Epidermal Growth Factor Receptor-dependent and -independent Cell-signaling Pathways Originating from the Urokinase Receptor

124

We have previously demonstrated the effectiveness of adenovirus-mediated expression of antisense urokinase-type plasminogen activator receptor (uPAR) and matrix metalloproteinase-9 (MMP-9) in inhibiting tumor invasion in vitro and ex vivo. However, the therapeutic effect of the adenovirus-mediated antisense approach was shown to be transient and required potentially toxic, high viral doses. In contrast, RNA interference (RNAi)-mediated gene targeting may be superior to the traditional antisense approach, because the target mRNA is completely degraded and the molar ratio of siRNA required to degrade the target mRNA is very low. Here, we have examined the siRNA-mediated target RNA degradation of uPAR and MMP-9 in human glioma cell lines. Using RNAi directed toward uPAR and MMP-9, we achieved specific inhibition of uPAR and MMP-9. This bicistronic construct (pUM) inhibited the formation of capillary-like structures in both in vitro and in vivo models of angiogenesis. We demonstrated that blocking the expression of these genes results in significant inhibition of glioma tumor invasion in Matrigel and spheroid invasion assay models. RNAi for uPAR and MMP-9 inhibited cell proliferation, and significantly reduced the levels of phosphorylated forms of MAPK, ERK, and AKT signaling pathway molecules when compared with parental and empty vector/scrambled vector-transfected SNB19 cells. Furthermore, using RNAi to simultaneously target two proteases resulted in total regression of pre-established intracerebral tumor growth. Our results provide evidence that the use of hairpin siRNA expression vectors for uPAR and MMP-9 may provide an effective tool for cancer therapy. RNA interference (RNAi)1 is a sequence-specific, post-transcriptional gene silencing mechanism, which is triggered by double-stranded RNA and causes the degradation of mRNA homologous in sequence to the double-stranded RNA (1-3). This is an ancient and ubiquitous antiviral system used by organisms to maintain the integrity of the genome, to defend cells against viral infection, and to regulate expression of cellular genes (4). RNAi depends upon the formation of doublestrand RNA (double-stranded RNA) whose antisense strand is complementary to the transcript of a targeted gene. Recently, it has been shown that sequence-specific inhibition RNAi can also be induced in mammalian cells (4, 5). In one implementation of RNAi, selective degradation of target mRNAs in mammalian cells is achieved by transfection with double-stranded, short interfering RNAs (siRNAs), leading to rapid and efficient degradation of the target (4). These siRNAs were shown to avoid the well documented nonspecific effects triggered by longer double-stranded RNAs in mammalian cells.Glioblastoma multiforme is a highly malignant primary central nervous system neoplasm, which is extremely refractory to therapy. One property that makes glioblastoma resistant to treatment is the tendency of the tumor cells to invade normal brain tissue (6). Invasiveness is thus considered to be a major determinant of ...

Section: Figmentioning

confidence: 99%

Specific Interference of Urokinase-type Plasminogen Activator Receptor and Matrix Metalloproteinase-9 Gene Expression Induced by Double-stranded RNA Results in Decreased Invasion, Tumor Growth, and Angiogenesis in Gliomas

Lakka¹,

Gondi²,

Dinh³

et al. 2005

124

“…Binding of ligands to LRP1, in Schwann cells, activates Rac1; however, in fibroblasts, LRP1 inhibits Rac1 activation by decreasing the cell surface abundance of uPAR (28,29,38). The indirect pathway, in which LRP1 regulates Rac1 downstream of uPAR is active only when cells are cultured on VN.…”

Section: Discussionmentioning

confidence: 99%

“…In fibroblasts that are plated on VN, LRP1 inhibits Rac1 activation downstream of the urokinase receptor (uPAR) by facilitating uPAR endocytosis (29,38). Because our studies were performed with Schwann cells cultured on FN and not VN, regulation of uPAR did not contribute to the effects of LRP1 on Rac1 activation.…”

Section: Lrp1 Regulates Rac1 In An Extracellularmentioning

confidence: 98%

Low Density Lipoprotein Receptor-related Protein (LRP1) Regulates Rac1 and RhoA Reciprocally to Control Schwann Cell Adhesion and Migration

Mantuano

Gonias

et al. 2010

Self Cite

LDL receptor-related protein (LRP1) is expressed by Schwann cells in vivo mainly after injury to the peripheral nervous system (PNS). Schwann cells in primary culture, which provide a model of Schwann cells in the injured PNS, also express abundant LRP1. Herein, we show that LRP1 gene-silencing or treatment with receptor-associated protein (RAP) promotes Schwann cell adhesion and inhibits cell migration on fibronectin. LRP1 genesilencing also resulted in the formation of prominent focal adhesions and actin stress fibers. These changes, which were induced by loss of LRP1 expression or activity, were explained mechanistically by an increase in activated RhoA, coupled with a decrease in activated Rac1. Known LRP1 ligands, including matrix metalloprotease-9, tissue-type plasminogen activator, and In the mature, uninjured peripheral nervous system (PNS), 2 Schwann cells are typically immobile, providing trophic support and in some cases, ensheathing and myelinating axons (1). PNS injury triggers Schwann cell dedifferentiation, allowing these cells to survive, detach from axonal connections, proliferate, and migrate (2). Dedifferentiation is essential for PNS regeneration (3). Many exogenous factors have been reported to stimulate migration of dedifferentiated Schwann cells, including neurotrophin-3 (NT-3), neuregulin-1, and insulinlike growth factor-I (4 -6). These factors promote migration, at least in part, by activating the Rho family GTPases, Rac1 and Cdc42. Rac1 promotes dynamic actin remodeling, lamellipodia formation and random cell migration (7,8). Cdc42 controls cell polarity and allows for directionally persistent cell migration (9). In Schwann cells, the ability of Rac1 to promote cell migration may be counteracted by high levels of activated RhoA and its downstream effector, Rho kinase (10). RhoA activation may be inhibited by Rac1 and Rac1 activation may be inhibited downstream of Rho kinase, allowing for orchestration of these GTPases in the control of cell morphology and migration (11)(12)(13)(14). In development, Schwann cell Rac1 facilitates radial axonal sorting and myelination (15).Gene products that control transformation of the Schwann cell phenotype in PNS injury remain incompletely characterized. We recently identified low density lipoprotein receptorrelated protein (LRP1) as a receptor expressed by Schwann cells mainly after PNS injury (16). LRP1 is a 600-kDa type I transmembrane protein in the LDL receptor gene family, originally characterized as an endocytic receptor, but currently recognized also for its role in cell signaling (17, 18). Diverse proteins implicated in PNS injury, including matrix metalloprotease-9 (MMP-9), tissue-type plasminogen activator (tPA), and activated ␣ 2 -macroglobulin (␣ 2 M) bind to LRP1 and activate Akt and ERK/MAP kinase in Schwann cells in vitro and in the injured . By its effect on cell signaling, LRP1 promotes Schwann cell survival and migration (16,19).Although the increase in Schwann cell migration, observed when cells are treated with MMP-9, has been attr...

“…In HEp3, COS-7, and MCF-7 cells, EGFR tyrosine kinase antagonists block uPA-induced ERK activation (20,26). The EGFR co-immunoprecipitates with uPAR and with ␤1 integrin (20); however, the mechanism by which the EGFR supports uPAR-initiated ERK activation remains unclear.…”

mentioning

confidence: 99%

Dynamic Assembly of the Urokinase-type Plasminogen Activator Signaling Receptor Complex Determines the Mitogenic Activity of Urokinase-type Plasminogen Activator

Thomas²,

Marozkina³

et al. 2005

Self Cite

The urokinase-type plasminogen activator (uPA) receptor (uPAR) functions in concert with co-receptors, including integrins, FPR-like receptor-1/lipoxin A4 receptor, and the epidermal growth factor receptor (EGFR), to initiate cell signaling. uPAR co-receptors may be dynamically organized into a multiprotein signaling receptor complex. In Chinese hamster ovary-K1 (CHO-K1) cells, uPA-binding to uPAR activates ERK/ MAP kinase, even though these cells do not express the EGFR; however, when CHO-K1 cells are transfected to express the EGFR, ERK activation becomes EGFR-dependent. In this study, we demonstrate that ERK activation in response to uPA follows equivalent biphasic kinetics in EGFR-expressing and -deficient CHO-K1 cells. In both cell types, the response is pertussis toxinsensitive; however, uPA promotes cell proliferation exclusively in the EGFR-expressing cells. uPA-induced mitogenic activity requires activation of both STAT5b and ERK. STAT5b was tyrosine-phosphorylated, in response to uPA, only in EGFR-expressing cells. uPA-induced cell proliferation was blocked by dominant-negative MEK1, dominant-negative STAT5b, and by expression of an EGFR that is mutated at Tyr-845, which is essential for STAT5b activation. In two cell culture models of uPAstimulated breast cancer growth, MDA-MB 468 cells treated with uPA and MCF-7 cells treated with uPAplasminogen activator inhibitor-1 complex, proliferation was completely inhibited when EGFR expression or activity was blocked. We conclude that expression and assembly of uPAR co-receptors in a specific cell type determines the response to uPA. The EGFR selectively cooperates with uPAR to mediate mitogenesis.Receptor transactivation provides a mechanism by which an expanded continuum of signaling pathways may be activated in response to a single ligand. The epidermal growth factor receptor (EGFR) 1 plays a prominent role in transactivation events initiated by G protein-coupled receptors (GPCRs), integrins, and cytokine receptors (1-3). Transactivated EGFR frequently is responsible for activating Ras and extracellular signal-regulated kinase/MAP kinase (ERK) (1-3). EGFR transactivation may be ligand-dependent, in which case membrane-associated metalloproteases release EGFR ligands from their transmembrane anchors (4, 5), or ligand-independent, due to the activity of intracellular kinases, such as c-Src, which cause EGFR tyrosine phosphorylation (6). The urokinase receptor (uPAR) has two distinct ligands, urokinase-type plasminogen activator (uPA) and vitronectin, which activate distinct cell-signaling pathways. Vitronectin activates the small GTPase, Rac1, and thereby regulates downstream factors involved in new actin polymerization (7-9). uPA activates multiple kinases and, perhaps most importantly, ERK (10 -13). ERK activation occurs in response to low uPAR fractional occupancy, even in cells that express minimum amounts of uPAR, accounting for the role of uPAR autocrine signaling in determining the basal level of activated ERK in many cells (9,13,14). In MCF-7 breast ...