Expanding the Chemical Space of Transforming Growth Factor-ß (TGFß) Receptor Type II Degraders with 3,4-Disubstituted Indole Derivatives.

The TGFß type II receptor (TßRII) is a central player in all TGFß signaling downstream events, has been linked to cancer progression, and thus, has emerged as an auspicious anti-TGFß strategy. Especially its targeted degradation presents an excellent goal for effective TGFß pathway inhibition. Here, cellular structure-activity relationship (SAR) data from the TßRII degrader chemotype 1 was successfully transformed into predictive ligand-based pharmacophore models that allowed scaffold hopping. Two distinct 3,4-disubstituted indoles were identified from virtual screening: tetrahydro-4-oxo-indole 2 and indole-3-acetate 3. Design, synthesis, and screening of focused amide libraries confirmed 2r and 3n as potent TGFß inhibitors. They were validated to fully recapitulate the ability of 1 to selectively degrade TßRII, without affecting TßRI. Consequently, 2r and 3n efficiently blocked endothelial-to-mesenchymal transition and cell migration in different cancer cell lines while not perturbing the microtubule network. Hence, 2 and 3 present novel TßRII degrader chemotypes that will (1) aid target deconvolution efforts and (2) accelerate proof-of-concept studies for small-molecule-driven TßRII degradation in vivo.

Acetylenic tricyclic bis-(cyano enone) interacts with Cys 374 of actin, a residue necessary for stress fiber formation and cell migration.

The migratory and invasive potential of tumour cells relies on the actin cytoskeleton. We previously demonstrated that the tricyclic compound, TBE-31, inhibits actin polymerization and here we further examine the precise interaction between TBE-31 and actin. We demonstrate that iodoacetamide, a cysteine (Cys) alkylating agent, interferes with the ability of TBE-31 to interact with actin. In addition, in silico analysis identified Cys 217, Cys 272, Cys 285 and Cys 374 as potential binding sites for TBE-31. Using mass spectrometry analysis, we determined that TBE-31 associates with actin with a stoichiometric ratio of 1:1. We mutated the identified cysteines of actin to alanine and performed a pull-down analysis with a biotin labeled TBE-31 and demonstrated that by mutating Cys 374 to alanine the association between TBE-31 and actin was significantly reduced, suggesting that TBE-31 binds to Cys 374. A characterization of the NIH3T3 cells overexpressing eGFP-actin-C374A showed reduced stress fiber formation, suggesting Cys 374 is necessary for efficient incorporation into filamentous actin. Furthermore, migration of eGFP-Actin-WT expressing cells were observed to be inhibited by TBE-31, however fewer eGFP-Actin-C374A expressing cells were observed to migrate compared to the cells expressing eGFP-Actin-WT in the presence or absence of TBE-31. Taken together, our results suggest that TBE-31 binds to Cys 374 of actin to inhibit actin stress fiber formation and may potentially be a mechanism through which TBE-31 inhibits cell migration.

Transforming growth factor-ß in tumour development.

Transforming growth factor-ß (TGFß) is a ubiquitous cytokine essential for embryonic development and postnatal tissue homeostasis. TGFß signalling regulates several biological processes including cell growth, proliferation, apoptosis, immune function, and tissue repair following injury. Aberrant TGFß signalling has been implicated in tumour progression and metastasis. Tumour cells, in conjunction with their microenvironment, may augment tumourigenesis using TGFß to induce epithelial-mesenchymal transition, angiogenesis, lymphangiogenesis, immune suppression, and autophagy. Therapies that target TGFß synthesis, TGFß-TGFß receptor complexes or TGFß receptor kinase activity have proven successful in tissue culture and in animal models, yet, due to limited understanding of TGFß biology, the outcomes of clinical trials are poor. Here, we review TGFß signalling pathways, the biology of TGFß during tumourigenesis, and how protein quality control pathways contribute to the tumour-promoting outcomes of TGFß signalling.

Prolonged proteasome inhibition antagonizes TGFß1-dependent signalling by promoting the lysosomal-targeting of TGFß receptors.

Impairing autophagy disrupts transforming growth factor beta 1 (TGFß1) 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 TGFß1-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 TGFß-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 TGFß receptor type II (TGFßRII) levels. However, prolonged (8-24 h) proteasome inhibition decreased TGFß1-dependent R-Smad phosphorylation and steady-state TGFßRI and TGFßRII levels. Furthermore, proteasome inhibition blunted TGFß-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 TGFßRII endocytosis and trafficking to lysosomes and we conclude that prolonged proteasome inhibition disrupts TGFß signalling outcomes through altered TGFß receptor trafficking.

Autophagy regulates transforming growth factor ß signaling and receptor trafficking.

Transforming growth factor beta (TGFß) stimulates tumorigenesis by inducing epithelial to mesenchymal transition (EMT) and cell migration. TGFß 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 TGFß 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 TGFß1-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 TGFß receptors and their trafficking to early/late endosomal and lysosomal compartments. The effects of autophagy inhibition on TGFß 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 TGFß signaling by regulating receptor trafficking, resulting in impaired Smad2/Smad3 phosphorylation and nuclear accumulation.

Canonical and Non-canonical TGFß Signaling Activate Autophagy in an ULK1-Dependent Manner.

The mechanism(s) in which transforming growth factor beta 1 (TGFß) modulates autophagy in cancer remain unclear. Here, we characterized the TGFß signaling pathways that induce autophagy in non-small cell lung cancer cells, using cells lines stably expressing GFP-LC3-RFP-LC3ΔG constructs that measure autophagic flux. We demonstrated that TGFß1 increases Unc 51-like kinase 1 (ULK1) protein levels, 5' adenosine monophosphate-activated protein kinase (AMPK)-dependent ULK1 phosphorylation at serine (S) 555 and ULK1 complex formation but decreases mechanistic target of rapamycin (mTOR) activity on ULK1. Further analysis revealed that the canonical Smad4 pathway and the non-canonical TGFß activated kinase 1/tumor necrosis factor receptor-associated factor 6/P38 mitogen activated protein kinase (TAK1-TRAF6-P38 MAPK) pathway are important for TGFß1-induced autophagy. The TAK1-TRAF6-P38 MAPK pathway was essential for downregulating mTOR S2448 phosphorylation, ULK1 S555 phosphorylation and autophagosome formation. Furthermore, although siRNA-mediated Smad4 silencing did not alter mTOR-dependent ULK1 S757 phosphorylation, it did reduce AMPK-dependent ULK1 S555 phosphorylation and autophagosome formation. Additionally, Smad4 silencing and inhibiting the TAK1-TRAF6-P38 MAPK pathway decreased autophagosome-lysosome co-localization in the presence of TGFß. Our results suggest that the Smad4 and TAK1-TRAF6-P38 MAPK signaling pathways are essential for TGFß-induced autophagy and provide specific targets for the inhibition of TGFß in tumor cells that utilize autophagy in their epithelial-mesenchymal transition program.

Molecular mechanisms of mammalian autophagy.

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.

p62/Sequestosome 1 regulates transforming growth factor beta signaling and epithelial to mesenchymal transition in A549 cells.

Transforming growth factor beta (TGFß) receptor trafficking regulates many TGFß-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 TGFß receptor trafficking, TGFß1-dependent Smad2 phosphorylation and EMT in A549 NSCLC cells. Using immunofluorescence microscopy, p62/SQSTM1 was observed to co-localize with TGFß 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 TGFß1-dependent EMT marker expression, actin stress fiber formation and A549 cell migration. We further observed that Smad4-independent TGFß1 signaling decreased p62/SQSTM1 protein levels via a proteasome-dependent mechanism. Although p62/SQSTM1 silencing did not impede TGFß-dependent autophagy, our results suggest that p62/SQSTM1 may aid in maintaining A549 cells in an epithelial state and TGFß1 decreases p62/SQSTM1 prior to inducing EMT and autophagy.

Assessing methods to quantitatively validate TGFß-dependent autophagy.

Transforming growth factor beta (TGFß) promotes tumorigenesis by suppressing immune surveillance and inducing epithelial to mesenchymal transition (EMT). TGFß may augment tumorigenesis by activating autophagy, which protects cancer cells from chemotherapy and promotes invasive and anti-apoptotic properties. Here, we assess how TGFß1 modulates autophagy related (ATG) gene expression and ATG protein levels. We also assessed microtubule-associated protein light chain 3 (LC3) lipidation, LC3 puncta formation and autophagosome-lysosome co-localization in non-small cell lung cancer (NSCLC) cell lines. These experimental approaches were validated using pharmacological autophagy inhibitors (chloroquine and spautin-1) and an autophagy activator (MG132). We found that TGFß1, chloroquine and MG132 had little effect on ATG protein levels but increased LC3 lipidation, LC3 puncta formation and autophagosome-lysosome co-localization. Since similar outcomes were observed using chloroquine and MG132, we concluded that several techniques employed to assess TGFß-dependent autophagy may not differentiate between the activation of autophagy versus lysosomal inhibition. Thus, NSCLC cell lines stably expressing a GFP-LC3-RFP-LC3ΔG autophagic flux probe were used to assess TGFß-mediated autophagy. Using this approach, we observed that TGFß, MG132 and serum starvation increased autophagic flux, whereas chloroquine and spautin-1 decreased autophagic flux. Finally, we demonstrated that ATG5 and ATG7 are critical for TGFß-dependent autophagy in NSCLC cells. The application of this model will fuel future experiments to characterize TGFß-dependent autophagy, which is necessary to understand the molecular processes that link, TGFß, autophagy and tumorigenesis.

SMURF2 and SMAD7 induce SARA degradation via the proteasome.

TGFß-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 TGFß 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 TGFß 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 TGFß-dependent Smad2 membrane recruitment and phosphorylation, as assessed by subcellular fractionation and western blotting. Furthermore, siRNA targeting SARA decreased TGFß-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 TGFß signalling is regulated by interplay between SARA and Smurf2/Smad7 complexes.

The synthetic oleanane triterpenoid CDDO-Me binds and inhibits pyruvate kinase M2.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Mechanisms of neuroprotection against ischemic insult by stress-inducible phosphoprotein-1/prion protein complex.

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 α7 nicotinic acetylcholine receptors (α7nAChR). Given that α7nAChR 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 α7nAChR, 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 α7nAChR as well as the ALK2 receptor may be activated in neurons by increased levels of STI1. This signaling pathway protects neurons from ischemic insults.

Synthetic triterpenoids inhibit GSK3ß activity and localization and affect focal adhesions 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 (GSK3ß), a kinase that regulates many cellular processes, including cell adhesion dynamics, is a triterpenoid-binding protein. Furthermore, triterpenoids were observed to inhibit GSK3ß activity and increase cellular focal adhesion size. To further examine whether these effects on focal adhesions in triterpenoid-treated cells were GSK3ß-dependent, GSK3ß inhibitors (lithium chloride and SB216763) were used to examine cell adhesion and morphology as well as cell migration. Our results showed that GSK3ß 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 GSK3ß.

Spatial and Temporal Regulation of Receptor Tyrosine Kinase Activation and Intracellular Signal Transduction.

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 acetylenic tricyclic bis(cyano enone), TBE-31, targets microtubule dynamics and cell polarity in migrating cells.

Cell migration is dependent on the microtubule network for structural support as well as for the proper delivery and positioning of polarity proteins at the leading edge of migrating cells. Identification of drugs that target cytoskeletal-dependent cell migration and protein transport in polarized migrating cells is important in understanding the cell biology of normal and tumor cells and can lead to new therapeutic targets in disease processes. Here, we show that the tricyclic compound TBE-31 directly binds to tubulin and interferes with microtubule dynamics, as assessed by end binding 1 (EB1) live cell imaging. Interestingly, this interference is independent of in vitro tubulin polymerization. Using immunofluorescence microscopy, we also observed that TBE-31 interferes with the polarity of migratory cells. The polarity proteins Rac1, IQGAP and Tiam1 were localized at the leading edge of DMSO-treated migrating cell, but were observed to be in multiple protrusions around the cell periphery of TBE-31-treated cells. Finally, we observed that TBE-31 inhibits the migration of Rat2 fibroblasts with an IC50 of 0.75 µM. Taken together, our results suggest that the inhibition of cell migration by TBE-31 may result from the improper maintenance of cell polarity of migrating cells.

Modulation of type II TGF-ß receptor degradation by integrin-linked kinase.

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

aPKC alters the TGFß response in NSCLC cells through both Smad-dependent and Smad-independent pathways.

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 SARA­Smad2­Smad4 association and observed that knockdown of aPKC increased SARA (also known as ZFYVE9) levels and SARA­Smad2 complex formation, increased cytoplasmic retention of Smad2 and reduced Smad2­Smad4 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 acetylenic tricyclic bis(cyano enone), TBE-31 inhibits non-small cell lung cancer cell migration through direct binding with actin.

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 TGFß-dependent EMT. Interestingly, TBE-31 does not interfere with TGFß-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.

TGFß in endosomal signaling.

The transforming growth factor beta (TGFß) 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 TGFß 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 TGFß 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 TGFß receptor trafficking to signaling outcome, this chapter will focus on strategies to isolate membrane rafts and techniques to follow the trafficking of cell-surface TGFß receptors and provide examples of functional readouts to assess TGFß signal transduction.

Par6 is phosphorylated by aPKC to facilitate EMT.

The conserved polarity proteins Par6 and aPKC regulate cell polarization processes. However, increasing evidence also suggests that they play a role in oncogenic progression. During tumor progression, epithelial to mesenchymal transition (EMT) delineates an evolutionary conserved process that converts stationary epithelial cells into mesenchymal cells, which have an acquired ability for independent migration and invasion. In addition to signaling pathways that alter genetic programes that trigger the loss of cell-cell adhesion, alternative pathways can alter cell plasticity to regulate cell-cell cohesion and increase invasive potential. One such pathway involves TGFß-induced phosphorylation of Par6. In epithelial cells, Par6 phosphorylation results in the dissolution of junctional complexes, cytoskeletal remodelling, and increased metastatic potential. Recently, we found that aPKC can also phosphorylate Par6 to drive EMT and increase the migratory potential of non-small cell lung cancer cells. This result has implications with respect to homeostatic and developmental processes involving polarization, and also with respect to cancer progression-particularly since aPKC has been reported to be an oncogenic regulator in various tumor cells.