Activation Of Src Family Kinases Research Articles

Histoplasma capsulatum-Induced Cytokine Secretion in Lung Epithelial Cells Is Dependent on Host Integrins, Src-Family Kinase Activation, and Membrane Raft Recruitment.

Histoplasma capsulatum var. capsulatum is a dimorphic fungus that causes histoplasmosis, a human systemic mycosis with worldwide distribution. In the present work, we demonstrate that H. capsulatum yeasts are able to induce cytokine secretion by the human lung epithelial cell line A549 in integrin- and Src-family kinase (SFK)-dependent manners. This conclusion is supported by small interfering RNA (siRNA) directed to α3 and α5 integrins, and PP2, an inhibitor of SFK activation. siRNA and PP2 reduced IL-6 and IL-8 secretion in H. capsulatum-infected A549 cell cultures. In addition, α3 and α5 integrins from A549 cells were capable of associating with H. capsulatum yeasts, and this fungus promotes recruitment of these integrins and SFKs to A549 cell membrane rafts. Corroborating this finding, membrane raft disruption with the cholesterol-chelator methyl-β-cyclodextrin reduced the levels of integrins and SFKs in these cell membrane domains. Finally, pretreatment of A549 cells with the cholesterol-binding compound, and also a membrane raft disruptor, filipin, significantly reduced IL-6 and IL-8 levels in A549-H.capsulatum cultures. Taken together, these results indicate that H. capsulatum yeasts induce secretion of IL-6 and IL-8 in human lung epithelial cells by interacting with α3 and α5 integrins, recruiting these integrins to membrane rafts, and promoting SFK activation.

Entamoeba histolytica Cysteine Proteinase 5 Evokes Mucin Exocytosis from Colonic Goblet Cells via αvβ3 Integrin.

Critical to the pathogenesis of intestinal amebiasis, Entamoeba histolytica (Eh) induces mucus hypersecretion and degrades the colonic mucus layer at the site of invasion. The parasite component(s) responsible for hypersecretion are poorly defined, as are regulators of mucin secretion within the host. In this study, we have identified the key virulence factor in live Eh that elicits the fast release of mucin by goblets cells as cysteine protease 5 (EhCP5) whereas, modest mucus secretion occurred with secreted soluble EhCP5 and recombinant CP5. Coupling of EhCP5-αvβ3 integrin on goblet cells facilitated outside-in signaling by activating SRC family kinases (SFK) and focal adhesion kinase that resulted in the activation/phosphorlyation of PI3K at the site of Eh contact and production of PIP3. PKCδ was activated at the EhCP5-αvβ3 integrin contact site that specifically regulated mucin secretion though the trafficking vesicle marker myristoylated alanine-rich C-kinase substrate (MARCKS). This study has identified that EhCP5 coupling with goblet cell αvβ3 receptors can initiate a signal cascade involving PI3K, PKCδ and MARCKS to drive mucin secretion from goblet cells critical in disease pathogenesis.

MP44-18 INHIBITION OF SMOOTH MUSCLE CONTRACTION BY THE INHIBITOR FOR CYTOHESIN FAMILIY GUANOSINE NUCLEOTIDE EXCHANGE FACTORS, SECIN H3 IN THE HYPERPLASTIC HUMAN PROSTATE

INTRODUCTION AND OBJECTIVES: In patients with benign prostatic hyperplasia, smooth muscle contraction and stromal growth in the prostate may both contribute to urethral obstruction and voiding symptoms. Although a context between control of smooth muscle tone and prostate growth may be assumed, molecular mechanisms underlying this connection still represent an enigma. Here, we addressed the role of Src family kinases (SFKs) for contraction of human prostate smooth muscle, and for growth of human stromal cells. METHODS: SFK expression was assessed by RT-PCR, Western blot, and immunofluorescence. Effects of two different SFK inhibitors, AZM475271 and PP2, on contraction were assessed in human prostate tissues obtained from radical prostatectomy. Effects on proliferation, actin organization, and cytotoxicity were examined in cultured stromal cells (WPMY-1). RESULTS: C-Src and Lck were detected by RT-PCR and Western blot in human prostate tissues and WPMY1 cells. Colocalization with calponin in immunofluorescence stainings of prostate sections indicated location to smooth muscle cells. AZM475271 (10 mM) and PP2 (10 mM) inhibited SFK activity in prostate tissues and WPMY-1 cell, being assessed as phospho-SFK content. Both inhibitors significantly inhibited contraction of prostate strips by norepinephrine, by the a1-adrenergic agonist phenylephrine, and by electric field stimulation. This may be explained by cytoskeletal deorganization, which was observed by phalloidin staining of actin filaments in response to AZM475271 and PP2 in WPMY-1 cells. This was paralleled by reduced proliferation of WPMY-1 cells, as indicated by EdU assay. Cytotoxicity was mainly observed at higher concentrations (50 mM or more) and longer incubation, but was minor at 10 mM for 24 h. CONCLUSIONS: Synchronized regulation of smooth muscle tone and growth by SFK-dependent control may explain their concomitant contributions to bladder outlet obstruction in the hyperplastic prostate. Targeting smooth muscle contraction and growth in the prostate at once by single agents may be principally possible.

Nuclear expression of Lyn, a Src family kinase member, is associated with poor prognosis in renal cancer patients.

Background8000 cases of renal cancer are diagnosed each year in the UK, with a five-year survival rate of 50 %. Treatment options are limited; a potential therapeutic target is the Src family kinases (SFKs). SFKs have roles in multiple oncogenic processes and promote metastases in solid tumours. The aim of this study was to investigate SFKs as potential therapeutic targets for clear cell renal cell carcinoma (ccRCC).MethodsSFKs expression was assessed in a tissue microarray consisting of 192 ccRCC patients with full clinical follow-up. SFK inhibitors, dasatinib and saracatinib, were assessed in early ccRCC cell lines, 786-O and 769-P and a metastatic ccRCC cell line, ACHN (± Src) for effects on protein expression, apoptosis, proliferation and wound healing.ResultsHigh nuclear expression of Lyn and the downstream marker of activation, paxillin, were associated with decreased patient survival. Conversely, high cytoplasmic expression of other SFK members and downstream marker of activation, focal adhesion kinase (FAK) were associated with increased patient survival. Treatment of non-metastatic 786-O and 769-P cells with dasatinib, dose dependently reduced SFK activation, shown via SFK (Y419) and FAK (Y861) phosphorylation, with no effect in metastatic ACHN cells. Dasatinib also increased apoptosis, while decreasing proliferation and migration in 786-O and 769-P cell lines, both in the presence and absence of Src protein.ConclusionsOur data suggests that nuclear Lyn is a potential therapeutic target for ccRCC and dasatinib affects cellular functions associated with cancer progression via a Src kinase independent mechanism.

Abstract P1-03-02: Estrogens contribute to cytokine upregulation and cancer stem cell recruitment upon breast cancer contact with mature human mammary adipocytes: Effects of estrogen type and adipocyte donor weight

Abstract Consequences of the obesity epidemic on cancer morbidity and mortality are not fully appreciated. While obesity confers increased cancer risk and worse outcome, mechanisms thereof are not fully known. We show prolonged co-culture of fat cells (human adipocyte stem cells, differentiated adipocytes or mature adipocytes) from breast tissue together with breast cancer lines or cultured primary dissociated human breast tumor cells increases secretion of six different pro-inflammatory cytokines, each of which contributes to tumor progression through cancer stem cell recruitment. Prolonged exposure to fat cells or to each cytokine increases the proportion of cells that form mammosphere and express ALDH1 activity in vitro and that can initiate primary orthotopic tumors and metastasis in vivo. Adipocyte and cytokine exposures activate Src, and Src family kinase activity leads to induction of embryonic transcription factors that upregulate miR302b. miR302b induction is Sox2-dependent, promotes cytokine-driven sphere formation, and in turn, stimulates cMYC and SOX2 expression. Src is not only activated by adipocyte or cytokine exposures, it is also required to sustain cytokine induction, since Src inhibitors decrease cytokine production after co-culture. Cytokine upregulation was much greater after co-culture of ER+ breast cancer cells with mature, aromatase positive, adipocytes than with adipocyte stem cells. Cytokine induction was estrogen regulated. The mechanisms of cytokine induction, ER-coactivation and effects of different estrogenic ligands will be presented. Present data illuminate the increased risk of breast cancer after menopause, particularly in obese women and the increased breast cancer mortality with obesity: cancer cell invasion into local fat, in the presence of high local aromatase and intracellular estrogen would establish feed-forward loops to activate Src, maintain pro-inflammatory cytokine production and increase tumor initiating cell abundance, tumor growth and metastasis. These data link obesity related pro-inflammatory cytokines to Src activation and cancer initiating cell abundance, and provide a novel rationale for Src inhibitors together with endocrine therapy for breast cancer. Citation Format: Slingerland J, Picon-Ruiz M, Jang K, Morata-Tarifa C, Pan C, Besser A, Kim M, Ince TA, Howard GA, El-Ashry D. Estrogens contribute to cytokine upregulation and cancer stem cell recruitment upon breast cancer contact with mature human mammary adipocytes: Effects of estrogen type and adipocyte donor weight. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-03-02.

Activation of zebrafish Src family kinases by the prion protein is an amyloid-β-sensitive signal that prevents the endocytosis and degradation of E-cadherin/β-catenin complexes in vivo.

BackgroundPrions and amyloid-β (Aβ) oligomers trigger neurodegeneration by hijacking a poorly understood cellular signal mediated by the prion protein (PrP) at the plasma membrane. In early zebrafish embryos, PrP-1-dependent signals control cell-cell adhesion via a tyrosine phosphorylation-dependent mechanism.ResultsHere we report that the Src family kinases (SFKs) Fyn and Yes act downstream of PrP-1 to prevent the endocytosis and degradation of E-cadherin/β-catenin adhesion complexes in vivo. Accordingly, knockdown of PrP-1 or Fyn/Yes cause similar zebrafish gastrulation phenotypes, whereas Fyn/Yes expression rescues the PrP-1 knockdown phenotype. We also show that zebrafish and mouse PrPs positively regulate the activity of Src kinases and that these have an unexpected positive effect on E-cadherin-mediated cell adhesion. Interestingly, while PrP knockdown impairs β-catenin adhesive function, PrP overexpression enhances it, thereby antagonizing its nuclear, wnt-related signaling activity and disturbing embryonic dorsoventral specification. The ability of mouse PrP to influence these events in zebrafish embryos requires its neuroprotective, polybasic N-terminus but not its neurotoxicity-associated central region. Remarkably, human Aβ oligomers up-regulate the PrP-1/SFK/E-cadherin/β-catenin pathway in zebrafish embryonic cells, mimicking a PrP gain-of-function scenario.ConclusionsOur gain- and loss-of-function experiments in zebrafish suggest that PrP and SFKs enhance the cell surface stability of embryonic adherens junctions via the same complex mechanism through which they over-activate neuroreceptors that trigger synaptic damage. The profound impact of this pathway on early zebrafish development makes these embryos an ideal model to study the cellular and molecular events affected by neurotoxic PrP mutations and ligands in vivo. In particular, our finding that human Aβ oligomers activate the zebrafish PrP/SFK/E-cadherin pathway opens the possibility of using fish embryos to rapidly screen for novel therapeutic targets and compounds against prion- and Alzheimer's-related neurodegeneration. Altogether, our data illustrate PrP-dependent signals relevant to embryonic development, neuronal physiology and neurological disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-016-0076-5) contains supplementary material, which is available to authorized users.

Abstract A39: A role for src kinase in estrogen receptor-positive breast cancer

Abstract Estrogen (E2) stimulation promotes proliferation in estrogen receptor-positive (ER+) breast cancer, which accounts for 60-75% of all breast cancer. E2 signals through the ER via three pathways: the classical genomic pathway involving transcriptional activation of the ER, crosstalk between ER and other transcription factors, and “non-genomic” signaling cascades, which do not involve the transcriptional activity of the ER. This “non-genomic” pathway likely involves Src family kinases (SFKs) and has been implicated in cell cycle progression in fibroblasts engineered to express the ER (Castoria 1999). While not typically amplified or mutationally activated in ER+ breast cancer, Src is hyperactive in most breast cancers. To test whether SFK activity is required for G1/S cell cycle progression in breast cancer cells, we pretreated quiescent ER+ MCF7 or ZR75-1 cells with a selective SFK inhibitor (SU11333) prior to stimulation with E2. Inhibition of SFK activity blocked progression from G1 to S phase. Previous data from our laboratory has demonstrated that G1/S progression in response to peptide growth factor stimulation of quiescent fibroblasts is dependent upon SFK-mediated stabilization of myc mRNA (Bromann 2005). We therefore tested whether SFK activity also regulates myc expression in breast cancer cells. Our data suggest that SFK activity is required for accumulation of myc mRNA in quiescent MCF7s after E2 stimulation, and that E2 stimulated stabilization of myc mRNA is dependent on SFK activity. We are currently investigating the mechanism involved in stabilizing myc mRNA levels.

Src family kinases suppress differentiation of brown adipocytes and browning of white adipocytes.

Brown adipocytes and beige adipocytes can expend energy, generate heat, and increase whole-body energy expenditure. The detailed mechanisms of adipogenesis and thermogenesis of these cells are still obscure. Here, we show that Src family kinases (SFKs) regulate both brown adipogenesis and browning of white adipocytes. To identify factors involved in brown adipogenesis, we first examined the effect of several chemical inhibitors on the differentiation of brown preadipocytes isolated from mouse brown adipose tissue (BAT) and found that treatment with PP2, the specific inhibitor of SFKs, promoted the differentiation. Another inhibitor of SFKs, PP1, also promoted the brown adipogenesis, whereas an inactive analogue of PP2, PP3, did not. Moreover, over-expression of C-terminal Src kinase (CSK), the negative regulator of SFKs, also promoted brown adipogenesis. Next, we examined the effect of inhibition of SFKs on the differentiation of white preadipocytes isolated from white adipose tissue (WAT). Our results showed that either PP2 treatment or CSK-over-expression generated Ucp1-positive beige adipocytes, thus inducing browning of white adipocytes. Finally, our analysis showed that the expression levels and activity of SFKs in WAT were much higher than in BAT. These results taken together suggest that SFKs regulate differentiation and browning of fat cells in vivo.

ERα Signaling Is Required for TrkB-Mediated Hippocampal Neuroprotection in Female Neonatal Mice after Hypoxic Ischemic Encephalopathy(1,2,3).

Male neonate brains are more susceptible to the effects of perinatal asphyxia resulting in hypoxia and ischemia (HI)-related brain injury. The relative resistance of female neonatal brains to adverse consequences of HI suggests that there are sex-specific mechanisms that afford females greater neuroprotection and/or facilitates recovery post-HI. We hypothesized that HI preferentially induces estrogen receptor α (ERα) expression in female neonatal hippocampi and that ERα is coupled to Src family kinase (SFK) activation that in turn augments phosphorylation of the TrkB and thereby results in decreased apoptosis. After inducing the Vannucci’s HI model on P9 (C57BL/6J) mice, female and male ERα wild-type (ERα+/+) or ERα null mutant (ERα−/−) mice received vehicle control or the selective TrkB agonist 7,8-dihydroxyflavone (7,8-DHF). Hippocampi were collected for analysis of mRNA of ERα and BDNF, protein levels of ERα, p-TrkB, p-src, and cleaved caspase 3 (c-caspase-3) post-HI. Our results demonstrate that: (1) HI differentially induces ERα expression in the hippocampus of the female versus male neonate, (2) src and TrkB phosphorylation post-HI is greater in females than in males after 7,8-DHF therapy, (3) src and TrkB phosphorylation post-HI depend on the presence of ERα, and (4) TrkB agonist therapy decreases the c-caspase-3 only in ERα+/+ female mice hippocampus. Together, these observations provide evidence that female-specific induction of ERα expression confers neuroprotection with TrkB agonist therapy via SFK activation and account for improved functional outcomes in female neonates post-HI.

Pharmacological Inactivation of Src Family Kinases Inhibits LPS-Induced TNF-α Production in PBMC of Patients with Behçet's Disease.

Behçet's disease (BD) is a multisystemic chronic inflammatory disease characterized by relapsing oral and genital ulcers, uveitis, and skin lesions. The pathogenesis of BD is still unknown. Aberrant production of some cytokines/chemokines plays an important role in the pathogenesis of various inflammatory diseases. Revealing a key signaling regulatory mechanism involved in proinflammatory cytokines/chemokines production is critical for understanding of the pathogenesis of BD. The aim of this study was to determine the role of Src family kinases (SFKs) in production of some LPS-induced proinflammatory cytokines/chemokines in peripheral blood mononuclear cells (PBMC) of active BD patients. Chemical inhibition of SFKs activity impaired LPS-induced TNF-α production in PBMC of active BD patients, suggesting that modulating SFKs activity may be a potential target for BD treatment.

Integrins Form an Expanding Diffusional Barrier that Coordinates Phagocytosis

Phagocytosis is initiated by lateral clustering of receptors, which in turn activates Src-family kinases (SFKs). Activation of SFKs requires depletion of tyrosine phosphatases from the area of particle engagement. We investigated how the major phosphatase CD45 is excluded from contact sites, using single-molecule tracking. The mobility of CD45 increased markedly upon engagement of Fcγ receptors. While individual CD45 molecules moved randomly, they were displaced from the advancing phagocytic cup by an expanding diffusional barrier. By micropatterning IgG, the ligand of Fcγ receptors, we found that the barrier extended well beyond the perimeter of the receptor-ligand engagement zone. Second messengers generated by Fcγ receptors activated integrins, which formed an actin-tethered diffusion barrier that excluded CD45. The expanding integrin wave facilitates the zippering of Fcγ receptors onto the target and integrates the information from sparse receptor-ligand complexes, coordinating the progression and ultimate closure of the phagocytic cup.

Atomic force microscopy and graph analysis to study the P-cadherin/SFK mechanotransduction signalling in breast cancer cells.

Physical forces mediated by cell-cell adhesion molecules, as cadherins, play a crucial role in preserving normal tissue architecture. Accordingly, altered cadherins' expression has been documented as a common event during cancer progression. However, in most studies, no data exist linking pro-tumorigenic signaling and variations in the mechanical balance mediated by adhesive forces. In breast cancer, P-cadherin overexpression increases in vivo tumorigenic ability, as well as in vitro cell invasion, by activating Src family kinase (SFK) signalling. However, it is not known how P-cadherin and SFK activation impact cell-cell biomechanical properties. In the present work, using atomic force microscopy (AFM) images, cell stiffness and cell-cell adhesion measurements, and undirected graph analysis based on microscopic images, we have demonstrated that P-cadherin overexpression promotes significant alterations in cell's morphology, by decreasing cellular height and increasing its area. It also affects biomechanical properties, by decreasing cell-cell adhesion and cell stiffness. Furthermore, cellular network analysis showed alterations in intercellular organization, which is associated with cell-cell adhesion dysfunction, destabilization of an E-cadherin/p120ctn membrane complex and increased cell invasion. Remarkably, inhibition of SFK signaling, using dasatinib, reverted the pathogenic P-cadherin induced effects by increasing cell's height, cell-cell adhesion and cell stiffness, and generating more compact epithelial aggregates, as quantified by intercellular network analysis. In conclusion, P-cadherin/SFK signalling induces topological, morphological and biomechanical cell-cell alterations, which are associated with more invasive breast cancer cells. These effects could be further reverted by dasatinib treatment, demonstrating the applicability of AFM and cell network diagrams for measuring the epithelial biomechanical properties and structural organization.

<b>Enhancement of Src Family Kinase Activity is Essential for p38 MAP Kinase-Mediated Dedifferentiation Signal of Parotid Acinar Cells </b>

<b>Enhancement of Src Family Kinase Activity is Essential for p38 MAP Kinase-Mediated Dedifferentiation Signal of Parotid Acinar Cells </b>

Src Family Kinases in Brain Edema After Acute Brain Injury.

Brain edema, the first stage of intracranial hypertension, has been associated with poor prognosis and increased mortality after acute brain injury such as ischemic stroke, intracranial hemorrhage (ICH), and traumatic brain injury (TBI). Acute brain injury often initiates release of many molecules, including glutamate, adenosine, thrombin, oxyhemoglobin, cytokines, reactive oxygen species (ROS), damage-associated molecular pattern molecules (DAMPs), and others. Most of these molecules activate Src family kinases (SFKs), a family of proto-oncogenic non-receptor tyrosine kinases, resulting in blood-brain barrier (BBB) disruption and brain edema at the acute stage after brain injury. However, SFKs also contribute to BBB self-repair and brain edema resolution in the chronic stage that follows brain injury. In this review, we summarize possible pathways through which SFKs are implicated in both brain edema formation and its eventual resolution.

Investigation of the Mechanism of Idelalisib Resistance in the Follicular Lymphoma WSU-Fsccl Cell Line

Idelalisib (Zydelig®), a first-in-class, selective, oral inhibitor of PI3Kd, is approved in the US and EU for the treatment of patients with follicular lymphoma (FL) who have received at least 2 prior systemic therapies based on the outcome of a phase 2 clinical trial demonstrating an ORR of 54% (Gopal AK, et al. ASH 2014, Abstract 1708; Zydelig® SmPC, Mar 2015). Complete responses are rare (6%) and patients ultimately relapse leading to an urgent need to understand mechanisms of resistance (MOR) to idelalisib. We report here the MOR identified from 3 sets of the transformed FL cell line WSU-FSCCL which were made resistant to idelalisib by continuous in vitro exposure.Methods: Idelalisib resistance was established by continuous passaging of a clonal isolate of WSU-FSCCL in the presence of 1 μM idelalisib. Growth inhibition to idelalisib or other inhibitors was performed using CellTiter Glo viability assay (Promega) at 96 h. Clonal isolates of idelalisib sensitive (FSCCLS) and idelalisib resistant (FSCCLR) cell lines were generated through two rounds of single cell limiting dilution. Characterization of mutations and gene expression in FSCCLS and FSCCLR clones was done by whole exome sequencing (WES, GENEWIZ) and RNA-Seq (Expression Analysis), respectively. Whole cell lysates were analyzed by Simple Western using Peggy Sue (Protein Simple) or SDS-PAGE and Western blot.Results: WSU-FSCCL were highly sensitive to idelalisib and the pan-PI3K inhibitor GDC-0941 with an EC50 of 140 and 180 nM, respectively, indicating that cell viability is driven by PI3Kd, while profiling of BTK inhibitors showed continued lack of activity. FSCCLR showed a loss of sensitivity to idelalisib (1 μM) with a maximal growth inhibition of 16% vs. 85% for parental line (FSCCLS). WES analysisrevealed PIK3CA resistance mutations in 3 independently generated sets of FSCCLR single cell clones, while no PIK3CD, PIK3CB, or PIK3CG resistance mutations were observed. PIK3CA was mutated at N345K (11/11 clones), P539R (3/3 clones) and E970K (3/3 clones). PI3K isoform profiling showed no alterations in expression of any of the four p110 isoforms. Idelalisib activity on PIK3CA mutant clone viability was dramatically shifted (EC50 >5 μM). The sensitivity of PIK3CA mutant FSCCLR to the PI3Kα- specific inhibitor BYL719 was not increased compared to FSCCLS (EC50 > 1.5 μM).Interestingly the combination of idelalisib treatment with BYL719 (0.5 μM) restored the sensitivity of FSCCLR to a concentration in the range of parental cells (EC50 = 0.066 μM, Figure 1). A second set of FSCCLR clones (set 2) were PI3KCA WT and had a 2.7-fold upregulation of PTEN. A survey of compensatory pathway activation revealed upregulation of Src family kinase (SFK) phosphorylation (p-SFK Y416) and specifically of p-Hck Y411 and p-Lyn Y396. SFK phosphorylation was sensitive to treatment with the SFK inhibitor dasatinib. Set 2 FSCCLR were slightly less sensitive to dasatinib (EC50 = 0.058 μM) compared to FSCCLS (EC50 = 0.034 μM). Interestingly, addition of 30 nM dasatinib (EC25 in FSCCLS) to FSCCLR increased the sensitivity of set 2 FSCCLR to idelalisib (EC50 = 0.95 μM) (Figure 2). FSCCLR clones were more resistant to the Syk inhibitor entospletinib (ENTO) (EC50 > 10 μM) as compared to FSCCLS (EC50 = 0.18 μM). Profiling of idelalisib activity on set 2 FSCCLR clones showed resistance to idelalisib as expected (EC50 > 10, μM); the addition of entospletinib at a clinically relevant concentration (0.68 μM), resulted in greater sensitivity than use of either single agent alone (EC50 = 2.27 μM ) (Figure 3). RNA-Seq analysis of the FSCCLR PIK3CA WT single cell clones additionally revealed that a subset of clones (set 3) upregulated a set of WNT pathway genes. Western blot analysis of set 3 FSCCLR showed upregulation of downstream markers of the canonical WNT pathway, including LEF1/TCF, c-Jun, β-catenin, c-Myc and p-GSK3β.Conclusions: Mechanisms of idelalisib resistance in the follicular lymphoma WSU-FSCCL cell line were independent of alterations in p110d but included the likely gain of function mutations in the PIK3CA gene and activation of SFK and WNT pathways. The sensitivity to idelalisib in the resistant clones could be re-established by the combination of idelalisib with inhibitors of compensatory pathways: BYL719 in PIK3CA mutant clones and dasatinib or entospletinib in SFK activated clones. [Display omitted] [Display omitted] [Display omitted] DisclosuresSorensen:Gilead Sciences: Employment, Other: Share holder. Meadows:Gilead Sciences: Employment, Other: Share holder. Yahiaoui:Gilead Sciences: Employment, Other: Share holder. Li:Gilead Sciences: Employment, Other: Share holder. Yue:Gilead Sciences: Employment, Other: Share holder. Kashishian:Gilead Sciences: Employment, Other: Share holder. Queva:Gilead Sciences: Other: Share holder. Tannheimer:Gilead Sciences: Employment, Other: Share holder.

Abstract LB-B02: Acquisition of stem cell-like properties accompanying with Src family kinase/Focal adhesion kinase activation contributes to acquired resistance to afatinib in lung cancer cells

Abstract [Background] Most NSCLC patients harboring activating EGFR mutations benefit from treatment with EGFR-TKIs, but the final clinical efficacy of EGFR-TKIs varies because of development of tumor resistant to EGFR-TKIs. Multiple kinase-targeted 2nd generation TKIs such as afatinib have been developed to overcome drug resistance to the 1st generation TKIs. Afatinib is an irreversible multitargeted TKI targeting EGFR including T790M, HER2 and HER4. To develop further personalized therapeutics and drug resistance modifiers, we should understand the molecular based mechanism of drug resistance to 2nd as well as 3rd generation TKIs. In our present study, we present a novel finding that acquisition of cancer stem-like cells properties accompanying with activation of residual Src family kinase (SFK)/focal adhesion kinase (FAK) is responsible for the survival of afatinib-resistant lung cancer cells when expression of targeted EGFR, HER2 and HER4 is abraded. [Materials and methods] We have established afatinib-resistant subclones from human lung cancer cell lines, HCC827, harboring activating EGFR mutation after stepwise exposure to afatinib. The afatinib-resistant clones do not harbor T790M, K-ras mutation or PTEN loss. We have characterized biochemical properties of drug-resistant subclones as compared with their drug-sensitive counterparts by Western blot and RT-PCR. ALDEFLUOR kit was used to isolate cell populations with the stem cell specific ALDH enzymatic activity. [Results] We characterized two independent afatinib-resistant subclones, BR1-8 and BR2-3. [1] Afatinib-resistant subclones showed markedly decreased expression of EGFR, HER2, HER3, c-Met and PDGFRβ as compared with their parental cell lines, HCC827; [2] Akt phosphorylation in afatinib-resistant subclones was not suppressed by afatinib; [3] SFK and FAK were activated in resistant subclones, and combination of afatinib with c-Src siRNA or with dasatinib moderately suppressed cell growth and AKT phosphorylation; [4] Combination of afatinib with siFAK moderately blocked cell growth of resistant subclones. [5] Cancer stem like cell marker, ALDH1, positive cell population increased in afatinib-resistant sublines compared with HCC827 cell lines. [Conclusions] Together, our present study presents that increase of cancer stem like cells and SFK/FAK activation could be a mechanism responsible for acquired resistance to afatinib. We would further discuss the association of ALDH1 expression and SFK/FAK activation with afatinib resistance in lung cancer. Citation Format: Yuichi Murakami, Kahori Sonoda, Koichi Azuma, Kosuke Watari, Michihiko Kuwano, Mayumi Ono. Acquisition of stem cell-like properties accompanying with Src family kinase/Focal adhesion kinase activation contributes to acquired resistance to afatinib in lung cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B02.

Alternate RASSF1 Transcripts Control SRC Activity, E-Cadherin Contacts, and YAP-Mediated Invasion

SummaryTumor progression to invasive carcinoma is associated with activation of SRC family kinase (SRC, YES, FYN) activity and loss of cellular cohesion. The hippo pathway-regulated cofactor YAP1 supports the tumorigenicity of RAS mutations but requires both inactivation of hippo signaling and YES-mediated phosphorylation of YAP1 for oncogenic activity. Exactly how SRC kinases are activated and hippo signaling is lost in sporadic human malignancies remains unknown. Here, we provide evidence that hippo-mediated inhibition of YAP1 is lost upon promoter methylation of the RAS effector and hippo kinase scaffold RASSF1A. We find that RASSF1A promoter methylation reduces YAP phospho-S127, which derepresses YAP1, and actively supports YAP1 activation by switching RASSF1 transcription to the independently transcribed RASSF1C isoform that promotes Tyr kinase activity. Using affinity proteomics, proximity ligation, and real-time molecular visualization, we find that RASSF1C targets SRC/YES to epithelial cell-cell junctions and promotes tyrosine phosphorylation of E-cadherin, β-catenin, and YAP1. RASSF1A restricts SRC activity, preventing motility, invasion, and tumorigenesis in vitro and in vivo, with epigenetic inactivation correlating with increased inhibitory pY527-SRC in breast tumors. These data imply that distinct RASSF1 isoforms have opposing functions, which provide a biomarker for YAP1 activation and explain correlations of RASSF1 methylation with advanced invasive disease in humans. The ablation of epithelial integrity together with subsequent YAP1 nuclear localization allows transcriptional activation of β-catenin/TBX-YAP/TEAD target genes, including Myc, and an invasive phenotype. These findings define gene transcript switching as a tumor suppressor mechanism under epigenetic control.

Inhibition of Lyn is a promising treatment for mantle cell lymphoma with bortezomib resistance.

Although proteasome inhibition with bortezomib (BTZ) is a validated treatment for relapsed or refractory mantle cell lymphoma (MCL), many patients show resistance to BTZ. However, the molecular mechanism of BTZ resistance in MCL has not been elucidated. In the present study, we investigated BTZ-resistant MCL cells in vitro and in vivo. We demonstrate that BTZ-resistant MCL cells showed highly increased expression of the B-cell receptor (BCR) components CD79A and CD19. Activation of the BCR signaling pathway enhanced the activity of Src family kinases (SFKs), especially Lyn, and downstream kinases PI3K/AKT/mTOR in BTZ-resistant MCL cells. Depletion of CD79A and Lyn significantly reduced several kinase activities involved in PI3K signaling, leading to inhibition of proliferation. In addition, the SFKs inhibitor dasatinib inhibited the proliferation of BTZ-resistant cells, preventing the binding of CD19 with Lyn and PI3K p85. We also verified our findings with the mouse xenograft tumor model. Dasatinib treatment significantly decreased tumor size in the mouse bearing BTZ-resistant MCL cells, but not in the mouse bearing BTZ-sensitive MCL cells. Collectively, our data show that overexpression of the BCR and its activated signaling confers BTZ resistance in MCL cells. Thus, targeting BCR signaling with dasatinib could be a novel therapeutic approach for patients with MCL that has relapsed or is refractory to treatment with BTZ.

Ionizing Radiation Induces Phosphorylation of Caveolin-1 on Tyr14

Caveolin-1 (CAV1) is a 22 kDa membrane protein associated with caveolae in the cell membrane. It is involved in endocytosis, modulation of signal transduction, and has been reported to mediate radioprotection. Various stimuli such as oxidative stress, insulin, or osmotic shock are known to induce phosphorylation of caveolin-1 on Tyr14. This event is an important prerequisite for CAV1 function in the formation of caveolae and the interaction with multiple signaling molecules. The aim of the present study was to test if ionizing radiation (IR) induces phosphorylation of CAV1 on Tyr14 similar to oxidative stress, and to test the role of stress signaling kinases p38, Src family kinases (SFK), and c-Jun N-terminal kinase (JNK). The human lymphoblastoid cell line TK6, which does not express endogenous CAV1, was transduced with two different lentiviral vectors, containing either the cDNA of wtCAV1 or the cDNA of CAV1Y14F (substitution of Tyr by Phe on position 14) connected by the IRES element of EMCV with the cDNA of EGFP. Oxidative stress in TK6-CAV1 and TK6-CAV1Y14F was induced by treatment with 1 mM - 50 mM H2O2 for 20 min at 37 °C; cell irradiation was performed using 6 MV X-ray (6.7 Gy/min, Elekta Synergy). Cell lysates were prepared at different time points (5 min to 8 h) after IR. Specific kinase inhibitors were used to explore the functional relevance of kinase activation for CAV1 phosphorylation after H2O2 or IR. Phosphorylation was detected by Western blotting using phospho-specific antibodies, quantified and normalized to the unphosphorylated form. Mean values and SEM of at least three independent experiments were calculated. Phosphorylation of CAV1, p38, SFK and JNK in TK6-CAV1 was markedly increased by H2O2 concentrations in the range of 5 mM – 20 mM. Activation of p38, SFK and JNK was similar in TK6-CAV1Y14F and thus independent of Tyr14 phosphorylation of CAV1. Radiation doses of 2 Gy and 4 Gy clearly increased phosphorylation of CAV1, p38, SFK and JNK with different kinetics. While maximum CAV1 and SFK phosphorylation occurred early after irradiation (15 min – 1 h), activation of p38 and JNK peaked at the later time points (1 h – 3 h). Inhibition of p38, SFK and JNK revealed SFK as the major kinases for CAV1 phosphorylation on Tyr14 after induction of oxidative stress. Induction of CAV1 phosphorylation on Tyr14 and activation of stress-associated signaling molecules indicated the activation of similar signaling proteins in response to H2O2 treatment and IR with H2O2 -induced phosphorylation being more potent. Activation of SFK was an important factor for the phosphorylation of CAV1 after treatment with H2O2 in the TK6 cells used here. Studies on the role of the different kinases and phosphorylation of CAV1 regarding proliferation and apoptosis after irradiation are in progress.

Multi-Inhibitory Effects of A2A Adenosine Receptor Signaling on Neutrophil Adhesion Under Flow.

A2A adenosine receptor (A2AAR) signaling negatively regulates inflammatory responses in many disease models, but the detailed mechanisms remain unclear. We used the selective A2AAR agonist, ATL313, to examine how A2AAR signaling affects human and murine neutrophil adhesion under flow. Treating neutrophils with ATL313 inhibited selectin-induced, β2 integrin-dependent slow rolling and chemokine-induced, β2 integrin-dependent arrest on ICAM-1. ATL313 inhibited selectin-induced β2 integrin extension, which supports slow rolling, and chemokine-induced hybrid domain "swing-out," which supports arrest. Furthermore, ATL313 inhibited integrin outside-in signaling as revealed by reduced neutrophil superoxide production and spreading on immobilized anti-β2 integrin Ab. ATL313 suppressed selectin-triggered activation of Src family kinases (SFKs) and p38 MAPK, chemokine-triggered activation of Ras-related protein 1, and β2 integrin-triggered activation of SFKs and Vav cytoskeletal regulatory proteins. ATL313 activated protein kinase A and its substrate C-terminal Src kinase, an inhibitor of SFKs. Treating neutrophils with a protein kinase A inhibitor blocked the actions of ATL313. In vivo, ATL313-treated neutrophils rolled faster and arrested much less frequently in postcapillary venules of the murine cremaster muscle after TNF-α challenge. Furthermore, ATL313 markedly suppressed neutrophil migration into the peritoneum challenged with thioglycollate. ATL313 did not affect A2AAR-deficient neutrophils, confirming its specificity. Our findings provide new insights into the anti-inflammatory mechanisms of A2AAR signaling and the potential utility of A2AAR agonists in inflammatory diseases.