Hypoxic Gene Research Articles

Application of Cluster Analysis of Time Evolution for Magnetic Resonance Imaging -Derived Oxygen Extraction Fraction Mapping: A Promising Strategy for the Genetic Profile Prediction and Grading of Glioma.

Background: The intratumoral heterogeneity of oxygen metabolism and angiogenesis are core hallmarks of glioma, unveiling that genetic aberrations associated with magnetic resonance imaging (MRI) phenotypes may aid in the diagnosis and treatment of glioma.Objective: To explore the predictability of MRI-based oxygen extraction fraction (OEF) mapping using cluster analysis of time evolution (CAT) for genetic profiling and glioma grading.Methods: Ninety-one patients with histopathologically confirmed glioma were examined with CAT for quantitative susceptibility mapping and quantitative blood oxygen level–dependent magnitude-based OEF mapping and dynamic contrast-enhanced (DCE) MRI. Imaging biomarkers, including oxygen metabolism (OEF) and angiogenesis [volume transfer constant, cerebral blood volume (CBV), and cerebral blood flow], were investigated to predict IDH mutation, O6-methylguanine-DNA-methyltransferase (MGMT) promoter methylation status, receptor tyrosine kinase (RTK) subgroup, and differentiation of glioblastoma (GBM) vs. lower-grade glioma (LGG). The corresponding DNA sequencing was also obtained. Results were compared with DCE-MRI using receiver operating characteristic (ROC) analysis.Results: IDH1-mutated LGGs exhibited significantly lower OEF and hypoperfusion than IDH wild-type tumors (all p < 0.01). OEF and perfusion metrics showed a tendency toward higher values in MGMT unmethylated GBM, but only OEF retained significance (p = 0.01). Relative prevalence of RTK alterations was associated with increased OEF (p = 0.003) and perfusion values (p < 0.05). ROC analysis suggested OEF achieved best performance for IDH mutation detection [area under the curve (AUC) = 0.828]. None of the investigated parameters enabled prediction of MGMT status except OEF with a moderate AUC of 0.784. Predictive value for RTK subgroup was acceptable by using OEF (AUC = 0.764) and CBV (AUC = 0.754). OEF and perfusion metrics demonstrated excellent performance in glioma grading. Moreover, mutational landscape revealed hypoxia or angiogenesis-relevant gene signatures were associated with specific imaging phenotypes.Conclusion: CAT for MRI-based OEF mapping is a promising technology for oxygen measurement and along with perfusion MRI can predict genetic profiles and tumor grade in a non-invasive and clinically relevant manner.Clinical Impact: Physiological imaging provides an in vivo portrait of genetic alterations in glioma and offers a potential strategy for non-invasively selecting patients for individualized therapies.

Understanding the role of potential pathways and its components including hypoxia and immune system in case of oral cancer

There are a few biological functions or phenomenon which are universally associated with majority of the cancers and hypoxia and immune systems are among them. Hypoxia often occurs in most of the cancers which helps the cells in adapting different responses with respect to the normal cells which may be the activation of signaling pathways which regulate proliferation, angiogenesis, and cell death. Similar to it, immune signaling pathways are known to play critical roles in cancers. Moreover, there are a number of genes which are known to be associated with these hypoxia and immune system and appear to direct affect the tumor growth and propagations. Cancer is among the leading cause of death and oral cancer is the tenth-leading cause due to cancer death. In this study, we were mainly interested to understand the impact of alteration in the expression of hypoxia and immune system-related genes and their contribution to head and neck squamous cell carcinoma. Moreover, we have collected the genes associated with hypoxia and immune system from the literatures. In this work, we have performed meta-analysis of the gene and microRNA expression and mutational datasets obtained from public database for different grades of tumor in case of oral cancer. Based on our results, we conclude that the critical pathways which dominantly enriched are associated with metabolism, cell cycle, immune system and based on the survival analysis of the hypoxic genes, we observe that the potential genes associated with head and neck squamous cell carcinoma and its progression are STC2, PGK1, P4HA1, HK1, SPIB, ANXA5, SERPINE1, HGF, PFKM, TGFB1, L1CAM, ELK4, EHF, and CDK2.

Hypoxic gene expression is a prognostic factor for disease free survival in a cohort of locally advanced squamous cell cancer of the uterine cervix

Introduction Tumour hypoxia in locally advanced squamous cervical cancer (LACC) has been shown to be of substantial prognostic importance. The aims of the present study were therefore to investigate if hypoxia could be identified by a newly validated hypoxic gene expression classifier and used as a prognostic factor for disease free survival (DFS). Material and methods Paraffin embedded biopsies were obtained from 190 patients with LACC with squamous cell carcinoma treated 2005–2016 with chemo-radiation and image guided adaptive brachytherapy. Analysis of hypoxia was successful in 183 patients (96%). Hypoxic classification of tumours into ‘more’ or ‘less’ hypoxic was based on 15 genes using the same method as in a prospective head and neck cancer trial (NCT02661152). HPV was genotyped using INNO-LiPA. Local tumour invasion was evaluated by the T-score. Primary endpoint was DFS analysed by Kaplan-Meier and Cox regression. Events were death of any cause, persistent disease, or recurrence. Results The FIGO2009 stage distribution was IB–IIA 9%, IIB 64%, and III–IVA 27%, and mean T-score was 7.2. Pathological nodes were present in 53%. Median observation time was 5.2 years. Local control rate at 5 years was 96%, and pelvic (loco-regional) control 91%. Overall, 36% of the tumours were classified as ‘more’ hypoxic. The frequency of ‘more’ hypoxic tumours increased with local tumour intrusion (30% for T-score 0–9 vs. 55% for T-score ≥10, p = 0.004). Hypoxia was associated with decreased DFS in univariate, HR 1.71 (1.04–2.82), and multivariate analysis, HR 1.75 (1.04–2.92), and the effect was particularly observed among tumours with a T-score ≥10. HPV 16/18 was not associated with improved DFS in neither in univariate nor in multivariate analysis. Conclusion Hypoxic gene expression is a prominent prognostic factor for DFS in LACC with SCC histology and should be considered for treatment stratification in clinical trials.

Vascular characteristics and expression of hypoxia genes in Tibetan pigs' hearts.

BackgroundTibetan pigs have exhibited unique characteristics from low‐altitudes pigs and adapted well to the Qinghai‐Tibet Plateau.ObjectivesThe current study was undertaken to investigate the hypoxic adaptation of heart in Tibetan pigs.MethodsThe hearts of Tibetan pigs and Landrace pigs raised at high or low altitudes were compared using 3D casting technology, scanning electron microscopy and real‐time quantitative PCR (qRT‐PCR).ResultsWe found that the ratio of the major axis to the minor axis and the density of the heart were significantly higher in Tibetan pigs than in Landrace pigs (p < 0.05). Tibetan pigs had larger diameters and higher densities of arterioles than Landrace pigs (p < 0.05), and these features have a similar variation with the expression of vascular endothelial growth factor (VEGF). The cardiac expression levels of hypoxia‐inducible factor‐1α (HIF‐1α) and endothelial nitric oxide synthase (eNOS) were significantly higher in pigs reared at high altitudes than in those reared at low altitudes (p < 0.05). In contrast, Egl nine homolog 1 (EGLN1) had the opposite trend with respect to HIF‐1α and eNOS and was related to red blood cell (RBC) counts. Notably, the expressions of erythropoietin (EPO) and endothelial PAS domain‐containing protein 1 (EPAS1) were significantly higher in Landrace pigs kept at high altitudes than in the others (p < 0.05) and were associated with haemoglobin.ConclusionsThese findings show that the regulation of the heart function of Tibetan pigs in a hypoxic environment is manifested at various levels to ensure the circulation of blood under extreme environmental conditions.

A Hypoxia Signature for Predicting Prognosis and Tumor Immune Microenvironment in Adrenocortical Carcinoma.

Adrenocortical carcinoma (ACC) is a rare malignancy with dismal prognosis. Hypoxia is one of characteristics of cancer leading to tumor progression. For ACC, however, no reliable prognostic signature on the basis of hypoxia genes has been built. Our study aimed to develop a hypoxia-associated gene signature in ACC. Data of ACC patients were obtained from TCGA and GEO databases. The genes included in hypoxia risk signature were identified using the Cox regression analysis as well as LASSO regression analysis. GSEA was applied to discover the enriched gene sets. To detect a possible connection between the gene signature and immune cells, the CIBERSORT technique was applied. In ACC, the hypoxia signature including three genes (CCNA2, COL5A1, and EFNA3) was built to predict prognosis and reflect the immune microenvironment. Patients with high-risk scores tended to have a poor prognosis. According to the multivariate regression analysis, the hypoxia signature could be served as an independent indicator in ACC patients. GSEA demonstrated that gene sets linked to cancer proliferation and cell cycle were differentially enriched in high-risk classes. Additionally, we found that PDL1 and CTLA4 expression were significantly lower in the high-risk group than in the low-risk group, and resting NK cells displayed a significant increase in the high-risk group. In summary, the hypoxia risk signature created in our study might predict prognosis and evaluate the tumor immune microenvironment for ACC.

Developmental programming of DNA methylation and gene expression patterns is associated with extreme cardiovascular tolerance to anoxia in the common snapping turtle

BackgroundEnvironmental fluctuation during embryonic and fetal development can permanently alter an organism’s morphology, physiology, and behaviour. This phenomenon, known as developmental plasticity, is particularly relevant to reptiles that develop in subterranean nests with variable oxygen tensions. Previous work has shown hypoxia permanently alters the cardiovascular system of snapping turtles and may improve cardiac anoxia tolerance later in life. The mechanisms driving this process are unknown but may involve epigenetic regulation of gene expression via DNA methylation. To test this hypothesis, we assessed in situ cardiac performance during 2 h of acute anoxia in juvenile turtles previously exposed to normoxia (21% oxygen) or hypoxia (10% oxygen) during embryogenesis. Next, we analysed DNA methylation and gene expression patterns in turtles from the same cohorts using whole genome bisulfite sequencing, which represents the first high-resolution investigation of DNA methylation patterns in any reptilian species.ResultsGenome-wide correlations between CpG and CpG island methylation and gene expression patterns in the snapping turtle were consistent with patterns observed in mammals. As hypothesized, developmental hypoxia increased juvenile turtle cardiac anoxia tolerance and programmed DNA methylation and gene expression patterns. Programmed differences in expression of genes such as SCN5A may account for differences in heart rate, while genes such as TNNT2 and TPM3 may underlie differences in calcium sensitivity and contractility of cardiomyocytes and cardiac inotropy. Finally, we identified putative transcription factor-binding sites in promoters and in differentially methylated CpG islands that suggest a model linking programming of DNA methylation during embryogenesis to differential gene expression and cardiovascular physiology later in life. Binding sites for hypoxia inducible factors (HIF1A, ARNT, and EPAS1) and key transcription factors activated by MAPK and BMP signaling (RREB1 and SMAD4) are implicated.ConclusionsOur data strongly suggests that DNA methylation plays a conserved role in the regulation of gene expression in reptiles. We also show that embryonic hypoxia programs DNA methylation and gene expression patterns and that these changes are associated with enhanced cardiac anoxia tolerance later in life. Programming of cardiac anoxia tolerance has major ecological implications for snapping turtles, because these animals regularly exploit anoxic environments throughout their lifespan.

Status of hypoxia-inducible factor-1α expression in non-small cell lung cancer.

According to the latest statistics from WHO for all cancers, lung cancer tops the list with a 14.5% prevalence and a 22% death rate in men, similar to the prevalence in women, which is 13.8%. It is also the number one killer of cancer in China, with 40 in every 100,000 people suffering from lung cancer. HIF-1α is widely present in human cells in hypoxic environments. It regulates the body's response to hypoxia, cell oxygen balance, and hypoxia gene expression; participates in the proliferation and apoptosis of non-small cell lung cancer cells; participates in the invasion, metastasis, and neovascularization of tumor tissues; and affects the treatment and prognosis of non-small cell lung cancer. In view of the role of HIF-1α in the occurrence and development of non-small cell lung cancer, blocking HIF-1α by use of a single medication or combination chemotherapy has become a research hotspot. This review summarizes the role of HIF-1α in non-small cell lung cancer and provides new ideas for the treatment of this cancer type by synthesizing the research results of various authors.

Identification of a Hypoxia-Related Molecular Classification and Hypoxic Tumor Microenvironment Signature for Predicting the Prognosis of Patients with Triple-Negative Breast Cancer.

PurposeThe hypoxic tumor microenvironment was reported to be involved in different tumorigenesis mechanisms of triple-negative breast cancer (TNBC), such as invasion, immune evasion, chemoresistance, and metastasis. However, a systematic analysis of the prognostic prediction models based on multiple hypoxia-related genes (HRGs) has not been established in TNBC before in the literature. We aimed to develop and verify a hypoxia gene signature for prognostic prediction in TNBC patients.MethodsThe RNA sequencing profiles and clinical data of TNBC patients were generated from the TCGA, GSE103091, and METABRIC databases. The TNBC-specific differential HRGs (dHRGs) were obtained from differential expression analysis of hypoxia cultured TNBC cell lines compared with normoxic cell lines from the GEO database. Non-negative matrix factorization (NMF) method was then performed on the TNBC patients using the dHRGs to explore a novel molecular classification on the basis of the dHRG expression patterns. Prognosis-associated dHRGs were identified by univariate and multivariate Cox regression analysis to establish the prognostic risk score model.ResultsBased on the expressions of 205 dHRGs, all the patients in the TCGA training cohort were categorized into two subgroups, and the patients in Cluster 1 demonstrated worse OS than those in Cluster 2, which was validated in two independent cohorts. Additionally, the effects of somatic copy number variation (SCNV), somatic single nucleotide variation (SSNV), and methylation level on the expressions of dHRGs were also analyzed. Then, we performed Cox regression analyses to construct an HRG-based risk score model (3-gene dHRG signature), which could reliably discriminate the overall survival (OS) of high-risk and low-risk patients in TCGA, GSE103091, METABRIC, and BMCHH (qRT-PCR) cohorts.ConclusionsIn this study, a robust predictive signature was developed for patients with TNBC, indicating that the 3-gene dHRG model might serve as a potential prognostic biomarker for TNBC.

Transcriptomic profiling of Gh/Igf system reveals a prompted tissue-specific differentiation and novel hypoxia responsive genes in gilthead sea bream

A customized PCR-array was used for the simultaneous gene expression of the Gh/Igf system and related markers of muscle growth, and lipid and energy metabolism during early life stages of gilthead sea bream (60–127 days posthatching). Also, transcriptional reprogramming by mild hypoxia was assessed in fingerling fish with different history trajectories on O2 availability during the same time window. In normoxic fish, the expression of almost all the genes in the array varied over time with a prompted liver and muscle tissue-specific differentiation, which also revealed temporal changes in the relative expression of markers of the full gilthead sea bream repertoire of Gh receptors, Igfs and Igf-binding proteins. Results supported a different contribution through development of ghr and igf subtypes on the type of action of GH via systemic or direct effects at the local tissue level. This was extensive to Igfbp1/2/4 and Igfbp3/5/6 clades that clearly evolved through development as hepatic and muscle Igfbp subtypes, respectively. This trade-off is however very plastic to cope changes in the environment, and ghr1 and igfbp1/3/4/5 emerged as hypoxic imprinting genes during critical early developmental windows leading to recognize individuals with different history trajectories of oxygen availability and metabolic capabilities later in life.

A combined hypoxia and immune gene signature for predicting survival and risk stratification in triple-negative breast cancer.

Background: Increasing evidence showed that the clinical significance of the interaction between hypoxia and immune status in tumor microenvironment. However, reliable biomarkers based on the hypoxia and immune status in triple-negative breast cancer (TNBC) have not been well established. This study aimed to explore a gene signature based on the hypoxia and immune status for predicting prognosis, risk stratification, and individual treatment in TNBC.Methods: Hypoxia-related genes (HRGs) and Immune-related genes (IRGs) were identified using the weighted gene co-expression network analysis (WGCNA) method and the single-sample gene set enrichment analysis (ssGSEA Z-score) with the transcriptomic profiles from Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) cohort. Then, prognostic hypoxia and immune based genes were identified in TNBC patients from the METABRIC (N = 221), The Cancer Genome Atlas (TCGA) (N = 142), and GSE58812 (N = 107) using univariate cox regression model. A robust hypoxia-immune based gene signature for prognosis was constructed using the least absolute shrinkage and selection operator (LASSO) method. Based on the cross-cohort prognostic hypoxia–immune related gene signature, a comprehensive index of hypoxia and immune was developed and two risk groups with distinct hypoxia–immune status were identified. The prognosis value, hypoxia and immune status, and therapeutic response in different risk groups were analyzed. Furthermore, a nomogram was constructed to predict the prognosis for individual patients, and an independent cohort from the gene expression omnibus (GEO) database was used for external validation.Results: Six cross-cohort prognostic hypoxia–immune related genes were identified to establish the comprehensive index of hypoxia and immune. Then, patients were clustered into high- and low-risk groups based on the hypoxia–immune status. Patients in the high-risk group showed poorer prognoses to their low-risk counterparts, and the nomogram we constructed yielded favorable performance to predict survival and risk stratification. Besides, the high-risk group had a higher expression of hypoxia-related genes and correlated with hypoxia status in tumor microenvironment. The high-risk group had lower fractions of activated immune cells, and exhibited lower expression of immune checkpoint markers. Furthermore, the ratio of complete response (CR) was greatly declined, and the ratio of breast cancer related events were significantly elevated in the high-risk group.Conclusion: The hypoxia–immune based gene signature we constructed for predicting prognosis was developed and validated, which may contribute to the optimization of risk stratification for prognosis and personalized treatment in TNBC patients.

OC-0279 Hypoxic gene expression is a prognostic factor for survival in locally advanced SCC cervical cancer

OC-0279 Hypoxic gene expression is a prognostic factor for survival in locally advanced SCC cervical cancer

OC-0066 Heterogeneity of a hypoxia gene signature within the dominant tumor lesion in prostate cancer

OC-0066 Heterogeneity of a hypoxia gene signature within the dominant tumor lesion in prostate cancer

Gene expression profiles of myopic mouse scleral fibroblasts: a bioinformatics analysis based on single-cell RNA sequencing

To investigate the changes in gene expression profiles of mouse scleral fibroblasts after myopia using single-cell RNA sequencing technology and explore the mechanism of dysfunction of the scleral fibroblasts in myopia. Normal healthy C57BL/6J mice were randomly divided into negative control group and myopia model group (n=6), and in the latter group, form deprivation myopia was induced in the right eye using translucent goggles. Single cell capture was performed in the right eye to obtain the scleral fibroblasts for RNA sequencing. The differentially expressed genes (DEGs) were screened and GO and KEGG analyses were carried out for functionally significant enrichment analysis. Comparison of the gene expression profiles identified a total of 169 DEGs between the myopia model group and the negative control group (P>0.05), including 112 up-regulated and 57 down-regulated genes. GO function analysis showed that the DEGs were involved in leukocyte aggregation, differentiation and adhesion and other inflammation-related terms; ATP metabolism and binding, redox process, oxidative stress response, oxidative phosphorylation and other GO terms related to hypoxia; protein folding, protein transport, negative regulation of protein metabolism, endoplasmic reticulum (ER), ER cavity, ER stress and other biological processes related to protein and ER stress. KEGG analysis analysis showed that the significantly enriched pathways of the DEGs involved mainly the TCA cycle, oxidative phosphorylation, PPAR signaling, oxidative phosphorylation and other pathways related to hypoxia; MAPK signaling pathway related to inflammation; leukocyte transendothelial transport; and protein-related Parkinson's disease, Huntington's disease, protein digestion and absorption pathways. The dysfunction of the scleral fibroblasts occurs in myopia through complex mechanisms involving inflammation, hypoxia, protein regulation, and ER stress-related gene expression and pathway regulation.

Multi-Omics Profiling Identifies Risk Hypoxia-Related Signatures for Ovarian Cancer Prognosis.

BackgroundOvarian cancer (OC) has the highest mortality rate among gynecologic malignancy. Hypoxia is a driver of the malignant progression in OC, which results in poor prognosis. We herein aimed to develop a validated model that was based on the hypoxia genes to systematically evaluate its prognosis in tumor immune microenvironment (TIM).ResultsWe identified 395 hypoxia-immune genes using weighted gene co-expression network analysis (WGCNA). We then established a nine hypoxia-related genes risk model using least absolute shrinkage and selection operator (LASSO) Cox regression, which efficiently distinguished high-risk patients from low-risk ones. We found that high-risk patients were significantly related to poor prognosis. The high-risk group showed unique immunosuppressive microenvironment, lower antigen presentation, and higher levels of inhibitory cytokines. There were also significant differences in somatic copy number alterations (SCNAs) and mutations between the high- and low-risk groups, indicating immune escape in the high-risk group. Tumor immune dysfunction and exclusion (TIDE) and SubMap algorithms showed that low-risk patients are significantly responsive to programmed cell death protein-1 (PD-1) inhibitors.ConclusionsIn this study, we highlighted the clinical significance of hypoxia in OC and established a hypoxia-related model for predicting prognosis and providing potential immunotherapy strategies.

ECOA-4. Hypoxia alters the DNA methylation profile of glioblastoma tumor cells

Abstract Glioblastoma (GBM) is the deadliest and most vascularized brain tumor in adults; however, blood circulation is highly inefficient in these tumors, contributing to areas of cell death (necrosis) within the tumor, which is likely due to oxygen deprivation (hypoxia). Hypoxia plays a major role in tumor growth, invasion, and resistance to therapy. Hypoxic stress has been linked to several changes that are fundamental to the malignant progression of GBM and other tumor types. Pimonidazole (PIMO) is an exogenous marker of hypoxia that is used to delineate hypoxic regions in several tumor types. To date, a clear hypoxia gene signature has not been specifically described for GBM. We hypothesize that specific cellular pathways are differentially regulated in hypoxic tumor niches and can serve as novel actionable targets for treatment-resistant tumor cells in GBM. Over the past 3 years, we have administered PIMO to 35 patients with primary IDH1/2 wild-type GBM and isolated PIMO-positive and PIMO-negative tumor cells by laser capture microdissection using a PIMO-specific antibody on frozen tumor specimens. Total genomic DNA was isolated and subjected to DNA methylation profiling using the Illumina Infinium Methylation EPIC array. Our preliminary results suggest that PIMO-positive (hypoxic) tumor cells display a distinct DNA methylation profile that corresponds to changes in expression of a set of genes associated with immune regulation, angiogenesis, and proliferation. Furthermore, multiple CpG sites within the promoter of some genes are differentially methylated in hypoxic cells, suggesting these genes may be epigenetically regulated under hypoxia. In conclusion, our results indicate that hypoxia is associated with distinct epigenetic alterations in tumor cells which may alter how these cells respond to low oxygen levels and can further be utilized to uncover the epigenomic vulnerabilities of hypoxic tumor cells in GBM.

Constructe a novel 5 hypoxia genes signature for cervical cancer

BackgroundHypoxia, which affects the development, metastasis and prognosis of cancer, represents a key feature of cancer. This study describe a hypoxia risk factor model, with predicting the prognosis of cervical cancer.MethodsBased on hypoxia pathway related genes, we divided cervical cancer samples into high and low expression groups. A cox analysis was then performed. Genes from these cervical cancer samples showing a significant impact on OS were selected for cluster analysis to obtain two subtypes. The TPM dataset of TCGA was divided into training and validation sets. For the training set, a lasso analysis was conducted as based on cox analysis of meaningful genes and a risk factor model was constructed. The constructed model was verified in internal and external data sets. Finally, RT-PCR, immunohistochemistry were used to detect the expression of relative genes or proteins and functional assays were used to evaluate the biological function of signature genes.ResultsTwo molecular subtypes were obtained, Cluster2 vs Cluster1.These subtypes were obtained by clustering with a total of 149 DEGs (Differential expressed genes) being in line with this standard, of which 27 were up-regulated and 122 were down-regulated. The five genes with lambda = 0.0571 were selected to construct the model, the RiskScore = AK4*0.042 + HK2*0.021 + P4HA1*0.22 + TGFBI*0.1 + VEGFA*0.077. Further, in order to verify the signature, we used TCGA-test and GSE44001 chip datasets to test, and finally got a good risk prediction effect in those datasets. Moreover, the result of RT-PCR and immunohistochemistry demonstrated that AK4, HK2, P4HA1, TGFBI and VEGFA were all highly expressed in these cervical cancer tissue samples. The functional study shown that expression of AK4, HK2, P4HA1, TGFBI and VEGFA can regulate the proliferation, migration, and invasion ability of cervical cancer cells in vitro.ConclusionsIn summary, we developed a 5-gene signature prognostic hierarchical system based on the hypoxic pathway of cervical cancer, which is independent of clinical characteristics. And also conducted experimental verifications on these signature gene. Therefore, we propose that use of this classifier as a molecular diagnostic test can provide an effective means for evaluating the prognostic risk of cervical cancer patients, and provide potential targets for the treatment of cervical cancer patients.

Targeting HIF-activated collagen prolyl 4-hydroxylase expression disrupts collagen deposition and blocks primary and metastatic uveal melanoma growth.

Uveal melanoma (UM) is the most prevalent primary intraocular malignancy in adults, and patients that develop metastases (~50%) survive <1 year, highlighting the urgent need for new therapies. TCGA has recently revealed that a hypoxia gene signature is associated with poor UM patient prognosis. Here we show that expression of hypoxia-regulated collagen prolyl-4-hydroxylase genes P4HA1 and P4HA2 is significantly upregulated in UM patients with metastatic disease and correlates with poor prognosis, suggesting these enzymes might be key tumor drivers. We targeted hypoxia-induced expression of P4HA1/2 in UM with KCN1, a hypoxia inducible factor-1 (HIF-1) pathway inhibitor and found potent inhibition of primary and metastatic disease and extension of animal survival, without overt side effects. At the molecular level, KCN1 antagonized hypoxia-induced expression of P4HA1 and P4HA2, which regulate collagen maturation and deposition in the extracellular matrix. The treatment decreased prolyl hydroxylation, induced proteolytic cleavage and rendered a disordered structure to collagen VI, the main collagen produced by UM, and reduced UM cell invasion. Together, these data demonstrate that extracellular collagen matrix formation can be targeted in UM by inhibiting hypoxia-induced P4HA1 and P4HA2 expression, warranting further development of this strategy in patients with uveal melanoma.

Abstract 1613: A hypoxia-driven transcriptional program characterizes acquired resistance to immune checkpoint inhibitors in lung cancer

Abstract Immune checkpoint inhibitors (ICIs) that block negative regulators of T cell function can generate lasting responses in lung cancer patients, yet only a minority of tumors respond to therapy and ~50% of patients whose tumors initially respond eventually develop acquired resistance. Mechanisms of acquired resistance to ICIs in lung cancer are not fully understood and strategies to overcome resistance are lacking. To address this challenge, we developed a new mouse model of acquired resistance to ICIs by knocking out the DNA mismatch repair gene Msh2 in Kras mutant mouse lung adenocarcinoma cells, leading to a higher mutation burden in these cells. Unlike Msh2 WT controls, Msh2 KO tumors transplanted into syngeneic mice are sensitive to ICIs. Importantly, chronic treatment with αPD-1 or αPD-1 + αCTLA-4 led to Msh2 KO tumors that initially responded to therapy but then developed acquired resistance. Tumors remained resistant to ICIs upon transplantation to new hosts, suggesting that tumor-intrinsic programs drive resistance. Gene set enrichment analysis (GSEA) of single cell RNA sequencing data revealed that tumors with acquired resistance to ICIs downregulated antigen processing and presentation gene signatures compared to tumors that were regressing on therapy. Specifically, tumor cells with acquired resistance downregulated expression of MHC class II, which was confirmed by flow cytometry, and this was accompanied by a decrease in expression of Stat1, Irf1, and Ciita, which drive MHC-II expression in response to IFNγ. Depletion of CD4+ T cells during αPD-1 + αCTLA-4 treatment abrogated the response to therapy, suggesting a functional role for tumor cell MHC-II expression. GSEA also revealed that tumors with acquired resistance to ICIs upregulated hypoxia and glycolysis gene signatures compared to tumors responding to therapy. Consistent with these results, resistant tumor cells had increased hypoxia, detected by pimonidazole staining, compared to tumor cells responding to therapy. Tumor infiltrating lymphocytes have been shown to be excluded from hypoxic tumor regions, suggesting that hypoxia in the tumor microenvironment may contribute to acquired resistance to ICIs by excluding T cells from these regions. Moreover, we found that Msh2 KO tumor cells upregulated MHC-II in response to IFNγ under normoxic in vitro conditions, but had lower MHC-II expression under hypoxia. Our data suggest that tumor hypoxia may contribute to both tumor-cell intrinsic transcriptional programs, such as alterations in antigen processing and presentation machinery, as well as tumor-cell extrinsic effects, such as exclusion of T cells from the tumor microenvironment, which together may impact the development of acquired resistance to immune checkpoint blockade. Our results can inform strategies to overcome acquired resistance, addressing a critical unmet need in cancer therapy. Citation Format: Camila Robles-Oteiza, Katherine Hastings, Susan M. Kaech, Katerina Politi. A hypoxia-driven transcriptional program characterizes acquired resistance to immune checkpoint inhibitors in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1613.

Abstract 2655: Abundance of intra-tumoral angiogenesis is associated with attenuated tumor aggressive biology, tumor microenvironment, as well as worse survival in gastric cancer patients

Abstract Background: Across the world, gastric cancer is the third leading cause of cancer deaths. There is an urgent need to develop a predictive biomarker that help guide treatment decision-making to precisely address gastric cancer progression and metastasis. Angiogenesis is the creation of de novo blood vessels from existing ones, and it is a cornerstone of cancer as it paves the way for cancer progression and metastasis. Angiogenic factors such as vascular endothelial growth factor (VEGF) and endothelial cell growth factor (EGF) are produced by cancer and stromal cells in many types of cancers including gastric cancer. We hypothesized that intra-tumoral angiogenesis plays a role in gastric cancer aggressiveness. Methods: The angiogenesis level of tumors from 1,348 gastric cancer patients in multiple cohorts was measured using the Gene Set Variation Analysis (GSVA) method to score tumor transcriptomes with the Hallmark angiogenesis gene set. Results: Gastric cancers with high angiogenesis score were significantly associated with high expression of multiple angiogenesis-related genes including VEGF-related, endothelial cell marker-related, and vascular stability-related genes in the TCGA and GSE84437 cohorts. Furthermore, in both cohorts, the angiogenesis score was moderately correlated with not only vessel-related stromal cells (endothelial cells [spearman r = 0.557, 0.473], lymphatic [r = 0.520, 0.597] and microvascular endothelial cells [r = 0.470, 0.460], and pericytes [r = 0.555, 0.301]), but also fibroblasts (r = 0.485, 0.467) and chondrocytes (r = 0.565, 0.601) in the tumor microenvironment. Tumors with high angiogenesis score were associated with a lower fraction of Th1, Th2, and dendritic cells, and a higher fraction of M1 macrophages compared with low angiogenesis score tumors. Gastric cancer with a high angiogenesis score was enriched with coagulation, epithelial mesenchymal transition, hypoxia, and TGF-β signaling gene sets in the TCGA, GSE84437, and GSE26253 cohorts. Interestingly, a high angiogenesis score was significantly associated with greater AJCC stage and T-category, but not N-category. Finally, patients with a high score had significantly shorter survival in three cohorts (overall survival: p = 0.002 in the TCGA, p &amp;lt; 0.001 in the GSE84437, and disease-free survival: p = 0.011 in GSE26253). Conclusion: The angiogenesis score can be a useful tool to assess intra-tumoral angiogenesis, which is associated with clinical outcomes in gastric cancer patients. Citation Format: Masanori Oshi, Tae Hee Kim, Vikas Satyananda, Fernando A. Angarita, Yoshihisa Tokumaru, Li Yan, Ryusei Matsuyama, Itaru Endo, Kazuaki Takabe. Abundance of intra-tumoral angiogenesis is associated with attenuated tumor aggressive biology, tumor microenvironment, as well as worse survival in gastric cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2655.

Identification and Validation of Autophagy-Related Gene Nomograms to Predict the Prognostic Value of Patients with Cervical Cancer.

Autophagy is a process of engulfing one's own cytoplasmic proteins or organelles and coating them into vesicles, fusing with lysosomes to form autophagic lysosomes, and degrading the contents it encapsulates. Increasing studies have shown that autophagy disorders are closely related to the occurrence of tumors. However, the prognostic role of autophagy genes in cervical cancer is still unclear. In this study, we constructed risk signatures of autophagy-related genes (ARGs) to predict the prognosis of cervical cancer. The expression profiles and clinical information of autophagy gene sets were downloaded from TCGA and GSE52903 queues as training and validation sets. The normal cervical tissue expression profile data from the UCSC XENA website (obtained from GTEx) were used as a supplement to the TCGA normal cervical tissue. Univariate COX regression analysis of 17 different autophagy genes was performed with the consensus approach. Tumor samples from TCGA were divided into six subtypes, and the clinical traits of the six subtypes had different distributions. Further absolute shrinkage and selection operator (LASSO) and multivariable COX regression yielded an autophagy genetic risk model consisting of eight genes. In the training set, the survival rate of the high-risk group was lower than that of the low-risk group (p < 0.0001). In the validation set, the AUC area of the receiver operating characteristic (ROC) curve was 0.772 for the training set and 0.889 for the verification set. We found that high and low risk scores were closely related to TNM stage (p < 0.05). The nomogram shows that the risk score combined with other indicators, such as G, T, M, and N, better predicts 1-, 3-, and 5-year survival rates. Decline curve analysis (DCA) shows that the risk model combined with other indicators produces better clinical efficacy. Immune cells with an enrichment score of 28 showed statistically significant differences related to high and low risk. GSEA enrichment analysis showed the main enrichment being in KRAS activation, genes defining epithelial and mesenchymal transition (EMT), raised in response to the low oxygen level (hypoxia) gene and NF-kB in response to TNF. These pathways are closely related to the occurrence of tumors. Our constructed autophagy risk signature may be a prognostic tool for cervical cancer.