Delta-like ligand 4-mediated Notch signaling controls proliferation of second heart field progenitor cells by regulating Fgf8 expression.

The role played by the Notch pathway in cardiac progenitor cell biology remains to be elucidated. Delta-like ligand 4 (Dll4), the arterial-specific Notch ligand, is expressed by second heart field (SHF) progenitors at time-points that are crucial in SHF biology. Dll4-mediated Notch signaling is required for maintaining an adequate pool of SHF progenitors, such that Dll4 knockout results in a reduction in proliferation and an increase in apoptosis. A reduced SHF progenitor pool leads to an underdeveloped right ventricle (RV) and outflow tract (OFT). In its most severe form, there is severe RV hypoplasia and poorly developed OFT resulting in early embryonic lethality. In its milder form, the OFT is foreshortened and misaligned, resulting in a double outlet right ventricle. Dll4-mediated Notch signaling maintains Fgf8 expression by transcriptional regulation at the promoter level. Combined heterozygous knockout of Dll4 and Fgf8 demonstrates genetic synergy in OFT alignment. Exogenous supplemental Fgf8 rescues proliferation in Dll4 mutants in ex-vivo culture. Our results establish a novel role for Dll4-mediated Notch signaling in SHF biology. More broadly, our model provides a platform for understanding oligogenic inheritance that results in clinically relevant OFT malformations.

GRP78 haploinsufficiency suppresses acinar-to-ductal metaplasia, signaling, and mutant Kras-driven pancreatic tumorigenesis in mice.

Pancreatic ductal adenocarcinoma (PDAC) remains a highly lethal disease in critical need of new therapeutic strategies. Here, we report that the stress-inducible 78-kDa glucose-regulated protein (GRP78/HSPA5), a key regulator of endoplasmic reticulum homeostasis and PI3K/AKT signaling, is overexpressed in the acini and PDAC of Pdx1-Cre;KrasG12D/+;p53f/+ (PKC) mice as early as 2 mo, suggesting that GRP78 could exert a protective effect on acinar cells under stress, as during PDAC development. The PKC pancreata bearing wild-type Grp78 showed detectable PDAC by 3 mo and rapid subsequent tumor growth. In contrast, the PKC pancreata bearing a Grp78f/+ allele (PKC78f/+ mice) expressing about 50% of GRP78 maintained normal sizes during the early months, with reduced proliferation and suppression of AKT, S6, ERK, and STAT3 activation. Acinar-to-ductal metaplasia (ADM) has been identified as a key tumor initiation mechanism of PDAC. Compared with PKC, the PKC78f/+ pancreata showed substantial reduction of ADM as well as pancreatic intraepithelial neoplasia-1 (PanIN-1), PanIN-2, and PanIN-3 and delayed onset of PDAC. ADM in response to transforming growth factor α was also suppressed in ex vivo cultures of acinar cell clusters isolated from mouse pancreas bearing targeted heterozygous knockout of Grp78 (c78f/+ ) and subjected to 3D culture in collagen. We further discovered that GRP78 haploinsufficiency in both the PKC78f/+ and c78f/+ pancreata leads to reduction of epidermal growth factor receptor, which is critical for ADM initiation. Collectively, our studies establish a role for GRP78 in ADM and PDAC development.

Histological Validation of 11Carbon-Acetate Positron Emission Tomography/Computerized Tomography in Detecting Lymph Node Metastases in Prostate Cancer.

PURPOSE: Conventional imaging cannot definitively detect nodal metastases of prostate cancer. We histologically validated C-acetate positron emission tomography/computerized tomography to identify nodal metastases, examining prostate cancer factors that influence detection rates. MATERIALS AND METHODS: Patients with C-acetate avid positron emission tomography/computerized tomography imaged pelvic/retroperitoneal lymph nodes underwent high extended robotic lymphadenectomy. A standardized mapping template comprising 8 predetermined anatomical regions was dissected during lymphadenectomy, allowing for matched, region based analysis and comparison of imaging and histological data. RESULTS: In 25 patients a total of 2,149 lymph nodes were excised (mean 86 per patient, range 27 to 136) and 528 (22%) harbored metastases (mean 21 positive nodes per patient, range 0 to 109). A total of 174 anatomical regions had matching imaging histological data. C-acetate positron emission tomography/computerized tomography accurately identified 48 node-positive regions and accurately ruled out 88 regions as metastasis-free. C-acetate sensitivity, specificity, and positive and negative predictive values were 67%, 84%, 74% and 79%, respectively. An increasing, histologically measured metastatic lesion size in long axis diameter of 5 or less, 6 to 10, 11 to 15, 16 to 20 and 21 mm or greater correlated with improved C-acetate detection rates of 45%, 62%, 81%, 89% and 100%, respectively. Each standard uptake value unit increase correlated with a 1.9 mm increase in nodal long axis diameter (p <0.001) and a 1.2 mm increase in short axis diameter (p <0.001). Positive C-acetate positron emission tomography/computerized tomography findings correlated with histological lymph node size (long axis diameter 12 mm and short axis diameter 6 mm), metastatic lesion size (long axis diameter 11 mm and short axis diameter 6 mm) and extranodal extension (positive 88% vs false-negative 58%, p = 0.005). CONCLUSIONS: C-acetate positron emission tomography/computerized tomography can identify prostate cancer metastatic nodal disease. However, it underestimates the true cephalad extent of nodal involvement, performing better in the pelvis than in the retroperitoneum. Standard uptake value, histological nodal size, intranodal metastasis size and extranodal extension correlate with cancer bearing nodes.

PET Imaging of Dll4 Expression in Glioblastoma and Colorectal Cancer Xenografts Using (64)Cu-Labeled Monoclonal Antibody 61B.

Delta-like ligand 4 (Dll4) expressed in tumor cells plays a key role to promote tumor growth of numerous cancer types. Based on a novel antihuman Dll4 monoclonal antibody (61B), we developed a (64)Cu-labeled probe for positron emission tomography (PET) imaging of tumor Dll4 expression. In this study, 61B was conjugated with the (64)Cu-chelator DOTA through lysine on the antibody. Human IgG (hIgG)-DOTA, which did not bind to Dll4, was also prepared as a control. The Dll4 binding activity of the probes was evaluated through the bead-based binding assay with Dll4-alkaline phosphatase. The resulting PET probes were evaluated in U87MG glioblastoma and HT29 colorectal cancer xenografts in athymic nude mice. Our results demonstrated that the 61B-DOTA retained (77.2 ± 3.7) % Dll4 binding activity of the unmodified 61B, which is significantly higher than that of hIgG-DOTA (0.06 ± 0.03) %. Confocal microscopy analysis confirmed that 61B-Cy5.5, but not IgG-Cy5.5, predominantly located within the U87MG and HT29 cells cytoplasm. U87MG cells showed higher 61B-Cy5.5 binding as compared to HT29 cells. In U87MG xenografts, 61B-DOTA-(64)Cu demonstrated remarkable tumor accumulation (10.5 ± 1.7 and 10.2 ± 1.2%ID/g at 24 and 48 h postinjection, respectively). In HT29 xenografts, tumor accumulation of 61B-DOTA-(64)Cu was significantly lower than that of U87MG (7.3 ± 1.3 and 6.6 ± 1.3%ID/g at 24 and 48 h postinjection, respectively). The tumor accumulation of 61B-DOTA-(64)Cu was significantly higher than that of hIgG-DOTA-(64)Cu in both xenografts models. Immunofluorescence staining of the tumor tissues further confirmed that tumor accumulation of 61B-Cy5.5 was correlated well with in vivo PET imaging data using 61B-DOTA-(64)Cu. In conclusion, 61B-DOTA-(64)Cu PET probe was successfully synthesized and demonstrated prominent tumor uptake by targeting Dll4. 61B-DOTA-(64)Cu has great potential to be used for noninvasive Dll4 imaging, which could be valuable for tumor detection, Dll4 expression level evaluation, and Dll4-based treatment monitoring.

Expression of the EPHB4 receptor tyrosine kinase in head and neck and renal malignancies--implications for solid tumors and potential for therapeutic inhibition.

Solid malignancies are often characterized by overexpression of various receptor tyrosine kinases (RTKs) against which many targeted therapies are currently in use and in active development. EPHB4 has recently emerged as a frequently overexpressed RTK in many types of cancer. Here, we demonstrate expression patterns of EPHB4 in two solid malignancies: squamous cell carcinoma of the head and neck (HNSCC) and renal cell carcinoma (RCC), by immunohistochemical analysis. We demonstrate the first association between EPHB4 expression and progression of HNSCC from normal tissue to dysplasia and to cancer. Interestingly, most RCC subtypes exhibited expression patterns that were opposite from that found in HNSCC, possibly owing to their unique biology and high degree of organ and tumor vasculature. Taken together, these results suggest a possible role for EPHB4 as a therapeutic target in these malignancies.

Design, synthesis, and validation of Axl-targeted monoclonal antibody probe for microPET imaging in human lung cancer xenograft.

Accumulating experimental evidence indicates that overexpression of the oncogenic receptor tyrosine kinase, Axl, plays a key role in the tumorigenesis and metastasis of various types of cancer. The objective of this study is to design a novel imaging probe based on the monoclonal antibody, h173, for microPET imaging of Axl expression in human lung cancer. A bifunctional chelator, DOTA, was conjugated to h173, followed by radiolabeling with (64)Cu. The binding of DOTA-h173 to the Axl receptor was first evaluated by a cell uptake assay and flow cytometry analysis using human lung cancer cell lines. The probe (64)Cu-DOTA-h173 was further evaluated by microPET imaging, and ex vivo histology studies in the Axl-positive A549 tumors. In vitro cellular study showed that Axl probe, (64)Cu-DOTA-h173, was highly immuno-reactive with A549 cells. Western blot analysis confirmed that Axl is highly expressed in the A549 cell line. For microPET imaging, the A549 xenografts demonstrated a significantly higher (64)Cu-DOTA-h173 uptake compared to the NCI-H249 xenograft (a negative control model). Furthermore, (64)Cu-DOTA-h173 uptake in A549 is significantly higher than that of (64)Cu-DOTA-hIgG. Immuno-fluorescence staining was consistent with the in vivo micro-PET imaging results. In conclusion, (64)Cu-DOTA-h173 could be potentially used as a probe for noninvasive imaging of Axl expression, which could collect important information regarding tumor response to Axl-targeted therapeutic interventions.

Improved efficacy of pembrolizumab combined with soluble EphB4-albumin in HPV-negative EphrinB2 positive head neck squamous cell carcinoma.

OBJECTIVE: Patients with relapsed or metastatic head and neck squamous cell carcinoma (HNSCC) after primary local therapy have low response rates with cetuximab, systemic chemotherapy or check point inhibitor therapy. Novel combination therapies with the potential to improve outcomes for patients with HNSCC is an area of high unmet need. METHODS: This is a phase II single-arm clinical trial of locally advanced or metastatic HNSCC patients treated with a combination of soluble EphB4-human serum albumin (sEphB4-HSA) fusion protein and pembrolizumab after platinum-based chemotherapy with up to 2 prior lines of treatment. The primary endpoints were safety and tolerability and the primary efficacy endpoint was overall response rate (ORR). Secondary endpoints included progression free survival (PFS) and overall survival (OS). HPV status and EphrinB2 expression were evaluated for outcome. RESULTS: Twenty-five patients were enrolled. Median follow up was 40.4 months (range 9.8 - 40.4). There were 6 responders (ORR 24%). There were 5 responders in the 11 HPV-negative and EphrinB2 positive patients, (ORR 45%) with 2 of these patients achieving a complete response (CR). The median PFS in HPV-negative/EphrinB2 positive patients was 3.2 months (95% CI 1.1, 7.3). Median OS in HPV-negative/EphrinB2 positive patients was 10.9 months (95% CI 2.0, 13.7). Hypertension, transaminitis and fatigue were the most common toxicities. DISCUSSION: The combination of sEphB4-HSA and pembrolizumab has a favorable toxicity profile and favorable activity particularly among HPV-negative EphrinB2 positive patients with HNSCC.

EphB4 as a therapeutic target in mesothelioma.

BACKGROUND: Malignant pleural mesothelioma (MPM) often develops decades following exposure to asbestos. Current best therapy produces a response in only half of patients, and the median survival with this therapy remains under a year. A search for novel targets and therapeutics is underway, and recently identified targets include VEGF, Notch, and EphB4-Ephrin-B2. Each of these targets has dual activity, promoting tumor cell growth as well as tumor angiogenesis. METHODS: We investigated EphB4 expression in 39 human mesothelioma tissues by immunohistochemistry. Xenograft tumors established with human mesothelioma cells were treated with an EphB4 inhibitor (monomeric soluble EphB4 fused to human serum albumin, or sEphB4-HSA). The combinatorial effect of sEphB4-HSA and biologic agent was also studied. RESULTS: EphB4 was overexpressed in 72% of mesothelioma tissues evaluated, with 85% of epithelioid and 38% of sarcomatoid subtypes demonstrating overexpression. The EphB4 inhibitor sEphB4-HSA was highly active as a single agent to inhibit tumor growth, accompanied by tumor cell apoptosis and inhibition of PI3K and Src signaling. Combination of sEphB4-HSA and the anti-VEGF antibody (Bevacizumab) was superior to each agent alone and led to complete tumor regression. CONCLUSION: EphB4 is a potential therapeutic target in mesothelioma. Clinical investigation of sEphB4-HSA as a single agent and in combination with VEGF inhibitors is warranted.

Targeting the EphB4 receptor for cancer diagnosis and therapy monitoring.

Accumulating evidence suggests that EphB4 plays key roles in cancer progression in numerous cancer types. In fact, therapies focusing on EphB4 have become potentially important components of various cancer treatment strategies. However, tumor sensitivity to EphB4 suppression may not be uniform for different cancers. In this study, we developed near-infrared fluorescence (NIRF) probes for EphB4 targeted imaging, based on EphB4-specific humanized monoclonal antibody hAb47. NIRF dye Cy5.5 was introduced to hAb47 either through the reaction with amino groups (named hAb47-Cy5.5) or sulfhydryl groups (named hAb47-Cy5.5-Mal). The resulting probes were evaluated in both HT-29 xenograft and the mAb131 (anti-EphB4) treated models. Although these methods lead to modifications of both the heavy chain and light chain of the antibody, the majority of the EphB4 binding affinity was maintained (81.62 ± 2.08% for hAb47-Cy5.5 and 77.14 ± 2.46% for hAb47-Cy5.5-Mal, respectively). hAb47-Cy5.5 was then chosen for in vivo NIRF imaging of EphB4 expression. In HT29 colorectal tumor xenografts, hAb47-Cy5.5 demonstrated significantly higher tumor uptake compared with that of the hIgG-Cy5.5 control, which was further confirmed by immunofluorescent staining. Moreover, hAb47-Cy5.5 successfully imaged the decreased EphB4 expression (confirmed by Western blot) in EphB4-targeted immunotherapy using another EphB4-specific antibody, mAb131. Collectively, hAb47-Cy5.5 could be used as a specific NIRF contrast agent for noninvasive imaging of EphB4 expression, which may predict whether an individual tumor would likely respond to EphB4 targeted interventions, as well as monitor the therapeutic response.

EphB4-targeted imaging with antibody h131, h131-F(ab')2 and h131-Fab.

Accumulating evidence suggests that overexpression of the tyrosine kinase receptor EphB4, a mediator of vascular development, is a novel target for tumor diagnosis, prognosis and therapy. Noninvasive imaging of EphB4 expression could therefore be valuable for evaluating disease course and therapeutic efficacy at the earliest stages of anti-EphB4 treatment. In this study, we systematically investigated the use of anti-EphB4 antibody h131 (150 kDa) and its fragments (h131-F(ab')2, 110 kDa; h131-Fab, 50 kDa) for near-infrared fluorescence (NIRF) imaging of EphB4 expression in vivo. h131-F(ab')2 and h131-Fab were produced through pepsin and papain digestion of h131 respectively, whose purity was confirmed by FPLC and SDS-PAGE. After conjugation with Cy5.5, in vivo characteristics of h131, h131-F(ab')2 and h131-Fab were evaluated in EphB4-positive HT29 tumor model. Although h131-Cy5.5 demonstrated highest tumor uptake among these probes, its optimal tumor uptake level was obtained at 2 days post injection (p.i.). For h131-Fab-Cy5.5, maximum tumor uptake was achieved at 4 h p.i. However, no significant difference was observed between h131-Fab-Cy5.5 and hIgG-Fab-Cy5.5, indicating the tumor accumulation was mainly caused by passive targeting. In contrast, h131-F(ab')2-Cy5.5 demonstrated prominent tumor uptake at 6 h p.i. The target specificity was confirmed by hIgG-F(ab')2-Cy5.5 control and immunofluorescent staining. Collectively, h131-F(ab')2 exhibited prominent and specific tumor uptake at early time points, which suggests it is a promising agent for EphB4-targeted imaging.

EphrinB2 Inhibition and Pembrolizumab in Metastatic Urothelial Carcinoma.

PURPOSE: Patients with metastatic urothelial carcinoma have poor prognosis after failure of standard first-line chemotherapy. Immune check point programmed death 1-programmed death ligand 1 antibodies have low response rates and thus there exists a major unmet need. MATERIALS AND METHODS: In this phase II trial, patients with metastatic urothelial carcinoma that recurred or progressed after platinum-based chemotherapy received soluble EphB4-human serum albumin (sEphB4-HSA) in combination with pembrolizumab. The primary end points were tolerability and overall survival (OS). The secondary end points were progression-free survival (PFS), objective response rate (ORR), duration of response, and toxicity. The expression of sEphB4-HSA target EphrinB2 was correlated with outcomes. RESULTS: Seventy patients were enrolled. The median follow up was 22.9 months (range, 1.3-54.7). The regimen had acceptable toxicity. In the intent-to-treat analysis (N = 70), the median OS was 14.6 months (95% CI, 9.2 to 21.5). Twenty-six (37%) patients had an objective response (95% CI, 26 to 48). The median PFS was 4.1 (95% CI, 1.5 to 5.7) months. Forty-six (66%) patients expressed EphrinB2, and among them, the median OS was 21.5 months (95% CI, 12.4 to not reached), the ORR was 52% (95% CI, 37 to 67), including a complete response rate of 24% (11 of 46; 95% CI, 12 to 36). The median PFS was 5.7 (95% CI, 2.7 to 27.9) months. Response was maintained at 6, 12, and 24 months in 88%, 74%, and 69% of the patients, respectively. CONCLUSION: The combination of sEphB4-HSA and pembrolizumab appears synergistic with improved OS and ORR compared with historical data for programmed death 1/programmed death ligand 1 monotherapy.

Inhibition of Notch signaling by Dll4-Fc promotes reperfusion of acutely ischemic tissues.

Notch pathway regulates vessel development and maturation. Dll4, a high-affinity ligand for Notch, is expressed predominantly in the arterial endothelium and is induced by hypoxia among other factors. Inhibition of Dll4 has paradoxical effects of reducing the maturation and perfusion in newly forming vessels while increasing the density of vessels. We hypothesized that partial and/or intermittent inhibition of Dll4 may lead to increased vascular response and still allow vascular maturation to occur. Thus tissue perfusion can be restored rapidly, allowing quicker recovery from ischemia or tissue injury. Our studies in two different models (hindlimb ischemia and skin flap) show that inhibition of Dll4 at low dose allows faster recovery from vascular and tissue injury. This opens a new possibility for Dll4 blockade's therapeutic application in promoting recovery from vascular injury and restoring blood supply to ischemic tissues.

Induction, regulation, and biologic function of Axl receptor tyrosine kinase in Kaposi sarcoma.

Axl is an oncogenic receptor tyrosine kinase that plays multiple roles in tumorigenesis and metastasis of many cancers. This study is the first to demonstrate that Axl is induced in Kaposi sarcoma and Kaposi sarcoma herpesvirus (KSHV) transformed endothelial cells. Conditionally, expression of one KSHV latency protein vFLIP induces Axl expression in endothelial cells. This induction can be blocked by nuclear factor-kappaB inhibitor, consistent with the known vFLIP mechanism of action. KS cell lines lacking KSHV also have elevated Axl expression, which probably resulted from hypomethylation of AXL promoter. Axl activation activates downstream phosphoinositol-3 kinase signaling, and Axl knockdown by siRNA impairs phosphoinositol-3 kinase signaling. Furthermore, Axl knockdown inhibits KS cell growth and invasion. To explore the potential for translation of these findings, we generated monoclonal antibodies to block the biologic functions of Axl. MAb173, which induces receptor degradation, showed activity in vitro to inhibit KS cell invasion. Moreover, in vivo xenograft studies with KS cells with or without KSHV infection showed that MAb173 reduced tumor growth, increased tumor cell apoptosis, and markedly decreased Axl protein level in tumors. Axl thus has a potential role in KS pathogenesis and is a candidate for prognostic and therapeutic investigations.

KSHV-induced notch components render endothelial and mural cell characteristics and cell survival.

Kaposi sarcoma-associated herpesvirus (KSHV) infection is essential to the development of Kaposi sarcoma (KS). Notch signaling is also known to play a pivotal role in KS cell survival and lytic phase entrance of KSHV. In the current study, we sought to determine whether KSHV regulates Notch components. KSHV-infected lymphatic endothelial cells showed induction of receptors Notch3 and Notch4, Notch ligands Dll4 and Jagged1, and activated Notch receptors in contrast to uninfected lymphatic endothelial cells. In addition, KSHV induced the expression of endothelial precursor cell marker (CD133) and mural cell markers (calponin, desmin, and smooth muscle alpha actin), suggesting dedifferentiation and trans-differentiation. Overexpression of latency proteins (LANA, vFLIP) and lytic phase proteins (RTA, vGPCR, viral interleukin-6) further supported the direct regulatory capacity of KSHV viral proteins to induce Notch receptors (Notch2, Notch3), ligands (Dll1, Dll4, Jagged1), downstream targets (Hey, Hes), and endothelial precursor CD133. Targeting Notch pathway with gamma-secretase inhibitor and a decoy protein in the form of soluble Dll4 inhibited growth of KSHV-transformed endothelial cell line. Soluble Dll4 was also highly active in vivo against KS tumor xenograft. It inhibited tumor cell growth, induced tumor cell death, and reduced vessel perfusion. Soluble Dll4 is thus a candidate for clinical investigation.

Novel EphB4 monoclonal antibodies modulate angiogenesis and inhibit tumor growth.

EphB4 receptor tyrosine kinase and its cognate ligand EphrinB2 regulate induction and maturation of newly forming vessels. Inhibition of their interaction arrests angiogenesis, vessel maturation, and pericyte recruitment. In addition, EphB4 is expressed in the vast majority of epithelial cancers and provides a survival advantage to most. Here, we describe two anti-EphB4 monoclonal antibodies that inhibit tumor angiogenesis and tumor growth by two distinct pathways. MAb131 binds to fibronectin-like domain 1 and induces degradation of human EphB4, but not murine EphB4. MAb131 inhibits human endothelial tube formation in vitro and growth of human tumors expressing EphB4 in vivo. In contrast, MAb47 targets fibronectin-like domain 2 of both human and murine EphB4 and does not alter EphB4 receptor levels, but inhibits angiogenesis and growth of both EphB4-positive and EphB4-negative tumors in a mouse s.c. xenograft model. Combination of MAb47 and bevacizumab enhances the antitumor activity and induces tumor regression. Indeed, humanized antibodies hAb47 and hAb131 showed similar affinity for EphB4 and retained efficacy in the inhibition of primary tumor development and experimental metastasis.

The role of Ephs, Ephrins, and growth factors in Kaposi sarcoma and implications of EphrinB2 blockade.

Kaposi sarcoma (KS) is associated with human herpesvirus (HHV)-8 and is dependent on the induction of vascular endothelial growth factors (VEGFs). VEGF regulates genes that provide arterial or venous identity to endothelial cells, such as the induction of EphrinB2, which phenotypically defines arterial endothelial cells and pericytes, and represses EphB4, which defines venous endothelial cells. We conducted a comprehensive analysis of the Eph receptor tyrosine kinases to determine which members are expressed and therefore contribute to KS pathogenesis. We demonstrated limited Eph/Ephrin expression; notably, the only ligand highly expressed is EphrinB2. We next studied the biologic effects of blocking EphrinB2 using the extracellular domain of EphB4 fused with human serum albumin (sEphB4-HSA). sEphB4-HSA inhibited migration and invasion of the KS cells in vitro in response to various growth factors. Finally, we determined the biologic effects of combining sEphB4-HSA and an antibody to VEGF. sEphB4-HSA was more active than the VEGF antibody, and combination of the 2 had at least additive activity. sEphB4-HSA reduced blood vessel density, pericyte recruitment, vessel perfusion, and increased hypoxia, with an associated increase in VEGF and DLL4 expression. The combination of sEphB4-HSA and VEGF antibody is a rational treatment combination for further investigation.

Tetraspanin18 regulates angiogenesis through VEGFR2 and Notch pathways.

The VEGF pathway is critically required for vasculogenesis, the formation of the primary vascular network. It is also required for angiogenesis resulting in sprouting and pruning of vessels to generate mature arborizing structures. The Notch pathway is essential for arterial-venous specification and the maturation of nascent vessels. We have determined that Tspan18, a member of the Tetraspanin family, is expressed in developing vessels but not in mature vasculature in zebrafish and mouse wound healing. Moreover, reduction at Tspan18 level resulted in aberrant vascular patterning, impaired vessel stability and defective arterial-venous specification. Tspan18 deficiency reduced VEGF, VEGFR2, Notch3 and EphrinB2, and increased EphB4, VEGFR3, Semaphorin3, Neuropilin and PlexinD1 expression. Furthermore, vascular defects of Tspan18 deficiency could be rescued by ectopic expression of VEGFR2 and Notch, but not by knockdown of Semaphorin or Plexin. Functional studies showed that knockdown of Tspan18 led to reduced endothelial cell migration, invasion and tube formation. Tspan18 has dynamic expression, regulates vascular development and maturation in the embryo with re-expression in adult life in wound healing.

Quantitative Tyrosine Phosphoproteome Profiling of AXL Receptor Tyrosine Kinase Signaling Network.

Overexpression and amplification of AXL receptor tyrosine kinase (RTK) has been found in several hematologic and solid malignancies. Activation of AXL can enhance tumor-promoting processes such as cancer cell proliferation, migration, invasion and survival. Despite the important role of AXL in cancer development, a deep and quantitative mapping of its temporal dynamic signaling transduction has not yet been reported. Here, we used a TMT labeling-based quantitative proteomics approach to characterize the temporal dynamics of the phosphotyrosine proteome induced by AXL activation. We identified >1100 phosphotyrosine sites and observed a widespread upregulation of tyrosine phosphorylation induced by GAS6 stimulation. We also detected several tyrosine sites whose phosphorylation levels were reduced upon AXL activation. Gene set enrichment-based pathway analysis indicated the activation of several cancer-promoting and cell migration/invasion-related signaling pathways, including RAS, EGFR, focal adhesion, VEGFR and cytoskeletal rearrangement pathways. We also observed a rapid induction of phosphorylation of protein tyrosine phosphatases, including PTPN11 and PTPRA, upon GAS6 stimulation. The novel molecules downstream of AXL identified in this study along with the detailed global quantitative map elucidating the temporal dynamics of AXL activation should not only help understand the oncogenic role of AXL, but also aid in developing therapeutic options to effectively target AXL.

PD-1 Blockade on Tumor Microenvironment-Resident ILC2s Promotes TNF-α Production and Restricts Progression of Metastatic Melanoma.

While pulmonary ILC2s represent one of the major tissue-resident innate lymphoid cell populations at steady state and are key drivers of cytokine secretion in their occupational niche, their role in pulmonary cancer progression remains unclear. As the programmed cell death protein-1 (PD-1) plays a major role in cancer immunotherapy and immunoregulatory properties, here we investigate the specific effect of PD-1 inhibition on ILC2s during pulmonary B16 melanoma cancer metastasis. We demonstrate that PD-1 inhibition on ILC2s suppresses B16 tumor growth. Further, PD-1 inhibition upregulates pulmonary ILC2-derived TNF-α production, a cytotoxic cytokine that directly induces cell death in B16 cells, independent of adaptive immunity. Together, these results highlight the importance of ILC2s and their anti-tumor role in pulmonary B16 cancer progression during PD-1 inhibitory immunotherapy.

A Novel DNA Methylation Signature as an Independent Prognostic Factor in Muscle-Invasive Bladder Cancer.

BACKGROUND: Muscle-invasive bladder cancer (MIBC) accounts for approximately 20% of all urothelial bladder carcinomas (UBC) at time of diagnosis, and up to 30% of patients with non-muscle invasive UBC will progress to MIBC over time. An increasing body of evidence has revealed a strong correlation between aberrant DNA methylation and tumorigenesis in MIBC. RESULTS: Using The Cancer Genome Atlas (TCGA) molecular data for 413 patients, we described a DNA methylation-based signature as a prognostic factor for overall survival (OS) in MIBC patients. By using a least absolute shrinkage and selection operator (LASSO) model, differentially methylated regions were first identified using multiple criteria followed by survival and LASSO analyses to identify DNA methylation probes related to OS and build a classifier to stratify patients with MIBC. The prognostic value of the classifier, referred to as risk score (RS), was validated in a held-out testing set from the TCGA MIBC cohort. Finally, receiver operating characteristic (ROC) analysis was used to compare the prognostic accuracy of the models built with RS alone, RS plus clinicopathologic features, and clinicopathologic features alone. We found that our seven-probe classifier-based RS stratifies patients into high- and low-risk groups for overall survival (OS) in the testing set (n = 137) (AUC at 3 years, 0.65; AUC at 5 years, 0.65). In addition, RS significantly improved the prognostic model when it was combined with clinical information including age, smoking status, Tumor (T) stage, and Lymph node metastasis (N) stage. CONCLUSIONS: The DNA methylation-based RS can be a useful tool to predict the accuracy of preoperative and/or post-cystectomy models of OS in MIBC patients.