Chemoinvasion Assay Research Articles

Statins may have double-edged effects in patients with lung adenocarcinoma after lung resection.

Purpose: The epithelial to mesenchymal transition (EMT) is pivotal for driving metastasis and recurrence in lung cancer. Some in vitro reports have shown that statins suppress EMT by inactivating mutant p53 functions. Several clinical trials of conventional treatments with statins have been performed, but the effect of these drugs on prognosis is still uncertain. The purpose of this study is to examine the impact of statins on EMT and the prognosis of patients with lung adenocarcinoma.Materials and methods: Morphological changes were evaluated and EMT markers (E-cadherin, vimentin) were analyzed by Western blotting in p53-overexpressing H1650 and mutant p53-harboring H1975 lung adenocarcinoma cells, with and without simvastatin administration. The invasive ability of these cells was analyzed in a Matrigel chemoinvasion assay. A total of 250 lung adenocarcinoma specimens were also collected from patients who underwent surgery in our institute. EMT markers in these tumor specimens were evaluated by immunostaining and p53 mutation status was determined by direct sequencing. Associations among EMT status, p53 mutation status, and statin use were evaluated, and prognosis was analyzed using a marginal structural model.Results: Mutant p53 induced EMT and increased the invasive ability of H1650 cells. Simvastatin restored the epithelial phenotype and decreased the invasive ability of both H1650 and H1975 cells. Statin administration was associated with inactivation of EMT only in patients with mutant p53, which was consistent with the in vitro results. Moreover, in patients with mutant p53, statin users had significantly better survival than non-statin users. In contrast, statins significantly worsened the prognosis of patients with wild type p53 (HR 2.10, 95% CI 1.14–3.85).Conclusion: Statins suppress EMT and change the prognosis of patients with lung adenocarcinoma in a p53 mutation-dependent manner.

Abstract 3202: Radiation increases invasive activity of breast cancer cells via altering lysosome exocytosis

Abstract Radiotherapy is a standard treatment for many localized solid cancers. However, previous studies have shown that radiation may increase the invasive activity of cancer cells and potentially distant metastasis. Recently, lysosome exocytosis has been linked to cancer cell invasiveness and progression. In this study, we evaluate the role of lysosome exocytosis on invasive activity of breast cancer cells upon radiation.We used both human and murine breast cancer cell lines (MDA-MB-231 and 4T1). The cells were treated with a single radiation dose of 4 Gy. Cell invasive activity was measured by matrigel chemoinvasion assay. Lysosome exocytosis was quantified by fluorescein isothiocyanate conjugated dextran (FITC-dextran) intake assay, cell-surface lysosomal associated membrane protein 1 (LAMP1) expression and cellular lysosome distribution assay. To validate the lysosome function, lysosome inhibitors, bafilomycin A1 and chloroquine were used. Short hairpin RNA (shRNA) was used to knockdown ARL8B, which is a small GTPase protein that regulates lysosome distribution and exocytosis. The invasive activity and lysosome exocytosis of tested breast cancer cell lines were increased after radiation treatment. Treatment with lysosome inhibitor bafilomycin A1 or chloroquine decreased the invasive activity of cancer cells, with or without radiation treatment. The protein level of ARL8B increased in the lysosome fraction upon radiation. Down-regulation of ARL8B with shRNA led to a decrease in lysosome exocytosis with a concomitant inhibition radiation induced invasive activity of the breast cancer cells without affecting the basal invasiveness. In addition, overexpression of ARL8B increased the invasive activity of breast cancer cells, which was similar to the result obtained after radiation.In summary, radiation enhances lysosome exocytosis in breast cancer cells that can lead to their increased invasive activity. Our findings provide a novel mechanism to understand cancer invasion after radiotherapy and suggest novel approaches to counteract this undesirable effect of radiotherapy in the future. Citation Format: Ping-Hsiu Wu, Yasuhito Onodera, Amato J. Giaccia, Quynh-Thu Le, Hiroki Shirato, Jin-Min Nam. Radiation increases invasive activity of breast cancer cells via altering lysosome exocytosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3202.

Enterolactone modulates the ERK/NF-κB/Snail signaling pathway in triple-negative breast cancer cell line MDA-MB-231 to revert the TGF-β-induced epithelial-mesenchymal transition.

Objective:Triple-negative breast cancer (TNBC) is highly metastatic, and there is an urgent unmet need to develop novel therapeutic strategies leading to the new drug discoveries against metastasis. The transforming growth factor-β (TGF-β) is known to promote the invasive and migratory potential of breast cancer cells through induction of epithelial–mesenchymal transition (EMT) via the ERK/NF-κB/Snail signaling pathway, leading to breast cancer metastasis. Targeting this pathway to revert the EMT would be an attractive, novel therapeutic strategy to halt breast cancer metastasis.Methods:Effects of enterolactone (EL) on the cell cycle and apoptosis were investigated using flow cytometry and a cleaved caspase-3 enzyme-linked immunosorbent assay (ELISA), respectively. Effects of TGF-β induction and EL treatment on the functional malignancy of MDA-MB-231 breast cancer cells were investigated using migration and chemo-invasion assays. The effects of EL on EMT markers and the ERK/NF-κB/Snail signaling pathway after TGF-β induction were studied using confocal microscopy, quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blot, and flow cytometry.Results:Herein, we report that EL exhibits a significant antimetastatic effect on MDA-MB-231 cells by almost reverting the TGF-β-induced EMT in vitro. EL downregulates the mesenchymal markers N-cadherin and vimentin, and upregulates the epithelial markers E-cadherin and occludin. It represses actin stress fiber formation via inhibition of mitogen-activated protein kinase p-38 (MAPK-p38) and cluster of differentiation 44 (CD44). EL also suppresses ERK-1/2, NF-κB, and Snail at the mRNA and protein levels. Conclusions:Briefly, EL was found to inhibit TGF-β-induced EMT by blocking the ERK/NF-κB/Snail signaling pathway, which is a promising target for breast cancer metastasis therapy.

Evaluation of Suppressive Effects of Tranilast on the Invasion/Metastasis Mechanism in a Murine Pancreatic Cancer Cell Line.

Numerous studies have investigated the mechanism of the antitumor effect of tranilast, well known as an antiallergic drug. Herein, we investigated the mechanism of the antitumor effects of tranilast using murine PAN 02 cell line. In an allograft mouse model, the number of metastatic sites in the liver was counted. Wound healing and chemoinvasion assay were performed to evaluate migration and invasive ability of PAN 02, respectively. Activities of matrix metalloproteinases (MMPs) were evaluated by gelatin zymography. The expression of cofactors in the activation of MMP-2 was assessed by immunohistochemical staining at the front of metastasis. The number of metastatic sites was reduced in tranilast-treated groups. Migration ability and tumor invasiveness were significantly inhibited by tranilast in a dose-dependent manner. Gelatin zymography revealed inhibition of MMP-2 activity. Immunohistochemical staining showed remarkable attenuation of tissue inhibitor of metalloproteinase (TIMP-) 2 expression in tranilast-treated groups. Tissue inhibitor of metalloproteinase 2 is necessary for MMP-2 activation with interaction between membrane type 1-MMP and proMMP-2. These results suggested that tranilast may inhibit MMP-2 activation through attenuating TIMP-2 expression, resulting in inhibition of tumor invasion and metastasis. Our results showed possibility of tranilast in clinical application for novel cancer therapy.

Ezrin Is Associated with Disease Progression in Ovarian Carcinoma.

ObjectiveEzrin and p130Cas are structural proteins with an important role in signaling pathways and have been shown to promote cancer dissemination. We previously reported on overexpression of both ezrin and p130Cas in breast carcinoma effusions compared to primary carcinomas. Since ovarian and breast carcinomas share the ability to disseminate by forming malignant effusions, we sought to study the role of these molecules in ovarian carcinoma (OC).MethodsOC cell lines were cultured in two different 3-dimensional conditions, on alginate scaffolds and as spheroids, which served as models for solid tumor and malignant effusions, respectively. shRNA was used to reduce protein expression in the cells. The malignant potential was evaluated by chemo-invasion assay, branching capacity on Matrigel and rate of proliferation. Subsequently, clinical specimens of high-grade serous carcinoma effusions, ovarian tumors and solid metastases were analyzed for ezrin and p130Cas expression.ResultsHigher ezrin expression was found in cells composing the spheroids compared to their counterparts cultured on alginate scaffold and in clinical samples of malignant effusions compared to solid tumors. In addition, reduced Ezrin expression impaired the invasion ability and the branching capacity of OC cells to a greater extent than reduced p130Cas expression. However, ezrin and p130Cas expression in effusions was unrelated to clinical outcome.ConclusionsThe 3-dimensional cell cultures were found to mimic the different tumor sites and be applicable as a model. The in vitro results concur with the clinical specimen analysis, suggesting that in OC, the role of ezrin in disease progression is more pronounced than that of p130Cas.

Extracellular Matrix Invasion in Metastases and Angiogenesis: Commentary on the Matrigel "Chemoinvasion Assay".

Invasive and metastatic cells must cross the basement membrane's extracellular matrix to disseminate to distant sites. Although in the eighties the concept was well established, no easy in vitro functional assay was available. Working in Hynda Kleinman's and George Martin's laboratory at NIH (Bethesda, MD), where the reconstituted basement membrane Matrigel was discovered, I had the intuition that Matrigel coating of migration filters could represent a valid tool to mimic in vitro biological matrix barriers. The "chemoinvasion assay" using Matrigel in Boyden blind-well chambers was developed in 1985-1986 and published in Cancer Research in 1987. It was a rapid and easy tool for studying invasion, a crucial step in cancer metastasis. Since its conception, the assay has been employed for studies on the metastatic process, angiogenesis, and for the screening of drugs that are potentially able to decrease cell invasion. It was adapted to be easily employed as a routine assay and commercialized. In that historical article, we also described the use of thick layers of Matrigel for the study of morphogenesis of invasive cells, a simple and visual assay, adaptable to reproduce collective cell migration in vitro To date, in its diverse optimized variants, the chemoinvasion assay is still widely used, contributing to novel data production. In the era of precision medicine and next-generation sequencing, the cheap, fast, and reproducible chemoinvasion assay may have further developments, including possible applications in the investigations on cancer stem cells, immunity and immune modulators, applications with siRNA silencing, selection of aggressive cell populations, and phenotypes and genetic evaluations. Cancer Res; 76(16); 4595-7. ©2016 AACR.See related article by Albini A et al., Cancer Res 1987;47:3239-45Visit the Cancer Research 75(th) Anniversary timeline.

Abstract 1582: Tumor-associated stromal cells increase malignancy of human colorectal cancers triggering EMT induction

Abstract In the last decades it has been demonstrated that the stroma surrounding tumor not only provides mechanical support, but profoundly influences the outcome of the primary tumor and its possible relapse, impacting on proliferation, metastatic progression and resistance to therapies. Major components of the stroma in most types of human carcinomas are the Tumor-Associated Stromal Cells (TASC). Different sources have been proposed for this population, ranging from the resident fibroblasts to bone marrow-derived mesenchymal stromal cells (BM-MSC) recruited to the trans-differentiated epithelial and endothelial cells, but the scenario is still unclear. Moreover, TASC have an intrinsic heterogeneity, combined also with the presence of multiple subpopulations, and they share several markers with other cell subsets of the stroma, thus making the identification of this population, based on the expression of specific markers, an arduous task. In this intricate context, we aim to address phenotypic and functional characterization of TASC isolated from colorectal cancer specimens, and to analyze TASC-mediated effects on CRC development and progression in vivo. TASC were characterized for phenotype and differentiation capacity. Human CRC cells were cultured in the presence or absence of TASC for five days. After co-culture tumor cells were sorted by flow cytometry and evaluated for the expression of EMT-related genes by Real Time PCR and for in vitro invasiveness by chemoinvasion assay. Furthermore, their tumorigenicity and the metastatic potential were assessed upon subcutaneous and intracoecally injection in NOD/SCID mice. Developed tumors, metastatic foci and circulating tumor cells were assessed by histology, flow cytometry and Imaging Flow Cytometry. Our results indicate that TASC resemble BM-MSC in morphology and phenotype. They also comprise a multipotent subpopulation that is able to differentiate into adipogenic and osteogenic lineage. After co-culture with CRC cells, TASC express membrane-bound TGF-β, through which they are capable to trigger epithelial-to-mesenchymal transition (EMT). Upon subcutaneous injection in NOD/SCID mice, tumor cells co-cultured or admixed with TASC before injection, show a faster growth kinetic and develop larger tumor masses as compared to tumor cells alone. Furthermore developed tumor masses are characterized by a higher vessel density. Notably, upon intracoecal injection TASC presence or conditioning leads to a higher metastatic formation in lungs and livers and an increased number of circulating tumor cells in the peripheral blood, most importantly comprising cells undergoing EMT. Our data clearly show that the stromal component in the human colorectal cancer microenvironment increases the malignancy of tumor cells, by providing pro-mitogenic factors and by triggering EMT initiation in vitro and in vivo, thus promoting tumorigenicity and metastasis formation in vivo. Citation Format: Valentina Mele, Valeria Governa, Manuele G. Muraro, Jesus F. Glaus Garzon, Lubor Borsig, Silvio Daester, Raoul Droeser, Daniel Oertli, Markus Zuber, Raffaele Rosso, Giulio C. Spagnoli, Giandomenica Iezzi. Tumor-associated stromal cells increase malignancy of human colorectal cancers triggering EMT induction. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1582.

Abstract 2370: Tumor-associated stromal cells increase malignancy of human colorectal cancers triggering the induction of Epithelial-to-mesenchymal transition

Abstract During tumor formation, normal tissue microenvironment is transformed in an “altered” niche, composed of non-malignant supporting cells, which influence the homeostasis of cancer cells via paracrine regulators. Tumor-associated stromal cells (TASC) are the prominent stromal elements in most types of human carcinomas including colorectal cancer (CRC). The differentiation of TASC from other cell types, such as resident stromal cells or bone marrow-derived mesenchymal stem cells (BM-MSC) is mainly mediated by factors produced during the crosstalk with malignant cells. TASC produce various extracellular matrix proteins, chemokines, and other tumor-promoting factors which affect vascularization, proliferation, tumor cell invasiveness and survival. Emerging data suggests that they also play a critical role in determining response to therapy. For instance TASC-derived factors may contribute to the development of a protective milieu by influencing cell-cell/cell-matrix interactions, cancer cell survival, and suppression of anti-tumor immune responses. Moreover, physical contact between TASC and malignant cells supports tumor cell survival via activation of anti-apoptotic pathways or inducing epithelial-to-mesenchymal transition (EMT). We previously showed that BM-MSC are capable of triggering EMT in CRC cells, leading to increased tumor cell aggressiveness in vitro and in vivo. However, the role played by the stromal component and the processes induced on CRC cells remain to be fully elucidated. For this purpose we have addressed phenotypic and functional characterization of TASC in vitro and we have analyzed TASC-mediated effects on CRC development and progression in vivo. TASC were characterized for phenotype and differentiation capacity. Human CRC cells were cultured in the presence or absence of TASC for five days. After co-culture tumor cells were sorted by flow cytometry and evaluated for the expression of EMT-related genes by Real Time PCR and for in vitro invasiveness by chemoinvasion assay. Furthermore, their tumorigenicity was assessed upon injection in NOD/SCID mice and developing tumors were analyzed. Our results indicate that TASC resemble BM-MSC in morphology and phenotype. They comprise a multipotent subpopulation that is able to differentiate into adipogenic and osteogenic lineage. After co-culture with CRC cells they express membrane-bound TGF-β, through which they are capable to initiate EMT in tumor cells. Moreover CRC cells acquire a more invasive phenotype after co-culture with TASC. Upon subcutaneous injection in NOD/SCID mice, tumor cells co-cultured with TASC show a significantly faster growth kinetic and develop significantly larger tumor masses as compared to tumor cells alone. Thus our data show that the stromal component of CRC increases the tumor cells malignancy triggering EMT induction. Citation Format: Valentina Mele, Manuele Giuseppe Muraro, Raoul Droeser, Daniel Oertli, Markus Zuber, Raffaele Rosso, Ivan Martin, Michael Heberer, Giulio C. Spagnoli, Giandomenica Iezzi. Tumor-associated stromal cells increase malignancy of human colorectal cancers triggering the induction of Epithelial-to-mesenchymal transition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2370. doi:10.1158/1538-7445.AM2015-2370

MicroRNA-425-5p is upregulated in human gastric cancer and contributes to invasion and metastasis in vitro and in vivo.

Emerging evidence indicates that microRNAs are involved in the development and progression of gastric cancer (GC), functioning as tumor suppressors or oncogenes. The aim of the current study was to explore the potential mechanism of microRNA-425-5p (miR-425-5p) in GC. Using reverse transcription quantitative polymerase chain reaction, miR-425-5p expression was detected in GC cell lines (SGC-7901, MKN-28, MKN-45, BGC-823 and AGS) and the GES-1 cell line. Cell proliferation, soft agar colony formation, flow cytometry, haptotactic migration and matrigel chemoinvasion assays were used to test the proliferation, apoptosis, invasion and migration of BGC-823 cells transfected with miR-425-5p mimics or an inhibitor. Female BALB/c mice were randomly divided into three groups. Each experimental group contained five mice. BGC 823 cells were stably transfected with miR-425-5p inhibitor, wild type or negative control and were injected into the mice through the tail vein. It was found that miR-425-5p expression was significantly upregulated in all the GC cell lines when compared with the GES-1 cell line. Downregulation of miR-425-5p expression inhibited GC cell proliferation, invasion and migration. In the in vivo studies, downregulation of miR-425-5p expression suppressed pulmonary metastasis in the nude mice. Thus, miR-425-5p was demonstrated to have the potential to become a novel metastasis-associated gene; however, the mechanisms associated with these effects require further study. In the future, the development of miR-425-5p as a novel therapeutic strategy for the treatment of GC may be possible.

Pro-angiogenic activity of notoginsenoside R1 in human umbilical vein endothelial cells in vitro and in a chemical-induced blood vessel loss model of zebrafish in vivo.

This study aimed at investigating whether notoginsenoside R1 (R1), a unique saponin found in Panax notoginseng could promote angiogenic activity on human umbilical vein endothelial cells (HUVECs) and elucidate their potential molecular mechanisms. In addition, vascular restorative activities of R1 was assessed in a chemically-induced blood vessel loss model in zebrafish. The in vitro angiogenic effect of R1 was compared with other previously reported angiogenic saponins Rg1 and Re. The HUVECs proliferation in the presence of R1 was determined by cell proliferation kit II (XTT) assay. R1, Rg1 and Re-induced HUVECs invasion across polycarbonate membrane was stained with Hoechst-33342 and quantified microscopically. Tube formation assay using matrigelcoated wells was performed to evaluate the pro-angiogenic actions of R1. In order to understand the mechanism underlying the pro-angiogenic effect, various pathway inhibitors such as SU5416, wortmannin (wort) or L-Nω-nitro- L-arginine methyl ester hydrochloride (L-NAME), SH-6 were used to probe the possible involvement of signaling pathway in the R1 mediated HUVECs proliferation. In in vivo assays, zebrafish embryos at 21 hpf were pre-treated with vascular endothelial growth factor (VEGF) receptor kinase inhibitor II (VRI) for 3 h only and subsequently post-treated with R1 for 48 h, respectively. The intersegmental vessels (ISVs) in zebrafish were assessed for the restorative effect of R1 on defective blood vessels. R1 could stimulate the proliferation of HUVECs. In the chemoinvasion assay, R1 significantly increased the number of cross-membrane HUVECs. In addition, R1 markedly enhanced the tube formation ability of HUVECs. The proliferative effects of these saponins on HUVECs were effectively blocked by the addition of SU5416 (a VEGF-KDR/Flk-1 inhibitor). Similarly, pre-treatment with wort [a phosphatidylinositol 3-kinase (PI3K)-kinase inhibitor], L-NAME [an endothelial nitric oxide synthase (eNOS) inhibitor] or SH-6 (an Akt pathway inhibitor) significantly abrogated the R1 induced proliferation of HUVECs. In chemicallyinduced blood vessel loss model in zebrafish, R1 significantly rescue the damaged ISVs. R1, similar to Rg1 and Re, had been showed pro-angiogenic action, possibly via the activation of the VEGF-KDR/Flk-1 and PI3K-Akt-eNOS signaling pathways. Our findings also shed light on intriguing pro-angiogenic effect of R1 under deficient angiogenesis condition in a pharmacologic-induced blood vessels loss model in zebrafish. The present study in vivo and in vitro provided scientific evidence to explain the ethnomedical use of Panax notoginseng in the treatment of cardiovascular diseases, traumatic injuries and wound healing.

Antisense Oligonucleotides Targeted to KRAS Gene Could Inhibit the Tumor Growth, Invasion, and MMP-2 and MMP-9 Expression in Hamster Pancreatic Cancer Model In Vitro and In Vivo

Introduction: To clarify the effectiveness of this gene therapy in hamster experimental cancer model. Methods: HaP-T1, a cell line derived from nitrosamine-induced pancreatic cancer was used. Transfection assay with ASO was performed. MTT and MTT agarose assays were done. Chemoinvasion assay was performed. MMP-2 and MMP-9 production by the cell lines was determined by gelatin zymography. For in vivo experiments, subcutaneously resected tumors were implanted orthotopically in Syrian golden hamsters, which were divided in 3 groups: positive control (PC), sense-treated hamsters (STH), and antisense-treated hamsters (ATH). Oligonucleotides were administered for 2 weeks. Follow-up was done. Five animals of each group were sacrificed at days 10, 17, 24, 31, and 38 to study the local response and metastatic sites. Five animals of each group were left to study the survival time. Specimens were studied histopathologically. Orthotopic pancreatic tumor MMP production was measured by gelatin zymography. Results: ASO inhibited the tumoral growth and invasiveness. They downregulated active forms of MMP-2 and MMP-9 in a dose-dependent manner in vitro. Positive controls, STH, and ATH survived in average 72.7, 74.3, and 82.7 days, respectively. Spontaneous lymph node metastases were found from 31 days in ATH group, while PC and STH groups showed metastases and direct invasion to adjacent organs from 17 days. After death, metastatic sites were similar in the 3 groups. ASO downregulated the activation of MMP-9 more than MMP-2 in vivo. Conclusion: These experiments suggest that ASO targeted KRAS gene may be a good choice in the management of pancreatic cancer because of the suppression of tumor growth and invasiveness in vitro and in vivo. ASO also downregulated the activation of MMP-9 and MMP-2 in vitro and in vivo.

Effect of Tip30 expression on the invasion and metastasis of hepatoma cells

To investigate the effect of Tip30 on the invasion and metastasis of hepatoma cells. Recombinant plasmid pAAV-Tip30 was transfected to HepG2 cells by polylactic-coglycolic acid (PLGA) nanoparticles. RT-PCR was used to detect the mRNA expression of matrix metalloproteinases (MMP)-2, MMP-9 and epidermal growth factor receptor (EGFR). MMP- 2 and MMP-9 protein level in the tumor tissue was detected by Western blot. The proliferation, of hepatoma cells was evaluated by MTS assay and clone formation method. The adhesion and invasion of hepatoma cells were evaluated by adhesion assay and Transwell migration assay, respectively. With RT-PCR and Western blot, we found Tip30 decreased the expression of MMP-2 and MMP-9. MTS assay and cell clone formation method showed inhibited proliferation of the HepG2 cells. Chemo invasion assays showed that Tip30 decreased the invasiveness of hepatoma cells. Tip30 attenuated the binding of liver cancer cell line to human umbilical vein endothelial cells and fibronectin. The invasion and metastasis of hepatoma cells can be inhibited by Tip30 gene in vitro.

Preuss Award 121 Leptomeningeal Dissemination Cascade in Medulloblastoma

INTRODUCTION: Medulloblastomas (MBs) are a diverse set of tumors in which variation in treatment response is determined by the genes that drive tumor growth. A defining characteristic of MB is leptomeningeal dissemination (LMD) to the brain and spinal cord, which is inversely correlated with survival. We have developed a mouse model as well as in-vitro experiments to further elucidate the LMD cascade. METHODS: We selected ARNT, Eras and RabGDI2 as target genes for our mouse model (n = 80) after a random mutagenesis model implied LMD potential. We also evaluated these candidate genes in-vitro using assays including in-vitro scratch assay with DAOY cells and matrigel chemoinvasion assay with SHH-GNP cells to study their metastatic traits including migration, invasiveness, and proliferation. RESULTS: The in-vivo studies revealed 2 new LMD-promoting genes: ARNT/HIF1ß and RabGDI2. ARNT increased LMD by 6 times and RabGDI by 5 times over the baseline Sonic Hedgehog (SHH) MB model. Also, ARNT was found to be the most potent driver of LMD when compared to all genes evaluated in the past 5 years. The in-vitro studies revealed that ARNT and Eras stimulated migration in the 2 cell lines analyzed. RabGDI2 increased migration of SHH-GNPs only. ARNT and RabGDI2 enhanced invasiveness, but Eras had no effect. No gene stimulated proliferation in either cell lines. These results exemplify the complex interactions, with each gene playing a specific metastatic role, in the LMD cascade. CONCLUSION: We have created a systematic approach to analyzing LMD genes by testing them in-vivo to determine the LMD potential followed by characterizing their specific metastatic role in-vitro. We plan to utilize this in-vivo/in-vitro system to further elucidate genes of the LMD cascade and develop a comprehensive model of the medulloblastoma metastasis.

Use of the tumor repressor DEDD as a prognostic marker of cancer metastasis.

DEDD, a member of a family of death effector domain-containing proteins, plays crucial roles in mediating apoptosis, regulating cell cycle, and inhibiting cell mitosis. Our recent work demonstrates that DEDD is a novel tumor repressor, which impedes metastasis by reversing the epithelial-mesenchymal transition (EMT) process in breast and colon cancers. DEDD expression therefore may represent a prognostic marker and potential therapeutic target for the prevention and treatment of cancer metastasis. To reveal the anti-metastatic roles of DEDD in these cancer cells, a number of experiments, including immunohistochemistry, the establishment of stably overexpressing or silencing cancer cells, chemoinvasion assay, soft agar assay, protein degradation, and protein-protein interaction were used in our in vitro and in vivo studies. This chapter focuses on the details of these experiments to provide references for the researchers to investigate the function of a gene in the regulation of tumor metastasis.

Effects of ameloblastoma-associated fibroblasts on the proliferation and invasion of tumor cells.

Ameloblastoma is the most common odontogenic tumor, however, the molecular pathology, especially the role of the tumor stroma, is still unclear. To investigate the effects of the ameloblatoma-associated fibroblast (AAFs) on the proliferation and invasion of tumor cells. Cell culture, ELISA, proliferation and invasion assays. The cocultivations and three-dimensional organotypic cultures of the AAFs and the tumor cells were performed. The gingival fibroblasts (GFs) were used as the control. The levels of TGF-β and HGF in the conditioned media were analyzed using ELISA technique. The MTT proliferation assays and Boyden chamber chemoinvasion assays were also performed. ANOVA. Both AAFs and GFs stimulated tumor cell growth. The TGF-β level in the AAF group was more than those of GF group, whereas the HGF levels were not different. The AAF conditioned media also stimulated tumor cell proliferation and invasion more than the GF conditioned media. However, no difference in the thickness and morphology between the AAF and GF groups could be demonstrated in the organotypic models. Both AAFs and GFs support the proliferation of the tumor cells in cocultivation experiment and three-dimensional organotypic cultures. However, the AAFs have a tendency to stimulate the proliferation and induce the invasion more than GFs. Increased TGF-β levels in the AAF condition media suggested the possible role of this growth factor in the ameloblastoma biology.

A new anti-angiogenic small molecule, G0811, inhibits angiogenesis via targeting hypoxia inducible factor (HIF)-1α signal transduction

Regulation of hypoxia inducible factor (HIF)-1α stabilization, which in turn contributes to adaptation of tumor cells to hypoxia has been highlighted as a promising therapeutic target in angiogenesis-related diseases. We have identified a new small molecule, G0811, as a potent angiogenesis inhibitor that targets HIF-1α signal transduction. G0811 suppressed HIF-1α stability in cancer cells and inhibited in vitro and in vivo angiogenesis, as validated by tube formation, chemoinvasion, and chorioallantoic membrane (CAM) assays. In addition, G0811 effectively decreased the expression of vascular endothelial growth factor (VEGF), which is one of target genes of HIF-1α. However, G0811 did not exhibit anti-proliferative activities or toxicity in human umbilical vein endothelial cells (HUVECs) at effective doses. These results demonstrate that G0811 could be a new angiogenesis inhibitor that acts by targeting HIF-1α signal transduction pathway.

Abstract P1-05-08: Targeting integrin signaling suppresses invasive recurrence in a three-dimensional model of radiation treated ductal carcinoma in situ

Abstract Background: DCIS (ductal carcinoma in situ) is comprised of cancerous cells that are contained within the milk duct and separated from the stroma by a basement membrane. Akt activation is implicated in breast cancer progression and also upregulated in ∼30% of DCIS lesions. We have previously shown that β1-integrin inhibition enhanced the efficacy of ionizing radiation (IR) in invasive breast cancer, via an Akt-mediated effect. Radiation therapy (RT) is commonly used to treat DCIS, resulting in a decrease in recurrence risk by ∼50%. However, DCIS still recurs in a substantial number of patients, and as invasive disease half of the time. This study aimed to identify the effects of IR on Akt-driven DCIS, and possible mechanisms underlying invasive progression in surviving cells. Materials and Methods: To model DCIS, we took advantage of the ability of non-invasive mammary epithelial cells, MCF10A that form duct-like structures in 3-dimensional laminin-rich extracellular matrix (3D lrECM), and overexpressed an activated myristoylated form of the serine/threonine kinase, Akt. The 3D lrECM cultured MCF10A-Akt cells show filled lumen and retained basal polarity, characteristic of DCIS lesions in vivo. After DCIS structures formed in 3D lrECM, cultures were exposed to 8 Gy IR. Confocal microscopy analysis, western blot analysis, matrigel chemoinvasion assay and matix degradation assay were performed to characterize the IR effect on luminal or basal cells of the DCIS like structures. Results: MCF10A-Akt structures post-8 Gy IR shows a significant increase in apoptosis measured by cleaved caspase-3 (n = 3, P < 0.05). When we propagated cells post-IR in 3D lrECM, an invasive phenotype emerged in a sub-population of survivors (n = 3, P < 0.01). We also confirmed that inhibitory antibodies to β1-integrin suppressed the invasive phenotype that was induced by 8 Gy IR. In addition to this, invasion activity emerged by 8 Gy IR was inhibited by nuclear factor-kappaB (NF-κB) inhibitor JSH-23 (n = 3, P < 0.01). Finally, inhibition of β1-integrins or NF-κB translocation completely suppresses the formation of invasive colonies. Discussion: In present study, we found that inhibition of β1-integrins abrogates the emergence of an invasive phenotype among surviving clones. Our current data suggest that regulation of β1-integrin signaling via NF-κB plays an important role in the emergence of invasive disease after radiation, and may be an important clinical target. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-08.

Combined effect of dehydroxymethylepoxyquinomicin and gemcitabine in a mouse model of liver metastasis of pancreatic cancer

Activation of nuclear factor-κB (NF-κB) has been implicated in metastasis of pancreatic cancer. We investigated the effects of the novel NF-κB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) on the inhibition of liver metastasis of pancreatic cancer in a mouse model of clinical liver metastasis. Nude mice were xenografted by intra-portal-vein injection with the human pancreatic adenocarcinomas cell line AsPC-1 via small laparotomy. Mice were treated with DHMEQ and gemcitabine (GEM), alone or in combination. The combination of GEM+DHMEQ showed a stronger antitumor effect than either monotherapy. Apoptosis induction in the metastatic foci was greatest in the DHMEQ+GEM group. Significant reductions in the numbers of neovessels were also seen in the DHMEQ and/or GEM groups. Cell growth inhibition assays revealed no synergistic effect of combination therapy, although each monotherapy had an individual cytotoxic effect. Combination therapy produced the greatest inhibition of tumor cell invasiveness in chemoinvasion assay. In addition, combination therapy significantly down-regulated the expression level of matrix metalloproteinase (MMP)-9 mRNA in AsPC-1 cells. DHMEQ also markedly down-regulated interleukin-8 and MMP-9, while GEM caused moderate down-regulation of vascular endothelial growth factor in metastatic foci, demonstrated by quantitative reverse transcription-polymerase chain reaction. These results demonstrate that DHMEQ can exert anti-tumor effects by inhibiting angiogenesis and tumor cell invasion, and by inducing apoptosis. Combination therapy with DHMEQ and GEM also showed potential efficacy. DHMEQ is a promising drug for the treatment of advanced pancreatic cancer.

Abstract 3840: Evaluation of the synergetic effect of a combination of dehydroxymethylepoxyquinomicin and gemcitabine on liver metastasis of pancreatic cancer

Abstract Activation of the nuclear transcription factor-kappaB (NF-κB) has been implicated in the progression and metastasis of pancreatic cancer. This study was designed to evaluate the effects of a novel NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ) in inhibition of liver metastasis of pancreatic cancer in a clinical liver metastasis-mimicking mouse model in which cancer cells were intra-portal injected under small laparotomy. Moreover, the synergistic effect of combination therapy with DHMEQ and gemcitabine (GEM) was investigated. Cytotoxicity of DHMEQ and GEM was evaluated by cell growth inhibition assay. Both DHMEQ and GEM monotherapies inhibited cell growth of the human pancreas cell line AsPC-1. However, there was no synergistic effect in the combination therapy. For the in vivo study, male BALB/c nude mice were xenografted with intra-portal vein injection of AsPC-1. Mice were divided into four groups: 1) control (without any treatment), 2) GEM-treated group, 3) DHMEQ-treated group, and 4) combined treatment group (GEM+DHMEQ). Mice were fed for four weeks, and the number of liver metastases, induction of apoptosis in metastasis focuses and angiogenesis were investigated after sacrifice. The number of liver metastasis was decreased significantly in GEM group, DHMEQ group and GEM+DHMEQ group compared with control group. Significant inductions of apoptosis were revealed in GEM group and DHMEQ group compared with control group, in which apoptosis bodies in metastasis sections were counted by using the TUNEL method. Moreover, combination therapy of DHMEQ+GEM resulted in the strongest inductions of apoptosis. Tumor vessels were identified by immunohistochemical staining of tumor metastasis sections with an antibody against CD31. There was a large reduction in the number of CD31-positive vessels in the tumor of DHMEQ or/and GEM-treated mice as compared with control group. However, there was an inconsistency in the results of cell growth inhibition assay (in vitro) and mice metastasis model (in vivo). Thus we evaluated effect of combination therapy in the invasive potential of tumor cells by using chemoinvasion assay. The invasion ability was significantly inhibited in the GEM group, DHMEQ group and GEM+DHMEQ group. Furthermore, real-time PCR was performed to evaluate expression of mRNA of MMP-9. The expression levels of MMP-9 were inhibited in respective groups in the same order. In conclusion, the anti-tumor effects of combination therapy were significant, as evidenced by angiogenesis inhibition, apoptosis induction and invasiveness inhibition rather than cell growth inhibition. 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 3840. doi:1538-7445.AM2012-3840

Effect of Cordyceps sinensis on TIMP-1 secretion from mouse melanoma cell

Abstract Cordyceps sinensis is a Chinese medicinal fungus traditionally used in cancer treatments. In a previous study, we investigated the antimetastatic activity of Cordyceps sinensis (WECS) extract using liver metastatic model mice injected with B16-F0 mouse melanoma cells into the spleen. WECS reduced the number of metastatic nodules of B16-F0 cells in the liver of C57BL/6 mice, and significantly prolonged survival of the mice. Furthermore, we examined the effects of WECS on hepatocyte growth factor (HGF)-accelerated invasion of B16-F0 cells using a chemo-invasion assay in vitro. WECS was shown to significantly reduce HGF-accelerated B16-F0 cell invasion. In the present study, we investigated the effect of WECS on Tissue Inhibitor of Metalloproteinase (TIMP)-1 secretion from B16-F0 cells in order to identify clues to the mechanism underlying the anti-invasive action of WECS. As a result, WECS significantly increased the secretion of TIMP-1 from B16-F0 cells. Moreover, we investigated the effect of cordycepin (3′-deoxyadenosine), a component of WECS, on TIMP-1 secretion from B16-F0 cells to potentially identify the pharmacologically active ingredient in WECS extract. Cordycepin was shown to significantly accelerate the release of TIMP-1 from cells. These findings suggest that WECS exerts anti-invasive activity, in part by increasing TIMP-1 secretion from melanoma cells, and that cordycepin potentially functions as the effective component.