Levels Of Receptor Tyrosine Kinases Research Articles

The prognostic and predictive values of differential expression of exosomal receptor tyrosine kinases and associated with the PI3K/AKT/mTOR signaling in breast cancer patients undergoing neoadjuvant chemotherapy.

Cancer cell-derived exosomes are the mediator of the tumor microenvironment and the molecular content of exosomes presents a promising prognostic or predictive marker in tumor progression and the treatment response of cancer patients. The aim of this study was to identify the expression levels of receptor tyrosine kinases (RTKs) and AKT1 and mTOR before and after neoadjuvant chemotherapy (NACT) in the exosomes of BC patients compared with healthy females. After isolating exosomes in the serum of 25 BC patients and characterization by flow cytometry, the mRNA levels of FGFR2, FGFR3, PDGFRB, AKT1 and mTOR in the exosomes were analyzed by RT-PCR. Our preliminary findings showed that FGFR2, PDGFRB, AKT1 and mTOR levels were significantly upregulated in BC patients before NACT compared with the healthy group (p < 0.05). Furthermore, the mRNA levels PDGFRB and AKT1 were significantly down-regulated after NACT compared with control. PDGFRB expression level could predict pathological non-response and significantly correlated with tumor size after NACT. Therefore, especially FGFR2, PDGFRB and AKT1 could be a therapeutic target as a prognostic marker, whereas PDGFRB may be a promising predictive indicator of therapy response in BC patients. However, the prognostic or predictive role of RTKs and PI3K/AKT/mTOR signaling in the exosomes should be further investigated in a large patient population.

Dual targeting of FGFR3 and ERBB3 enhances the efficacy of FGFR inhibitors in FGFR3 fusion-driven bladder cancer

BackgroundMutations and fusions in Fibroblast Growth Factor Receptor 3 (FGFR3) occur in 10–20% of metastatic urothelial carcinomas and confer sensitivity to FGFR inhibitors. However, responses to these agents are often short-lived due to the development of acquired resistance. The objective of this study was to identify mechanisms of resistance to FGFR inhibitors in two previously uncharacterised bladder cancer cell lines harbouring FGFR3 fusions and assess rational combination therapies to enhance sensitivity to these agents.MethodsAcquired resistance to FGFR inhibitors was generated in two FGFR3 fusion harbouring cell lines, SW780 (FGFR3-BAIAP2L1 fusion) and RT4 (FGFR3-TACC3 fusion), by long-term exposure to the FGFR inhibitor BGJ398. Changes in levels of receptor tyrosine kinases were assessed by phospho-RTK arrays and immunoblotting. Changes in cell viability and proliferation were assessed by the Cell-Titre Glo assay and by propidium iodide staining and FACS analysis.ResultsLong term treatment of FGFR3-fusion harbouring SW780 and RT4 bladder cancer cell lines with the FGFR inhibitor BGJ398 resulted in the establishment of resistant clones. These clones were cross-resistant to the clinically approved FGFR inhibitor erdafitinib and the covalently binding irreversible FGFR inhibitor TAS-120, but remained sensitive to the MEK inhibitor trametinib, indicating resistance is mediated by alternate activation of MAPK signalling. The FGFR inhibitor-resistant SW780 and RT4 lines displayed increased expression of pERBB3, and strikingly, combination treatment with an FGFR inhibitor and the ATP-competitive pan-ERBB inhibitor AZD8931 overcame this resistance. Notably, rapid induction of pERBB3 and reactivation of pERK also occurred in parental FGFR3 fusion-driven lines within 24 h of FGFR inhibitor treatment, and combination treatment with an FGFR inhibitor and AZD8931 delayed the reactivation of pERBB3 and pERK and synergistically inhibited cell proliferation.ConclusionsWe demonstrate that increased expression of pERBB3 is a key mechanism of adaptive resistance to FGFR inhibitors in FGFR3-fusion driven bladder cancers, and that this also occurs rapidly following FGFR inhibitor treatment. Our findings demonstrate that resistance can be overcome by combination treatment with a pan-ERBB inhibitor and suggest that upfront combination treatment with FGFR and pan-ERBB inhibitors warrants further investigation for FGFR3-fusion harbouring bladder cancers.

Cionin, a vertebrate cholecystokinin/gastrin homolog, induces ovulation in the ascidian Ciona intestinalis type A

Cionin is a homolog of vertebrate cholecystokinin/gastrin that has been identified in the ascidian Ciona intestinalis type A. The phylogenetic position of ascidians as the closest living relatives of vertebrates suggests that cionin can provide clues to the evolution of endocrine/neuroendocrine systems throughout chordates. Here, we show the biological role of cionin in the regulation of ovulation. In situ hybridization demonstrated that the mRNA of the cionin receptor, Cior2, was expressed specifically in the inner follicular cells of pre-ovulatory follicles in the Ciona ovary. Cionin was found to significantly stimulate ovulation after 24-h incubation. Transcriptome and subsequent Real-time PCR analyses confirmed that the expression levels of receptor tyrosine kinase (RTK) signaling genes and a matrix metalloproteinase (MMP) gene were significantly elevated in the cionin-treated follicles. Of particular interest is that an RTK inhibitor and MMP inhibitor markedly suppressed the stimulatory effect of cionin on ovulation. Furthermore, inhibition of RTK signaling reduced the MMP gene expression in the cionin-treated follicles. These results provide evidence that cionin induces ovulation by stimulating MMP gene expression via the RTK signaling pathway. This is the first report on the endogenous roles of cionin and the induction of ovulation by cholecystokinin/gastrin family peptides in an organism.

Resveratrol reduced the detrimental effects of malondialdehyde on human endothelial cells.

Introduction: According to the statistics, vascular injury occurs during the onset of diabetic changes after the production of several byproducts. Many authorities have focused to find an alternative therapy for diabetic patients. In this study, we investigated the therapeutic effects of natural polyphenol like resveratrol on human endothelial cells exposed to malondialdehyde for 48 hours. Methods: Human Umbilical Vein Endothelial Cells were randomly classified into four groups;control, malondialdehyde (2.5 mM), resveratrol (100 μM), and cells received the combined regime for 48 hours. Cell viability was determined by 3-(4, 5-dimethyl thiazol-2-yl) 2, 5-diphenyl-tetrazoliumbromide (MTT) assay. Griess reaction was performed to measure the content of Nitric oxide (NO).Apoptosis was studied by using real-time polymerase chain reaction (RT-PCR) and western blotting assays. Levels of receptor tyrosine kinases like VEGFR-1, -2, Tie-1, and -2 were analyzed by enzyme-linked immunosorbent assay(ELISA). The affinity of resveratrol and malondialdehyde to serum albumin was measured by Surface Plasmon Resonance Assay. Any changes in chromatin remodeling were detected by PCR array analysis. Results: Resveratrol reduced cytotoxicity and NO content inside cells induced by malondialdehyde(MDA) (P < 0.05). Endothelial cell apoptosis was decreased by the reduction of pro-apoptotic factor Bax and increase of Bcl-2 following the incubation with resveratrol (P < 0.05). MDA-induced receptor tyrosine kinases increase was inhibited by resveratrol and reached near-to-normal levels (P < 0.05).Surface Plasmon Resonance revealed a higher affinity of resveratrol to albumin compared to the malondialdehyde-albumin complex. Polymerase chain reaction (PCR) array revealed the potency of resveratrol in chromatin remodeling following the treatment with malondialdehyde (P < 0.05). Conclusion: Based on our findings, resveratrol has the potential to decrease diabetic vascular injury induced by lipid byproducts such as MDA.

High Levels of Receptor Tyrosine Kinases in CCM3-Deficient Cells Increase Their Susceptibility to Tyrosine Kinase Inhibition.

Cerebral cavernous malformations (CCMs) are vascular malformations that can be the result of the deficiency of one of the CCM genes. Their only present treatment is surgical removal, which is not always possible, and an alternative pharmacological strategy to eliminate them is actively sought. We have studied the effect of the lack of one of the CCM genes, CCM3, in endothelial and non-endothelial cells. By comparing protein expression in control and CCM3-silenced cells, we found that the levels of the Epidermal Growth Factor Receptor (EGFR) are higher in CCM3-deficient cells, which adds to the known upregulation of Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) in these cells. Whereas VEGFR2 is upregulated at the mRNA level, EGFR has a prolonged half-life. Inhibition of EGFR family members in CCM3-deficient cells does not revert the known cellular effects of lack of CCM genes, but it induces significantly more apoptosis in CCM3-deficient cells than in control cells. We propose that the susceptibility to tyrosine kinase inhibitors of CCM3-deficient cells can be harnessed to kill the abnormal cells of these lesions and thus treat CCMs pharmacologically.

Immuno-PET Detects Changes in Multi-RTK Tumor Cell Expression Levels in Response to Targeted Kinase Inhibition.

Receptor tyrosine kinase (RTK) coexpression facilitates tumor resistance due to redundancies in the phosphatidylinositol-3'-kinase/protein kinase B and KRAS/extracellular-signal-regulated kinase signaling pathways, among others. Crosstalk between the oncogenic RTK hepatocyte growth factor receptor (MET), epidermal growth factor receptor (EGFR), and human epidermal growth factor receptor 2 (HER2) are involved in tumor resistance to RTK-targeted therapies. Methods: In a relevant renal cell carcinoma patient-derived xenograft model, we use the 89Zr-labeled anti-RTK antibodies (immuno-PET) onartuzumab, panitumumab, and trastuzumab to monitor MET, EGFR, and HER2 protein levels, respectively, during treatment with agents to which the model was resistant (cetuximab) or sensitive (INC280 and trametinib). Results: Cetuximab treatment resulted in continued tumor growth, as well as an increase in all RTK protein levels at the tumor in vivo on immuno-PET and ex vivo at the cellular level. Conversely, after dual MET/mitogen-activated protein kinase inhibition, tumor growth was significantly blunted and corresponded to a decrease in RTK levels. Conclusion: These data show the utility of RTK-targeted immuno-PET to annotate RTK changes in protein expression and inform tumor response to targeted therapies.

Nintedanib ameliorates animal model of dermatitis

Nintedanib, a receptor tyrosine kinase (RTK) inhibitor has been developed as therapeutics for idiopathic pulmonary fibrosis and non-small lung cancer. We found that the expression levels of RTK, especially VEGFR1 is increased in skin biopsies of dermatitis patients from multiple independent datasets. Moreover, VEGFR1 is highly expressed by infiltrated cells in dermis from oxazolone (OXA) treated mice. Interestingly, nintedanib alleviates dermatitis symptom in OXA-induced animal model. Especially, levels of epidermis thickness, infiltrated immune cells including mast cells and eosinophils were decreased from mice cotreated with nintedanib and OXA compared with OXA treated mice. Moreover, serum IgE and Th2 cytokines including IL-4 and IL-13 were decreased by nintedanib treatment. These results suggest an evidence that nintedanib alleviates animal model of dermatitis.

Elevated plasma tyrosine kinases VEGF-D and HER4 in heart failure patients decrease after heart transplantation in association with improved haemodynamics

Receptor tyrosine kinases (RTKs) are implicated in cardiovascular growth and remodelling. We aimed to identify the plasma levels of RTKs and related proteins and their association with haemodynamic alterations in heart failure (HF) and related pulmonary hypertension (PH) following heart transplantation (HT). Using proximity extension assay, 28 RTKs and related proteins were analysed in plasma from 20 healthy controls and 26 HF patients before and 1-year after HT. In end-stage HF, out of 28 RTKs, plasma vascular endothelial growth factor-D (VEGF-D) and human epidermal growth factor-4 (HER4) were elevated compared to controls (p < 0.001), but decreased (p < 0.0001) and normalised after HT. Following HT, plasma changes (Δ) of VEGF-D correlated with Δmean pulmonary artery pressure (rs = 0.65, p = 0.00049), Δpulmonary artery wedge pressure (rs = 0.72, p < 0.0001), Δpulmonary arterial compliance (PAC) (rs = − 0.52, p = 0.0083) and Δpulmonary vascular resistance (PVR) (rs = 0.58, p = 0.0032). ΔHER4 correlated with Δmean right atrial pressure (rs = 0.51, p = 0.012), ΔNT-proBNP (rs = 0.48, p = 0.016) and Δcardiac index (rs = − 0.56, p = 0.0044). In HF patients following HT, normalisation of VEGF-D reflected reversal of passive pulmonary congestion and restored PAC and PVR; whereas the normalisation of HER4 reflected decreased volume overload and improved cardiac function. The precise function of these proteins, their potential clinical use and pathophysiological relation in HF and related PH remain to be elucidated.

FLT3 Is Sumoylated and Localizes to the Nucleus in Acute Myeloid Leukemia

FLT3 is a receptor tyrosine kinase (RTK) that is mutated and constitutively active in 30% of patients with acute myeloid leukemia (AML). Notably, mutations in FLT3 serve as poor prognostic indicator in AML patients. Despite its clinical relevance, the mechanisms of FLT3 subcellular trafficking remain poorly understood. Apart from the canonical downstream signaling cascades within the cytoplasm, accumulating data reveal direct nuclear translocation of RTKs. This alternative signaling pathway suggests that upon nuclear entry, RTKs can influence transcription and regulate gene expression. Interestingly, increased nuclear RTK levels have been linked to advanced tumor stages and poor clinical outcome. Whether or not the same phenomenon occurs in oncogenic FLT3 remains to be elucidated. This study focuses on establishing whether FLT3 undergoes nuclear translocation in AML. Moreover, we aim to understand the underlying subcellular trafficking mechanisms involved. First, the intracellular location of FLT3 was investigated in different human AML cell lines, stably transfected murine cell lines, as well as in primary patient samples. Subcellular fractionation protocols were optimized for each cell type and validated using protein markers for different cellular organelles. The cells were fractionated into cytoplasmic and nuclear fractions and analyzed by immunoblotting, immunofluorescent staining, and confocal imaging. Cells expressing FLT3 internal tandem duplication (FLT3-ITD) mutation show predominant cytoplasmic localization of FLT3. Interestingly, a basal level of FLT3-ITD was also detected in the nuclear fraction independent of ligand stimulation. To determine whether the same thing holds true for wild-type FLT3, AML cell lines which express wild-type FLT3 (FLT3-WT) were stimulated with its ligand for different time periods. FLT3-WT receptor was scarcely present in the nucleus under basal conditions. However, upon stimulation, a transient increase in nuclear FLT3 was observed. Using cell surface biotin labeling, we could verify that the detected nuclear FLT3 comes from the cell surface. Our results further show that nuclear FLT3 is phosphorylated to a certain degree, but that receptor activation is not required for nuclear translocation to occur. To investigate the subcellular trafficking mechanisms involved, the cells were treated with various small molecule inhibitors of cellular trafficking. Nuclear FLT3 levels were attenuated upon inhibition of clathrin and importin β, but stabilized by proteasomal inhibition. Modification of proteins by small ubiquitin-like modifier (SUMO) is a post-translational modification mainly observed in nuclear proteins. Since some RTKs require SUMOylation for nuclear entry, we investigated if this was also true for FLT3. Using immunoprecipitation, we could demonstrate that nuclear FLT3 gets SUMOylated by SUMO-1. To determine whether FLT3 SUMOylation is physiologically relevant, detection of endogenous SUMOylated FLT3 was done without overexpression of components of the SUMO machinery. Taken together, the present study demonstrates a previously uncharacterized post-translational modification and intracellular localization of FLT3. These data provide novel insights on the pathogenesis of AML and possibly new clues to improve targeted treatment of the disease. Additional in vitro studies and clinical data are necessary to establish the functional relevance of FLT3 translocation. On-going studies involve chromatin immunoprecipitation followed by sequencing (ChIP-seq) to gain further understanding on the biological function of nuclear FLT3. Ultimately, we will validate its clinical significance by analyzing nuclear FLT3 in AML patient samples and correlating this with patient outcome. Disclosures Ronnstrand: Acrivon Therapeutics: Consultancy.

Comprehensive biomarker analyses identifies HER2, EGFR, MET RNA expression and thymidylate synthase 5'UTR SNP as predictors of benefit from S-1 adjuvant chemotherapy in Japanese patients with stage II/III gastric cancer.

Purpose: A comprehensive molecular analysis was conducted to identify prognostic and predictive markers for adjuvant S-1 chemotherapy in stage II/III Japanese gastric cancer (GC) patients and to evaluate their potential suitability for alternative cytotoxic or targeted drugs.Experimental Design: We investigated genetic polymorphisms of enzymes potentially involved in 5-fluoruracil (5-FU) metabolism as well as platinum resistance, previously identified genomic subtypes potentially predicting 5-FU benefit, and mRNA expression levels of receptor tyrosine kinases and KRAS as potential treatment targets in a single institution cohort of 252 stage II/III GC patients treated with or without S-1 after D2 gastrectomy.Results: 88% and 62% GC had a potentially 5-FU sensitive phenotype by SNP analyses of TS 3'UTR, and TS 5'UTR, respectively. 24%, 46%, 40%, 5%, and 44% GC had a potentially platinum sensitive phenotype by SNP analyses of GSTP1, ERCC1 rs11615, ERCC1 rs3212986, ERCC2, and XRCC1, respectively. High HER2, EGFR, FGFR2, or MET mRNA expression was observed in 49%, 66%, 72%, and 54% GC, respectively. High HER2 expression was the only significant prognosticator (HR=3.912, 95%CI: 1.706-8.973, p=0.0005). High HER2 (p=0.031), low EGFR (p=0.124), high MET (p=0.165) RNA expression, and TS 5'UTR subtype 2R/2R, 2R/3C, or 3C (p=0.058) were significant independent predictors for S-1 resistance.Conclusions: The present study suggests that platinum-based or RTK targeted agents could be alternative treatment options for a substantial subgroup of Japanese GC patients currently treated with S-1. HER2, EGFR, MET, and TS 5'UTR SNP appear to be promising predictive markers for S-1 resistance warranting validation in an independent GC series.

Abstract 3995: Identification of receptor tyrosine kinases (RTKs) expression in tumor-infiltrating lymphocytes (TILs) and peripheral blood mononuclear cells (PBMC)

Abstract Aim of this study is to discover expression levels of RTKs (VEGFR1, VEGFR2, PDGFRa, PDGFRb) in CD45+ population of PMBC and TILs isolated from patients with clear cell renal cell cancer (RCC). Here we present preliminary data. Species of tumors and blood were prospective collected from 9 patients with RCC after radical nephrectomy or tumor resection from July to October 2016. TILs isolation was performed under standard protocol with slight modification. Briefly, tumor was shredded with scissors on small pieces of 2-3 mm and twice washed in RPMI 1640 media by centrifugation for 10 min at 1500 rpm. After centrifugation, pellet was diluted RPMI 1640 with enzyme mix (Collagenase and Dispase II) on Petri dish and placed on shaker-thermostat andincubate sample for 12h at 37C with continuous shaking. After incubation, cell suspension was passed thought a cell strainer, mesh size 70 μm, placed on a 50 mL tube and washed twice washed in RPMI 1640 media by centrifugation. For TILs isolation cell suspension was placed on Ficoll-Paque Plus and isolated by centrifugation for 25 min at 1500 rpm. Cell pellet was resuspended in RPMI 1640 media. PMBC was isolated from whole blood using the Ficoll-Paque method. Isolated TIL and PMBC were prepared for flow cytometry at typical protocol with or without permeabilization. Cells were stained with anti-CD45 FITC-conjugated antibodies (Novus), primary anti-VEGFR1, anti-VEGFR2, anti-PDGFRa, anti-PDGFRb (Abcam) mouse antibodies and Alexa-568 conjuated secondary anti-mouse antibodies (Invitrogen) and analyzed on BD Canto II flow cytometer. Expression of RTKs on CD45+ cell population were evaluated. Among PMBC 86±9% of cells was CD45-positive and expressed RTKs (10±1% cells were positive for VEGFR1 and VEGFR2, and 6±2% cells were positive for PDGRFa and PDGFRb). TILs contained 65±18% of CD45-positive cells and did not express RTKs on the cell surface (less than 1% of cells), but intracellular staining showed presence of RTKs: 8±2% of cells were VEGFR1 or VEGFR2-positive, 20±8% of cells were PDGFRa or PDGFRb-positive. Preliminary data demonstrate extracellular expression of RTKs on PBMC and intracellular expression of RTKs in TILs. The study will be continued. Citation Format: Dmitry Khochenkov, Maria Volkova, Ilya Tsimafeyeu. Identification of receptor tyrosine kinases (RTKs) expression in tumor-infiltrating lymphocytes (TILs) and peripheral blood mononuclear cells (PBMC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3995. doi:10.1158/1538-7445.AM2017-3995

Deep Coverage of Global Protein Expression and Phosphorylation in Breast Tumor Cell Lines Using TMT 10-plex Isobaric Labeling.

Labeling peptides with isobaric tags is a popular strategy in quantitative bottom-up proteomics. In this study, we labeled six breast tumor cell lysates (1.34 mg proteins per channel) using 10-plex tandem mass tag reagents and analyzed the samples on a Q Exactive HF Quadrupole-Orbitrap mass spectrometer. We identified a total of 8,706 proteins and 28,186 phosphopeptides, including 7,394 proteins and 23,739 phosphosites common to all channels. The majority of technical replicates correlated with a R2 ≥ 0.98, indicating minimum variability was introduced after labeling. Unsupervised hierarchical clustering of phosphopeptide data sets successfully classified the breast tumor samples into Her2 (epidermal growth factor receptor 2) positive and Her2 negative groups, whereas mRNA abundance did not. The tyrosine phosphorylation levels of receptor tyrosine kinases, phosphoinositide-3-kinase, protein kinase C delta, and Src homology 2, among others, were significantly higher in the Her2 positive than the Her2 negative group. Despite ratio compression in MS2-based experiments, we demonstrated the ratios calculated using an MS2 method are highly correlated (R2 > 0.65) with ratios obtained using MS3-based quantitation (using a Thermo Orbitrap Fusion mass spectrometer) with reduced ratio suppression. Given the deep coverage of global and phosphoproteomes, our data show that MS2-based quantitation using TMT can be successfully used for large-scale multiplexed quantitative proteomics.

Uncarboxylated Osteocalcin Induces Antitumor Immunity against Mouse Melanoma Cell Growth.

Because of the poor response to chemotherapy and radiation therapy, new treatment approaches by immune-based therapy involving activated T cells are required for melanoma. We previously reported that the uncarboxylated form of osteocalcin (GluOC), derived from osteoblasts, potentially suppresses human prostate cancer cell proliferation by direct suppression of cell growth. However, the mechanisms in vivo have not been elucidated. In this study, we found that GluOC suppressed tumor growth of B16 mouse melanoma transplants in C57Bl/6N wild-type mice. Our data demonstrated that GluOC suppressed cell growth by downregulating phosphorylation levels of receptor tyrosine kinases and inducing apoptosis in vitro. Additionally, stimulation of primary mouse splenocytes with concanavalin A, a polyclonal T-cell mitogen, in the presence of GluOC increased T cell proliferation and their interferon-γ production. Taken together, we demonstrate that GluOC exerts multiple antitumor effects not only in vitro, but also in vivo through cellular immunostimulatory effects against B16 mouse melanoma cells.

Lipid Raft Integrity Is Required for Survival of Triple Negative Breast Cancer Cells.

PurposeLipid rafts are cholesterol enriched microdomains that colocalize signaling pathways involved in cell proliferation, metastasis, and angiogenesis. We examined the effect of methyl-β-cyclodextrin (MβCD)-mediated cholesterol extraction on the proliferation, adhesion, invasion, and angiogenesis of triple negative breast cancer (TNBC) cells.MethodsWe measured cholesterol and estimated cell toxicity. Detergent resistant membrane (DRM) and non-DRM fractions were separated using the OptiPrep gradient method. Cell cycles stages were analyzed by flow cytometry, apoptosis was assessed using the TdT-mediated dUTP nick end-labeling assay, and metastasis was determined using a Matrigel invasion assay. Neo-vessel pattern and levels of angiogenic modulators were determined using an in vitro angiogenesis assay and an angiogenesis array, respectively.ResultsThe present study found that the cholesterol-depleting agent MβCD, efficiently depleted membrane cholesterol and caused concentration dependent (0.1–0.5 mM) cytotoxicity compared to nystatin and filipin III in TNBC cell lines, MDA-MB 231 and MDA-MB 468. A reduced proportion of caveolin-1 found in DRM fractions indicated a cholesterol extraction-induced disruption of lipid raft integrity. MβCD inhibited 52% of MDA-MB 231 cell adhesion on fibronectin and 56% of MDA-MB 468 cell adhesion on vitronectin, while invasiveness of these cells was decreased by 48% and 52% respectively, following MβCD treatment (48 hours). MβCD also caused cell cycle arrest at the G2M phase and apoptosis in MDA-MB 231 cells (25% and 58% cells, respectively) and in MDA-MB 468 cells (30% and 38% cells, respectively). We found that MβCD treated cells caused a 52% and 58% depletion of neovessel formation in both MDA-MB 231 and MDA-MB 468 cell lines, respectively. This study also demonstrated that MβCD treatment caused a respective 2.6- and 2.5-fold depletion of tyrosine protein kinase receptor (TEK) receptor tyrosine kinase levels in both TNBC cell lines.ConclusionMβCD-induced cholesterol removal enhances alterations in lipid raft integrity, which reduces TNBC cell survival.

Motor and Sensory Deficits in the teetering Mice Result from Mutation of the ESCRT Component HGS.

Neurons are particularly vulnerable to perturbations in endo-lysosomal transport, as several neurological disorders are caused by a primary deficit in this pathway. In this report, we used positional cloning to show that the spontaneously occurring neurological mutation teetering (tn) is a single nucleotide substitution in hepatocyte growth factor-regulated tyrosine kinase substrate (Hgs/Hrs), a component of the endosomal sorting complex required for transport (ESCRT). The tn mice exhibit hypokenesis, muscle weakness, reduced muscle size and early perinatal lethality by 5-weeks of age. Although HGS has been suggested to be essential for the sorting of ubiquitinated membrane proteins to the lysosome, there were no alterations in receptor tyrosine kinase levels in the central nervous system, and only a modest decrease in tropomyosin receptor kinase B (TrkB) in the sciatic nerves of the tn mice. Instead, loss of HGS resulted in structural alterations at the neuromuscular junction (NMJ), including swellings and ultra-terminal sprouting at motor axon terminals and an increase in the number of endosomes and multivesicular bodies. These structural changes were accompanied by a reduction in spontaneous and evoked release of acetylcholine, indicating a deficit in neurotransmitter release at the NMJ. These deficits in synaptic transmission were associated with elevated levels of ubiquitinated proteins in the synaptosome fraction. In addition to the deficits in neuronal function, mutation of Hgs resulted in both hypermyelinated and dysmyelinated axons in the tn mice, which supports a growing body of evidence that ESCRTs are required for proper myelination of peripheral nerves. Our results indicate that HGS has multiple roles in the nervous system and demonstrate a previously unanticipated requirement for ESCRTs in the maintenance of synaptic transmission.

PHLPP phosphatases are master regulators of cellular receptor tyrosine kinase levels (802.19)

Growth factor receptor levels are aberrantly high in diverse cancers, driving the proliferation and survival of tumor cells. Understanding the molecular basis for this aberrant elevation has profound clinical applications. Here we show that the PH domain leucine‐rich repeat protein phosphatase (PHLPP) suppresses the steady‐state levels of receptor tyrosine kinases (RTKs) by a novel mechanism unrelated to its previously described function as the hydrophobic motif phosphatase for AKT, protein kinase C and S6 kinase. Specifically, genetic depletion and pharmacological studies reveal that PHLPP1 and PHLPP2 promote histone deacetylase (HDAC)‐dependent repression of the transcription of diverse growth factor receptors, including the EGF receptor. These data uncover a novel, and much broader, role for PHLPP in regulation of growth factor signaling beyond its direct inactivation of AKT: by suppressing RTK levels, PHLPP dampens the downstream signaling output of two major oncogenic pathways, the PI3 kinase/AKT and the RAS/ERK pathways. Our data are consistent with a model in which PHLPP is a master regulator of the RTK signaling output of cells.

A Novel AKT1 Mutant Amplifies an Adaptive Melanoma Response to BRAF Inhibition

BRAF inhibitor (BRAFi) therapy leads to remarkable anti melanoma responses, but the initial tumor shrinkage is commonly incomplete, providing a nidus for subsequent disease progression. Adaptive signaling may underlie early BRAFi resistance and influence the selection pattern for genetic variants, causing late, acquired resistance. We show here that BRAFi (or BRAFi + MEKi) therapy in patients frequently led to rebound phosphorylated AKT (p-AKT) levels in their melanomas early on-treatment. In cell lines, BRAFi treatment led to rebound levels of receptor tyrosine kinases (RTK; including PDGFRβ), phosphatidyl (3,4,5)-triphosphate (PIP3), pleckstrin homology domain recruitment, and p-AKT. PTEN expression limited this BRAFi-elicited PI3K-AKT signaling, which could be rescued by the introduction of a mutant AKT1 (Q79K) known to confer acquired BRAFi resistance. Functionally, AKT1(Q79K) conferred BRAFi resistance via amplification of BRAFi-elicited PI3K-AKT signaling. In addition, mitogen-activated protein kinase pathway inhibition enhanced clonogenic growth dependency on PI3K or AKT. Thus, adaptive or genetic upregulation of AKT critically participates in melanoma survival during BRAFi therapy.

Abstract 1323: Identification of conditions of lapatinib effectiveness in prostate cancer.

Abstract ABSTRACT Lapatinib has been used in multiple clinical trials of either hormone-naïve men with prostate cancer (PCa) or in patients who have progressed to castration resistant prostate cancer (CRPC) following androgen deprivation therapy (ADT). Although lapatinib as a single-agent showed no overall positive effect in hormone-naive PCa, a small subset of unselected CRPC patients experienced PSA decline ∼50% with lapatinib treatment, with one patient remaining on this drug &amp;gt;45 months (Whang, Y.E. et al, 2011. Urol Oncol). The current investigation had been undertaken to determine whether lapatinib may benefit any select group of patients with PCa. Studies in vitro and in animal models supported the observation that lapatinib was ineffective in the presence of androgens; however, in low-androgen conditions, only androgen-dependent LNCaP PCa cells, but not CRPC cells, responded to lapatinib. Since lapatinib is a dual inhibitor of the receptor tyrosine kinase (RTK) epidermal growth factor receptor (EGFR) and a closely related member, ErbB2, hence we investigated the effect of this drug on the EGFR family. Comparison of the various cell lines that did or did not respond to lapatinib demonstrated that those that did not respond to lapatinib had very high inherent EGFR and ErbB3 activity, whereas LNCaP cells did not. More importantly, unlike its CRPC sublines C4-2 and LNCaP-AI, which were less responsive to lapatinib compared to LNCaP, the parental line experienced an increase in ErbB3 during androgen deprivation. Overexpression of ErbB3 induced resistance to lapatinib even in LNCaP cells, indicating an increase in the levels of RTKs as a potential cause for lapatinib resistance. Indeed, lapatinib treatment resulted in an overall increase in the levels of EGFR and/or ErbB2 in all the cell lines tested, even in LNCaP cells that do respond to lapatinib. Thus, our data demonstrate that (i) lapatinib is ineffective in PCa expressing high ErbB3 and (ii) among those with low intrinsic ErbB3, lapatinib is effective only in tumors where ErbB3 levels increase upon androgen withdrawal. Our results show that the critical window of opportunity for employing lapatinib and other dual EGFR/ErbB2 inhibitors is when ErbB3 levels are rising, but not once they have already increased. Since ErbB3 can be detected in the serum of patients with PCa, the increase of this RTK during ADT should be tested in future studies as a predictive marker of lapatinib response. Citation Format: Maitreyee K. Jathal, Benjamin A. Mooso, Leandro S. D'Abronzo, Salma Siddiqui, Liqun Chen, Elyse van Spyck, Anisha Madhav, Paramita M. Ghosh. Identification of conditions of lapatinib effectiveness in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1323. doi:10.1158/1538-7445.AM2013-1323

Abstract 227: Overexpression of EphB1 leads to Stat3 phosphorylation

Abstract The Receptor Tyrosine Kinase (RTK) family comprises some important disease drivers. Over-expressed or mutated RTKs in disease may lead to aberrant phosphorylation of intracellular signaling molecules. Therefore it is desirable to monitor simultaneously the activation levels of RTKs and signaling nodes. Here we used a multiplexed antibody array to monitor the phosphorylation of 28 RTKs and 11 intracellular signaling molecules simultaneously. We have observed a strong signal on a spot corresponding to phospho-STAT3 Tyr705 with lysates derived from CHO cells transiently over-expressing EphB1. The strong Phospho-STAT3 signal was not seen in non-transfected cells or in lysates made form cells transfected with several other Eph family members. Western blotting with phospho-specific antibodies showed an increase in STAT3 phosphorylation on Tyr705 in CHO cells upon EphB1 ectopic expression. The amounts of Tyr705 STAT3 phosphorylation positively correlated with the levels of EphB1 protein. Co-precipitation experiments showed that EphB1 and STAT3 interact both in CHO cells and MCF-7 breast cancer cells. Phosphorylated JAK2 was not found in these immuno-precipitates suggesting an involvement of another tyrosine kinase in STAT3 phosphorylation in cells over-expressing EphB1. Consistently with these observations, a potent JAK1 and JAK2 inhibitor INCB018424 inhibited the basal but not the EphB1-induced STAT3 Tyr705 phosphorylation in transfected CHO cells. These results suggest that EphB1 over-expression may bypass the requirement in Jak kinase and lead to STAT3 activation either directly or through another receptor-tethered kinase. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 227. doi:1538-7445.AM2012-227

Abstract B34: Increase in EGFR level through SLUG-induced repression of dynactin 5 in triple-negative breast cancer cells

Abstract Aggressive breast cancers of the triple negative type (TNBCs) are often characterized with higher levels of receptor tyrosine kinases (RTKs; e.g. EGFR) and G-protein coupled receptors (GPCRs; e.g. PAR1). These receptors are activated by their agonists, induce signal transduction and then are attenuated by signal uncoupling and retrograde recycling endosome transport. We report here that the levels of several of these receptor proteins are elevated and hyperactivated on the surface of aggressive breast cancer cells that have high levels of the transcriptional repressor protein SLUG. Chip-on-chip analysis to identify genes that are repressed by SLUG in the breast cancer cells revealed one of the proteins that are critical for the retrograde recycling transport of the RTKs and GPCRs, namely dynactin 5 (DCTN5). DCTN5 (aka p25) is a member of the dynactin complex. DCTN5 is a subunit of the pointed-end subcomplex of dynactin that is thought to interact with membranous cargo. Interestingly, DCTN5 is expressed from the reverse activity of the bidirectional promoter of human PALB2 (partner and localizer of BRCA2) gene whose product regulates the functions of the tumor suppressor protein BRCA2. We found that SLUG inhibits both PALB2 and DCTN5 gene expression through chromatin remodeling in human breast cancer cells. We hypothesize that the malignant breast cancer cells acquire highly aggressive and metastatic characteristics in part due to the higher levels and hyperactivation of EGFR on their surface. The hyperactivity of EGFR occurs through the SLUG-mediated repression of DCTN5 which mediates the internalization and thus, degradation of activated receptors. We evaluated the role of DCTN5 in the determination of aggressiveness in TNBC cells. We postulate that maintenance of normal levels DCTN5 in the TNBC cells through the inhibition of the functions of SLUG will control the invasive growth of the breast tumors. Supported in parts by the DOD-CDMRP grants BC050641, BC086542 and BC103645. Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):B34.