Biosafety Oversight and Compliance: What do you Mean, I have to Fill Out Another Form?!

This unit is an overview of biosafety compliance and oversight in the United States. Specific attention is given to the oversight of the Institutional Biosafety Committee (IBC) and how the purview of the IBC may overlap with other local committees, such as the Institutional Animal Care and Use Committee (IACUC) for animal research and the Institutional Review Board (IRB) for research on human subjects. Requirements for the Federal Select Agent Program and Dual Use Research of Concern (DURC) are also briefly reviewed for those working with materials and experiments covered under these regulations. This unit serves as a guide for new and established investigators who are navigating the regulatory world and how regulatory oversight applies to their research.

Interleukin-1 beta and transforming growth factor-beta 3 cooperate to activate matrix metalloproteinase expression and invasiveness in A549 lung adenocarcinoma cells.

Cytokines present in the tumor microenvironment can promote the invasiveness and metastatic potential of cancer cells. We therefore investigated the effects of interleukin-1 beta (IL-1B) and transforming growth factor beta-3 (TGFB3) on the non-small cell lung carcinoma (NSCLC) cell line A549. We found that these cytokines synergistically activated matrix metalloproteinase (MMP)-1, MMP-3, and MMP-10 gene expression in these cells through mitogen-activated protein kinase (MAPK)-dependent pathways. Consistent with this, both cytokines stimulated epithelial to mesenchymal transition and MAPK-dependent invasion through Matrigel™. These studies identify IL-1B and TGFB3 as pro-invasive factors in NSCLC and potential therapeutic targets for tumor progression.

AUF1/hnRNP D represses expression of VEGF in macrophages.

Vascular endothelial growth factor (VEGF) is a regulator of vascularization in development and is a key growth factor in tissue repair. In disease, VEGF contributes to vascularization of solid tumors and arthritic joints. This study examines the role of the mRNA-binding protein AUF1/heterogeneous nuclear ribonucleoprotein D (AUF1) in VEGF gene expression. We show that overexpression of AUF1 in mouse macrophage-like RAW-264.7 cells suppresses endogenous VEGF protein levels. To study 3' untranslated region (UTR)-mediated regulation, we introduced the 3' UTR of VEGF mRNA into a luciferase reporter gene. Coexpression of AUF1 represses VEGF-3' UTR reporter expression in RAW-264.7 cells and in mouse bone marrow-derived macrophages. The C-terminus of AUF1 contains arginine-glycine-glycine (RGG) repeat motifs that are dimethylated. Deletion of the RGG domain of AUF1 eliminated the repressive effects of AUF1. Surprisingly, expression of an AUF1-RGG peptide reduced endogenous VEGF protein levels and repressed VEGF-3' UTR reporter activity in RAW-264.7 cells. These findings demonstrate that AUF1 regulates VEGF expression, and this study identifies an RGG peptide that suppresses VEGF gene expression.

Orphan nuclear receptor NR4A2 induces synoviocyte proliferation, invasion, and matrix metalloproteinase 13 transcription.

OBJECTIVE: To address the role of the nuclear receptor 4A (NR4A) family of orphan nuclear receptors in synoviocyte transformation, hyperplasia, and regulation of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in models of inflammatory arthritis. METHODS: NR4A messenger RNA levels in synovial tissue and primary synoviocytes were measured by quantitative reverse transcription-polymerase chain reaction (RT-PCR). NR4A2 was stably overexpressed in normal synoviocytes, and cell proliferation, survival, anchorage-independent growth, migration, and invasion were monitored in vitro. MMP and TIMP expression levels were analyzed by quantitative RT-PCR, and MMP-13 promoter activity was measured using reporter assays. Stable depletion of endogenous NR4A levels was achieved by lentiviral transduction of NR4A short hairpin RNA (shRNA), and the effects on proliferation, migration, and MMP-13 expression were analyzed. RESULTS: NR4A2 was expressed at elevated levels in normal, OA, and RA synovial tissue and in primary RA synoviocytes. Tumor necrosis factor α (TNFα) rapidly and selectively induced expression of NR4A2 in synoviocytes. Ectopic expression of NR4A2 in normal synoviocytes significantly increased proliferation and survival, promoted anchorage-independent growth, and induced migration and invasion. MMP-13 gene expression was synergistically induced by NR4A2 and TNFα, while expression of TIMP-2 was antagonized. NR4A2 directly transactivated the proximal MMP-13 promoter, and a point mutation in the DNA binding domain of NR4A2 abolished transcriptional activation. Depletion of endogenous NR4A receptors with shRNA reduced synoviocyte proliferation, migration, and MMP-13 expression. CONCLUSION: The orphan nuclear receptor NR4A2 is a downstream mediator of TNFα signaling in synovial tissue. NR4A2 transcriptional activity contributes to the hyperplastic and invasive phenotype of synoviocytes that leads to cartilage destruction, suggesting that this receptor may show promise as a therapeutic target in inflammatory arthritis.

Interleukin-1ß mediates metalloproteinase-dependent renal cell carcinoma tumor cell invasion through the activation of CCAAT enhancer binding protein ß.

Effective treatment of metastatic renal cell carcinoma (RCC) remains a major medical concern, as these tumors are refractory to standard therapies and prognosis is poor. Although molecularly targeted therapies have shown some promise in the treatment of this disease, advanced RCC tumors often develop resistance to these drugs. Dissecting the molecular mechanisms underlying the progression to advanced disease is necessary to design alternative and improved treatment strategies. Tumor-associated macrophages (TAMs) found in aggressive RCC tumors produce a variety of inflammatory cytokines, including interleukin-1ß (IL-1ß). Moreover, the presence of TAMs and high serum levels of IL-1ß in RCC patients correlate with advanced disease. We hypothesized that IL-1ß in the tumor microenvironment promotes the development of aggressive RCC tumors by directing affecting tumor epithelial cells. To address this, we investigated the role of IL-1ß in mediating RCC tumor cell invasion as a measure of tumor progression. We report that IL-1ß induced tumor cell invasion of RCC cells through a process that was dependent on the activity of matrix metalloproteinases (MMPs) and was independent of migration rate. Specifically, IL-1ß induced the expression of MMP-1, MMP-3, MMP-10, and MT1-MMP in a mechanism dependent on IL-1ß activation of the transcription factor CCAAT enhancer binding protein ß (CEBPß). Consistent with its role in MMP gene expression, CEBPß knockdown significantly reduced invasion, but not migration, of RCC tumor cells. These results identify the IL-1ß /CEBPß/MMP pathway as a putative target in the design of anti-metastatic therapies for the treatment of advanced RCC.

CCAAT-enhancer-binding protein beta activation of MMP-1 gene expression in SW1353 cells: independent roles of extracellular signal-regulated and p90/ribosomal S6 kinases.

CCAAT-enhancer-binding protein beta (CEBPB) is a pluripotent transcription factor that controls inflammation, proliferation, and differentiation. We recently reported a role for CEBPB during matrix metalloproteinase (MMP) gene expression, but the mechanisms involved are poorly understood. To address this we interrogated CEBPB-dependent MMP-1 and MMP-13 gene activation in the SW1353 chondrosarcoma cell line, a well-established model of MMP gene regulation in mesenchymal cells. IL-1B treatment increased CEBPB expression in SW1353 cells over a 24-h period and knockdown of CEBPB with shRNA abrogated IL-1B-dependent MMP-1 and MMP-13 gene activation. Exogenous expression of the CEBPB isoforms LAP1 or LAP2 was sufficient to induce MMP-1 mRNA levels comparable to IL-1B-induced expression, while the truncated LIP isoform repressed IL-1B-induced MMP-1. Although exogenous CEBPB expression induced MMP-13 mRNA, the response was less robust than was observed for MMP-1. CEBPB is activated by the extracellular-regulated kinases (ERK) and RSK kinases in response to oncogenes and growth factors. We found that the MEK inhibitor U0126 and the RSK inhibitor BI-D1870 both reduced IL-1B-dependent MMP-1 gene expression in SW1353 cells. Although ERK is known to phosphorylate CEBPB on threonine 235, this residue was not required for CEBPB-dependent activation of MMP-1. In contrast, the RSK target serine 321 was required for LAP1 and LAP2-dependent activation of MMP-1. These findings establish CEBPB as a critical intermediate for IL-1B-dependent MMP gene activation and assign specific roles for the ERK and RSK kinases in this pathway.

PTEN suppression of YY1 induces HIF-2 activity in von-Hippel-Lindau-null renal-cell carcinoma.

Despite recent advances in cancer therapies, metastatic renal cell carcinoma (RCC) remains difficult to treat. Most RCCs result from inactivation of the von Hippel Lindau (VHL) tumor suppressor, leading to stable expression of Hypoxia-Inducible Factor-alpha (HIF-1alpha, -2alpha, -3alpha) and the induction of downstream target genes, including those responsible for angiogenesis and metastasis. While VHL is inactivated in the majority of RCC cases, expression of the PTEN tumor suppressor is reduced in about 30% of cases. PTEN functions to antagonize PI3K/Akt/mTOR signaling, thereby controlling cell growth and survival. Activation of PI3K/Akt/mTOR leads to increased HIF-1alpha expression in certain cancer cells, supporting the rationale of using mTOR inhibitors as anti-cancer agents. Notably, HIF-2alpha, rather than HIF-1alpha, has been shown to play a critical role in renal tumorigenesis. To investigate whether HIF-2alpha is similarly regulated by the PI3K pathway in VHL(-/-)RCC cells, we manipulated PI3K signaling using PTEN overexpression and siRNA knockdown studies and pharmacologic inhibition of PI3K or Akt. Our data support a novel role for wild-type PTEN in promoting HIF-2alpha activity in VHL null RCC cells. This mechanism is unique to the cellular environment in which HIF-2alpha expression is deregulated, resulting from the loss of VHL function. Our data show that PTEN induces HIF-2alpha transcriptional activity by inhibiting expression of Yin Yang 1 (YY1), which acts as a novel corepressor of HIF-2alpha. Further, PTEN suppression of YY1 is mediated through antagonism of PI3K signaling. We conclude that wild-type PTEN relieves the repressive nature of YY1 at certain HIF-2alpha target promoters and that this mechanism may promote early renal tumorigenesis resulting from VHL inactivation by increasing HIF-2alpha activity.

Assessment of local proteolytic milieu as a factor in tumor invasiveness and metastasis formation: in vitro collagen degradation and invasion assays.

Matrix invasion by a tumor cell requires the degradation of components in the extracellular matrix (ECM) as one of the initial steps in the metastatic process. Tumors cells achieve ECM invasion primarily through the overexpression of matrix metalloproteinases (MMPs), a family of enzymes that function to degrade ECM proteins. In this chapter, an in vitro collagen degradation assay and a modified collagen invasion assay system are described. The collagen degradation assay is a simple method to measure the ability of tumor cells to degrade type I collagen, the main constituent of the stromal compartment, in a 3-D matrix environment. The modified collagen invasion assay system enables researchers to study the effects of transient overexpression and/or targeted knockdown (as with siRNAs) of a given gene on collagen invasion of tumor cells in a real-time format.

Tumor cell invasion of von Hippel Lindau renal cell carcinoma cells is mediated by membrane type-1 matrix metalloproteinase.

BACKGROUND: Metastatic renal cell carcinoma (RCC) remains the leading cause of mortality in patients with clear cell RCC arising from mutations in the von Hippel Lindau (VHL) tumor suppressor. Successful RCC tumor suppression by VHL requires the negative regulation of hypoxia inducible factor alpha (HIF alpha) protein and its downstream targets. Thus, identification of HIF target genes responsible for RCC tumor progression will aid in the development of therapies for this disease. We previously identified membrane type-1 matrix metalloproteinase (MT1-MMP) as a transcriptional target of HIF-2alpha in RCC cells null for VHL and showed that MT1-MMP is overexpressed in these cells. MT1-MMP is a key regulator of tumor progression through its functions as a matrix-degrading enzyme, as well as its ability to cleave factors, such as adhesion molecules and other MMPs. The aim of this study was to investigate the contribution of MT1-MMP to the invasive potential of RCC cells using in vitro type I collagen degradation and invasion assays. RESULTS: We evaluated RCC cells wild-type (WT8) and null (pRc-9) for VHL for invasive characteristics and showed that the pRc-9 cells demonstrated a greater propensity for both invasion and degradation of a type I collagen matrix. Furthermore, overexpression of either HIF-2alpha or MT1-MMP in the poorly invasive cell line, WT8, promoted collagen degradation and invasion of these cells. Finally, using RNAi, we show that inhibition of MT1-MMP suppresses tumor cell invasion of RCC cells. CONCLUSION: Our results suggest that MT1-MMP is a major mediator of tumor cell invasiveness and type I collagen degradation by VHL RCC cells that express either MT1-MMP or HIF-2alpha. As such, MT1-MMP may represent a novel target for anti-invasion therapy for this disease.

Identification of membrane type-1 matrix metalloproteinase as a target of hypoxia-inducible factor-2 alpha in von Hippel-Lindau renal cell carcinoma.

Metastatic renal cell carcinoma (RCC) resulting from the hereditary loss of the von Hippel-Lindau (VHL) tumor suppressor gene is the leading cause of death in VHL patients due to the deleterious effects of the metastatic tumor(s). VHL functions in the destruction of the alpha subunits of the heterodimeric transcription factor, hypoxia-inducible factor (HIF-1 alpha and HIF-2 alpha), in normoxic conditions. When VHL function is lost, HIF-alpha protein is stabilized, and target hypoxia-inducible genes are transcribed. The process of tumor invasion and metastasis involves the destruction of the extracellular matrix, which is accomplished primarily by the matrix metalloproteinase (MMP) family of enzymes. Here, we describe a connection between the loss of VHL tumor suppressor function and the upregulation of membrane type-1 MMP (MT1-MMP) gene expression and protein. Specifically, MT1-MMP is upregulated in VHL-/- RCC cells through an increase in gene transcription, which is mediated by the cooperative effects of the transcription factors, HIF-2 and Sp1. Further, we identify a functional HIF-binding site in the proximal promoter of MT1-MMP. To our knowledge, this is the first report to show direct regulation of MT1-MMP by HIF-2 and to provide a direct link between the loss of VHL tumor suppressor function and an increase in MMP gene and protein expression.