High Expression Of Vascular Endothelial Growth Factor A Research Articles

Abstract 2569: VEGFA gene expression is associated with cell proliferation, less immune cell infiltration, and worse survival, but better response to chemotherapy and immunotherapy

Abstract Introduction: Vascular endothelial growth factor-A (VEGFA) is a well-known factor that induces angiogenesis, one of the hallmarks of cancer, which generates new blood vessels in the tumor microenvironment (TME) and provides conduit for cancer to metastasize. Interestingly, an anti-VEGFA drug (Avastin) failed to show benefit in clinical trials for breast cancer (BC). In this study we investigated the clinical relevance of VEGFA gene expression in BC. Methods: A total of 7336 BC patients from eight independent cohorts, TCGA (n= 1073), METABRIC (n= 1903), SCAN-B (GSE96058, n= 3069), GSE20194 (n=278), GSE34138 (n=178), GSE163882 (n=222), GSE25066 (n=508), and ISPY2 (GSE173839, n= 105) were analyzed. These cohorts were divided into high and low VEGFA gene expression groups using the median. Results: High VEGFA expression in BC was associated with worse overall survival in SCAN-B cohort which was validated in METABRIC cohort with overall, disease-specific, and disease-free survival (all p<0.02). VEGFA expression was higher in Triple Negative BC (TNBC) but was lower in BC with lymph node metastasis. High VEGFA BC was associated with significantly high intratumoral heterogeneity, homologous recombination defect, silent and non-silent mutations, and SNV neoantigens in TCGA, and was also associated with higher cell proliferation, evidenced by higher Nottingham histological grade, higher Ki67 gene expression, and enrichment of all the Hallmark cell proliferation-related gene sets: Mitotic spindle, G2M Checkpoint, E2F Targets, MYC Targets v1 and v2, and mTorC1 signaling, consistently, in SCAN-B, METABRIC, and TCGA cohorts. Reflecting its enhanced growth, high VEGFA expression was associated with less infiltration of fibroblasts and adipocytes (all p<0.05). Surprisingly, VEGFA expression did not enrich angiogenesis gene set in any of the cohorts, nor was related with infiltrations of blood or lymphatic vascular endothelial cells, besides pericytes. But high VEGFA BC was associated with significantly less infiltrations of anti-cancer immune cells; CD8, CD4, and dendritic cells were all significantly less, and T helper type 1 cells and regulatory T cells were significantly higher consistently in SCAN-B, METABRIC, and TCGA cohorts. VEGFA expression was significantly higher in BC that achieved pathological complete response after Anthracycline and Taxane based neoadjuvant therapy in both ER+/HER2- and TNBC subtypes in the GSE25066 cohort, and after immunotherapy in ER+/HER2- subtype, but not TNBC, in the ISPY2 clinical trial cohort. This was not validated in GSE20194, GSE34138, GSE163882 cohorts. Conclusion: Our research indicates that high VEGFA BC is associated with high cell proliferation, less immune cell infiltration, and worse survival, but better response to Anthracycline and Taxane based chemotherapy, and immunotherapy. Citation Format: Pia Sharma, Kohei Chida, Kenichi Hakamada, Kazuaki Takabe. VEGFA gene expression is associated with cell proliferation, less immune cell infiltration, and worse survival, but better response to chemotherapy and immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2569.

MicroRNA-299-3p inhibits cell proliferation, motility, invasion and angiogenesis via VEGFA in upper tract urothelial carcinoma.

Upper tract urothelial carcinoma (UTUC) is a rare tumor with extraordinarily different features between Eastern and Western countries. Vascular endothelial growth factor-A (VEGFA) was originally identified as a secreted signaling protein and regulator of vascular development and cancer progression. In this study, we aimed to elucidate the molecular mechanisms underlying the regulation of VEGFA by microRNA in UTUC. VEGFA expression was evaluated by immunohistochemistry in 140 human UTUC tissue samples. Next, we assessed the regulatory relationship between VEGFA and miR-299-3p by real-time PCR, western blotting, ELISA and dual-luciferase reporter assays using two UTUC cell lines. The role of miR-299-3p/VEGFA in cell proliferation, motility, invasion, and tube formation was analyzed in vitro. High VEGFA expression was significantly associated with tumor stage, grade, distant metastasis and cancer-related death and correlated with poor progression-free and cancer-specific survival. VEGFA knockdown repressed proliferation, migration, invasion and angiogenesis in UTUC cell lines. miR-299-3p significantly reduced VEGFA protein expression and miR-299-3p overexpression inhibited VEGFA mRNA and protein expression by directly targeting its 3'-UTR. Functional studies indicated that VEGFA overexpression reversed the miR-299-3p-mediated suppression of tumor cell proliferation, migration, invasion and angiogenesis. In addition, miR-299-3p/VEGFA suppressed cellular functions in UTUC by modulating the expression of P18 and cyclin E2. Our findings suggest that miR-299-3p possibly suppresses UTUC cell proliferation, motility, invasion and angiogenesis via VEGFA. VEGFA may act as a prognostic predictor, and both VEGFA and miR-299-3p could be potential therapeutic targets for UTUC.

Clinico-pathological characteristics of IGFR1 and VEGF-A co-expression in early and locally advanced-stage lung adenocarcinoma.

Although targeted therapies and immunotherapy have achieved significant clinical benefits in patients with certain pathological types of lung cancer. However, prognosis for patients with lung adenocarcinoma still remains unsatisfactory. It is of extremely importance to find ideal prognostic indicators to predict the prognosis of lung adenocarcinoma patients, especially for patients with early and locally advanced-stage lung adenocarcinoma. The purpose of this study is to elucidate the significance of Insulin-like growth factor receptor 1 (IGFR1) and Vascular endothelial growth factor A (VEGF-A) expression in predicting progression-free survival (PFS) and overall survival (OS) in patients with early and locally advanced-stage lung adenocarcinoma. In this study, IGFR1 and VEGF-A expression on 119 specimens of patients early and locally advanced-stage lung adenocarcinoma were analyzed by immunohistochemistry with an H-score system. Both high IGFR1 expression and VEGF-A expression patients were resulted in 59 (49.6%) separately. The numbers and proportions of IGFR1-&VEGF-A- subgroup, IGFR1-&VEGF-A+ subgroup, IGFR1+&VEGF-A- subgroup and IGFR1+&VEGF-A+ subgroup are 23 (19.3%), 37 (31.1%), 37 (31.1%) and 22 (18.5%) respectively. High IGFR1 expression was significantly associated with both poor PFS and OS of all patients in a univariate analysis. Multivariable analysis showed that patients with IGFR1+&VEGF-A+ expression exhibited a worst PFS and OS in the subgroup of lung adenocarcinoma patients with EGFR mutation. These results suggest that IGFR1+&VEGF-A+ is expected to be a disadvantageous factor for prognosis in the subgroup of EGFR mutation in patients with early and locally advanced-stage lung adenocarcinoma. What's more, this study may provide the theoretical possibility to screen optimal population for a combination therapy with anti-VEGF and anti-IGFR1 in patients with early and locally advanced-stage lung adenocarcinoma.

RETRACTED ARTICLE: KRAS activation in gastric cancer stem-like cells promotes tumor angiogenesis and metastasis

Our previous work showed that KRAS activation in gastric cancer cells leads to activation of an epithelial-to-mesenchymal transition (EMT) program and generation of cancer stem-like cells (CSCs). Here we analyze how this KRAS activation in gastric CSCs promotes tumor angiogenesis and metastasis. Gastric cancer CSCs were found to secrete pro-angiogenic factors such as vascular endothelial growth factor A (VEGF-A), and inhibition of KRAS markedly reduced secretion of these factors. In a genetically engineered mouse model, gastric tumorigenesis was markedly attenuated when both KRAS and VEGF-A signaling were blocked. In orthotropic implant and experimental metastasis models, silencing of KRAS and VEGF-A using shRNA in gastric CSCs abrogated primary tumor formation, lymph node metastasis, and lung metastasis far greater than individual silencing of KRAS or VEGF-A. Analysis of gastric cancer patient samples using RNA sequencing revealed a clear association between high expression of the gastric CSC marker CD44 and expression of both KRAS and VEGF-A, and high CD44 and VEGF-A expression predicted worse overall survival. In conclusion, KRAS activation in gastric CSCs enhances secretion of pro-angiogenic factors and promotes tumor progression and metastasis.

Targeted VEGFA therapy in regulating early acute kidney injury and late fibrosis.

Damage to peritubular capillaries is a key process that contributes to acute kidney injury (AKI) progression. Vascular endothelial growth factor A (VEGFA) plays a critical role in maintaining the renal microvasculature. However, the physiological role of VEGFA in various AKI durations remains unclear. A severe unilateral ischemia‒reperfusion injury model was established to provide an overview of VEGFA expression and the peritubular microvascular density from acute to chronic injury in mouse kidneys. Therapeutic strategies involving early VEGFA supplementation protecting against acute injury and late anti-VEGFA treatment for fibrosis alleviation were analyzed. A proteomic analysis was conducted to determine the potential mechanism of renal fibrosis alleviation by anti-VEGFA. The results showed that two peaks of extraglomerular VEGFA expression were observed during AKI progression: one occurred at the early phase of AKI, and the other occurred during the transition to chronic kidney disease (CKD). Capillary rarefaction progressed despite the high expression of VEGFA at the CKD stage, and VEGFA was associated with interstitial fibrosis. Early VEGFA supplementation protected against renal injury by preserving microvessel structures and counteracting secondary tubular hypoxic insults, whereas late anti-VEGFA treatment attenuated renal fibrosis progression. The proteomic analysis highlighted an array of biological processes related to fibrosis alleviation by anti-VEGFA, which included regulation of supramolecular fiber organization, cell-matrix adhesion, fibroblast migration, and vasculogenesis. These findings establish the landscape of VEGFA expression and its dual roles during AKI progression, which provides the possibility for the orderly regulation of VEGFA to alleviate early acute injury and late fibrosis.

Abstract 2451: KRAS activation in gastric cancer stem-like cells promotes tumor angiogenesis and metastasis

Abstract Our previous work showed that KRAS activation in gastric cancer cells leads to activation of an epithelial-to-mesenchymal transition (EMT) program and generation of cancer stem-like cells (CSCs). Here we analyze how this KRAS activation in gastric CSCs promotes tumor angiogenesis and metastasis. Gastric cancer CSCs were found to secrete pro-angiogenic factors such as vascular endothelial growth factor A (VEGF-A), and inhibition of KRAS markedly reduced secretion of these factors. In a genetically engineered mouse model, gastric tumorigenesis was markedly attenuated when both KRAS and VEGF-A signaling were blocked. In orthotropic implant and experimental metastasis models, silencing of KRAS and VEGF-A using shRNA in gastric CSCs abrogated primary tumor formation, lymph node metastasis, and lung metastasis far greater than individual silencing of KRAS or VEGF-A. Analysis of gastric cancer patient samples using RNA sequencing revealed a clear association between high expression of the gastric CSC marker CD44 and expression of both KRAS and VEGF-A, and high CD44 and VEGF-A expression predicted worse overall survival. In conclusion, KRAS activation in gastric CSCs enhances secretion of pro-angiogenic factors and promotes tumor progression and metastasis. Citation Format: Changhwan Yoon, Jun Lu, Yukyung Jun, Bang- Jin Kim, Jacob Till, Jong Hyun Kim, Sara Keshavjee, Sandra Ryeom, Sam Yoon. KRAS activation in gastric cancer stem-like cells promotes tumor angiogenesis and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2451.

VEGFA promotes the occurrence of PLA2R-associated idiopathic membranous nephropathy by angiogenesis via the PI3K/AKT signalling pathway

BackgroundThe M-type phospholipase A2 receptor (PLA2R)-associated idiopathic membranous nephropathy (IMN) is a common immune-related disease in adults. Vascular endothelial growth factor A (VEGFA) is the key mediator of angiogenesis, which leads to numerous kidney diseases. However, the role of VEGFA in IMN is poorly understood.MethodsIn the present study, we downloaded the microarray data GSE115857 from Gene Expression Omnibus (GEO). The differentially expressed genes (DEGs) were identified with R software. The cytoHubba plug-in were used to identify hub genes from the protein–protein interaction network. Gene set enrichment analysis (GSEA) was used to identify signalling pathway in IMN. CCK8 was performed to assess the cell viability in human vascular endothelial cells (HVECs). Then, passive Heymann nephritis (PHN) was induced in rats by a single tail vein injection of anti-Fx1A antiserum. Animals treated with VEGFA inhibitor bevacizumab (BV), with saline as a positive control. Proteinuria was evaluated by biochemical measurements. Immunohistochemistry and immunofluorescence was used to evaluate relative proteins expression. Electron microscopy was performed to observe the thickness of the glomerular basement membrane (GBM).ResultsWe revealed 3 hub genes, including one up-regulated gene VEGFA and two down-regulated genes JUN and FOS, which are closely related to the development of PLA2R-associated IMN. Pathway enrichment analysis found that the biological process induced by VEGFA is associated with PI3K/Akt signalling. GSEA showed that the signalling pathway of DEGs in GSE115857 was focused on angiogenesis, in which VEGFA acts as a core gene. We confirmed the high expression of VEGFA, PI3K, and AKT in IMN renal biopsy samples with immunohistochemistry. In HVECs, we found that BV suppresses cell viability in a time and dose dependent manner. In vivo, we found low dose of BV attenuates proteinuria via inhibiting VEGFA/PI3K/AKT signalling. Meanwhile, low dose of BV alleviates the thickening of the GBM.ConclusionVEGFA/PI3K/AKT signalling may play significant roles in the pathogenesis of IMN, which may provide new targets for the treatment of IMN.

Vascular endothelial growth factor-A is an Immunohistochemical biomarker for the efficacy of bevacizumab-containing chemotherapy for duodenal and jejunal adenocarcinoma

BackgroundThe efficacy and safety of bevacizumab-containing chemotherapy for patients with metastatic duodenal and jejunal adenocarcinoma (mDJA) are unclear. The present study aimed to evaluate the efficacy of bevacizumab and to explore immunohistochemical markers that can predict the efficacy of bevacizumab for patients with mDJA.MethodsThis multicentre study included patients with histologically confirmed small bowel adenocarcinoma who received palliative chemotherapy from 2008 to 2017 at 15 hospitals. Immunostaining was performed for vascular endothelial growth factor-A (VEGF-A), TP53, Ki67, β-catenin, CD10, MUC2, MUC5AC, MUC6, and mismatch repair proteins.ResultsA total of 74 patients were enrolled, including 65 patients with mDJA and 9 with metastatic ileal adenocarcinoma. Patients with mDJA who received platinum-based chemotherapy with bevacizumab as first-line treatment tended to have a longer progression-free survival and overall survival than those treated without bevacizumab (P = 0.075 and 0.077, respectively). Multivariate analysis extracted high VEGF-A expression as a factor prolonging progression-free survival (hazard ratio: 0.52, 95% confidence interval: 0.30–0.91). In mDJA patients with high VEGF-A expression, those who received platinum-based chemotherapy with bevacizumab as a first-line treatment had significantly longer progression-free survival and tended to have longer overall survival than those treated without bevacizumab (P = 0.025 and P = 0.056, respectively), whereas no differences were observed in mDJA patients with low VEGF-A expression.ConclusionImmunohistochemical expression of VEGF-A is a potentially useful biomarker for predicting the efficacy of bevacizumab-containing chemotherapy for patients with mDJA.

Receptor-Interacting Serine/Threonine-Protein Kinase-2 as a Potential Prognostic Factor in Colorectal Cancer.

Background and objectives: Receptor-interacting serine/threonine-protein kinase-2 (RIPK2) is an important mediator in different pathways in the immune and inflammatory response system. RIPK2 was also shown to play different roles in different cancer types; however, in colorectal cancer (CRC), its role is not well established. This study aims at identifying the role of RIPK2 in CRC progression and survival. Materials and methods: Data of patients and mRNA protein expression level of genes associated with CRC (RIPK2, tumor necrosis factor (TNF), TRAF1, TRAF7, KLF6, interlukin-6 (Il6), interlukin-8 (Il8), vascular-endothelial growth factor A (VEGFA), MKI67, TP53, nuclear factor-kappa B (NFKB), NFKB2, BCL2, XIAP, and RELA) were downloaded from the PrognoScan online public database. Patients were divided between low and high RIPK2 expression and different CRC characteristics were studied between the two groups. Survival curves were evaluated using a Kaplan–Meier estimator. The Pearson correlation was used to study the correlation between RIPK2 and the other factors. Statistical analysis was carried out using SPSS version 25.0. The Human Protein Atlas was also used for the relationship between RIPK2 expression in CRC tissues and survival. Differences were considered statistically significant at p < 0.05. Results: A total of 520 patients were downloaded from the PrognoScan database, and RIPK2 was found to correlate with MKI67, TRAF1, KLF6, TNF, Il6, Il8, VEGFA, NFKB2, BCL2, and RELA. High expression of RIPK2 was associated with high expression of VEGFA (p < 0.01) and increased mortality (p < 0.01). Conclusions: In this study, RIPK2 is shown to be a potential prognostic factor in CRC; however, more studies are needed to assess and verify its potential role as a prognostic marker and in targeted therapy.

High Expression of Solute Carrier Family 2 Member 1 (SLC2A1) in Cancer Cells Is an Independent Unfavorable Prognostic Factor in Pediatric Malignant Peripheral Nerve Sheath Tumor.

Malignant peripheral nerve sheath tumor (MPNST) in children is a rare mesenchymal malignancy developing predominantly in the setting of neurofibromatosis type 1. The prognosis in advanced MPNST is poor therefore new prognostic markers are highly needed for optimal therapeutic decisions. In many solid tumors, the bidirectional interactions between hypoxia and inflammation in the tumor microenvironment via functions of tumor-associated cells, like neutrophils, lymphocytes and macrophages, have been investigated recently. There is no data whether in MPNST hypoxic microenvironment may translate into systemic inflammation, which is a well-established factor for worse prognosis in cancer patients. Therefore, we investigated the prognostic significance of markers of tumor hypoxia and systemic inflammation in 26 pediatric malignant peripheral nerve sheath tumors (MPNST). Tumor tissue microarrays were stained for hypoxia-inducible factor-1α (HIF1A), solute carrier family 2 member 1 (SLC2A1, also known as glucose transporter 1 (GLUT1)), carbonic anhydrase 9 (CA9), and vascular endothelial growth factor A (VEGFA) and classified into low- or high-expression groups. Baseline complete blood counts and C-reactive protein (CRP) levels were collected for all cases. Neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and lymphocyte-to-monocyte ratio (LMR) were calculated from age-adjusted complete blood count parameters. Both 10-year RFS and OS were significantly lower in patients with high NLR values (17% vs. 75%, p = 0.009, q = 0.018; and 31% vs. 100%, p = 0.0077, q = 0.014; respectively). Ten-year-OS was significantly lower in patients with high expression of SLC2A1 (20.00% vs. 94%, p < 0.001, log-rank), high expression of HIF1A (23% vs. 79%, p = 0.016, log-rank), and CRP higher than 31 mg/L (11% vs. 82%, p = 0.003, q = 0.009). Cox’s proportional hazard regression analysis revealed that high expression of SLC2A1 (HR = 3.31, 95% CI = 1.08–10.09, p = 0.036) and VEGFA (HR = 4.40, 95% CI = 0.95–20.34, p = 0.058) were the independent factors predicting relapse, whereas high SLC2A1 was identified as the independent risk factor for death (HR = 12.20, 95% CI = 2.55–58.33, p = 0.002). Patients with high expression of hypoxic markers and low or high NLR/CRP values had the highest events rate, patients with low hypoxic markers and high NLR/CRP had intermediate events rate, while patients with low hypoxic markers and low NLR/CRP had the lowest events rate. SLC2A1 and VEGFA are promising novel prognostic factors in pediatric MPNST. Correlations between hypoxic and systemic inflammatory markers suggest the interplay between local tumor hypoxia and systemic inflammation.

Correlation of APE1 with VEGFA and CD163+ macrophage infiltration in bladder cancer and their prognostic significance.

The present study sought to estimate the applicability of apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1), vascular endothelial growth factor A (VEGFA) expression and CD163+ tumor-associated macrophage (TAM) ratio as prognostic factors in bladder cancer (BCa). A total of 127 patients with bladder urothelial cancer who underwent radical cystectomy at Daping Hospital were recruited between January 2013 and January 2017, including 45 cases of non-muscle invasive BCa (NMIBC) and 82 of MIBC. Immunohistochemical detection of APE1, VEGFA and CD163, as well as multiple immunofluorescence staining for APE1, VEGFA, CD163 and CD34, were performed on tissue samples. For APE1 and VEGFA, the staining was graded based on intensity (0–3), while CD163 was graded (0–3) based on the percentage of positively stained cells. The prognostic value of APE1, VEGF and CD163 was assessed using Kaplan-Meier and Cox regression analysis. The results suggested that in BCa, high APE1 expression was associated with high VEGFA expression and more infiltration of CD163+ TAM. Furthermore, high expression of APE1 was associated with lymphovascular invasion of BCa, as well as reduced survival time. This indicates that APE1 may be associated with CD163+ TAM infiltration in BCa, with VEGFA as a possible influencing factor.

Extracellular Vesicles Derived From Human Adipose-Derived Stem Cell Prevent the Formation of Hypertrophic Scar in a Rabbit Model.

BackgroundPreventing scar formation during wound healing has important clinical implications. Numerous studies have indicated that adipose-derived stem cell culture mediums, which are rich in cytokines and extracellular vesicles (EVs), regulate matrix remodeling and prevent scar formation after wound healing. Therefore, using a rabbit scar model, we tried to demonstrate which factor in adipose-derived stem cell culture mediums plays a major role in preventing scar formation (EVs or cytokines), as well as revealing the underlying mechanism.MethodsHuman adipose-derived stem cells (hASCs) were isolated from the subcutaneous adipose tissue of a healthy female donor. The surface CD markers of third-passage hASCs were analyzed by flow cytometry. The adipogenic differentiation capacity of the hASCs was detected using Oil O staining. A cultured medium of third- to five-passage hASCs was collected for EV and EV-free medium isolations. Extracellular vesicles were characterized using transmission electron microscopy, NanoSight, and the Western blotting for surface markers CD63, TSG101, and Alix. The EV-free medium was characterized by Western blotting for vascular endothelial growth factor A (VEGFA), platelet derived growth factor B (PDGFB), and transforming growth factor β 1 (TGFβ1). Eight-millimeter-diameter wounds were created on the ventral side of both ears of 16 New Zealand rabbits. A total of 0.1 mL of the human adipose-derived stem cell–extracellular vesicle (hASC-EV) or EV-free medium was locally injected into wounds made on the right ears during wound healing. Meanwhile, equal amounts of phosphate buffer saline were injected into the left ears as a control. Biopsies of the wounded skin and surrounding tissue were excised on postoperative day 28 and subjected to hematoxylin and eosin (H&E), Masson, and α-SMA immunofluorescence staining. The protein expression of α-SMA and collagen I in both scar tissues and the normal skin were evaluated via Western blotting.ResultsThe hASCs expressed high levels of 49d, CD90, CD105, and CD73 but did not express CD34 or CD45. The hASCs differentiated into adipocytes under an adipogenic induction medium. Under transmission electron microscopy, the hASC-EVs were circular, bilayer membrane vesicles and approximately 95% of the particles were between 50 and 200 nm in size. The hASC-EVs expressed the same surface markers as EVs, including CD63, TSG101, and Alix and displayed little expression of VEGFA, PDGFB, and TGFβ1. The EV-free medium had a high expression of VEGFA, PDGFB, and TGFβ1 but displayed no expression of CD63, TSG101, and Alix. In vivo, the hASC-EV treatment prevented the formation of hypertrophic scars on postoperative day 28 and suppressed collagen deposition and myofibroblast aggregation. However, the EV-free medium did not prevent the formation of hypertrophic scars on the same time point and had little effect on collagen deposition and myofibroblast aggregation when compared with the control group.ConclusionsOur study suggests that hASCs are associated with preventive scar formation therapy because of paracrine EVs rather than cytokines. A local injection of hASC-EVs during wound healing efficiently prevented hypertrophic scar formation, which may have a clinically beneficial antiscarring effect.

Long non-coding RNA NEAT1 promotes colorectal cancer progression by regulating miR-205-5p/VEGFA axis.

Long non-coding RNAs (lncRNAs) play key roles in tumorigenesis. It has been reported that the lncRNA nuclear-enriched abundant transcript 1 (NEAT1) may act as an oncogenic regulator in several cancers. However, the biological mechanism of action of NEAT1, particularly the miRNA sponge role in colorectal cancer (CRC), has not been fully elucidated. In our study, the expression of NEAT1, miR-205-5p, and vascular endothelial growth factor A (VEGFA) in CRC cell lines were assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. Cell proliferation was detected by Cell Counting Kit-8 (CCK-8) assay. Cell migration and invasion were examined by wound healing and transwell assays, respectively. RNA-binding protein immunoprecipitation (RIP), and dual-luciferase and RNA pull-down assays were conducted to determine the correlation between miR-205-5p and NEAT1 or VEGFA. VEGFA, matrix metalloproteinase (MMP)2, and MMP9 protein and mRNA expression were measured by western blotting and RT-qPCR analysis, respectively. Our results demonstrated high expression of NEAT1 and VEGFA and low expression of miR-205-5p in CRC cell lines. The RIP and dual-luciferase assays confirmed miR-205-5p as a target of NEAT1. In addition, VEGFA was identified as a direct target of miR-205-5p. Inhibition of NEAT1 or overexpression of miR-205-5p was able to repress VEGFA expression. Moreover, downregulation of NEAT1 and VEGFA inhibited cell proliferation, migration, and invasion. NEAT1 overexpression facilitated tumor growth by modulating miR-205-5p. Taken together, lncRNA NEAT1 was found to be upregulated in CRC cell lines, promoting CRC cell proliferation, migration, and invasion through regulating the miR-205-5p/VEGFA signaling pathway. These findings suggest that NEAT1 may be a promising biomarker in CRC diagnosis and treatment.

High expression of VEGFA in MSCs promotes tendon-bone healing of rotator cuff tear via microRNA-205-5p.

To explore the role of vascular endothelial growth factor A (VEGFA) in tendon-bone healing of rotator cuff tear (RCT) and to investigate its possible mechanism. Mesenchymal stem cells (MSCs) were transfected with pcDNA-VEGFA. The viability of MSCs was detected by cell counting kit-8 (CCK-8) assay. Expression levels of type I and type II collagen in MSCs were detected by quantitative Real time-polymerase chain reaction (qRT-PCR). RCT was constructed in rats. Meanwhile, all rats were divided into MSCs group and MSCs-pcDNA-VEGFA group, respectively. Biomechanical test was performed to detect ultimate load of failure and stiffness in RCT rats. Dual-luciferase reporter gene assay was conducted to analyze the binding condition between microRNA-205-5p and VEGFA, which was further verified by Western blot and qRT-PCR. VEGFA overexpression significantly promoted viability and proliferation of MSCs. Expression levels of type I and type II collagen were significantly upregulated after VEGFA overexpression in MSCs. Biomechanical test showed that VEGFA overexpression in RCT rats remarkably elevated ultimate load of failure and stiffness. Dual-luciferase reporter gene assay elucidated that VEGFA was the target gene of microRNA-205-5p. Furthermore, VEGFA negatively regulated microRNA-205-5p expression. VEGFA promotes tendon-bone healing of RCT via inhibiting microRNA-205-5p expression.

PO-315 Intertumor heterogeneity in vascularity and response to bevacizumab treatment in artificial melanoma brain metastases

IntroductionPatients diagnosed with melanoma brain metastases have few treatment options and poor prognosis, and antiangiogenic agents targeting the vascular endothelial growth factor A (VEGF-A) could represent a potential treatment strategy. The purpose of this preclinical investigation was to study the vascularisation pattern and the effect of the VEGF-A targeting agent bevacizumab in artificial brain metastases established from four human melanoma cell lines with different angiogenic and invasive properties.Material and methodsA-07, D-12, R-18, and U-25 cells transfected with GFP were injected intracerebrally in nude mice treated with bevacizumab (10 mg/ml) or vehicle. Treatment was initiated one day before tumour cell injection, and continued twice a week until the mice became moribund. Moribund mice were killed and autopsied, and the brain was evaluated by fluorescence imaging or by histological examination. Expression and secretion of factors involved in angiogenesis and invasion was assessed by quantitative PCR and ELISA.Results and discussionsThe melanoma cells showed a preference for growth in the meninges and ventricles after intracerebral injection, and intertumor heterogeneity in the aggressiveness of meningeal tumours reflected differences in angiogenic activity and expression of VEGF-A and interleukin 8 (IL-8). In contrast, growth and invasion of the brain parenchyma relied primarily on vascular co-option. The response to bevacizumab treatment depended on the angiogenic signature of the tumour cells and on the intracranial growth site. Bevacizumab treatment resulted in delayed meningeal tumour growth and prolonged survival in cell lines showing high VEGF-A expression and high angiogenic activity in the meninges, whereas no difference in survival was observed between bevacizumab-treated and vehicle-treated mice in cell lines showing low VEGF-A expression and lower angiogenic activity in the meninges.ConclusionThe melanoma cell lines showed different response to bevacizumab treatment, and these differences reflected differences in intracranial vascularisation patterns and in expression of VEGF-A.

HIF-1α contributes to hypoxia adaptation of the naked mole rat.

Background/AimsNaked mole rats (NMRs) spend their lives in burrow systems containing very low levels of oxygen, indicating long-term hypoxic exposure, and suggesting that pathological changes caused by hypoxia are attenuated or absent in this hypoxia-tolerant species. The mechanisms underlying NMRs hypoxia tolerance remain poorly understood. In this study, we explored whether hypoxia inducible factor 1α (HIF-1α), and vascular endothelial growth factor A (VEGFA) play a role in NMRs adaption to hypoxia.MethodsPrimary hepatic stellate cells (HSCs) isolated from NMRs and mice were treated with 50 μM YC-1, 50 μM KC7F2 or VEGFA siRNA. HIF-1α or VEGFA expression was detected by Western blot and real-time PCR. Apoptosis was determined by flow cytometry. The expression of autophagy markers (LC3 and p62) was detected by Western blot.ResultsOur results showed that HIF-1α and VEGFA expression in NMRs was significantly higher than in hypoxia-sensitive mice. Inhibition of HIF-1α expression induced apoptosis in both NMR and mouse HSCs following hypoxia. However, blocking VEGFA transcription results in a significant increase of apoptosis in both NMR and mouse HSCs before and after hypoxia. In addition, NMR HSCs displayed higher levels of autophagy (ratio of LC3ΙΙ/LC3Ι = 9.6) than mouse HSCs (relative ratio of LC3ΙΙ/ LC3Ι = 4.9) under hypoxic conditions.ConclusionWe conclude that HIF-1α activation may be an important mechanism for hypoxia adaption. However, high expression of VEGFA follows HIF-1α activation in NMRs.

ADAM9 promotes lung cancer progression through vascular remodeling by VEGFA, ANGPT2, and PLAT

Lung cancer has a very high prevalence of brain metastasis, which results in a poor clinical outcome. Up-regulation of a disintegrin and metalloproteinase 9 (ADAM9) in lung cancer cells is correlated with metastasis to the brain. However, the molecular mechanism underlying this correlation remains to be elucidated. Since angiogenesis is an essential step for brain metastasis, microarray experiments were used to explore ADAM9-regulated genes that function in vascular remodeling. The results showed that the expression levels of vascular endothelial growth factor A (VEGFA), angiopoietin-2 (ANGPT2), and tissue plasminogen activator (PLAT) were suppressed in ADAM9-silenced cells, which in turn leads to decreases in angiogenesis, vascular remodeling, and tumor growth in vivo. Furthermore, simultaneous high expression of ADAM9 and VEGFA or of ADAM9 and ANGPT2 was correlated with poor prognosis in a clinical dataset. These findings suggest that ADAM9 promotes tumorigenesis through vascular remodeling, particularly by increasing the function of VEGFA, ANGPT2, and PLAT.

The Neuropilins and Their Ligands in Hematogenous Metastasis of Salivary Adenoid Cystic Carcinoma—An Immunohistochemical Study

We investigated the expression of neuropilin-1 (NRP1), neuropilin-2 (NRP2), vascular endothelial growth factor-A (VEGF-A), semaphorin-3A (Sema-3A), and semaphorin-3F (Sema-3F) in normal salivary gland (NSG) tissue, nonmetastatic salivary adenoid cystic carcinoma (SACC), and metastatic SACC to better understand their role in intratumoral angiogenesis and hematogenous metastasis of SACC. The study included 60 SACC patients, equally divided between nonmetastatic SACC and metastatic SACC. We used 30 NSG samples as the control. The expression of cytokines was studied by immunohistochemistry and compared using the integrated optical density. The relationship between NRP1, NRP2, VEGF-A, and Sema-3A expression and microvessel density (MVD) was analyzed in the 3 groups. In metastatic SACC, the expression levels of NRP1 and VEGF-A were significantly greater than those in nonmetastatic SACC and NSG. The expression of Sema-3A and Sema-3F was significantly lower in metastatic SACC than that in nonmetastatic SACC and NSG (P<.0001). No significant differences were found in NRP2 expression among the 3 groups (P=.43). The MVD of metastatic SACC was significantly greater than that of nonmetastatic SACC and NSG (P<.0001). However, the lymphatic vessel density of the 3 groups was not significantly different statistically. The relationship between MVD and NRP1 or VEGF-A showed a significant positive correlation (P<.0001, for both). However, a significant negative correlation was found between the MVD and Sema-3A or Sema-3F expression (P<.0001, for both). Hematogenous metastasis of SACC is correlated with high expression of NRP1 and VEGF-A and low expression of Sema-3A and Sema-3F. The increased numbers of microvessels induced by VEGF-A signaling, combined with NRP1, could be one of the key reasons leading to the enhanced hematogenous metastasis in SACC.

Glucose Transporter 1 (SLC2A1) and Vascular Endothelial Growth Factor A (VEGFA) Predict Survival After Resection of Colorectal Cancer Liver Metastasis.

To investigate the individual and combined prognostic value of HIF1α, SLC2A1, and vascular endothelial growth factor A (VEGFA) in a multi-institutional cohort of patients with resected colorectal cancer liver metastasis (CRCLM). In the majority of patients with CRCLM, resection seems not to be curative, despite its curative intent. Overexpression of hypoxia-inducible factor 1α (HIF1α), glucose transporter 1 (SLC2A1; also known as GLUT1), and VEGFA has been associated with tumor progression and poor prognosis of patients with colorectal cancer (CRC). Tissue microarrays were generated using CRCLM and patient-matched primary CRC from patients who underwent CRCLM resection between 1990 and 2010. Prognostic value of HIF1α, SLC2A1, and VEGFA was determined by immunohistochemistry. A 500-fold cross-validated hazard rate ratio (HRRav) for overall survival was calculated. HIF1α, SLC2A1, and VEGFA expression could be evaluated in 328, 350, and 335 patients, respectively. High SLC2A1 expression was associated with good prognosis (HRRav, 0.67; P (HRR >1) < 0.01) and high VEGFA expression to poor prognosis (HRRav, 1.84; P (HRR < 1) = 0.02), also after multivariate analysis including established clinicopathological prognostic variables (HRRav, 0.67; P (HRR > 1) < 0.01 and HRRav, 1.50; P (HRR < 1) = 0.02, respectively). SLC2A1 showed prognostic value particularly in patients treated with systemic therapy (P < 0.01), whereas the prognostic value of VEGFA expression was mainly observed in patients not treated with systemic therapy (P < 0.01). Prognosis was especially poor in patients with both low SLC2A1 and high VEGFA expression (P < 0.01). HIF1α expression was not associated with survival. SLC2A1 and VEGFA expression are prognostic molecular biomarkers for patients with CRCLM with added value to established clinicopathological variables.

Abstract B03: The predictive value of microvessel density, vascular proliferation index and markers of VEGF associated angiogenesis in patients with metastatic melanoma treated with bevacizumab monotherapy

Abstract Background: Angiogenesis is a hallmark of cancer and essential for tumor growth and metastases. As previously published, clinical benefit was observed in 31% of patients with metastatic melanoma who were treated with bevacizumab monotherapy. Response evaluation was done according to the RECIST guidelines; overall response (OR) defines patients with complete or partial response, clinical benefit (CB) includes also stable disease for at least 6 months. The main objective of this study was to identify predictive markers of anti-angiogenic treatment. Here, we focus on the correlation between microvessel density and treatment response. Methods: 35 patients were enrolled in an open-label single arm phase II clinical trial at Haukeland University Hospital, Norway, and were treated with bevacizumab 10mg/kg q14 until disease progression or intolerable toxicity (ClinicalTrials.gov: NCT00139360). Paraffin embedded tissue of primary tumors and metastases as well as blood samples were available for investigation. Tissue sections were double stained with antibodies for Factor VIII and Ki67. The sections were screened for areas with the highest density of Factor VIII positive vessels. For assessment of microvessel density (MVD), the number of Factor VIII positive vascular units within the tumor was registered in three high power fields (HPFs). Microvessels with co-expression of Ki67 in the nucleus and Factor VIII in the cytoplasm of endothelial cells (ECs) were defined as proliferating vessels (pMVD) and were counted in the same HPFs. The ratio between pMVD and MVD was defined as vascular proliferation index (VPI). In addition, the presence of glomeroid microvascular proliferations (GMPs) was registered in tissue sections stained with Factor VIII mono. GMPs, a marker for VEGF associated angiogenesis, were defined as focal glomerulus-like aggregates of multi-layered Factor VIII positive ECs with a minimum of 15 cells. Associations with response as well as with various angiogenic factors in tissue and blood samples were analyzed. Results: High pMVD and VPI were significantly associated with high expression of vascular endothelial growth factor A (VEGF-A) in primary melanomas (Mann-Whitney U test, p=0.023; p=0.032). In addition, high MVD in metastases was associated with high expression of VEGF-A in metastases and low VEGF-A concentration in plasma (Mann-Whitney U test, p=0.036; p=0.046). However, expression of VEGF-A in primary melanomas or metastases was not associated with treatment response. All primary melanomas with GMPs show expression of VEGF-A in ECs (Chi-square test p=0.029). High MVD in primary tumors predicted CB (Mann-Whitney U test, p=0.042). However, this correlation was not observed in the metastases. Still, neither MVD in primary melanomas nor in metastases was correlated with OR to bevacizumab monotherapy in patients with metastatic melanoma. VPI and pMVD in primary melanomas or metastases were not correlated to any kind of treatment response. Similarly, the presence of GMP in primary melanomas or metastases did not show any correlation to treatment response. Conclusion: Although a significant association was present between MVD in primary lesions and CB, neither MVD, pMVD or VEGF expression seems to be robust predictive markers of response to bevacizumab monotherapy for metastatic melanoma. Further investigation is ongoing to identify predictive biomarkers in this trial. Citation Format: Cornelia Schuster, Lars A. Akslen, Oddbjørn Straume. The predictive value of microvessel density, vascular proliferation index and markers of VEGF associated angiogenesis in patients with metastatic melanoma treated with bevacizumab monotherapy. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr B03.