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.

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.

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.

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.