Transforming Growth Factor-β1-dependent Urokinase Up-regulation and Promotion of Invasion Are Involved in Src-MAPK-dependent Signaling in Human Ovarian Cancer Cells

Abstract

Urokinase-type plasminogen activator (uPA) has been implicated in tumor cell invasion and metastasis. We reported previously that transforming growth factor (TGF)-␤1 induces a dose-and time-dependent up-regulation of uPA mRNA and protein in highly invasive human ovarian cancer cell line HRA, leading to invasion. To further elucidate the mechanism of the invasive effect of TGF-␤1, we investigated which signaling pathway transduced by TGF-␤1 is responsible for this effect. Here, we show that 1) nontoxic concentrations of TGF-␤1 activated Src kinase; 2) TGF-␤1 rapidly phosphorylates ERK1/2 and Akt, but not p38; 3) pharmacological Src inhibitor PP2 or antisense (AS) c-Src oligodeoxynucleotide (ODN) treatment reduced TGF-␤1-induced phosphorylation of ERK1/2 and Akt by 85-90% compared with controls; 4) pharmacological inhibition of MAPK by PD98059 abrogated TGF-␤1-mediated Akt stimulation, whereas TGF-␤1-induced ERK1/2 stimulation was not inhibited by PI3K inhibitor LY294002 or AS-PI3K ODN transfection; 5) up-regulation of uPA mRNA in response to TGF-␤1 was almost totally blocked by PP2 and PD98059 and partially (ϳ55%) by LY294002; 6) TGF-␤1-induced uPA mRNA up-regulation was inhibited by treatment with AS ODNs to c-Src or PI3K by 90 or 60%, respectively, compared with control ODN treatment; and 7) blockade of the release of the transcription factor NF-B by pyrrolidinedithiocarbamate reduced the TGF-␤1-induced activation of the uPA gene by ϳ65%. In addition, curcumin, a blocker of the transcriptional factor AP-1, partially (35%) canceled this effect. Taken together, these data support a role for TGF-␤1 activation of two distinct pathways (Src-MAPK-PI3K-NF-B-dependent and Src-MAPK-AP-1-dependent) for TGF-␤1-dependent uPA up-regulation and promotion of invasion.The processes of ovarian cancer dissemination are characterized by altered local proteolysis, cellular proliferation, cell attachment, and invasion, suggesting that the urokinase-type plasminogen activator (uPA) 1 could be involved in the pathogenesis of peritoneal dissemination (1). uPA is a serine protease associated with various pathological conditions including tumor invasion and metastasis (2). One of the factors regulating the metastatic process is considered to be transforming growth factor-␤ (TGF-␤), which is a multifunctional cytokine that elicits numerous cellular effects pertinent to the metastatic process (1). TGF-␤ regulates a wide range of physiological and pathological cellular processes, including cell growth, differentiation, invasion, migration, mesenchymal transition, extracellular matrix synthesis, and cell death in many cell types including ovarian cancer cells (3). Recent data demonstrated that TGF-␤ specifically stimulates up-regulation of uPA mRNA and protein in certain types of neoplastic cells (1). The cellular mechanism(s) of the TGF-␤-induced uPA-dependent tumor invasion and metastasis has been extensively studied. The identity of the signaling pathway(s) involved in the TGF-␤-induced uPA up-regulation (4) remains less known. In a well...

Phosphatidylinositol 3-kinase/Akt regulates the balance between plasminogen activator inhibitor-1 and urokinase to promote migration of SKOV-3 ovarian cancer cells

“…However, in further support of our results, a recent study showed a correlation between decreased phosphorylated Akt levels and decreased invasion in SKOV-3 cells [42]. Likewise, the regulation of uPA expression and activity by the PI3K/Akt pathway that we showed confirmed previously published results [12][13][14][15]39,40]. Finally, Venugopal et al [23] showed in an in vivo study that plasma PAI-1 was up-regulated in Akt-deficient mice (protein kinase B or PKBα −/− ), which would attenuate the PI3K/Akt signaling pathway.…”

“…By contrast, expression of constitutively active Akt caused the opposite effects on SKOV-3 cells: an increase in phosphorylated Akt levels correlated with a decrease in PAI-1 expression and an increase in wound-induced migration. The changes in SKOV-3 cell migration that accompanied the increase (constitutively active Akt adenovirus) or decrease (LY294002 and wortmannin treatment, Akt siRNA) in active Akt levels were similar to previously published studies [11][12][13][14][15][36][37][38][39][40][41]. It will be important to further these observations using different ovarian cancer cell lines, especially those that are not dependent on PI3K/Akt for migration and invasion.…”

“…3). As previously shown by others [12][13][14], we also found an accompanying decrease in the amount of uPA secreted (55-70%) when SKOV-3 cells were treated with LY294002 (Fig. 3).…”

“…The PI3K/Akt pathway regulates uPA expression; selective inhibition of the PI3K/Akt pathway in numerous cell types decreases uPA expression and/ or activity with a subsequent decrease in cell invasion [12][13][14]. Urokinase itself has been reported to stimulate PI3K activity and activates the downstream effectors Akt [15,16] and Rac1 [17].…”

Hypoxia induced by benign intestinal epithelial cells is associated with cyclooxygenase-2 expression in stromal cells through AP-1-dependent pathway

“…In contrast, the DNA-binding activity of NF-kB (Figure 7), SP1 and CREB (data not shown) were not changed in the hypoxic condition. Furthermore, to confirm the role of AP-1 pathway in COX-2 induction in co-cultured NIH3T3 cells, the 'dense' co-culture plates with Intestine-407 cells were treated with 20 mM of curcumin, which inhibits AP-1 activity (Huang et al, 1991;Lu et al, 1994;Tanaka et al, 2004;Park et al, 2005; Figure 7), for 24 h. As depicted in Figure 6, COX-2 expression under the 'dense' co-cultured condition was Hypoxia induces stromal cyclooxygenase-2 Y Uenoyama et al significantly reduced to the same expression level with gentle shaking, although curcumin did not alter cell number and viability (data not shown).…”