Linda C. Meade-Tollin

Invasion and metastasis of a mammary tumor involves TGF-β signaling

Several studies have correlated escape from TGF‐β‐mediated cell cycle arrest with the tumorigenic phenotype. Most often, this escape from growth control has been linked to dysfunctional TGF‐β receptors or defects in the TGF‐β‐mediated SMAD signaling pathway. In this report, we found that highly metastatic 4T1 mammary carcinoma cells express functional TGF‐β receptors capable of initiating SMAD‐mediated transcription, yet are not growth inhibited by TGF‐β1. We further observed that TGF‐β directly contributes to the metastatic behavior of this cell line. Exposure to TGF‐β caused 4T1 cells to undergo morphological changes associated with the metastatic phenotype and invade more readily through collagen coated matrices. Furthermore, expression of a dominant negative truncated type II receptor diminished TGF‐β signaling and significantly restricted the ability of 4T1 cells to establish distant metastases. Our results suggest that regardless of 4T1 resistance to TGF‐β‐mediated growth inhibition, TGF‐β signaling is required for tumor invasion and metastases formation. Int. J. Cancer 91:76–82, 2001.

Expression of multiple matrix metalloproteinases and urokinase type plasminogen activator in cultured Kaposi sarcoma cells

Kaposis sarcoma (KS) cells are considered to be of endothelial origin. KS lesions are characterized by hyperproliferation and an invasive phenotype. We have determined that KS cell cultures constitutively secrete multiple forms of several matrix metalloproteinases (MMPs) and an altered form of urokinase plasminogen activator (uPA) by zymogram and Western analysis of the culture media. MMPs are a family of secreted endoproteinases which degrade components of the extracellular matrix. Their enhanced expression and activity are strongly correlated with cellular processes involving tissue remodeling and invasion. The KS cells secrete increased levels of gelatinase A and B and a high molecular weight uPA in vitro when compared with non-KS endothelial or epithelial cells. Multiple forms of gelatinases A and B were observed on gelatin zymograms. Caseinolytic bands observed were confirmed by Western blot analysis to be due to stromelysin activity, whereas matrilysin was not detected by casein zymography. Western blot analysis also detected secretion of interstitial collagenase and high molecular weight uPA. Gelatinolytic activity with the mobility of gelatinase B was detected on gelatin zymograms, but not by Western analysis. This unusual constitutive expression pattern of MMPs and uPA by KS cells in vitro is characterized by elevated levels of gelatinase A, gelatinase B, interstitial collagenase, stromelysin and a high molecular weight form of uPA, and the lack of expression of matrilysin. These secreted MMPs, taken together, are capable of digesting a broad range of components of the extracellular matrix. This unusual pattern is likely to contribute to the characteristic hyperproliferative and invasive phenotype of KS lesions.

Loss of p53 and overexpression of EphA2 predict poor prognosis for ovarian cancer patients.

Analysis of EphA2 Expression and Mutant p53 in Ovarian Carcinoma William M. Merritt, Premal H. Thaker, Charles N. Landen Jr., Michael T. Deavers, Mavis S. Fletcher, Yvonne G. Lin, Liz Y. Han, Aparna A. Kamat, Rosemarie Schmandt, David M. Gershenson, Michael S. Kinch and Anil K. Sood Volume: 5 | Issue: 10 | Pages: 1357 - 1360

In vivo inhibition of cysteine proteinases delays the onset of growth of human pancreatic cancer explants

An animal model was used to study the effects of oral treatment with a small molecular selective inhibitor of cysteine proteinases, Z-Phe-Arg-fluoromethylketone (Z-Phe-Arg-FMK) on primary tumour development. Poorly differentiated rapidly growing and moderately differentiated slowly growing human pancreatic tumours were implanted in the neck of nude mice that were orally treated or not with the inhibitor. Growth rates of the tumours were determined during 38 days after implantation. The poorly differentiated tumours were not affected by treatment with the inhibitor. Development of the moderately differentiated tumours was inhibited significantly by Z-Phe-Arg-FMK treatment. Moreover, the amount of stroma was increased and the volume of cancer cells was reduced in the moderately differentiated tumours that had grown in the treated animals. Reduction in size of the tumours was not achieved by reduction in growth rate but in a delay of the onset of growth. It is concluded that cysteine proteinases play a transient role at the start of tumour development only when cancer cells are surrounded by stroma as was the case in the moderately differentiated but not in the poorly differentiated pancreatic tumours. However, this role of cysteine proteinases can easily be taken over by other proteinases.

Fusion of HepG2 cells with mesenchymal stem cells increases cancer-associated and malignant properties: An in vivo metastasis model

In the present study, we have tested the hypothesis that fusion between an altered cell and a mesenchymal stem cell produces a hybrid cell with enhanced characteristics associated with metastatic cancer cells, and we have developed a flexible model for investigating the mechanisms of metastasis. Human HepG2 cells with low metastatic potential were induced to fuse with rat bone marrow mesenchymal stem cells, and the progeny were compared with the parental cells for possession of enhanced in vitro and in vivo characteristics of malignant cells. Compared to the parental cells, the fused cells exhibited enhanced expression of E-cadherin, vimentin, Twist, Snail, matrix metalloproteinase 2 and 9 activities, aneuploidy and enhanced in vitro invasion and migration. In an in vivo xenograft assay, the fused cells generated increased numbers of metastatic liver and lung lesions. This model system is a flexible tool for investigation of the mechanisms of stem cell fusion in carcinogenesis and metastasis and for the discovery of new therapeutic targets to inhibit metastasis.

Time lapse phase contrast video microscopy of directed migration of human microvascular endothelial cells on matrigel

Migration of microvascular endothelial cells is an early and critical step in angiogenesis. Formation of branching and polygonal cellular aggregates by endothelial cells on matrigel has often been considered to be an in vitro model for angiogenesis, although formation of lumens has not always been confirmed. The dynamics of migration of living cells of a human dermal microvascular endothelial cell line (HMEC-1) on a reconstituted basement membrane matrix have been captured in real time using time lapse video microscopy. The cells exhibit periods of quiescence and directed rapid migration by formation of extensions towards a specific target cell. Cells repeatedly extend flexible protrusions from the cell body both within the plane of the matrix and out of the plane of the matrix into the incubation medium. Connections between protrusions and target cells are made frequently, but not all cells which start to form protrusions achieve connections with other cells. Some of these migrating cells which do not connect arrest before reaching the target, or arrest and retract to their origin. After formation of multicellular polygonal structures, the structures contract to form amorphous clusters of fused cells without visible effects on the underlying matrix. The study demonstrates that time lapse video microscopy is a simple but very useful approach to monitor the dynamics of movements which vary in speed and frequency during migration of living cells.

A comparison of levels of intrinsic single strand breaks/alkali labile sites associated with human melanoma cell invasion

Intrinsic levels of protein-free single strand breaks/alkali-labile sites in human melanoma cell populations of varying in vitro invasive capacity have been assayed with DNA filter elution methodology. DNA from two human melanoma cell lines, A375P and C8161, and from a subpopulation selected from A375P, A375P-5, were assayed to test the hypothesis that increased levels of DNA damage may be associated with the phenotype of increased invasive and metastatic capacities. The elution profiles obtained reveal statistically significant increases in the level of single strand breaks and/or alkali-labile sites (SSB/ALS) which correlate with increasing invasive and metastatic capacities. The increased levels of SSB/ALS in A375P-5 observed in freshly selected cells decline as these cells are maintained in culture. The stability of this A375P-5 phenotype correlates with previously reported levels of double minute chromosomes, an indicator of genomic instability. Alterations in average intrinsic levels of cellular lesions are therefore an additional factor to be considered in the phenotypic characterization of invasive and metastatic tumor cells and may reflect or contribute to the genomic instability characteristic of tumor cell populations.