Drp1/Fis1-mediated mitochondrial fragmentation leads to lysosomal dysfunction in cardiac models of Huntington's disease.

Huntington's disease (HD) is a fatal hereditary neurodegenerative disorder, best known for its clinical triad of progressive motor impairment, cognitive deficits and psychiatric disturbances, is caused by CAG-repeat expansion in exon 1 of Huntingtin (HTT). However, in addition to the neurological disease, mutant HTT (mHTT), which is ubiquitously expressed in all tissues, impairs other organ systems. Not surprisingly, cardiovascular dysautonomia as well as the deterioration of circadian rhythms are among the earliest detectable pathophysiological changes in individuals with HD. Mitochondrial dysfunction in the brain and skeletal muscle in HD has been well documented, as the disease progresses. However, not much is known about mitochondrial abnormalities in the heart. In this study, we describe a role for Drp1/Fis1-mediated excessive mitochondrial fission and dysfunction, associated with lysosomal dysfunction in H9C2 expressing long polyglutamine repeat (Q73) and in human iPSC-derived cardiomyocytes transfected with Q77. Expression of long polyglutamine repeat led to reduced ATP production and mitochondrial fragmentation. We observed an increased accumulation of damaged mitochondria in the lysosome that was coupled with lysosomal dysfunction. Importantly, reducing Drp1/Fis1-mediated mitochondrial damage significantly improved mitochondrial function and cell survival. Finally, reducing Fis1-mediated Drp1 recruitment to the mitochondria, using the selective inhibitor of this interaction, P110, improved mitochondrial structure in the cardiac tissue of R6/2 mice. We suggest that drugs focusing on the central nervous system will not address mitochondrial function across all organs, and therefore will not be a sufficient strategy to treat or slow down HD disease progression.

Phorbol myristate acetate-mediated stimulation of transcytosis and apical recycling in MDCK cells.

We observed that phorbol myristate acetate (PMA) stimulates transcytosis of the polymeric immunoglobulin receptor (pIgR) in MDCK cells. Apical release of pre-endocytosed ligand (dimeric IgA) bound to the pIgR can be stimulated twofold within 7 min of addition of PMA while recycling of the ligand from the basal surface is not affected. In addition, apical surface delivery of pIgR and cleavage of its ectodomain to secretory component (SC) is also stimulated by PMA. The recycling of apically internalized ligand back to the apical surface is similarly stimulated. These results suggest that the stimulation of apical delivery is from an apical recycling compartment. The effect of PMA suggests that protein kinase C (PKC) is involved in the regulation of pIgR trafficking in MDCK cells. To test this we down regulated PKC activity by pre-treating cells with PMA for 16 h and observed that transcytosis could no longer be stimulated by PMA. Western blots show that the PKC isozymes alpha and to a lesser extent epsilon, are depleted from MDCK cells which have been pre-treated with PMA for 16 h and that treatment of MDCK cells with PMA for 5 min causes a dramatic translocation of the PKC alpha isozyme and a partial translocation of the epsilon isozyme from the cytosol to the membrane fraction of cell homogenates. This translocation suggests that the alpha and/or epsilon isozymes may be involved in PMA mediated stimulation of transcytosis. A mutant pIgR in which serines 664 and 726, the major sites of phosphorylation, are replaced by alanine is stimulated to transcytose by PMA, suggesting that phosphorylation of pIgR at these sites is not required for the effect of PMA. These results suggest that PMA-mediated stimulation of pIgR transcytosis may involve the activation of PKC alpha and/or epsilon, and that this stimulation occurs independently of the major phosphorylation sites on the pIgR. Finally, PMA stimulates transcytosis of basolaterally internalized transferrin, suggesting that PMA acts to generally stimulate delivery of endocytosed proteins to the apical surface.

Signal transduction by the polymeric immunoglobulin receptor suggests a role in regulation of receptor transcytosis.

Many membrane traffic events that were previously thought to be constitutive recently have been found to be regulated by a variety of intracellular signaling pathways. The polymeric immunoglobulin receptor (pIgR) transcytoses dimeric IgA (dIgA) from the basolateral to the apical surface of polarized epithelial cells. Transcytosis is stimulated by binding of dIgA to the pIgR, indicating that the pIgR can transduce a signal to the cytoplasmic machinery responsible for membrane traffic. We report that dIgA binding to the pIgR causes activation of protein kinase C (PKC) and release of inositol 1,4,5-trisphosphate (IP3). The IP3 causes an elevation of intracellular Ca. Artificially activating PKC with phorbol myristate acetate or poisoning the calcium pump with thapsigargin stimulates transcytosis of pIgR, while the intracellular Ca chelator BAPTA-AM inhibits transcytosis. Our data suggest that ligand-induced signaling by the pIgR may regulate membrane traffic via well-known second messenger pathways involving PKC, IP3, and Ca. This may be a model of a general means by which membrane traffic is regulated by receptor-ligand interaction and signaling pathways.

Localization of protein kinases by anchoring proteins: a theme in signal transduction.

A fundamental question in signal transduction is how stimulation of a specific protein kinase leads to phosphorylation of particular protein substrates throughout the cell. Recent studies indicate that specific anchoring proteins located at various sites in the cell compartmentalize the kinases to their sites of action. Inhibitors of the interactions between kinases and their anchoring proteins inhibit the functions mediated by the kinases. These data indicate that the location of these anchoring proteins provides some of the specificity of the responses mediated by each kinase and suggest that inhibitors of the interaction between the kinases and their anchoring proteins may be useful as therapeutic agents.

Islet cell survival during isolation improved through protein kinase C epsilon activation.

Strategies inhibiting cell death signaling pathways may enhance the availability of islet transplantation for patients with type 1 diabetes mellitus. The epsilon isoform of protein kinase C (PKC epsilon) has been shown to have an anti-apoptotic effect in many cell types. The present study investigated whether activation of PKC epsilon may improve the yield of functional islet cells for transplantation. Islet cells were isolated from wild-type BALB/c mice preconditioned with either a PKC epsilon activator (psi epsilon RACK) or a TAT carrier control peptide and further treated with the same agents during isolation and in vitro for either 0, 1, 16, or 40 hours. Islet cells were assessed at each time point for viability, apoptosis, and function. psi epsilon RACK-treated islets showed significantly decreased islet cell death up to 40 hours after isolation compared with TAT-treated control islets. Beta-cell function in response to high glucose challenge remained unchanged.

Peptide modulators of protein-protein interactions in intracellular signaling.

Signal transduction cascades involve multiple enzymes and are orchestrated by selective protein-protein interactions that are essential for the progression of intracellular signaling events. Modulators of these protein-protein interactions have been used to dissect the role of individual components of each signaling cascade. We describe several methods that have been developed for the identification of peptides that inhibit the interaction between signaling proteins and hence selectively modulate their functions. Such peptide modulators provide important tools for basic research and have great potential as leads for the development of new classes of therapeutic drugs.

Cardiotrophic effects of protein kinase C epsilon: analysis by in vivo modulation of PKCepsilon translocation.

Protein kinase C (PKC) is a key mediator of many diverse physiological and pathological responses. Although little is known about the specific in vivo roles of the various cardiac PKC isozymes, activation-induced translocation of PKC is believed to be the primary determinant of isozyme-specific functions. Recently, we have identified a catalytically inactive peptide translocation inhibitor (epsilonV1) and translocation activator (psiepsilonRACK [receptors for activated C kinase]) specifically targeting PKCepsilon. Using cardiomyocyte-specific transgenic expression of these peptides, we combined loss- and gain-of-function approaches to elucidate the in vivo consequences of myocardial PKCepsilon signaling. As expected for a PKCepsilon RACK binding peptide, confocal microscopy showed that epsilonV1 decorated cross-striated elements and intercalated disks of cardiac myocytes. Inhibition of cardiomyocyte PKCepsilon by epsilonV1 at lower expression levels upregulated alpha-skeletal actin gene expression, increased cardiomyocyte cell size, and modestly impaired left ventricular fractional shortening. At high expression levels, epsilonV1 caused a lethal dilated cardiomyopathy. In contrast, enhancement of PKCepsilon translocation with psiepsilonRACK resulted in selectively increased beta myosin heavy chain gene expression and normally functioning concentric ventricular remodeling with decreased cardiomyocyte size. These results identify for the first time a role for PKCepsilon signaling in normal postnatal maturational myocardial development and suggest the potential for PKCepsilon activators to stimulate "physiological" cardiomyocyte growth.

Adaptor proteins in protein kinase C-mediated signal transduction.

Spatial and temporal organization of signal transduction is essential in determining the speed and precision by which signaling events occur. Adaptor proteins are key to organizing signaling enzymes near their select substrates and away from others in order to optimize precision and speed of response. Here, we describe the role of adaptor proteins in determining the specific function of individual protein kinase C (PKC) isozymes. These isozyme-selective proteins were called collectively RACKs (receptors for activated C-kinase). The role of RACKs in PKC-mediated signaling was determined using isozyme-specific inhibitors and activators of the binding of each isozyme to its respective RACK. In addition to anchoring activated PKC isozymes, RACKs anchor other signaling enzymes. RACK1, the anchoring protein for activated betaIIPKC, binds for example, Src tyrosine kinase, integrin, and phosphodiesterase. RACK2, the epsilonPKC-specific RACK, is a coated-vesicle protein and thus is involved in vesicular release and cell-cell communication. Therefore, RACKs are not only adaptors for PKC, but also serve as adaptor proteins for several other signaling enzymes. Because at least some of the proteins that bind to RACKs, including PKC itself, regulate cell growth, modulating their interactions with RACKs may help elucidate signaling pathways leading to carcinogenesis and could result in the identification of novel therapeutic targets.

Molecular transporters for peptides: delivery of a cardioprotective epsilonPKC agonist peptide into cells and intact ischemic heart using a transport system, R(7).

BACKGROUND: Recently, we reported a novel oligoguanidine transporter system, polyarginine (R(7)), which, when conjugated to spectroscopic probes (e.g., fluorescein) and drugs (e.g., cyclosporin A), results in highly water-soluble conjugates that rapidly enter cells and tissues. We report herein the preparation of the first R(7) peptide conjugates and a study of their cellular and organ uptake and functional activity. The octapeptide (psi)(epsilon)RACK was selected for this study as it is known to exhibit selective epsilon protein kinase C isozyme agonist activity and to reduce ischemia-induced damage in cardiomyocytes. However, (psi)(epsilon)RACK is not cell-permeable. RESULTS: Here we show that an R(7)-(psi)(epsilon)RACK conjugate readily enters cardiomyocytes, significantly outperforming (psi)(epsilon)RACK conjugates of the transporters derived from HIV Tat and from Antennapedia. Moreover, R(7)-(psi)(epsilon)RACK conjugate reduced ischemic damage when delivered into intact hearts either prior to or after the ischemic insult. CONCLUSIONS: Our data suggest that R(7) converts a peptide lead into a potential therapeutic agent for the ischemic heart.

Arachidonic acid protects neonatal rat cardiac myocytes from ischaemic injury through epsilon protein kinase C.

OBJECTIVES: Arachidonic acid is a second messenger which activates protein kinase C (PKC) and is released from the heart during ischaemic preconditioning. The purpose of this study was to examine the effect of arachidonic acid on activation of PKC in cardiac myocytes and the cellular consequences. METHODS: Neonatal rat cardiac myocytes were isolated and maintained in culture. Arachidonic acid-induced activation of PKC was examined by cell fractionation and western blot analysis. Contraction frequency was measured by visual inspection under a microscope. Ischaemia was simulated by subjecting cells to an atmosphere of lower than 0.5% oxygen in the absence of glucose and cell damage determined by release of cytosolic lactate dehydrogenase or direct cell viability assay. RESULTS: Arachidonic acid resulted in translocation of delta and epsilonPKC but not alpha, betaII, eta or zetaPKC isozymes, indicating activation of only delta and epsilonPKC. Arachidonic acid induced a dose-dependent decrease in spontaneous contraction rate of cardiac myocytes which was blocked by a selective peptide translocation inhibitor of epsilonPKC. Pretreatment with arachidonic acid partially protected cardiac myocytes against ischaemia. Down-regulation of PKC with 24 h 4beta-phorbol,12-myristate,13-acetate treatment, inhibition of PKC by chelerythrine and selective inhibition of epsilonPKC translocation all decreased the protective effect of arachidonic acid. Pretreatment with eicosapentaenoic acid or oleic acid also protected cardiac myocytes against ischaemia. CONCLUSIONS: These results demonstrate that arachidonic acid selectively activates delta and epsilonPKC in neonatal rat cardiac myocytes, leading to protection from ischaemia. We suggest this is a potential mechanism of PKC activation during PC. In addition, our results suggest that different classes of free fatty acid directly exert cardioprotection from ischaemic injury in cardiac myocytes.

Differential activation of protein kinase C isozymes by phorbol ester and collagen in human skin microvascular endothelial cells.

Human dermal microvascular endothelial cells participate in activities including inflammation, wound healing, and angiogenesis (neovascularization). Two stages of angiogenesis can be mimicked in vitro by two models of cultured foreskin human dermal microvascular endothelial cells: the differentiation of epithelioid endothelial cells to spindle-shaped mesenchymal-like cells induced by phorbol ester treatment; and the formation of vascular channels induced by exposing the luminal surface of endothelial cell monolayers to type I collagen gels. The mechanisms underlying these two processes, however, are largely unknown. Protein kinase C isozymes, which are activated by phorbol esters, are important mediators in the angiogenic process. In the current work, we identified the protein kinase C isozymes present in human dermal microvascular endothelial cells and determined which of the isozymes are activated in response to phorbol ester or to collagen treatments. Using western blot analysis, we found that microvascular endothelial cells contain at least six protein kinase C isozymes (alpha, beta, delta, epsilon, zeta, eta). Immunocytochemical studies demonstrated that the isozymes are located in distinct cellular compartments and that following treatment with phorbol 12-myristate 13-acetate or with a collagen gel overlay, most isozymes (protein kinase C alpha, beta1, betaII, delta, epsilon, eta) translocated to different parts of the cell. Moreover, for two of these isozymes (betaII and eta), the localization differs after phorbol 12-myristate 13-acetate treatment as compared with collagen treatment. These results demonstrate that agents that mimic two stages in the angiogenic process in vitro initiate diverse changes in the subcellular localization of specific protein kinase C isozymes and suggest a role for different isozymes in this process.

An anti-acetylcholine receptor monoclonal antibody cross-reacts with phosvitin.

Rabbit and mouse anti-Torpedo acetylcholine receptor antibodies cross-reacted partially with the highly phosphorylated protein, phosvitin. We have selected an anti-Torpedo acetylcholine receptor monoclonal antibody which binds specifically to phosvitin; this binding is inhibited by acetylcholine receptor. These findings suggest that a phosphorylated amino acid residue may be a part of the determinant on the acetylcholine receptor recognized by this monoclonal antibody.

Characterization of the binding and phosphorylation of cardiac calsequestrin by epsilon protein kinase C.

In this study, we report the cloning of the rat cardiac isoform of calsequestrin on the basis of its interaction with an epsilonprotein kinase C-unique sequence (epsilonV1) derived form the epsilonprotein kinase C regulatory domain. Calsequestrin binds activated epsilonprotein kinase C holoenzyme better than the inactive enzyme and nearly three times better than other protein kinase C isozymes. The interaction between epsilonprotein kinase C and calsequestrin is mediated by sequences in both the regulatory and kinase domains of the epsilonprotein kinase C. Finally, we show that calsequestrin is an epsilonprotein kinase C substrate in vitro and protein kinase C phosphorylation of calsequestrin leads to a decreased binding of epsilonprotein kinase C to calsequestrin.

Chronic ethanol-induced heterologous desensitization is mediated by changes in adenosine transport.

Chronic exposure of cultured cell lines to ethanol results in a heterologous desensitization of receptors coupled to adenylate cyclase via GS, the stimulatory guanine nucleotide regulatory protein. This heterologous desensitization is accompanied by a decrease in alpha S, the GTP-binding subunit of GS. Ethanol-induced accumulation of extracellular adenosine is required for the development of heterologous desensitization. To determine the mechanism underlying the ethanol-dependent increase in extracellular adenosine, we investigated the effects of ethanol on the nucleoside transporter responsible for the bidirectional transport of adenosine into and out of the cell. We found that ethanol specifically and non-competitively inhibited nucleoside uptake. Inhibition of adenosine uptake was primarily due to decreased influx via the nucleoside transporter. Thus, ethanol-induced increases in extracellular adenosine appear to be due to inhibition of adenosine influx. After chronic exposure to ethanol, cells became tolerant to the acute effects of ethanol, i.e. ethanol no longer inhibited uptake and, consequently, no longer increased extracellular adenosine concentration. Taken together with our previous studies, these results suggest that acute ethanol inhibition of adenosine influx leads to an increase in extracellular adenosine which in turn activates adenosine A2 receptors to increase cyclic AMP levels, leading to desensitization of receptor-dependent cyclic AMP signal transduction after chronic exposure to ethanol. We next determined whether the same effects of ethanol also occur in alcoholics. We isolated lymphocytes from alcoholics and non-alcoholics and found that alcoholics had a 75% decrease in basal and adenosine receptor-stimulated cyclic AMP production compared with non-alcoholics. To determine whether these differences were due to exposure to ethanol in vivo or to a possible genetic difference between alcoholics and non-alcoholics, we grew lymphocytes in culture in the absence of ethanol. Adenosine receptor-stimulated cyclic AMP levels were higher in alcoholics than non-alcoholics. Moreover, cultured cells from alcoholics were more sensitive to the effects of chronic alcohol on cyclic AMP signal transduction than cells from non-alcoholics. Our results suggest that the cyclic AMP signal transduction system may reflect a genetic alteration in alcoholics and that studies in cultured lymphocytes may allow us to identify individuals at risk of developing alcoholism.

Antigenic specificity of acetylcholine receptor in developing muscle. Studies with monoclonal antibodies.

Monoclonal antibodies (mcAbs) elicited against the nicotinic acetylcholine receptor (AChR) from Torpedo, were used to follow antigenic changes in AChR during muscle development. Newborn rat muscle and denervated mouse muscle were used as sources of extrajunctional AChR; adult innervated rat and mouse muscle were used as sources of junctional AChR. Most of the mcAbs tested reacted preferably, but not exclusively with extrajunctional AChR (EJR), as compared to junctional AChR (JR). None was found to react with only one of the two forms of AChR. We conclude that the anti-AChR monoclonal antibodies used in this study detect antigenic determinants which are shared by EJR and JR, but which probably undergo structural changes during muscle development.

The identification and purification of a mammalian-like protein kinase C in the yeast Saccharomyces cerevisiae.

We have purified a yeast protein kinase that is phospholipid-dependent and activated by Diacylglycerol (DAG) in the presence of Ca2+ or by the tumour-promoting agent tetradecanoyl-phorbol acetate (TPA). The properties of this enzyme are similar to those of the mammalian protein kinase C (PKC). The enzyme was purified using chromatography on DEAE-cellulose followed by hydroxylapatite. The latter chromatography separated the activity to three distinguishable sub-species, analogous to the mammalian PKC isoenzymes. The fractions enriched in PKC activity contain proteins that specifically bind TPA, are specifically phosphorylated in the presence of DAG and recognized by anti-mammalian PKC antibodies.

Developmental expression of protein kinase C subspecies in rat brain-pituitary axis.

We have examined the neonatal developmental expression of protein kinase C subspecies (PKCs) in rat brain, pituitary glands and cells by enzymatic activity assays, immunohistochemistry and Western blot analysis with type-specific antibodies. A very large increase (455%) was noticed in brain PKC activity during the first week of life with the particulate fraction (22% of total enzyme activity on day 1) increasing dramatically (900%) during the first week to 50% of enzyme activity. In contrast, the pituitary gland showed high activity on day 1 that decreased progressively to reach the lowest levels at 1 year of age. Paradoxically, the number of pituitary cells immunolabeled for PKC increases as a function of age. Western blot analysis showed only small changes in PKC alpha, PKC beta and PKC epsilon when brains from 6-day-old and 3-month-old female rats were compared, whereas PKC tau and PKC delta increased markedly during this period. On the other hand, brain PKC zeta decreased between 6 days and 3 months of age. Western blot analysis showed no major changes in pituitary PKC alpha, PKC beta and PKC zeta when 6-day-old and 3-month-old female rats were compared, while PKC tau was not detected. The major band of pituitary PKC delta (76 kDa) decreased markedly between 6 days and 3 months of age whereas the minor band (68 kDa) did not change.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of adenosine and adenosine transport in ethanol-induced cellular tolerance and dependence. Possible biologic and genetic markers of alcoholism.

Acute exposure to ethanol in culture inhibits adenosine uptake into cells, thereby increasing the concentration of extracellular adenosine. Extracellular adenosine then reacts with adenosine A2 receptors to stimulate intracellular cAMP production. During prolonged exposure to ethanol, the increase in cAMP is followed by the development of heterologous desensitization of receptors coupled to adenylyl cyclase via Gs, the stimulatory GTP-binding protein. Ethanol-induced heterologous desensitization appears to be due to a reduction in mRNA and protein for G alpha s, a subunit of Gs. This is an example of cellular dependence on ethanol. The important implication of these findings is that a selective inhibitory effect of ethanol on adenosine uptake can lead to desensitization of diverse receptors coupled to cAMP production. Such changes could contribute to the pleiotropic effects of ethanol in the brain and other organs. Prolonged exposure to ethanol also alters the nucleoside transport system. While ethanol inhibits adenosine uptake into naive cells, ethanol no longer inhibits adenosine uptake into cells that have adapted to ethanol. This resistance to ethanol inhibition appears to be a form of cellular tolerance to ethanol. Thus, there appears to be a synergism between ethanol-induced heterologous desensitization of receptor-stimulated cAMP production (cellular dependence) and resistance to ethanol inhibition of adenosine uptake (cellular tolerance), because both lead to reduced intracellular levels of cAMP. Our studies on cAMP signal transduction in cell culture are directly relevant to the pathophysiology of human alcoholism. Heterologous desensitization of cAMP production is demonstrable in lymphocytes taken from actively drinking alcoholics; this measurement appears to be a biologic marker of active alcohol consumption. In addition, regulation of adenosine receptor-dependent cAMP production may be altered in patients at risk to develop alcoholism because of genetic factors. Thus, lymphocytes from alcoholics cultured many generations in the absence of ethanol show increased adenosine receptor-dependent cAMP production and increased sensitivity to ethanol-induced heterologous desensitization. These persistent phenotypic abnormalities in cell culture could be used as genetic markers for alcoholism. Studies are under way to test this possibility.

Prolonged phorbol ester treatment down-regulates protein kinase C isozymes and increases contraction rate in neonatal cardiac myocytes.

We have determined the effects of chronic exposure to the protein kinase C (PKC) activating drug 4-beta phorbol 12-myristate-13-acetate (PMA) on PKC isozymes and the rate of spontaneous contraction in neonatal rat cardiac myocytes in culture. Western blot analyses revealed that a two day exposure to 0.1-1 nM PMA increased the total amount of delta PKC, whereas, 100 nM PMA concentrations caused a complete down-regulation of the alpha PKC and an 80 kDa zeta PKC-like protein. In addition, 100 nM PMA treatment for 2 days did not result in complete down-regulation of the beta, delta, and epsilon PKC isozymes in Western blot and immunocytochemical studies. We also found a PKC-mediated enhancement of the rate of contraction in these cells following prolonged exposure to PMA (1-100nM). Our studies suggest that this enhancement of contraction rate may be mediated by the beta, delta, or epsilon PKC isozymes. A better understanding of the role(s) of PKC isozymes in the modulation of cardiac functions may reveal selective targets for therapies of cardiac disorders.

Proteins kinase Cɛ is required for non-small cell lung carcinoma growth and regulates the expression of apoptotic genes.

Protein kinase C (PKC)ɛ, a member of the novel PKC family, has key roles in mitogenesis and survival in normal and cancer cells. PKCɛ is frequently overexpressed in epithelial cancers, particularly in lung cancer. Using a short-hairpin RNA approach, here we established that PKCɛ is required for non-small cell lung carcinoma (NSCLC) growth in vitro as well as tumor growth when inoculated into athymic mice. Moreover, sustained delivery of a PKCɛ-selective inhibitor peptide, ɛV1-2, reduced xenograft growth in mice. Both RNA interference depletion and pharmacological inhibition of PKCɛ caused a marked elevation in the number of apoptotic cells in NSCLC tumors. PKCɛ-depleted NSCLC cells show elevated expression of pro-apoptotic proteins of the Bcl-2 family, caspase recruitment domain-containing proteins and tumor necrosis factor ligands/receptor superfamily members. Moreover, a Gene Set Enrichment Analysis revealed that a vast majority of the genes changed in PKCɛ-depleted cells were also deregulated in human NSCLC. Our results strongly suggest that PKCɛ is required for NSCLC cell survival and maintenance of NSCLC tumor growth. Therefore, PKCɛ may represent an attractive therapeutic target for NSCLC.