Receptor Tyrosine Kinase Tie2 Research Articles

Comprehensive Analysis of TEK Variants in Patients With Vascular Malformations.

Pathogenic variants in the receptor tyrosine kinase TIE2, encoded by TEK, are known to cause vascular malformations (VMs). In this study, we retrospectively reviewed the deidentified data generated through clinical NGS testing in our laboratory and found 88 VM cases with a total of 107 clinically significant TEK variants. Among those, 23 unique variants at the amino acid level were identified, including five novel (p.Cys1040Arg, p.Arg1099PhefsTer12, p.Glu1109Ter, p.Phe1111LeufsTer7, p.Phe1111ValfsTer7) and 18 previously published variants. Missense variants were identified more often in the tyrosine kinase domain, while all nonsense/frameshift variants were clustered in the C-terminal tail (CTT). In addition, most variants occurred as solitary alterations, whereas certain variants always co-occurred with a second TEK variant. Five patterns of TEK variants (P1-P5) were identified: (P1) Arg849 + another variant; (P2) Tyr897 + another variant; (P3) Leu914 single variants; (P4) Arg915/918 single variants; and (P5) CTT single /co-occurring variants. This study provides the most comprehensive view of pathogenic TEK variants in VMs to date.

Heterogeneity of endothelial VE-PTP downstream polarization, Tie2 activation, junctional claudin-5, and permeability in the aorta and vena cava

Endothelial cells of mammalian blood vessels have multiple levels of heterogeneity along the vascular tree and among different organs. Further heterogeneity results from blood flow turbulence and variations in shear stress. In the aorta, vascular endothelial protein tyrosine phosphatase (VE-PTP), which dephosphorylates tyrosine kinase receptor Tie2 in the plasma membrane, undergoes downstream polarization and endocytosis in endothelial cells exposed to laminar flow and high shear stress. VE-PTP sequestration promotes Tie2 phosphorylation at tyrosine992 and endothelial barrier tightening. The present study characterized the heterogeneity of VE-PTP polarization, Tie2-pY992 and total Tie2, and claudin-5 in anatomically defined regions of endothelial cells in the mouse descending thoracic aorta, where laminar flow is variable and IgG extravasation is patchy. We discovered that VE-PTP and Tie2-pY992 had mosaic patterns, unlike the uniform distribution of total Tie2. Claudin-5 at tight junctions also had a mosaic pattern, whereas VE-cadherin at adherens junctions bordered all endothelial cells. Importantly, the amounts of Tie2-pY992 and claudin-5 in aortic endothelial cells correlated with downstream polarization of VE-PTP. VE-PTP and Tie2-pY992 also had mosaic patterns in the vena cava, but claudin-5 was nearly absent and extravasated IgG was ubiquitous. Correlation of Tie2-pY992 and claudin-5 with VE-PTP polarization supports their collective interaction in the regulation of endothelial barrier function in the aorta, yet differences between the aorta and vena cava indicate additional flow-related determinants of permeability. Together, the results highlight new levels of endothelial cell functional mosaicism in the aorta and vena cava, where blood flow dynamics are well known to be heterogeneous.

A ligand-independent Tie2-activating antibody reduces vascular leakage in models of Clarkson disease.

Vascular dysfunction resulting from endothelial hyperpermeability is a common and important feature of critical illness due to sepsis, trauma, and other conditions associated with acute systemic inflammation. Clarkson disease [monoclonal gammopathy-associated idiopathic systemic capillary leak syndrome (ISCLS)] is a rare, orphan disorder marked by spontaneous and recurrent episodes of hypotensive shock and peripheral edema due to widespread vascular leakage in peripheral tissues. Mortality from acute flares approaches 30% due to lack of effective therapies. We evaluated a monoclonal antibody (4E2) specific for the endothelial receptor tyrosine kinase Tie2 in ISCLS models. 4E2 activated Tie2 in ISCLS patient-derived endothelial cells and reduced baseline and proinflammatory mediator-induced barrier dysfunction. 4E2 also reduced mortality and/or vascular leakage associated with systemic histamine challenge or influenza infection in the SJL/J mouse model of ISCLS. These findings support a critical role for Tie2 dysregulation in ISCLS and highlight a viable therapeutic approach to this catastrophic disorder.

Platelet Angiopoietin-1 Protects Against Experimental Lung Metastasis By Limiting Tumor Cell Extravasation

In addition to their fundamental roles in hemostasis, platelets can contribute to both tumor growth and metastasis. However, despite being a reservoir for pro-angiogenic and metastatic cytokines, platelets also harbor negative regulators of tumor progression. One such regulator is angiopoietin-1 (Angpt1), an angiogenic cytokine that preserves vascular integrity by promoting cell-cell endothelial interactions downstream of its receptor, the receptor tyrosine kinase Tie-2. In addition, Angpt1 also protects against tumor cell metastasis through an unidentified mechanism. Although platelets are believed to be the primary source of Angpt1 in circulation, the contributions of platelet-derived Angpt1 to tumor growth and metastasis have not been investigated. In this study, we demonstrate that platelet Angpt1 is selectively upregulated in the spontaneous PyMT breast cancer mouse model, but not pre-clinical models of melanoma or non-malignant inflammation (colitis), suggesting a distinct contribution of platelet Angpt1 in breast cancer. To test whether deletion of platelet Angpt1 affects tumor growth or metastasis, we generated mice selectively lacking Angpt1 in megakaryocytes and their platelet progeny (Angpt1 fl/flPF4 Cre, referred to as Angpt1 Plt KO). Plasma Angpt1 was undetectable in Angpt1 Plt KO mice, comparable to mice deficient in the megakaryocyte differentiation factor thrombopoietin, which display approximately 10% of platelet counts. Taken together, these findings establish that platelets are the principal source of circulating Angpt1. In tumor implantation studies, orthotopic tumor growth and vascularization were unaffected in Angpt1 Plt KO mice in pre-clinical models of breast cancer and melanoma. However, Angpt1 Plt KO mice had increased experimental metastasis during both early (3-24 hours) and late (10 days, Figure 1) stages of tumor colonization in both breast cancer and melanoma models, revealing an important role for platelet Angpt1 in restraining the latter stages of tumor metastasis following tumor engraftment. Next, we sought to mechanistically determine how platelet Angpt1 limits tumor cell metastasis. Static adhesion assays demonstrated that platelets lacking Angpt1 did not affect tumor cell attachment to endothelial cells when compared to platelets containing Angpt1. However, Angpt1 Plt KO mice had increased tumor cell retention and extravasation in their lungs following experimental metastasis (3-24 hours), suggesting that platelet Angpt1 limits endothelial permeabilization and the trans-endothelial migration of tumor cells out of blood vessels. Most importantly, all intravascular tumor cells were found within platelet-rich microthrombi immediately after sequestration in the lungs. We therefore conclude that localized Angpt1 released by activated platelets at the site of tumor cell arrest likely reprograms the surrounding endothelium to limit successful tumor cell extravasation. These findings reveal an unexpected role for platelets in restricting tumor cell metastasis.

Targeting the microenvironment in the treatment of arteriovenous malformations

Extracranial arteriovenous malformations (AVMs) are regarded as rare diseases and are prone to complications such as pain, bleeding, relentless growth, and high volume of shunted blood. Due to the high vascular pressure endothelial cells of AVMs are exposed to mechanical stress. To control symptoms and lesion growth pharmacological treatment strategies are urgently needed in addition to surgery and interventional radiology. AVM cells were isolated from three patients and exposed to cyclic mechanical stretching for 24 h. Thalidomide and bevacizumab, both VEGF inhibitors, were tested for their ability to prevent the formation of circular networks and proliferation of CD31+ endothelial AVM cells. Furthermore, the effect of thalidomide and bevacizumab on stretched endothelial AVM cells was evaluated. In response to mechanical stress, VEGF gene and protein expression increased in patient AVM endothelial cells. Thalidomide and bevacizumab reduced endothelial AVM cell proliferation. Bevacizumab inhibited circular network formation of endothelial AVM cells and lowered VEGF gene and protein expression, even though the cells were exposed to mechanical stress. With promising in vitro results, bevacizumab was used to treat three patients with unresectable AVMs or to prevent regrowth after incomplete resection. Bevacizumab controlled bleeding, pulsation, and pain over the follow up of eight months with no patient-reported side effects. Overall, mechanical stress increases VEGF expression in the microenvironment of AVM cells. The monoclonal VEGF antibody bevacizumab alleviates this effect, prevents circular network formation and proliferation of AVM endothelial cells in vitro. The clinical application of bevacizumab in AVM treatment demonstrates effective symptom control with no side effects.Graphical abstractMechanical stress increases VEGF expression in endothelial AVM cells, possibly causing the VEGF upregulation in the microenvironment of AVM cells. The resulting RAS/RAF/MEK/ERK signaling in leads to progression of fast-flow malformations. The monoclonal VEGF-A antibody bevacizumab alleviates this effect, prevents circular network formation and proliferation of AVM endothelial cells in vitro. Sporadically occurring slow-flow malformations (LMs, VMs) have mutations in TEK or PIK3CA. TEK encodes the endothelial receptor tyrosine kinase TIE2. Sporadic extracranial fast-flow malformations (AVMs) show mutations in KRAS, BRAF and MAP2K1, which encodes the dual specificity mitogen-activated protein kinase MEK1. Combined targeting of the molecular causes of the disease could be key to achieve symptom control and reduce lesion growth. Orange: gain-of-function; Blue, circled with orange: enhanced signaling.

Gene Expression Profiles of Long-Chain Acyl-Coenzyme A Dehydrogenase, Nuclear Distribution C-Containing Protein 3, and Receptor Tyrosine Kinase Tie-1 in Swimming Larva of Sea Cucumber Apostichopus japonicus.

The sea cucumber, Apostichopus japonicus, is one of the most valuable aquatic species. The color of body wall and appearance are important for the value of sea cucumbers. To examine expression pattern of long-chain acyl-coenzyme A dehydrogenase (LCAD), nuclear distribution C-containing protein 3 (NUDCD3), and receptor tyrosine kinase Tie-1 (TIE1), previously reported as differently expressed genes during the pigmentation of sea cucumber, we analyzed the temporal profiles of LCAD, NUDCD3, and TIE1 mRNAs in LED-exposed and light-shielded A. japonicus. Real-time quantitative PCR revealed that the LCAD, NUDCD3, and TIE1 mRNAs from the juveniles at 40-60 days post-fertilization (dpf) exhibited increasing patterns as compared to those of an early developmental larva (6-dpf). At 60-dpf juveniles, the LCAD and TIE1 mRNA levels of LED-exposed individuals were higher than those of light-shielded ones, whereas at 40-dpf and 50-dpf juveniles, the NUDCD3 mRNA expression was higher in the light-shielded condition (p<0.05). In the pigmented juveniles (90-dpf), the LCAD and TIE1 mRNA levels tended to show higher levels in red individuals than those in green ones, but there was a conversely higher level of NUDCD3 mRNA in green larva. In situ examination of LCAD and NUDCD3 mRNAs in light-shielded 6-dpf larva revealed that both genes are mainly expressed in the internal organs compared to the body surface. Together, these results may provide insights into the differential gene expression of LCAD, NUDCD3, and TIE1 during pigmentation process of the sea cucumber.

Inhaled aptamer therapy: A hopeful therapy for lung disease

Inhaled aptamer therapy: A hopeful therapy for lung disease

Von Willebrand factor binds to angiopoietin-2 within endothelial cells and after release from Weibel–Palade bodies

BackgroundThe von Willebrand factor (VWF) is a multimeric plasma glycoprotein essential for hemostasis, inflammation, and angiogenesis. The majority of VWF is synthesized by endothelial cells (ECs) and stored in Weibel–Palade bodies (WPB). Among the range of proteins shown to co-localize to WPB is angiopoietin-2 (Angpt-2), a ligand of the receptor tyrosine kinase Tie-2. We have previously shown that VWF itself regulates angiogenesis, raising the hypothesis that some of the angiogenic activity of VWF may be mediated by its interaction with Angpt-2. MethodsStatic-binding assays were used to probe the interaction between Angpt-2 and VWF. Binding in media from cultured human umbilical vein ECs s and in plasma was determined by immunoprecipitation experiments. Immunofluorescence was used to detect the presence of Angpt-2 on VWF strings, and flow assays were used to investigate the effect on VWF function. ResultsStatic-binding assays revealed that Angpt-2 bound to VWF with high affinity (KD,app ∼3 nM) in a pH and calcium-dependent manner. The interaction was localized to the VWF A1 domain. Co-immunoprecipitation experiments demonstrated that the complex persisted following stimulated secretion from ECs and was present in plasma. Angpt-2 was also visible on VWF strings on stimulated ECs. The VWF–Angpt-2 complex did not inhibit the binding of Angpt-2 to Tie-2 and did not significantly interfere with VWF-platelet capture. ConclusionsTogether, these data demonstrate a direct binding interaction between Angpt-2 and VWF that persists after secretion. VWF may act to localize Angpt-2; further work is required to establish the functional consequences of this interaction.

Shear stress control of vascular leaks and atheromas through Tie2 activation by VE‐PTP sequestration

Vascular endothelial protein tyrosine phosphatase (VE‐PTP) influences endothelial barrier function by regulating the activation of tyrosine kinase receptor Tie2. We determined whether this action is linked to the development of atherosclerosis by examining the influence of arterial shear stress on VE‐PTP, Tie2 activation, plasma leakage, and atherogenesis. We found that exposure to high average shear stress led to downstream polarization and endocytosis of VE‐PTP accompanied by Tie2 activation at cell junctions. In aortic regions with disturbed flow, VE‐PTP was not redistributed away from Tie2. Endothelial cells exposed to high shear stress had greater Tie2 activation and less macromolecular permeability than regions with disturbed flow. Deleting endothelial VE‐PTP in VE‐PTPiECKO mice increased Tie2 activation and reduced plasma leakage in atheroprone regions. ApoE−/− mice bred with VE‐PTPiECKO mice had less plasma leakage and fewer atheromas on a high‐fat diet. Pharmacologic inhibition of VE‐PTP by AKB‐9785 had similar anti‐atherogenic effects. Together, the findings identify VE‐PTP as a novel target for suppression of atherosclerosis.

The effects and mechanisms of ghrelin upon angiogenesis in human coronary artery endothelial cells under hypoxia

Ghrelin, an endogenous ligand of the growth hormone secretagogue receptor (GHSR), has been found to stimulate angiogenesis both in vivo and in vitro. However, the effect of ghrelin upon angiogenesis, and the corresponding mechanisms of ghrelin therein, in human coronary artery endothelial cells (HCAECs) under hypoxia is still unknown. Our study found that ghrelin significantly increased HCAECs proliferation, migration, in vitro angiogenesis, and microvessel sprouting from the aortic ring under hypoxic conditions. The ghrelin-induced angiogenic process was accompanied by vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2) and endothelial-specific receptor tyrosine kinase (Tie2) expressions. In addition, this angiogenic effect was almost completely inhibited by Ang-2 RNAi and Tie2 RNAi. Pretreatment with the GHSR1a blocker [D-Lys3]-GHRP-6 abolished ghrelin-induced VEGF, Ang-1, Ang-2 and Tie2 expressions and in vitro angiogenesis. In conclusion, this is the first demonstration that ghrelin stimulates HCAECs in vitro angiogenesis through GHSR1a-mediated VEGF, Ang-1, Ang-2 and Tie2 pathways under hypoxic conditions. It indicated that ghrelin might play an important role in myocardial angiogenesis after ischemic injury.

Cooperation of Angiopoietin-2 and Angiopoietin-4 in Schlemm's Canal Maintenance.

PurposeDefects in the iridocorneal angle tissues, including the trabecular meshwork (TM) and Schlemm's canal (SC), impair aqueous humor flow and increase the intraocular pressure (IOP), eventually resulting in glaucoma. Activation of endothelial tyrosine kinase receptor Tie2 by angiopoietin-1 (Angpt1) has been demonstrated to be essential for SC formation, but roles of the other two Tie2 ligands, Angpt2 and Angpt4, have been controversial or not yet characterized, respectively.MethodsAngpt4 expression was investigated using genetic cell fate mapping and reporter mice. Congenital deletion of Angpt2 and Angpt4 and tamoxifen-inducible deletion of Angpt1 in mice were used to study the effects of Angpt4 deletion alone and in combination with the other angiopoietins. SC morphology was examined with immunofluorescent staining. IOP measurements, electron microscopy, and histologic evaluation were used to study glaucomatous changes.Results Angpt4 was postnatally expressed in the TM. While Angpt4 deletion alone did not affect SC and Angpt4 deletion did not aggravate Angpt1 deletion phenotype, absence of Angpt4 combined with Angpt2 deletion had detrimental effects on SC morphology in adult mice. Consequently, Angpt2−/−;Angpt4−/− mice displayed glaucomatous changes in the eye. Mice with Angpt2 deletion alone showed only moderate SC defects, but Angpt2 was necessary for proper limbal vasculature development. Mechanistically, analysis of Tie2 phosphorylation suggested that Angpt2 and Angpt4 cooperate as agonistic Tie2 ligands in maintaining SC integrity.ConclusionsOur results indicated an additive effect of Angpt4 in SC maintenance and Tie2 activation and a spatiotemporally regulated interplay between the angiopoietins in the mouse iridocorneal angle.

Combined treatment of marizomib and cisplatin modulates cervical cancer growth and invasion and enhances antitumor potential in vitro and in vivo.

Proteasome inhibition is an attractive approach for anticancer therapy. Cisplatin (cis-diamminedichloroplatinum, CDDP) is widely used as a standard chemotherapy drug in the treatment of solid malignant tumors, such as cervical cancer, ovarian cancer, colorectal cancer, and lung cancer. However, the development of CDDP resistance largely limits its clinical application. Proteasome inhibitors may enhance traditional chemotherapy agent-induced cytotoxicity and apoptosis. Marizomib (NPI-0052, salinosporamide A, Mzb), a second-generation proteasome inhibitor, shows synergistic anticancer activity with some drugs. Currently, the effect of Mzb on cervical cancer cell proliferation remains unclear. In this study, we explored the role of Mzb in three cervical cancer cell lines, HeLa, CaSki, and C33A, representing major molecular subtypes of cervical cancer and xenografts. We found that Mzb alone showed noteworthy cytotoxic effects, and its combination with CDDP resulted in more obvious cytotoxicity and apoptosis in cervical cancer cell lines and xenografts. In order to investigate the mechanism of this effect, we probed whether Mzb alone or in combination with CDDP had a better antitumor response by enhancing CDDP-induced angiopoietin 1 (Ang-1) expression and inhibiting the expression of TEK receptor tyrosine kinase (Tie-2) in the Ang-1/Tie-2 pathway, FMS-like tyrosine kinase 3 ligand (Flt-3L) and stem cell factor (SCF) as identified by a cytokine antibody chip test. The results suggest that Mzb has better antitumor effects on cervical cancer cells and can sensitize cervical cancer cells to CDDP treatment both in vitro and in vivo. Accordingly, we conclude that the combination of CDDP with Mzb produces synergistic anticancer activity and that Mzb may be a potential effective drug in combination therapy for cervical cancer patients.

Further Evidence of Neuroprotective Effects of Recombinant Human Erythropoietin and Growth Hormone in Hypoxic Brain Injury in Neonatal Mice.

Experimental in vivo data have recently shown complementary neuroprotective actions of rhEPO and growth hormone (rhGH) in a neonatal murine model of hypoxic brain injury. Here, we hypothesized that rhGH and rhEPO mediate stabilization of the blood–brain barrier (BBB) and regenerative vascular effects in hypoxic injury to the developing brain. Using an established model of neonatal hypoxia, neonatal mice (P7) were treated i.p. with rhGH (4000 µg/kg) or rhEPO (5000 IU/kg) 0/12/24 h after hypoxic exposure. After a regeneration period of 48 h or 7 d, cerebral mRNA expression of Vegf-A, its receptors and co-receptors, and selected tight junction proteins were determined using qRT-PCR and ELISA. Vessel structures were assessed by Pecam-1 and occludin (Ocln) IHC. While Vegf-A expression increased significantly with rhGH treatment (p < 0.01), expression of the Vegfr and TEK receptor tyrosine kinase (Tie-2) system remained unchanged. RhEPO increased Vegf-A (p < 0.05) and Angpt-2 (p < 0.05) expression. While hypoxia reduced the mean vessel area in the parietal cortex compared to controls (p < 0.05), rhGH and rhEPO prevented this reduction after 48 h of regeneration. Hypoxia significantly reduced the Ocln+ fraction of cortical vascular endothelial cells. Ocln signal intensity increased in the cortex in response to rhGH (p < 0.05) and in the cortex and hippocampus in response to rhEPO (p < 0.05). Our data indicate that rhGH and rhEPO have protective effects on hypoxia-induced BBB disruption and regenerative vascular effects during the post-hypoxic period in the developing brain.

In vitro inhibition of cancer angiogenesis and migration by a nanobody that targets the orphan receptor Tie1.

The human signaling molecules Tie1 and Tie2 receptor tyrosine kinases (RTKs) play important pathophysiological roles in many diseases, including different cancers. The activity of Tie1 is mediated mainly through the downstream angiopoietin-1 (Ang1)-dependent activation of Tie2, rendering both Tie 1 and the Tie1/Tie2/Ang1 axis attractive putative targets for therapeutic intervention. However, the development of inhibitors that target Tie1 and an understanding of their effect on Tie2 and on the Tie1/Tie2/Ang1 axis remain unfulfilled tasks, due, largely, to the facts that Tie1 is an orphan receptor and is difficult to produce and use in the quantities required for immune antibody library screens. In a search for a selective inhibitor of this orphan receptor, we sought to exploit the advantages (e.g., small size that allows binding to hidden epitopes) of non-immune nanobodies and to simultaneously overcome their limitations (i.e., low expression and stability). We thus performed expression, stability, and affinity screens of yeast-surface-displayed naïve and predesigned synthetic (non-immune) nanobody libraries against the Tie1 extracellular domain. The screens yielded a nanobody with high expression and good affinity and specificity for Tie1, thereby yielding preferential binding for Tie1 over Tie2. The stability, selectivity, potency, and therapeutic potential of this synthetic nanobody were profiled using in vitro and cell-based assays. The nanobody triggered Tie1-dependent inhibition of RTK (Tie2, Akt, and Fak) phosphorylation and angiogenesis in endothelial cells, as well as suppression of human glioblastoma cell viability and migration. This study opens the way to developing nanobodies as therapeutics for different cancers associated with Tie1 activation.

In vitro culture of hematopoietic stem cell niche using angiopoietin-1-coupled alginate hydrogel

Stem cells exist and maintain their quiescence and pluripotency in stem cell niche. Here, we hypothesized that regulation of cell-cell interactions using a polymeric scaffold as synthetic extracellular matrix (ECM) could be critical in creating a hematopoietic stem cell (HSC) niche in vitro. Angiopoietin-1 (Ang1) binds to the tyrosine kinase receptor (Tie2), and regulation of the Tie2/Ang1 interaction is important in maintaining the quiescence of HSCs in vivo. Alginate hydrogel was thus modified with Ang1 as a synthetic ECM to mimic the HSC niche. Long-term HSCs (CD34−, CD135−, and CD150+) were isolated from mouse femurs and cultured on Ang1-modified alginate hydrogel. The percentage of LT-HSCs in G0 phase was 46.8 ± 1.8%, which was comparable to that of LT-HSCs co-cultured with osteoblasts (46.8 ± 2.1%). Ang1-coupled alginate gels were useful to provide a niche for HSC quiescence without a co-culture system. Polymeric scaffolds containing biomimetic and cell-instructive characteristics for stem cell phenotype regulation might help create HSC niches in vitro and be useful to engineer tissues and transplant stem cells.

Abstract P1-08-36: Racial disparity in post-neoadjuvant chemotherapy residual breast tumor microenvironment

Abstract Background: Black women with residual estrogen receptor (ER)-positive breast cancer after neoadjuvant chemotherapy (NAC) have worse distant recurrence free survival (DRFS) compared to White women. Distant metastases result from motile breast cancer stem cells (BCSCs) disseminating through intravasation portals on blood vessels called tumor microenvironment of metastasis (TMEM) doorways. Each TMEM doorway is composed of an actin-regulatory protein (Mena) expressing cancer cell, a macrophage expressing high levels of receptor tyrosine kinase TIE2, and an angiopoietin-expressing endothelial cell. High TMEM doorway density predicts metastatic potential in patients with ER+/HER2- breast cancer. The function of TMEM doorways can be pharmacologically blocked using TIE2 inhibitors. We hypothesized that racial disparity in DRFS in patients with residual ER-positive disease post-NAC is due to difference in density of TMEM doorways, BCSCs, and motile cells in the residual breast tumor microenvironment (TME). Methods: We performed a retrospective, multi-institutional study of the TME in the residual disease post-NAC from 200 patients. Triple immunohistochemistry of formalin-fixed paraffin-embedded (FFPE) tissue sections was used to visualize TMEM doorways. Immunofluorescent staining of FFPE tissue sections for SOX9 and MenaINV was used to visualize BCSCs and motile cells, respectively. The density of TMEM doorways, BCSC and motile cells was determined by automated image analysis. The relationship between the density of TMEM doorways, stem and motile cells, and DRFS was examined using Kaplan Meier log-rank test. Comparisons were made for the entire cohort, and separately for the two most prevalent molecular subtypes: ER+/HER2- and triple negative (TN). The analysis included 200 samples of residual breast cancer post-NAC: 100 from Black and 100 from White women. Results: Black compared to White women were more likely to develop distant recurrence (49% vs 34%, p=0.04), to receive mastectomy (70% vs 51%, p=0.009), and to have TN subtype (35% vs 21%, p&amp;lt;0.0001). Median time-to-follow-up was 70 months (range 2.8-160.7), and was similar between Black and White patients (67.7 vs 71.0 months, p=0.7). There was no racial disparity in DRFS in the overall cohort or TN subtype, but there was a trend towards worse survival in Black women in the ER+/HER2- subtype (Log-Rank p=0.09). TMEM doorway density was higher in Black women in the entire cohort (p=0.001) and in the ER+/HER2- subtype (p=0.01), while there was no difference in the TN subtype. TMEM doorway density was prognostic for DRFS in all patients (Log-Rank p=0.009) and the ER+/HER2- subtype (Log-Rank p=0.001), but not in the TN subtype. There was a trend towards higher BCSC density in the entire cohort (p=0.06) and the ER+/HER2- subtype (p=0.09), but there was no difference in the TN subtype. There was no racial disparity in motile cell density in the entire cohort as well as in the two subtypes. BCSC density and motile cell density were not prognostic for DRFS. Conclusion: Worse DRFS in Black, compared to White, women with ER-positive residual breast cancer post-NAC may be due to differences in TMEM doorway and BCSC density. Further studies are necessary to determine if the outcome of patients with residual disease and high TMEM doorway density post-NAC could be improved by blocking TMEM doorway function with TIE2 inhibitors. Citation Format: Gina Kim, Saeed Asiry, Isabelle Oktay, Yu Lin, Xianjun Ye, Esther Cheng, Nurfiza Ladak, John Condeelis, Esther Adler, Paula Ginter, Timothy D'Alfonso, David Entenberg, Maja Oktay. Racial disparity in post-neoadjuvant chemotherapy residual breast tumor microenvironment [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-08-36.

Targeting Tie2 in the Tumor Microenvironment: From Angiogenesis to Dissemination.

Simple SummaryThe dissemination of cancer cells from their original location to distant organs where they grow, a process called metastasis, causes more than 90% of cancer deaths. The identification of the molecular mechanisms of metastasis and the development of anti-metastatic therapies are essential to increase patient survival. In recent years, targeting the tumor microenvironment has become a promising avenue to prevent both tumor growth and metastasis. As the tumor microenvironment contains not only cancer cells but also blood vessels, immune cells, and other non-cancerous cells, it is naïve to think that therapy only affects a single cell type in this complex environment. Here we review the importance, and ways to inhibit the function, of one therapeutic target: the receptor Tie2. Tie2 is a receptor present on the cell surface of several cell types within the tumor microenvironment and regulates tumor angiogenesis, growth, and metastasis to distant organs.The Tie2 receptor tyrosine kinase is expressed in vascular endothelial cells, tumor-associated macrophages, and tumor cells and has been a major focus of research in therapies targeting the tumor microenvironment. The most extensively studied Tie2 ligands are Angiopoietin 1 and 2 (Ang1, Ang2). Ang1 plays a critical role in vessel maturation, endothelial cell migration, and survival. Ang2, depending on the context, may function to disrupt connections between the endothelial cells and perivascular cells, promoting vascular regression. However, in the presence of VEGF-A, Ang2 instead promotes angiogenesis. Tie2-expressing macrophages play a critical role in both tumor angiogenesis and the dissemination of tumor cells from the primary tumor to secondary sites. Therefore, Ang-Tie2 signaling functions as an angiogenic switch during tumor progression and metastasis. Here we review the recent advances and complexities of targeting Tie2 signaling in the tumor microenvironment as a possible anti-angiogenic, and anti-metastatic, therapy and describe its use in combination with chemotherapy.

Intrinsic differences in the mechanisms of Tie2 binding to angiopoietins exploited by directed evolution to create an Ang2-selective ligand trap

Angiopoietins Ang1 and Ang2 are secreted ligands for the endothelial receptor tyrosine kinase Tie2 essential for vascular development and maintenance. Ang1 acts as an agonist to maintain normal vessel function, whereas Ang2 acts as a Tie2 antagonist. Ang2 is increased in macular edema, sepsis, and other conditions, in which it blocks Ang1-mediated signaling, causing vascular dysfunction and contributing to disease pathology. Therefore, Ang2 is an attractive therapeutic target. Previously, we reported a Tie2 ectodomain variant that selectively binds Ang2 and acts as soluble ligand trap to sequester Ang2; however, the mechanism of Ang2-binding selectivity is unknown. In the present study, we used directed protein evolution to enhance Ang2-binding affinity of this Tie2 ectodomain trap. We examined contributions of individual residues in the ligand-binding interface of Tie2 to Ang1 and Ang2 binding. Surprisingly, different combinations of Tie2 residues were found to bind each ligand, with hydrophobic residues binding both ligands and polar residues contributing selectively to either Ang1 or Ang2 binding. Our analysis also identified a single Tie2 residue, His168, with a pivotal role in both Ang1 and Ang2 binding, enabling competition between binding ligands. In summary, this study reports an enhanced-affinity Ang2-selective ligand trap with potential for therapeutic development and reveals the mechanism behind its selectivity. It also provides the first analysis of contributions of individual Tie2 residues to Ang1 and Ang2 binding and identifies selectivity-determining residues that could be targeted in the future design of small molecule and other inhibitors of Ang2 for the treatment of vascular dysfunction.

Vascular Endothelial Protein Tyrosine Phosphatase Regulates Endothelial Function

Vascular endothelial protein tyrosine phosphatase (VE-PTP) is a receptor-type PTP (RPTP), predominantly expressed in vascular endothelial cells. It regulates embryonic and tumor angiogenesis and controls vascular permeability and homeostasis in inflammation. Major substrates are the tyrosine kinase receptor Tie-2 and the adhesion molecule VE-cadherin. This review describes how VE-PTP controls vascular functions by its various substrates and the therapeutic potential of VE-PTP in various pathophysiological settings.

Adaptor Protein ShcD/SHC4 Interacts with Tie2 Receptor to Synergistically Promote Glioma Cell Invasion.

Gliomas are characterized by diffuse infiltration of tumor cells into surrounding brain tissue, and this highly invasive nature contributes to disease recurrence and poor patient outcomes. The molecular mechanisms underlying glioma cell invasion remain incompletely understood, limiting development of new targeted therapies. Here, we have identified phosphotyrosine adaptor protein ShcD as upregulated in malignant glioma and shown that it associates with receptor tyrosine kinase Tie2 to facilitate invasion. In human glioma cells, we find that expression of ShcD and Tie2 increases invasion, and this significant synergistic effect is disrupted with a ShcD mutant that cannot bind Tie2 or hyperphosphorylate the receptor. Expression of ShcD and/or Tie2 further increases invadopodia formation and matrix degradation in U87 glioma cells. In a coculture model, we show that U87-derived tumor spheroids expressing both ShcD and Tie2 display enhanced infiltration into cerebral organoids. Mechanistically, we identify changes in focal adhesion kinase phosphorylation in the presence of ShcD and/or Tie2 in U87 cells upon Tie2 activation. Finally, we identify a strong correlation between transcript levels of ShcD and Tie2 signaling components as well as N-cadherin in advanced gliomas and those with classical or mesenchymal subtypes, and we show that elevated expression of ShcD correlates with a significant reduction in patient survival in higher grade gliomas with mesenchymal signature. Altogether, our data highlight a novel Tie2-ShcD signaling axis in glioma cell invasion, which may be of clinical significance. IMPLICATIONS: ShcD cooperates with Tie2 to promote glioma cell invasion and its elevated expression correlates with poor patient outcome in advanced gliomas.