Signaling-induced systematic repression of miRNAs uncovers cancer vulnerabilities and targeted therapy sensitivity.

Targeted therapies are effective in treating cancer, but success depends on identifying cancer vulnerabilities. In our study, we utilize small RNA sequencing to examine the impact of pathway activation on microRNA (miRNA) expression patterns. Interestingly, we discover that miRNAs capable of inhibiting key members of activated pathways are frequently diminished. Building on this observation, we develop an approach that integrates a low-miRNA-expression signature to identify druggable target genes in cancer. We train and validate our approach in colorectal cancer cells and extend it to diverse cancer models using patient-derived in vitro and in vivo systems. Finally, we demonstrate its additional value to support genomic and transcriptomic-based drug prediction strategies in a pan-cancer patient cohort from the National Center for Tumor Diseases (NCT)/German Cancer Consortium (DKTK) Molecularly Aided Stratification for Tumor Eradication (MASTER) precision oncology trial. In conclusion, our strategy can predict cancer vulnerabilities with high sensitivity and accuracy and might be suitable for future therapy recommendations in a variety of cancer subtypes.

ADMA elevation does not exacerbate development of diabetic nephropathy in mice with streptozotocin-induced diabetes mellitus.

BACKGROUND AND AIMS: Cardiovascular disease is nowadays the major cause of mortality and morbidity worldwide. The risk of developing cardiovascular disease is significantly increased in patients with diabetic nephropathy. It has been suggested that asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthases (NOS), may play an important role in the pathogenesis of diabetic nephropathy. ADMA is mainly metabolized by dimethylarginine dimethylaminohydrolase 1 (DDAH1). The goal of this study was to test the hypothesis that elevation of systemic ADMA levels by knocking out DDAH1 would exacerbate functional and structural glomerular abnormalities in a murine model of diabetic nephropathy. METHODS: Streptozotocin (STZ) was used to induce diabetes in adult DDAH1 knock-out and wild type mice. Healthy mice served as controls. Mice were sacrificed after 20 weeks of diabetes. Plasma ADMA levels were assessed by isotope-dilution tandem mass spectrometry and albumin by ELISA. Kidneys were used for FACS analysis and were also stained for markers of inflammation, cell proliferation, glomerular cells and cell matrix. RESULTS: STZ led to development of diabetes mellitus in all injected animals. Deficiency of DDAH1 led to a significant increase in plasma ADMA levels in healthy and diabetic mice. The diabetic state itself did not influence systemic ADMA levels. Diabetic mice of both genotypes developed albuminuria and had increased glomerulosclerosis index. There were no changes in desmin expression, glomerular cell proliferation rate, matrix expansion and expression of Mac-2 antigen in the diabetic mice of both genotypes as compared to the healthy ones. CONCLUSIONS: In summary, STZ-induced diabetes led to the development of early features of diabetic nephropathy. Deficiency of DDAH1 and subsequent increase in systemic ADMA levels did not exacerbate these changes, indicating that ADMA is not the major mediator of diabetic nephropathy in this experiment model.

Kidney and liver are the main organs of expression of a key metabolic enzyme alanine:glyoxylate aminotransferase 2 in humans.

BACKGROUND: The metabolic syndrome is a cluster of cardiovascular risk factors and is highly predictive for development of cardiovascular diseases. An association between elevated plasma levels of the endogenous inhibitor of nitric oxide synthases asymmetric dimethylarginine (ADMA) and risk of cardiovascular diseases has been demonstrated in numerous epidemiological studies. ADMA can be catabolized by dimethylarginine dimethylaminohydrolase (DDAH) or metabolized through a much less understood alternative pathway by alanine:glyoxylate aminotransferase 2 (AGXT2) with the formation of α-keto-δ-(N,N-dimethylguanidino)valeric acid (ADGV). Previous RT-PCR and Western Blot studies suggested that Agxt2 is expressed in the mouse kidney and liver at comparable levels, while Northern Blot and in-situ RNA-hybridisation experiments demonstrated that the kidney is the main organ of Agxt2 expression in rats. Given this discrepancy, the goal of the current study was to analyse the expression of AGXT2 in human tissues. MATERIAL AND METHODS: We analyzed AGXT2 expression in human tissues from a normal tissue bank by RT-PCR and further validated the results by Western Blot. We also performed immunohistochemical staining for AGXT2 and double fluorescent staining with an anti-AGXT2 antibody and a monoclonal anti-mitochondrial antibody. RESULTS: We saw the strongest expression of AGXT2 in the kidney and liver and confirmed this results on protein level. By IHC staining we were able to show that AGXT2 is present in the convoluted tubule in the kidney and in the liver hepatocytes. The double fluorescent staining revealed mitochondrial localization of AGXT2. CONCLUSIONS: Our current data suggest that both hepatocytes and kidney tubular epithelial cells are the major sources of AGXT2 in humans. We also demonstrated the mitochondrial localization of human AGXT2 enzyme.

Microarray analysis for delineating the gene expression in biopsies of gastrocnemius muscle of patients with chronic critical limb ischaemia compared with non-ischaemic controls.

BACKGROUND: Microarray analysis has been carried out in this pilot study to compare delineated gene expression profiles in the biopsies of skeletal muscle taken from patients with chronic critical limb ischaemia (CLI) and non-ischaemic control subjects. PATIENTS AND METHODS: Biopsy of gastrocnemius muscle was obtained from six patients with unreconstructed CLI referred for surgical major amputation. As control, biopsies of six patients undergoing elective knee arthroplasty without evidence of peripheral arterial occlusive disease were taken. The differences in gene expression associated with angiogenic processes in specimens obtained from ischaemic and non-ischaemic skeletal muscle were confirmed by quantitative real-time polymerase chain reaction (PCR) analysis. RESULTS: Compared with non-ischaemic skeletal muscle biopsy of chronic-ischaemic skeletal muscle contained 55 significantly up-regulated and 45 down-regulated genes, out of which 64 genes had a known genetic product. Tissue samples of ischaemic muscle were characterized by increased expression of cell survival factors (e. g. tissue factor pathway inhibitor 2) in combination with reduced expression of cell proliferation effectors (e. g. microfibrillar-associated protein 5 and transferrin receptor). The expression of growth factors (e. g. early growth response 3 and chemokine receptor chemokine C-X-C motif ligand 4) which play a central role in arterial and angiogenic processes and anti-angiogenetic factors (e. g. pentraxin 3) were increased in chronic ischaemic skeletal muscle. An increased expression of extracellular matrix proteins (e. g. cysteine-rich angiogenic inducer 61) was also observed. CONCLUSIONS: Gene expression profiles in biopsies of gastrocnemius muscle in patients with chronic critical limb ischaemia showed an increase in pro-survival factors, extracellular matrix protein deposition, and impaired proliferation, compared with non-ischaemic controls. Further studies are required to analyse the endogenous repair mechanism.

ADMA reduction does not protect mice with streptozotocin-induced diabetes mellitus from development of diabetic nephropathy.

BACKGROUND AND AIMS: Cardiovascular disease is the major cause of morbidity and mortality in the world. Diabetes and its complications, such as diabetic nephropathy, dramatically increase cardiovascular risk. Association studies suggest that asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthases, plays a role in the pathogenesis of diabetic nephropathy. The major pathway of ADMA catabolism is hydrolysis by dimethylarginine dimethylaminohydrolase 1 (DDAH1). The goal of this study was to test the hypothesis that lowering ADMA by overexpression of DDAH1 protects from development of diabetic nephropathy. METHODS: Diabetes was induced with streptozotocin (STZ) in wild type and DDAH1 transgenic mice. Healthy mice served as controls. Mice were sacrificed after 20 weeks of diabetes. ADMA levels were assessed by isotope-dilution tandem mass spectrometry, creatinine by standard laboratory methods and albumin by ELISA. Kidney tissues were stained for markers of glomerular cells, cell matrix, inflammation and cell proliferation. RESULTS: STZ led to development of diabetes in all injected mice. Transgenic overexpression of DDAH1 led to a decrease in plasma ADMA levels in healthy animals. Diabetic state itself did not lead to elevation of plasma ADMA levels. Diabetic mice of both genotypes developed albuminuria (27 and 25 vs. 9 and 6 µg albumin/mg creatinine) (p < 0.01). There were no changes in glomerular matrix expansion, podocyte injury, inflammatory or proliferative response. CONCLUSIONS: STZ-induced diabetes led to the development of early features of diabetic nephropathy. Overexpression of DDAH1 and lowering of systemic ADMA levels did not prevent these changes, indicating that ADMA is not the major mediator of the early diabetic changes reflected by this experimental model.

A Novel Pathway for Metabolism of the Cardiovascular Risk Factor Homoarginine by alanine:glyoxylate aminotransferase 2.

Low plasma concentrations of L-homoarginine are associated with an increased risk of cardiovascular events, while homoarginine supplementation is protective in animal models of metabolic syndrome and stroke. Catabolism of homoarginine is still poorly understood. Based on the recent findings from a Genome Wide Association Study we hypothesized that homoarginine can be metabolized by alanine:glyoxylate aminotransferase 2 (AGXT2). We purified human AGXT2 from tissues of AGXT2 transgenic mice and demonstrated its ability to metabolize homoarginine to 6-guanidino-2-oxocaproic acid (GOCA). After incubation of HepG2 cells overexpressing AGXT2 with isotope-labeled homoarginine-d4 we were able to detect labeled GOCA in the medium. We injected wild type mice with labeled homoarginine and detected labeled GOCA in the plasma. We found that AGXT2 knockout (KO) mice have higher homoarginine and lower GOCA plasma levels as compared to wild type mice, while the reverse was true for AGXT2 transgenic (Tg) mice. In summary, we experimentally proved the presence of a new pathway of homoarginine catabolism - its transamination by AGXT2 with formation of GOCA and demonstrated that endogenous AGXT2 is required for maintenance of homoarginine levels in mice. Our findings may lead to development of novel therapeutic approaches for cardiovascular pathologies associated with homoarginine deficiency.

Diabetes-linked transcription factor HNF4α regulates metabolism of endogenous methylarginines and ß-aminoisobutyric acid by controlling expression of alanine-glyoxylate aminotransferase 2.

Elevated levels of circulating asymmetric and symmetric dimethylarginines (ADMA and SDMA) predict and potentially contribute to end organ damage in cardiovascular diseases. Alanine-glyoxylate aminotransferase 2 (AGXT2) regulates systemic levels of ADMA and SDMA, and also of beta-aminoisobutyric acid (BAIB)-a modulator of lipid metabolism. We identified a putative binding site for hepatic nuclear factor 4 α (HNF4α) in AGXT2 promoter sequence. In a luciferase reporter assay we found a 75% decrease in activity of Agxt2 core promoter after disruption of the HNF4α binding site. Direct binding of HNF4α to Agxt2 promoter was confirmed by chromatin immunoprecipitation assay. siRNA-mediated knockdown of Hnf4a led to an almost 50% reduction in Agxt2 mRNA levels in Hepa 1-6 cells. Liver-specific Hnf4a knockout mice exhibited a 90% decrease in liver Agxt2 expression and activity, and elevated plasma levels of ADMA, SDMA and BAIB, compared to wild-type littermates. Thus we identified HNF4α as a major regulator of Agxt2 expression. Considering a strong association between human HNF4A polymorphisms and increased risk of type 2 diabetes our current findings suggest that downregulation of AGXT2 and subsequent impairment in metabolism of dimethylarginines and BAIB caused by HNF4α deficiency might contribute to development of cardiovascular complications in diabetic patients.

Acetylation of asymmetric and symmetric dimethylarginine: an undercharacterized pathway of metabolism of endogenous methylarginines.

BACKGROUND: Increased levels of asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are associated with cardiovascular and renal diseases. We and others have shown that both ADMA and SDMA can be Nα-acetylated to form asymmetric and symmetric Nα-acetyldimethylarginine (Ac-ADMA and Ac-SDMA). The current study further investigated this undercharacterized metabolic pathway. METHODS: ADMA and SDMA were infused in C57/BL6 mice for 3 days using osmotic minipumps. Half of the mice underwent bilateral nephrectomy 24 h before completion of the infusion. Plasma and tissue levels of Ac-ADMA and Ac-SDMA were detected by liquid chromatography-tandem mass spectrometry. RESULTS: ADMA and SDMA infusion resulted in a 3.6-fold increase in plasma Ac-ADMA and a 21-fold increase in plasma Ac-SDMA levels, respectively. Plasma Ac-ADMA and Ac-SDMA levels were dramatically increased after bilateral nephrectomy. The highest baseline tissue concentrations of Ac-ADMA and Ac-SDMA in wild-type mice were detected in the liver, kidney, small intestine, pancreas and spleen. Incubation of the tissue lysates with ADMA and SDMA resulted in increased levels of the corresponding Nα-acetylated products only in the liver, kidney and small intestine. CONCLUSIONS: Our results show that overload of ADMA or SDMA leads to an increase in plasma Ac-ADMA and Ac-SDMA levels. This observation is consistent with the hypothesis that Ac-ADMA and Ac-SDMA are formed directly from ADMA and SDMA in vivo. The increase in plasma Ac-ADMA and Ac-SDMA concentrations after bilateral nephrectomy suggests that both compounds are predominantly eliminated via the kidneys. We demonstrated that acetylation of ADMA and SDMA occurs primarily in the liver, kidney and small intestine.

Role of alanine:glyoxylate aminotransferase 2 in metabolism of asymmetric dimethylarginine in the settings of asymmetric dimethylarginine overload and bilateral nephrectomy.

BACKGROUND: Asymmetric and symmetric dimethylarginines (ADMA and SDMA) predict complications and mortality in cardiovascular and renal diseases. Alanine:glyoxylate aminotransferase 2 (AGXT2) can metabolize both ADMA and SDMA; however, this metabolic pathway is still poorly understood. The goal of our study was to test the hypothesis that AGXT2 is compensatory upregulated in the settings of ADMA overload and bilateral nephrectomy. METHODS: ADMA was infused for 3 days using osmotic minipumps in mice. Half of the mice underwent bilateral nephrectomy 24 h before the end of the infusion. RESULTS: Infusion of ADMA caused a 3- to 4-fold increase in plasma and urine ADMA levels and a 2- to 3-fold increase in plasma and urine levels of the ADMA-specific metabolite of AGXT2 α-keto-δ-(N,N-dimethylguanidino)valeric acid (DMGV). Bilateral nephrectomy led to an ∼4-fold increase of plasma SDMA levels, but did not change plasma ADMA levels. Interestingly, plasma levels of DMGV were elevated 32-fold in the mice, which underwent bilateral nephrectomy. Neither bilateral nephrectomy nor ADMA infusion caused upregulation of AGXT2 expression or activity. CONCLUSIONS: Our data demonstrate that short-term elevation of systemic levels of ADMA leads to a dramatic increase of DMGV formation without upregulation of AGXT2 expression or activity, which suggests that AGXT2-mediated pathway of ADMA metabolism is not saturated under normal conditions and may play a major role in the maintenance of ADMA homeostasis in the setting of local or systemic elevation of ADMA levels.

Stromal fibroblasts in colorectal liver metastases originate from resident fibroblasts and generate an inflammatory microenvironment.

Cancer-associated stromal fibroblasts (CAFs) are the main cellular constituents of reactive stroma in primary and metastatic cancer. We analyzed phenotypical characteristics of CAFs from human colorectal liver metastases (CLMs) and their role in inflammation and cancer progression. CAFs displayed a vimentin(+), alpha-smooth-muscle actin(+), and Thy-1(+) phenotype similar to resident portal-located liver fibroblasts (LFs). We demonstrated that CLMs are inflammatory sites showing stromal expression of interleukin-8 (IL-8), a chemokine related to invasion and angiogenesis. In vitro analyses revealed a striking induction of IL-8 expression in CAFs and LFs by tumor necrosis factor-alpha (TNF-alpha). The effect of TNF-alpha on CAFs is inhibited by the nuclear factor-kappaB inhibitor parthenolide. Conditioned medium of CAFs and LFs similarly stimulated the migration of DLD-1, Colo-678, HuH7 carcinoma cells, and human umbilical vein endothelial cells in vitro. Pretreatment of CAFs with TNF-alpha increased the chemotaxis of Colo-678 colon carcinoma cells by conditioned medium of CAFs; however, blockage of IL-8 activity showed no inhibitory effect. In conclusion, these data raise the possibility that the majority of CAFs in CLM originate from resident LFs. TNF-alpha-induced up-regulation of IL-8 via nuclear factor-kappaB in CAFs is an inflammatory pathway, potentially permissive for cancer invasion that may represent a novel therapeutic target.

Imatinib mesylate inhibits proliferation and modulates cytokine expression of human cancer-associated stromal fibroblasts from colorectal metastases.

Recent data have expanded the concept that cancer-associated stromal fibroblasts (CAFs) play an important role in tumor invasion and angiogenesis. Here, we show that platelet-derived growth factor (PDGF) is a mitogen for human CAFs isolated from hepatic metastases of colorectal cancer. The tyrosine kinase inhibitor imatinib mesylate (1 microM) abrogated the PDGF-induced DNA synthesis, and furthermore counteracted an inhibitory effect of PDGF on the expression of alpha-smooth muscle actin (alpha-SMA). High-dose imatinib mesylate (10 microM) decreased the viability of CAFs in vitro independent from co-stimulation with PDGF. Interestingly, imatinib mesylate (10 microM) strikingly induced the expression of the pro-inflammatory and pro-angiogenic cytokines interleukin (IL)-6 and IL-8, and mildly stimulated the release of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Our results suggest that imatinib mesylate, due to its anti-proliferative activity, may be effective in combination with other substances for the treatment of colorectal metastasis progression.