Membrane-based Antibody Array Research Articles

AT2R activation increases in vitro angiogenesis in pregnant human uterine artery endothelial cells.

Angiogenesis is vital during pregnancy for remodeling and enhancing vasodilation of maternal uterine arteries, and increasing uterine blood flow. Abnormal angiogenesis is associated with decreased uteroplacental blood flow and development of pregnancy disorders such as gestational hypertension, preeclampsia, fetal growth restriction, preterm delivery, stillbirth, and miscarriage. The mechanisms that contribute to normal angiogenesis remain obscure. Our previous studies demonstrated that expression of the angiotensin type 2 receptor (AT2R) is increased while the angiotensin type 1 receptor (AT1R) is unchanged in the endothelium of uterine arteries, and that AT2R-mediated pregnancy adaptation facilitates enhanced vasodilation and uterine arterial blood flow. However, the role of AT2R in regulating angiogenesis during pregnancy has never been studied. This study examines whether or not AT2R activation induces angiogenesis and, if so, what mechanisms are involved. To this end, we used primary human uterine artery endothelial cells (hUAECs) isolated from pregnant and nonpregnant women undergoing hysterectomy. The present study shows that Compound 21, a selective AT2R agonist, induced proliferation of pregnant-hUAECs, but not nonpregnant-hUAECs, in a concentration-dependent manner, and that this C21-induced mitogenic effect was blocked by PD123319, a selective AT2R antagonist. The mitogenic effects induced by C21 were inhibited by blocking JNK—but not ERK, PI3K, and p38—signaling pathways. In addition, C21 concentration dependently increased cell migration and capillary-like tube formation in pregnant-hUAECs. The membrane-based antibody array showed that C21 increased expression of multiple angiogenic proteins, including EGF, bFGF, leptin, PLGF, IGF-1, and angiopoietins. Our qPCR analysis demonstrates that C21-induced increase in expression of these angiogenic proteins correlates with a proportional increase in mRNA expression, indicating that AT2R activates angiogenic proteins at the transcriptional level. In summary, the present study shows that AT2R activation induces angiogenesis of hUAECs in a pregnancy-specific manner through JNK-mediated pathways with associated transcriptional upregulation of multiple proangiogenic proteins.

CTEN Induces Tumour Cell Invasion and Survival and Is Prognostic in Radiotherapy-Treated Head and Neck Cancer.

Simple SummaryC-terminal tensin-like, or CTEN, is a cytoskeletal protein that is expressed highly in head and neck cancer (HNSCC). We studied CTEN function using gene knockdown and found that CTEN contributes to HNSCC progression in several ways, promoting tumour cell invasion and also cell survival. Notably, CTEN expression protects tumour cells from radiation-induced apoptosis and consistent with this, we found that CTEN expression predicts for survival in patients treated with radiotherapy (but not surgery), suggesting that CTEN may have utility as a predictive marker of radiotherapy resistance.Head and neck squamous cell carcinoma (HNSCC) is a heterogenous disease treated with surgery and/or (chemo) radiotherapy, but up to 50% of patients with late-stage disease develop locoregional recurrence. Determining the mechanisms underpinning treatment resistance could identify new therapeutic targets and aid treatment selection. C-terminal tensin-like (CTEN) is a member of the tensin family, upregulated in several cancers, although its expression and function in HNSCC are unknown. We found that CTEN is commonly upregulated in HNSCC, particularly HPV−ve tumours. In vitro CTEN was upregulated in HPV−ve (n = 5) and HPV+ve (n = 2) HNSCC cell lines. Stable shRNA knockdown of CTEN in vivo significantly reduced tumour growth (SCC-25), and functional analyses in vitro showed that CTEN promoted tumour cell invasion, colony formation and growth in 3D-culture (SCC-25, Detroit 562). RNA sequencing of SCC-25 cells following CTEN siRNA knockdown identified 349 differentially expressed genes (logFC > 1, p < 0.05). Gene ontology analysis highlighted terms relating to cell locomotion and apoptosis, consistent with in vitro findings. A membrane-based antibody array confirmed that CTEN regulated multiple apoptosis-associated proteins, including HSP60 and cleaved caspase-3. Notably, in a mixed cohort of HPV+ve and HPV−ve HNSCC patients (n = 259), we found a significant, independent negative association of CTEN with prognosis, limited to those patients treated with (chemo)radiotherapy, not surgery, irrespective of human papillomavirus (HPV) status. These data show that CTEN is commonly upregulated in HNSCC and exerts several functional effects. Its potential role in modulating apoptotic response to therapy suggests utility as a predictive biomarker or radio-sensitising target.

Microvesicles from Human Immortalized Cell Lines of Endothelial Progenitor Cells and Mesenchymal Stem/Stromal Cells of Adipose Tissue Origin as Carriers of Bioactive Factors Facilitating Angiogenesis.

Endothelial progenitor cells (EPCs) and mesenchymal stem/stromal cells (MSCs) are associated with maintaining tissue homeostasis and tissue repair. Both types of cells contribute to tissue regeneration through the secretion of trophic factors (alone or in the form of microvesicles). This study investigated the isolation and biological properties of microvesicles (MVs) derived from human immortalized MSC line HATMSC1 of adipose tissue origin and EPC line. The human immortalized cell line derived from the adipose tissue of a patient with venous stasis was established in our laboratory using the hTERT and pSV402 plasmids. The human EPC line originating from cord blood (HEPC-CB.1) was established in our previous studies. Microvesicles were isolated through a sequence of centrifugations. Analysis of the protein content of both populations of microvesicles, using the Membrane-Based Antibody Array and Milliplex ELISA showed that isolated microvesicles transported growth factors and pro- and antiangiogenic factors. Analysis of the miRNA content of isolated microvesicles revealed the presence of proangiogenic miRNA (miR-126, miR-296, miR-378, and miR-210) and low expression of antiangiogenic miRNA (miR-221, miR-222, and miR-92a) using real-time RT-PCR with the TaqMan technique. The isolated microvesicles were assessed for their effect on the proliferation and proangiogenic properties of cells involved in tissue repair. It was shown that both HEPC-CB.1- and HATMSC1-derived microvesicles increased the proliferation of human endothelial cells of dermal origin and that this effect was dose-dependent. In contrast, microvesicles had a limited impact on the proliferation of fibroblasts and keratinocytes. Both types of microvesicles improved the proangiogenic properties of human dermal endothelial cells, and this effect was also dose-dependent, as shown in the Matrigel assay. These results confirm the hypothesis that microvesicles of HEPC-CB.1 and HATMSC1 origin carry proteins and miRNAs that support and facilitate angiogenic processes that are important for cutaneous tissue regeneration.

CXCR4-overexpressing Mesenchymal Stem Cells exhibit increased cell migration and cytokine secretion

Background & Aim The regenerative and immunomodulatory properties of Mesenchymal Stem Cells (MSCs) have been demonstrated extensively in vitro, and in vivo in various types of pre-clinical models. However, in clinical settings, while the safety profile of MSCs has been well-established, the efficacy data obtained in pre-clinical studies hasn't been recapitulated in most cases where MSCs have been administered to human subjects. Many factors are thought to hinder MSC-based therapies; low potency and retention, and limited homing to the site of injury. In the current study, we sought to engineer bone-marrow-derived MSCs (BMSCs) with improved migratory properties so as to facilitate homing to the site of injury. Methods, Results & Conclusion We utilized a genetic approach in which BMSCs were transfected with CXCR4 mRNA. CXCR4, a G-protein coupled receptor highly expressed on the surface of multiple cell types is best known for its role in hematopoiesis and leukocyte chemotaxis. We showed that transfection of CXCR4 mRNA into BMSCs for eight hours resulted in over 60% of cells positive for CXCR4 protein expression, as determined by flow cytometry. Less than 1% of un-transfected BMSCs expresses CXCR4 on their surface. The expression of CXCR4 was maintained post-thaw when transfected BMSCs were recovered from LN2 Storage. Transfected cells also maintained their viability post-thaw. In terms of functionality, we demonstrated that BMSCs expressing CXCR4 were more migratory compared to their naive counterparts lacking CXCR4. This effect on migration was augmented when CXCR4-BMSCs were induced to migrate towards SDF-1α, the cognate ligand for CXCR4. In addition to the effects on directed cell migration, with the use of a membrane-based antibody array, we showed that compared to control BMSCs, CXCR4-BMSCs secrete greater quantities of MCP-1, a key chemokine that regulates macrophage migration and infiltration. In summary, we have shown that transfection of BMSCs with CXCR4 mRNA increases directed cell migration and alters cytokine secretion. This is a potential strategy for generating BMSCs with superior homing properties for use in clinical settings.

Proteome characterization of small extracellular vesicles from spared nerve injury model of neuropathic pain

Exosomes are 30–150 nm extracellular vesicles mediating intercellular communication. Disease states can alter exosome composition affecting the message carried and thereby, its functional impact. The objective of this study was to identify proteins present in these vesicles in a mouse model of neuropathic pain induced by spared nerve injury (SNI). Small extracellular vesicles (sEVs) were purified from serum four weeks after SNI surgery and the protein composition was determined using tandem mass spectrometry and cytokine array. Proteomic analysis detected 274 gene products within sEVs. Of these, 24 were unique to SNI model, 100 to sham surgery control and five to naïve control samples. In addition to commonly expressed sEVs proteins, multiple members of serpin and complement family were detected in sEVs. Cytokine profiling using a membrane-based antibody array showed significant upregulation of complement component 5a (C5a) and Intercellular Adhesion Molecule 1 (ICAM-1) in sEVs from SNI model compared to sham control. We observed a differential distribution of C5a and ICAM-1 within sEVs and serum between sham and SNI, indicating changes from local or paracrine to long distance signaling under neuropathic pain. Our studies suggest critical roles for cargo sorting of vesicular proteins in mediating signaling mechanisms underlying neuropathic pain. SignificanceApproximately 100 million U.S. adults are burdened by chronic pain. Neuropathic pain resulting from injury or dysfunction of the nervous system is challenging to treat. Unlike acute pain that resolves over time, chronic pain persists resulting in changes in the peripheral and central nervous system. The transport of biomolecular cargo comprised of proteins and RNAs by small extracellular vesicles (sEVs) including exosomes has been proposed to be a fundamental mode of intercellular communication. To obtain insights on the role of exosome-mediated information transfer in the context of neuropathic pain, we investigated alterations in protein composition of sEVs in a mouse model of neuropathic pain induced by spared nerve injury (SNI). Our studies using mass spectrometry and cytokine array show that sEVs from SNI model harbor unique proteins. We observed an upregulation of C5a and ICAM-1 in exosomes from SNI model compared to control. There was a differential distribution of C5a and ICAM-1 within exosomes and serum, between control and SNI suggesting a switch from local to long distance signaling. Our studies suggest critical roles for cargo sorting of vesicular proteins in mediating signaling under neuropathic pain.

ErbB2 and EphA3 as a novel prognostic and therapeutic target of Recurrent Non-functioning Pituitary Adenomas: A pilot study

Non-functioning pituitary adenomas (NFPA) are 30% of all pituitary adenomas. Although benign in nature but they may be invasive and recurrent. Markers of recurrence are needed to guide patient management. Recteptor Tyrosine Kinases (RTK) may sereve as therapeutic marker for recurrence as they can targeted by already available tyrosine kinase inhibitors. To examine differential RTK phosphorylation pattern of recurrent NFPAs, we recruited 20 patients and grouped them as non-recurrent (n=10), and recurrent (n=10). Recurrence was determined by follow-up of 15 years. We then performed a membrane-based antibody array (ARY001B) for the determination of the relative phosphorylation of 49 tyrosine kinases in recurrent NFPAs. As the experiment was replicated on two set of membranes, each tyrosine kinase was represented in quadruples. Student’s t-test was performed to compare the means between two groups. We found ErbB2, PDGFR beta, SCFR, Trk, VEGFR1, VEGFR2, EphA3, and Alk significantly hyperphosphorylated in recurrent NFPAs. Out of these eight hyperphosphorylated tyrosine kinases ErbB2 and EphA3 were 1.6 (p=0.01) and 1.9 (p=0.002) times hyperphosphorylated in recurrent NFPAs. This result indicates that EphA3 may be an effective therapeutic target in recurrent NFPAs.

The antifibrotic drug pirfenidone inhibits spondyloarthritis fibroblast-like synoviocytes and osteoblasts in vitro

BackgroundThe pathogenesis of spondyloarthritis (SpA) involves both inflammation and new bone formation in the spine. In line with this, the disease has been characterized as both inflammatory and fibrotic. The current treatment dampens inflammation while new bone formation can progress. Therefore, there is an unmet therapeutic need for the treatment of new bone formation in SpA. Fibrosis is mediated by myofibroblasts and new bone formation is the result of increased osteoblast mineralization and decreased osteoclast resorption. Here, we evaluate the potential effect of the newly approved anti-fibrotic agent pirfenidone (PFD) on fibrosis and new bone formation in cell culture models of SpA.MethodsFibroblast-like synoviocytes (FLSs) were isolated from SpA patients (n = 6) while the osteoblast cell line Saos-2 was purchased. The cells were cultured with PFD at 0.25 0.5, or 1.0 mg/ml. The proliferation of FLSs was analyzed with light microscopy and flow cytometry. The differentiation and activation of FLSs was assessed with flow cytometry, a membrane-based antibody array and enzyme-linked immunosorbant assays. The mineralization capacity of osteoblasts was studied with an assay measuring deposition of hydroxyapatite.ResultsPFD reduced the Ki67 expression 7.1-fold in untreated FLSs (p = 0.001) and 11.0-fold in FLSs stimulated with transforming growth factor beta (TGFβ), tumor necrosis factor alpha (TNFα), and interferon gamma (IFNγ) (p = 0.022). There were no statistically significant changes in membrane expression of alpha smooth muscle actin (αSMA), intercellular adhesion molecule 1 (ICAM-1), or human leukocyte antigen DR (HLA-DR). In supernatants from FLSs stimulated with TGFβ, TNFα, and IFNγ, PFD decreased the secretion of 3 of 12 proteins more than 2-fold in the membrane-based antibody array. The changes in secretion of monocyte chemoattractant protein 1 (MCP-1) and chitinase-3-like protein 1 (CHI3L1, YKL-40) were validated with ELISA. PFD decreased the secretion of both Dickkopf-related protein 1 (DKK1) (p = 0.006) and osteoprotegerin (OPG) (p = 0.02) by SpA FLSs stimulated with TGFβ, TNFα, and IFNγ. Finally, PFD inhibited the deposition of hydroxyapatite by osteoblasts in a dose-dependent manner (p = 0.0001).ConclusionsPFD inhibited SpA FLS proliferation and function and osteoblast mineralization in vitro. This encourages studies of the in vivo effect of PFD in SpA.

Abstract 196: The effect of modulated electro-hyperthermia on B16-F10 melanoma tumor growth

Abstract The objective of the present study was to examine the effect of modulated electro-hyperthermia (mEHT) on B16-F10 mouse melanoma cell line in vitro and in vivo. mEHT induces tumor-selective heat shock at ~42°C and is used as adjuvant to conventional cancer therapy. For in vitro comparison of the effect of mEHT to conventional hyperthermia (convHT), cells grown on coverslips were treated using mEHT or convHT in water-bath at 42°C for 1x60 min. Changes in gene expression, viability and invasiveness were measured. For in vivo studies, B16-F10 cells mixed with Matrigel were injected subcutaneously into C57Bl/6 mice as the tumors were exposed to 42°C for 30 minutes with mEHT every second day for three times. Tumors were removed 48 hours post treatment, weighed and digested to single-cell suspension. The supernatant of tumor cells and the plasma of tumor-bearing mice were used to screen a membrane-based antibody array representing a panel of 111 cytokines. The tumor cells were stained for flow cytometry to determine the changes induced on melanoma cells and tumor-infiltrating leukocytes. As a marker of in vitro treatment we measured HSPA2 expression with qPCR. Similar expression patterns were induced both by mEHT and convHT. Three hours after treatment the peak levels of HSPA2 were 3 times higher as compared to the untreated control in a time-course experiment. Next, we showed that in parallel to the upregulation of proapoptotic genes Puma, Bak-1 and Bax, downregulation of prosurvival genes XIAP, Bcl-2, Bcl-XL was induced both by convHT and mEHT with similar kinetics and magnitude. Furthermore, cell viability decreased and the invasivness was impaired as measured by scratch assay by both treatment types. The in vivo mEHT of primary melanoma tumors resulted in considerable tumor size reduction. Flow cytometry analysis revealed the increase of MHC class I on the surface of melanoma cells and concomitant activation of CD8+ cytotoxic T lymphocytes in response to treatment. Increased number of CD4+ T cells and Gr1+ granulocytes was detected, although changes in the various subpopulations need further analysis. F4/80+CD11b+ macrophages and mature DC-s were slightly elevated in the treated tumors. The cytokine array analysis shows that several cytokines responsible for leukocyte recruitment were enriched in the treated tumor (SDF-1, osteopontin, IL-1). Complement activation was induced by mEHT both in the tumor microenvironment and the plasma, indicating an acute inflammatory response to treatment. Furthermore, along the short pentraxins, CRP and SAP, the long pentraxin PTX3 was elevated in the tumor and plasma of the treated mice, suggesting that mEHT induced the tumor clearance. Taken together, our results demonstrate that in vitro mEHT and convHT produce comparable effects, whereas the in vivo mEHT treatment was effective in reducing the tumor size by enhancing immune recognition of B16-F10. This study was supported by NVKP 16-1- 2016-0042 grant. Citation Format: Balázs Besztercei, Enikő Major, Anett Benedek, Zoltán Benyó, Andrea Balogh. The effect of modulated electro-hyperthermia on B16-F10 melanoma tumor growth [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 196.

CD140b (PDGFRβ) signaling in adipose-derived stem cells mediates angiogenic behavior of retinal endothelial cells.

Adipose-derived stem cells (ASCs) are multipotent mesenchymal progenitor cells that have functional and phenotypic overlap with pericytes lining microvessels in adipose tissue. The role of CD140b [platelet-derived growth factor receptor- β (PDGFR-β)], a constitutive marker expressed by ASCs, in the angiogenic behavior of human retinal endothelial cells (HREs) is not known. CD140b was knocked down in ASCs using targeted siRNA and lipofectamine transfection protocol. Both CD140b+ and CD140b- ASCs were tested for their proliferation (WST-1 reagent), adhesion (laminin-1 coated plates), and migration (wound-scratch assay). Angiogenic effect of CD140b+ and CD140b- ASCs on HREs was examined by co-culturing ASCs:HREs in 12:1 ratio for 6 days followed by visualization of vascular network by Isolectin B4 staining. The RayBio® Membrane-Based Antibody Array was used to assess differences in human cytokines released by CD140b+ or CD140b- ASCs. Knockdown of CD140b in ASCs resulted in a significant 50% decrease in proliferation rate, 25% decrease in adhesion ability to Laminin-1, and 50% decrease in migration rate, as compared to CD140b+ ASCs. Direct contact of ASCs expressing CD140b+ with HREs resulted in robust vascular network formation that was significantly reduced with using CD140b- ASCs. Of the 80 proteins tested, 45 proteins remained unchanged (>0.5-<1.5 fold), 6 proteins including IL-10 downregulated (<0.5 fold) and 29 proteins including IL-16 & TNF-β were upregulated (>1.5 fold) in CD140b- ASCs compared to CD140b+ ASCs. Our data demonstrate a substantial role for CD140b in the intrinsic abilities of ASCs and their angiogenic influence on HREs. Future studies are needed to fully explore the signaling of CD140b in ASCs in vivo for retinal regeneration.

PDGFA/PDGFR\u03b1-regulated GOLM1 promotes human glioma progression through activation of AKT

BackgroundGolgi Membrane Protein 1 (GOLM1), a protein involved in the trafficking of proteins through the Golgi apparatus, has been shown to be oncogenic in a variety of human cancers. Here, we examined the role of GOLM1 in the development of human glioma.MethodsqRT-PCR, immunohistochemistry, and western blot analysis were performed to evaluate GOLM1 levels in cell lines and a cohort of primary human glioma and non-neoplastic brain tissue samples. Glioma cell lines were modified with lentiviral constructs expressing short hairpin RNAs targeting GOLM1 or overexpressing the protein to assess function in proliferation, viability, and migration and invasion in vitro using EdU, CCK8, clone-forming, Transwell assays, 3D tumor spheroid invasion assay and in vivo in orthotopic implantations. Protein lysates were used to screen a membrane-based antibody array to identify kinases mediated by GOLM1. Specific inhibitors of PDGFRα (AG1296) and AKT (MK-2206) were used to examine the regulation of PDGFA/PDGFRα on GOLM1 and the underlying pathway respectively.ResultsqRT-PCR, immunohistochemistry and western blot analysis revealed GOLM1 expression to be elevated in glioma tissues and cell lines. Silencing of GOLM1 attenuated proliferation, migration, and invasion of U251, A172 and P3#GBM (primary glioma) cells, while overexpression of GOLM1 enhanced malignant behavior of U87MG cells. We further demonstrated that activation of AKT is the driving force of GOLM1-promoted glioma progression. The last finding of this research belongs to the regulation of PDGFA/PDGFRα on GOLM1, while GOLM1 was also a key element of PDGFA/PDGFRα-mediated activation of AKT, as well as the progression of glioma cells.ConclusionsPDGFA/PDGFRα-regulated GOLM1 promotes glioma progression possibly through activation of a key signaling kinase, AKT. GOLM1 interference may therefore provide a novel therapeutic target and improve the efficacy of glioma treatment, particularly in the case of the proneural molecular subtype of human glioma.

Abstract 2971: The prognostic relevance of inflammatory cytokines and growth factors elaborated from adipose-derived stem cells in breast cancer

Abstract Introduction: Given the high rates of obesity and aggressive breast cancers, it is paramount to understand how adipose tissue supports the tumor microenvironment. We previously found that adipose-derived stem cells cultured with triple-negative tumor cells enhanced the expression of numerous pro-inflammatory cytokines. This finding is significant as it could explain, in part, how obesity contributes to worse outcomes in breast cancer. To further expand this study, we probed the Cancer Genome Atlas (TCGA) breast invasive carcinoma dataset for the expression profiles of the inflammatory cytokines we found to be highly up-regulated in vitro. We predicted TCGA data would demonstrate a correlation between cytokine overexpression and worse clinical scenarios. Methods: Cytokines and growth factors were measured in the media of adipose stromal cells and MDA-MB-231 tumor cells via membrane-based antibody array. To validate clinical significance, we compared each cytokine’s mRNA expression profile, available from primary tumor samples of TCGA breast invasive carcinoma dataset (n = 1,005), to indices of tumor progression and survival status. For survival analysis, samples were dichotomized as high or low expression by individual cytokines (z score ≥ 1.5). Cytokine and growth factor expression was further stratified according to molecular subtype of breast cancer (luminal A, luminal B, HER2, triple negative) and receptor status. Ingenuity pathway analysis (IPA) was performed to identify candidate markers related to obesity or inflammation. Results: High levels of FGF7 and CCL5 protein were found in the conditioned media of tumor and adipose stem cell co-cultures and together they showed a significant impact on breast cancer survival within TCGA data. FGF7, CCL5, together with CCL2, IL6, and IL6R were further analyzed for expression across molecular subtypes of breast cancer. IL6R, CCL5, and CCL2 expression varied significantly between breast cancer subtypes, with mean expression highest in triple-negative tumors. After dichotomizing the samples for estrogen receptor status, IL6R, CCL5, and CCL2 showed significantly higher mean expression in ER negative tumors. Greater expression of these cytokines in tumors was correlated with reduced patient survival in triple-negative tumors and ER negative subtypes. IPA analysis indicated IL6, IL6R, CCL5, and CCL2, but not FGF7, cooperate through inflammatory pathways to promote tumor progression. Conclusions: Genomic data analysis supports in vitro findings that IL6R, CCL5, and CCL2 overexpression, either independently or synergistically, predict a worse prognosis in breast cancer. For adipose-driven, hormone receptor negative breast tumors, these cytokines and growth factors could serve as novel therapeutic targets. (Supported by NIH P20GM103434 and NIGMS U54GM104942) Citation Format: Nicole Werwie, Kelsey Sadlek, Eric Lundstrom, Daniel Berrebi, Gerald Hobbs, Linda Vona-Davis. The prognostic relevance of inflammatory cytokines and growth factors elaborated from adipose-derived stem cells in breast cancer [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 2971. doi:10.1158/1538-7445.AM2017-2971

Coactivation of Receptor Tyrosine Kinases in Malignant Mesothelioma as a Rationale for Combination Targeted Therapy

To identify new therapeutic approaches in malignant mesothelioma (MM), we examined the expression and activation of receptor tyrosine kinases (RTKs) and the effects of specific RTK inhibitors and the mammalian target of rapamycin (mTOR) inhibitor rapamycin; the latter being of special interest in MM given the recent linkage between NF2 loss and mTOR activation. We performed a screen for mutated or activated RTKs in 14 MM cell lines and 70 primary tumors. Expression of phosphorylated RTKs was analyzed by Western blotting and a membrane-based antibody array in normal growth conditions and after treatment by specific inhibitors. MET and epidermal growth factor receptor (EGFR) mutations were screened by sequencing. MET, hepatocyte growth factor, insulin-like growth factor 1 receptor, and EGFR expression were studied by Western blotting, immunohistochemistry, enzyme-linked immunosorbent assay, and by Affymetrix expression microarrays. Profiling of the phosphorylation status of 42 RTKs showed prominent coactivation of MET and EGFR in 8 of 14 (57%) MM cell lines. MET, EGFR, and insulin-like growth factor 1 receptor were the main RTKs activated after mTOR inhibition and contributed to AKT feedback activation. Knockdown of MET by RNA interference inhibited not only the phosphorylation of MET but also that of EGFR. Conversely, stimulation with hepatocyte growth factor increased both phospho-MET and phospho-EGFR. The combination of PHA-665752 and the EGFR inhibitor, erlotinib, suppressed cell growth more than either agent alone in three of six cell lines tested. Finally, combinations of rapamycin and different RTK inhibitors were more active than either drug alone in 12 of 13 cell lines. Combination targeting of kinase signaling pathways is more effective than single agents in most MM.