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PDF - 16K, Tri- and Penta-cyclic compounds to not quench pyrene actin florescence intensity. To ascertain that the addition of TBE-31 or CDDO-Im would not quench pyrene-actin fluorescence intensity, pyrene actin polymerization was carried out as described in the Materials section, measured (-) and then DMSO, TBE-31 or CDDO-Im were added to the samples and re-measured (+) for fluorescence intensity. Note that the fluorescence intensity did not appreciably change after vehicle or drug addition.

AVI - 10062K, Confluent monolayers of H1299 lung tumor cells were scratched and incubated at 37 degrees C in media containing either vehicle (DMSO), 1 microM TBE-31 or 1 microM CDDO-Im for 18 h. Representative bright field microscopy images were taken at 10x magnification every 15 min using an Olympus IX81 microscope to create a movie.

PDF - 8699K, Single cell tracking of NIH 3T3 cells was carried out using time-lapse microscopy with an Olympus IX81 inverted microscope equipped with a custom chamber which allowed us to maintain a temperature of 37 degrees C and an atmosphere containing 5% CO2. Bright field images were collected from cells incubated with vehicle (DMSO), 1 microM TBE-31 or 0.5 microM CDDO-Im at 10 min intervals over 18 h. Distance of migration was determined by tracking the positions of cell nuclei over 18 h using the MtrackJ plugin (32) for imageJ software (33).

The migratory and invasive potential of the epithelial-derived tumor cells depends on epithelial-to-mesenchymal transition (EMT) as well as the reorganization of the cell cytoskeleton. Here, we show that the tricyclic compound acetylenic tricyclic bis(cyano enone), TBE-31, directly binds to actin and inhibits linear and branched actin polymerization in vitro. Furthermore, we observed that TBE-31 inhibits stress fiber formation in fibroblasts as well as in non–small cell lung cancer cells during TGFβ-dependent EMT. Interestingly, TBE-31 does not interfere with TGFβ-dependent signaling or changes in E-cadherin and N-cadherin protein levels during EMT. Finally, we observed that TBE-31 inhibits fibroblast and non–small cell lung tumor cell migration with an IC50 of 1.0 and 2.5 μmol/L, respectively. Taken together, our results suggest that TBE-31 targets linear actin polymerization to alter cell morphology and inhibit cell migration. Cancer Prev Res; 7(7); 727–37. ©2014 AACR.

The ubiquitin-proteasome pathway (UPP) and autophagy play integral roles in cellular homeostasis. As part of their normal life cycle, most proteins undergo ubiquitination for some form of redistribution, localization and/or functional modulation. However, ubiquitination is also important to the UPP and several autophagic processes. The UPP is initiated after specific lysine residues of short-lived, damaged or misfolded proteins are conjugated to ubiquitin, which targets these proteins to proteasomes. Autophagy is the endosomal/lysosomal-dependent degradation of organelles, invading microbes, zymogen granules and macromolecules such as protein, carbohydrates and lipids. Autophagy can be broadly separated into three distinct subtypes termed microautophagy, chaperone-mediated autophagy and macroautophagy. Although autophagy was once thought of as non-selective bulk degradation, advancements in the field have led to the discovery of several selective forms of autophagy. Here, we focus on the mechanisms of primary and selective mammalian autophagy pathways and highlight the current knowledge gaps in these molecular pathways.

Impairing autophagy disrupts transforming growth factor beta 1 (TGFβ1) signalling and epithelial-mesenchymal transition (EMT) in non-small cell lung cancer (NSCLC). Since autophagy and proteasome-mediated degradation are interdependent, we investigated how prolonged downregulation of proteasomal catalytic activity affected TGFβ1-dependent signalling and EMT. Proteasome-dependent degradation was inhibited in A549 and H1299 NSCLC cells using MG132 and lactacystin, which are reversible and irreversible proteasome inhibitors, respectively. We observed that inhibiting proteasomal activity for 24 h decreased TGFβ-dependent nuclear accumulation of Smad2/3. Time course studies were then carried out to characterize the time frame of this observation. Short-term (8 h) proteasome inhibition resulted in increased receptor regulated Smad (R-Smad) phosphorylation and steady-state TGFβ receptor type II (TGFβRII) levels. However, prolonged (8-24 h) proteasome inhibition decreased TGFβ1-dependent R-Smad phosphorylation and steady-state TGFβRI and TGFβRII levels. Furthermore, proteasome inhibition blunted TGFβ-dependent E- to N-Cadherin shift, stress fiber formation, and increased cellular apoptosis via the TAK-1-TRAF6-p38 MAPK pathway. Interestingly, proteasome inhibition also increased autophagic flux, steady-state microtubule-associated protein light chain 3B-II and active uncoordinated 51-like autophagy activating kinase 1 levels, and co-localization of lysosomes with autophagy cargo proteins and autophagy-related proteins. Finally, we observed that proteasome inhibition increased TGFβRII endocytosis and trafficking to lysosomes and we conclude that prolonged proteasome inhibition disrupts TGFβ signalling outcomes through altered TGFβ receptor trafficking.

Transforming growth factor beta (TGFβ) stimulates tumorigenesis by inducing epithelial to mesenchymal transition (EMT) and cell migration. TGFβ signaling is regulated by the endocytosis of cell surface receptors and their subcellular trafficking into the endo-lysosomal system. Here we investigated how autophagy, a cellular quality control network that delivers material to lysosomes, regulates TGFβ signaling pathways that induce EMT and cell migration. We impaired autophagy in non-small cell lung cancer cells using chloroquine, spautin-1, ULK-101, or small interfering RNA (siRNA) targeting autophagy-related gene (ATG)5 and ATG7 and observed that inhibiting autophagy results in a decrease in TGFβ1-dependent EMT transcription factor and cell marker expression, as well as attenuated stress fiber formation and cell migration. This correlated with decreased internalization of cell surface TGFβ receptors and their trafficking to early/late endosomal and lysosomal compartments. The effects of autophagy inhibition on TGFβ signaling were investigated by Smad2/Smad3 phosphorylation and cellular localization using western blotting, subcellular fractionation, and immunofluorescence microscopy. We observed that inhibiting autophagy decreased the amount and timeframe of Smad2/Smad3 signaling. Taken together, our results suggest that inhibiting autophagy attenuates pro-tumorigenic TGFβ signaling by regulating receptor trafficking, resulting in impaired Smad2/Smad3 phosphorylation and nuclear accumulation.

Transforming growth factor beta (TGFβ) receptor trafficking regulates many TGFβ-dependent cellular outcomes including epithelial to mesenchymal transition (EMT). EMT in A549 non-small cell lung cancer (NSCLC) cells has recently been linked to the regulation of cellular autophagy. Here, we investigated the role of the autophagy cargo receptor, p62/sequestosome 1 (SQSTM1), in regulating TGFβ receptor trafficking, TGFβ1-dependent Smad2 phosphorylation and EMT in A549 NSCLC cells. Using immunofluorescence microscopy, p62/SQSTM1 was observed to co-localize with TGFβ receptors in the late endosome. Small interfering RNA (SiRNA)-mediated silencing of p62/SQSTM1 resulted in an attenuated time-course of Smad2 phosphorylation but did not alter Smad2 nuclear translocation. However, p62/SQSTM1 silencing promoted TGFβ1-dependent EMT marker expression, actin stress fiber formation and A549 cell migration. We further observed that Smad4-independent TGFβ1 signaling decreased p62/SQSTM1 protein levels via a proteasome-dependent mechanism. Although p62/SQSTM1 silencing did not impede TGFβ-dependent autophagy, our results suggest that p62/SQSTM1 may aid in maintaining A549 cells in an epithelial state and TGFβ1 decreases p62/SQSTM1 prior to inducing EMT and autophagy.

TGFβ-dependent signal transduction is facilitated by Smad anchor for receptor activation (SARA) and inhibited by the inhibitory-Smad, Smad7, which recruits the E3 ubiquitin ligase, Smurf2, to catalyze the degradation of TGFβ receptors. Since the signalling and degradation pathways target active receptor complexes, we assessed if SARA and Smurf2/Smad7 interact and if Smad7/Smurf2 would affect SARA steady state levels. We observed that the Smurf2/Smad7 complex induces a decrease of SARA steady state levels in a process that is dependent on the HECT ubiquitin E3 ligase activity of Smurf2 but is independent of SARA associating with TGFβ receptors or Smad2. We observed that Smurf2/Smad7-dependent reduction of SARA levels is dependent on proteasome activity, as the pharmacological inhibition of the proteasome using MG132 blocked degradation of SARA. When we assessed the functional outcome of reducing endogenous SARA levels via siRNA-mediated silencing, we observed that siRNA directed at SARA decreased both TGFβ-dependent Smad2 membrane recruitment and phosphorylation, as assessed by subcellular fractionation and western blotting. Furthermore, siRNA targeting SARA decreased TGFβ-dependent epithelial to mesenchymal transition, as measured by cellular E- and N-Cadherin protein levels, and the reorganization of actin from cortical actin to stress fiber formation. These data describe a previously undescribed mechanism where the robustness of the TGFβ signalling is regulated by interplay between SARA and Smurf2/Smad7 complexes.

Stress-inducible phosphoprotein 1 (STI1) acts as a neuroprotective factor in the ischemic brain and its levels are increased following ischemia. Previous work has suggested that some of these STI1 actions in a stroke model depend on the recruitment of bone marrow-derived stem cells to improve outcomes after ischemic insult. However, STI1 can directly increase neuroprotective signaling in neurons by engaging with the cellular prion protein (PrPC) and activating 7 nicotinic acetylcholine receptors (alpha 7nAChR). Given that alpha 7nAChR activation has also been involved in neuroprotection in stroke, it is possible that STI1 can have direct actions on neurons to prevent deleterious consequences of ischemic insults. Here, we tested this hypothesis by exposing primary neuronal cultures to 1-h oxygen-glucose deprivation (OGD) and reperfusion and assessing signaling pathways activated by STI1/PrPC. Our results demonstrated that STI1 treatment significantly decreased apoptosis and cell death in mouse neurons submitted to OGD in a manner that was dependent on PrPC and alpha 7nAChR, but also on the activin A receptor 1 (ALK2), which has emerged as a signaling partner of STI1. Interestingly, pharmacological inhibition of the ALK2 receptor prevented neuroprotection by STI1, while activation of ALK2 receptors by bone morphogenetic protein 4 (BMP4) either before or after OGD was effective in decreasing neuronal death induced by ischemia. We conclude that PrPC/STI1 engagement and its subsequent downstream signaling cascades involving alpha 7nAChR as well as the ALK2 receptor may be activated in neurons by increased levels of STI1. This signaling pathway protects neurons from ischemic insults.

Epidermal growth factor (EGF) and insulin receptor tyrosine kinases (RTKs) exemplify how receptor location is coupled to signal transduction. Extracellular binding of ligands to these RTKs triggers their concentration into vesicles that bud off from the cell surface to generate intracellular signaling endosomes. On the exposed cytosolic surface of these endosomes, RTK autophosphorylation selects the downstream signaling proteins and lipids to effect growth factor and polypeptide hormone action. This selection is followed by the recruitment of protein tyrosine phosphatases that inactivate the RTKs and deliver them by membrane fusion and fission to late endosomes. Coincidentally, proteinases inside the endosome cleave the EGF and insulin ligands. Subsequent inward budding of the endosomal membrane generates multivesicular endosomes. Fusion with lysosomes then results in RTK degradation and downregulation. Through the spatial positioning of RTKs in target cells for EGF and insulin action, the temporal extent of signaling, attenuation, and downregulation is regulated.

The M2 isoform of the glycolytic enzyme pyruvate kinase (PKM2) is one of the key components in the Warburg effect, and an important regulator of cancer cell metabolism. Elevated PKM2 expression is a hallmark of numerous tumor types, making it a promising target for cancer therapy. Migration of H1299 lung tumor cells treated with synthetic oleanane triterpenoid derivatives CDDO-Me and CDDO-Im was monitored using scratch and transwell assays. Direct binding and inhibition of PKM2 activity by CDDO-Me was demonstrated by pull-down and activity assays. PKM2 localization in the absence and presence of CDDO-Me or CDDO-Im was determined by subcellular fractionation and immunofluorescence microscopy. Involvement of PKM2 in tumor cell migration was assessed using a stable PKM2 knockdown cell line. We demonstrate that migration of H1299 lung tumor cells is inhibited by CDDO-Me and CDDO-Im in scratch and transwell assays. CDDO-Me binds directly and specifically to recombinant PKM2, leading to a reduction of its catalytic activity. PKM2 knockdown cells exhibit significantly lower migration compared to control cells when subjected to glucose and oxygen deprivation, but not under regular conditions. The results suggest that PKM2 expression in a tumor-like environment contributes to cell migration, and that PKM2 activity can be down regulated by synthetic triterpenoid derivatives.

Epithelial-to-mesenchymal transition (EMT) is controlled by cellular signaling pathways that trigger the loss of cell-cell adhesion and lead to the restructuring of the cell cytoskeleton. Transforming growth factor β (TGF-β) has been shown to regulate cell plasticity through the phosphorylation of Par6 on a conserved serine residue (S345) by the type II TGF-β receptor. We show here that atypical protein kinase C (aPKC) is an essential component to this signaling pathway in non-small-cell lung cancer (NSCLC) cells. We show that the aPKC, PKCι, interacts with TGF-β receptors through Par6 and that these proteins localize to the leading edge of migrating cells. Furthermore, Par6 phosphorylation on serine 345 by TGF-β receptors is enhanced in the presence of aPKC. aPKC kinase activity, as well as an association with Par6, were found to be important for Par6 phosphorylation. In effect, small interfering RNA-targeting aPKC reduces TGF-β-induced RhoA and E-cadherin loss, cell morphology changes, stress fiber production, and the migration of NSCLC cells. Interestingly, reintroduction of a phosphomimetic Par6 (Par6-S345E) into aPKC-silenced cells rescues both RhoA and E-cadherin loss with TGF-β stimulation. In conclusion, our results suggest that aPKCs cooperate with TGF-β receptors to regulate phospho-Par6-dependent EMT and cell migration.

Synthetic triterpenoids are a class of anti-cancer compounds that target many cellular functions, including apoptosis and cell growth in both cell culture and animal models. We have shown that triterpenoids inhibit cell migration in part by interfering with Arp2/3-dependent branched actin polymerization in lamellipodia (To et al., 2010). Our current studies reveal that Glycogen Synthase Kinase 3 beta (GSK3β), a kinase that regulates many cellular processes, including cell adhesion dynamics, is a triterpenoid-binding protein. Furthermore, triterpenoids were observed to inhibit GSK3β activity and increase cellular focal adhesion size. To further examine whether these effects on focal adhesions in triterpenoid-treated cells were GSK3β-dependent, GSK3β inhibitors (lithium chloride and SB216763) were used to examine cell adhesion and morphology as well as cell migration. Our results showed that GSK3β inhibitors also altered cell adhesion sizes. Moreover, these inhibitors blocked cell migration and displaced proteins at the leading edge of migrating cells, consistent with what was observed in triterpenoid-treated cells. Therefore, the triterpenoids may affect cell migration via a mechanism that targets and alters the activity and localization of GSK3β.

Cutaneous responses to injury, infection, and tumor formation involve the activation of resident dermal fibroblasts and subsequent transition to myofibroblasts. The key for induction of myofibroblast differentiation is the activation of transforming growth factor-β (TGF-β) receptors and stimulation of integrins and their associated proteins, including integrin-linked kinase (ILK). Cross-talk processes between TGF-β and ILK are crucial for myofibroblast formation, as ILK-deficient dermal fibroblasts exhibit impaired responses to TGF-β receptor stimulation. We now show that ILK associates with type II TGF-β receptors (TβRII) in ligand- and receptor kinase activity–independent manners. In cells with targeted Ilk gene inactivation, cellular levels of TβRII are decreased, through mechanisms that involve enhanced ubiquitination and proteasomal degradation. Partitioning of TGF-β receptors into membrane has been linked to proteasome-dependent receptor degradation. We found that interfering with membrane raft formation in ILK-deficient cells restored TβRII levels and signaling. These observations support a model whereby ILK functions in fibroblasts to direct TβRII away from degradative pathways during their differentiation into myofibroblasts.

Endoglin is an accessory receptor for transforming growth factor beta (TGFbeta) in endothelial cells, essential for vascular development. Its pivotal role in angiogenesis is underscored in Endoglin null (Eng-/-) murine embryos, which die at mid-gestation (E10.5) from impaired yolk sac vessel formation. Moreover, mutations in endoglin and the endothelial-specific TGFbeta type I receptor, ALK1, are linked to hereditary hemorrhagic telangiectasia. To determine the role of endoglin in TGFbeta pathways, we derived murine endothelial cell lines from Eng+/+ and Eng-/- embryos (E9.0). Whereas Eng+/+ cells were only partially growth inhibited by TGFbeta, Eng-/- cells displayed a potent anti-proliferative response. TGFbeta-dependent Smad2 phosphorylation and Smad2/3 translocation were unchanged in the Eng-/- cells. In contrast, TGFbeta treatment led to a more rapid activation of the Smad1/5 pathway in Eng null cells that was apparent at lower TGFbeta concentrations. Enhanced activity of the Smad1 pathway in Eng-/- cells was reflected in higher expression of ALK1-dependent genes such as Id1, Smad6, and Smad7. Analysis of cell surface receptors revealed that the TGFbeta type I receptor, ALK5, which is required for ALK1 function, was increased in Eng-/- cells. TGFbeta receptor complexes were less numerous but displayed a higher binding affinity. These results suggest that endoglin modulates TGFbeta signaling in endothelial cells by regulating surface TGFbeta receptors and suppressing Smad1 activation. Thus an altered balance in TGFbeta receptors and downstream Smad pathways may underlie defects in vascular development and homeostasis.

Transforming growth factor b (TGFb) signaling controls many cellular responses including proliferation, epithelial to mesenchymal transition and apoptosis, through the activation of canonical (Smad) as well as non-canonical (e.g., Par6) pathways. Previous studies from our lab have demonstrated that aPKC inhibition regulates TGFb receptor trafficking and signaling. Here, we report that downstream TGFb-dependent transcriptional responses in aPKC-silenced NSCLC cells were reduced compared with those of control cells, despite a temporal extension of Smad2 phosphorylation. We assessed SARA–Smad2–Smad4 association and observed that knockdown of aPKC increased SARA (also known as ZFYVE9) levels and SARA–Smad2 complex formation, increased cytoplasmic retention of Smad2 and reduced Smad2–Smad4 complex formation, which correlated with reduced Smad2 nuclear translocation. Interestingly, we also detected an increase in p38 MAPK phosphorylation and apoptosis in aPKC-silenced cells, which were found to be TRAF6-dependent. Taken together, our results suggest that aPKC isoforms regulate Smad and non-Smad TGFb pathways and that aPKC inhibition sensitizes NSCLC cells to undergo TGFb dependent apoptosis.

Transforming growth factor b (TGFb) signaling controls many cellular responses including proliferation, epithelial to mesenchymal transition and apoptosis, through the activation of canonical (Smad) as well as non-canonical (e.g., Par6) pathways. Previous studies from our lab have demonstrated that aPKC inhibition regulates TGFb receptor trafficking and signaling. Here, we report that downstream TGFb-dependent transcriptional responses in aPKC-silenced NSCLC cells were reduced compared with those of control cells, despite a temporal extension of Smad2 phosphorylation. We assessed SARA–Smad2–Smad4 association and observed that knockdown of aPKC increased SARA (also known as ZFYVE9) levels and SARA–Smad2 complex formation, increased cytoplasmic retention of Smad2 and reduced Smad2–Smad4 complex formation, which correlated with reduced Smad2 nuclear translocation. Interestingly, we also detected an increase in p38 MAPK phosphorylation and apoptosis in aPKC-silenced cells, which were found to be TRAF6-dependent. Taken together, our results suggest that aPKC isoforms regulate Smad and non-Smad TGFb pathways and that aPKC inhibition sensitizes NSCLC cells to undergo TGFb dependent apoptosis.

The migratory and invasive potential of the epithelial-derived tumor cells depends on epithelial-to-mesenchymal transition (EMT) as well as the reorganization of the cell cytoskeleton. Here, we show that the tricyclic compound acetylenic tricyclic bis(cyano enone), TBE-31, directly binds to actin and inhibits linear and branched actin polymerization in vitro. Furthermore, we observed that TBE-31 inhibits stress fiber formation in fibroblasts as well as in non–small cell lung cancer cells during TGFβ-dependent EMT. Interestingly, TBE-31 does not interfere with TGFβ-dependent signaling or changes in E-cadherin and N-cadherin protein levels during EMT. Finally, we observed that TBE-31 inhibits fibroblast and non–small cell lung tumor cell migration with an IC50 of 1.0 and 2.5 μmol/L, respectively. Taken together, our results suggest that TBE-31 targets linear actin polymerization to alter cell morphology and inhibit cell migration. Cancer Prev Res; 7(7); 727–37. ©2014 AACR.

The reovirus type 3 S1 gene product (type 3 hemagglutinin; HA3) is the viral protein responsible for binding to a mammalian cell-surface receptor. It has been shown that HA3 binding to its receptor inhibits cell growth, even in the continuous presence of serum mitogens. Here, receptor-mediated signal transduction leading to growth arrest was studied after binding with synthetic or recombinant ligands in the absence of viral infection. Receptor ligation caused rapid inactivation of p21(ras), a decrease in Raf phosphorylation and in mitogen-activated protein kinase (MAPK) enzymatic activity, and G1 cell cycle arrest. Transfection and expression of constitutively active v-Has-ras prevented the G1 arrest, indicating that inactivation of p21(ras) is causative. Interestingly, v-Has-ras expression also decreased the efficiency of reoviridae replication, suggesting that inactivation of p21(ras) signals is required at some step of the viral cycle. This study may define new mechanisms regulating cell growth and support the approach of using viral proteins to identify and study cellular receptors. Synthetic receptor ligands with antiproliferative properties may be useful in drug development with the aim of blocking mitosis.

The transforming growth factor beta (TGFβ) signaling pathway is important for normal cell homeostasis and has critical roles in apoptosis, cell-cycle arrest, and cellular differentiation (reviewed in Massague, 2008 ). In the classical TGFβ pathway, the endosomal trafficking of receptors has a direct outcome on signal transduction—receptors internalized via clathrin-mediated endocytosis enter the early endosome and propagate signaling, while those internalized via membrane rafts are targeted for degradation. Recently, there have been a number of articles that have identified TGFβ receptor-binding proteins that direct receptor endocytosis and/or intracellular trafficking and affect signal output ( Atfi et al., 2007; Bauge, Girard, Leclercq, Galera, & Boumediene, 2012; Bizet et al., 2011, 2012; Chen et al., 2007; Gunaratne, Benchabane, & Di Guglielmo, 2012; Hao et al., 2011; McLean, Bhattacharya, & Di Guglielmo, 2013; Zhao et al., 2012 ). Given the importance of TGFβ receptor trafficking to signaling outcome, this chapter will focus on strategies to isolate membrane rafts and techniques to follow the trafficking of cell-surface TGFβ receptors and provide examples of functional readouts to assess TGFβ signal transduction.

The transforming growth factor beta (TGFβ) signaling pathway is important for normal cell homeostasis and has critical roles in apoptosis, cell-cycle arrest, and cellular differentiation (reviewed in Massague, 2008). In the classical TGFβ pathway, the endosomal trafficking of receptors has a direct outcome on signal transduction-receptors internalized via clathrin-mediated endocytosis enter the early endosome and propagate signaling, while those internalized via membrane rafts are targeted for degradation. Recently, there have been a number of articles that have identified TGFβ receptor-binding proteins that direct receptor endocytosis and/or intracellular trafficking and affect signal output (Atfi et al., 2007; Bauge, Girard, Leclercq, Galera,Boumediene, 2012; Bizet et al., 2011, 2012; Chen et al., 2007; Gunaratne, Benchabane,Di Guglielmo, 2012; Hao et al., 2011; McLean, Bhattacharya,Di Guglielmo, 2013; Zhao et al., 2012). Given the importance of TGFβ receptor trafficking to signaling outcome, this chapter will focus on strategies to isolate membrane rafts and techniques to follow the trafficking of cell-surface TGFβ receptors and provide examples of functional readouts to assess TGFβ signal transduction.

Epithelial-to-mesenchymal transition (EMT) is controlled by cellular signaling pathways that trigger the loss of cell-cell adhesion and lead to the restructuring of the cell cytoskeleton. Transforming growth factor β (TGF-β) has been shown to regulate cell plasticity through the phosphorylation of Par6 on a conserved serine residue (S345) by the type II TGF-β receptor. We show here that atypical protein kinase C (aPKC) is an essential component to this signaling pathway in non-small-cell lung cancer (NSCLC) cells. We show that the aPKC, PKCι, interacts with TGF-β receptors through Par6 and that these proteins localize to the leading edge of migrating cells. Furthermore, Par6 phosphorylation on serine 345 by TGF-β receptors is enhanced in the presence of aPKC. aPKC kinase activity, as well as an association with Par6, were found to be important for Par6 phosphorylation. In effect, small interfering RNA-targeting aPKC reduces TGF-β-induced RhoA and E-cadherin loss, cell morphology changes, stress fiber production, and the migration of NSCLC cells. Interestingly, reintroduction of a phosphomimetic Par6 (Par6-S345E) into aPKC-silenced cells rescues both RhoA and E-cadherin loss with TGF-β stimulation. In conclusion, our results suggest that aPKCs cooperate with TGF-β receptors to regulate phospho-Par6-dependent EMT and cell migration.

Wound healing is an intricate biological process in which the skin, or any other tissue, repairs itself after injury. Normal wound healing relies on the appropriate levels of cytokines and growth factors to ensure that cellular responses are mediated in a coordinated manner. Among the many growth factors studied in the context of wound healing, transforming growth factor beta (TGF-β) is thought to have the broadest spectrum of effects.Many of the molecular mechanisms underlying the TGF-β/Smad signaling pathway have been elucidated, and the role of TGF-β in wound healing has been well characterized. Targeting the TGF-β signaling pathway using therapeutic agents to improve wound healing and/or reduce scarring has been successful in pre-clinical studies.Although TGF-β isoforms (β1, β2, β3) signal through the same cell surface receptors, they display distinct functions during wound healingUnderstanding the differences in TGF-β isoform signaling at the molecular level and identification of novel components of the TGF-β signaling pathway that critically regulate wound healing may lead to the discovery of potential therapeutic targets for treatment of impaired wound healing and pathological scarring.

β-Arrestins play critical roles in chemotaxis and cytoskeletal reorganization downstream of several receptor types, including G protein-coupled receptors (GPCRs), which are targets for greater than 50% of all pharmaceuticals. Among them, receptors for lysophosphatidic acid (LPA), namely LPA(1) are overexpressed in breast cancer and promote metastatic spread. We have recently reported that β-arrestin2 regulates LPA(1)-mediated breast cancer cell migration and invasion, although the underlying molecular mechanisms are not clearly understood. We show here that LPA induces activity of the small G protein, Rap1 in breast cancer cells in a β-arrestin2-dependent manner, but fails to activate Rap1 in non-malignant mammary epithelial cells. We found that Rap1A mRNA levels are higher in human breast tumors compared to healthy patient samples and Rap1A is robustly expressed in human ductal carcinoma in situ and invasive tumors, in contrast to the normal mammary ducts. Rap1A protein expression is also higher in aggressive breast cancer cells (MDA-MB-231 and Hs578t) relative to the weakly invasive MCF-7 cells or non-malignant MCF10A mammary cells. Depletion of Rap1A expression significantly impaired LPA-stimulated migration of breast cancer cells and invasiveness in three-dimensional Matrigel cultures. Furthermore, we found that β-arrestin2 associates with the actin binding protein IQGAP1 in breast cancer cells, and is necessary for the recruitment of IQGAP1 to the leading edge of migratory cells. Depletion of IQGAP1 blocked LPA-stimulated breast cancer cell invasion. Finally, we have identified that LPA enhances the binding of endogenous Rap1A to β-arrestin2, and also stimulates Rap1A and IQGAP1 to associate with LPA(1). Thus our data establish novel roles for Rap1A and IQGAP1 as critical regulators of LPA-induced breast cancer cell migration and invasion.

The Transforming Growth Factor beta (TGFβ) signaling pathway is necessary for a variety of normal cellular processes. However, the distinct mechanisms involved in TGFβ receptor turnover and the effect on signal transduction have yet to be fully elucidated. We have previously shown that TβRIII is able to interact with the TβRII/TβRI complex to increase clathrin-dependent endocytosis and receptor half-life. Others have shown that βarrestin2 binds TβRIII to mediate TβRII/TβRIII endocytosis. To further understand the mechanism regulating TGFβ receptor signaling, we evaluated the role of βarrestin2 in TGFβ receptor signal transduction, half-life and trafficking. We have found that TβRII binds βarrestin2 in the absence of TβRIII. Furthermore, using immunofluorescence microscopy we show that βarrestin2 traffics to the early endosome with TβRII. We investigated the effect of loss of βarrestin2 on TβRII dynamics and found that loss of βarrestin2 increases steady-state levels of TβRII at the cell surface. The interaction of TβRII with βarrestin2 is involved in modulating TGFβ signal transduction, as loss of βarrestin2 increases the phosphorylation of p38 and modestly affects pSmad levels. Using a luciferase assay to assess TGFβ-dependent transcription we show that loss of βarrestin2 decreases Smad-dependent TGFβ-stimulated transcription. Furthermore, loss of βarrestin2 increases p38 signal transduction, which correlated with increased cell death via apoptosis. Overall, our results suggest a role for βarrestin2 in the regulation of Smad-dependent and independent TGFβ pathways.

Extravillous trophoblasts (EVT) of the human placenta invade the uterine decidua and its arteries to ensure successful placentation. We previously identified two decidua-derived molecules, TGF-β and a TGF-β-binding proteoglycan decorin (DCN), as negative regulators of EVT proliferation, migration, and invasiveness and reported that DCN acts via multiple tyrosine kinase receptors [epidermal growth factor-receptor (EGF-R), IGF receptor-1 (IGFR1), and vascular endothelial growth factor 2 receptor (VEGFR-2)]. Because binding of DCN toVEGFR-2 has never been reported earlier, present study explored this binding, the approximate location of VEGFR-2-binding site in DCN, and its functional role in our human first trimester EVT cell line HTR-8/SVneo. Based on far-Western blotting and coimmunoprecipitation studies, we report that DCN binds both native (EVT expressed) and recombinant VEGFR-2 and that this binding is abrogated with a VEGFR-2 blocking antibody, indicating an overlap between the ligand-binding and the DCN-binding domains of VEGFR-2. We determined that 125I-labeled VEGF-E (a VEGFR-2 specific ligand) binds EVT with a dissociation constant (K d) of 566 pM, and DCN displaced this binding with an inhibition constant (K i) of 3.93-5.78 nM, indicating a 7- to 10-fold lower affinity of DCN for VEGFR-2. DCN peptide fragments derived from the leucine rich repeat 5 domain that blocked DCN-VEGFR-2 interactions or VEGF-E binding in EVT cells also blocked VEGF-A- and VEGF-E-induced EVT cell proliferation and migration, indicative of functional VEGFR-2-binding sites of DCN. Finally, DCN inhibited VEGF-E-induced EVT migration by interfering with ERK1/2 activation. Our findings reveal a novel role of DCN as an antagonistic ligand for VEGFR-2, having implications for pathophysiology of preeclampsia, a trophoblast hypoinvasive disorder in pregnancy, and explain its antiangiogenic function. © 2011 by The Endocrine Society.

Transforming growth factor beta (TGFβ) signaling is linked to the membrane trafficking of TGFβ receptors. The Protein Kinase C (PKC) family of serine/threonine kinases have been implicated in modulating the endocytic processes of various receptors. The present study investigated whether PKC activity plays a role in the trafficking, and signaling of TGFβ receptors, and further explored which PKC isoforms may be responsible for altered TGFβ signaling patterns. Using immunofluorescence microscopy and 125 I-TGFβ internalization assays, we show that the pharmacological inhibition of PKC activity alters TGFβ receptor trafficking and delays TGFβ receptor degradation. Consistent with these findings, we demonstrate that PKC inhibition extends TGFβ-dependent Smad2 phosphorylation. Previous studies have shown that PKCζ associates with TGFβ receptors to modulate cell plasticity. We therefore used siRNA directed at the atypical PKC isoforms to investigate if reducing PKCι and PKCζ protein levels would delay TGFβ receptor degradation and extend TGFβ signaling. Our findings suggest that atypical PKC isoforms regulate TGFβ signaling by altering cell surface TGFβ receptor trafficking and degradation.

Synthetic triterpenoids are anti-tumor agents that affect numerous cellular functions including apoptosis and growth inhibition. Here, we used mass spectrometric and protein array approaches and uncovered that triterpenoids associate with proteins of the actin cytoskeleton, including actin-related protein 3 (Arp3). Arp3, a subunit of the Arp2/3 complex, is involved in branched actin polymerization and the formation of lamellipodia. 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO)-Im and CDDO-Me were observed to 1) inhibit the localization of Arp3 and actin at the leading edge of cells, 2) abrogate cell polarity, and 3) inhibit Arp2/3-dependent branched actin polymerization. We confirmed our drug effects with siRNA targeting of Arp3 and observed a decrease in Rat2 cell migration. Taken together, our data suggest that synthetic triterpenoids target Arp3 and branched actin polymerization to inhibit cell migration.

The TGFbeta (transforming growth factor beta) pathway is an essential cell signalling pathway that is implicated in both normal developmental processes, such as organogenesis, and pathological disorders, such as cancer and fibrosis. There are three prototypical TbetaRs (TGFbeta receptors): TbetaRI (TbetaR type I), TGbetaRII (TbetaR type II) and TGFbetaRIII (TbetaR type III, also known as betaglycan). Whereas the role of TbetaRII and TbetaRI in TGFbeta signal propagation has been established, the contribution of TbetaRIII to TGFbeta signalling is less well understood. At the cell surface, TbetaRI and TbetaRII receptors can be internalized by clathrin-mediated endocytosis and clathrin-independent membrane-raft-dependent endocytosis. Interestingly, the endocytic route of the receptors plays a direct role in TGFbeta-dependent Smad signal transduction; receptors endocytosed via clathrin-mediated endocytosis activate Smad signalling, whereas receptors endocytosed via membrane rafts are targeted for degradation. The objective of the present study was to evaluate the contribution of TbetaRIII to TbetaRII and TbetaRI membrane partitioning, receptor half-life and signalling. Using sucrose-density ultracentrifugation to isolate membrane-raft fractions, we show that TbetaRIII recruits both TbetaRII and TbetaRI to non-raft membrane fractions. Immunofluorescence microscopy analysis demonstrated that overexpression of TbetaRIII affects intracellular trafficking of TbetaRII by recruiting TbetaRII to EEA1 (early endosome antigen 1)- and Rab5-positive early endosomes. Using 125I-labelled TGFbeta1 to follow cell-surface receptor degradation we show that overexpression of TbetaRIII also extends the receptor half-life of the TbetaRII-TbetaRI complex. Interestingly, we also show, using a luciferase reporter assay, that TbetaRIII increases basal TGFbeta signalling. As numerous pathologies show aberrant activation of TGFbeta signalling, the present study illustrates that TbetaRIII may represent a novel therapeutic target.

Cell-surface TGFbeta (transforming growth factor beta) receptors partition into membrane rafts and the caveolin-positive endocytic compartment by an unknown mechanism. In the present study, we investigated the determinant in the TGFbeta type II receptor (TbetaRII) that is necessary for membrane raft/caveolar targeting. Using subcellular fractionation and immunofluorescence microscopy techniques, we demonstrated that the extracellular domain of TbetaRII mediates receptor partitioning into raft and caveolin-positive membrane domains. Pharmacological perturbation of glycosylation using tunicamycin or the mutation of Mgat5 [mannosyl(alpha-1,6)-glycoprotein beta-1,6-N-acetylglucosaminyltransferase V] activity interfered with the raft partitioning of TbetaRII. However, this was not due to the glycosylation state of TbetaRII, as a non-glycosylated TbetaRII mutant remained enriched in membrane rafts. This suggested that other cell-surface glycoproteins associate with the extracellular domain of TbetaRII and direct their partitioning in membrane raft domains. To test this we analysed a GMCSF (granulocyte/macrophage colony-stimulating factor)-TbetaRII chimaeric receptor, which contains a glycosylated GMCSF extracellular domain fused to the transmembrane and intracellular domains of TbetaRII. This chimaeric receptor was found to be largely excluded from membrane rafts and caveolin-positive structures. Our results indicate that the extracellular domain of TbetaRII mediates receptor partitioning into membrane rafts and efficient entrance into caveolin-positive endosomes.

The anti-tumor synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO)-imidazolide (CDDO-Im) ectopically activates the transforming growth factor beta (TGFbeta)-Smad pathway and extends the duration of signaling by an undefined mechanism. Here we show that CDDO-Imdependent persistence of Smad2 phosphorylation is independent of Smad2 phosphatase activity and correlates with delayed TGFbeta receptor degradation and trafficking. Altered TGFbeta trafficking parallels the dispersal of EEA1-positive endosomes from the perinuclear region of CDDO-Im-treated cells. The effect of CDDO-Im on the EEA1 compartment led to an analysis of the cytoskeleton, and we observed that CDDO-Im alters microtubule dynamics by disrupting the microtubule-capping protein, Clip-170. Interestingly, biotinylated triterpenoid was found to localize to the polarity complex at the leading edge of migrating cells. Furthermore, CDDO-Im disrupted the localization of IQGAP1, PKCzeta, Par6, and TGFbeta receptors from the leading edge of migrating cells and inhibited TGFbeta-dependent cell migration. Thus, the synthetic triterpenoid CDDO-Im interferes with TGFbeta receptor trafficking and turnover and disrupts cell migration by severing the link between members of the polarity complex and the microtubule network.

The conjugation of ubiquitin to proteins involves a cascade of activating (E1), conjugating (E2), and ubiquitin-ligating (E3) type enzymes that commonly signal protein destruction. In TGFbeta signaling the inhibitory protein Smad7 recruits Smurf2, an E3 of the C2-WW-HECT domain class, to the TGFbeta receptor complex to facilitate receptor degradation. Here, we demonstrate that the amino-terminal domain (NTD) of Smad7 stimulates Smurf activity by recruiting the E2, UbcH7, to the HECT domain. A 2.1 A resolution X-ray crystal structure of the Smurf2 HECT domain reveals that it has a suboptimal E2 binding pocket that could be optimized by mutagenesis to generate a HECT domain that functions independently of Smad7 and potently inhibits TGFbeta signaling. Thus, E2 enzyme recognition by an E3 HECT enzyme is not constitutively competent and provides a point of control for regulating the ubiquitin ligase activity through the action of auxiliary proteins.

The Golgi enzyme β1,6 N-acetylglucosaminyltransferase V (Mgat5) is up-regulated in carcinomas and promotes the substitution of N-glycan with poly N-acetyllactosamine, the preferred ligand for galectin-3 (Gal-3). Here, we report that expression of Mgat5 sensitized mouse cells to multiple cytokines. Gal-3 cross-linked Mgat5-modified N-glycans on epidermal growth factor and transforming growth factor–β receptors at the cell surface and delayed their removal by constitutive endocytosis. Mgat5 expression in mammary carcinoma was rate limiting for cytokine signaling and consequently for epithelial-mesenchymal transition, cell motility, and tumor metastasis. Mgat5 also promoted cytokine-mediated leukocyte signaling, phagocytosis, and extravasation in vivo. Thus, conditional regulation of N-glycan processing drives synchronous modification of cytokine receptors, which balances their surface retention against loss via endocytosis.

Endocytosis of cell surface receptors is an important regulatory event in signal transduction. The transforming growth factor beta (TGF-beta) superfamily signals to the Smad pathway through heteromeric Ser-Thr kinase receptors that are rapidly internalized and then downregulated in a ubiquitin-dependent manner. Here we demonstrate that TGF-beta receptors internalize into both caveolin- and EEA1-positive vesicles and reside in both lipid raft and non-raft membrane domains. Clathrin-dependent internalization into the EEA1-positive endosome, where the Smad2 anchor SARA is enriched, promotes TGF-beta signalling. In contrast, the lipid raft-caveolar internalization pathway contains the Smad7-Smurf2 bound receptor and is required for rapid receptor turnover. Thus, segregation of TGF-beta receptors into distinct endocytic compartments regulates Smad activation and receptor turnover.

Lung fibrosis is often treated with corticosteroids to reduce the inflammatory response, however, no effective treatment options exist for the underlying disease. An important player in the fibrotic cascade is the cytokine, transforming growth factor beta (TGFβ). TGFβ is converted from an inactive procytokine complex to active TGFβ by enzymes such as thrombospondin-1 (TSP-1). It is therefore presumed that TSP-1 deficient mice would fare better to bleomycin-induced pulmonary fibrosis because TGFβ would not be efficiently converted to the active form. Interestingly, a recent article by Ezzie and colleagues shows that TSP-1 deficiency does not protect mice from systemic bleomycin challenge. Indeed, they find the opposite, as TSP-1-null mice appear to exhibit greater lung fibrosis than wild type mice, although similar TGFβ signaling was observed in the lungs of both mouse strains.

Receptor-mediated endocytosis and subsequent endosomal proteolysis of [125I]TyrA14-[HisA8,HisB4,GluB10,HisB27]insulin ([125I]TyrA14-H2 analogue), an insulin analogue exhibiting a high affinity for the insulin receptor, has been studied in liver parenchymal cells by quantitative subcellular fractionation and compared with that of wild-type [125I]TyrA14-insulin. Whereas the kinetics of uptake of the H2 analogue by liver was not different from that of insulin, the H2 analogue radioactivity after the 2 min peak declined significantly more slowly. A significant retention of the H2 analogue compared with insulin in both plasma membrane and endosomal fractions was observed and corresponded to decreased processing and dissociation of the H2 analogue. Cell-free endosomes preloaded in vivo with radiolabelled ligands and incubated in vitro processed insulin and extraluminally released insulin intermediates at a 2–3-fold higher rate than the H2 analogue. In vitro proteolysis of both non-radiolabelled and monoiodinated molecules by endosomal lysates showed a decreased response to the endosomal proteolytic machinery for the H2 analogue. However, in cross-linking and competition studies the H2 analogue exhibited an affinity for insulin-degrading enzyme identical with that of wild-type insulin. Brij-35-permeabilized endosomes revealed a 2-fold higher rate of dissociation of insulin from internalized receptors compared with the H2 analogue. After the administration of a saturating dose of both ligands, a rapid and reversible ligand-induced translocation of insulin receptor was observed, but without receptor loss. The H2 analogue induced a higher receptor concentration and tyrosine autophosphorylation of the receptor β subunit in endosomes. Moreover, a prolonged temporal interaction of the in vivo injected H2 analogue with receptor was observed by direct binding assays performed on freshly prepared subcellular fractions. These results indicate that endosomal proteolysis for the H2 analogue is slowed as a result of an increased residence time of the analogue on the insulin receptor and a low affinity of endosomal acidic insulinase for the dissociated H2 molecule.

Synthetic triterpenoids are a class of anti-tumor compounds that target various cellular functions including apoptosis, growth inhibition, anti-inflammation and cytoprotection. Previously, we have shown that triterpenoids also inhibit cell migration and the objective of our current study is to examine the molecular mechanisms. Since cellular adhesion dynamics play a critical role in cell migration, we investigated the effects of triterpenoids on focal adhesion morphology. Using immunofluorescence microscopic analysis of focal adhesion markers, paxillin and Focal Adhesion Kinase (FAK), we found that cells that were treated with synthetic triterpenoids possessed enlarged focal adhesions. Interestingly, numerous focal adhesion related proteins including IQGAP1, paxillin, FAK and Glycogen Synthase Kinase 3 (GSK3) were identified as drug-binding proteins using pull down and mass spectrometry approaches. Therefore, we investigated if triterpenoids target these proteins and affect cell adhesion turnover to inhibit cell migration. We found that focal adhesion re-establishment was altered by triterpenoids while pre-existing focal adhesions remained largely unaffected. To further explore the underlying mechanism of triterpenoids on GSK3β dependent cell adhesion dynamics, we analyzed GSK3β activity. We found that triterpenoids stimulate the phosphorylation of GSK3β at serine 9, however, both FAK and paxillin phosphorylation was largely unaffected by the triterpenoids. In parallel studies, we observed that focal adhesion staining was enlarged with cells that were treated with the GSK3β inhibitors, lithium chloride and SB216763. In addition, GSK3β inhibition affected the localization of not only GSK3β but also IQGAP1, an important scaffolding protein involved in cell polarity. From our results, we postulate that synthetic triterpenoids may affect cell migration by targeting GSK3β, which in turns, inhibits its activity and displaces its localization from the leading edge. More importantly, GSK3β inhibition by the triterpenoids and by GSK3β inhibitors also lead to the displacement of IQGAP1 from the leading edge, which further suggests that triterpenoids may act via a GSK3β mechanism to affect focal adhesions and cell migration. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 466. doi:1538-7445.AM2012-466

Transforming growth factor beta (TGFβ) regulates many important cellular processes including growth control and cell migration. In epithelial tumor cells, TGFβ signaling is fundamentally altered and induces epithelial to mesenchymal transition (EMT) leading to increased migratory potential. We have recently reported that the inhibition of atypical PKC isoforms (aPKC) extends TGFβ induced Smad signaling by altering receptor trafficking. Here we report that siRNA targeting of aPKC in A549 lung adenocarcinoma cells extends temporal P-Smad2 signaling but does not alter the transcript levels of Snail and Slug, as assessed by qPCR analysis. Interestingly, TGFβ was not observed to have as robust an increase in MMP9 and PAI-1 levels in aPKC-silenced cells compared to cells transfected with control siRNA. To further analyze the functional consequences, we assessed EMT by immunofluorescence microscopy analysis as well as immunoblotting for epithelial and mesenchymal cell markers. We found that TGFβ-dependent EMT was significantly reduced in aPKC silenced cells. We also uncovered that in addition to increased P-Smad2 signaling, P38 MAPK phosphorylation was increased and extended in aPKC silenced cells. Finally, we discovered that aPKC phosphorylates Par6 on a conserved serine residue that has been shown to regulate EMT and metastasis in breast cancer. Taken together, we believe that aPKC regulates several aspects of TGFβ signaling, including P-Smad2, P-P38 and P-Par6 levels. Consequently, convergence of these signals in aPKC silenced cells leads to reduced EMT and migratory potential. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 175. doi:1538-7445.AM2012-175

Transforming growth factor beta (TGFβ) is an essential cytokine for tissue homeostasis. Its signalling pathway is under intense study as it is commonly dysregulated in many cancers, including pancreatic cancer, non-small cell lung cancer, and colorectal cancer. The TGFβ signalling cascade is propagated by the binding of ligand to cell-surface serine-threonine kinase receptors, which leads to the phosphorylation of receptor regulated Smad (R-Smad) proteins. There are three main ligands capable of activating the classical TGFβ pathway: TGFβ1, TGFβ2 and TGFβ3. These ligands show distinct spatial and temporal expression patterns and have non-overlapping functions in vivo. Extensive studies have been conducted on the role of TGFβ1 in the tumour microenvironment, and the role of TGFβ3 has largely been thought to be the same as TGFβ1 though there are few studies to support this claim. Interestingly in the wound microenvironment, TGFβ1 and TGFβ3 have vastly different signalling outcomes: TGFβ1 promotes the formation of a scar while TGFβ3 induces scar-free wound resolution. The objective of the present study was to evaluate the signalling capacity of TGFβ1 and TGFβ3 in non-small cell lung cancer cells. Our studies show that TGFβ3 is less potent at initiating Smad2 phosphorylation than TGFβ1, resulting in reduced transcriptional activity, as assessed by microarray analysis. We also observed that TGFβ3 is less effective than TGFβ1 at altering cell-cell adhesions. In order to assess the differences in signalling potential, we investigated TGFβ receptor engagement at the cell surface using radiolabelled TGFβ ligands. We observed that the ratio of activated receptors in signalling complexes is altered in the presence of TGFβ3, which may lead to a decreased signalling capacity. Future studies will evaluate the capacity of TGFβ3 to induce cancer cell migration and invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3019. doi:1538-7445.AM2012-3019

Kisspeptins (Kp), peptide products of the Kisspeptin-1 (KISS1) gene are endogenous ligands for a G protein-coupled receptor 54 (GPR54). Previous findings have shown that KISS1 acts as a metastasis suppressor in numerous cancers in humans. However, recent studies have demonstrated that an increase in KISS1 and GPR54 expression in human breast tumors correlates with higher tumor grade and metastatic potential. At present, whether or not Kp signaling promotes breast cancer cell invasiveness, required for metastasis and the underlying mechanisms, is unknown. We have found that kisspeptin-10 (Kp-10), the most potent Kp, stimulates the invasion of human breast cancer MDA-MB-231 and Hs578T cells using Matrigel-coated Transwell chamber assays and induces the formation of invasive stellate structures in three-dimensional invasion assays. Furthermore, Kp-10 stimulated an increase in matrix metalloprotease (MMP)-9 activity. We also found that Kp-10 induced the transactivation of epidermal growth factor receptor (EGFR). Knockdown of the GPCR scaffolding protein, β-arrestin 2, inhibited Kp-10-induced EGFR transactivation as well as Kp-10 induced invasion of breast cancer cells via modulation of MMP-9 secretion and activity. Finally, we found that the two receptors associate with each other under basal conditions, and FRET analysis revealed that GPR54 interacts directly with EGFR. The stability of the receptor complex formation was increased upon treatment of cells by Kp-10. Taken together, our findings suggest a novel mechanism by which Kp signaling via GPR54 stimulates breast cancer cell invasiveness.