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In JoVE (1)
Other Publications (29)
- The Journal of Cell Biology
- The Journal of Biological Chemistry
- The Journal of Pharmacology and Experimental Therapeutics
- The Journal of Biological Chemistry
- Methods in Molecular Biology (Clifton, N.J.)
- Cellular Signalling
- FEBS Letters
- Cancer Letters
- American Journal of Physiology. Renal Physiology
- The FEBS Journal
- Neoplasia (New York, N.Y.)
- The Journal of Pharmacology and Experimental Therapeutics
- Toxicological Sciences : an Official Journal of the Society of Toxicology
- Molecular Biology of the Cell
- Biochemical Pharmacology
- FEBS Letters
- Journal of Medicinal Chemistry
- Toxicological Sciences : an Official Journal of the Society of Toxicology
- American Journal of Physiology. Renal Physiology
- The Journal of Pharmacology and Experimental Therapeutics
- Neoplasia (New York, N.Y.)
- Cancer Biology & Therapy
- Neoplasia (New York, N.Y.)
- Molecular and Cellular Pharmacology
- Molecular and Cellular Biochemistry
- The Journal of Pharmacology and Experimental Therapeutics
- The Journal of Pharmacology and Experimental Therapeutics
- The Journal of Pharmacology and Experimental Therapeutics
- The Biochemical Journal
Articles by Raymond R. Mattingly in JoVE
JoVE Clinical and Translational Medicine
MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression
1Department of Pharmacology, Wayne State University, 2Barbara Ann Karmanos Cancer Institute, Wayne State University
We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.
Other articles by Raymond R. Mattingly on PubMed
High RhoA Activity Maintains the Undifferentiated Mesenchymal Cell Phenotype, Whereas RhoA Down-regulation by Laminin-2 Induces Smooth Muscle Myogenesis
Round embryonic mesenchymal cells have the potential to differentiate into smooth muscle (SM) cells upon spreading/elongation (Yang, Y., K.C. Palmer, N. Relan, C. Diglio, and L. Schuger. 1998. Development. 125:2621-2629; Yang, Y., N.K. Relan, D.A. Przywara, and L. Schuger. 1999. Development. 126:3027-3033; Yang, Y., S. Beqaj, P. Kemp, I. Ariel, and L. Schuger. 2000. J. Clin. Invest. 106:1321-1330). In the developing lung, this process is stimulated by peribronchial accumulation of laminin (LN)-2 (Relan, N.K., Y. Yang, S. Beqaj, J.H. Miner, and L. Schuger. 1999. J. Cell Biol. 147:1341-1350). Here we show that LN-2 stimulates bronchial myogenesis by down-regulating RhoA activity. Immunohistochemistry, immunoblotting, and reverse transcriptase-PCR indicated that RhoA, a small GTPase signaling protein, is abundant in undifferentiated embryonic mesenchymal cells and that its levels decrease along with SM myogenesis. Functional studies using agonists and antagonists of RhoA activation and dominant positive and negative plasmid constructs demonstrated that high RhoA activity was required to maintain the round undifferentiated mesenchymal cell phenotype. This was in part achieved by restricting the localization of the myogenic transcription factor serum response factor (SRF) mostly to the mesenchymal cell cytoplasm. Upon spreading on LN-2 but not on other main components of the extracellular matrix, the activity and level of RhoA decreased rapidly, resulting in translocation of SRF to the nucleus. Both cell elongation and SRF translocation were prevented by overexpression of dominant positive RhoA. Once the cells underwent SM differentiation, up-regulation of RhoA activity induced rather than inhibited SM gene expression. Therefore, our studies suggest a novel mechanism whereby LN-2 and RhoA modulate SM myogenesis.
Mechanism of 17-beta-estradiol-induced Erk1/2 Activation in Breast Cancer Cells. A Role for HER2 AND PKC-delta
Activation of mitogen-activated protein kinase (Erk/MAPK) is a critical signal transduction event for estrogen (E(2))-mediated cell proliferation. Recent studies from our group and others have shown that persistent activation of Erk plays a major role in cell migration and tumor progression. The signaling mechanism(s) responsible for persistent Erk activation are not fully characterized, however. In this study, we have shown that E(2) induces a slow but persistent activation of Erk in MCF-7 breast carcinoma cells. The E(2)-induced Erk activation is dependent on new protein synthesis, suggesting that E(2)-induced growth factors play a major role in Erk activation. When MCF-7 cells were treated with E(2) in the presence of an anti-HER-2 monoclonal antibody (herceptin), 60-70% of E(2)-induced Erk activation is blocked. In addition, when untreated MCF-7 cells were exposed to conditioned medium from E(2)-treated cells, Erk activity was significantly enhanced. Furthermore Erk activity was blocked by an antibody against HER-2 or by heregulin (HRG) depletion from the conditioned medium through immunoprecipitation. In contrast, epidermal growth factor receptor (Ab528) antibody only blocked 10-20% of E(2)-induced Erk activation, suggesting that E(2)-induced Erk activation is predominantly mediated through the secretion of HRG and activation of HER-2 by an autoctine/paracrine mechanism. Inhibition of PKC-delta-mediated signaling by a dominant negative mutant or the relatively specific PKC-delta inhibitor rottlerin blocked most of the E(2)-induced Erk activation but had no effect on TGF alpha-induced Erk activation. By contrast inhibition of Ras, by inhibition of farnesyl transferase (Ftase-1) or dominant negative (N17)-Ras, significantly inhibited both E(2)- and TGF alpha-induced Erk activation. This evaluation of downstream signaling revealed that E(2)-induced Erk activation is mediated by a HRG/HER-2/PKC-delta/Ras pathway that could be crucial for E(2)-dependent growth-promoting effects in early stages of tumor progression.
Potent Suppression of Proliferation of A10 Vascular Smooth Muscle Cells by Combined Treatment with Lovastatin and 3-allylfarnesol, an Inhibitor of Protein Farnesyltransferase
Statins, which inhibit 3-hydroxy-3-methylglutaryl-CoA reductase and thus the synthesis of cholesterol, are remarkably effective in the treatment of cardiovascular disease. In addition to their favorable effect on lipid profile, these drugs may also prevent the proliferation of vascular smooth muscle that is characteristic of atherosclerosis. We hypothesize that statins prevent the post-translational prenylation, and thus inhibit the function, of critical small GTPases in vascular smooth muscle cells. We have therefore assayed the effect of lovastatin on both the growth of A10 vascular smooth muscle cells and the status of their Ras and RhoB proteins. We find that < or =1 microM lovastatin potently inhibits the proliferation of A10 cultures, and higher concentrations (> or =3 microM) induce apoptosis. We have also tested the effect of 3-allylfarnesol (3-alFOH), an inhibitor of farnesyl transferase (FTI). The data show that although > or =10 microM 3-alFOH is required for a cytostatic effect, the action of 3 microM 3-alFOH can be greatly potentiated by even nanomolar levels of lovastatin. We also find that lovastatin and 3-alFOH exhibit synergism to cause the up-regulation and relocalization of RhoB from the membrane to cytosolic compartments. This relocalization of RhoB, which is presumed to reflect an inhibition of its prenylation, correlates with the proapoptotic activities of combined 3-alFOH and lovastatin treatment. These data suggest that RhoB may be a valuable pharmacological target in cardiovascular disease, and that combinations of statins and certain FTIs may be of value in treatment of disorders that are characterized by excess cell proliferation.
Phosphorylation of the Ras-GRF1 Exchange Factor at Ser916/898 Reveals Activation of Ras Signaling in the Cerebral Cortex
The Ras-GRF1 exchange factor, which is regulated by increases in intracellular calcium and the release of G beta gamma subunits from heterotrimeric G proteins, plays a critical role in the activation of neuronal Ras. Activation of G protein-coupled receptors stimulates an increase in the phosphorylation of Ras-GRF1 at certain serine residues. The first of these sites to be identified, Ser(916) in the mouse sequence (equivalent to Ser(898) in the rat sequence), is required for full activation of the Ras exchange factor activity of Ras-GRF1 by muscarinic receptors. We demonstrate here that Ras-GRF1 is highly expressed in rat brain compared with the Sos exchange factor and that there is an increase in incorporation of (32)P into Ser(898) of brain Ras-GRF1 following activation of protein kinase A. Phosphorylation of Ras-GRF1 at Ser(916) is also required for maximal induction of Ras-dependent neurite outgrowth in PC12 cells. A novel antibody (termed 2152) that selectively recognizes Ras-GRF1 when it is phosphorylated at Ser(916/898) confirmed the regulated phosphorylation of Ras-GRF1 by Western blotting in both model systems of transfected COS-7 and PC12 cells and also of the endogenous protein in rat forebrain slices. Indirect confocal immunofluorescence of transfected PC12 cells using antibody 2152 demonstrated reactivity only under conditions in which Ras-GRF1 was phosphorylated at Ser(916/898). Confocal immunofluorescence of cortical slices of rat brain revealed widespread and selective phosphorylation of Ras-GRF1 at Ser(898). In the prefrontal cortex, there was striking phosphorylation of Ras-GRF1 in the dendritic tree, supporting a role for Ras activation and signal transduction in neurotransmission in this area.
Mitogen-activated Protein Kinase Signaling in Drug-resistant Neuroblastoma Cells
Widespread inherent or acquired resistance to cytotoxic drugs is a major limitation to chemotherapy. There are many mechanisms that contribute to such resistance. In neuroblastomas there is evidence that acquired drug resistance may be associated with altered response to growth factor signals. The ubiquitous mitogen-activated protein kinase (MAPk) cascade, which transmits growth factor signals from the cell membrane to the nucleus, provides a principal mechanism for regulation of cell cycle progression and proliferation. We have shown that there is a relationship between acquired drug resistance in human neuroblastoma cells to doxorubicin, a topoisomerase-2 inhibitor, and to MDL-28842, an inhibitor of S-adenosylhomocysteine hydrolase, and reductions in the activation and nuclear translocation of MAPk.
Cell Surface Receptors Activate P21-activated Kinase 1 Via Multiple Ras and PI3-kinase-dependent Pathways
p21-activated kinases (PAKs) were the first identified mammalian members of a growing family of Ste20-like serine-threonine protein kinases. In this study, we show that PAK1 can be stimulated by carbachol, lysophosphatidic acid (LPA), epidermal growth factor (EGF), and phorbol 12-myristate 13-acetate (PMA) by multiple independent and overlapping pathways. Dominant-negative Ras, Rac, and Cdc42 inhibited PAK1 activation by all of these agonists, while active Rac1 and Cdc42 were sufficient to maximally activate PAK1 in the absence of any treatment. Active Ras induced only a weak activation of PAK1 that could be potentiated by muscarinic receptor stimulation. Studies using inhibitors of the EGF receptor tyrosine kinase, phosphatidylinositol 3-kinase (PI3-kinase) and protein kinase C (PKC) revealed that all of the cell surface agonists could activate PAK1 through pathways independent of PKC, that EGF stimulated a PI3-kinase dependent pathway to stimulate PAK1, and that muscarinic receptor stimulation of PAK1 was predominantly mediated through this EGF-R-dependent mechanism. Activation of PAK1 by LPA was independent of PI3-kinase and the EGF receptor, but was inhibited by dominant-negative RhoA. These results identify multiple Ras-dependent pathways to activation of PAK1.
Gbetagamma Subunits Stimulate P21-activated Kinase 1 (PAK1) Through Activation of PI3-kinase and Akt but Act Independently of Rac1/Cdc42
The p21-activated kinase (PAK) family is homologous to the yeast sterile 20 (Ste20) and regulates a wide variety of cellular responses, including cell morphology, proliferation, and survival. In this study we examined the activation of PAK1 by Gbetagamma subunits. Co-transfection of COS7 cells with Gbeta1gamma2 or Gbeta1gamma5 was sufficient to induce agonist-independent activation of PAK1. Expression of dominant/negative Rac, Cdc42, or Ras did not inhibit this Gbetagamma-dependent activation. Wortmannin, which inhibits phosphoinositide 3-kinase (PI3-kinase) activity, and expression of a dominant/negative form of Akt were sufficient to abrogate the activation of PAK1 that was induced by Gbetagamma. These results reveal that stimulation of PAK1 by Gbetagamma can occur via a PI3-kinase and Akt pathway that does not require Rac1 or Cdc42.
TNF-alpha-mediated Apoptosis in Normal Human Prostate Epithelial Cells and Tumor Cell Lines
In this study we compared the role of TNF-alpha in the regulation of growth and apoptosis in normal human prostate epithelial cells (NP) and prostate tumor cell lines PC3 and LNCap. The NP and PC3 cells were resistant whereas the LNCap cell line was highly sensitive to TNF-alpha induced growth arrest and apoptosis. The resistance of NP and PC3 cells was mediated via an NF-kB survival pathway as treatment of resistant cells with TNF-alpha was accompanied by phosphorylation of I-kBalpha and translocation of NF-kB to the nucleus. TNF-alpha did not induce phosphorylation of I-kB in the sensitive LNCap cells. The sensitivity of LNCap cells involved a cysteine protease cascade as Z-VAD-CH2 F reversed the sensitivity of LNCap cells and induced resistance to TNF-alpha. The differences in susceptibilities to TNF-alpha induced apoptosis of NP and certain prostate tumor cells offer intriguing possibilities for the treatment of prostate cancer without affecting the normal prostate tissue.
Angiotensin II Directly Stimulates Activity and Alters the Phosphorylation of Na-K-ATPase in Rat Proximal Tubule with a Rapid Time Course
We present evidence that Na-K-ATPase in the rat proximal tubule is directly activated by ANG II much faster than previously observed. Specifically, we show that a 2-min exposure to 0.1 and 1 nM ANG II slowed the rate of intracellular sodium accumulation in response to an increase in extracellular sodium added in the presence of gramicidin D. From these data, we show that ANG II directly stimulates Na-K-ATPase activity at rate-limiting concentrations of intracellular sodium. Under these same conditions, exposing proximal tubules to ANG II altered the amount of 32P incorporated into multiple phosphopeptides generated from a tryptic digest of the alpha-subunit of Na-K-ATPase. Na-K-ATPase was isolated from whole cell lysates by means of a ouabain-affinity column and then separated into its individual subunits by SDS-PAGE. Na-K-ATPase bound to the column in its E2 conformation and was eluted by altering its conformation to E1 using Na+ATP. Na-K-ATPase isolated from cells treated with ANG II eluted more easily from the ouabain-affinity column than Na-K-ATPase isolated from control cells, suggesting that ANG II decreased the affinity of Na-K-ATPase for ouabain. Thus ANG II rapidly stimulated the activity of Na-K-ATPase in 2 min or less by a mechanism that could involve changes in phosphorylation and conformation of Na-K-ATPase. We suggest that the physiological role for rapid direct activation of Na-K-ATPase is greater control of intracellular sodium during sodium reabsorption.
Endogenous Expression and Protein Kinase A-dependent Phosphorylation of the Guanine Nucleotide Exchange Factor Ras-GRF1 in Human Embryonic Kidney 293 Cells
We have previously reported the Ras-dependent activation of the mitogen-activated protein kinases p44 and p42, also termed extracellular signal-regulated kinases (ERK)1 and 2 (ERK1/2), mediated through Gs-coupled serotonin receptors transiently expressed in human embryonic kidney (HEK) 293 cells. Whereas Gi- and Gq-coupled receptors have been shown to activate Ras through the guanine nucleotide exchange factor (GEF) called Ras-GRF1 (CDC25Mm) by binding of Ca2+/calmodulin to its N-terminal IQ domain, the mechanism of Ras activation through Gs-coupled receptors is not fully understood. We report the endogenous expression of Ras-GRF1 in HEK293 cells. Serotonin stimulation of HEK293 cells transiently expressing Gs-coupled 5-HT7 receptors induced protein kinase A-dependent phosphorylation of the endogenous human Ras-GRF1 on Ser927 and of transfected mouse Ras-GRF1 on Ser916. Ras-GRF1 overexpression increased basal and serotonin-stimulated ERK1/2 phosphorylation. Mutations of Ser916 inhibiting (Ser916Ala) or mimicking (Ser916Asp/Glu) phosphorylation did not alter these effects. However, the deletion of amino acids 1-225, including the Ca2+/calmodulin-binding IQ domain, from Ras-GRF1 reduced both basal and serotonin-stimulated ERK1/2 phosphorylation. Furthermore, serotonin treatment of HEK293 cells stably expressing 5-HT7 receptors increased [Ca2+]i, and the serotonin-induced ERK1/2 phosphorylation was Ca2+-dependent. Therefore, both cAMP and Ca2+ may contribute to the Ras-dependent ERK1/2 activation after 5-HT7 receptor stimulation, through activation of a guanine nucleotide exchange factor with activity towards Ras.
Active P21-activated Kinase 1 Rescues MCF10A Breast Epithelial Cells from Undergoing Anoikis
The protein kinase, PAK1, is overexpressed in human breast cancer and may contribute to malignancy through induction of proliferation and invasiveness. In this study, we examined the role of PAK1 in the survival of detached MCF10A breast epithelial cells to test whether it may also regulate the early stages of neoplasia. MCF10A cells undergo anoikis, as measured by the cleavage of caspase 3 and poly(ADP-ribose) polymerase (PARP), after more than 8 hours of detachment. Endogenous Akt, PAK1, and BAD are phosphorylated in attached MCF10A cells, but these phosphorylation events are all lost during the first 8 hours of detachment. Expression of constitutively active PAK1 or Akt suppresses the cleavage of caspase 3 and PARP in detached MCF10A cells. Co-overexpression of active PAK1 with dominant-negative Akt, or of active Akt with dominant-negative PAK1, still suppresses anoikis. Thus, Akt and PAK1 enhance survival through pathways that are at least partially independent. PAK1-dependent regulation of anoikis is likely to occur early in the apoptotic cascade as expression of dominant-negative PAK1 increased the cleavage of the upstream caspase 9, while constitutively active PAK1 inhibited caspase 9 activation. These results support a role for activated PAK1 in the suppression of anoikis in MCF10A epithelial cells.
The Mitogen-activated Protein Kinase/extracellular Signal-regulated Kinase Kinase Inhibitor PD184352 (CI-1040) Selectively Induces Apoptosis in Malignant Schwannoma Cell Lines
Type 1 neurofibromatosis (NF1) is a common autosomal dominant disorder that results in neuroectodermal tumors. The NF1 tumor-suppressor gene encodes neurofibromin, which includes a GTPase-activating domain for Ras inactivation. Affinity purification showed N-Ras to be the predominant activated isoform of Ras in two independent neurofibrosarcoma cell lines from NF1 patients (lines ST88-14 and NF90-8). These NF1 cells also demonstrated increased constitutive activity of the extracellular signal-regulated kinases 1 and 2 (ERK1,2) mitogen-activated protein (MAP) kinases compared with a sporadic malignant schwannoma cell line that maintains neurofibromin expression (STS-26T). Thus, MAP kinase kinase (MEK) inhibitors may be a rational approach to NF1 therapy. The MEK inhibitors PD98059 [2'-amino-3'-methoxyflavone], PD184352 (also called CI-1040) [2-(2-chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzamide], and U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene] all produced concentration-dependent suppression of the proliferation of the three cell lines. Individual MEK inhibitors had similar effects in all three cell lines. However, only the antiproliferative effects of PD184352 correlated closely with the elimination of ERK1,2 MAP kinase activities. PD98059 was primarily cytostatic, whereas U0126 and PD184352 were cytotoxic. Only PD184352 induced apoptosis in all three lines, as indicated by morphology, activation of DEVDase, procaspase-3 cleavage, and the appearance of populations having sub-G(0)/G(1) DNA contents. The differential effects of the MEK inhibitors on cell survival were not dependent on p53 status or effects on the ERK5 pathway. PD184352 was also proapoptotic to primary rat Schwann cells. Hence, although PD184352 effectively killed neurofibrosarcoma cells, its effects on normal Schwann cells may limit its usefulness in the clinic.
Inorganic Mercury Inhibits the Activation of LAT in T-cell Receptor-mediated Signal Transduction
Little is known as to the molecular mechanisms involved with mercury intoxication at very low levels. Although the mechanism is not known, animal studies have nevertheless shown that low levels of mercury may target the immune system. Inorganic mercury (Hg2+) at very low (but non-toxic) levels can disrupt immune system homeostasis, in that genetically susceptible rodents develop idiosyncratic autoimmune disease, which is associated with defective T-cell function. T lymphocyte function is intimately coupled to the T-cell receptor. We have previously reported that on a molecular level, low concentrations of Hg2+ disrupt signaling from the T-cell receptor by interfering with activation of Ras and ERK MAP kinase. In this report we expand upon those results by showing that in T lymphocytes exposed to low concentration of Hg2+, Ras fails to become properly activated because upstream of Ras in the T cell signal transduction pathway, the important scaffolding element Linker for Activation of T Cells (LAT) fails to become properly phosphorylated. Hypo-phosphorylation of LAT occurs, because upstream of LAT, the LAT reactive tyrosine kinase ZAP-70 is also not properly activated in Hg2+ treated cells.
The Ras-GRF1 Exchange Factor Coordinates Activation of H-Ras and Rac1 to Control Neuronal Morphology
The Ras-GRF1 exchange factor has regulated guanine nucleotide exchange factor (GEF) activity for H-Ras and Rac1 through separate domains. Both H-Ras and Rac1 activation have been linked to synaptic plasticity and thus could contribute to the function of Ras-GRF1 in neuronal signal transduction pathways that underlie learning and memory. We defined the effects of Ras-GRF1 and truncation mutants that include only one of its GEF activities on the morphology of PC12 phaeochromocytoma cells. Ras-GRF1 required coexpression of H-Ras to induce morphological effects. Ras-GRF1 plus H-Ras induced a novel, expanded morphology in PC12 cells, which was characterized by a 10-fold increase in soma size and by neurite extension. A truncation mutant of Ras-GRF1 that included the Ras GEF domain, GRFdeltaN, plus H-Ras produced neurite extensions, but did not expand the soma. This neurite extension was blocked by inhibition of MAP kinase activation, but was independent of dominant-negative Rac1 or RhoA. A truncation mutant of Ras-GRF1 that included the Rac GEF domains, GRFdeltaC, produced the expanded phenotype in cotransfections with H-Ras. Cell expansion was inhibited by wortmannin or dominant-negative forms of Rac1 or Akt. GRFdeltaC binds H-Ras.GTP in both pulldown assays from bacterial lysates and by coimmunoprecipitation from HEK293 cells. These results suggest that coordinated activation of H-Ras and Rac1 by Ras-GRF1 may be a significant controller of neuronal cell size.
Molecular Targets for Emerging Anti-tumor Therapies for Neurofibromatosis Type 1
Neurofibromatosis type 1 (NF1) is the most common cancer predisposition syndrome. NF1 patients present with a constellation of clinical manifestations and have an increased risk of developing certain benign and malignant tumors. This disease results from mutation within the gene encoding neurofibromin, a GTPase activating protein (GAP) for Ras. Functional loss of this protein compromises Ras inactivation, which leads to the aberrant growth and proliferation of neural crest-derived cells and, ultimately, tumor formation. Current management of NF1-associated malignancy involves radiation, surgical excision, and cytotoxic drugs. The limited success of these strategies has fueled researchers to further elucidate the molecular changes that drive tumor formation and progression. This discussion will highlight how intracellular signaling molecules, cell-surface receptors, and the tumor microenvironment constitute potential therapeutic targets, which may be relevant not only to NF1-related malignancy but also to other human cancers.
Epac- and Rap- Independent ERK1/2 Phosphorylation Induced by Gs-coupled Receptor Stimulation in HEK293 Cells
Serotonin activates Ras and Ras-dependent ERK1/2 phosphorylation in HEK293 cells expressing G(s)-coupled 5-HT(4) or 5-HT(7) serotonin receptors through unknown mechanisms. Both Epac/Rap-dependent and -independent pathways for Ras-dependent ERK1/2 activation have been suggested. Epac overexpression or Epac-specific 8-CPT-2'-O-Me-cAMP did not cause ERK1/2 phosphorylation, despite Rap activation. The data did not support a role for PLCepsilon or DAG-dependent Ras GEFs of the Ras-GRP family in Ras-dependent ERK1/2 phosphorylation. However, serotonin stimulated phosphorylation of endogenous and recombinant Ras-GRF1, increased [Ca(2+)](i) and caused Ca(2+)- and calmodulin-dependent ERK1/2 phosphorylation. Different signalling pathways seem to be utilised by G(s)-coupled receptors in various isolates of HEK293 cells.
Synthesis, Biochemical, and Cellular Evaluation of Farnesyl Monophosphate Prodrugs As Farnesyltransferase Inhibitors
Certain farnesyl diphosphate (FPP) analogs are potent inhibitors of the potential anticancer drug target protein farnesyltransferase (FTase), but these compounds are not suitable as drug candidates. Thus, phosphoramidate prodrug derivatives of the monophosphate precursors of FPP-based FTase inhibitors have been synthesized. The monophosphates themselves were significantly more potent inhibitors of FTase than the corresponding FPP analogs. The effects of the prodrug 5b (a derivative of 3-allylfarnesyl monophosphate) have been evaluated on prenylation of RhoB and on the cell cycle in a human malignant schwannoma cell line (STS-26T). In combination treatments, 1-3 microM 5b plus 1 microM lovastatin induced a significant inhibition of RhoB prenylation, and a combination of these drugs at 1 microM each also resulted in significant cell cycle arrest in G1. Indeed, combinations as low as 50 nM lovastatin + 1 microM 5c or 250 nM lovastatin + 50 nM 5c were highly cytostatic in STS-26T cell culture.
Low and Nontoxic Inorganic Mercury Burdens Attenuate BCR-mediated Signal Transduction
The ubiquitous environmental heavy metal contaminant mercury (Hg) is a potent immunomodulator that has been implicated as a factor contributing to autoimmune disease. However, the mechanism(s) whereby Hg initiates or perpetuates autoimmune responses, especially at the biochemical/molecular level, remain poorly understood. Recent work has established a relationship between impaired B-cell receptor (BCR) signal strength and autoimmune disease. In previous studies, we have shown that in mouse WEHI-231 B cells, noncytotoxic concentrations of inorganic mercury (Hg(+2)) interfered with BCR-mediated growth control, suggesting that BCR signal strength was impaired by Hg(+2). Extracellular signal-regulated kinase (ERK) 1,2 mitogen-activated protein kinase (MAPK) is responsible for the activation of several transcription factors in B cells. Phosphorylation of ERK serves as an essential node of signal integration for the BCR. Thus, the magnitude of ERK activation serves as an operational metric for BCR signal strength. Using Western blotting and phospho-specific flow cytometry, we now show that the kinetics and magnitude of BCR-mediated activation of ERK-MAPK are markedly attenuated in WEHI-231 cells and splenic B cells that have been exposed to low and nontoxic burdens of Hg(+2). However, Hg(+2) does not seem to act directly on ERK-MAPK but rather on an upstream element or elements of the BCR signal transduction pathway, above the level of the key protein tyrosine kinase Syk. Our data suggest that the site of action of Hg(+2) may very well be localized on the plasma membrane. These findings support a connection between Hg(+2) and attenuated BCR signal strength in the etiology of autoimmune disease.
Angiotensin II Stimulates Elution of Na-K-ATPase from a Digoxin-affinity Column by Increasing the Kinetic Response to Ligands That Trigger the Decay of E2-P
We earlier observed that treating rat proximal tubules with concentrations of angiotensin II (ANG II) that directly stimulate Na-K-ATPase activity changed how Na-K-ATPase subsequently eluted from an ouabain-affinity column. In this study we tested whether ANG II increases the rate of elution in response to ligands that trigger the decay of E(2)-P, which implies a change in functional properties of Na-K-ATPase, or by decreasing the amount subsequently eluted with SDS, which suggests a change in how Na-K-ATPase interacts with other proteins. We utilized a new digoxin-affinity column and novel lines of opossum kidney (OK) cells that coexpress the rat AT(1a) receptor and either the wild-type rat alpha(1)-isoform of Na-K-ATPase or a truncation mutant missing the first 32 amino acids of its NH(2) terminus. We characterized how rat kidney microsomes bind to and elute from the digoxin-affinity column and demonstrated that they are heterogeneous in the rate at which they release digoxin in response to ligands that trigger the decay of E(2)-P. Incubating OK cells with ANG II stimulated the ensuing elution of wild-type rat alpha(1)-subunit by increasing the kinetic response to ligands that cause a decay of E(2)-P without affecting the amount later eluted with SDS. In contrast, ANG II had no effect on the kinetic response of the truncation mutant but decreased the amount eluted with SDS. These data suggest that ANG II regulates both the kinetic properties of Na-K-ATPase and its interaction with other proteins by a mechanism(s) involving its NH(2) terminus.
Induction of Apoptosis in Neurofibromatosis Type 1 Malignant Peripheral Nerve Sheath Tumor Cell Lines by a Combination of Novel Farnesyl Transferase Inhibitors and Lovastatin
Neurofibromatosis type 1 (NF1) is a genetic disorder that is driven by the loss of neurofibromin (Nf) protein function. Nf contains a Ras-GTPase-activating protein domain, which directly regulates Ras signaling. Numerous clinical manifestations are associated with the loss of Nf and increased Ras activity. Ras proteins must be prenylated to traffic and functionally localize with target membranes. Hence, Ras is a potential therapeutic target for treating NF1. We have tested the efficacy of two novel farnesyl transferase inhibitors (FTIs), 1 and 2, alone or in combination with lovastatin, on two NF1 malignant peripheral nerve sheath tumor (MPNST) cell lines, NF90-8 and ST88-14. Single treatments of 1, 2, or lovastatin had no effect on Ras prenylation or MPNST cell proliferation. However, low micromolar combinations of 1 or 2 with lovastatin (FTI/lovastatin) reduced Ras prenylation in both MPNST cell lines. Furthermore, this FTI/lovastatin combination treatment reduced cell proliferation and induced an apoptotic response as shown by morphological analysis, procaspase-3/-7 activation, loss of mitochondrial membrane potential, and accumulation of cells with sub-G(1) DNA content. Little to no detectable toxicity was observed in normal rat Schwann cells following FTI/lovastatin combination treatment. These data support the hypothesis that combination FTI plus lovastatin therapy may be a potential treatment for NF1 MPNSTs.
P21-Activated Kinase 1 Coordinates Aberrant Cell Survival and Pericellular Proteolysis in a Three-dimensional Culture Model for Premalignant Progression of Human Breast Cancer
Overexpression of p21-activated kinase 1 (PAK1) occurs during the progression of human breast cancer. We have investigated the role of PAK1 in the premalignant progression of the MCF10 series of human breast epithelial cell lines. Levels of PAK1 expression and activation increased with premalignant progression, and expression of dominant-negative (DN) PAK1 reduced both cell proliferation and migration/invasion. In three-dimensional (3D) overlay cultures in reconstituted basement membrane, the MCF10 series produced an in vitro model for premalignant progression. MCF10AneoT cells formed a hyperplastic morphology in which some spheroids developed abnormal lumens. The MCF10.AT1 line exhibited an atypical hyperplastic morphology of abnormal spheroid clusters that did not form lumens. The MCF10.DCIS cells exhibited dysplastic growth. Expression of DN-PAK1 promoted lumen formation in 3D-cultured MCF10A, NeoT, and AT1 structures, suggesting partial reversion of the premalignant phenotype, but did not affect the atypical budding of AT1 structures or the dysplastic growth of ductal carcinoma in situ structures. Aberrant proteolysis is another important characteristic of breast cancer progression and invasion. DN-PAK1 or knock-down of PAK1 reduced pericellular proteolysis of DQ-collagen IV in the 3D cultures. Treatment of cells with an inhibitor of Rac1 also reduced pericellular proteolysis, and this reduction was reversed by the expression of activated PAK1. Our conclusion is that overexpressed and activated PAK1 may be a key coordinator of aberrant cell survival and proteolysis in breast cancer progression.
Suppression of Proliferation of Two Independent NF1 Malignant Peripheral Nerve Sheath Tumor Cell Lines by the Pan-ErbB Inhibitor CI-1033
Neurofibromatosis Type 1 (NF1) is characterized by the abnormal proliferation of neuroectodermal tissues and the development of certain tumors, particularly neurofibromas, which may progress into malignant peripheral nerve sheath tumors (MPNSTs). Effective pharmacological therapy for the treatment of NF1 tumors is currently unavailable and the prognosis for patients with MPNSTs is poor. Loss of neurofibromin correlates with increased expression of the epidermal growth factor receptor (EGFR) and ErbB2 tyrosine kinases and these kinases have been shown to promote NF1 tumor-associated pathologies in vivo. We show here that while NF1 MPNST cells have higher EGFR expression levels and are more sensitive to EGF when compared to a non-NF1 MPNST cell line, the ability of the EGFR inhibitor gefitinib to selectively inhibit NF1 MPNST cell proliferation is marginal. We also show that NF1 MPNST proliferation correlates with activated ErbB2 and can be suppressed by nanomolar concentrations of the pan-ErbB inhibitor CI-1033 (canertinib). Consequently, targeting both EGFR and ErbB2 may prove an effective strategy for suppressing NF1 MPNST tumor growth in vivo.
Restoration of E-cadherin Cell-cell Junctions Requires Both Expression of E-cadherin and Suppression of ERK MAP Kinase Activation in Ras-transformed Breast Epithelial Cells
E-cadherin is a main component of the cell-cell adhesion junctions that play a principal role in maintaining normal breast epithelial cell morphology. Breast and other cancers that have up-regulated activity of Ras are often found to have down-regulated or mislocalized E-cadherin expression. Disruption of E-cadherin junctions and consequent gain of cell motility contribute to the process known as epithelial-to-mesenchymal transition (EMT). Enforced expression of E-cadherin or inhibition of Ras-signal transduction pathway has been shown to be effective in causing reversion of EMT in several oncogene-transformed and cancer-derived cell lines. In this study, we investigated MCF10A human breast epithelial cells and derivatives that were transformed with either activated H-Ras or N-Ras to test for the reversion of EMT by inhibition of Ras-driven signaling pathways. Our results demonstrated that inhibition of mitogen-activated protein kinase (MAPK) kinase, but not PI3-kinase, Rac, or myosin light chain kinase, was able to completely restore E-cadherin cell-cell junctions and epithelial morphology in cell lines with moderate H-Ras expression. In MCF10A cells transformed by a high-level expression of activated H-Ras or N-Ras, restoration of E-cadherin junction required both the enforced reexpression of E-cadherin and suppression of MAPK kinase. Enforced expression of E-cadherin alone did not induce reversion from the mesenchymal phenotype. Our results suggest that Ras transformation has at least two independent actions to disrupt E-cadherin junctions, with effects to cause both mislocalization of E-cadherin away from the cell surface and profound decrease in the expression of E-cadherin.
Working Together: Farnesyl Transferase Inhibitors and Statins Block Protein Prenylation
Farnesyl transferase inhibitors (FTIs) have so far proved to have limited value as single agents in clinical trials. This PharmSight will focus on the use of a novel group of FTIs that are most effective in vitro when used in combination with the "statin" class of anti-hypercholesterolemic agents, which also block protein prenylation. We recently showed that these novel FTIs in combination with lovastatin reduce Ras prenylation and induce an apoptotic response in malignant peripheral nerve sheath cells. The combination of statins with these new FTIs may produce profound synergistic cytostatic and cytotoxic effects against a variety of tumors and other proliferative disorders. Since statins are well tolerated in the clinic, we suggest that this combination approach should be tested in in vivo models.
The Role of Neurofibromin in N-Ras Mediated AP-1 Regulation in Malignant Peripheral Nerve Sheath Tumors
Plexiform neurofibromas commonly found in patients with Neurofibromatosis type I (NF1) have a 5% risk of being transformed into malignant peripheral nerve sheath tumors (MPNST). Germline mutations in the NF1 gene coding for neurofibromin, which is a Ras GTPase activating protein (RasGAP) and a negative regulator of Ras, result in an upregulation of the Ras pathway. We established a direct connection between neurofibromin deficiency and downstream effectors of Ras in cell lines from MPNST patients by demonstrating that knockdown of NF1 expression using siRNA in a NF1 wild type MPNST cell line, STS-26T, activates the Ras/ERK1,2 pathway and increases AP-1 binding and activity. We believe this is the first time the transactivation of AP-1 has been linked directly to neurofibromin deficiency in a disease relevant MPNST cell line. Previously, we have shown that N-Ras is constitutively activated in cell lines derived from independent MPNSTs from NF1 patients. We therefore sought to analyze the role of the N-Ras pathway in deregulating AP-1 transcriptional activity. We show that STS-26T clones conditionally expressing oncogenic N-Ras show increased phosphorylated ERK1,2 and phosphorylated JNK expression concomitant with increased AP-1 activity. MAP kinase pathways (ERK1,2 and JNK) were further examined in ST88-14, a neurofibromin-deficient MPNST cell line. The basal activity of ERK1,2 but not JNK was found to increase AP-1 activity. These experiments further confirmed the link between the loss of neurofibromin and increased activity of Ras/MAP kinase pathways and the activation of downstream transcriptional mechanisms in MPNSTs from NF1 patients.
Three-dimensional Overlay Culture Models of Human Breast Cancer Reveal a Critical Sensitivity to Mitogen-activated Protein Kinase Kinase Inhibitors
Tumor cells that are grown in three-dimensional (3D) cell culture exhibit relative resistance to cytotoxic drugs compared with their response in conventional two-dimensional (2D) culture. We studied the effects of targeted agents and doxorubicin on 2D and 3D cultures of human breast cell lines that represent the progression from normal epithelia (modeled by MCF10A cells) through hyperplastic variants to a dysplastic/carcinoma phenotype (MCF10.DCIS cells), variants transformed by expression of activated Ras, and also a basal-subtype breast carcinoma cell line (MDA-MB-231). The results showed the expected relative resistance to the cytotoxic agent doxorubicin in 3D cultures, with greater resistance in normal and hyperplastic cells than in carcinoma models. However, the response to the targeted inhibitors was more complex. Inhibition of mitogen-activated protein kinase kinase (MEK) by either 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0126) or 2-(2-chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzamide (CI-1040, PD184352) produced a similar inhibition of the growth of all the MCF10 cell lines in 2D. In 3D culture, the normal and hyperplastic models exhibited some resistance, whereas the carcinoma models became far more sensitive to MEK inhibition. Increased sensitivity to MEK inhibition was also seen in MDA-MB-231 cells grown in 3D compared with 2D. In contrast, inhibition of phosphatidylinositol 3'-kinase activity by wortmannin had no significant effect on the growth of any of the cells in either 2D or 3D. Our conclusion is that 3D culture models may not only model the relative resistance of tumor cells to cytotoxic therapy but also that the 3D approach may better identify the driving oncogenic pathways and critical targeted inhibitors that may be effective treatment approaches.
A Novel Geranylgeranyl Transferase Inhibitor in Combination with Lovastatin Inhibits Proliferation and Induces Autophagy in STS-26T MPNST Cells
Prenylation inhibitors have gained increasing attention as potential therapeutics for cancer. Initial work focused on inhibitors of farnesylation, but more recently geranylgeranyl transferase inhibitors (GGTIs) have begun to be evaluated for their potential antitumor activity in vitro and in vivo. In this study, we have developed a nonpeptidomimetic GGTI, termed GGTI-2Z [(5-nitrofuran-2-yl)methyl-(2Z,6E,10E)-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraenyl 4-chlorobutyl(methyl)phosphoramidate], which in combination with lovastatin inhibits geranylgeranyl transferase I (GGTase I) and GGTase II/RabGGTase, without affecting farnesylation. The combination treatment results in a G(0)/G(1) arrest and synergistic inhibition of proliferation of cultured STS-26T malignant peripheral nerve sheath tumor cells. We also show that the antiproliferative activity of drugs in combination occurs in the context of autophagy. The combination treatment also induces autophagy in the MCF10.DCIS model of human breast ductal carcinoma in situ and in 1c1c7 murine hepatoma cells, where it also reduces proliferation. At the same time, there is no detectable toxicity in normal immortalized Schwann cells. These studies establish GGTI-2Z as a novel geranylgeranyl pyrophosphate derivative that may work through a new mechanism involving the induction of autophagy and, in combination with lovastatin, may serve as a valuable paradigm for developing more effective strategies in this class of antitumor therapeutics.
Aborted Autophagy and Nonapoptotic Death Induced by Farnesyl Transferase Inhibitor and Lovastatin
Exposure of the human malignant peripheral nerve sheath tumor cell lines STS-26T, ST88-14, and NF90-8 to nanomolar concentrations of both lovastatin and farnesyl transferase inhibitor (FTI)-1 but not to either drug alone induced cell death. ST88-14 and NF90-8 cells underwent apoptosis, yet dying STS-26T cells did not. FTI-1 cotreatment induced a strong and sustained autophagic response as indicated by analyses of microtubule-associated protein-1 light chain 3 (LC3)-II accumulation in STS-26T cultures. Extensive colocalization of LC3-positive punctate spots was observed with both lysosome-associated membrane protein (LAMP)-1 and LAMP-2 (markers of late endosomes/lysosomes) in solvent or FTI-1 or lovastatin-treated STS-26T cultures but very little colocalization in lovastatin/FTI-1-cotreated cultures. The absence of colocalization in the cotreatment protocol correlated with loss of LAMP-2 expression. Autophagic flux studies indicated that lovastatin/FTI-1 cotreatment inhibited the completion of the autophagic program. In contrast, rapamycin induced an autophagic response that was associated with cytostasis but maintenance of viability. These studies indicate that cotreatment of STS-26T cells with lovastatin and FTI-1 induces an abortive autophagic program and nonapoptotic cell death.
Angiotensin II-dependent Phosphorylation at Ser-11/Ser-18 and Ser-938 Shift the E2 Conformations of Rat Kidney Na,K-ATPase
Kidney plasma membranes, which contain a single a-1 isoform of Na,K-ATPase, simultaneously contain two sub-conformations of EP2, differing in their rate of digoxin release in response to Na and ATP. Treating cells with angiotensin II (Ang II) somehow changes the conformation of both, because it differentially inhibits the rate of digoxin release. We test if Ang II regulates release by increasing phosphorylation at Ser-11/Ser-18 and Ser-938. Opossum kidney cells co-expressing the AT1a receptor and either alpha-1.wild-type, alpha-1.S11A/S18A or alpha-1.S938A were treated ± 10 nM Ang II for 5 min, increasing phosphorylation at the three sites. Na,K-ATPase was bound to digoxin-affinity columns in the presence of Na, ATP, and Mg. A solution containing 30 mM NaCl and 3 mM ATP eluted ~20% of bound untreated Na,K-ATPase (Population #1). Pre-treating cells with Ang II slowed the elution of Population #1 in alpha-1.wild-type and alpha-1.S938A, but not alpha-1.S11A/S18A cells. Another 50% of bound Na,K-ATPase (Population #2) was subsequently eluted in two phases by a solution containing 150 mM NaCl and 3 mM ATP. Ang II increased the initial rate and slowed the second phase in alpha-1.wild-type, but not a-1.S938A cells. Thus, Ang II changes the conformation of two forms of EP2 via differential phosphorylation.
