VEGFR Pathways Research Articles

Immunotherapy in colorectal cancer: an unmet need deserving of change.

The efficacy of immunotherapy in colorectal cancer is currently restricted to patients with metastatic colorectal cancer presenting deficient mismatch repair (dMMR) or microsatellite instability (MSI). So far, neither immunotherapy agents as monotherapy nor immunotherapy-based combinations have shown benefit for metastatic colorectal cancer with proficient mismatch repair (pMMR) or microsatellite stability (MSS), which are mostly immunologically cold. 1 Ganesh K Stadler ZK Cercek A et al. Immunotherapy in colorectal cancer: rationale, challenges and potential. Nat Rev Gastroenterol Hepatol. 2019; 16: 361-375 Crossref PubMed Scopus (480) Google Scholar However, the immune landscape of colorectal cancer is complex and heterogenous, and the development of these treatment strategies must consider, not only the histological context, but also its potential distinctive features. 2 Guinney J Dienstmann R Wang X et al. The consensus molecular subtypes of colorectal cancer. Nat Med. 2015; 21: 1350-1356 Crossref PubMed Scopus (2403) Google Scholar In The Lancet Oncology, Carlotta Antoniotti and colleagues 3 Antoniotti C Rossini D Pietrantonio F et al. Upfront FOLFOXIRI plus bevacizumab with or without atezolizumab in the treatment of patients with metastatic colorectal cancer (AtezoTRIBE): a multicentre, open-label, randomised, controlled, phase 2 trial. Lancet Oncol. 2022; (published online May 27.)https://doi.org/10.1016/S1470-2045(22)00274-1 Summary Full Text Full Text PDF PubMed Google Scholar report the results of the addition of atezolizumab to front-line FOLFOXIRI (fluorouracil, leucovorin, oxaliplatin, and irinotecan) plus bevacizumab (the atezolizumab group) compared with FOLFOXIRI plus bevacizumab (the control group) in patients with metastatic colorectal cancer from the phase 2 AtezoTRIBE trial, irrespective of tumour microsatellite status. The superiority of FOLFOXIRI plus bevacizumab over FOLFIRI (leucovorin, fluorouracil, and irinotecan) plus bevacizumab as front-line treatment of metastatic colorectal cancer was previously demonstrated in the phase 3 TRIBE trial. Drawing from this rationale, in this multicentre, academic study, the investigators raised the question of whether the restoration of the anticancer immunity through inhibition of the PD-1–PD-L1 axis enhances efficacy of an upfront intensified chemotherapy regimen. The underlying hypothesis was that chemotherapy can induce tumour cells to release more neoantigens, leading to immunogenic cell death and activation of CD8 T lymphocytes, while bevacizumab-mediated vasculature normalisation upon VEGF or VEGFR blockade promotes increased tumour-infiltrating lymphocytes and activation of effector immune cells. 4 Kanterman J Sade-Feldman M Biton M et al. Adverse immunoregulatory effects of 5FU and CPT11 chemotherapy on myeloid-derived suppressor cells and colorectal cancer outcomes. Cancer Res. 2014; 74: 6022-6035 Crossref PubMed Scopus (113) Google Scholar , 5 Terme M Pernot S Marcheteau E et al. VEGFA–VEGFR pathway blockade inhibits tumor-induced regulatory T-cell proliferation in colorectal cancer. Cancer Res. 2013; 73: 539-549 Crossref PubMed Scopus (413) Google Scholar Given that no significant benefit has been derived from the combination of immunotherapy with non-intensified regimens, 6 Grothey A Tabernero J Arnold D et al. Fluoropyrimidine (FP) + bevacizumab (BEV) + atezolizumab vs FP/BEV in BRAFwt metastatic colorectal cancer (mCRC): findings from Cohort 2 of MODUL—a multicentre, randomized trial of biomarker-driven maintenance treatment following first-line induction therapy. Ann Oncol. 2018; 29: viii714-viii715 Google Scholar , 7 Lenz HJ Parikh AR Spigel DR et al. Nivolumab (NIVO) + 5-fluorouracil/leucovorin/oxaliplatin (mFOLFOX6)/bevacizumab (BEV) versus mFOLFOX6/BEV for first-line (1L) treatment of metastatic colorectal cancer (mCRC): phase 2 results from CheckMate 9X8. Proc Am Soc Clin Oncol. 2022; 40 (abstr).: 8 Crossref PubMed Google Scholar the use of FOLFOXIRI is a rational partner in an untreated RAS-mutated enriched metastatic colorectal cancer population. Upfront FOLFOXIRI plus bevacizumab with or without atezolizumab in the treatment of patients with metastatic colorectal cancer (AtezoTRIBE): a multicentre, open-label, randomised, controlled, phase 2 trialThe addition of atezolizumab to first-line FOLFOXIRI plus bevacizumab is safe and improved progression-free survival in patients with previously untreated metastatic colorectal cancer. Full-Text PDF

Treatment of malignant pleural effusion in non-small cell lung cancer with VEGF-directed therapy

Background Vascular endothelial growth factor (VEGF) is a critical regulator of malignant pleural effusion (MPE) in non-small-cell lung cancer (NSCLC). Bevacizumab (BEV) and apatinib (APA) are novel VEGF blockers that inhibit lung cancer cell proliferation and the development of pleural effusion. Methods In this study, we established Lewis lung cancer (LLC) xenograft mouse models to compare the therapeutic effect of APA and BEV in combination with cisplatin (CDDP) against MPE. The anti-tumour and anti-angiogenic effects of this combination therapy were evaluated by 18F-FDG PET/CT imaging, TUNEL assay and Immunohistochemistry. Results The triple drug combination significantly prolonged the overall survival of the tumour-bearing mice by reducing MPE and glucose metabolism and was more effective in lowering VEGF/soluble VEGFR-2 levels in the serum and pleural exudates compared to either of the monotherapies. Furthermore, CDDP + APA + BEV promoted in vivo apoptosis and decreased microvessel density. Conclusions Mechanistically, LLC-induced MPE was inhibited by targeting the VEGF-MEK/ERK pathways. Further studies are needed to establish the synergistic therapeutic effect of these drugs in NSCLC patients with MPE. KEY MESSAGES Combined treatment of MPE with apatinib, bevacizumab and cisplatin can prolong the survival time of mice, reduce the content of MPE, decrease the SUVmax of thoracic tumour tissue, down-regulate the content of VEGF and sVEGFR-2 in serum and pleural fluid, and promote the apoptosis of tumour cells. Angiogenesis and MPE formation can be inhibited by down-regulation of HIF-1α, VEGF, VEGFR-2, MEK1 and MMP-2 molecular signalling pathway proteins.

Evaluation of antitumor effects of VEGFR-2 inhibitor F16in a colorectal xenograft model.

Colorectal cancer(CRC) is the third most prevalent type of cancer in the United States. The treatment options for cancer include surgery, chemotherapy, radiation, and/or targeted therapy, which show significant improvement in overall survival. Among the various available treatments, antagonizing VEGF/VEGFR-2 pathways have shown effectiveness in limiting colorectal cancer growth and improving clinical outcomes. In this regard, we hypothesized that F16, a novel VEGFR-2 inhibitor, would control colorectal cancer growth by blocking the VEGFR-2 singling pathway in both in vitro and in vivo conditions. Therefore, the current study was aimed to analyze the efficacy of F16 on the growth of Colo 320DM cells under in vitro and in vivo conditions. Human RT2 profiler PCR array analysis results clearly showed that angiogenesis and anti-apoptosis-related gene expressions were significantly reduced in HUVEC cells after F16 (5μM) treatment. In addition, Western blot results revealed that F16 attenuated the downstream signaling of the VEGFR-2 pathway in HUVEC cells by up-regulating the p53 and p21 levels and down-regulating the p-AKT and p-FAK levels. Accordingly, F16 confirmed potent cytotoxic effects against the cell viability of Colo 320DM tumors, with an IC50 valueof 9.52 ± 1.49µM. Furthermore, treatment of mice implanted with Colo 320DM xenograft tumors showed a significant reduction in tumor growth and increases in survival rate compared to controls. Immunohistochemistry analysis of tumor tissues showeda reduction in CD31 levels also in F16 treated groups. These results justify further evaluation of F16 as a potential new therapeutic agent for treating colorectal cancers.

The lymph node transcriptome of unicentric and idiopathic multicentric Castleman disease.

Castleman disease is a polyclonal lymphoproliferative disorder characterized by unicentric or multicentric lymphadenopathy with characteristic histomorphological features, in addition to variable inflammatory symptomatology. The molecular mechanisms and etiologies of unicentric Castleman disease (UCD) and idiopathic multicentric Castleman disease (iMCD) are poorly understood, and identification of targetable disease mediators remains an unmet clinical need. We performed whole exome sequencing on lymph node biopsies from patients with UCD and iMCD and compared the transcriptomic profiles to that of benign control lymph nodes. We identified significantly upregulated genes in UCD (n=443), iMCD (n=316) or both disease subtypes (n=51) and downregulated genes in UCD (n=321), iMCD (n=105) or both (n=10). The transcriptomes of UCD and iMCD showed enrichment and upregulation of elements of the complement cascade. By immunohistochemistry, C4d deposits indicative of complement activation were found to be present in UCD and iMCD, mostly within abnormally regressed germinal centers, but also in association with plasma cell clusters, endothelial cells and stroma cell proliferations. Other enriched gene sets included collagen organization, S1P3 pathway and VEGFR pathway in UCD; and humoral response, oxidative phosphorylation and proteosome in iMCD. Analysis of cytokine transcripts showed upregulation of CXCL13 but not IL6 in UCD and iMCD. Among angiogenic mediators, the VEGFR1 ligand placental growth factor (PGF) was upregulated in both disease subtypes. We hereby report for the first time the whole lymph node transcriptomes of UCD and iMCD, underscoring findings that could aid in the discovery of targetable disease mediators.

Abstract P5-06-03: Intratumoral lymphatic endothelial cell infiltration reflects lymphangiogenesis and lymph node metastasis, but is counterbalanced by immune response and better cancer biology in breast cancer tumor microenvironment

Abstract Background: Unfavorable vasculogenesis, such as lymphangiogenesis and hemoangiogenesis are known to be pivotal of cancer progression. Our group reported that S1P pathway link chronic inflammation, such as obesity, and cancer progression by inducing lymphangiogenesis and hemoangiogenesis in breast cancer. During the past decade, complex cytokine network interactions between cancer cells and stromal cells have been found to mediate tumor-associated lymphangiogenesis and hemoangiogenesis. Thus, it is crucial to understand cancer biology as a tumor microenvironment, rather than focusing solely on cancer cells. To this end, we hypothesized that amount of intratumoral lymphatic endothelial cells (iLEC) reflect lymphangiogenesis and associate with poor clinical characteristics and outcomes. Method: We analyzed a total of 4145 breast cancer patient’s clinical and transcriptome data from The Cancer Genome Atlas (TCGA), and GSE96058 datasets. Intratumoral lymphatic endothelial cells (iLEC) and all the other immune cells were determined by xCell algorithm, and a median cutoff was adopted to divide high and low groups. Result: First we performed gene set enrichment assay using Hallmark and PID collections from MSigDB and found that high iLEC breast cancer enriched Lymphangiogenesis-related gene sets (lymph angiogenesis, S1P meta, S1P1, and S1P3 pathway) and hemoangiogenesis-related gene sets (Angiogenesis, VEGFR1 pathway, and VEGFR2 pathway) (all FDR<0.25). Microvascular endothelial cells were also predominant in high iLEC tumors (p < 0.001). Even though iLEC was significantly higher in patients with lymph node metastasis consistently in both TCGA and GSE96058 cohorts, there was no survival difference by the amount of iLEC in neither of the cohorts. High iLEC breast cancer also enriched cancer stem cell-related gene sets (notch signaling, hedgehog signaling, epithelial mesenchymal transition, and Wnt β catenin signaling) and metabolic pathways (bile acid metabolism, fatty acid metabolism), and S1P2 pathway (all FDR<0.25). Stromal cells such as Adipocytes, Preadipocytes and Fibroblasts, were also more prominent in the high iLEC group (all p < 0.001). The immune-related gene sets (allograft rejection, IL2 STAT5 pathway, IL6 Jak STAT3 signaling, inflammatory response, TNFα signaling via NFkB, and complement) and cytolytic activity, reflecting anti-tumor immunity, were elevated in high iLEC group of both cohorts (all FDR<0.25, p < 0.001), but anti-cancer immune cells were not significantly infiltrated. Interestingly, cell proliferation-related gene set (E2F target, G2M checkpoint, MYC target v1) and mTORc1 signaling gene set was strongly enriched in low iLEC breast cancer (all FDR<0.25). Furthermore, Ki67 expression and histological grade as well as aggressive cancer subtype were negatively correlated with iLEC fraction (all p<0.002, r <-0.29). Finally, we found that in the TCGA cohort, low iLEC breast cancer was significantly associated with intratumor heterogeneity, silent and nonsilent mutation rate, fraction altered, and homologous recombination deficiency, based on scoring by Thorrson et al (all p < 0.001). Despite the association with high TIL regional fraction and IFN-γ Response, low iLEC breast cancer was infiltrated by pro-cancer immune cells, such as Th2, Treg, and B-cells (all p < 0.001). Conclusion: High iLEC breast cancer reflected lymphangiogenesis and were associated with cancer stem cell-related genes and lymph node metastasis, while low iLEC breast cancers were associated with accelerated cell proliferation and pro-cancer immune cell infiltrations. Unfavorable cancer characteristics on both high and low iLEC groups may explain the lack of difference in patient survival. Citation Format: Rongrong Wu, Masanori Oshi, Mariko Asaoka, Akimitsu Yamada, Yamato Takabe, Li Yan, Itaru Endo, Takashi Ishikawa, Kazuaki Takabe. Intratumoral lymphatic endothelial cell infiltration reflects lymphangiogenesis and lymph node metastasis, but is counterbalanced by immune response and better cancer biology in breast cancer 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 P5-06-03.

CaMKII inhibitor KN-93 impaired angiogenesis and aggravated cardiac remodelling and heart failure via inhibiting NOX2/mtROS/p-VEGFR2 and STAT3 pathways.

Persistent cardiac Ca2+/calmodulin‐dependent Kinase II (CaMKII) activation was considered to promote heart failure (HF) development, some studies believed that CaMKII was a target for therapy of HF. However, CaMKII was an important mediator for the ischaemia‐induced coronary angiogenesis, and new evidence confirmed that angiogenesis inhibited cardiac remodelling and improved heart function, and some conditions which impaired angiogenesis aggravated ventricular remodelling. This study aimed to investigate the roles and the underlying mechanisms of CaMKII inhibitor in cardiac remodelling. First, we induced cardiac remodelling rat model by ISO, pre‐treated by CaMKII inhibitor KN‐93, evaluated heart function by echocardiography measurements, and performed HE staining, Masson staining, Tunel staining, Western blot and RT‐PCR to test cardiac remodelling and myocardial microvessel density; we also observed ultrastructure of cardiac tissue with transmission electron microscope. Second, we cultured HUVECs, pre‐treated by ISO and KN‐93, detected cell proliferation, migration, tubule formation and apoptosis, and carried out Western blot to determine the expression of NOX2, NOX4, VEGF, VEGFR2, p‐VEGFR2 and STAT3; mtROS level was also measured. In vivo, we found KN‐93 severely reduced myocardial microvessel density, caused apoptosis of vascular endothelial cells, enhanced cardiac hypertrophy, myocardial apoptosis, collagen deposition, aggravated the deterioration of myocardial ultrastructure and heart function. In vitro, KN‐93 inhibited HUVECs proliferation, migration and tubule formation, and promoted apoptosis of HUVECs. The expression of NOX2, NOX4, p‐VEGFR2 and STAT3 were down‐regulated by KN‐93; mtROS level was severely reduced by KN‐93. We concluded that KN‐93 impaired angiogenesis and aggravated cardiac remodelling and heart failure via inhibiting NOX2/mtROS/p‐VEGFR2 and STAT3 pathways.

Design, synthesis and evaluation of anticancer activity of new pyrazoline derivatives by down-regulation of VEGF: Molecular docking and apoptosis inducing activity

Two series of pyrazoline compounds were designed and synthesized as antiproliferative agents by VEGFR pathway inhibition. All synthesized compounds were screened by the National Cancer Institute (NCI), Bethesda, USA for anticancer activity against 60 human cancer cell lines. Compound 3f exhibited the highest anticancer activity on the ovarian cell line (OVCAR-4) with IC50 = 0.29 μM and on the breast cell line (MDA-MB-468) with IC50 = 0.35 μM. It also exhibited the highest selectivity index (SI = 74). Compound 3f caused cell cycle arrest in OVCAR-4 cell line at the S phase which consequently inhibited cell proliferation and induced apoptosis. Moreover, 3f showed potent down-regulation of VEGF and p-VEGFR-2. Docking studies showed that compound 3f interacts in a similar pattern to axitinib on the VEGFR-2 receptor. The same compound was also able to fit into the gorge of STAT3 binding site, the transcription factor for VEGF, which explains the VEGF down-regulation.

The immunomodulatory activity of lenvatinib prompts the survival of patients with advanced hepatocellular carcinoma.

BackgroundLenvatinib is a novel multiple receptor tyrosine kinase inhibitor used for hepatocellular carcinoma (HCC) treatment. Although its main function is to suppress VEGFR and FGFR pathway, its immunomodulatory activity in HCC is not elucidated. Thus, this study aimed to investigate the immunomodulatory capability of lenvatinib in HCC.Material and methodsTotally 47 patients with HCC were enrolled in this study, and the immune cells and serum cytokine profiles before initiation of treatment and after 1 and 3 months were measured. The immune checkpoint receptors on the immune cells were also evaluated. Kaplan–Meier survival estimate and log rank tests were used to assess the prognostic value.ResultThe frequency of T helper (Th) cells and T regulatory (Treg) cells reduced after lenvatinib treatment, while cytotoxic T lymphocyte (CTL) cells increased significantly. The cytokine profiles showed IL‐2, IL‐5, IFN‐γ increased; other cytokines including IL‐6, IL‐10, TNF‐ α and TNF‐ β decreased with lenvatinib therapy. Furthermore, the PD‐1 and TIM‐3 expressed on CTL had greatly decreased; the expression of TIM‐3 and CTLA‐4 was reduced on Treg cells as well. Besides, the new index CTL/Treg ratio was created, and low ratio was associated with the unfavorable outcome of HCC patients.ConclusionOur results confirmed that lenvatinib is capable of improving patients’ immune status, saving the effector cells from exhaustion status and inhibiting the number and function of immunosuppressive cells. The novel index CTL/Treg ratio qualifies as a predictor for the outcome of patients with lenvatinib therapy.

Mesenchymal stromal cells enhance self-assembly of a HUVEC tubular network through uPA-uPAR/VEGFR2/integrin/NOTCH crosstalk

Endothelial cells (ECs) degrade the extracellular matrix of vessel walls and contact surrounding cells to facilitate migration during angiogenesis, leading to formation of an EC-tubular network (ETN). Mesenchymal stromal cells (MSC) support ETN formation when co-cultured with ECs, but the mechanism is incompletely understood. We examined the role of the urokinase-type plasminogen activator (uPA) system, i.e. the serine protease uPA, its inhibitor PAI-1, receptor uPAR/CD87, clearance by the low-density lipoprotein receptor-related protein (LRP1) and their molecular partners, in the formation of ETNs supported by adipose tissue-derived MSC. Co-culture of human umbilical vein ECs (HUVEC) with MSC increased mRNA expression levels of uPAR, MMP14, VEGFR2, TGFβ1, integrin β3 and Notch pathway components (Notch1 receptor and ligands: Dll1, Dll4, Jag1) in HUVECs and uPA, uPAR, TGFβ1, integrin β3, Jag1, Notch3 receptor in MSC. Inhibition at several steps in the activation process indicates that uPA, uPAR and LRP1 cross-talk with αv-integrins, VEGFR2 and Notch receptors/ligands to mediate ETN formation in HUVEC-MSC co-culture. The urokinase system mediates ETN formation through the coordinated action of uPAR, uPA's catalytic activity, its binding to uPAR and its nuclear translocation. These studies identify potential targets to help control aberrant angiogenesis with minimal impact on healthy vasculature.

Immunomodulatory Effects of Lenvatinib Plus Anti-Programmed Cell Death Protein 1 in Mice and Rationale for Patient Enrichment in Hepatocellular Carcinoma.

Lenvatinib is an effective drug in advanced HCC. Its combination with the anti-PD1 (programmed cell death protein 1) immune checkpoint inhibitor, pembrolizumab, has generated encouraging results in phase Ib and is currently being tested in phase III trials. Here, we aimed to explore the molecular and immunomodulatory effects of lenvatinib alone or in combination with anti-PD1. We generated three syngeneic models of HCC in C57BL/6J mice (subcutaneous and orthotopic) and randomized animals to receive placebo, lenvatinib, anti-PD1, or combination treatment. Flow cytometry, transcriptomic, and immunohistochemistry analyses were performed in tumor and blood samples. A gene signature, capturing molecular features associated with the combination therapy, was used to identify a subset of candidates in a cohort of 228 HCC patients who might respond beyond what is expected for monotherapies. In mice, the combination treatment resulted in tumor regression and shorter time to response compared to monotherapies (P<0.001). Single-agent anti-PD1 induced dendritic and T-cell infiltrates, and lenvatinib reduced the regulatory T cell (Treg) proportion. However, only the combination treatment significantly inhibited immune suppressive signaling, which was associated with the TGFß pathway and induced an immune-active microenvironment (P<0.05 vs. other therapies). Based on immune-related genomic profiles in human HCC, 22% of patients were identified as potential responders beyond single-agent therapies, with tumors characterized by Treg cell infiltrates, low inflammatory signaling, and VEGFR pathway activation. Lenvatinib plus anti-PD1 exerted unique immunomodulatory effects through activation of immune pathways, reduction of Treg cell infiltrate, and inhibition of TGFß signaling. A gene signature enabled the identification of ~20% of human HCCs that, although nonresponding to single agents, could benefit from the proposed combination.

Inhibition of CD146 attenuates retinal neovascularization via vascular endothelial growth factor receptor 2 signalling pathway in proliferative diabetic retinopathy.

To investigate the expression of CD146 and its role in proliferative diabetic retinopathy (PDR). Enzyme linked immunosorbent assay was performed to analyse the expression and relationship of sCD146, vascular endothelial growth factor (VEGF), sVEGFR1 and sVEGFR2 in vitreous specimens from PDR and idiopathic epiretinal membranes (IERM) or idiopathic macular hole patients. The location of CD146 in ERMs was detected by immunofluorescence. The oxygen-induced retinopathy (OIR) mice model was established and the adeno-associated virus expressing a shRNA of CD146 (AAV1-shCD146-GFP) was administered via intravitreal injection. The effect of AAV1-shCD146-GFP was explored by immunofluorescence, Western blot and quantitative real-time PCR. The levels of sCD146 in vitreous specimens from PDR patients and CD146 in retinas from OIR mice were significantly increased. Immunofluorescence showed that CD146 was co-located with CD31, VEGF, VEGFR1 and VEGFR2, respectively. Intravitreal injection of AAV1-shCD146-GFP could dramatically reduce the formation of neovascularization and non-perfusion area by inhibiting VEGFR2 phosphorylation. Our results indicated that CD146 was involved in the development of retinal neovascularization via VEGFR2 pathway. Anti-CD146 may be an innovative or adjuvant therapy, which provides a new direction for the treatment of PDR and other ocular neovascular diseases.

1245P Efficacy and safety of apatinib plus EGFR-TKI in advanced non-small cell lung cancer with EGFR-TKI resistance

Dual blockade of both EGFR and VEGFR pathways in EGFR-mutant NSCLC has shown enhanced antitumor efficacy versus EGFR-TKIs alone. Apatinib is an orally effective VEGFR-2 tyrosine kinase inhibitor (TKI). Previous studies have shown that apatinib (a TKI against VEGFR-2) combined with EGFR-TKI might prevent progression of the disease. This study aims to evaluate the efficacy and safety of apatinib plus EGFR-TKIs compared with chemotherapy for EGFR-TKI resistant NSCLC patients.

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

Simple SummaryAXL is a receptor tyrosine kinase belonging to the TAM (Tyro3, Axl and Mer) family. The AXL protein plays an important role in promoting cancer development, such as proliferation, migration, invasion and survival of cancer cells. In this study, we used mass spectrometry-based proteomics to quantify the cancer signaling regulated by AXL activation. Our study identified more than 1000 phosphotyrosine sites and discovered that activation of AXL can upregulate multiple cancer-promoting and cell migration/invasion-related signaling pathways. We also observed significant crosstalk as evidenced by rapid phosphorylation of multiple receptor tyrosine kinases and protein tyrosine phosphatases, including PTPN11 and PTPRA, upon GAS6 stimulation. These discoveries should serve as a potentially useful resource for studying AXL functions as well as for the development of effective therapeutic options to target AXL.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.

Nephrotoxicity of Anti-Angiogenic Therapies.

The use of inhibitors of vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor 2 (VEGFR2) signaling for the treatment of cancer has increased over the last decade. This signaling pathway plays a fundamental role in angiogenesis and also in kidney physiology. The emergence of anti-angiogenic therapies has led to adverse nephrotoxic effects, despite improving the outcomes of patients. In this review, we will present the different anti-angiogenic therapies targeting the VEGFR pathway in association with the incidence of renal manifestations during their use. In addition, we will discuss, in detail, the pathophysiological mechanisms of frequent renal diseases such as hypertension, proteinuria, renal dysfunction, and electrolyte disorders. Finally, we will outline the cellular damage described following these therapies.

Clinical and genomic characteristics of metabolic syndrome in colorectal cancer.

Metabolic syndrome (MetS) is characterized by a group of metabolic disturbances which leads to the enhanced risk of cancer development. Elucidating the mechanisms between these two pathologies is essential to identify the potential therapeutic molecular targets for colorectal cancer (CRC). 716 colorectal patients from the First and Second Affiliated Hospital of Wenzhou Medical University were involved in our study and metabolic disorders were proven to increase the risk of CRC. The prognostic value of the MetS factors was analyzed using the Cox regression model and a clinical MetS-based nomogram was established. Then by using multi-omics techniques, the distinct molecular mechanism of MetS genes in CRC was firstly systematically characterized. Strikingly, MetS genes were found to be highly correlated with the effectiveness of targeted chemotherapy administration, especially for mTOR and VEGFR pathways. Our results further demonstrated that overexpression of MetS core gene IL6 would promote the malignancy of CRC, which was highly dependent on mTOR-S6K signaling. In conclusion, we comprehensively explored the clinical value and molecular mechanism of MetS in the progression of CRC, which may serve as a candidate option for cancer management and therapy in the future.

Beneficial Actions of Orostachys japonica and Its Compounds against Tumors via MAPK Signaling Pathways.

Tumors are one of the most life-threatening diseases, and a variety of cancer treatment options have been continuously introduced in order to overcome cancer and improve conventional therapy. Orostachys japonica (O. japonica), which is a perennial plant belonging to the genus Orostachys of the Crassulaceae family, has been revealed to exhibit pharmacological properties against various tumors in numerous studies. The present review aimed to discuss the biological actions and underlying molecular mechanisms of O. japonica and its representative compounds—kaempferol and quercetin—against tumors. O. japonica reportedly has antiproliferative, anti-angiogenic, and antimetastatic activities against various types of malignant tumors through the induction of apoptosis and cell cycle arrest, a blockade of downstream vascular endothelial growth factor (VEGF)-VEGFR2 pathways, and the regulation of epithelial-to-mesenchymal transition. In addition, emerging studies have highlighted the antitumor efficacy of kaempferol and quercetin. Interestingly, it was found that alterations of the mitogen-activated protein kinase (MAPK) signaling cascades are involved in the pivotal mechanisms of the antitumor effects of O. japonica and its two compounds against cancer cell overgrowth, angiogenesis, and metastasis. In summary, O. japonica could be considered a preventive and therapeutic medicinal plant which exhibits antitumor actions by reversing altered patterns of MAPK cascades, and kaempferol and quercetin might be potential components that can contribute to the efficacy and underlying mechanism of O. japonica.

Protection against light-induced retinal degeneration via dual anti-inflammatory and anti-angiogenic functions of thrombospondin-1.

Retinal photodamage is a high-risk factor for age-related macular degeneration (AMD), the leading cause of irreversible blindness worldwide. However, both the pathogenesis and effective therapies for retinal photodamage are still unclear and debated. The anti-inflammatory effects of thrombospondin-1 on blue light-induced inflammation in ARPE-19 cells and in retinal inflammation were evaluated. Furthermore, the anti-angiogenic effects of thrombospondin-1 on human microvascular endothelial cells (hMEC-1 cells) and a laser-induced choroidal neovascularisation (CNV) mouse model were evaluated. in vitro experiments, including western blotting, immunocytochemistry, migration assays and tube formation assays, as well as in vivo experiments, including immunofluorescence, visual electrophysiology, spectral-domain optical coherence tomography, and fluorescein angiography, were employed to evaluate the anti-inflammatory and anti-angiogenic effects of thrombospondin-1. Specific effects of blue light-induced retinal inflammation and pathological angiogenesis were reflected by up-regulation of pro-inflammatory factors and activation of angiogenic responses, predominantly regulated by the NF-κB and VEGFR2 pathways respectively. During the blue light-induced pathological progress, THBS-1 derived from retinal pigment epithelium down-regulated proteomics and biological assays. Thrombospondin-1 treatment also suppressed inflammatory infiltration and neovascular leakage. The protective effect of Thrombospondin-1 was additionally demonstrated by a substantial rescue of visual function. Mechanistically, thrombospondin-1 reversed blue light-induced retinal inflammation and angiogenesis by blocking the activated NF-κB and VEGFR2 pathways, respectively. Thrombospondin-1, with dual anti-inflammatory and anti-neovascularisation properties, is a promising agent for protection against blue light-induced retinal damage and retinal degenerative disorders which are pathologically associated with inflammatory and angiogenic progress. This article is part of a themed issue on Inflammation, Repair and Ageing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.9/issuetoc.

Virtual Prediction of Lycopene and Quercetin Effects on Angiogenesis Through VEGFR-2 Pathway

Angiogenesis is a complex process that is required for cancer cells to perform metastasis. The binding of a growth factor such as VEGF to its receptor is one of the factors to trigger angiogenesis through VEGFR-2 pathway. This study is conducted to analyze the effect of lycopene and quercetin, which are compounds found in watermelon (Citrullus lanatus) on angiogenesis through VEGFR-2 pathway. The study was carried out in silico. Ligands were obtained from PubChem and prepared using PyRx while the protein was obtained from PDB and prepared using BIOVIA Discovery Studio 2019. The docking was done by using HEX 8.0.0 and the results were visualized using BIOVIA Discovery Studio 2019. Lycopene and quercetin were able to bind with VEGFR-2 to interrupt the binding of VEGFA. The presence of lycopene and quercetin also lowers the binding strength of VEGFA with VEGFR-2 as they can affect interactions between VEGFA and VEGFR-2 at 4 and 5 amino acid residues by changing the type of interactions to make the binding strength weaker. The binding of lycopene and quercetin have the potential to interrupt the downstream pathway of angiogenesis through VEGFR-2 pathway.

Vascular NRP2 triggers PNET angiogenesis by activating the SSH1-cofilin axis

BackgroundAngiogenesis is a critical step in the growth of pancreatic neuroendocrine tumors (PNETs) and may be a selective target for PNET therapy. However, PNETs are robustly resistant to current anti-angiogenic therapies that primarily target the VEGFR pathway. Thus, the mechanism of PNET angiogenesis urgently needs to be clarified.MethodsDataset analysis was used to identify angiogenesis-related genes in PNETs. Immunohistochemistry was performed to determine the relationship among Neuropilin 2 (NRP2), VEGFR2 and CD31. Cell proliferation, wound-healing and tube formation assays were performed to clarify the function of NRP2 in angiogenesis. The mechanism involved in NRP2-induced angiogenesis was detected by constructing plasmids with mutant variants and performing Western blot, and immunofluorescence assays. A mouse model was used to evaluate the effect of the NRP2 antibody in vivo, and clinical data were collected from patient records to verify the association between NRP2 and patient prognosis.ResultsNRP2, a VEGFR2 co-receptor, was positively correlated with vascularity but not with VEGFR2 in PNET tissues. NRP2 promoted the migration of human umbilical vein endothelial cells (HUVECs) cultured in the presence of conditioned medium PNET cells via a VEGF/VEGFR2-independent pathway. Moreover, NRP2 induced F-actin polymerization by activating the actin-binding protein cofilin. Cofilin phosphatase slingshot-1 (SSH1) was highly expressed in NRP2-activating cofilin, and silencing SSH1 ameliorated NRP2-activated HUVEC migration and F-actin polymerization. Furthermore, blocking NRP2 in vivo suppressed PNET angiogenesis and tumor growth. Finally, elevated NRP2 expression was associated with poor prognosis in PNET patients.ConclusionVascular NRP2 promotes PNET angiogenesis by activating the SSH1/cofilin/actin axis. Our findings demonstrate that NRP2 is an important regulator of angiogenesis and a potential therapeutic target of anti-angiogenesis therapy for PNET.

Inhibitory effects of Paris saponin I, II, Ⅵ and Ⅶ on HUVEC cells through regulation of VEGFR2, PI3K/AKT/mTOR, Src/eNOS, PLCγ/ERK/MERK, and JAK2-STAT3 pathways

Rhizoma Paris is a popular Chinese medicine in clinics. It contains four main saponins which are its major bioactive compounds. These saponins are Paris saponin I, II, VI and VII (PSI, PSII, PSVI and PSVII, respectively). Up to now, the research using HUVEC cells to evaluate the anti-angiogenic activity of four saponins is blank. The purpose of this study was to evaluate the anti-angiogenic properties (also known as angiotoxicity) of the four saponins in Rhizoma Paris on vascular endothelial cells-HUVEC cells, and to investigate the underlying mechanism, which has not been studied before. In this study, MTT assay, Lactate dehydrogenase (LDH) assay, wound healing experiments, transwell cell invasion assay, tubule formation experiment, DAPI staining, AV-PI double staining, and cell cycle analysis were used to determine the effects of Paris saponins. The results showed that, with increases in concentrations of PSI, PSII, PSVI and PSVII, the viability of HUVEC cells decreased significantly. In addition, four saponins dose-dependent enhanced LDH release and inhibited HUVEC cell migration, invasion, and angiogenesis. In terms of mechanism, PSI significantly inhibited protein expression in multiple signaling pathways. In particular, with the VEGF2 as the target, it activate the downstream PI3K / AKT / mTOR, SRC / eNOS, P38, PLCγ / ERK / MERK and JAK2/STAT3 signaling pathways. In conclusion, PSI, PSII, PSVI and PSVII can inhibit endothelial cell proliferation, migration and invasion, block endothelial cell cycle, induce endothelial cell apoptosis, act on protein expression in several anti-angiogenic signaling pathways, and finally inhibit angiogenesis in vitro. This study provides further data support for the clinical application of Paris saponins as antiangiogenic drugs.