Differential roles of Src in transforming growth factor-ß regulation of growth arrest, epithelial-to-mesenchymal transition and cell migration in pancreatic ductal adenocarcinoma cells

Abstract

Abstract. Both transforming growth factor (TGF)-ß and the non-receptor tyrosine kinase Src play major roles during tumorigenesis by regulating cell growth, epithelial-to-mesenchymal transition (EMT), migration/invasion and metastasis, but little is known about the signaling crosstalk between them. To interfere with Src function in vitro and in vivo many studies have employed the pharmacologic Src inhibitors PP2 and PP1. Both agents have recently been shown to be powerful inhibitors of TGF-ß receptor type I/ALK5 and type II. As this situation prohibited any definite conclusions with respect to the relative contribution of TGF-ß vs. Src signaling, we decided to reappraise a potential role of Src in TGF-ß1-mediated cellular responses using RNA and dominant-negative (dn) interference to block Src expression and function, respectively. In TGF-ß-responsive pancreatic ductal adenocarcinoma (PDAC) cells, we show that Src is activated by TGF-ß1 and that its specific inhibition strongly attenuated basal proliferation and enhanced TGF-ß1-mediated growth arrest. However, Src inhibition was unable to impair TGF-ß1-controlled EMT as evidenced by cell morphology and regulation of the epithelial marker E-cadherin. Despite its dispensibility for TGF-ß-induced EMT, specific inhibition of Src dramatically reduced basal and TGF-ß1-induced cell migration in Panc-1 cells as measured with a novel real-time migration assay (xCELLigence DP system). Biochemically, dnSrc inhibition failed to block TGF-ß1/ALK5-induced activation of Smad2 and Smad3, but partially inhibited transcriptional activation of TGF-ß/Smad-responsive reporter genes, and effectively blocked basal and TGF-ß1-induced activation of p38 MAPK. Together, the data provide evidence for a role of Src in the regulation of basal proliferation as well as in basal and TGF-ß1-mediated cell motility but not EMT in TGF-ß-responsive pancreatic (tumor) cells. IntroductionSrc kinases are non-receptor intracellular tyrosine kinases that mediate a variety of cellular responses. The prototype member p60Src is the product of the proto-oncogene c-Src, and is the cellular homolog of the Rous sarcoma virus transforming protein v-Src. Src kinases are involved in numerous signaling pathways controlling proliferation, migration, adhesion, and angiogenesis (1). Src together with · V ß3 integrin promotes anchorage-independent growth and metastasis (2) and recent data have revealed that transient activation of Src by immortalized mammary epithelial cells triggered an inflammatory response that resulted in the initiation and maintenance of cellular transformation and a switch from a non-transformed to a transformed phenotype (3). Src mediates signaling from many types of receptors including receptor tyrosine kinases (EGF-R), integrins, and G-protein-coupled receptors such as proteinase-activated receptors (PARs) (4). Src is required for EGF-R stimulated DNA synthesis and has been reported to stimulate IGF-I-dependent cell proliferation by increasing IGF-I receptor number (5). Moreover, overexpression...

The Signaling Mechanism of TGF-β1 Induced Bovine Mammary Epithelial Cell Apoptosis

“…But the molecular mechanism involved in TGF-β 1 -regulated Ca 2+ concentration alterations via ALK-5-Smad2/3 and Ca 2+ activating apoptosis-regulating factors remains to be further explored. Recently, the signal pathway of TGF-β has become a target for cancer treatment (Lv et al, 2010; Ungefroren et al, 2011), and inhibiting TGF-β 1 I-type receptor kinase selectively can inhibit the proliferation and transfer of mammary cancer cells of mouse, and activating TGF-β 1 I-type receptors (ALK-5) kinase can improve survivability of mammary epithelial cells and promote the progress of breast tumor (Ge et al, 2006; Muraoka-Cook et al, 2006). It was shown in this study that SB-431542 could block the signal pathway of TGF-β, therefore SB-431542, a TGF-β 1 I-type receptors kinase inhibitor, is a potential new drug in treating metastatic breast tumors.…”

The role of TGF-β and its crosstalk with RAC1/RAC1b signaling in breast and pancreas carcinoma

“…The complexity of TGF-β signaling in EMT induction is also reflected in the various interactions of TGF-β signaling with at least six other signaling pathways (RTK signaling, cytokine signaling, Wnt/β-catenin signaling, Notch signaling, Sonic hedgehog signaling, Hippo signaling) in regulating the expression and/or activity of transcription factors that elicit the EMT [26]. Crosstalk with RTK signaling involves common intracellular mediators with a known role in driving proliferation and cell motility/metastasis such as Ki-RAS [53], SRC [54], and p53 [55, 56], all of which have been implicated in TGF-β-induced EMT. TGF-β signaling has also been shown to promote metastatic colonization and mesenchymal-epithelial transition (MET) by inhibiting Twist1 [57] (Fig.…”

TGFβ1 rapidly activates Src through a non-canonical redox signaling mechanism

“…Src activation is responsible for many TGF-β-mediated effects (43-45). The mechanism underlying Src activation by TGF-β however, has been unclear.…”

“…Many effects of TGF-β are mediated through Src-mediated signaling pathways (43-45). How TGF-β activates Src is not completely clear.…”