PKB Activation Research Articles

The GPCR OGR1 (GPR68) mediates diverse signalling and contraction of airway smooth muscle in response to small reductions in extracellular pH

Previous studies have linked a reduction in pH in airway, caused by either environmental factors, microaspiration of gastric acid or inflammation, with airway smooth muscle (ASM) contraction and increased airway resistance. Neural mechanisms have been shown to mediate airway contraction in response to reductions in airway pH to < 6.5; whether reduced extracellular pH (pHo) has direct effects on ASM is unknown. Intracellular signalling events stimulated by reduced pHo in human cultured ASM cells were examined by immunoblotting, phosphoinositide hydrolysis and calcium mobilization assays. ASM cell contractile state was examined using magnetic twisting cytometry. The expression of putative proton-sensing GPCRs in ASM was assessed by real-time PCR. The role of ovarian cancer G protein-coupled receptor 1 (OGR1 or GPR68) in acid-induced ASM signalling and contraction was assessed in cultures subjected to siRNA-mediated OGR1 knockdown. ASM cells responded to incremental reductions in pHo (from pH 8.0 to pH 6.8) by activating multiple signalling pathways, involving p42/p44, PKB, PKA and calcium mobilization. Coincidently, ASM cells contracted in response to decreased pHo with similar 'dose'-dependence. Real-time PCR suggested OGR1 was the only proton-sensing GPCR expressed in ASM cells. Both acid-induced signalling (with the exception of PKB activation) and contraction were significantly attenuated by knockdown of OGR1. These studies reveal OGR1 to be a physiologically relevant GPCR in ASM cells, capable of pleiotropic signalling and mediating contraction in response to small reductions in extracellular pH. Accordingly, ASM OGR1 may contribute to asthma pathology and represent a therapeutic target in obstructive lung diseases.

Integrin-linked kinase at a glance

Integrins are a large family of cell–extracellular matrix (ECM) and cell–cell adhesion molecules that regulate development and tissue homeostasis by controlling cell migration, survival, proliferation and differentiation ([Hynes, 2002][1]). They are non-covalently associated heterodimers

Protein kinase Cδ but not PKCα is involved in insulin‐induced glucose metabolism in hepatocytes

The liver is a major insulin-responsive tissue responsible for glucose regulation. One important mechanism in this phenomenon is insulin-induced glycogen synthesis. Studies in our laboratory have shown that protein kinase Cs delta (PKCδ) and alpha (α) have important roles in insulin-induced glucose transport in skeletal muscle, and that their expression and activity are regulated by insulin. Their importance in glucose regulation in liver cells is unclear. In this study we investigated the possibility that these isoforms are involved in the mediation of insulin-induced glycogen synthesis in hepatocytes. Studies were done on rat hepatocytes in primary culture and on the AML-12 (alpha mouse liver) cell line. Insulin increased activity and tyrosine phosphorylation of PKCδ within 5 min. In contrast, activity and tyrosine phosphorylation of PKCα were not increased by insulin. PKCδ was constitutively associated with IR, and this was increased by insulin stimulation. Suppression of PKCδ expression by transfection with RNAi, or overexpression of kinase dead (dominant negative) PKCδ reduced both the insulin-induced activation of PKB/Akt and the phosphorylation of glycogen synthase kinase 3 (GSK3) and reduced significantly insulin-induced glucose uptake. In addition, treatment of primary rat hepatocytes with rottlerin abrogated insulin-induced increase in glycogen synthesis. Neither overexpression nor inhibition of PKCα appeared to alter activation of PKB, phosphorylation of GSK3 or glucose uptake in response to insulin. We conclude that PKCδ, but not PKCα, plays an essential role in insulin-induced glucose uptake and glycogenesis in hepatocytes.

Glucagon‐like peptide‐1 signals to PKB and mTORC1 via the activation of the insulin‐like growth factor receptor (IGF1R) in islets of Langerhans

ObjectivesGlucagon‐like peptide‐1(GLP‐1) stimulates β‐cell proliferation and protects cells against cytokine induced apoptosis via activation of mTORC1 and PKB. However, it is not fully understood how GLP1 receptor activation leads to the activation of PKB/mTORC1 although it has been reported that this is mediated by EGFR transactivation. Therefore, study aims to investigate the transduction pathways by which GLP‐1 regulates PKB/mTORC1 activation.ResultsWe showed that that AG1478 (an EGFR inhibitor), at a dose that effectively blocks EGF‐stimulated Erk phosphorylation, does not affect GLP1‐stimulated PKB or mTORC1 activation. However, diazoxide, an inhibitor of exocytosis, significantly reduced GLP1‐induced PKB and mTORC1 activation. siRNA mediated knock‐down of insulin receptor expression was unable to inhibit GLP1‐stimulated PKB or mTORC1 activation. In contrast, siRNA mediated knockdown of the IGF1 receptor effectively blocked GLP1‐stimulated PKB and mTORC1 activation.ConclusionsAcute activation of PKB and mTORC1 by GLP1 is mediated via the activation of the IGF1 receptor, presumably through the autocrine effect of IGF.

Endothelin‐1 (ET‐1)‐induced Early Growth Response Factor‐1 Expression in Vascular Smooth Muscle Cells (VSMC) requires c‐SRC and ERK1/2 activation

Egr‐1 transcription factor plays an important role in vascular biology. Following activation, Egr‐1 is expressed in the nucleus and can regulate transcription of several genes implicated in the development of vascular disease (VD). A potential role of aberrant ET‐1 signaling is thought to contribute to the development of VD, such as atherosclerosis, through the hyperactivation of growth promoting pathways, including PI3K/PKB and MAPK pathways, as well as regulation of multiple transcription factors. We have previously shown that several protein tyrosine kinases are upstream regulators of ET‐1‐induced activation of PKB in VSMC, however, a role of ET‐1 on Egr‐1 expression and its modulation by signaling components activated by ET‐1 remains unclear. Therefore, we have investigated the involvement of IGF‐1R, c‐Src and MAPKs in ET‐1‐induced Egr‐1 expression. ET‐1 time‐dependently enhanced Egr‐1 protein levels in VSMC. AG1024 and PP2, selective pharmacological inhibitors of IGF‐1R and c‐Src, respectively, inhibited ET‐1‐induced Egr‐1 expression. PD98059, a selective inhibitor of MEK and ERK1/2, also inhibited ET‐1‐ induced Egr‐1 expression in VSMC, however, SB202190, a highly selective inhibitor of p38mapk, failed to do so. In summary, these data demonstrate that ET‐1 potently induces the expression of Egr‐ 1 through a pathway implicating IGF‐1R and c‐Src, as well as ERK1/2 in VSMC. (Supported by CIHR grants)

Anti-diabetic activity of extract from Persea americana Mill. leaf via the activation of protein kinase B (PKB/Akt) in streptozotocin-induced diabetic rats

Ethnopharmacological relevanceThe leaves of Persea americana Mill. (Lauraceae) have been popularly used in the treatment of diabetes in countries in Latin America and Africa. Aim of the studyTo investigate the hypoglycaemic properties and to determine the molecular mechanism by which the hydroalcoholic extract of the leaves of Persea americana reduce blood glucose levels in streptozotocin (STZ)-induced diabetes in rats via the enzymatic pathway of protein kinase B (PKB/Akt). MethodsThe hydroalcoholic extract of the leaves of Persea americana (0.15 and 0.3g/kg/day), vehicle and metformin (0.5g/kg/day) were administered orally to STZ-diabetic rats (n=7/group) for 4 weeks. Changes in body weight, food and water intake, fasting glucose levels and oral glucose tolerance were evaluated. Phosphorylation and the expression of PKB in the liver and soleus muscle were determined by Western blot. ResultsThe hydroalcoholic extract of the leaves of Persea americana reduced blood glucose levels and improved the metabolic state of the animals. Additionally, PKB activation was observed in the liver and skeletal muscle of treated rats when compared with untreated rats. ConclusionThe results indicate that the hydroalcoholic extract of the leaves of Persea americana has anti-diabetic properties and possibly acts to regulate glucose uptake in liver and muscles by way of PKB/Akt activation, restoring the intracellular energy balance.

Ursolic acid induces apoptosis in human leukaemia cells and exhibits anti-leukaemic activity in nude mice through the PKB pathway.

Ursolic acid (UA) has been extensively used as an anti-leukaemic agent in traditional Chinese medicine. In the present study, we investigated the ability of UA to induce apoptosis in human leukaemia cells in relation to its effects on caspase activation, Mcl-1 down-regulation and perturbations in stress-induced signalling pathways such as PKB and JNK. Leukaemia cells were treated with UA after which apoptosis, caspase activation, PKB and JNK signalling pathways were evaluated. The anti-tumour activity of UA was evaluated using xenograft mouse model. UA induced apoptosis in human leukaemia cells in a dose- and time-dependent manner; this was associated with caspase activation, down-regulation of Mcl-1 and inactivation of PKB accompanied by activation of JNK. Enforced activation of PKB by a constitutively active PKB construct prevented UA-mediated JNK activation, Mcl-1 down-regulation, caspase activation and apoptosis. Conversely, UA lethality was potentiated by the PI3-kinase inhibitor LY294002. Interruption of the JNK pathway by pharmacological or genetic (e.g. siRNA) attenuated UA-induced apoptosis. Furthermore, UA-mediated inhibition of tumour growth in vivo was associated with induction of apoptosis, inactivation of PKB as well as activation of JNK. Collectively, these findings suggest a hierarchical model of UA-induced apoptosis in human leukaemia cells in which UA induces PKB inactivation, leading to JNK activation and culminating in Mcl-1 down-regulation, caspase activation and apoptosis. These findings indicate that interruption of PKB/JNK pathways may represent a novel therapeutic strategy in haematological malignancies.

P070 Activation of the Th9/Th17 pathway in inflammatory bowel disease patients failing anti-TNF therapy

primed, but not IL8 stimulated PMN from CD patients was observed. Conclusions: Although initiation of apoptosis in CD PMN is normal as determined by Annexin V staining, an enhanced survival signal provided by GM-CSF-induced PKB activation may lead to a decreased cleaving of caspases, thereby rescuing PMN from the apoptotic pathway. In toto, our results suggest an enhanced GM-CSF-mediated PMN survival, which may play a role in the chronic intestinal inflammation in CD.

P069 Anti-TNF induced regulatory macrophages display high levels of autophagy

primed, but not IL8 stimulated PMN from CD patients was observed. Conclusions: Although initiation of apoptosis in CD PMN is normal as determined by Annexin V staining, an enhanced survival signal provided by GM-CSF-induced PKB activation may lead to a decreased cleaving of caspases, thereby rescuing PMN from the apoptotic pathway. In toto, our results suggest an enhanced GM-CSF-mediated PMN survival, which may play a role in the chronic intestinal inflammation in CD.

Chronic effects of palmitate overload on nutrient-induced insulin secretion and autocrine signalling in pancreatic MIN6 beta cells.

BackgroundSustained exposure of pancreatic β cells to an increase in saturated fatty acids induces pleiotropic effects on β-cell function, including a reduction in stimulus-induced insulin secretion. The objective of this study was to investigate the effects of chronic over supply of palmitate upon glucose- and amino acid-stimulated insulin secretion (GSIS and AASIS, respectively) and autocrine-dependent insulin signalling with particular focus on the importance of ceramide, ERK and CaMKII signalling.Principal FindingsGSIS and AASIS were both stimulated by >7-fold resulting in autocrine-dependent activation of protein kinase B (PKB, also known as Akt). Insulin release was dependent upon nutrient-induced activation of calcium/calmodulin-dependent protein kinase II (CaMKII) and extracellular signal-regulated kinase (ERK) as their pharmacological inhibition suppressed GSIS/AASIS significantly. Chronic (48 h, 0.4 mM) palmitate treatment blunted glucose/AA-induced activation of CaMKII and ERK and caused a concomitant reduction (∼75%) in GSIS/AASIS and autocrine-dependent activation of PKB. This inhibition could not be attributed to enhanced mitochondrial fatty acid uptake/oxidation or ceramide synthesis, which were unaffected by palmitate. In contrast, diacylglycerol synthesis was elevated suggesting increased palmitate esterification rather than oxidation may contribute to impaired stimulus-secretion coupling. Consistent with this, 2-bromopalmitate, a non-oxidisable palmitate analogue, inhibited GSIS as effectively as palmitate.ConclusionsOur results exclude changes in ceramide content or mitochondrial fatty acid handling as factors initiating palmitate-induced defects in insulin release from MIN6 β cells, but suggest that reduced CaMKII and ERK activation associated with palmitate overload may contribute to impaired stimulus-induced insulin secretion.

PINCH Proteins Regulate Cardiac Contractility by Modulating Integrin-Linked Kinase-Protein Kinase B Signaling

Integrin-linked kinase (ILK) is an essential component of the cardiac mechanical stretch sensor and is bound in a protein complex with parvin and PINCH proteins, the so-called ILK-PINCH-parvin (IPP) complex. We have recently shown that inactivation of ILK or β-parvin activity leads to heart failure in zebrafish via reduced protein kinase B (PKB/Akt) activation. Here, we show that PINCH proteins localize at sarcomeric Z disks and costameres in the zebrafish heart and skeletal muscle. To investigate the in vivo role of PINCH proteins for IPP complex stability and PKB signaling within the vertebrate heart, we inactivated PINCH1 and PINCH2 in zebrafish. Inactivation of either PINCH isoform independently leads to instability of ILK, loss of stretch-responsive anf and vegf expression, and progressive heart failure. The predominant cause of heart failure in PINCH morphants seems to be loss of PKB activity, since PKB phosphorylation at serine 473 is significantly reduced in PINCH-deficient hearts and overexpression of constitutively active PKB reconstitutes cardiac function in PINCH morphants. These findings highlight the essential function of PINCH proteins in controlling cardiac contractility by granting IPP/PKB-mediated signaling.

Increased oxidative metabolism is associated with erythroid precursor expansion in β 0-thalassaemia/Hb E disease

Erythropoiesis in β 0-thalassaemia/Hb E patients, the most common variant form of β-thalassaemia in Southeast Asia, is characterized by accelerated differentiation and over-expansion of erythroid precursor cells. The mechanism driving this accelerated expansion and differentiation remain unknown. To address this issue a proteomic analysis was undertaken to firstly identify proteins differentially expressed during erythroblast differentiation and a second analysis was undertaken to identify proteins differentially expressed between β 0-thalassaemia/Hb E erythroblasts and control erythroblasts. The majority of proteins identified as being differentially expressed between β 0-thalassaemia/Hb E and control erythroblasts were constituents of the glycolysis/TCA pathway and levels of oxidative stress correlated with the degree of erythroid expansion. A model was constructed linking these observations with previous studies showing increased phosphorylation of ERK1/2 in thalassemic erythroblasts which predicted the increased activation of PKA, PKB and PKC which Western analysis confirmed. Inhibition of PKA or PKC reduced β 0-thalassaemia/Hb E erythroblast differentiation and/or expansion. We propose that increased expansion and differentiation of β 0-thalassaemia/Hb E erythroblasts occur as a result of feedback loops acting through increased oxidative metabolism.

SCFAs Induce Mouse Neutrophil Chemotaxis through the GPR43 Receptor

Short chain fatty acids (SCFAs) have recently attracted attention as potential mediators of the effects of gut microbiota on intestinal inflammation. Some of these effects have been suggested to occur through the direct actions of SCFAs on the GPR43 receptor in neutrophils, though the precise role of this receptor in neutrophil activation is still unclear. We show that mouse bone marrow derived neutrophils (BMNs) can chemotax effectively through polycarbonate filters towards a source of acetate, propionate or butyrate. Moreover, we show that BMNs move with good speed and directionality towards a source of propionate in an EZ-Taxiscan chamber coated with fibrinogen. These effects of SCFAs were mimicked by low concentrations of the synthetic GPR43 agonist phenylacetamide-1 and were abolished in GPR43−/− BMNs. SCFAs and phenylacetamide-1 also elicited GPR43-dependent activation of PKB, p38 and ERK and these responses were sensitive to pertussis toxin, indicating a role for Gi proteins. Phenylacetamide-1 also elicited rapid and transient activation of Rac1/2 GTPases and phosphorylation of ribosomal protein S6. Genetic and pharmacological intervention identified important roles for PI3Kγ, Rac2, p38 and ERK, but not mTOR, in GPR43-dependent chemotaxis. These results identify GPR43 as a bona fide chemotactic receptor for neutrophils in vitro and start to define important elements in its signal transduction pathways.

PKB signaling and atrogene expression in skeletal muscle of aged mice

The purpose of this study was to determine if PKB signaling is decreased and contractile protein degradation is increased in extensor digitorum longus (EDL) and soleus (SOL) muscles from middle-aged (MA) and aged (AG) mice. We also examined the effect of age on atrogene expression in quadriceps muscle. PKB activity, as assessed by Thr(308) and Ser(473) phosphorylation, was significantly higher in EDL and SOL muscles from AG than MA mice. The age-related increase in PKB activity appears to be due to an increase in expression of the kinase, as PKB-α and PKB-β levels were significantly higher in EDL and SOL muscles from AG than MA mice. The phosphorylation of forkhead box 3a (FOXO3a) on Thr(32), a PKB target, was significantly higher in EDL muscles from AG than MA mice. The rate of contractile protein degradation was similar in EDL and SOL muscles from AG and MA mice. Atrogin-1 and muscle-specific RING finger protein 1 (MuRF-1) mRNA levels did not change in muscles from AG compared with MA mice, indicating that ubiquitin-proteasome proteolysis does not contribute to sarcopenia. A significant decrease in Bcl-2 and 19-kDa interacting protein 3 (Bnip3) and GABA receptor-associated protein 1 (Gabarap1) mRNA was observed in muscles from AG compared with MA mice, which may contribute to age-related contractile dysfunction. In conclusion, the mechanisms responsible for sarcopenia are distinct from experimental models of atrophy and do not involve atrogin-1 and MuRF-1 or enhanced proteolysis. Finally, a decline in autophagy-related gene expression may provide a novel mechanism for impaired contractile function and muscle metabolism with advancing age.

Apoptosis induced by overall metabolic stress converges on the Bcl-2 family proteins Noxa and Mcl-1

Apoptosis provoked by glucose shortage in dividing T cells is mediated via the BH3-only protein Noxa and inhibition of its binding partner Mcl-1. It is unknown how signals from cellular metabolism can affect the balance between Mcl-1 and Noxa and to what extent other Bcl-2 members are involved in this apoptosis cascade. Here, we defined the mechanism underlying apoptosis in relation to various types of metabolic stress. First, we established that the Noxa/Mcl-1 balance is regulated by glucose deprivation as well as by general metabolic stress, via changes in proteasome-mediated degradation of Mcl-1. Second, in contrast with cytokine-deprivation, no transcriptional modulation of Mcl-1, Puma, Bim or Noxa was observed during glucose deprivation. Third, no changes in PKB or GSK3 activity occurred and no clear role for AMPK was detected. Fourth, apoptosis triggered by nutrient deprivation was executed without signs of overt autophagy and independent of ROS production or p38 MAP kinase activity. Lastly, apoptosis under nutrient limitation could also be delayed by knock-down of Bim or overexpression of Bcl-2. In conclusion, Noxa functions in a specific apoptotic pathway that integrates overall nutrient stress, independent from attenuated PI3K/PKB signaling and without clear involvement of autophagy.

Curcumin Reduces Neoplasms in Colitis-Associated Cancer via Effects on Ilk, Beta-Catenin, Cyclin D, NF-kappa B and Pkb Activation

Curcumin Reduces Neoplasms in Colitis-Associated Cancer via Effects on Ilk, Beta-Catenin, Cyclin D, NF-kappa B and Pkb Activation

Sepsis and Mechanical Ventilation Restrain Translation Initiation in Skeletal Muscle by Inducing AMPK‐associated TSC2 Restriction of mTOR Signaling in Pigs

In skeletal muscle, AMP‐activated protein kinase (AMPK) acts as a cellular energy sensor of AMP:ATP and modulates translation by repressing mammalian target of rapamycin (mTOR) activation. Endotoxin (LPS)‐induced sepsis reduces muscle protein synthesis by blunting translation initiation. We hypothesize that mechanical ventilation and sepsis restrain translation in skeletal muscle by activation of AMPK. Neonatal pigs (n=8/group) were subjected to mechanical ventilation for 9 h in the absence (MV) and presence of LPS (LPSMV). AMPK and translation activation signals were determined in longissimus dorsi muscle. Plasma insulin levels rose with MV and were greatest in LPSMV pigs. In muscle, despite higher PKB activation, MV and LPS additively reduced mTOR phosphorylation and decreased the phosphorylation of tuberous sclerosis complex 2 (TSC2) on Thr1462. AMPK phosphorylation increased during MV and was augmented further in LPSMV pigs. TSC2 phosphorylation on the AMPK site, Ser1387, increased in MVLPS. MV decreased eIF4G·eIF4E association, which was further decreased by LPS. These findings suggest that both MV and sepsis increase AMPK activation in peripheral skeletal muscle, leading to inhibition of TSC2‐mediated mTOR‐dependent translation signaling when MV and sepsis coexist. NIH AR51563, NIH AR44474, and USDA 6250‐51000‐040.

Functional interactions between PI3-kinase and NF-κB signaling pathways promote joint destruction in rheumatoid arthritis

Background and objectivesRheumatoid arthritis (RA) synovium is characterised by hyperplasia of fibroblast-like synoviocytes (FLS), which display properties of transformed cells. Regulation of proto-oncogenic phosphatidylinositol 3-kinase (PI3-K) signaling by components of...

Phosphoinositide 3-kinase as a novel functional target for the regulation of the insulin signaling pathway by SIRT1

The protein deacetylase SIRT1, and its activator resveratrol, exert beneficial effects on glucose metabolism. Different SIRT1 targets have been identified, including PTP1B, AMPK, FOXO, PGC-1α and IRS2. The latter may underscore a tight link between SIRT1 and insulin signaling components. However, whether SIRT1 has a direct effect on insulin resistance and whether resveratrol acts directly or indirectly in this context is still a matter of controversy and this question has not been addressed in muscle cells.Here, we show that SIRT1 protein expression is decreased in muscle biopsies and primary myotubes derived from type 2 diabetic patients, suggesting a contribution of diminished SIRT1 in the determination of muscle insulin resistance. To investigate the functional impact of SIRT1 on the insulin pathway, the activation of insulin downstream effector PKB was evaluated after SIRT1 inactivation by RNAi, SIRT1 overexpression, or resveratrol treatments. In muscle cells and HEK293 cells, downregulation of SIRT1 reduced, while overexpression increased, insulin-induced PKB activatory phosphorylation. Further molecular characterisation revealed that SIRT1 interacts in an insulin-independent manner with the PI3K adapter subunit p85. We then investigated whether resveratrol may improve insulin signaling in muscle cells via SIRT1, or alternative targets. Incubation of muscle cells with resveratrol reverted the insulin-resistant state induced by prolonged TNFα or insulin treatment. Resveratrol-dependent improvement of insulin-resistance occurred through inhibition of serine phosphorylation of IRS1/2, implicating resveratrol as a serine kinase inhibitor. Finally, a functional interaction between PI3K and SIRT1 was demonstrated in C. elegans, where constitutively active PI3K – mimicking increased IIS signaling – lead to shortened lifespan, while removal of sir-2.1 abolished PI3K-induced lifespan shortening.Our data identify SIRT1 as a positive modulator of insulin signaling in muscle cells through PI3K, and this mechanism appears to be conserved from C. elegans through humans.

Specific posttranslational modification regulates early events in mammary carcinoma formation

The expression of an enzyme, GnT-V, that catalyzes a specific posttranslational modification of a family of glycoproteins, namely a branched N-glycan, is transcriptionally up-regulated during breast carcinoma oncogenesis. To determine the molecular basis of how early events in breast carcinoma formation are regulated by GnT-V, we studied both the early stages of mammary tumor formation by using 3D cell culture and a her-2 transgenic mouse mammary tumor model. Overexpression of GnT-V in MCF-10A mammary epithelial cells in 3D culture disrupted acinar morphogenesis with impaired hollow lumen formation, an early characteristic of mammary neoplastic transformation. The disrupted acinar morphogenesis of mammary tumor cells in 3D culture caused by her-2 expression was reversed in tumors that lacked GnT-V expression. Moreover, her-2-induced mammary tumor onset was significantly delayed in the GnT-V null tumors, evidence that the lack of the posttranslational modification catalyzed by GnT-V attenuated tumor formation. Inhibited activation of both PKB and ERK signaling pathways was observed in GnT-V null tumor cells. The proportion of tumor-initiating cells (TICs) in the mammary tumors from GnT-V null mice was significantly reduced compared with controls, and GnT-V null TICs displayed a reduced ability to form secondary tumors in NOD/SCID mice. These results demonstrate that GnT-V expression and its branched glycan products effectively modulate her-2-mediated signaling pathways that, in turn, regulate the relative proportion of tumor initiating cells and the latency of her-2-driven tumor onset.