Angiogenesis-associated Genes Research Articles

An angiogenesis-associated gene-based signature predicting prognosis and immunotherapy efficacy of head and neck squamous cell carcinoma patients

ObjectivesTo develop a model that can assist in the diagnosis and prediction of prognosis for head and neck squamous cell carcinoma (HNSCC).Materials and methodsData from TCGA and GEO databases were used to generate normalized gene expression data. Consensus Cluster Plus was used for cluster analysis and the relationship between angiogenesis-associated gene (AAG) expression patterns, clinical characteristics and survival was examined. Support vector machine (SVM) and least absolute shrinkage and selection operator (LASSO) analyzes and multiple logistic regression analyzes were performed to determine the diagnostic model, and a prognostic nomogram was constructed using univariate and multivariate Cox regression analyses. ESTIMATE, XCELL, TIMER, QUANTISEQ, MCPCOUNTER, EPIC, CIBERSORT-ABS, CIBERSORT algorithms were used to assess the immune microenvironment of HNSCC patients. In addition, gene set enrichment analysis, treatment sensitivity analysis, and AAGs mutation studies were performed. Finally, we also performed immunohistochemistry (IHC) staining in the tissue samples.ResultsWe classified HNSCC patients into subtypes based on differences in AAG expression from TCGA and GEO databases. There are differences in clinical features, TME, and immune-related gene expression between two subgroups. We constructed a HNSCC diagnostic model based on nine AAGs, which has good sensitivity and specificity. After further screening, we constructed a prognostic risk signature for HNSCC based on six AAGs. The constructed risk score had a good independent prognostic significance, and it was further constructed into a prognostic nomogram together with age and stage. Different prognostic risk groups have differences in immune microenvironment, drug sensitivity, gene enrichment and gene mutation.ConclusionWe have constructed a diagnostic and prognostic model for HNSCC based on AAG, which has good performance. The constructed prognostic risk score is closely related to tumor immune microenvironment and immunotherapy response.

Identification of an angiogenesis-related risk score model for survival prediction and immunosubtype screening in multiple myeloma.

Multiple myeloma (MM) is an incurable B-cell malignancy, but with the emergence of immunotherapy, a potential cure is hopeful. The individualized interaction between the tumor and bone marrow (BM) microenvironment determines the response to immunotherapy. Angiogenesis is a constant hallmark of the BM microenvironment in MM. However, little is known about the potency ability of angiogenesis-associated genes (AAGs) to regulate the immune microenvironment of MM patients. We comprehensively dissected the associations between angiogenesis and genomic landscapes, prognosis, and the immune microenvironment by integrating 36 AAGs. Immunohistochemistry was performed to verify the correlation between angiogenic factor expression and patient prognosis. Single-sample gene set enrichment analysis was applied to quantify the relative abundance of 28 infiltrating cells. The AAG score was constructed using the least absolute shrinkage and selection operator Cox regression model. Angiogenesis was closely correlated with MM patient prognosis, and the mutation intensity of the AAGs was low. Immunohistochemistry confirmed that high microvessel density predicted poor prognosis. Three AAG clusters and two gene clusters with distinct clinical outcomes and immune characteristics were identified. The established AAG_score model performed well in predicting patient prognosis and active immunotherapy response. The high-AAG_score subgroup was characterized by reduced immune cell infiltration, poor prognosis, and inactive immunotherapy response. Multivariate analyses indicated that the AAG_score was strongly robust and independent among the prognostic variables. This study revealed that angiogenesis is significantly related to MM patient prognosis and immune phenotype. Evaluating the AAG signature was conducive to predicting patient response to immunotherapy and guiding more efficacious immunotherapy strategies.

Abstract A066: Machine learning elucidates biology of response within and outside the mechanisms of action of therapeutic agents in the I-SPY2 breast cancer TRIAL

Abstract Background: Machine learning (ML) in translational medicine has led to prediction of clinical outcomes and identification of new biomarkers. We employ ML in prediction of pathologic complete response (pCR) in high-risk breast cancer patients in the neoadjuvant I-SPY2 TRIAL where not all novel agents have strong predictive biomarkers. Leveraging a ML approach using progressively expanded candidate genes, we explore the limitations of using only known mechanisms of action in predicting pCR, and the extent to which biology outside known drug action improves response prediction in the first 10 arms of the trial. Methods: ML random forest models were developed in I-SPY2 patients (n=982) with pre-treatment gene expression and pCR data across 10 treatment arms (PMID: 35623341), including inhibitors of HER2: neratinib (N), pertuzumab (P), TDM1/P; AKT (MK-2206); IGF1R (ganitumab); HSP90 (ganetespib); PARP/DNA repair (veliparib/carboplatin, VC); ANG1/2 (trebananib, T); immune checkpoints (PD1-inh); and Control (Ctr). Each HR/HER2 receptor/treatment arm subset (m=27) was evaluated independently. We employed a three-pronged feature-selection approach using (1) genes restricted to known mechanism of action of individual I-SPY2 agents (k=10 to 88 genes); (2) genes expanded to include targeted pathways for all 10 agents/combinations (k=282); and (3) an unbiased whole genome approach (k=17,990). Samples were partitioned with 75% used for training and cross-validation, and 25% held out as test sets. Predictive ML models were defined as those with performance ≥ 0.90 based on different performance metrics (e.g., AUC, sensitivity, specificity). Results: For each of the 27 subtype-treatment subsets, at least one high performing model was identified. In 6 subtype-treatment subsets, mechanism of action genes were sufficient to predict pCR: AKT/PI3K/HER genes in HR+HER2- N and HR-HER2+ P; DNA repair genes in HR+HER2- VC; angiogenesis-associated genes in HR+HER2+ T; and immune-associated genes in both HR+HER2- and HR-HER2- PD1-inh subsets. Expanded targeted pathway models were required to identify predictive models in 8 additional subtype-treatment pairs from the N, T-DM1/P, MK-2206, VC, T, and HER2+ Ctr arms, with significant contribution of DNA repair, immune, and HSP90 genes for multiple arms. A genome-wide approach was required for the remaining 13 subtype-treatment pairs with no previous models from the N, P, MK-2206, ganitumab, ganetespib, T, and HER2- Ctr arms. Even for subtype-treatment pairs where mechanism of action gene sets was sufficient for reasonable models, expanded gene sets resulted in improved performance. For instance, metabolism genes improved model performance for HR-HER2+ in N and Ctr, and for HR+HER2- in the PD1-inh arm; and mitochondrial and protein folding dysfunction genes improved response prediction in HR-HER2- in the ganetespib arm. Conclusion: Our study identifies mechanism of action biomarkers associated with response to each drug and elucidates possible off-target effects contributing to observed drug sensitivity and resistance. Citation Format: Rosalyn W. Sayaman, Denise M. Wolf, Christina Yau, Julia Wulfkhule, Emanuel F. Petricoin, Lamorna Brown-Swigart, Tam Binh Bui, Gillian L. Hirst, Diane Heditsian, W. Fraser Symmans, Angela DeMichele, Mark LaBarge, Laura J. Esserman, Laura van ‘t Veer. Machine learning elucidates biology of response within and outside the mechanisms of action of therapeutic agents in the I-SPY2 breast cancer TRIAL [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A066.

Clinical significance and immune landscape of angiogenesis-related genes in bladder cancer.

Angiogenesis is a major promotor of tumor progression and metastasis. Nevertheless, it is undetermined how angiogenesis-related genes (ARGs) influence bladder cancer. The profiles of bladder cancer gene expression were collected from the TCGA-BLCA cohort. The LASSO regression analysis was used to build an angiogenesis-related signature (ARG_score) with the prognostic ARGs. Verification analyses were conducted across the GSE48075 dataset to demonstrate the robustness of the signature. Differences between the two risk groups based on clinical outcomes, immune landscape, mutation status, chemotherapeutic effectiveness for anticancer drugs, and immunotherapy efficacy were analyzed. A nomogram was developed to improve the clinical efficacy of this predictive tool. The expression levels of model genes in normal bladder epithelial cell lines (SV-HUC-1) and bladder cancer cell lines (T24 and 5637) were detected by qRT-PCR assay. Four angiogenesis-associated gene signature was constructed based on the LASSO regression algorithm. The signature showed independent risk factors of overall survival for bladder cancer, validated using two external survival datasets. Additionally, we built a prognostic nomogram to improve the practicality of the ARG_score. High-risk individuals showed stronger immunocyte infiltration, immune-related functions, elevated expression of immune checkpoints, reduced TIDE score, and higher combined IPS-PD-1 and IPS-CTLA4 scores, suggesting a heightened responsiveness to immune checkpoint inhibitors. Furthermore, patients with low and high risk showed distinct responsiveness to anticancer drugs. The expression levels of 5 model genes (COL5A2, JAG1, MSX1, OLR1, and STC) were significantly increased in bladder cancer cell lines (T24 and 5637) compared with the normal bladder epithelial cell line SV-HUC-1. The model constructed based on ARGs may have wide application in predicting outcomes and therapeutic responses.

Adipose-derived Mesenchymal Stem Cells are Ideal for the Cell-based Treatment of Refractory Wounds: Strong Potential for Angiogenesis.

Although Mesenchymal Stem Cells (MSCs)-based therapy has been proposed as a promising strategy for the treatment of chronic lower-extremity ulcers, their optimal sources, amounts, and delivery methods are urgently needed to be determined. In this study, we compared the heterogeneity of the human MSCs derived from bone marrow (BMSCs), umbilical cord (UCMSCs), and adipose tissue (ADSCs) in accelerating wound healing and promoting angiogenesis and explored the underlying mechanism. Briefly, a diabetic rat model with a full-thickness cutaneous wound on the dorsal foot was developed. The wound was topically administered with three types of MSCs. Additionally, we carried out in vitro and in vivo analysis of the angiogenic properties of the MSCs. Moreover, the molecular mechanism of the heterogeneity of the MSCs derived from the three tissues was explored by transcriptome sequencing. When compared with the BMSCs- and UCMSCs-treated groups, the ADSCs-treated group exhibited markedly accelerated healing efficiency, characterized by increased wound closure rates, enhanced angiogenesis, and collagen deposition at the wound site. The three types of MSCs formed three-dimensional capillary-like structures and promoted angiogenesis in vitro and in vivo, with ADSCs exhibiting the highest capacity for tube formation and pro-angiogenesis. Furthermore, transcriptome sequencing revealed that ADSCs had higher expression levels of angiogenesis-associated genes. Our findings indicate that MSCs-based therapy accelerates the healing of ischemia- and diabetes-induced lower-extremity ulcers and that adipose tissue-derived MSCs might be ideal for therapeutic angiogenesis and treatment of chronic ischemic wounds.

A Pan-cancer Analysis Reveals the Tissue Specificity and Prognostic Impact of Angiogenesis-associated Genes in Human Cancers

Introduction: Angiogenesis is one of the hallmarks of cancer and can impact the processes of cancer initiation, progression, and response to therapy. Background: Anti-angiogenic therapy is thus an encouraging therapeutic option to treat cancers, but the detailed angiogenic mechanisms and the association between angiogenesis and clinical outcome remain unknown in different cancers. Methods: Here, we systematically assess the impacts of 82 angiogenesis-associated genes (AAGs) in tumor tissue specificity and prognosis across 16 cancer types. Results: Results demonstrate that the expression patterns of the 82 AAGs can reflect the tumor tissue specificity, and high expressions of up-regulated AAGs are significantly associated with poor prognosis of cancer. We further define a prognostic score for predicting overall survival (OS) based on the expressions of up-regulated AAGs and confirm its reliable predictive ability. Results indicate that a low prognostic score demonstrates a superior OS and vice versa. Conclusion: The results of this study will contribute to the understanding of different tumor angiogenesis mechanisms in various tissues and cancer-personalized anti-angiogenic treatment. The code of our analysis can be accessed at https://github.com/ZhenshenBao/AAGs_analysis.git.

Immune Cell Infiltration and Clinical Significance of Angiogenesis-related Genes in Lung Adenocarcinoma.

Angiogenesis is one of the hallmarks of cancer. However, the role of molecular subtypes of angiogenesis-associated genes (AAGs) in the tumor immune microenvironment (TIME) of lung adenocarcinoma (LUAD) remains unclear. The expression of AAGs in patients with LUAD were studied. Consensus clustering was performed to identify new AAG-associated molecular subgroups. The TIME and immune status of the subgroups were analyzed. Functional enrichment analysis was performed on the differentially expression genes among the clustered subgroups to analyze their relationship with AAGs. Furthermore, a prognostic risk model and clinical nomogram associated with survival time were constructed. Risk scores of drug sensitivity, immune checkpoint molecules, tumor mutational burden, and tumor cell stemness were analyzed. Finally, a series of in vitro experiments were performed to investigate the role of dickkopf WNT signaling pathway inhibitor 1 (DKK1) in LUAD. Two molecular subgroups with significantly different survival rates and TIME were identified. Immune checkpoint scores were higher in the subgroup with a worse prognosis. Moreover, differentially expressed genes were enriched in cell-cycle regulation, protein metabolism, and the immune microenvironment. The risk model and clinical nomogram constructed based on AAGs accurately predicted the prognosis of patients with LUAD. Patients with high-risk scores were less sensitive to chemotherapy but more sensitive to immunotherapy. DKK1 was highly expressed in basal cells and luminal cells. In addition, the knockdown of DKK1 reduced LUAD cell proliferation, invasion, and migration. Models based on AAGs can play an important role in predicting LUAD prognosis and immunotherapy effects. We further characterized the angiogenesis of TIME and studied the AAG DKK1. Our findings provide a theoretical basis for antitumor strategies targeting angiogenesis.

Endocan Knockdown Down-Regulates the Expression of Angiogenesis-Associated Genes in Il-1ß Activated Chondrocytes.

Endocan is a small soluble proteoglycan (PG) known to be involved in inflammation and angiogenesis. Increased endocan expression was found in the synovia of arthritic patients and chondrocytes stimulated with IL-1ß. Considering these findings, we aimed to investigate the effects of endocan knockdown on the modulation of pro-angiogenic molecules expression in a model of IL-1ß-induced inflammation in human articular chondrocytes. Endocan, VEGF-A, MMP-9, MMP-13, and VEGFR-2 expression was measured in both normal and endocan knockdown chondrocytes stimulated with IL-1ß. VEGFR-2 and NF-kB activation were also measured. Results have shown that endocan, VEGF-A, VEGFR-2, MMP-9, and MMP-13 were significantly up-regulated during IL-1ß-induced inflammation; interestingly, the expression of such pro-angiogenic molecules and NF-kB activation were significantly reduced by endocan knockdown. These data support the hypothesis that endocan released by activated chondrocytes may be involved in the mechanisms that stimulate cell migration and invasion, as well as angiogenesis, in the pannus of arthritic joints.

Hypoxia preconditioned DPSC-derived exosomes regulate angiogenesis via transferring LOXL2

Hypoxia was proved to enhance the angiogenesis of stem cells. However, the mechanism of the angiogenic potential in hypoxia-pretreated dental pulp stem cells (DPSCs) is poorly understood. We previously confirmed that hypoxia enhances the angiogenic potential of DPSC-derived exosomes with upregulation of lysyl oxidase-like 2 (LOXL2). Therefore, our study aimed to illuminate whether these exosomes promote angiogenesis via transfer of LOXL2. Exosomes were generated from hypoxia-pretreated DPSCs (Hypo-Exos) stably silencing LOXL2 after lentiviral transfection and characterized with transmission electron microscopy, nanosight and Western blot. The efficiency of silencing was verified using quantitative real-time PCR (qRT-PCR) and Western blot. CCK-8, scratch and transwell assays were conducted to explore the effects of LOXL2 silencing on DPSCs proliferation and migration. Human umbilical vein endothelial cells (HUVECs) were co-incubated with exosomes to assess the migration and angiogenic capacity through transwell and matrigel tube formation assays. The relative expression of angiogenesis-associated genes was characterized by qRT-PCR and Western blot. LOXL2 was successfully silenced in DPSCs and inhibited DPSC proliferation and migration. LOXL2 silencing in Hypo-Exos partially reduced promotion of HUVEC migration and tube formation and inhibited the expression of angiogenesis-associated genes. Thus, LOXL2 is one of various factors mediating the angiogenic effects of Hypo-Exos.

An Angiogenic Gene Signature for Prediction of the Prognosis and Therapeutic Responses of Hepatocellular Carcinoma

Among cancer-related deaths worldwide, hepatocellular carcinoma (HCC) ranks second. The hypervascular feature of most HCC underlines the importance of angiogenesis in therapy. This study aimed to identify the key genes which could characterize the angiogenic molecular features of HCC and further explore therapeutic targets to improve patients' prognosis. Public RNAseq and clinical data are from TCGA, ICGC, and GEO. Angiogenesis-associated genes were downloaded from the GeneCards database. Then, we used multi-regression analysis to generate a risk score model. This model was trained on the TCGA cohort (n = 343) and validated on the GEO cohort (n = 242). The predicting therapy in the model was further evaluated by the DEPMAP database. We developed a fourteen-angiogenesis-related gene signature that was distinctly associated with overall survival (OS). Through the nomograms, our signature was proven to possess a better predictive role in HCC prognosis. The patients in higher-risk groups displayed a higher tumor mutation burden (TMB). Interestingly, our model could group subsets of patients with different sensitivities to immune checkpoint inhibitors (ICIs) and Sorafenib. We also predicted that Crizotinib, an anti-angiogenic drug, might be more sensitive to these patients with high-risk scores by the DEPMAP. The inhibitory effect of Crizotinib in human vascular cells was obvious in vitro and in vivo. This work established a novel HCC classification based on the gene expression values of angiogenesis genes. Moreover, we predicted that Crizotinib might be more effective in the high-risk patients in our model.

Curcumin Nanofibers: A Novel Approach to Enhance the Anticancer Potential and Bioavailability of Curcuminoids.

Development of novel treatment methods for cancer is needed given the limitations of current treatment methods, including side effects and chemotherapeutic resistance, which may provide new hope to cancer patients. Cancer is the second leading cause of global mortality. Curcumin, the active ingredient of turmeric, has been used since ancient times for various therapeutic purposes. Several studies have identified its activity against cancer. Despite the established anticancer activity of curcumin, its low aqueous solubility and bioavailability are barriers to its effectiveness. In an attempt to solve this problem, many studies have formulated curcumin nanofiber preparations using a variety of methods. Electrospinning is a simple and affordable method for the production of nanofibers. Studies have shown increased curcumin bioavailability in nanofibers resulting from their high surface/volume ratio and porosity. We have undertaken a detailed review of studies on the anticancer effects of curcumin nanofibers. Curcumin acts by inhibiting various biological cancer pathways, including NF-κB, mTOR, complex I, cytokines, expression of p-p65, Ki67, and angiogenesis-associated genes. It also induces apoptosis through activation of caspase pathways and ROS production in cancer cells. Curcumin-loaded PLA50/PVP50/Cur15 nanofibers were investigated in breast cancer, one of the most studied cancers, and was shown to have significant effects on the widely used HeLa-cell line. Most of the studies undertaken have been performed in cell lines in vitro, while relatively few animal studies have been reported. More preclinical and clinical studies are needed to evaluate the anticancer activity of curcumin nanofibers. Amongst studies undertaken, a variety of curcumin nanofibers of various formulations have been shown to suppress a variety of cancer types. Overall, curcumin nanofibers have been found to be more efficient than free curcumin. Thus, curcumin nanofibers have been observed to improvise cancer treatment, offering great potential for effective cancer management. Further studies, both in vitro and in vivo, involving curcumin nanofibers have the potential to benefit cancer management.

Abstract PR010: Single-cell sequencing elucidates the effects of chemotherapy on cancer cell heterogeneity and the tumor microenvironment of human pancreatic adenocarcinoma

Abstract The tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC) is a complex ecosystem that drives tumor progression. Improving our understanding of the PDAC TME and its role in response to therapy via in-depth single cell characterization will have broad clinical implications for biomarker development and therapeutic design. In this study, we performed single-cell RNA sequencing on freshly collected human PDAC samples of primary (n=16) or metastatic (n=11) origin, either before (n=20) or after (n=7) chemotherapy. We found a heterogeneous mixture of basal and classical Moffitt cancer cell subtypes in all samples, along with distinct cancer-associated fibroblast (CAF) and tumor-associated macrophage (TAM) subpopulations. We identified the major CAF subpopulations as inflammatory CAFs (iCAFs) and myofibroblastic CAFs (myCAFs); within these subpopulations were a very few cells expressing immunogenic features which have previously been associated with antigen presenting CAFs (apCAFs). Tumor-associated macrophages (TAMs) could be categorized into two major subpopulations, C1QC+ TAMs or SPP1+ TAMs, each with distinct functional characteristics. For example, phagocytosis-associated gene sets were enriched in C1QC+ TAMs, while angiogenesis-associated gene sets were enriched in SPP1+ TAMs. Comparison of naïve and chemotherapy treated primary PDAC samples revealed that classical and basal-like cancer cells exhibited similar transcriptional responses to chemotherapy; this contrasts with some previous reports which posited a shift towards a basal-like transcriptional program among treated samples. We further noted that treated samples evinced fewer ligand-receptor interactions, particularly between TIGIT on CD8+ T cells and its ligand on cancer cells. We also identified TIGIT, not PD1, as the major inhibitory checkpoint molecule of CD8+ T cells in the PDAC TME. Altogether, our results suggest that chemotherapy impacts the PDAC TME and may further reduce response to immunotherapy. Citation Format: Daniel Weissinger, Emily A. Kawaler, Gregor Werba, Ende Zhao, Despoina Kalfakakou, Surajit Dhara, Grace Oh, Xiaohong Jing, Nina Beri, Lauren Khanna, Tamas Gonda, Paul E. Oberstein, Cristina Hajdu, Cynthia Loomis, Adriana Heguy, Mara H. Sherman, Amanda W. Lund, Theodore H. Welling, Igor Dolgalev, Aristotelis Tsirigos, Diane M. Simeone. Single-cell sequencing elucidates the effects of chemotherapy on cancer cell heterogeneity and the tumor microenvironment of human pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR010.

Distinctive effects of SGLT2 inhibitors on angiogenesis in zebrafish embryos

Sodium glucose cotransporter 2 (SGLT2) inhibitor canagliflozin has been found to increase the risk for lower-limb amputations in type 2 diabetics about two-fold. Conversely, empagliflozin and dapagliflozin do not display a similar effect. A question arises whether the increased risk for minor amputations is associated only with canagliflozin or whether it is a class effect of SGLT2 inhibitors. Defective angiogenesis has a role in amputations. We compared the effects of empagliflozin, dapagliflozin and canagliflozin on angiogenesis in vivo using zebrafish model, and in vitro using human umbilical vein endothelial cells (HUVECs). The effects of SGLT2 inhibitors on the formation of intersegmental blood vessels (ISVs) of the zebrafish embryos were clarified. Additionally, transcriptome analysis was performed to explore whether putative angiogenesis-associated genes are differentially regulated by SGLT2 inhibitors. The effects of SGLT2 inhibitors on the viability of HUVECs were examined. We noticed that especially empagliflozin and also dapagliflozin significantly accelerated the formation of ISVs of zebrafish embryos. In contrast, canagliflozin was not able to stimulate ISV formation, and at high concentration, it was lethal to the embryos. Transcriptome analysis demonstrated that in empagliflozin-treated embryos compared to canagliflozin-treated embryos seven genes previously shown to contribute to angiogenesis were upregulated, and four downregulated. Canagliflozin at high concentrations, but not empagliflozin or dapagliflozin, decreased the viability of HUVECs and disrupted their capability to sprout. SGLT2 inhibitors differed in their effects on angiogenic processes in zebrafish embryos and on the viability of HUVECs suggesting that the risk of SGLT2 inhibitors for peripheral amputations likely differs.

The clinical significance, immune infiltration, and tumor mutational burden of angiogenesis-associated lncRNAs in kidney renal clear cell carcinoma.

BackgroundPoor prognosis of kidney renal clear cell carcinoma (KIRC) is often related to angiogenesis. The lncRNAs that regulate angiogenesis could also affect the prognosis of KIRC. It is meaningful for us to use lncRNAs related to angiogenesis to construct a generic, individualized prognostic signature for patients with KIRC.MethodsWe identified eight angiogenesis-associated genes (AAGs) by differential expression analysis and univariate Cox regression from The Cancer Genome Atlas dataset, including 537 KIRC samples and 72 normal samples. In total, 23 prognostic lncRNAs were screened out after Pearson correlation analysis and univariate Cox regression analysis. Then, we performed least absolute shrinkage and selection operator (LASSO) regression and multivariate Cox regression to establish a four-AAG-related lncRNA prognostic signature.ResultsThe risk score was calculated for each KIRC patients by using a four-AAG-related lncRNA prognostic signature. We divided the KIRC patients into high- and low-risk groups by the median of the risk score. It was confirmed that the AAG-related lncRNA prognostic signature has good prognostic value for KIRC patients by time-dependent receiver operating characteristic and Kaplan–Meier survival analysis. We identified 3,399 differentially expressed genes between the high- and low-risk groups and performed their functional enrichment analyses. The AAG-related lncRNA prognostic signature was an independent prognostic predictor for KIRC patients and was used to perform a combined nomogram. We reevaluated them in terms of survival, clinic characteristics, tumor-infiltrating immune cells and tumor mutation burden.ConclusionOur research indicates that the AAG-related lncRNA prognostic signature is a promising and potential independent prognostic indicator for KIRC patients. Then, it could offer new insights into the prognosis assessment and potential treatment strategies of KIRC patients.

Abstract 3204: Differential expression of angiogenesis-associated genes in smoker and non-smoker lung adenocarcinoma patients

Abstract Tobacco smoke (TS) is one of the most prominent risk factors for lung and several other malignancies. Toxic chemicals present in TS induce DNA damage leading to carcinogenic mutations. Moreover, TS can also cause epigenetic and gene expression changes in tumor and other cells of the tumor microenvironment, especially through its addictive component nicotine. We and others have reported that nicotine promotes tumor angiogenesis via its direct impact on the endothelial cells or by altering the cancer cell secretome. In this study, we wanted to explore the association of smoking habits with the differential expression of angiogenesis-related genes in lung adenocarcinoma (LUAD) patients. Gene Set Enrichment Analysis (GSEA) identified a set of 48 angiogenesis-related genes that were surveyed for differential expression in smoker versus non-smoker patients in The Cancer Genomic Atlas (TCGA) database using an interactive web application, UALCAN. Gender and age of the LUAD patients were considered as confounding variables since patients of different age groups and gender can have biological and physiological differences, which could influence gene expression regardless of the smoking habits. We identified a total of fourteen differentially-expressed genes, of which four (HTATIP2, NCL, SPHK1, and VEGFA) were overexpressed and ten (AGGF1, AMOT, C1GALT1, ERAP1, NPR1, PML, RNH1, SHH, SPINL5, and TNFSF12) were downregulated in LUAD of smokers than those of non-smokers. Five of the differentially-expressed genes (HTATIP2, NCL NPR1, PML, and TNFSF12) exhibited significantly different transcript levels in males versus females and three (SPHK1, AGGF1, and SPINK5) showed varying pattern of expression in LUAD of different age group patients. Literature survey found prior published evidence for an association of two of the fourteen differentially expressed genes (NCL and NPR) with nicotine exposure. Together, our findings identify smoking-associated angiogenic gene signatures that could be potential effectors of nicotine signaling that promotes cancer angiogenesis and should be validated further by conducting laboratory investigations. Citation Format: Shubhangi Singh, Santanu Dasgupta, Elba A. Turbat-Herrera, Seema Singh, Ajay P. Singh. Differential expression of angiogenesis-associated genes in smoker and non-smoker lung adenocarcinoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3204.

Fusion Peptide-Engineered Polyetheretherketone Implants with Photo-Assisted Anti-Pathogen and Enhanced Angiogenesis for in vivo Osseointegrative Fixation

Owing to its superior mechanical and physicochemical characteristics, polyetheretherketone (PEEK) is considered a potent orthopedic material. Yet, poor bactericidal, osseointegrative and angiogenic properties of bioinert PEEK collectively hamper its clinical adoption. Herein, we present a strategy for developing multifunctional hierarchical coatings bearing a synthetic fusion peptide and polydopamine/silver nanoparticles (pDA/AgNPs) bio-heterojunctions (bio-HJs), in an attempt to empower PEEK with triple-modal abilities, which prevents bacterial infection, enhances ossification and promotes re-vascularization for physiological osseointegrative fixation. By harnessing the adhesion of bioinspired pDA, AgNPs are facilely immobilized onto peripheral surface and incorporated into interior cavity of micro/nano-porous sulfonated PEEK (SP) to form hierarchical pDA/AgNPs heterostructures. The bio-HJs coatings exhibit robust and repeatable bacteria-eradicating ability, assisted by near-infrared illumination, due to the synergistic effects of the metal ion/photothermal therapy. The fusion peptide-engineered PEEK exhibits osteogenesis activity in terms of promoting osteoblast proliferation, spreading, boosting the synthesis and secretion of osteogenesis-related genes. Besides, the fusion peptide-engineered PEEK also has the ability to promote HUVECs growth, reproduction, and tube formation, facilitating the synthesis and secretion of angiogenesis-associated genes. Moreover, the results of the in vivo experiments further corroborate the in vivo osteointegration and angiogenic abilities of materials. Expectantly, the multifunctional coatings with the presented triple-therapeutic effects represent a prospective clinical solution to address the current clinical challenges associated with the use of PEEK implants.

A Crucial Angiogenesis-Associated Gene MEOX2 Could Be a Promising Biomarker Candidate for Breast Cancer.

BackgroundAngiogenesis plays a critical role in the growth and metastasis of breast cancer and angiogenesis inhibition has become an effective strategy for cancer therapy. Our study aimed to clarify the key candidate genes and pathways related to breast cancer angiogenesis.MethodsDifferentially expressed genes (DEGs) in the raw breast cancer (BRCA) gene dataset from the Cancer Genome Atlas (TCGA) database were identified and gene ontology analysis of the DEGs was performed. Hub genes were subsequently determined using the Gene Expression Omnibus database. The expression of the mesenchyme homeobox 2 (MEOX2) in breast cancer cells and tissues was assessed by quantification real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC), respectively. The prognostic value of the MEOX2 gene in breast cancer tissue was evaluated with the Kaplan-Meier plotter.ResultsA total of 61 angiogenesis-related DEGs were identified in the TCGA dataset, among which the gene MEOX2 was significantly down-regulated. GO functional annotation and pathway enrichment analyses showed that MEOX2 was significantly enriched in the regulation of vasculature development. The IHC results confirmed that MEOX2 expression was repressed in breast cancer tissues and the relatively low level indicated the tissue was densely vascularized. Moreover, MEOX2 expression was significantly elevated in breast cancer cells after treatment with cisplatin (DDP) and epirubicin (EPI). Finally, the Kaplan-Meier plotter confirmed that higher expression levels of MEOX2 were related to better overall survival.ConclusionOur study revealed that the angiogenesis-associated gene MEOX2 can be used as a novel biomarker for breast cancer diagnosis and clinical therapy.

The transplantation of rapamycin-treated senescent human mesenchymal stem cells with enhanced proangiogenic activity promotes neovascularization and ischemic limb salvage in mice.

Few therapies can reverse the proangiogenic activity of senescent mesenchymal stromal/stem cells (MSCs). In this study, we investigated the effects of rapamycin on the proangiogenic ability of senescent human umbilical cord MSCs (UCMSCs). An in vitro replicative senescent cell model was established in cultured UCMSCs. We found that late passage (P25 or later) UCMSCs (LP-UCMSCs) exhibited impaired proangiogenic abilities. Treatment of P25 UCMSCs with rapamycin (900 nM) reversed the senescent phenotype and notably enhanced the proangiogenic activity of senescent UCMSCs. In a nude mouse model of hindlimb ischemia, intramuscular injection of rapamycin-treated P25 UCMSCs into the ischemic limb significantly promoted neovascularization and ischemic limb salvage. We further analyzed the changes in the expression of angiogenesis-associated genes in rapamycin-primed MSCs and found higher expression of several genes related to angiogenesis, such as VEGFR2 and CTGF/CCN2, in primed cells than in unprimed MSCs. Taken together, our data demonstrate that rapamycin is a potential drug to restore the proangiogenic activity of senescent MSCs, which is of importance in treating ischemic diseases and tissue engineering.

Contribution of Endothelial Laminin-Binding Integrins to Cellular Processes Associated with Angiogenesis.

Endothelial cells engage extracellular matrix and basement membrane components through integrin-mediated adhesion to promote angiogenesis. Angiogenesis involves the sprouting of endothelial cells from pre-existing vessels, their migration into surrounding tissue, the upregulation of angiogenesis-associated genes, and the formation of new endothelial tubes. To determine whether the endothelial laminin-binding integrins, α6β4, and α3β1 contribute to these processes, we employed RNAi technology in organotypic angiogenesis assays, as well in migration assays, in vitro. The endothelial depletion of either α6β4 or α3β1 inhibited endothelial sprouting, indicating that these integrins have non-redundant roles in this process. Interestingly, these phenotypes were accompanied by overlapping and distinct changes in the expression of angiogenesis-associated genes. Lastly, depletion of α6β4, but not α3β1, inhibited migration. Taken together, these results suggest that laminin-binding integrins regulate processes associated with angiogenesis by distinct and overlapping mechanisms.

Molecular Characteristics, Clinical Significance, and Cancer Immune Interactions of Angiogenesis-Associated Genes in Gastric Cancer.

BackgroundImmunotherapy has evolved as a critical option to treat diverse cancers. The active response to immunotherapy relies on the unique interaction between cancer and the tumor microenvironment (TME). Angiogenesis is one of the hallmarks of cancer. However, the association between angiogenesis and clinical outcome, immune cell infiltration, and immunotherapy remains unknown in gastric cancer (GC).MethodsWe systematically assessed 36 angiogenesis-associated genes (AAGs) and comprehensively identified the correlation between angiogenesis and transcriptional patterns, prognosis, and immune cell infiltration. The AAG_score was applied to quantify the angiogenesis subtypes of each patient. We then evaluated their values in prognostic prediction and therapeutic responses in GC.ResultsWe discussed the mutations of AAGs in GC specimens from genetic levels and identified their expression patterns from TCGA and GEO cohorts. We determined two different molecular subtypes and observed that AAG mutations were related to patients’ clinicopathological characteristics, prognosis, and infiltrating TME. Next, an AAG_score for predicting overall survival (OS) was established and its reliable predictive ability in GC patients was confirmed. Furthermore, we created a highly reliable nomogram to facilitate the clinical viability of the AAG_score. A low AAG_score, characterized by elevated microsatellite instability-high, mutation burden, and immune activation, demonstrated a superior OS. Additionally, the AAG_score was remarkedly correlated with the cancer stem cell index and drug susceptibility.ConclusionCollectively, we identified a prognostic AAG signature for GC patients. This signature may contribute to clarifying the characteristics of TME and enable the exploration of more potent immunotherapy strategies.