Receptor Tyrosine Kinase Phosphorylation Research Articles

No Sugar Added: A New Strategy to Inhibit Glioblastoma Receptor Tyrosine Kinases.

A novel inhibitor of N-linked glycosylation (NGI-1) inhibits the glycosylation and phosphorylation of multiple receptor tyrosine kinases in glioblastoma (GBM). NGI-1 sensitizes multiple models of GBM to chemotherapy and radiation in vitro and in vivo and may be especially effective in GBMs that retain active PTEN.See related article by Baro et al., p. 784.

Boswellia frereana suppresses HGF-mediated breast cancer cell invasion and migration through inhibition of c-Met signalling

BackgroundHepatocyte growth factor (HGF) plays a pivotal role in breast cancer cell motility, invasion and angiogenesis. These pro-metastatic events are triggered through HGF coupling and activation of the c-Met receptor. Reports have demonstrated that HGF/c-Met signalling plays an important part in breast cancer progression and that their expression is linked to poor patient outcome. In the present study, we investigated the anti-metastatic potential of an extract from traditional Somalian frankincense, Boswellia frereana, on human breast cancer cells. In addition, we also examined the effect of this Boswellia frereana extract (BFE) upon HGF-mediated stimulation of the c-Met receptor.MethodsTwo triple negative human breast cancer cell lines, BT549 and MDA-MB-231, were utilised in the study to examine the effect of BFE on tumour cell proliferation, migration, matrix-adhesion, angiogenesis and invasion. Cell migration was investigated using a Cell IQ time-lapsed motion analysis system; while tumour cell–matrix adhesion, angiogenesis and invasion were assessed through Matrigel-based in vitro assays. Breast cancer cell growth and spheroid formation was examined through proliferation assay and 3D non-scaffold cell culture techniques. Western Blotting was employed to determine the phosphorylation status of the c-Met receptor tyrosine kinase following BFE treatment and subsequent HGF stimulation.ResultsFollowing HGF treatment, the breast cancer cells displayed a significant increase in migration, matrix adhesion, vessel/tubule formation, invasion and c-Met activation. HGF did not appear to have any bearing on the proliferation rate or spheroid formation of these breast cancer cells. The addition of the BFE extract quenched the HGF-enhanced migratory, angiogenic and invasive potential of these cells. Further study revealed that BFE inhibited c-Met receptor tyrosine kinase phosphorylation within these breast cancer cells.ConclusionsOur findings reveal that BFE was able to significantly suppress the influence of HGF in breast cancer cell motility and invasion in vitro, through the ability of BFE to reduce HGF/c-Met signalling events. Therefore, these results indicate that BFE could play a novel role in the treatment of breast cancer.

BMX-Mediated Regulation of Multiple Tyrosine Kinases Contributes to Castration Resistance in Prostate Cancer.

Prostate cancer responds to therapies that suppress androgen receptor (AR) activity (androgen deprivation therapy, ADT) but invariably progresses to castration-resistant prostate cancer (CRPC). The Tec family nonreceptor tyrosine kinase BMX is activated downstream of PI3K and has been implicated in regulation of multiple pathways and in the development of cancers including prostate cancer. However, its precise mechanisms of action, and particularly its endogenous substrates, remain to be established. Here, we demonstrate that BMX expression in prostate cancer is suppressed directly by AR via binding to the BMX gene and that BMX expression is subsequently rapidly increased in response to ADT. BMX contributed to CRPC development in cell line and xenograft models by positively regulating the activities of multiple receptor tyrosine kinases through phosphorylation of a phosphotyrosine-tyrosine (pYY) motif in their activation loop, generating pYpY that is required for full kinase activity. To assess BMX activity in vivo, we generated a BMX substrate-specific antibody (anti-pYpY) and found that its reactivity correlated with BMX expression in clinical samples, supporting pYY as an in vivo substrate. Inhibition of BMX with ibrutinib (developed as an inhibitor of the related Tec kinase BTK) or another BMX inhibitor BMX-IN-1 markedly enhanced the response to castration in a prostate cancer xenograft model. These data indicate that increased BMX in response to ADT contributes to enhanced tyrosine kinase signaling and the subsequent emergence of CRPC, and that combination therapies targeting AR and BMX may be effective in a subset of patients.Significance: The tyrosine kinase BMX is negatively regulated by androgen and contributes to castration-resistant prostate cancer by enhancing the phosphorylation and activation of multiple receptor tyrosine kinases following ADT. Cancer Res; 78(18); 5203-15. ©2018 AACR.

Production via good manufacturing practice of exofucosylated human mesenchymal stromal cells for clinical applications

BackgroundThe regenerative and immunomodulatory properties of human mesenchymal stromal cells (hMSCs) have raised great hope for their use in cell therapy. However, when intravenously infused, hMSCs fail to reach sites of tissue injury. Fucose addition in α(1,3)-linkage to terminal sialyllactosamines on CD44 creates the molecule known as hematopoietic cell E-/L-selectin ligand (HCELL), programming hMSC binding to E-selectin that is expressed on microvascular endothelial cells of bone marrow (BM), skin and at all sites of inflammation. Here we describe how this modification on BM-derived hMSCs (BM-hMSCs) can be adapted to good manufacturing practice (GMP) standards. MethodsBM-hMSCs were expanded using xenogenic-free media and exofucosylated using α(1,3)-fucosyltransferases VI (FTVI) or VII (FTVII). Enforced fucosylation converted CD44 into HCELL, and HCELL formation was assessed using Western blot, flow cytometry and cell-binding assays. Untreated (unfucosylated), buffer-treated and exofucosylated BM-hMSCs were each analyzed for cell viability, immunophenotype and differentiation potential, and E-selectin binding stability was assessed at room temperature, at 4°C, and after cryopreservation. Cell product safety was evaluated using microbiological testing, karyotype analysis, and c-Myc messenger RNA (mRNA) expression, and potential effects on genetic reprogramming and in cell signaling were analyzed using gene expression microarrays and receptor tyrosine kinase (RTK) phosphorylation arrays. ResultsOur protocol efficiently generates HCELL on clinical-scale batches of BM-hMSCs. Exofucosylation yields stable HCELL expression for 48 h at 4°C, with retained expression after cell cryopreservation. Cell viability and identity are unaffected by exofucosylation, without changes in gene expression or RTK phosphorylation. DiscussionThe described exofucosylation protocol using xenogenic-free reagents enforces HCELL expression on hMSCs endowing potent E-selectin binding without affecting cell viability or native phenotype. This described protocol is readily scalable for GMP-compliant clinical production.

Synthesis, Evaluation for Cytotoxicity and Molecular Docking Studies of Benzo[c]furan-Chalcones for Potential to Inhibit Tubulin Polymerization and/or EGFR-Tyrosine Kinase Phosphorylation.

A series of 2-arylbenzo[c]furan-chalcone hybrids 3a–y have been synthesized and evaluated for antiproliferative effects against the human breast cancer (MCF-7) cell line and for its potential to induce apoptosis and also to inhibit tubulin polymerization and/or epidermal growth factor receptor-tyrosine kinase (EGFR-TK) phosphorylation. Most of these compounds exhibited moderate to significant antigrowth effects in vitro against the MCF-7 cell line when compared to the reference standard actinomycin D. The capabilities of the most cytotoxic benzofuran-chalcone hybrids 3b and 3i, to induce apoptosis, have been evaluated by Annexin V-Cy3 SYTOX staining and caspase-3 activation. The experimental and molecular docking results suggest that the title compounds have the potential to exhibit inhibitory effects against tubulin polymerization and epidermal growth factor receptor tyrosine kinase (EGFR-TK) phosphorylation. The modeled structures of representative compounds displayed hydrophobic interactions as well as hydrogen and/or halogen bonding with the protein residues. These interactions are probably responsible for the observed increased binding affinity for the two receptors and their significant antigrowth effect against the MCF-7 cell line.

Stemness Phenotype in Tamoxifen Resistant Breast Cancer Cells May be Induced by Interactions Between Receptor Tyrosine Kinases and ERα-66.

Tamoxifen is widely administered for patients with estrogen receptor-positive breast cancer. Despite many patients benefiting from Tamoxifen as an effective anti-hormonal agent in adjuvant therapy, a noticeable number of patients tend to develop resistance. The aim of this study was to shed light upon the molecular mechanisms associated with Tamoxifen resistance which can help improve current treatment strategies available for stimulating responsiveness and combating resistance. Relevant articles were obtained from PubMed and google scholar, nearly all dated from 2010 to 2017. Articles were screened to select the ones meeting the objective. The molecular interactions in the resistant network were extracted from the appropriate articles. The mechanisms of developing Tamoxifen resistance were briefly outlined. Overactivation of Receptor Tyrosine Kinases (RTKs) pathways, commonly known as alternative growth cascades, is one of the main players in acquired cancer cell stemness, which can induce unrestricted proliferation in the presence of Tamoxifen. There are seven recent patents including 6291496B1 as an anti-HER2, 8143226B2 as an inhibitor of RTK phosphorylation, 9062308B2 as an anti-HOXB7, Lapatinib functioning as an anti-EGFR/HER2, Everolimus as an inhibitor of mTOR, Exemestane as an aromatase inhibitor and Perifosine as an AKT inhibitor. Altogether, it seems that tumor cells express a stemness phenotype which tends to override anti-hormonal adjuvant therapies. Since RTKs are overactivated and overexpressed in such cells, specialized targeted therapies suppressing RTKs would be a novel and effective way in restoring Tamoxifen sensitivity in resistant breast cancer tumor cells.

Abstract 899: Phosphorylated mTOR by signaling through EphB3 confers resistance to a selective FGFR inhibitor in SNU-16 gastric cancer cells

Abstract Because about 10% of gastric cancers show amplification of FGFR2, inhibition of FGFR2 activation has been regarded as one of therapeutic targets. AZD4547, a selective inhibitor of the FGFR1-3 tyrosine kinases, was developed to inhibit FGFR signaling; however, its efficacy is limited by emergency of the acquired resistance. We tried to clarify a resistance mechanism against the FGFR inhibitor. The cell line resistant against AZD4547 was established using SNU-16 (SNU16R), an FGFR2-amplified gastric cancer cell line, by culturing with increasing concentration of AZD4547. The expression levels of FGFR or phosphorylated FGFR (pFGFR), EphB3 and downstream signaling molecules, proteins involved in epithelial-mesenchymal transition (EMT) and apoptosis, were determined by Western blot. Cell viability was measured with EZ-Cytox kit reagent (DoGen, Seoul, Korea). Relative level of tyrosine phosphorylation of receptor tyrosine kinases (RTKs) was evaluated with Proteome ProfilerTM array by human phosphor-RTK kit (R&D Systems). Apoptosis was determined by Annexin V-FITC apoptosis detection kit (BioBud, Seoul, Korea). Cell cycle assay was performed using Beckman Coulter flow cytometer and WinMDI for determination of cell numbers in G1, S, G2/M phases. Here we show the following: 1) establishment of a gastric cancer cell line (SNU16) resistant to AZD4547 (SNU16R); 2) expression of EphB3 receptor in SNU16R and induction of apoptosis and cell cycle arrest as well as inhibition of cell proliferation and suppressed expression of EMT markers under the treatment EphB3 inhibitor in SNU16R; 3) demonstration of mTOR as a downstream signaling cascade molecule of EphB3 by showing increased expression of phosphorylated (p-) mTOR and its downstream molecules in SNU16R and suppressed expression of p-mTOR by the EphB3 inhibitor. Cell cycle arrest, inhibition of cell proliferation and suppressed expression of p-mTOR under treatment with everolimus were also shown. We concluded that phosphorylation of mTOR through activation of EphB3 receptor is one of the mechanisms that confer resistance to AZD 4547, a selective FGFR inhibitor, in gastric cancer cells. Citation Format: Suk-young Lee, YooJin Na, YoonA Jung, JungLim Kim, Sang Cheul Oh, Dae-Hee Lee. Phosphorylated mTOR by signaling through EphB3 confers resistance to a selective FGFR inhibitor in SNU-16 gastric cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 899.

Abstract 4781: Changes in intracellular signaling following chronic FGFR inhibition in urothelial bladder cancer models

Abstract Preclinical and clinical studies have validated the therapeutic potential of FGFR inhibition in urothelial bladder cancer (UBC) patients with FGFR genetic aberrations. Rogaratinib is a potent small-molecule pan-FGFR inhibitor being studied in phase I trials in UBC. Here, we studied the effects of chronic exposure of bladder cancer cells in vitro to FGFR inhibition by rogaratinib to identify changes in signaling and gene expression patterns that may identify possible drug combinations that may enhance efficacy of rogaratinib and help overcome inherent and/or acquired treatment resistance. Cell proliferation in response to rogaratinib was evaluated using crystal violet staining in a panel of 13 UBC cell lines. Continuous culture of two cell lines–JMSU1 and RT112–with either constant or increasing concentrations of rogaratinib in several independent approaches generated the resistant sublines, JMSU1-R1 to -R4 and RT112-R1 to -R4. In both cellular models rogaratinib resistance (defined as >30-fold [JMSU1] or >100-fold [RT112] difference in absolute IC50 of sublines vs. parental lines) arose reproducibly and with various treatment schedules. Morphologic changes were also observed. Transcriptomic (RNAseq) and proteomic (R&D Systems proteome profiler arrays) analyses of these cell lines or rogaratinib-treated versus untreated parental cells, respectively, revealed changes that co-occur with the development of resistance. Analysis of the phospho-proteome showed increased phosphorylation of several receptor tyrosine kinases compared to the parental cell lines. Phosphorylation levels varied among the 4 resistant sublines that were derived from the same parental line. This resulted in alteration of downstream signaling pathways in rogaratinib-treated sublines compared to parental cell lines. In conclusion, exposure of selected bladder cancer cell lines to rogaratinib resulted in development of resistance and changes in FGFR signaling and gene expression pathways that may identify strategies to optimize treatment with FGFR inhibitors. Citation Format: Isabel S. Jerchel, Atanas Kamburov, Ralf Lesche, Sabine Zitzmann-Kolbe, Alexander Walter, Peter Ellinghaus, Dominik Mumberg, Oliver Politz, Sylvia Gruenewald. Changes in intracellular signaling following chronic FGFR inhibition in urothelial bladder cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4781.

Abstract 3092: Redirection of human HER2-positive breast cancer cells

Abstract Breast cancer is the most common form of cancer among women, affecting one eigth of the female population. While advances in clinical management and therapeutic modalities have been achieved, serious limitations in the improvement of outcomes after the diagnosis remain, largely due to the complexity of the underlying biology. Studying the mechanism that makes cancer cells tumorigenic and how they can lose the ability to cause malignant formations is the first step towards understanding complex mechanisms of this disease. The normal mammary microenvironment can redirect cells to assume a normal mammary epithelial phenotype. This phenomenon also applies to tumorigenic cells; cancer cells lose their tumor-forming capacity and differentiate, assuming a normal, non-tumorigenic phenotype. This phenomenon has been termed “cancer cell redirection.” Comparing cells that went through this transformation with original cancer cells, features that identify tumorigenic activity can be acknowledged. We have established an in vitro model of cancer cell redirection, in which HER2+ breast cancer cells are grown alone or in co-culture with normal human breast epithelial cells. The ratios of cells used in co-cultures, 1:1 and 1:50 (cancer: normal) were previously established through in vivo and in vitro studies. Redirection was confirmed by immunostaining. The metric used to determine cancer cell redirection was the absence of receptor tyrosine kinase phosphorylation without a change in surface expression of the receptor. HER2 and P-HER2 expression were both present in the HER2+ cancer cells and in the: MCF10A and 1:1 co-cultures. However, HER2 expression was observed in 1:50 co-cultures, but an absence of P-HER2 was noted. Based on attenuation of receptor phosphorylation in the 1:50 co-cultures we can confirm the breast cancer cell redirection, which matches our previous results. Furthermore, alteration of HER2 dimerization patterns in redirected cells were investigated. The capacity of cancer and redirected cells to form tumors was examined with use of mammosphere assay. Formation of mammospheres has correlated to stem cell function and tumor-initiation capacity. The HER2+ breast cancer cell lines formed significantly higher numbers of spheres compared to the non-tumorigenic breast epithelial cells. Mammosphere formation is decreased in redirected cancer cells. Breast cancer stem cells are HER2+ regardless of pathological or genetic classification. This suggests that HER2 expression is not only a target in HER2+ breast cancer but should be targeted in all breast cancers to prevent breast cancer stem cells from recapitulating tumors following treatment. Our data demonstrates that by attenuating activity of HER2 theses cancer cells lose the capacity to form tumors in vivo and lose their cancer phenotype in vitro. Citation Format: Anastasia Frank-Kamenetskii, Julia M. Mook, Brian W. Booth. Redirection of human HER2-positive breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3092.

Direct Phosphorylation of SRC Homology 3 Domains by Tyrosine Kinase Receptors Disassembles Ligand-Induced Signaling Networks

Phosphotyrosine (pTyr) signaling has evolved into a key cell-to-cell communication system. Activated receptor tyrosine kinases (RTKs) initiate several pTyr-dependent signaling networks by creating the docking sites required for the assembly of protein complexes. However, the mechanisms leading to network disassembly and its consequence on signal transduction remain essentially unknown. We show that activated RTKs terminate downstream signaling via the direct phosphorylation of an evolutionarily conserved Tyr present in most SRC homology (SH) 3 domains, which are often part of key hub proteins for RTK-dependent signaling. We demonstrate that the direct EPHA4 RTK phosphorylation of adaptor protein NCK SH3s at these sites results in the collapse of signaling networks and abrogates their function. We also reveal that this negative regulation mechanism is shared by other RTKs. Our findings uncover a conserved mechanism through which RTKs rapidly and reversibly terminate downstream signaling while remaining in a catalytically active state on the plasma membrane.

Benzofuran–appended 4-aminoquinazoline hybrids as epidermal growth factor receptor tyrosine kinase inhibitors: synthesis, biological evaluation and molecular docking studies

A series of 2-arylbenzo[b]furan–appended 4-aminoquinazoline hybrids were prepared and evaluated for cytotoxicity in vitro against the human lung cancer (A549), colorectal adenocarcinoma (Caco-2), hepatocellular carcinoma (C3A) and cervical cancer (HeLa) cell lines. Compounds 10d and 10j exhibited significant cytotoxicity against the C3A and Caco-2 cell lines and induced apoptosis in these cell lines. Likewise, compounds 10d and 10e exhibited significant inhibitory activity towards epidermal growth factor receptor-tyrosine kinase phosphorylation (IC50 values of 29.3 nM and 31.1 nM, respectively) against Gefitinib (IC50 = 33.1 nM). Molecular docking of compounds 10 into EGFR-TK active site suggests that they bind to the region of EGFR like Gefitinib does.

Gramine attenuates EGFR-mediated inflammation and cell proliferation in oral carcinogenesis via regulation of NF-κB and STAT3 signaling

Gramine, a natural indole alkaloid found in Hordeum vulgare has been possesses anti-mutagenic properties. The aim of the present study was to evaluate the effect of gramine on inflammation and proliferation in 7,12-dimethylbenz[a]anthracene(DMBA)-induced hamster buccal pouch (HBP) carcinogenesis. Epidermal growth factor receptor (EGFR) tyrosine kinase phosphorylation trigged PI3K/Akt/mTOR and JAK signaling that activates NF-κB and STAT3. In contrast, gramine suppressed EGFR tyrosine kinase phosphorylation and simultaneously inhibiting PI3K/Akt/mTOR and JAK phosphorylation, thereby blockage NF-κB and STAT3 nuclear translocation. Attenuation of these oncogenic signals leads to downregulated iNOS, COX-2, TNF-α, IL-6, cyclin D1 and PCNA protein expressions. In addition, gramine enhanced the expression of the tumor suppressors p53, p21(WAF1/CIP1) and Gsk-3β by inhibiting MDM2. These results suggested that gramine exhibited anti-inflammation and anti-proliferation effects via suppressed EGFR/PI3K/Akt/mTOR/ IKK/NF-κB and JAK/STAT3 signaling pathways.

Microfluidic preparation of anchored cell membrane sheets for in vitro analyses and manipulation of the cytoplasmic face

Molecular networks on the cytoplasmic faces of cellular plasma membranes are critical research topics in biological sciences and medicinal chemistry. However, the selective permeability of the cell membrane restricts the researchers from accessing to the intact intracellular factors on the membrane from the outside. Here, a microfluidic method to prepare cell membrane sheets was developed as a promising tool for direct examination of the cytoplasmic faces of cell membranes. Mammalian cells immobilized on a poly(ethylene glycol)-lipid coated substrate were rapidly and efficiently fractured, with the sheer stress of laminar flow in microchannels, resulting in isolation of the bottom cell membrane sheets with exposed intact cytoplasmic faces. On these faces of the cell membrane sheets, both ligand-induced phosphorylation of receptor tyrosine kinases and selective enzymatic modification of a G-protein coupling receptor were directly observed. Thus, the present cell membrane sheet should serve as a unique platform for studies providing new insights into juxta-membrane molecular networks and drug discovery.

Activation Status of Receptor Tyrosine Kinases as an Early Predictive Marker of Response to Chemotherapy in Osteosarcoma

Receptor tyrosine kinases (RTKs) are membrane receptors that play a vital role in various biological processes, in particular, cell survival, cell proliferation, and cell differentiation. These cellular processes are composed of multitiered signaling cascades of kinases starting from ligand binding to extracellular domains of RTKs that activate the entire pathways through tyrosine phosphorylation of the receptors and downstream effectors. A previous study reported that, based on proteomics data, RTKs were a major candidate target for osteosarcoma. In this study, activation profiles of six candidate RTKs, including c-Met, c-Kit, VEGFR2, HER2, FGFR1, and PDGFRα, were directly examined from chemonaive fresh frozen tissues of 32 osteosarcoma patients using a multiplex immunoassay. That examination revealed distinct patterns of tyrosine phosphorylation of RTKs in osteosarcoma cases. Unsupervised hierarchical clustering was calculated using Pearson uncentered correlation coefficient to classify RTKs into two groups—Group A (c-Met, c-Kit, VEGFR2, and HER2) and Group B (FGFR1 and PDGFRα)—based on tyrosine phosphorylation patterns. Nonactivation of all Group A RTKs was associated with shorter overall survival in stage IIB osteosarcoma patients. Percentages of tumor necrosis in patients with inactive Group A RTKs were significantly lower than those in patients with at least one active Group A RTK. Paired primary osteosarcoma cells with fresh osteosarcoma tissue were extracted and cultured for cytotoxicity testing. Primary cells with active Group A RTKs tended to be sensitive to doxorubicin and cisplatin. We also found that osteosarcoma cells with active Group A RTKs were more proliferative than cells with inactive Group A RTKs. These findings indicate that the activation pattern of Group A RTKs is a potential risk stratification and chemoresponse predictor and might be used to guide the optimum chemotherapy regimen for osteosarcoma patients.

A novel multikinase inhibitor R8 exhibits potent inhibition on cancer cells through both apoptosis and autophagic cell death.

Chemotherapy is an important treatment for cancer patients, especially for those with unresectable lesions. Targeted therapy of cancer by specific inhibition of aberrant tyrosine kinase activities in cancer cells with chemically synthesized tyrosine kinase inhibitors (TKIs), shows better responses while less side effects than traditional chemotherapeutic drugs. It is common that cancer cells often exhibit deregulation of several tyrosine kinases simultaneously, multikinase TKIs (MKIs) therefore have greater advantages over single-target TKIs. Currently more MKIs are under developing for better efficacy for different types of cancer. In the present work, we evaluated the in vitro therapeutic potential of a novel MKI, namely R8, with comparison to the clinically available MKI Sunitinib. Results showed that R8 has stronger inhibition on six different types of cancer cell lines with lower IC50 than Sunitinib does. Cell cycle analysis showed that R8 induced significant G0/G1 arrest phase of lung cancer A549 and NCI-H226 cells. The inhibition was also confirmed by colony formation and migration assays in both lung cancer cell lines in a dose-dependent manner. R8 could significantly inhibit the phosphorylation of multiple receptor tyrosine kinases (RTKs) included PDGFRβ, VEGFR2, EGFR and C-Kit, leading to the down-regulation of PI3K-Akt-mTOR signaling. Further analysis revealed that R8 treatment induced more significant apoptosis than Sunitinib did, which might be the consequence of the autophagic cell death. In conclusion, this work suggested R8 to be a promising novel anticancer MKI, and provided the basis for further in vivo investigation on its potential in treatment of lung cancer.

Receptor tyrosine kinase expression and phosphorylation in canine nasal carcinoma

Preliminary studies have supported use of toceranib phosphate (Palladia®) in treatment of canine nasal carcinomas, though the mechanisms of its activity are unknown. This study evaluated sixteen canine nasal carcinoma and five normal nasal epithelium samples for expression and phosphorylation of known targets of toceranib [vascular endothelial growth factor receptor-2 (VEGR2), platelet derived growth factor alpha (PDGFR-α), platelet derived growth factor receptor beta (PDGFR-β), and stem cell factor receptor (c-KIT)] and epidermal growth factor receptor 1 (EGFR1) using immunohistochemistry, RT-PCR and a receptor tyrosine kinase (RTK) phosphorylation panel. Protein for VEGFR2 was expressed in all carcinomas, PDGFR-α was noted in 15/16, whereas PDGFR-β was detected in 3/16 samples, but showed significant stromal staining. Protein expression for c-KIT was present in 4/16 and EGFR1 was noted in 14/16 samples. Normal tissue showed variable protein expression of the RTKs. Messenger RNA for VEGFR2, PDGFR-β, and c-KIT were noted in all samples. Messenger RNA for PDGFR-α and EGFR1 were detected in 15/16 samples. All normal nasal tissue detected messenger RNA. Phosphorylation of VEGFR2, PDGFR-α, PDGFR-β and c-KIT was not observed in any carcinoma or normal nasal sample, but phosphorylation of EGFR1 was noted in 10/16 carcinoma and 3/5 normal samples. The absence of phosphorylated RTK targets of toceranib suggests any clinical effect of toceranib occurs through inhibition of alternative unidentified RTK pathways in canine nasal carcinomas. The observed protein and message expression and phosphorylation of EGFR1 in the nasal carcinoma samples merits further inquiry into EGFR1 as a therapeutic target for this cancer.

Abstract LB-018: Cleavage of SNX2 protein by initiator caspases promotes hepatocyte growth factor (MET) receptor tyrosine kinase signaling

Abstract Cell death induced by apoptosis involves the stepwise activation of initiator and executioner caspases. Unfolding of this cellular process concurs with caspase-mediated proteolysis of hundreds of proteins with the ultimate result of stopping crucial cell survival, including the intracellular endocytic receptor trafficking. During cancer progression, tumor cells often become resistance to normal death-inducing signals. Typically, this resistance involves a cellular block of the apoptotic cascade downstream of the initiator caspases. Thus, apoptosis-resistant cancer cells are endowed with the unique capacity of sustaining elevated activation of initiator caspases. Apoptosis resistance being critical for metastatic progression, it opens the question of whether the cleavage products of initiator caspases contribute to the metastatic properties of apoptosis-resistant cells. In a recent study, we identified the SNX1 and SNX2 proteins, which are members of the sorting nexin (SNX) family of proteins critical in the control of endosomal sorting, as initiator caspase substrates. In addition, we demonstrated that the cleavage of SNX2 abolished its endosomal sorting ability, as illustrated by loss of its association with the endosome-to-trans-Golgi network transport protein Vps35 and a delocalization its binding partner Vps26 from endosomes. Importantly, depletion of SNX2 in cells was shown to enhance hepatocyte growth factor-induced phosphorylation of the MET receptor tyrosine kinase (RTK) and that of Erk1/2 proteins. Furthermore, we identified reduced SNX2 mRNA and protein levels in colorectal carcinoma specimens when compared to adjacent normal tissues, and that low SNX2 mRNA expression in primary colorectal tumors correlated with poor survival of patients. Deregulation of RTK signaling, such as that of the epidermal and hepatocyte growth factor receptors, is known to contribute to metastatic progression. Signaling and biological activity of these cell surface receptors are tightly controlled by their internalization upon activation and trafficking into endosomes, where they are either recycled back to the plasma membrane or degraded in lysosomes. Thus, our novel discoveries suggest that the activation of initiator caspases in apoptosis-resistant cancer cells fosters cancer progression by selective cleavage of proteins involved in RTK endocytic trafficking. Note: This abstract was not presented at the meeting. Citation Format: Catherine M. Duclos, Audrey Champagne, Julie C. Carrier, Caroline Saucier, Christine L. Lavoie, Jean-Bernard Denault. Cleavage of SNX2 protein by initiator caspases promotes hepatocyte growth factor (MET) receptor tyrosine kinase signaling [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 LB-018. doi:10.1158/1538-7445.AM2017-LB-018

Abstract 1136: Heat shock protein 90 inhibitors suppress PDGFRA reactivation and other receptor tyrosine kinase activation important in drug resistant gastrointestinal stromal tumors

Abstract Background: Inhibition of KIT oncoproteins by imatinib induces clinical responses in most gastrointestinal stromal tumor (GIST) patients. However, many patients develop imatinib resistance due to secondary KIT mutations. The drug resistance mechanism has only been partially elucidated, and the sunitinib resistance mechanism is also unknown. In this study, we elucidate the new imatinib resistant mechanism and find effective inhibitors. Methods: We have established imatinib-resistant cells GIST-T1R by culturing cells with increasing concentrations of imatinib. We analyzed receptor tyrosine kinase and intracellular signal protein strongly expressed in resistant cells using Western blotting and Phosphorylation Array. Then, the antitumor effect was examined using an agent that inhibits strongly expressed molecules of the resistant cells. Results: The IC50 of imatinib in the sensitive cells(GIST-T1) and the resistant cells(GIST-T1R) was 10 nM and 20 μM, respectively, which demonstrated 2000-fold difference. These imatinib resistant cells were also cross-resistant to sunitinib. In western blotting, PDGFRA phosphorylation and protein expression were enhanced in GIST-T1R compared to GIST-T1. When GIST-T1 was treated with imatinib, KIT phosphorylation was suppressed but PDGFRA phosphorylation was enhanced. Imatinib treatment of GIST-T1R enhanced PDGFRA phosphorylation and protein expression as compared to before imatinib treatment. Phosphorylation of PDGFRA in these resistant cells could not be suppressed by regorafenib. Furthermore, in the protein phosphorylated array, in addition to phosphorylation of PDGFRA in resistant cells, phosphorylation of multiple RTKs was also enhanced. An HSP90 inhibitor AUY922 capable of simultaneously suppressing a plurality of proteins was used. AUY922 suppressed GIST-T1 and GIST-T1R cell proliferation at low concentrations and was more effective than regorafenib. Furthermore, AUY922 inhibited phosphorylation and protein expression of PDGFRA in drug-resistant cells, as well as phosphorylation of other several receptor tyrosine kinases. Conclusion: Activation of PDGFRA was considered to be important for the mechanism of drug resistance of GIST. Furthermore, since activation of multiple receptor tyrosine kinases can be observed simultaneously in drug resistance, it is considered that inhibiting a single target is not effective. Since HSP90 inhibition suppresses PDGFRA protein expression and possibly inhibits multiple target proteins at the same time, it is the most effective treatment for drug resistant GIST. Citation Format: Masahiro Yamamura, Akira Yamauchi, Naoki Katase, Makoto Okawaki, Yousuke Katata, Futoshi Kuribayashi, Junichi Kurebayashi, Yoshiyuki Yamaguchi. Heat shock protein 90 inhibitors suppress PDGFRA reactivation and other receptor tyrosine kinase activation important in drug resistant gastrointestinal stromal tumors [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 1136. doi:10.1158/1538-7445.AM2017-1136

Abstract 2086: Dominant role of receptor tyrosine kinase in the activation of MAPK signaling in BRAF non-V600 mutant cancers

Abstract BRAF mutations are found in ~8% of all cancers. In addition to the commonest mutation in kinase domain at the V600, a wide range of other missense mutations (non-V600) have been reported. Non-V600 mutant BRAF protein was also shown to enhance MAPK signaling, suggesting the effectiveness of MEK inhibitors. However, the existence of feedback mechanism in MAPK singling and the roles of receptor tyrosine kinases (RTKs) in these cells are not fully understood. To interrogate this, BRAF non-V600 cell lines with intermediate/impaired kinase activity were treated with MEK inhibitor. RTKs involved in the upregulation of P-MEK following MEK inhibition in BRAF non-V600E mutant cell lines were determined by phosphor-RTK arrays. The effect of combinatorial inhibition of RTKs and MEK in BRAF non-V600 cancer was assessed in vitro and in vivo. By analyzing drug screen data, we first found cells with BRAF non-V600E mutation were less sensitive to MEK inhibition compared with BRAF V600E mutant cell lines and almost comparable to RAS/RAF wild-type cell lines. MEK inhibition did not induce P-MEK suppression and it resulted in significant upregulation of p-MEK compared to BRAF V600E mutant cell lines. Phospho-RTK arrays in the presence or absence of trametinib displayed basal phosphorylation of RTKs including EGFR, MET, and IGF1R, although the phosphorylation level was not consistently changed following MEK inhibition. Among these RTKs, treatment with the EGFR inhibitor erlotinib led to more complete suppression of P-ERK upon trametinib treatment. Furthermore, we found that erlotinib monotherapy downregulated P-ERK in BRAF non-V600 mutant cell lines in contrast that inhibition of RTKs had no effect on MAPK signaling in BRAF V600 mutant cells. While erlotinib treatment led to MAPK inhibition both in intermediate active and impaired kinase active BRAF mutant cells, p-MEK and p-ERK were more strongly suppressed in impaired type BRAF non-V600 mutant cells. The combination of EGFR inhibitor and MEK inhibitor suppressed cell growth and achieved tumor shrinkage in mice xenograft. These results suggest that RTKs, especially EGFR, are dominantly involved in the regulation of MAPK signaling in BRAF non-V600 cancer cells. Note: This abstract was not presented at the meeting. Citation Format: Hiromichi Ebi, Hiroshi Kotani, Hidenori Kitai, Yuta Adachi, Seiji Yano. Dominant role of receptor tyrosine kinase in the activation of MAPK signaling in BRAF non-V600 mutant cancers [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 2086. doi:10.1158/1538-7445.AM2017-2086

MTORC1-independent autophagy regulates receptor tyrosine kinase phosphorylation in colorectal cancer cells via an mTORC2-mediated mechanism

The intracellular autophagic degradative pathway can have a tumour suppressive or tumour-promoting role depending on the stage of tumour development. Upon starvation or targeting of oncogenic receptor tyrosine kinases (RTKs), autophagy is activated owing to the inhibition of PI3K/AKT/mTORC1 signalling pathway and promotes survival, suggesting that autophagy is a relevant therapeutic target in these settings. However, the role of autophagy in cancer cells where the PI3K/AKT/mTORC1 pathway is constitutively active remains partially understood. Here we report a role for mTORC1-independent basal autophagy in regulation of RTK activation and cell migration in colorectal cancer (CRC) cells. PI3K and RAS-mutant CRC cells display basal autophagy levels despite constitutive mTORC1 signalling, but fail to increase autophagic flux upon RTK inhibition. Inhibition of basal autophagy via knockdown of ATG7 or ATG5 leads to decreased phosphorylation of several RTKs, in particular c-MET. Internalised c-MET colocalised with LAMP1-negative, LC3-positive vesicles. Finally, autophagy regulates c-MET phosphorylation via an mTORC2-dependent mechanism. Overall, our findings reveal a previously unappreciated role of autophagy and mTORC2 in regulation of oncogenic RTK activation, with implications for understanding of cancer cell signalling.