Brenda L. Petrella

Identification of Membrane Type-1 Matrix Metalloproteinase as a Target of Hypoxia-Inducible Factor-2α 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-1α and HIF-2α), in normoxic conditions. When VHL function is lost, HIF-α 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.

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

BackgroundMetastatic 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.ResultsWe 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.ConclusionOur 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.

AUF1/hnRNP D represses expression of VEGF in macrophages.

Vascular endothelial growth factor (VEGF) expression is regulated by sequence elements in the 3′ UTR of VEGF mRNA. AUF1/hnRNP D suppresses VEGF 3′ UTR–dependent expression. Peptides with arginine–glycine–glycine motifs derived from AUF1 also suppress VEGF expression.

PTEN suppression of YY1 induces HIF-2 activity in von hippel lindau null renal cell carcinoma

We are grateful to the following for their generous gifts: Dr. William G. Kaelin, Jr. for use of the WT8 and pRc-9 cells and the VHL expression construct; Dr. Richard Bruick for the HRE-luc reporter and the HIF-2

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.

Interleukin‐1β mediates metalloproteinase‐dependent renal cell carcinoma tumor cell invasion through the activation of CCAAT enhancer binding protein β

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‐1β (IL‐1β). Moreover, the presence of TAMs and high serum levels of IL‐1β in RCC patients correlate with advanced disease. We hypothesized that IL‐1β 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‐1β in mediating RCC tumor cell invasion as a measure of tumor progression. We report that IL‐1β 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‐1β induced the expression of MMP‐1, MMP‐3, MMP‐10, and MT1‐MMP in a mechanism dependent on IL‐1β activation of the transcription factor CCAAT enhancer binding protein β (CEBPβ). Consistent with its role in MMP gene expression, CEBPβ knockdown significantly reduced invasion, but not migration, of RCC tumor cells. These results identify the IL‐1β /CEBPβ/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. J. Cell. Physiol. 226: 3349–3354, 2011.

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.

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.

UNIT 1A.5 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.