Angiotensinogen, Angiotensin-Converting Enzyme, and Chymase Gene Polymorphisms as Biomarkers for Basal Cell Carcinoma Susceptibility.

INTRODUCTION: The intake of angiotensin-converting enzyme (ACE) inhibitors and specific antagonists of angiotensin II receptors, widely used as antihypertensive drugs, significantly reduces the risk of developing basal cell carcinoma (BCC), highlighting the possible tumorigenic role of angiotensin II (AngII). We present here the investigated genetic association between the development of BCC and functional DNA polymorphisms M235T, I/D, and A1903G in the genes of angiotensinogen (AGT), angiotensin-converting enzyme (ACE), and chymase (CMA1), which mediate AngII production levels. METHODS: DNA samples of 203 unrelated Greeks were studied, including 100 patients with BCC and 103 matched healthy controls. RESULTS: The MT genotype of the AGT-M235T polymorphism was significantly more prevalent in the patient group (78.0%) versus the healthy control group (28.3%; p < 0.001). The DD genotype of the ACE-I/D polymorphism was also increased in BCC patients (72.8%) compared to controls (46.2%; p = 0.001). The heterozygous AG genotype of CMA1-A1903G was significantly more frequent in the BCC group (86%) than in the healthy controls (50.5%; p < 0.001). CONCLUSIONS: The MT, DD, and AG genotypes of the AGT- M235T, ACE-I/D, and CMA1-A1903G polymorphisms, respectively, were significantly increased in frequency within the group of cancer patients compared to the healthy controls. All three genotypes correspond to increased enzyme levels or activity and result in increased levels of AngII; therefore, they may be potentially utilized as reliable biomarkers associated with an individual's increased risk for BCC development.

Functional Exploration of Conserved Sequences in the Distal Face of Angiotensinogen-Brief Report.

BACKGROUND: Angiotensinogen (AGT) is an essential component in the renin-angiotensin system. AGT has highly conserved sequences in the loop and ß-sheet regions among species; however, their functions have not been studied. METHODS: Adeno-associated viral vector (AAV) serotype 2/8 encoding mouse AGT with mutations of conserved sequences in the loop (AAV.loop-Mut), ß-sheet (AAV.ßsheet-Mut), or both regions (AAV.loop/ßsheet-Mut) was injected into male hepatocyte-specific AGT-deficient (hepAGT-/-) mice in an LDL (low-density lipoprotein) receptor-deficient background. AAV containing mouse wild-type AGT (AAV.mAGT) or a null vector (AAV.null) were used as controls. Two weeks after AAV administration, all mice were fed a western diet for 12 weeks. To determine how AGT secretion is regulated in hepatocytes, AAVs containing the above mutations were transducted into HepG2 cells. RESULTS: In hepAGT-/- mice infected with AAV.loop-Mut or ßsheet-Mut, plasma AGT concentrations, systolic blood pressure, and atherosclerosis were comparable to those in AAV.mAGT-infected mice. Interestingly, plasma AGT concentrations, systolic blood pressure, and atherosclerotic lesion size in hepAGT-/- mice infected with AAV.loop/ßsheet-Mut were not different from mice infected with AAV.null. In contrast, hepatic Agt mRNA abundance was elevated to a comparable magnitude as AAV.mAGT-infected mice. Immunostaining showed that AGT protein was accumulated in hepatocytes of mice infected with AAV.loop/ßsheet-Mut or HepG2 cells transducted with AAV.loop/ßsheet-Mut. Accumulated AGT was not located in the endoplasmic reticulum. CONCLUSIONS: The conserved sequences in either the loop or ß-sheet region individually have no effect on AGT regulation, but the conserved sequences in both regions synergistically contribute to the secretion of AGT from hepatocytes.

Pathologic HDAC1/c-Myc signaling axis is responsible for angiotensinogen transcription and hypertension induced by high-fat diet.

High-fat diet (HFD)-induced obesity is a cause of resistant hypertension. We have shown a possible link between histone deacetylases (HDACs) and renal angiotensinogen (Agt) upregulation in the HFD-induced hypertension, whereas the underlying mechanisms remain to be elucidated. Here, using a HDAC1/2 inhibitor romidepsin (FK228) and siRNAs, we determined roles of HDAC1 and HDAC2 in HFD-induced hypertension and found the pathologic signaling axis between HDAC1 and Agt transcription. Treatment with FK228 canceled the increased blood pressure of male C57BL/6 mice induced by HFD. FK228 also blocked upregulation of renal Agt mRNA, protein, angiotensin II (Ang II) or serum Ang II. Activation and nuclear accumulation of both HDAC1 and HDAC2 occurred in the HFD group. The HFD-induced HDAC activation was associated with an increase in deacetylated c-Myc transcription factor. Silencing of HDAC1, HDAC2 or c-Myc in HRPTEpi cells decreased Agt expression. However, only HDAC1 knockdown, but not HDAC2, increased c-Myc acetylation, suggesting selective roles in two enzymes. Chromatin immunoprecipitation assay revealed that HFD induced the binding of HDAC1 and deacetylated c-Myc at the Agt gene promoter. A putative c-Myc binding sequence in the promotor region was necessary for Agt transcription. Inhibition of c-Myc downregulated Agt and Ang II levels in kidney and serum, ameliorating HFD-induced hypertension. Thus, the abnormal HDAC1/2 in the kidney may be responsible for the upregulation of the Agt gene expression and hypertension. The results expose the pathologic HDAC1/c-myc signaling axis in kidney as a promising therapeutic target for obesity-associated resistant hypertension.

Angiotensinogen: More Than its Downstream Products: Evidence From Population Studies and Novel Therapeutics.

The renin-angiotensin-aldosterone system (RAAS) is a well-defined pathway playing a key role in maintaining circulatory homeostasis. Abnormal activation of RAAS contributes to development of cardiovascular disease, including heart failure, cardiac hypertrophy, hypertension, and atherosclerosis. Although several key RAAS enzymes and peptide hormones have been thoroughly investigated, the role of angiotensinogen-the precursor substrate of the RAAS pathway-remains less understood. The study of angiotensinogen single-nucleotide polymorphisms (SNPs) has provided insight into associations between angiotensinogen and hypertension, congestive heart failure, and atherosclerotic cardiovascular disease. Targeted drug therapy of RAAS has dramatically improved clinical outcomes for patients with heart failure, myocardial infarction, and hypertension. However, all such therapeutics block RAAS components downstream of angiotensinogen and elicit compensatory pathways that limit their therapeutic efficacy as monotherapy. Upstream RAAS targeting by an angiotensinogen inhibitor has the potential to be more efficacious in patients with suboptimal RAAS inhibition and has a better safety profile than multiagent RAAS blockade. Newly developed therapeutics that target angiotensinogen through antisense oligonucleotides or silencer RNA technologies are providing a novel perspective into the pathobiology of angiotensinogen and show promise as the next frontier in the treatment of cardiovascular disease.

Structural and dynamic investigation of non-synonymous variations in Renin-AGT complex revealed altered binding via hydrogen-bonding network reprogramming to accelerate the hypertension pathway.

Hypertension is one of the major issues worldwide and one of the main factors involved in heart and kidney failure. Angiotensinogen and renin are key components of the renin-angiotensin-aldosterone system, which plays an indispensable role in hypertension. The aim of this study was to find out the non-synonymous mutations and structure-based mutation-function correlation in the renin-AGT complex and reveal the most deleterious mutations to accelerated hypertension. In the current study, we employed computational modeling and molecular simulation approaches to demonstrate the impact of specific mutations in the REN-AGT interface in hypertension. Computational algorithms, that is, PhD-SNP, PolyPhen-1, MAPP, Sorting Intolerant from Tolerant, Screening of non-acceptable polymorphism, PredictSNP, PolyPhen-2, and Protein Analysis Through Evolutionary Relationships predicted 20 mutations as deleterious in AGT while only five mutations were confirmed as deleterious in the renin protein. Investigation of the bonding analysis revealed that two mutations S107L and V193F in renin altered the hydrogen-bonding paradigm at the interface site. Furthermore, exploration of structural-dynamic behaviors demonstrated by that these mutations also increases the structural stability to regulate the expression of disease pathway. The flexibility index of each residues and structural compactness analysis further validated the findings by portraying the difference in the dynamic behavior in contrast to the wild type. Binding energy calculations based on molecular mechanics/generalized Born surface area methods were used which further established the binding differences between the wild type, S107L, and V193F mutant variants. The total binding energy for wild type, S107L, and V193F was reported to be -27.79, -47.72, and -38.25, respectively. In conclusion, these two mutations increase the binding free energy alongside the docking score to enhance the binding between renin and AGT to overexpress this pathway in a hypertension disease condition. Patients with these mutations may be screened for potential therapeutic intervention.

Associations between AGT M235T Polymorphism and Cancer: An Updated Meta-Analysis.

We assessed the relationship between AGT gene M235T polymorphism and the susceptibility to cancer by performing an updated meta-analysis. This study retrospectively searched related articles in the electronic databases. Afterwards, we determined combined odds ratios (ORs) and related 95% confidence intervals (CIs) by the fixed- or random-effects model. The present meta-analysis enrolled altogether 9 articles. On the whole, the relationship between AGT M235T polymorphism and the cancer risk was not significant among the entire population (TT vs. MM: OR = 1.28, 95%CI = 0.80 - 2.04; TM vs. MM: OR = 0.90, 95%CI = 0.53 - 1.52; recessive model: OR = 1.13, 95%CI = 0.83 - 1.52; dominant model: OR = 0.93, 95%CI = 0.55 - 1.57). Subgroup analysis by ethnicity, cancer type, and study quality for the relationship between the AGT M235T polymorphism and cancer risk showed no significant association. According to findings in the present meta-analysis, AGT M235T polymorphism may not be related to cancer susceptibility.

Associations between AGT, MTHFR, and VEGF gene polymorphisms and preeclampsia in the Chinese population.

INTRODUCTION: Preeclampsia (PE) is a pregnancy-specific multisystemic syndrome. This study aimed to investigate the associations between angiotensinogen (AGT), methylenetetrahydrofolate reductase (MTHFR), vascular endothelial growth factor (VEGF) polymorphisms, and PE in the Han Chinese population. METHODS: We genotyped 26 single-nucleotide polymorphisms (SNP) in three genes by using QuantStudio™ 12 K Flex Real-Time PCR technology in 168 patients with PE and 204 healthy pregnant control subjects. The associations of tested polymorphisms with PE were analyzed at allele, genotype, and haplotype levels. RESULTS: A common coding variant in MTHFR, rs2274976, was significantly associated with increased risk of PE in both allelic and genotype models (P < 0.05). The heterozygous genotypes of rs699 (G/A vs G/G) in AGT gene and rs3025035 (C/T vs C/C) in VEGF gene showed weak associations with increased PE risk, whereas the mutant homozygous genotype of rs3024987 (TT vs C/C) and the heterozygous genotype of rs3025039 (C/T vs C/C) in VEGF gene displayed weak associations with decreased PE risk (P < 0.05). DISCUSSION: However, these weak associations lost significance after multiple testing correction. The results indicated that rs2274976 in MTHFR gene may contribute to the increased risk of PE in pregnant women. AGT and VEGF gene polymorphisms may not play a significant role in PE development.

Identification of AGT and CD44 in methotrexate-resistant colorectal cancer and reversal of methotrexate-resistance.

This study aims to screen out hub genes in 2 methotrexate-resistant colorectal cancer (CRC) cells (HT29 and Caco2), compared with parental CRC cells and reverse methotrexate-resistance in methotrexate-resistant CRC. GEO database and R software were utilized to analyze the gene expression profiles GSE11440 and GSE16066. Venn diagram was used to identify intersection differentially expressed genes (DEGs) between GSE11440 and GSE16066. Protein-protein interaction (PPI) was utilized to screen out central node genes. Hub genes were determined by volcano graphs, heatmaps and box plots. The functional enrichment analysis was exhibited with DAVID. The GEPIA was used to obtain survival curves to analyze association between patient prognosis and hub genes. Western blotting was used to detect the expressions of hub genes. CCK-8 assay was used to show MTX-resistant CRC cell viability following CD44 inhibitor (THIQ) and AGT inhibitor (O6-BG) treatments. In our results, there were 180 intersection DEGs between GSE11440 and GSE16066. CD44 and AGT were screened out as hub genes by PPI, heatmaps, volcano and box plots. In the 2 MTX-resistant CRC cells, the expressions of CD44 and AGT were up-regulated compared with parental CRC cells. The results of western blotting showed that CD44 and AGT were up-regulated in MTX-resistant HT29 and Caco2 cells compared with parental CRC cells. CCK-8 assay results showed that the combination of MTX with O6-BG or THIQ could significantly reduce the activity of MTX-resistant CRC cells. This research screened out CD44 and AGT in MTX-resistant CRC cells by bioinformatics and suggested that the combination of MTX with O6-BG or THIQ could enhance the sensitivity of MTX-resistant CRC cells to MTX. This research provides a new strategy for overcoming MTX-resistance in CRC.

Renin-Angiotensin System Single Nucleotide Polymorphisms Are Associated with Bladder Cancer Risk.

The renin-angiotensin system (RAS), besides being a major regulator of blood pressure, is also involved in tumor angiogenesis. Emerging evidence suggests a correlation between the use of pharmacologic RAS inhibitors and a delay in urothelial bladder cancer (BC) progression. However, it is unknown whether RAS gene variants may predispose to the development of BC. This study examined the association of RAS single nucleotide polymorphisms (SNPs) including AT1R rs5186, AT2R rs11091046, REN rs12750834, ANG rs4762, and ANG rs699 with the risk of developing non-invasive BC. Peripheral blood samples from 73 patients with T1 urothelial BC (66 men, seven women) and an equal number of healthy subjects (control group) were collected. The TT genotype of the REN rs12750834 SNP (OR: 2.8 [1.3-6.05], p = 0.008) and to a lesser extent the presence of the T allele (OR: 2.3 [1.2-4.48], p = 0.01) conferred a higher risk of BC. The highest risk for BC within SNP carriers of the RAS system was associated with the presence of the CC genotype (OR: 17.6 [7.5-41.35], p < 0.001) and C allele (OR: 17.7 [8.8-35.9], p < 0.001) of the ANG rs699 SNP. The presence of the AT2R rs11091046 SNP, particularly the AA genotype, was associated with a protective effect against developing BC (OR: 0.268 [0.126-057], p < 0.001). In conclusion, these results support the clinical utility of RAS gene SNPs AT2R rs11091046, REN rs12750834, and ANG rs699 in the genetic cancer risk assessment of patients and families with BC.

Genetic Predictors of Salt Sensitivity of Blood Pressure: The Additive Impact of 2 Hits in the Same Biological Pathway.

[Figure: see text].

AGT serves as a potential biomarker and drives tumor progression in colorectal carcinoma.

BACKGROUND: Colorectal carcinoma (CRC) is one of the most common aggressive tumors worldwide, and it is necessary to identify candidate biomarkers and therapeutic targets in CRC to improve patient outcomes. METHODS: The differentially expressed genes (DEGs) were obtained from CRC microarray. Functional enrichment was performed to explore the function of DEGs, and core genes were identified by Cytoscape. Then, the diagnosis and prognosis markers were identified by ROC curve and survival analyses. More importantly, a series of in vitro studies were conducted in CRC cells to explore the function of the selected biomarker. Further, the drug response was performed by Cancer Cell Line Encyclopedia (CCLE) and Cancer Therapy Response Portal (CTRP). In addition, the effect of drug on CRC cells was evaluated by functional experiments. RESULTS: The identified DEGs were mainly associated with the processes relating to tumorigenesis. 25 core genes were selected and angiotensinogen (AGT) was filtered out as a diagnosis and prognosis biomarker. Comprehensive in vitro experiments showed that AGT attributed to the proliferation, migration, and invasion of CRC cells, as well as angiogenesis of HUVECs induced by CRC conditional medium. Furthermore, drug response analysis implied that AGT expression was associated with isoliquiritigenins (ISL). Additionally, ISL could suppress the progression of CRC cells. CONCLUSIONS: AGT is identified as diagnosis and prognosis prediction of CRC. Moreover, AGT attributes to the progression of CRC. Additionally, AGT exhibits fine drug response to ISL, and ISL is also evaluated as potential therapy drug in CRC.

Linkage of blood pressure, obesity and diabetes mellitus with angiotensinogen gene (AGT 704T>C/rs699) polymorphism in hypertensive patients.

AIMS: The study aim was to analyse the frequency of polymorphic variants of angiotensinogen gene polymorphism (AGT 704T>C, rs699) in essential arterial hypertension (EAH) patients. METHODS: Seventy-two individuals with EAH and hypertension­mediated organ damage (stage 2), moderate, high or very high cardiovascular risks were involved in the case-control study. Among them, 70.84 % (51) were females and 29.16 % (21) were males; mean age was 59.87±7.98 y. The control group consisted of fifty practically healthy individuals at relevant age (49.13±6.28 y) and with relevant sex distribution (62 % were females, 38 % were males). AGT (704T>C) gene polymorphism was examined by RT-PCR. RESULTS: The distribution of genotypes in the study group was as follows: TT - 14 %, TC - 60 %, CC - 26 %, which corresponded to the distribution in the control group - 16 %, 54 % and 30 %, respectively, and did not deviate from the Hardy-Weinberg equilibrium. Smoking, type 2 diabetes mellitus (DM2) and obesity increased the relative risk of EAH in the examined population 2.5 times [OR=2.81; p=0.049], 3.75 times [OR=4.68; p=0.005] and almost twofold [OR=2.90; p=0.004], respectively. The probability of EAH increases fourfold with the angiotensin II elevation in the serum. Genotypes and alleles of the AGT (704T>C) gene were not significant risk factors for EAH and DM2 in the studied population. However, the TC-genotype (lesser T-allele) increases the risk of obesity in EAH patients more than 1.5 times [OR=2.93; p=0.03]. In addition, the T-allele increases the risk for blood pressure (BP) to elevate up to grade 2-3 [OR=3.64; p < 0.001]. CONCLUSIONS: One-way ANOVA analysis confirmed the AGT (704T>C) gene polymorphism to be associated with systolic and diastolic BP elevation (F=7.80; p < 0.001 and F=4.90; p=0.01, respectively), especially in TT-genotype carriers (p < 0.05), and with body mass index increase, albeit only in women (F=13.94; p < 0.001) (Tab. 4, Fig. 3, Ref. 26). Text in PDF www.elis.sk Keywords: angiotensinogen gene (AGT 704T>C), diabetes mellitus type 2, arterial hypertension, obesity, risk.

Boosting Auto-Induction of Recombinant Proteins in Escherichia coli with Glucose and Lactose Additives.

BACKGROUND: Auto-induction is a convenient way to produce recombinant proteins without inducer addition using lac operon-controlled Escherichia coli expression systems. Auto-induction can occur unintentionally using a complex culture medium prepared by mixing culture substrates. The differences in culture substrates sometimes lead to variations in the induction level. OBJECTIVES: In this study, we investigated the feasibility of using glucose and lactose as boosters of auto-induction with a complex culture medium. METHODS: First, auto-induction levels were assessed by quantifying recombinant GFPuv expression under the control of the T7 lac promoter. Effectiveness of the additive-containing medium was examined using ovine angiotensinogen (tac promoter-based expression) and Thermus thermophilus manganese-catalase (T7 lac promoter-based expression). RESULTS: Auto-induced GFPuv expression was observed with the enzymatic protein digest Polypepton, but not with another digest tryptone. Regardless of the type of protein digest, supplementing Terrific Broth medium with glucose (at a final concentration of 2.9 g/L) and lactose (at a final concentration of 7.6 g/L) was successful in obtaining an induction level similar to that achieved with a commercially available auto-induction medium. The two recombinant proteins were produced in milligram quantity of purified protein per liter of culture. CONCLUSION: The medium composition shown in this study would be practically useful for attaining reliable auto-induction for E. coli-based recombinant protein production.

Molecular basis of a redox switch: molecular dynamics simulations and surface plasmon resonance provide insight into reduced and oxidised angiotensinogen.

Angiotensinogen fine-tunes the tightly controlled activity of the renin-angiotensin system by modulating the release of angiotensin peptides that control blood pressure. One mechanism by which this modulation is achieved is via angiotensinogen's Cys18-Cys138 disulfide bond that acts as a redox switch. Molecular dynamics simulations of each redox state of angiotensinogen reveal subtle dynamic differences between the reduced and oxidised forms, particularly at the N-terminus. Surface plasmon resonance data demonstrate that the two redox forms of angiotensinogen display different binding kinetics to an immobilised anti-angiotensinogen monoclonal antibody. Mass spectrometry mapped the epitope for the antibody to the N-terminal region of angiotensinogen. We therefore provide evidence that the different redox forms of angiotensinogen can be detected by an antibody-based detection method.

DNA Methylation of the Angiotensinogen Gene, AGT, and the Aldosterone Synthase Gene, CYP11B2 in Cardiovascular Diseases.

Angiotensinogen (AGT) and aldosterone play key roles in the regulation of blood pressure and are implicated in the pathogenesis of cardiovascular diseases. DNA methylation typically acts to repress gene transcription. The aldosterone synthase gene CYP11B2 is regulated by angiotensin II and potassium. DNA methylation negatively regulates AGT and CYP11B2 expression and dynamically changes in response to continuous promoter stimulation of each gene. High salt intake and excess circulating aldosterone cause DNA demethylation around the CCAAT-enhancer-binding-protein (CEBP) sites of the CYP11B2 promoter region, thereby converting the phenotype of AGT expression from an inactive to an active state in visceral adipose tissue and heart. A close association exists between low DNA methylation at CEBP-binding sites and increased AGT expression in salt-sensitive hypertensive rats. Salt-dependent hypertension may be partially affected by increased cardiac AGT expression. CpG dinucleotides in the CYP11B2 promoter are hypomethylated in aldosterone-producing adenomas. Methylation of recognition sequences of transcription factors, including CREB1, NGFIB (NR4A1), and NURR1 (NR4A2) diminish their DNA-binding activity. The methylated CpG-binding protein MECP2 interacts directly with the methylated CYP11B2 promoter. Low salt intake and angiotensin II infusion lead to upregulation of CYP11B2 expression and DNA hypomethylation in the adrenal gland. Treatment with the angiotensin II type 1 receptor antagonist decreases CYP11B2 expression and leads to DNA hypermethylation. A close association between low DNA methylation and increased CYP11B2 expression are seen in the hearts of patients with hypertrophic cardiomyopathy. These results indicate that epigenetic regulation of both AGT and CYP11B2 contribute to the pathogenesis of cardiovascular diseases.

Renin-angiotensin system is involved in embryonic emergence of hematopoietic stem/progenitor cells.

Angiotensin-converting enzyme (ACE), a key element of the renin-angiotensin system (RAS), has recently been identified as a new marker of both adult and embryonic human hematopoietic stem/progenitor cells (HSPCs). However, whether a full renin-angiotensin pathway is locally present during the hematopoietic emergence is still an open question. In the present study, we show that this enzyme is expressed by hematopoietic progenitors in the developing mouse embryo. Furthermore, ACE and the other elements of RAS-namely angiotensinogen, renin, and angiotensin II type 1 (AT1) and type 2 (AT2) receptors-are expressed in the paraaortic splanchnopleura (P-Sp) and in its derivative, the aorta-gonad-mesonephros region, both in human and mouse embryos. Their localization is compatible with the existence of a local autocrine and/or paracrine RAS in these hemogenic sites. in vitro perturbation of the RAS by administration of a specific AT1 receptor antagonist inhibits almost totally the generation of blood CD45-positive cells from dissected P-Sp, implying that angiotensin II signaling is necessary for the emergence of hematopoietic cells. Conversely, addition of exogenous angiotensin II peptide stimulates hematopoiesis in culture, with an increase in the number of immature c-Kit+ CD41+ CD31+ CD45+ hematopoietic progenitors, compared to the control. These results highlight a novel role of local-RAS during embryogenesis, suggesting that angiotensin II, via activation of AT1 receptor, promotes the emergence of undifferentiated hematopoietic progenitors.

Parathyroid hormone type 1 receptor regulates osteosarcoma K7M2 Cell growth by interacting with angiotensinogen.

This study aimed to determine the interactions between parathyroid hormone type 1 receptor (PTHR1) and angiotensinogen (AGT) and the effects of these agents on osteosarcoma (OS). We constructed a stably transfected mouse OS K7M2 cell line (shPTHR1- K7M2) using shRNA and knocked down AGT in these cells using siRNA-AGT. The transfection efficiency and expression of AGT, chemokine C-C motif receptor 3 (CCR3), and chemokine (C-C motif) ligand 9 (CCL9) were determined using real-time quantitative PCR. Cell viability and colony formation were assessed using Cell Counting Kit-8 and crystal violet staining, respectively. Cell apoptosis and cycle phases were assessed by flow cytometry, and cell migration and invasion were evaluated using Transwell assays. Interference with PTHR1 upregulated the expression of AGT and CCR3, and downregulated that of CCL9, which was further downregulated by AGT knockdown. Cell viability, migration, invasion and colony formation were significantly decreased, while cell apoptosis was significantly increased in shPTHR1-K7M2, compared with those in K7M2 cells (P < .05 for all). However, AGT knockdown further inhibited cell viability after 72 h of culture but promoted cell migration and invasion. PTHR1 interference decreased and increased the numbers of cells in the G0/G1 and G2/M phases, respectively, compared with those in K7M2 cells. Angiotensinogen knockdown increased the number of cells in the G0/G1 phase compared with that in the shPTHR1-K7M2 cells. Therefore, PTHR1 affects cell viability, apoptosis, migration, invasion and colony formation, possibly by regulating AGT/CCL9 in OS cells.

Cancer Stem Cells in Metastatic Head and Neck Cutaneous Squamous Cell Carcinoma Express Components of the Renin-Angiotensin System.

We investigated the expression of components of the renin-angiotensin system (RAS) by cancer stem cell (CSC) subpopulations in metastatic head and neck cutaneous squamous cell carcinoma (mHNcSCC). Immunohistochemical staining demonstrated expression of prorenin receptor (PRR), angiotensin-converting enzyme (ACE), and angiotensin II receptor 2 (AT2R) in all cases and angiotensinogen in 14 cases; however, renin and ACE2 were not detected in any of the 20 mHNcSCC tissue samples. Western blotting showed protein expression of angiotensinogen in all six mHNcSCC tissue samples, but in none of the four mHNcSCC-derived primary cell lines, while PRR was detected in the four cell lines only. RT-qPCR confirmed transcripts of angiotensinogen, PRR, ACE, and angiotensin II receptor 1 (AT1R), but not renin or AT2R in all four mHNcSCC tissue samples and all four mHNcSCC-derived primary cell lines, while ACE2 was expressed in the tissue samples only. Double immunohistochemical staining on two of the mHNcSCC tissue samples showed expression of angiotensinogen by the SOX2+ CSCs within the tumor nests (TNs), and immunofluorescence showed expression of PRR and AT2R by the SOX2+ CSCs within the TNs and the peritumoral stroma (PTS). ACE was expressed on the endothelium of the tumor microvessels within the PTS. We demonstrated expression of angiotensinogen by CSCs within the TNs, PRR, and AT2R by the CSCs within the TNs and the PTS, in addition to ACE on the endothelium of tumor microvessels in mHNcSCC.

Cell autonomous angiotensin II signaling controls the pleiotropic functions of oncogenic K-Ras.

Oncogenic K-Ras (K-RasG12V) promotes senescence in normal cells but fuels transformation of cancer cells after the senescence barrier is bypassed. The mechanisms regulating this pleiotropic function of K-Ras remain to be fully established and bear high pathological significance. We find that K-RasG12V activates the angiotensinogen (AGT) gene promoter and promotes AGT protein expression in a Kruppel-like factor 6-dependent manner in normal cells. We show that AGT is then converted to angiotensin II (Ang II) in a cell-autonomous manner by cellular proteases. We show that blockade of the Ang II receptor type 1 (AT1-R) in normal cells inhibits oncogene-induced senescence. We provide evidence that the oncogenic K-Ras-induced synthesis of Ang II and AT1-R activation promote senescence through caveolin-1-dependent and nicotinamide adenine dinucleotide phosphate oxidase 2-mediated oxidative stress. Interestingly, we find that expression of AGT remains elevated in lung cancer cells but in a Kruppel-like factor 6-independent and high-mobility group AT-hook 1-dependent manner. We show that Ang II-mediated activation of the AT1-R promotes cell proliferation and anchorage-independent growth of lung cancer cells through a STAT3-dependent pathway. Finally, we find that expression of AGT is elevated in lung tumors of K-RasLA2-G12D mice, a mouse model of lung cancer, and human lung cancer. Treatment with the AT1-R antagonist losartan inhibits lung tumor formation in K-RasLA2-G12D mice. Together, our data provide evidence of the existence of a novel cell-autonomous and pleiotropic Ang II-dependent signaling pathway through which oncogenic K-Ras promotes oncogene-induced senescence in normal cells while fueling transformation in cancer cells.

Landscape of Germline Genetic Variants in AGT, MGMT, and TP53 in Mexican Adult Patients with Astrocytoma.

Astrocytoma is the most common type of primary brain tumor. The risk factors for astrocytoma are poorly understood; however, germline genetic variants account for 25% of the risk of developing gliomas. In this study, we assessed the risk of astrocytoma associated with variants in AGT, known by its role in angiogenesis, TP53, a well-known tumor suppressor and the DNA repair gene MGMT in a Mexican population. A case-control study was performed in 49 adult Mexican patients with grade II-IV astrocytoma. Sequencing of exons and untranslated regions of AGT, MGMT, and TP53 from was carried in an Ion Torrent platform. Individuals with Mexican Ancestry from the 1000 Genomes Project were used as controls. Variants found in our cohort were then assessed in a The Cancer Genome Atlas astrocytoma pan-ethnic validation cohort. Variants rs1926723 located in AGT (OR 2.74, 1.40-5.36 95% CI), rs7896488 in MGMT (OR 3.43, 1.17-10.10 95% CI), and rs4968187 in TP53 (OR 2.48, 1.26-4.88 95% CI) were significantly associated with the risk of astrocytoma after multiple-testing correction. This is the first study where the AGT rs1926723 variant, TP53 rs4968187, and MGMT rs7896488 were found to be associated with the risk of developing an astrocytoma.