesis. Chronic cadmium exposure of the MCF-10A cells yielded increased expression of matrix metalloproteinase-9, an enzyme that facilitates tumor cell invasion. These cells also formed cell mounds, indicating a loss of contact inhibition (the natural process of cell growth stopping once a certain density of cells is reached). When these transformed cells were implanted in mice, they formed highly aggressive tumors that demonstrated metastatic potential. Transformed MCF-10A cells remained negative for ER-α and ER-β and also lacked HER2 protein. However, metallothionein, typically overexpressed in ER-negative breast cancers, was elevated as were several other breast cancer markers. These characteristics collectively suggest that cadmium could be a risk factor for a basal-like breast cancer phenotype, which is clinically associated with a higher risk of relapse after treatment and lower survival rates. The precise mechanism by which cadmium may transform breast cells is unknown, but the results of this study suggest it is unlikely to be a metalloestrogenic effect via estrogen receptors. Although additional research is needed to define the mechanism, the current study provides strong evidence that cadmium may play a role in human breast cancer.

Chemoattractant Signaling between Tumor Cells and Macrophages Regulates Cancer Cell Migration, Metastasis and Neovascularization

Tumor-associated macrophages are known to influence cancer progression by modulation of immune function, angiogenesis, and cell metastasis, however, little is known about the chemokine signaling networks that regulate this process. Utilizing CT26 colon cancer cells and RAW 264.7 macrophages as a model cellular system, we demonstrate that treatment of CT26 cells with RAW 264.7 conditioned medium induces cell migration, invasion and metastasis. Inflammatory gene microarray analysis indicated CT26-stimulated RAW 264.7 macrophages upregulate SDF-1α and VEGF, and that these cytokines contribute to CT26 migration in vitro. RAW 264.7 macrophages also showed a robust chemotactic response towards CT26-derived chemokines. In particular, microarray analysis and functional testing revealed CSF-1 as the major chemoattractant for RAW 264.7 macrophages. Interestingly, in the chick CAM model of cancer progression, RAW 264.7 macrophages localized specifically to the tumor periphery where they were found to increase CT26 tumor growth, microvascular density, vascular disruption, and lung metastasis, suggesting these cells home to actively invading areas of the tumor, but not the hypoxic core of the tumor mass. In support of these findings, hypoxic conditions down regulated CSF-1 production in several tumor cell lines and decreased RAW 264.7 macrophage migration in vitro. Together our findings suggest a model where normoxic tumor cells release CSF-1 to recruit macrophages to the tumor periphery where they secrete motility and angiogenic factors that facilitate tumor cell invasion and metastasis.