Theresa D'Souza

Claudin-3 and Claudin-4 Expression in Ovarian Epithelial Cells Enhances Invasion and Is Associated with Increased Matrix Metalloproteinase-2 Activity

Claudin proteins form a large family of integral membrane proteins crucial for tight junction formation and function. Our previous studies have revealed that claudin-3 and claudin-4 proteins are highly overexpressed in ovarian cancer. To clarify the roles of claudins in ovarian tumorigenesis, we have generated human ovarian surface epithelial (HOSE) cells constitutively expressing wild-type claudin-3 and claudin-4. Expression of these claudins in HOSE cells increased cell invasion and motility as measured by Boyden chamber assays and wound-healing experiments. Conversely, small interfering RNA (siRNA)-mediated knockdown of claudin-3 and claudin-4 expression in ovarian cancer cell lines reduced invasion. Claudin expression also increased cell survival in HOSE cells but did not significantly affect cell proliferation. Moreover, the claudin-expressing ovarian epithelial cells were found to have increased matrix metalloproteinase-2 (MMP-2) activity indicating that claudin-mediated increased invasion might be mediated through the activation of MMP proteins. However, siRNA inactivation of claudins in ovarian cancer cell lines did not have a significant effect on the high endogenous MMP-2 activity present in these cells, showing that malignant cells have alternative or additional pathways to fully activate MMP-2. Taken together, our results suggest that claudin overexpression may promote ovarian tumorigenesis and metastasis through increased invasion and survival of tumor cells.

Filamin A-mediated Down-regulation of the Exchange Factor Ras-GRF1 Correlates with Decreased Matrix Metalloproteinase-9 Expression in Human Melanoma Cells

The actin-binding protein filamin A (FLNa) is associated with diverse cellular processes such as cell motility and signaling through its scaffolding properties. Here we examine the effect of FLNa on the regulation of signaling pathways that control the expression of matrix metalloproteinases (MMPs). The lack of FLNa in human M2 melanoma cells was associated with constitutive and phorbol ester-induced expression and secretion of active MMP-9 in the absence of MMP-2 up-regulation. M2 cells displayed stronger MMP-9 production and activity than their M2A7 counterparts where FLNa had been stably reintroduced. Using an MMP-9 promoter construct (pMMP-9-Luc), in vitro kinase assays, and genetic and pharmacological approaches, we demonstrate that FLNa mediated transcriptional down-regulation of pMMP-9-Luc by suppressing the constitutive hyperactivity of the Ras/MAPK extracellular signal-regulated kinase (ERK) cascade. Experimental evidence indicated that this phenomenon was associated with destabilization and ubiquitylation of Ras-GRF1, a guanine nucleotide exchange factor that activates H-Ras by facilitating the release of GDP. Ectopic expression of Ras-GRF1 was accompanied by ERK activation and elevated levels of MMP-9 in M2A7 cells, whereas a catalytically inactive dominant negative Ras-GRF1, which prevented ERK activation, reduced MMP-9 expression in M2 cells. Our results indicate that expression of FLNa regulates constitutive activation of the Ras/ERK pathway partly through a Ras-GRF1 mechanism to modulate the production of MMP-9.

Regulation of the CLDN3 gene in ovarian cancer cells.

The claudin (CLDN) genes encode a family of proteins involved in the formation and function of tight junctions. CLDN gene expression is frequently altered in several human cancers, and in particular, CLDN3 and CLDN4 are commonly overexpressed in ovarian cancer. However, the mechanisms leading to the deregulation of these genes in cancer remain unclear. In the present study, we have examined the CLDN3 promoter and have identified a minimal region containing an Sp1 site crucial for its activity. In addition, we find that the CLDN3 promoter is regulated through epigenetic processes. Cells that express high levels of CLDN3 exhibit low DNA methylation and high histone H3 acetylation of the critical CLDN3 promoter region, and the reverse is observed in cells that do not express this gene. CLDN3-negative cells can be induced to express CLDN3 through treatment with DNA methyltransferase or histone deacetylase inhibitors. Interestingly, in vitro binding experiments, as well as chip assays show that Sp1 binds the unmethylated promoter much more efficiently, providing a mechanism for CLDN3 silencing in non-expressing cells. Finally, siRNA-mediated knockdown of Sp1 led to a significant decrease of CLDN3 expression at both the mRNA and protein levels, demonstrating a crucial role for this transcription factor in the regulation of CLDN3. Our data provide a basis for CLDN3 expression in ovarian cancer cells, as well as a mechanism for the silencing of this promoter in tumors lacking expression of claudin-3.

Age-Related Changes of Claudin Expression in Mouse Liver, Kidney, and Pancreas

Tight junctions (TJs) play crucial roles in tissue homeostasis and inflammation through their roles in the control of paracellular transport and barrier function. There is evidence that these functions are compromised in older organisms, but the exact mechanisms leading to TJ deterioration are not well understood. Claudin proteins are a family of membrane proteins that constitute the structural barrier elements of TJs and therefore play a major role in their formation and function. Using immunohistochemistry and immunoblotting, we have studied the expression of six different claudin proteins (claudin-1, -2, -3, -4, -5, and -7) in three tissues (liver, kidney, and pancreas) of aging male and female mice. In general, we find an age-dependent decrease in the expression of several claudin proteins in all three tissues observed, although the exact changes are tissue specific. Our findings provide a possible basis for the decrease in tissue barrier function in older organisms.

Abstract 822: Simple and easy monitoring of tube formation and migration assays with the CytoSMARTTMLive Cell Imaging System

Movement of cells plays a critical role in the development of cancer. Analyzing the motility of cells in appropriate cell culture models is therefore an important tool for cancer researchers. Live cell imaging is particularly well suited to capture dynamic processes in cell culture. The CytoSMART™ Device is an easy-to-use, small and affordable live cell monitoring system suitable for the label-free analysis of cell motility within a standard cell culture incubator. Label-free approaches offer the benefit of cell analysis without potential cytotoxic or other side-effects of the used markers or dyes on the cells. This presentation shows the suitability of the CytoSMART™ Device for the analysis of different cancer-relevant assays. The formation of new blood vessels is required to ensure sufficient nutrient and oxygen supply and to allow solid tumors to grow beyond a certain size. This process can be mimicked in cell culture models in so-called tube formation assays. In this study, Human Umbilical Vein Endothelial Cells (HUVEC) were seeded on Engelbreth-Holm Swarm Sarcoma derived Basement Membrane Extract (BME). The resulting formation of endothelial tubes was monitored with the CytoSMART™ Device. Subsequently, the average length of the formed tubes as well as the number of closed tube circles was quantitatively determined. In addition, the impact of Suramin on tube formation was evaluated as an example for a tube formation inhibiting compound. The migration of cancer cells is also required for the growth and in particular the metastasis of tumors. In a first example, the closure of a so-called wound or scratch in a confluent monolayer of cancer cells was monitored with the CytoSMART™ Device. Determining the migration speed of the cells by measuring the speed of wound closure is a simple assay to determine the migration potential of cancer cells. Modifying the cancer cells, e.g. by knocking down specific genes with siRNA, can help to identify genes that play a role in cell migration. Compounds that are expected to reduce cell motility and therefore reduce the metastasis potential of cancer cells can be easily tested. In a second example, the invasion of cancer cells into a three-dimensional (3D) matrix was analyzed. Tumor cells were embedded in a cancer-relevant matrix and their invasion into the 3D matrix was documented with the CytoSMART™ Device. While this type of model is slightly more difficult in set-up and analysis compared to simple scratch assays, it may reflect better the in vivo situation where solid tumors develop within three-dimensional tissues. The images captured with the CytoSMART™ System were quantitatively analyzed using appropriate software. Overall, the CytoSMART™ System is an easy-to-use, small and affordable live cell imaging system suitable for the label-free analysis of different cancer-relevant assays. Citation Format: Stefanie Buesch, Sabine Schaepermeier, Theresa D9Souza, Bodo Ortmann, Claudia Schwartz, Jenny Schroeder. Simple and easy monitoring of tube formation and migration assays with the CytoSMART TM Live Cell Imaging System [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 822. doi:10.1158/1538-7445.AM2017-822

Abstract 5778: A three-dimensional RAFT™ co-culture as advanced model for breast cancer drug discovery

High-throughput screening (HTS) using two-dimensional (2D) cell culture models (2D HTS) is essential for rapid identification of drug candidates from chemical libraries. However, it often results in a large number of poorly qualified leads that exert extra burden on the downstream, preclinical animal studies and that cannot be translated into clinical success, because 2D culture cannot represent the complexity of the tumor microenvironment in vivo. The progression of tumors and their response to drugs in vivo are regulated by their interactions with neighboring cells, and the natural gradients of nutrients, cytokines, wastes, oxygen, which can be better mimicked with three-dimensional (3D) cell-culture models. We constructed a breast cancer model using the RAFTTM 3D Cell Culture System. The essential component of this system is a collagen matrix condensed to the physiologically-relevant collagen density by removing the majority of the liquid from the collagen hydrogel with specialized absorbers. Our 3D model is a co-culture of human mammary fibroblasts (HMFs), embedded in the RAFT™ Collagen matrix, and the MCF7 human breast cancer epithelial cells, overlaying on top of the matrix, to model the interaction of breast cancer cells and stromal cells in vivo. As non-tumorous control, human mammary epithelial cells (nHMECs), isolated from normal breast tissue, were used to replace MCF7 cells in such a co-culture. A medium formulation combining fibroblast medium FGM2™ and mammary epithelial cell medium MEGM™ was optimized to grow both cell types in the co-culture. The RAFT™ co-cultures were stained with standard immunocytochemistry protocol to reveal the morphology of the cells. After three days in culture, the HMFs, evenly interspersed in the matrix, fully stretched out in the collagen matrix, and the nHMECs grew to reach above 90% confluence on the collagen matrix. The cell proliferation in the co-culture was quantified with the ViaLight™ Plus Cell Proliferation and Cytotoxicity BioAssay, which measures cell viability with bioluminescent detection of cellular ATP. Based on this assay, the viabilities of the RAFT™ cultures of single cell types, as well as those of the co-cultures, increased from Day 1 to Day 3. Normal HMECs proliferated much faster than MCF7 cells on the matrix. We demonstrate here the feasibility of co-culturing HMFs and mammary epithelial cells, in the RAFT™ System, as a 3D breast cancer model. The efficacy of anti-cancer drugs can be assessed by measuring the cell viability with the ViaLight™ Assay. This 3D model can be used to bridge the gap between 2D HTS and preclinical animal studies. It may provide physiologically-relevant data and better prediction of the in vivo efficacy and dosage of the drug candidates identified in HTS, and reduce the burden of animal studies in breast cancer drug discovery. Citation Format: Ying Nie, Krista L. Garner, Theresa D9Souza. A three-dimensional RAFT™ co-culture as advanced model for breast cancer drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5778. doi:10.1158/1538-7445.AM2017-5778

Abstract 4673: The anti-proliferative effects of non-steroidal anti-inflammatory drugs (NSAIDs) diclofenac and indomethacin in ovarian cancer cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Epidemiological studies have shown that the regular use of non-steroidal anti-inflammatory (NSAIDs) drugs is associated with a reduced risk of various cancers. In addition, in vitro and preclinical mouse model experiments have demonstrated that NSAIDs decrease tumor initiation and/or progression of several cancers. In an attempt to clarify the mechanisms of tumor prevention by NSAIDs, we have studied the effects of two NSAIDs, diclofenac and indomethacin, in several ovarian cancer cell lines. Diclofenac and indomethacin treatment decreased cell growth by inducing cell cycle arrest and/or apoptosis. To identify the possible molecular pathways mediating the effects of NSAID treatment in ovarian cancer, ovarian cancer cell lines were treated with diclofenac or indomethacin and microarray analysis was performed. Among the genes differentially expressed in the different cell lines were genes involved in signaling, metabolism, and cell cycle regulation. Some of the genes found downregulated following diclofenac or indomethacin treatment are transcriptional target genes of E2F1 and E2F4, suggesting involvement of these transcription factors in the response of ovarian cancer cells to NSAID treatment. In addition, E2F1 was found downregulated at the protein level upon treatment with diclofenac and indomethacin. The levels of phosphorylated Rb protein also decreased upon treatment with NSAIDs, suggesting activation of Rb and involvement of the Rb/E2F pathway in the effects of NSAID treatment in ovarian cancer cells. In conclusion, NSAIDs diclofenac and indomethacin exert an anti-proliferative effect in ovarian cancer cells and we have identified a number of target genes and pathways that may be important for the NSAID-induced cell cycle arrest and apoptosis. Current efforts are focused on determining the exact roles of these genes and pathways in NSAID-mediated cancer 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 4673. doi:1538-7445.AM2012-4673

Abstract C96: The effect of non‐steroidal anti‐inflammatory drugs (NSAIDs) in ovarian cancer cells

Epidemiological and laboratory studies have suggested a protective role for non‐steroidal anti‐inflammatory drugs (NSAIDs) against various neoplasms, including ovarian cancer. In an attempt to clarify the mechanisms and molecular targets of tumor prevention by NSAIDs, we have studied the effects of multiple NSAIDs in several ovarian cancer cell lines. NSAID treatment decreased the viability of all the ovarian cancer cell lines examined. To determine the effect of NSAIDs on the cell cycle, flow cytometry analysis was performed. Cells treated with indomethacin, ibuprofen or celecoxib were arrested in G1 phase, while cells treated with diclofenac underwent an S/G2 arrest. In addition, induction of apoptosis was cell line specific. The levels of several cell cycle regulatory and apoptosis proteins were examined. The levels of phosphorylated Rb protein decreased and phospho‐p53 increased upon treatment with NSAIDs, suggesting an involvement of these proteins in the response of ovarian cancer cells to NSAID treatment. In order to further clarify the mechanisms of action of NSAIDs in ovarian cancer cells, gene expression profiling experiments were performed. Ovarian cancer cell lines Hey, OVCAR5, and UCI101 were treated with diclofenac, indomethacin, celecoxib, and ibuprofen, and microarray analysis was performed. A large number of gene expression changes were observed in the different cell lines treated with various NSAIDs. Among the most consistent and reproducible changes observed, S‐phase kinase associated protein 2 (Skp2) was found to be downregulated at the mRNA and protein levels in all cell lines with all NSAIDs tested. In addition, Damage inducible transcript‐4 (DDIT4) was found to be upregulated in all cell lines upon treatment with diclofenac, indomethacin and celecoxib. Skp2 forms part of the SCF ubiquitin ligase and is involved in the targeting for degradation of several proteins, including the cyclin dependent kinase inhibitors p21 and p27, therefore facilitating S‐phase entry. DDIT‐4, is transcriptionally activated upon p53 activation and it is known to inhibit mTOR signaling. In conclusion, we have identified a number of target genes that may be important for the NSAID‐induced cell cycle arrest and apoptosis and their exact roles are currently being investigated. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C96.