TGF-beta1-induced PAI-1 gene expression requires MEK activity and cell-to-substrate adhesion.

The type-1 inhibitor of plasminogen activator (PAI-1) is an important physiological regulator of extracellular matrix (ECM) homeostasis and cell motility. Various growth factors mediate temporal changes in the expression and/or focalization of PAI-1 and its protease target PAs, thereby influencing cell migration by barrier proteolysis and/or ECM adhesion modulation. TGF-beta1, in particular, is an effective inducer of matrix deposition/turnover, cell locomotion and PAI-1 expression. Therefore, the relationship between motility and PAI-1 induction was assessed in TGF-beta1-sensitive T2 renal epithelial cells. PAI-1 synthesis and its matrix deposition in response to TGF-beta1 correlated with a significant increase in cell motility. PAI-1 expression was an important aspect in cellular movement as PAI-1-deficient cells had significantly impaired basal locomotion and were unresponsive to TGF-beta1. However, the induced migratory response to this growth factor was complex. TGF-beta1 concentrations of 1-2 ng/ml were significantly promigratory, whereas lower levels (0.2-0.6 ng/ml) were ineffective and final concentrations > or =5 ng/ml inhibited T2 cell motility. This same growth factor range progressively increased PAI-1 transcript levels in T2 cells consistent with a bifunctional role for PAI-1 in cell migration. TGF-beta1 induced PAI-1 mRNA transcripts in quiescent T2 cells via an immediate-early response mechanism. Full TGF-beta1-stimulated expression required tyrosine kinase activity and involved MAPK/ERK kinase (MEK). MEK appeared to be a major mediator of TGF-beta1-dependent PAI-1 expression and T2 cell motility since PD98059 effectively attenuated both TGF-beta1-induced ERK1/2 activation and PAI-1 transcription as well as basal and growth factor-stimulated planar migration. Since MEK activation in response to growth factors is adhesion-dependent, it was important to determine whether cellular adhesive state influenced TGF-beta1-mediated PAI-1 expression in the T2 cell system. Cells maintained in suspension culture (i.e., over agarose underlays) in growth factor-free medium or treated with TGF-beta1 in suspension expressed relatively low levels of PAI-1 transcripts compared with the significant induction of PAI-1 mRNA evident in T2 cells upon stimulation with TGF-beta1 during adhesion to a fibronectin-coated substrate. Attachment to fibronectin alone (i.e., in the absence of added growth factor) was sufficient to initiate PAI-1 transcription, albeit at levels considerably lower than that induced by the combination of cell adhesion in the presence of TGF-beta1. T2 cells allowed to attach to vitronectin-coated surfaces also expressed PAI-1 transcripts but to a significantly reduced extent relative to cells adherent to fibronectin. Moreover, newly vitronectin-attached cells did not exhibit a PAI-1 inductive response to TGF-beta1, at least during the short 2 hour period of combined treatment. PAI-1 mRNA synthesis in response to substrate attachment, like TGF-beta1-mediated induction in adherent cultures, also required MEK activity as fibronectin-stimulated PAI-1 expression was effectively attenuated by the MEK inhibitor PD98059. These data indicate that cellular adhesive state modulates TGF-beta1 signaling to particular target genes (i.e., PAI-1) and that MEK is a critical mediator of the PAI-1(+)/promigratory phenotype switch induced by TGF-beta1 in T2 cells.

AsGM1+ NK cells prevent metastasis of invading LD-MCA-38 tumor cells in the nude mouse.

BACKGROUND: Although the liver is a potent tumor cell killing organ it is frequently the site of lethal metastases often signifying the endstage for patients with colorectal cancers. Enhancing hepatic-associated immunity remains elusive until the interactions among hepatic nonparenchymal cells (NPC) are deciphered. We sought to modulate the cellular components of the hepatic immune system of mice with anti-NK and anti-T-cell-neutralizing antibodies in order to determine the cell type most efficacious in preventing liver metastasis. MATERIALS AND METHODS: Liver-derived murine colon adenocarcinoma (LD-MCA-38) cells were injected into the ileocolic vein (ICV) of immunocompetent and immunodeficient C57BL/6 mice. Mice were pretreated 1 day prior to tumor cell injection with one of three antibodies: anti-AsGM1, Anti-NK1.1, or Anti-Thy1.2. On Day 21 laparotomy was performed to determine the extent of hepatic tumor foci. The number of hepatic tumor foci was recorded and compared by the Wilcoxon rank sum test. RESULTS: Mice pretreated with anti-AsGM1 or Anti-NK1.1 developed a massive increase in the number of hepatic tumor foci and decreased survival compared to the control treated mice. Pretreatment with anti-Thy1.2 antibody resulted in a significant decrease in the number of hepatic tumor foci. LD-MCA-38 tumor cells were unable to colonize the liver of C57BL/6 athymic nude mice; however, anti-AsGM1 antibody abolished this antimetastatic effect. There was no difference in the extent of hepatic metastasis and survival between immunodeficient C57BL/6 bg/bg and their conventional littermates bg/+. CONCLUSION: AsGM1+ NK cells exhibit a significant antitumor response in the absence of T-cells. The concept of stimulating NK cell activity and suppressing T-cell function may enhance liver-associated immunity and serve as a deterrent for blood-borne tumor cells metastasizing to the liver.

Low density lipoproteins and Lovastatin modulate the organ-specific transendothelial migration of primary and metastatic human colon adenocarcinoma cell lines in vitro.

Tumor cell arrest and tumor migration are two of the critical steps in the metastatic cascade. We hypothesized that these steps may be facilitated by the low density lipoprotein (LDL)-induced activation of microvessel endothelial cells (MVEC). The purpose of our study was to investigate the biological effects of an LDL-enriched milieu and the effects of the anticholesterol drug Lovastatin on metastatic behavior. The SW480 and SW620 are primary and metastatic human colonic adenocarcinoma cell lines derived from the same patient. We investigated the effect of LDL on adhesion and migration of the two tumor cell lines across human brain, lung, liver and dermal endothelial monolayers. Adhesion and migration assays were done before and after pretreatment of the MVEC or tumor cells with LDL (100 microg/ml) for 24 h. Although metastatic SW620 cells were more adherent to MVEC compared with primary SW480 cells, LDL pretreatment of SW480 and SW620 cells did not affect tumor cell adhesion to MVEC. In contrast, tumor cell migration was significantly increased across endothelial monolayers when MVEC were pretreated with LDL. Transendothelial cell migration was not significantly affected by pretreatment of the tumor cells with LDL. Lovastatin is an inhibitor of HMG-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis. It has been shown to have anti-tumor activity in vitro. We investigated the effect of Lovastatin on tumor cell kinetics and tumor cell migration across MVEC. Growth curves and migration assays were done before and after pretreatment of the tumor cells with Lovastatin (30 microg/ml). Migration assays were also done after treatment of unstimulated or LDL-stimulated MVEC (100 microg/ml) for 24 h with Lovastatin. Lovastatin inhibited the in vitro growth of the metastatic SW620 cell line to a greater extent than the invasive SW480E cell line. On the other hand, pretreatment of tumor cells with Lovastatin (30 microg/ml) did not suppress transendothelial tumor cell migration of tumor cells. Finally, Lovastatin given to mice effectively suppressed the number of MCA-26 tumor colonies in the liver of Balb/c mice compared with untreated mice.

Cellular effects of hypercholesterolemia in modulation of cancer growth and metastasis: a review of the evidence.

Hypercholesterolemia and increased cancer risk have been associated, particularly with the high fat diets characteristic of Western societies. We were interested in the possible association between preexisting hypercholesterolemia and the rapidity and extent of tumor metastases in these patients. To date there has been only a few studies that have suggested and explored this determinant of cancer metastases although it may play a role in a subset of patients who develop cancers. This article will review the literature on the effects of LDL-cholesterol on cell proliferation and differentiation and speculate on mechanisms of involvement of a hypercholesterolemic milieu on cancer progression and enhancement of metastatic potential.