Chick Chorioallantoic Membrane Assay: A 3D Animal Model for Study of Human Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma (NPC) is a highly invasive and metastatic head and neck cancer. However, mechanistic study of the invasion and metastasis of NPC has been hampered by the lack of proper in vivo models. We established an in vivo chick embryo chorioallantoic membrane (CAM) model to study NPC tumor biology. We found 100% micro-tumor formation 3 days after inoculation with NPC cell lines (4/4) or primary tumor biopsy tissue (35/35). The transplanted NPC micro-tumors grew on CAMs with extracellular matrix interaction and induced angiogenesis. In addition, the CAM model could be used to study the growth of transplanted NPC tumors and also several important steps of metastasis, including tumor invasion by detecting the extent of basement membrane penetration, tumor angiogenesis by analyzing the area of neo-vessels, and tumor metastasis by quantifying tumor cells in distant organs. We established and described a feasible, easy-to-manipulate and reliable CAM model for in vivo study of NPC tumor biology. This model closely simulates the clinical features of NPC growth, progression and metastasis and could help elucidate the biological mechanisms of the growth pattern and invasion of NPC cells and in quantitative assessment of angiogenesis and cell intravasation.     

Upregulation of SQLE Contributes to Poor Survival in Head and Neck Squamous Cell Carcinoma

Recently, increasing attention has been paid to the role of Squalene epoxidase (SQLE) in several types of cancers. However, its functional role in tumor progression of head and neck squamous cell carcinoma (HNSCC) is still unclear. We performed bioinformatic analyses and relative experiments to assess the potential mechanism of SQLE-mediated HNSCC malignancy. And the results showed that SQLE was significantly upregulated in tumor samples compared with peritumor samples. Mechanistically, miR-584-5p downregulation may lead to the upregulation of SQLE in HNSCC. Moreover, high SQLE expression in HNSCC was associated with TNM stage, distant metastasis, and poor survival, indicating that SQLE be involved in the progression of HNSCC. Furtherly, SQLE boosted proliferation, migration, invasion of HNSCC cells in vitro and in vivo. Bioinformatic studies showed that PI3K/Akt signaling participated in HNSCC progression mediated by SQLE overexpression, which is confirmed by in vitro and in vivo analysis. Particularly, treatment with terbinafine, an inhibitor of SQLE widely used in the treatment of fungal infections, showed a therapeutic influence on HNSCC. Our findings demonstrate that SQLE plays a vital role in HNSCC progression, providing research evidence for SQLE as a prospective HNSCC therapeutic target and for terbinafine as a candidate drug of HNSCC treatment in the future     

Epidermal Growth Factor Receptor Inhibition Reduces Angiogenesis via Hypoxia-Inducible Factor-1α and Notch1 in Head Neck Squamous Cell Carcinoma

Angiogenesis, a marker of cancer development, affects response to radiotherapy sensibility. This preclinical study aims to understand the receptor tyrosine kinase-mediated angiogenesis in head neck squamous cell carcinoma (HNSCC). The receptor tyrosine kinase activity in a transgenic mouse model of HNSCC was assessed. The anti-tumorigenetic and anti-angiogenetic effects of cetuximab-induced epidermal growth factor receptor (EGFR) inhibition were investigated in xenograft and transgenic mouse models of HNSCC. The signaling transduction of Notch1 and hypoxia-inducible factor-1α (HIF-1α) was also analyzed. EGFR was overexpressed and activated in the Tgfbr1/Pten deletion (2cKO) mouse model of HNSCC. Cetuximab significantly delayed tumor onset by reducing tumor angiogenesis. This drug exerted similar effects on heterotopic xenograft tumors. In the human HNSCC tissue array, increased EGFR expression correlated with increased HIF-1α and micro vessel density. Cetuximab inhibited tumor-induced angiogenesis in vitro and in vivo by significantly downregulating HIF-1α and Notch1. EGFR is involved in the tumor angiogenesis of HNSCC via the HIF-1α and Notch1 pathways. Therefore, targeting EGFR by suppressing hypoxia- and Notch-induced angiogenesis may benefit HNSCC therapy.     

CHL1 is involved in human breast tumorigenesis and progression

Neural cell adhesion molecules (CAM) play important roles in the development and regeneration of the nervous system. The L1 family of CAMs is comprised of L1, Close Homolog of L1 (CHL1, L1CAM2), NrCAM, and Neurofascin, which are structurally related trans-membrane proteins in vertebrates. Although the L1CAM has been demonstrated play important role in carcinogenesis and progression, the function of CHL1 in human breast cancer is limited. Here, we found that CHL1 is down-regulated in human breast cancer and related to lower grade. Furthermore, overexpression of CHL1 suppresses proliferation and invasion in MDA-MB-231 cells and knockdown of CHL1 expression results in increased proliferation and invasion in MCF7 cells in vitro. Finally, CHL1 deficiency promotes tumor formation in vivo. Our results may provide a strategy for blocking breast carcinogenesis and progression.     

Growth suppression of MCF-7 cancer cell-derived xenografts in nude mice by caveolin-1

Caveolin-1 is an essential structural constituent of caveolae membrane domains that has been implicated in mitogenic signaling and oncogenesis. However, the exact functional role of caveolin-1 still remains controversial. In this report, utilizing MCF-7 human breast adenocarcinoma cells stably transfected with caveolin-1 (MCF-7/cav-1 cells), we demonstrate that caveolin-1 expression dramatically inhibits invasion and migration of these cells. Importantly, in vivo experiments employing xenograft tumor models demonstrated that expression of caveolin-1 results in significant growth inhibition of breast tumors. Moreover, a dramatic delay in tumor progression was observed in MCF-7/cav-1 cells as compared with MCF-7 cells. Histological analysis of tumor sections demonstrated a marked decrease in the percentage of proliferating tumor cells (Ki-67 assay) along with an increase in apoptotic tumor cells (TUNEL assay) in MCF-7/cav-1-treated animals. Our current findings provide for the first time in vivo evidence that caveolin-1 can indeed function as a tumor suppressor in human breast adenocarcinoma derived from MCF-7 cells rather than as a tumor promoter.     

Role of EZH2 in oral squamous cell carcinoma carcinogenesis

Oral squamous cell carcinoma (OSCC) is a common human malignancy with high incidence rate and poor prognosis. Although the polycomb group protein enhancer of zeste homolog 2 (EZH2) plays a crucial role in cell proliferation and differentiation during the occurrence and development progress of several kinds of malignant tumors, the impact of EZH2 on the development and progression of OSCC is unclear. In this study, we demonstrate that EZH2 is overexpressed in OSCC cells and clinical tissue. With in vitro RNAi analysis, we generated stable EZH2 knocking down cell lines from two OSCC cell lines, with two sh-RNAs targeting to EZH2, respectively. We found that knocking down of EZH2 could decrease the proliferation ability and induce apoptosis of OSCC cells. Moreover, we demonstrated that of EZH2 inhibition decreased the migration and metastasis of OSCC cells. In conclusion, the results of the current study demonstrated an association between EZH2 expression and OSCC cell development. We recommend that EZH2 acts as an oncogene and plays an important role in OSCC carcinogenesis.     

Lysyl oxidase-like 2 represses Notch1 expression in the skin to promote squamous cell carcinoma progression

Lysyl oxidase-like 2 (LOXL2) is involved in a wide range of physiological and pathological processes, including fibrosis and tumor progression, implicating intracellular and extracellular functions. To explore the specific in vivo role of LOXL2 in physiological and tumor contexts, we generated conditional gain- and loss-of-function mouse models. Germ-line deletion of Loxl2 promotes lethality in half of newborn mice mainly associated to congenital heart defects, while Loxl2 overexpression triggers male sterility due to epididymal dysfunction caused by epithelial disorganization, fibrosis and acute inflammation. Remarkably, when challenged to chemical skin carcinogenesis, Loxl2-overexpressing mice increased tumor burden and malignant progression, while Loxl2-deficient mice exhibit the opposite phenotypes. Loxl2 levels in premalignant tumors negatively correlate with expression of epidermal differentiation markers and components of the Notch1 pathway. We show that LOXL2 is a direct repressor of NOTCH1. Additionally, we identify an exclusive expression pattern between LOXL2 and members of the canonical NOTCH1 pathway in human HNSCC. Our data identify for the first time novel LOXL2 roles in tissue homeostasis and support it as a target for SCC therapy.     

Luteolin Inhibits Human Prostate Tumor Growth by Suppressing Vascular Endothelial Growth Factor Receptor 2-Mediated Angiogenesis

Angiogenesis, the formation of new blood vessels from pre-existing vascular beds, is essential for tumor growth, invasion, and metastasis. Luteolin is a common dietary flavonoid found in fruits and vegetables. We studied the antiangiogenic activity of luteolin using in vitro, ex vivo, and in vivo models. In vitro studies using rat aortic ring assay showed that luteolin at non-toxic concentrations significantly inhibited microvessel sprouting and proliferation, migration, invasion and tube formation of endothelial cells, which are key events in the process of angiogenesis. Luteolin also inhibited ex vivo angiogenesis as revealed by chicken egg chorioallantoic membrane assay (CAM) and matrigel plug assay. Gelatin zymographic analysis demonstrated the inhibitory effect of luteolin on the activation of matrix metalloproteinases MMP-2 and MMP-9. Western blot analysis showed that luteolin suppressed VEGF induced phosphorylation of VEGF receptor 2 and their downstream protein kinases AKT, ERK, mTOR, P70S6K, MMP-2, and MMP-9 in HUVECs. Proinflammatory cytokines such as IL-1β, IL-6, IL-8, and TNF-α level were significantly reduced by the treatment of luteolin in PC-3 cells. Luteolin (10 mg/kg/d) significantly reduced the volume and the weight of solid tumors in prostate xenograft mouse model, indicating that luteolin inhibited tumorigenesis by targeting angiogenesis. CD31 and CD34 immunohistochemical staining further revealed that the microvessel density could be remarkably suppressed by luteolin. Moreover, luteolin reduced cell viability and induced apoptosis in prostate cancer cells, which were correlated with the downregulation of AKT, ERK, mTOR, P70S6K, MMP-2, and MMP-9 expressions. Taken together, our findings demonstrate that luteolin inhibits human prostate tumor growth by suppressing vascular endothelial growth factor receptor 2-mediated angiogenesis.     

Suppression of tumorigenicity by MicroRNA-138 through inhibition of EZH2-CDK4/6-pRb-E2F1 signal loop in glioblastoma multiforme

Deregulation of microRNAs (miRNAs) is implicated in tumor progression. We attempt to indentify the tumor suppressive miRNA not only down-regulated in glioblastoma multiforme (GBM) but also potent to inhibit the oncogene EZH2, and then investigate the biological function and pathophysiologic role of the candidate miRNA in GBM. In this study, we show that miRNA-138 is reduced in both GBM clinical specimens and cell lines, and is effective to inhibit EZH2 expression. Moreover, high levels of miR-138 are associated with long overall and progression-free survival of GBM patients from The Cancer Genome Atlas dataset (TCGA) data portal. Ectopic expression of miRNA-138 effectively inhibits GBM cell proliferation in vitro and tumorigenicity in vivo through inducing cell cycles G1/S arrest. Mechanism investigation reveals that miRNA-138 acquires tumor inhibition through directly targeting EZH2, CDK6, E2F2 and E2F3. Moreover, an EZH2-mediated signal loop, EZH2-CDK4/6-pRb-E2F1, is probably involved in GBM tumorigenicity, and this loop can be blocked by miRNA-138. Additionally, miRNA-138 negatively correlates to mRNA levels of EZH2 and CDK6 among GBM clinical samples from both TCGA and our small amount datasets. In conclusion, our data demonstrate a tumor suppressive role of miRNA-138 in GBM tumorigenicity, suggesting a potential application in GBM therapy.     

Hepatocyte growth factor inhibits anoikis by induction of activator protein 1-dependent cyclooxygenase-2. Implication in head and neck squamous cell carcinoma progression

Anoikis, also called suspension-induced apoptosis, plays an important role in tumor development, progression, and metastasis. Recently we found that hepatocyte growth factor (HGF) inhibited anoikis of human head and neck squamous cell carcinoma (HNSCC) cells by activating the extracellular signal-regulated kinase (ERK)-signaling pathway. However, the anti-apoptotic effectors that were regulated by the ERK-signaling pathway were unknown. Here we report that HGF-mediated inhibition of anoikis was dependent on activator protein-1 activity through the activation of the ERK-signaling pathway. Using a combination of microarray analysis and Northern blot analysis, we found that an anti-apoptotic gene cyclooxygenase-2 (cox-2) was induced by HGF in an activator protein-1-dependent fashion. Inhibition of Cox-2 activity partially abolished HGF-mediated cell survival, and overexpression of Cox-2 in HNSCC cells provided resistance against anoikis. Moreover, HNSCC cells stably expressing Cox-2 had aggressive tumor growth in a nude mouse model compared with control cells. Taken together, our results demonstrate that Cox-2 plays an important role in HGF-mediated anoikis resistance. HGF may stimulate the progression and growth of HNSCC in vivo by induction of Cox-2.     

Egr-1 is required for neu/HER2-induced mammary tumors

Egr-1 is known to function mainly as a tumor suppressor through direct regulation of multiple tumor suppressor genes. To determine the role of Egr-1 in breast tumors in vivo, we used mouse models of breast cancer induced by HER2/neu. We compared neu-overexpressing Egr-1 knockout mice (neu/Egr-1 KO) to neu-overexpressing Egr-1 wild type or heterozygote mice (neu/Egr-1 WT or neu/Egr-1 het) with regard to onset of tumor appearance and number of tumors per mouse. In addition, to examine the role of Egr-1 in vitro, we established neu/Egr-1 WT and KO tumor cell lines derived from breast tumors developed in each mouse. Egr-1 deletion delayed tumor development in vivo and decreased the rate of cell growth in vitro. These results suggest that Egr-1 plays an oncogenic role in HER2/neu-driven mammary tumorigenesis.     

Epithelial Membrane Protein-2 (EMP2) Activates Src Protein and Is a Novel Therapeutic Target for Glioblastoma

Despite recent advances in molecular classification, surgery, radiotherapy, and targeted therapies, the clinical outcome of patients with malignant brain tumors remains extremely poor. In this study, we have identified the tetraspan protein epithelial membrane protein-2 (EMP2) as a potential target for glioblastoma (GBM) killing. EMP2 had low or undetectable expression in normal brain but was highly expressed in GBM as 95% of patients showed some expression of the protein. In GBM cells, EMP2 enhanced tumor growth in vivo in part by up-regulating αvβ3 integrin surface expression, activating focal adhesion kinase and Src kinases, and promoting cell migration and invasion. Consistent with these findings, EMP2 expression significantly correlated with activated Src kinase in patient samples and promoted tumor cell invasion using intracranial mouse models. As a proof of principle to determine whether EMP2 could serve as a target for therapy, cells were treated using specific anti-EMP2 antibody reagents. These reagents were effective in killing GBM cells in vitro and in reducing tumor load in subcutaneous mouse models. These results support the role of EMP2 in the pathogenesis of GBM and suggest that anti-EMP2 treatment may be a novel therapeutic treatment.     

IGF-1 receptor inhibition by picropodophyllin in medulloblastoma

The insulin-like growth factor-1 receptor (Igf1r) is a multifunctional membrane-associated tyrosine kinase associated with regulation of transformation, proliferation, differentiation and apoptosis. Increased IGF pathway activity has been reported in human and murine medulloblastoma. Tumors from our genetically-engineered medulloblastoma mouse model over-express Igf1r, and thus this mouse model is a good platform with which to study the role of Igf1r in tumor progression. We hypothesize that inhibition of IGF pathway in medulloblastoma can slow or inhibit tumor growth and metastasis. To test our hypothesis, we tested the role of IGF in tumor growth in vitro by treatment with the tyrosine kinase small molecule inhibitor, picropodophyllin (PPP), which strongly inhibits the IGF pathway. Our results demonstrate that PPP-mediated downregulation of the IGF pathway inhibits mouse tumor cell growth and induces apoptotic cell death in vitro in primary medulloblastoma cultures that are most reflective of tumor cell behavior in vivo.     

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth,Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.     

Quercetin Inhibits Angiogenesis Mediated Human Prostate Tumor Growth by Targeting VEGFR- 2 Regulated AKT/mTOR/P70S6K Signaling Pathways

Angiogenesis is a crucial step in the growth and metastasis of cancers, since it enables the growing tumor to receive oxygen and nutrients. Cancer prevention using natural products has become an integral part of cancer control. We studied the antiangiogenic activity of quercetin using ex vivo, in vivo and in vitro models. Rat aortic ring assay showed that quercetin at non-toxic concentrations significantly inhibited microvessel sprouting and exhibited a significant inhibition in the proliferation, migration, invasion and tube formation of endothelial cells, which are key events in the process of angiogenesis. Most importantly, quercetin treatment inhibited ex vivo angiogenesis as revealed by chicken egg chorioallantoic membrane assay (CAM) and matrigel plug assay. Western blot analysis showed that quercetin suppressed VEGF induced phosphorylation of VEGF receptor 2 and their downstream protein kinases AKT, mTOR, and ribosomal protein S6 kinase in HUVECs. Quercetin (20 mg/kg/d) significantly reduced the volume and the weight of solid tumors in prostate xenograft mouse model, indicating that quercetin inhibited tumorigenesis by targeting angiogenesis. Furthermore, quercetin reduced the cell viability and induced apoptosis in prostate cancer cells, which were correlated with the downregulation of AKT, mTOR and P70S6K expressions. Collectively the findings in the present study suggest that quercetin inhibits tumor growth and angiogenesis by targeting VEGF-R2 regulated AKT/mTOR/P70S6K signaling pathway, and could be used as a potential drug candidate for cancer therapy.     

METCAM/MUC18 augments migration, invasion, and tumorigenicity of human breast cancer SK-BR-3 cells

Previous research has identified METCAM/MUC18, an integral membrane cell adhesion molecule (CAM) in the Ig-like gene super-family, as a promoter or a suppressor in the development of human breast cancer by MCF7, MDA-MB-231, and MDA-MB-468. To resolve these conflicting results we have investigated the role of this CAM in the progression of the three aforementioned cell lines plus one additional human breast cancer cell line, SK-BR-3. We transfected the SK-BR-3 cells with human METCAM/MUC18 cDNA to obtain G418-resistant clones, which expressed different levels of the protein and which were used to test the effect of human METCAM/MUC18 expression on in vitro motility, invasiveness, anchorage-independent colony formation in soft agar, disorganized growth in a 3D basement membrane culture assay, and in vivo tumorigenesis in athymic nude mice. Enforced METCAM/MUC18 expression increased in vitro motility, invasiveness, and anchorage-independent colony formation of SK-BR-3 cells and favored disorganized growth of the cells in 3D basement membrane culture. Enforced expression also increased tumorigenicity and final tumor weights of SK-BR-3 clones/cells after subcutaneous injection of the cells under the left third nipple of female athymic nude mice. To understand the mechanisms, we also determined the expression of several downstream key effectors in the tumors. Tumor cells from METCAM/MUC18 expressing clones exhibited elevated expression of an anti-apoptotic and survival index (Bcl2), an aerobic glycolysis index (LDH-A), and pro-angiogenesis indexes (VEGF and VAGFR2). We concluded that human METCAM/MUC18 promotes the development of breast cancer cells by increasing an anti-apoptosis and survival pathway and augmenting aerobic glycolysis and angiogenesis.     

A Novel Zebrafish Xenotransplantation Model for Study of Glioma Stem Cell Invasion

Invasion and metastasis of solid tumors are the major causes of death in cancer patients. Cancer stem cells (CSCs) constitute a small fraction of tumor cell population, but play a critical role in tumor invasion and metastasis. The xenograft of tumor cells in immunodeficient mice is one of commonly used in vivo models to study the invasion and metastasis of cancer cells. However, this model is time-consuming and labor intensive. Zebrafish (Danio rerio) and their transparent embryos are emerging as a promising xenograft tumor model system for studies of tumor invasion. In this study, we established a tumor invasion model by using zebrafish embryo xenografted with human glioblastoma cell line U87 and its derived cancer stem cells (CSCs). We found that CSCs-enriched from U87 cells spreaded via the vessels within zebrafish embryos and such cells displayed an extremely high level of invasiveness which was associated with the up-regulated MMP-9 by CSCs. The invasion of glioma CSCs (GSCs) in zebrafish embryos was markedly inhibited by an MMP-9 inhibitor. Thus, our zebrafish embryo model is considered a cost-effective approach tostudies of the mechanisms underlying the invasion of CSCs and suitable for high-throughput screening of novel anti-tumor invasion/metastasis agents.