Activation of the GTPase ARF6 regulates invasion of human vascular smooth muscle cells by stimulating MMP14 activity.

Hormones and growth factors stimulate vascular smooth muscle cells (VSMC) invasive capacities during the progression of atherosclerosis. The GTPase ARF6 is an important regulator of migration and proliferation of various cell types, but whether this small G protein can be activated by a variety of stimuli to promote invasion of VSMC remains unknown. Here, we aimed to define whether Platelet-derived growth factor (PDGF), a mitogenic stimulant of vascular tissues, and Angiotensin II (Ang II), a potent vasoactive peptide, can result in the activation of ARF6 in a human model of aortic SMC (HASMC). We demonstrate that these two stimuli can promote loading of GTP on this ARF isoform. Knockdown of ARF6 reduced the ability of both PDGF and Ang II to promote invasion suggesting that this GTPase regulates key molecular mechanisms mediating degradation of the extracellular matrix and migration. We report that PDGF-BB-mediated stimulation of ARF6 results in the activation of the MAPK/ERK1/2, PI3K/AKT and PAK pathways essential for invasion of HASMC. However, Ang II-mediated stimulation of ARF6 only promotes signaling through the MAPK/ERK1/2 and PAK pathways. These ARF6-mediated events lead to activation of MMP14, a membrane-bound collagenase upregulated in atherosclerosis. Moreover, ARF6 depletion decreases the release of MMP2 in the extracellular milieu. Altogether, our findings demonstrate that the GTPase ARF6 acts as a molecular switch to regulate specific signaling pathways that coordinate invasiveness of HASMC.

Discovery of a dual Ras and ARF6 inhibitor from a GPCR endocytosis screen.

Internalization and intracellular trafficking of G protein-coupled receptors (GPCRs) play pivotal roles in cell responsiveness. Dysregulation in receptor trafficking can lead to aberrant signaling and cell behavior. Here, using an endosomal BRET-based assay in a high-throughput screen with the prototypical GPCR angiotensin II type 1 receptor (AT1R), we sought to identify receptor trafficking inhibitors from a library of ~115,000 small molecules. We identified a novel dual Ras and ARF6 inhibitor, which we named Rasarfin, that blocks agonist-mediated internalization of AT1R and other GPCRs. Rasarfin also potently inhibits agonist-induced ERK1/2 signaling by GPCRs, and MAPK and Akt signaling by EGFR, as well as prevents cancer cell proliferation. In silico modeling and in vitro studies reveal a unique binding modality of Rasarfin within the SOS-binding domain of Ras. Our findings unveil a class of dual small G protein inhibitors for receptor trafficking and signaling, useful for the inhibition of oncogenic cellular responses.

Surface-Enhanced Raman Scattering Optophysiology Nanofibers for the Detection of Heavy Metals in Single Breast Cancer Cells.

Mercury(II) ions (Hg2+) and silver ions (Ag+) are two of the most hazardous pollutants causing serious damage to human health. Here, we constructed surface-enhanced Raman scattering (SERS)-active nanofibers covered with 4-mercaptopyridine (4-Mpy)-modified gold nanoparticles to detect Hg2+ and Ag+. Experimental evidence suggests that the observed spectral changes originate from the combined effect of (i) the coordination between the nitrogen on 4-Mpy and the metal ions and (ii) the 4-Mpy molecular orientation (from flatter to more perpendicular with respect to the metal surface). The relative intensity of a pair of characteristic Raman peaks (at ∼428 and ∼708 cm-1) was used to quantify the metal ion concentration, greatly increasing the reproducibility of the measurement compared to signal-on or signal-off detection based on a single SERS peak. The detection limit of this method for Hg2+ is lower than that for the Ag+ (5 vs 100 nM), which can be explained by the stronger interaction energy between Hg2+ and N compared to Ag+ and N, as demonstrated by density functional theory calculations. The Hg2+ and Ag+ ions can be masked by adding ethylenediaminetetraacetate and Cl-, respectively, to the Hg2+ and Ag+ samples. The good sensitivity, high reproducibility, and excellent selectivity of these nanosensors were also demonstrated. Furthermore, detection of Hg2+ in living breast cancer cells at the subcellular level is possible, thanks to the nanometric size of the herein described SERS nanosensors, allowing high spatial resolution and minimal cell damage.

Branched Au Nanoparticles on Nanofibers for Surface-Enhanced Raman Scattering Sensing of Intracellular pH and Extracellular pH Gradients.

The development of plasmonic-active nanosensors for surface-enhanced Raman scattering (SERS) sensing is important for gaining knowledge on intracellular and extracellular chemical processes, hypoxia detection, and label-free detection of neurotransmitters and metabolites, among other applications in cell biology. The fabrication of SERS nanosensors for optophysiology measurements using substrates such as nanofibers with a uniform distribution of plasmonic nanoparticles (NPs) remains a critical hurdle. We report here on a strategy using block copolymer brush-layer templating and ligand exchange for fabricating highly reproducible and stable SERS-active nanofibers with tip diameters down to 60 nm and covered with well-dispersed and uniformly distributed branched AuNPs, which have intrinsic hotspots favoring inherently high plasmonic sensitivity. Among the SERS sensors investigated, those with Au nanostars with short branches [AuNS(S)s] exhibit the greatest SERS sensitivity, as verified also by COMSOL Multiphysics simulations. Functionalization of the AuNS(S)s with the pH-sensitive molecule, 4-mercaptobenzoic acid, led to SERS nanosensors capable of quantifying pH over a linear range of 6.5-9.5, covering the physiological range. These pH nanosensors were shown to be able to detect the intracellular pH as well as extracellular pH gradients of in vitro breast cancer cells with minimal invasiveness and improved SERS sensitivity, along with a high spatial resolution capability.

Methods to Investigate the ß-Arrestin-Mediated Control of ARF6 Activation to Regulate Trafficking and Actin Cytoskeleton Remodeling.

ADP-ribosylation factors (ARF) are GTPases that act to control the activation of numerous signaling events and cellular responses. The ARF6 isoform, present at the plasma membrane, can be activated by the angiotensin II type 1 receptor (AT1R), a process dependent upon ß-arrestin recruitment to the activated receptor. Here, we describe classical methods used to assess ß-arrestin-dependent activation of ARF6 following agonist stimulation of cells. In addition, because ARF6 and ß-arrestin can form a complex, we describe the procedures used to detect the interaction of ß-arrestin with this GTPase.

Neurons and Microglia; A Sickly-Sweet Duo in Diabetic Pain Neuropathy.

Diabetes is a common condition characterized by persistent hyperglycemia. High blood sugar primarily affects cells that have a limited capacity to regulate their glucose intake. These cells include capillary endothelial cells in the retina, mesangial cells in the renal glomerulus, Schwann cells, and neurons of the peripheral and central nervous systems. As a result, hyperglycemia leads to largely intractable complications such as retinopathy, nephropathy, hypertension, and neuropathy. Diabetic pain neuropathy is a complex and multifactorial disease that has been associated with poor glycemic control, longer diabetes duration, hypertension, advanced age, smoking status, hypoinsulinemia, and dyslipidemia. While many of the driving factors involved in diabetic pain are still being investigated, they can be broadly classified as either neuron -intrinsic or -extrinsic. In neurons, hyperglycemia impairs the polyol pathway, leading to an overproduction of reactive oxygen species and reactive nitrogen species, an enhanced formation of advanced glycation end products, and a disruption in Na+/K+ ATPase pump function. In terms of the extrinsic pathway, hyperglycemia leads to the generation of both overactive microglia and microangiopathy. The former incites a feed-forward inflammatory loop that hypersensitizes nociceptor neurons, as observed at the onset of diabetic pain neuropathy. The latter reduces neurons' access to oxygen, glucose and nutrients, prompting reductions in nociceptor terminal expression and losses in sensation, as observed in the later stages of diabetic pain neuropathy. Overall, microglia can be seen as potent and long-lasting amplifiers of nociceptor neuron activity, and may therefore constitute a potential therapeutic target in the treatment of diabetic pain neuropathy.

ARF6 protects sister chromatid cohesion to ensure the formation of stable kinetochore-microtubule attachments.

Sister chromatid cohesion, facilitated by the cohesin protein complex, is crucial for the establishment of stable bipolar attachments of chromosomes to the spindle microtubules and their faithful segregation. Here, we demonstrate that the GTPase ARF6 prevents the premature loss of sister chromatid cohesion. During mitosis, ARF6-depleted cells normally completed chromosome congression. However, at the metaphase plate, chromosomes failed to establish stable kinetochore-microtubule attachments because of the impaired cohesion at centromeres. As a result, the spindle assembly checkpoint (SAC) was active and cyclin B ubiquitylation and degradation were blocked. Chromosomes and/or chromatids in these cells scattered gradually from the metaphase plate to the two poles of the cell or remained blocked at the metaphase plate for hours. Our study demonstrates that the small GTP-binding protein ARF6 is essential for maintaining centromeric cohesion between sister chromatids, which is necessary for the establishment of stable k-fibres, SAC satisfaction and the onset of anaphase.

ARF GTPases control phenotypic switching of vascular smooth muscle cells through the regulation of actin function and actin dependent gene expression.

Vascular smooth muscle cells (VSMC) can exhibit a contractile or a synthetic phenotype depending on the extracellular stimuli present and the composition of the extracellular matrix. Uncontrolled activation of the synthetic VSMC phenotype is however associated with the development of cardiovascular diseases. Here, we aimed to elucidate the role of the ARF GTPases in the regulation of VSMC dedifferentiation. First, we observed that the inhibition of the activation of ARF proteins with SecinH3, a blocker of the cytohesin ARF GEF family, reduced the ability of the cells to migrate and proliferate. In addition, this inhibitor also blocked expression of sm22α and αSMA, two contractile markers, at the transcription level impairing cell contractility. Specific knockdown of ARF1 and ARF6 showed that both isoforms were required for migration and proliferation, but ARF1 only regulated contractility through sm22α and αSMA expression. Expression of these VSMC markers was correlated with the degree of actin polymerization. VSMC treatment with SecinH3 as well as ARF1 depletion was both able to block the formation of stress fibres and focal adhesions, demonstrating the role of this GTPase in actin filament formation. Consequently, we observed that both treatments increased the ratio of G-actin to F-actin in these cells. The elevated amounts of cytoplasmic G-actin, acting as a signaling intermediate, blocked the recruitment of the Mkl1 (MRTF-A) transcription factor in the nucleus, demonstrating its involvement in the regulation of contractile protein expression. Altogether, these findings show for the first time that ARF GTPases are actively involved in VSMC phenotypic switching through the regulation of actin function in migration and proliferation, and the control of actin dependent gene regulation.

Arterial stiffness induced by carotid calcification leads to cerebral gliosis mediated by oxidative stress.

BACKGROUND: Arterial stiffness is a risk factor for cognitive decline and dementia. However, its precise effects on the brain remain unexplored. Using a mouse model of carotid stiffness, we investigated its effect on glial activation and oxidative stress. METHODS: Arterial stiffness was induced by the application of calcium chloride to the adventitial region of the right carotid. Superoxide anion production, NADPH activity and levels, as well as glial activation were examined with immunohistochemical and biochemical approaches, 2-week postcalcification. Antioxidant treatment was done with Tempol (1 mmol/l) administered in the drinking water during 2 weeks. RESULTS: The current study revealed that arterial stiffness increases the levels of the microglial markers ionized calcium-binding adapter molecule 1 and cluster of differentiation 68 in hippocampus, and of the astrocyte marker, s100 calcium binding protein ß in hippocampus and frontal cortex. The cerebral inflammatory effects of arterial stiffness were specific to the brain and not due to systemic inflammation. Treatment with Tempol prevented the increase in superoxide anion in mice with carotid stiffness and attenuated the activation of microglia and astrocytes in the hippocampus. To determine whether the increased oxidative stress derives from NADPH oxidase, superoxide anion production was assessed by incubating brain tissue in the presence of gp91ds-tat, a selective NADPH oxidase 2 inhibitor. This peptide inhibited superoxide anion production to a greater extent in the brains of mice with carotid calcification compared with controls. CONCLUSION: Carotid calcification leads to cerebral gliosis mediated by oxidative stress. Correcting arterial stiffness could offer a novel paradigm to protect the brain in populations where stiffness is prominent.

Correction: Ultra-low fouling alkylimidazolium modified surfaces for the detection of HER2 in breast cancer cell lysates.

Correction for 'Ultra-low fouling methylimidazolium modified surfaces for the detection of HER2 in breast cancer cell lysates' by Alexandra Aubé et al., Analyst, 2017, 142, 2343-2353.

Ultra-low fouling alkylimidazolium modified surfaces for the detection of HER2 in breast cancer cell lysates [corrected].

We synthesized novel ultra-low fouling ionic liquids and demonstrated their use with surface plasmon resonance (SPR) sensing for the analysis of HER2 in breast cancer cell lysates. Whilst biomarkers are commonly detected in serum, this remains challenging for cancer diagnosis due to their low concentrations in circulation and in some cases, there is a poor correlation between serum and tissue concentrations. Therefore, a cell lysate constitutes an interesting biosample for cancer diagnosis and typing, which has been largely unexploited for chemical biosensing of cancer biomarkers. However, high fouling of surfaces in contact with the cell lysate and the absence of effective surface chemistry to prevent fouling are currently limiting biomarker analysis in cell lysates. To address this challenge, we report the synthesis of 1-(carboxyalkyl)-3-(12-mercaptododecyl)-1H-imidazolium ionic liquids with different anions (Br-, BF4-, PF6-, ClO4-, and NTf2-) and ethyl and pentyl chains to form monolayers and analyse specific proteins from cell lysates. The most efficient ionic liquid monolayer, 1-(carboxyethyl)-3-(12-mercaptododecyl)-1H-imidazolium bromide, was able to eliminate the nonspecific adsorption (surface coverage of 2 ± 2 ng cm-2) of a concentrated cell lysate (protein concentration of ∼3.5 mg mL-1), which was significantly better than carboxy-PEG (surface coverage of 14 ± 7 ng cm-2), a benchmark monolayer commonly used to reduce nonspecific adsorption. These ionic liquid monolayers were modified with anti-HER2 and the detection of the HER2 breast cancer biomarker was carried out in crude breast cancer cell lysates, as shown with HER2-negative MCF-7 cells spiked with HER2 and with HER2 positive SK-BR-3 cells.

Application of constrained aza-valine analogs for Smac mimicry.

Constrained azapeptides were designed based on the Ala-Val-Pro-Ile sequence from the second mitochondria-derived activator of caspases (Smac) protein and tested for ability to induce apoptosis in cancer cells. Diels-Alder cyclizations and Alder-ene reactions on azopeptides enabled construction of a set of constrained aza-valine dipeptide building blocks, that were introduced into mimics using effective coupling conditions to acylate bulky semicarbazide residues. Evaluation of azapeptides 7-11 in MCF-7 breast cancer cells indicated aza-cyclohexanylglycyine analog 11 induced cell death more efficiently than the parent tetrapeptide likely by a caspase-9 mediated apoptotic pathway. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 235-244, 2016.

ADP-ribosylation factor 1 expression regulates epithelial-mesenchymal transition and predicts poor clinical outcome in triple-negative breast cancer.

Metastatic capacities are fundamental features of tumor malignancy. ADP-ribosylation factor (ARF) 1 has emerged as a key regulator of invasion in breast cancer cells. However, the importance of this GTPase, in vivo, remains to be demonstrated. We report that ARF1 is highly expressed in breast tumors of the most aggressive and advanced subtypes. Furthermore, we show that lowered expression of ARF1 impairs growth of primary tumors and inhibits lung metastasis in a murine xenograft model. To understand how ARF1 contributes to invasiveness, we used a poorly invasive breast cancer cell line, MCF7 (ER+), and examined the effects of overexpressing ARF1 to levels similar to that found in invasive cell lines. We demonstrate that ARF1 overexpression leads to the epithelial-mesenchymal transition (EMT). Mechanistically, ARF1 controls cell-cell adhesion through ß-catenin and E-cadherin, oncogenic Ras activation and expression of EMT inducers. We further show that ARF1 overexpression enhances invasion, proliferation and resistance to a chemotherapeutic agent. In vivo, ARF1 overexpressing MCF7 cells are able to form more metastases to the lung. Overall, our findings demonstrate that ARF1 is a molecular switch for cancer progression and thus suggest that limiting the expression/activation of this GTPase could help improve outcome for breast cancer patients.

The GTPase ARF6 Controls ROS Production to Mediate Angiotensin II-Induced Vascular Smooth Muscle Cell Proliferation.

High reactive oxygen species (ROS) levels and enhanced vascular smooth muscle cells (VSMC) proliferation are observed in numerous cardiovascular diseases. The mechanisms by which hormones such as angiotensin II (Ang II) acts to promote these cellular responses remain poorly understood. We have previously shown that the ADP-ribosylation factor 6 (ARF6), a molecular switch that coordinates intracellular signaling events can be activated by the Ang II receptor (AT1R). Whether this small GTP-binding protein controls the signaling events leading to ROS production and therefore Ang II-dependent VSMC proliferation, remains however unknown. Here, we demonstrate that in rat aortic VSMC, Ang II stimulation led to the subsequent activation of ARF6 and Rac1, a key regulator of NADPH oxidase activity. Using RNA interference, we showed that ARF6 is essential for ROS generation since in conditions where this GTPase was knocked down, Ang II could no longer promote superoxide anion production. In addition to regulating Rac1 activity, ARF6 also controlled expression of the NADPH oxidase 1 (Nox 1) as well as the ability of the EGFR to become transactivated. Finally, ARF6 also controlled MAPK (Erk1/2, p38 and Jnk) activation, a key pathway of VSMC proliferation. Altogether, our findings demonstrate that Ang II promotes activation of ARF6 to controls ROS production by regulating Rac1 activation and Nox1 expression. In turn, increased ROS acts to activate the MAPK pathway. These signaling events represent a new molecular mechanism by which Ang II can promote proliferation of VSMC.

ß-Arrestin-mediated Angiotensin II Signaling Controls the Activation of ARF6 Protein and Endocytosis in Migration of Vascular Smooth Muscle Cells.

Angiotensin II (Ang II) is a vasopressive hormone but is also a potent activator of cellular migration. We have previously shown that it can promote the activation of the GTPase ARF6 in a heterologous overexpressing system. The molecular mechanisms by which receptors control the activation of this small G protein remain, however, largely unknown. Furthermore, how ARF6 coordinates the activation of complex cellular responses needs to be further elucidated. In this study, we demonstrate that Ang II receptors engage ß-arrestin, but not Gq, to mediate ARF6 activation in HEK 293 cells. To further confirm the key role of ß-arrestin proteins, we overexpressed ß-arrestin2-(1-320), a dominant negative mutant known to block receptor endocytosis. We show that expression of this truncated construct does not support the activation of the GTPase nor cell migration. Interestingly, ß-arrestin2 can interact with the ARF guanine nucleotide exchange factor ARNO, although the C-terminally lacking mutant does not. We finally examined whether receptor endocytosis controlled ARF6 activation and cell migration. Although the clathrin inhibitor PitStop2 did not impact the ability of Ang II to activate ARF6, cell migration was markedly impaired. To further show that ARF activation regulates key signaling events leading to migration, we also examined MAPK activation. We demonstrate that this signaling axis is relevant in smooth muscle cells of the vasculature. Altogether, our findings show for the first time that Ang II receptor signaling to ß-arrestin regulates ARF6 activation. These proteins together control receptor endocytosis and ultimately cell migration.

The small GTPase ADP-Ribosylation Factor 1 mediates the sensitivity of triple negative breast cancer cells to EGFR tyrosine kinase inhibitors.

The clinical use of EGFR-targeted therapy, in triple negative breast cancer patients, has been limited by the development of resistance to these drugs. Although activated signaling molecules contribute to this process, the molecular mechanisms remain relatively unknown. We have previously reported that the small GTPase ADP-Ribosylation Factor 1 (ARF1) is highly expressed in invasive breast cancer cells and acts as a molecular switch to activate EGF-mediated responses. In this study, we aimed at defining whether the high expression of ARF1 limits sensitivity of these tumor cells to EGFR inhibitors, such as gefitinib. Here, we show that the knock down of ARF1 expression or activity decreased the dose and latency time required by tyrosine kinase inhibitors to induce cell death. This may be explained by the observation that the depletion of ARF1 suppressed gefitinib-mediated activation of key mediators of survival such as ERK1/2, AKT and Src, while enhancing cascades leading to apoptosis such as the p38MAPK and JNK pathways, modifying the Bax/Bcl2 ratio and cytochrome c release. In addition, inhibiting ARF1 expression and activation also results in an increase in gefitinib-mediated EGFR internalization and degradation further limiting the ability of this receptor to promote its effects. Interestingly, we observed that gefitinib treatment resulted in the enhanced activation of ARF1 by promoting its recruitment to the receptor AXL, an important mediator of EGFR inhibition suggesting that ARF1 may promote its pro-survival effects by coupling to alternative mitogenic receptors in conditions where the EGFR is inhibited. Together our results uncover a new role for ARF1 in mediating the sensitivity to EGFR inhibition and thus suggest that limiting the activation of this GTPase could improve the therapeutic efficacy of EGFR inhibitors.

Investigation of the active turn geometry for the labour delaying activity of indolizidinone and azapeptide modulators of the prostaglandin F2α receptor.

On pursuing molecules that delay labour, so-called tocolytics, the prostaglandin F2α receptor (FP) was targeted, because of its role in the stimulation of uterine contractions leading to birth and preterm birth. Previously, both the indolizidinone PDC-113.824 (5) and the aza-glycinyl-proline analog 6 were shown to delay labour in mice by modulating the FP function, likely by an allosteric mechanism, which features biased signalling. The crystal structure and computational analyses of the indolizidin-2-one amino acid and aza-glycinyl-proline components of 5 and 6 in model peptides have shown them to adopt a geometry that mimics ideal type I and II'ß-turns. To elucidate the precise turn geometry for receptor recognition, analogs 1-4 have now been synthesized: macrocycle and pyrroloazepinone mimics 1 and 2 to mimic type I, and glycinyl-proline and d-alaninyl-proline analogs 3 and 4 to favour type II'ß-turn geometry. Notably, transannular cyclization of peptide macrocycle 13 has provided diastereoselectively pyrroloazepinone 15 by a novel route that provides effective access to mimics 1 and 2 by way of a common intermediate. Among the four analogs, none exhibited efficacy nor potency on par with 5 and 6; however, d-alaninyl-proline analog 4 proved superior to the other analogs in reducing PGF2α-induced myometrial contractions and inhibiting FP modulation of cell ruffling, a response dependent on the Gα12/RhoA/ROCK signaling pathway. Furthermore Gly-Pro analog 3 potentiated the effect of PGF2α on Gαq mediated ERK1/2 activation. Evidence that 4 adopted turn geometry was obtained by conformational analysis using NMR spectroscopy to characterize respectively the influence of solvent and temperature on the chemical shifts of the amide NH protons. Although mimicry of the type II' geometry by 3, 4, 5 and 6 may favour activity, distortion from ideal geometry by the indolizidinone and aza-glycinyl residues of the latter appears to enhance their biological effects.

ARF1 regulates adhesion of MDA-MB-231 invasive breast cancer cells through formation of focal adhesions.

Adhesion complex formation and disassembly is crucial for maintaining efficient cell movement. During migration, several proteins act in concert to promote remodeling of the actin cytoskeleton and we have previously shown that in highly invasive breast cancer cells, this process is regulated by small GTP-binding proteins of the ADP-ribosylation factor (ARF) family. These are overexpressed and highly activated in these cells. Here, we report that one mechanism by which ARF1 regulates migration is by controlling assembly of focal adhesions. In cells depleted of ARF1, paxillin is no longer colocalized with actin at focal adhesion sites. In addition, we demonstrate that this occurs through the ability of ARF1 to regulate the recruitment of key proteins such as paxillin, talin and FAK to ß1-integrin. Furthermore, we show that the interactions between paxillin and talin together and with FAK are significantly impaired in ARF1 knocked down cells. Our findings also indicate that ARF1 is essential for EGF-mediated phosphorylation of FAK and Src. Finally, we report that ARF1 can be found in complex with key focal adhesion proteins such as ß1-integrin, paxillin, talin and FAK. Together our findings uncover a new mechanism by which ARF1 regulates cell migration and provide this GTPase as a target for the development of new therapeutics in triple negative breast cancer.

Design and synthesis of novel azapeptide activators of apoptosis mediated by caspase-9 in cancer cells.

A set of azapeptides was designed based on the Ala-Val-Pro-Ile peptide (derived from Smac protein) to activate caspase-9 and induce apoptosis in breast cancer cells. The diversity-oriented synthesis of the aza-peptides 5-9 was accomplished by alkylation of the aza-residue of aza-Gly-Pro dipeptide 15 using potassium tert-butoxide and a range of different alkyl halides. The resulting protected aza-dipeptide building blocks were then introduced into mimics 5-9 using standard coupling conditions. Biological evaluation of 5-9 was performed in MDA-MB-231 breast cancer cells, and indicated that the aza-Gly and aza-Phe analogs 5 and 7 were most efficient in inducing cell death by a caspase-9 mediated apoptotic pathway. Revealing a relationship between azabicycloalkanone and aza peptide mimics, novel AVPI mimics were synthesized which exhibit utility for studying structure-activity relationships to develop leads for activating apoptosis in cancer cells.

The adaptor proteins p66Shc and Grb2 regulate the activation of the GTPases ARF1 and ARF6 in invasive breast cancer cells.

Signals downstream of growth factor receptors play an important role in mammary carcinogenesis. Recently, we demonstrated that the small GTPases ARF1 and ARF6 were shown to be activated downstream of the epidermal growth factor receptor (EGFR) and act as a key regulator of growth, migration, and invasion of breast cancer cells. However, the mechanism via which the EGFR recruits and activates ARF1 and ARF6 to transmit signals has yet to be fully elucidated. Here, we identify adaptor proteins Grb2 and p66Shc as important regulators mediating ARF activation. We demonstrate that ARF1 can be found in complex with Grb2 and p66Shc upon EGF stimulation of the basal-like breast cancer MDA-MB-231 cell line. However, we report that these two adaptors regulate ARF1 activation differently, with Grb2 promoting ARF1 activation and p66Shc blocking this response. Furthermore, we show that Grb2 is essential for the recruitment of ARF1 to the EGFR, whereas p66Shc hindered ARF1 receptor recruitment. We demonstrate that the negative regulatory role of p66Shc stemmed from its ability to block the recruitment of Grb2/ARF1 to the EGFR. Conversely, p66Shc potentiates ARF6 activation as well as the recruitment of this ARF isoform to the EGFR. Interestingly, we demonstrate that Grb2 is also required for the activation and receptor recruitment of ARF6. Additionally, we show an important role for p66Shc in modulating ARF activation, cell growth, and migration in HER2-positive breast cancer cells. Together, our results highlight a central role for adaptor proteins p66Shc and Grb2 in the regulation of ARF1 and ARF6 activation in invasive breast cancer cells.