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Introduction Dysregulated choline metabolism is a well-known feature of breast cancer, but the underlying mechanisms are not fully understood. In this study, the metabolomic and transcriptomic characteristics of a large panel of human breast cancer xenograft models were mapped, with focus on choline metabolism. Methods Tumor specimens from 34 patient-derived xenograft models were collected and divided in two. One part was examined using high-resolution magic angle spinning (HR-MAS) MR spectroscopy while another part was analysed using gene expression microarrays. Expression data of genes encoding proteins in the choline metabolism pathway were analysed and correlated to the levels of choline (Cho), phosphocholine (PCho) and glycerophosphocholine (GPC) using Pearson's correlation analysis. For comparison purposes, metabolic and gene expression data were collected from human breast tumors belonging to corresponding molecular subgroups. Results Most of the xenograft models were classified as basal-like (N = 19) or luminal B (N = 7). These two subgroups showed significantly different choline metabolic and gene expression profiles. The luminal B xenografts were characterized by a high PCho/GPC ratio while the basal-like xenografts were characterized by highly variable PCho/GPC ratio. Also, Cho, PCho and GPC levels were correlated to expression of several genes encoding proteins in the choline metabolism pathway, including choline kinase alpha (CHKA) and glycerophosphodiester phosphodiesterase domain containing 5 (GDPD5). These characteristics were similar to those found in human tumor samples. Conclusion The higher PCho/GPC ratio found in luminal B compared with most basal-like breast cancer xenograft models and human tissue samples do not correspond to results observed from in vitro studies. It is likely that microenvironmental factors play a role in the in vivo regulation of choline metabolism. Cho, PCho and GPC were correlated to different choline pathwayencoding genes in luminal B compared with basal-like xenografts, suggesting that regulation of choline metabolism may vary between different breast cancer subgroups. The concordance between the metabolic and gene expression profiles from xenograft models with breast cancer tissue samples from patients indicates that these xenografts are representative models of human breast cancer and represent relevant models to study tumor metabolism in vivo. [ABSTRACT FROM AUTHOR]

Background: In a previously published report we characterized the expression of the metastasis-associated proteins S100A4, osteopontin (OPN) and ephrin-A1 in a prospectively collected panel of non-small cell lung cancer (NSCLC) tumors. The aim of the present follow-up study was to investigate the prognostic impact of these potential biomarkers in the same patient cohort. In addition, circulating serum levels of OPN were measured and single nucleotide polymorphisms (SNP) in the -443 position of the OPN promoter were analyzed.Methods: Associations between immunohistochemical expression of S100A4, OPN and ephrin-A1 and relapse free and overall survival were examined using univariate and multivariate analyses. Serum OPN was measured by ELISA, polymorphisms in the -443 position of the tumor OPN promoter were analyzed by PCR, and associations between OPN levels and promoter polymorphisms and clinicopathological parameters and patient outcome were investigated.Results: High expression of OPN in NSCLC tumors was associated with poor patient outcome, and OPN was a strong, independent prognostic factor for both relapse free and overall survival. Serum OPN levels increased according to tumor pT classification and tumor size, and patients with OPN-expressing tumors had higher serum levels than patients with OPN-negative tumors. S100A4 was a negative prognostic factor in several subgroups of adenocarcinoma patients, but not in the overall patient cohort. There was no association between ephrin-A1 expression and patient outcome.Conclusions: OPN is a promising prognostic biomarker in NSCLC, and should be further explored in the selection of patients for adjuvant treatment following surgical resection. [ABSTRACT FROM AUTHOR]

Introduction: The phosphatidylinositol 3-kinase (PI3K) pathway is frequently activated in cancer cells through numerous mutations and epigenetic changes. The recent development of inhibitors targeting different components of the PI3K pathway may represent a valuable treatment alternative. However, predicting efficacy of these drugs is challenging, and methods for therapy monitoring are needed. Basal-like breast cancer (BLBC) is an aggressive breast cancer subtype, frequently associated with PI3K pathway activation. The objectives of this study were to quantify the PI3K pathway activity in tissue sections from xenografts representing basal-like and luminal-like breast cancer before and immediately after treatment with PI3K inhibitors, and to identify metabolic biomarkers for treatment response. Methods: Tumor-bearing animals (n = 8 per treatment group) received MK-2206 (120 mg/kg/day) or BEZ235 (50 mg/ kg/day) for 3 days. Activity in the PI3K/Akt/mammalian target of rapamycin pathway in xenografts and human biopsies was evaluated using a novel method for semiquantitative assessment of Aktser473 phosphorylation. Metabolic changes were assessed by ex vivo high-resolution magic angle spinning magnetic resonance spectroscopy. Results: Using a novel dual near-infrared immunofluorescent imaging method, basal-like xenografts had a 4.5-fold higher baseline level of pAktser473 than luminal-like xenografts. Following treatment, basal-like xenografts demonstrated reduced levels of pAktser473 and decreased proliferation. This correlated with metabolic changes, as both MK-2206 and BEZ235 reduced lactate concentration and increased phosphocholine concentration in the basal-like tumors. BEZ235 also caused increased glucose and glycerophosphocholine concentrations. No response to treatment or change in metabolic profile was seen in luminal-like xenografts. Analyzing tumor sections from five patients with BLBC demonstrated that two of these patients had an elevated pAktser473 level. Conclusion: The activity of the PI3K pathway can be determined in tissue sections by quantitative imaging using an antibody towards pAktser473. Long-term treatment with MK-2206 or BEZ235 resulted in significant growth inhibition in basal-like, but not luminal-like, xenografts. This indicates that PI3K inhibitors may have selective efficacy in basal-like breast cancer with increased PI3K signaling, and identifies lactate, phosphocholine and glycerophosphocholine as potential metabolic biomarkers for early therapy monitoring. In human biopsies, variable pAktser473 levels were observed, suggesting heterogeneous PI3K signaling activity in BLBC. [ABSTRACT FROM AUTHOR]

Background: The metastasis-promoting protein S100A4 activates the transcription factor NF-κB through the classical NF-κB activation pathway. The upstream signal transduction mechanisms leading to increased NF-κB activity are, however, incompletely characterized. Methods: The human osteosarcoma cell line II-11b was stimulated with recombinant S100A4 in the presence or absence of inhibitors of common signal transduction pathways, and NF-κB activity was examined using a luciferasebased reporter assay and phosphorylation of Iκ?Bα. mRNA expression was analyzed by real-time RT-PCR, protein expression was examined by Western blotting and IKK activity was measured using an in vitro kinase assay. The role of upstream kinases and the cell surface receptor RAGE was investigated by overexpression of dominant negative proteins and by siRNA transfection. Results: The Ser/Thr kinase inhibitors H-7 and staurosporine inhibited S100A4-induced Iκ?Bα phosphorylation and subsequent NF-κB activation. The protein tyrosine kinase inhibitor genistein and the phospholipase C inhibitor compound 48/80 had a partial inhibitory effect on Iκ?Bα phosphorylation, whereas inhibitors of protein kinase C, Gprotein coupled receptors and PI 3-kinases had no effect on the level of phosphorylation. Interestingly, S100A4 treatment induced activating phosphorylations of IKKα/β, but neither H-7 nor staurosporine was able to significantly inhibit IKK activation. Dominant negative MEKK1 or NIK did not inhibit S100A4-induced NF-κB activity, and S100A4 stimulation did not influence AKT phosphorylation. Furthermore, diminished expression of the putative S100 protein receptor RAGE did not affect the observed phosphorylation of Iκ?Bα. Conclusions: S100A4 activates NF-κB by inducing phosphorylation of IKKα/β, leading to increased Iκ?Bα phosphorylation. The Ser/Thr kinase inhibitors H-7 and staurosporine attenuated S100A4-induced NF-κB activation and inhibited IKK-mediated phosphorylation of Iκ?Bα. S100A4-induced NF-κB activation was independent of the putative S100 protein receptor RAGE and the Ser/Thr kinases MEKK1, NIK and AKT. These findings lead to increased understanding of S100A4 signaling, which may contribute to the identification of novel targets for anti-metastatic therapy. [ABSTRACT FROM AUTHOR]

Background: Increased concentrations of choline-containing compounds are frequently observed in breast carcinomas, and may serve as biomarkers for both diagnostic and treatment monitoring purposes. However, underlying mechanisms for the abnormal choline metabolism are poorly understood. Methods: The concentrations of choline-derived metabolites were determined in xenografted primary human breast carcinomas, representing basal-like and luminal-like subtypes. Quantification of metabolites in fresh frozen tissue was performed using high-resolution magic angle spinning magnetic resonance spectroscopy (HR MAS MRS). The expression of genes involved in phosphatidylcholine (PtdCho) metabolism was retrieved from whole genome expression microarray analyses. The metabolite profiles from xenografts were compared with profiles from human breast cancer, sampled from patients with estrogen/progesterone receptor positive (ER+/PgR+) or triple negative (ER-/PgR-/HER2-) breast cancer. Results: In basal-like xenografts, glycerophosphocholine (GPC) concentrations were higher than phosphocholine (PCho) concentrations, whereas this pattern was reversed in luminal-like xenografts. These differences may be explained by lower choline kinase (CHKA, CHKB) expression as well as higher PtdCho degradation mediated by higher expression of phospholipase A2 group 4A (PLA2G4A) and phospholipase B1 (PLB1) in the basal-like model. The glycine concentration was higher in the basal-like model. Although glycine could be derived from energy metabolism pathways, the gene expression data suggested a metabolic shift from PtdCho synthesis to glycine formation in basal-like xenografts. In agreement with results from the xenograft models, tissue samples from triple negative breast carcinomas had higher GPC/PCho ratio than samples from ER+/PgR+ carcinomas, suggesting that the choline metabolism in the experimental models is representative for luminal-like and basal-like human breast cancer. Conclusions: The differences in choline metabolite concentrations corresponded well with differences in gene expression, demonstrating distinct metabolic profiles in the xenograft models representing basal-like and luminallike breast cancer. The same characteristics of choline metabolite profiles were also observed in patient material from ER+/PgR+ and triple-negative breast cancer, suggesting that the xenografts are relevant model systems for studies of choline metabolism in luminal-like and basal-like breast cancer. [ABSTRACT FROM AUTHOR]

Background: High activity of cysteine proteases such as legumain and the cathepsins have been shown to facilitate growth and invasion of a variety of tumor types. In breast cancer, several recent studies have indicated that loss of the cysteine protease inhibitor cystatin E/M leads to increased growth and metastasis. Although cystatin E/M is normally expressed in the skin, its role in cysteine protease regulation and progression of malignant melanoma has not been studied. Methods: A panel of various non-melanoma and melanoma cell lines was used. Cystatin E/M and C were analyzed in cell media by immunoblotting and ELISA. Legumain, cathepsin B and L were analyzed in cell lysates by immunoblotting and their enzymatic activities were analyzed by peptide substrates. Two melanoma cell lines lacking detectable secretion of cystatin E/M were transfected with a cystatin E/M expression plasmid (pCST6), and migration and invasiveness were studied by a Matrigel invasion assay. Results: Cystatin E/M was undetectable in media from all established melanoma cell lines examined, whereas strong immunobands were detected in two of five primary melanoma lines and in two of six lines derived from patients with metastatic disease. Among the four melanoma lines secreting cystatin E/M, the glycosylated form (17 kD) was predominant compared to the non-glycosylated form (14 kD). Legumain, cathepsin B and L were expressed and active in most of the cell lines, although at low levels in the melanomas expressing cystatin E/M. In the melanoma lines where cystatin E/M was secreted, cystatin C was generally absent or expressed at a very low level. When melanoma cells lacking secretion of cystatin E/M were transfected with pCST6, their intracellular legumain activity was significantly inhibited. In contrast, cathepsin B activity was not affected. Furthermore, invasion was suppressed in cystatin E/M over-expressing melanoma cell lines as measured by the transwell Matrigel assay. Conclusions: These results suggest that the level of cystatin E/M regulates legumain activity and hence the invasive potential of human melanoma cells. [ABSTRACT FROM AUTHOR]

Background/aims: Breast cancer metastasis suppressor 1 (BRMS1) blocks metastasis in melanoma xenografts; however, its usefulness as a biomarker in human melanomas has not been widely studied. The goal was to measure BRMS1 expression in benign nevi, primary and metastatic melanomas and evaluate its impact on disease progression and prognosis.Methods: Paraffin-embedded tissue from 155 primary melanomas, 69 metastases and 15 nevi was examined for BRMS1 expression using immunohistochemistry. siRNA mediated BRMS1 down-regulation was used to study impact on invasion and migration in melanoma cell lines.Results: A significantly higher percentage of nevi (87%), compared to primary melanomas (20%) and metastases (48%), expressed BRMS1 in the nucelus (p < 0.0001). Strong nuclear staining intensity was observed in 67% of nevi, and in 9% and 24% of the primary and metastatic melanomas, respectively (p < 0.0001). Comparable cytoplasmic expression was observed (nevi; 87%, primaries; 86%, metastases; 72%). However, a decline in cytoplasmic staining intensity was observed in metastases compared to nevi and primary tumors (26%, 47%, and 58%, respectively, p < 0.0001). Score index (percentage immunopositive celles multiplied with staining intensity) revealed that high cytoplasmic score index (≥ 4) was associated with thinner tumors (p = 0.04), lack of ulceration (p = 0.02) and increased disease-free survival (p = 0.036). When intensity and percentage BRMS1 positive cells were analyzed separately, intensity remained associated with tumor thickness (p = 0.024) and ulceration (p = 0.004) but was inversely associated with expression of proliferation markers (cyclin D3 (p = 0.008), cyclin A (p = 0.007), and p21Waf1/Cip1 (p = 0.009)). Cytoplasmic score index was inversely associated with nuclear p-Akt (p = 0.013) and positively associated with cytoplasmic p-ERK1/2 expression (p = 0.033). Nuclear BRMS1 expression in ≥ 10% of primary melanoma cells was associated with thicker tumors (p = 0.016) and decreased relapse-free period (p = 0.043). Nuclear BRMS1 was associated with expression of fatty acid binding protein 7 (FABP7; p = 0.011), a marker of invasion in melanomas. In line with this, repression of BRMS1 expression reduced the ability of melanoma cells to migrate and invade in vitro.Conclusion: Our data suggest that BRMS1 is localized in cytoplasm and nucleus of melanocytic cells and that cellular localization determines its in vivo effect. We hypothesize that cytoplasmic BRMS1 restricts melanoma progression while nuclear BRMS1 possibly promotes melanoma cell invasion.Please see related article: http://www.biomedcentral.com/1741-7015/10/19. [ABSTRACT FROM AUTHOR]

Background: The metastasis-promoting protein S100A4 activates the transcription factor NF-kappaB through the classical NF-kappaB activation pathway. The upstream signal transduction mechanisms leading to increased NF-kappaB activity are, however, incompletely characterized.Methods: The human osteosarcoma cell line II-11b was stimulated with recombinant S100A4 in the presence or absence of inhibitors of common signal transduction pathways, and NF-kappaB activity was examined using a luciferase-based reporter assay and phosphorylation of IkappaBalpha. mRNA expression was analyzed by real-time RT-PCR, protein expression was examined by Western blotting and IKK activity was measured using an in vitro kinase assay. The role of upstream kinases and the cell surface receptor RAGE was investigated by overexpression of dominant negative proteins and by siRNA transfection.Results: The Ser/Thr kinase inhibitors H-7 and staurosporine inhibited S100A4-induced IkappaBalpha phosphorylation and subsequent NF-kappaB activation. The protein tyrosine kinase inhibitor genistein and the phospholipase C inhibitor compound 48/80 had a partial inhibitory effect on IkappaBalpha phosphorylation, whereas inhibitors of protein kinase C, G-protein coupled receptors and PI 3-kinases had no effect on the level of phosphorylation. Interestingly, S100A4 treatment induced activating phosphorylations of IKKalpha/beta, but neither H-7 nor staurosporine was able to significantly inhibit IKK activation. Dominant negative MEKK1 or NIK did not inhibit S100A4-induced NF-kappaB activity, and S100A4 stimulation did not influence AKT phosphorylation. Furthermore, diminished expression of the putative S100 protein receptor RAGE did not affect the observed phosphorylation of IkappaBalpha.Conclusions: S100A4 activates NF-kappaB by inducing phosphorylation of IKKalpha/beta, leading to increased IkappaBalpha phosphorylation. The Ser/Thr kinase inhibitors H-7 and staurosporine attenuated S100A4-induced NF-kappaB activation and inhibited IKK-mediated phosphorylation of IkappaBalpha. S100A4-induced NF-kappaB activation was independent of the putative S100 protein receptor RAGE and the Ser/Thr kinases MEKK1, NIK and AKT. These findings lead to increased understanding of S100A4 signaling, which may contribute to the identification of novel targets for anti-metastatic therapy. [ABSTRACT FROM AUTHOR]

Background: Increased concentrations of choline-containing compounds are frequently observed in breast carcinomas, and may serve as biomarkers for both diagnostic and treatment monitoring purposes. However, underlying mechanisms for the abnormal choline metabolism are poorly understood.Methods: The concentrations of choline-derived metabolites were determined in xenografted primary human breast carcinomas, representing basal-like and luminal-like subtypes. Quantification of metabolites in fresh frozen tissue was performed using high-resolution magic angle spinning magnetic resonance spectroscopy (HR MAS MRS). The expression of genes involved in phosphatidylcholine (PtdCho) metabolism was retrieved from whole genome expression microarray analyses. The metabolite profiles from xenografts were compared with profiles from human breast cancer, sampled from patients with estrogen/progesterone receptor positive (ER+/PgR+) or triple negative (ER-/PgR-/HER2-) breast cancer.Results: In basal-like xenografts, glycerophosphocholine (GPC) concentrations were higher than phosphocholine (PCho) concentrations, whereas this pattern was reversed in luminal-like xenografts. These differences may be explained by lower choline kinase (CHKA, CHKB) expression as well as higher PtdCho degradation mediated by higher expression of phospholipase A2 group 4A (PLA2G4A) and phospholipase B1 (PLB1) in the basal-like model. The glycine concentration was higher in the basal-like model. Although glycine could be derived from energy metabolism pathways, the gene expression data suggested a metabolic shift from PtdCho synthesis to glycine formation in basal-like xenografts. In agreement with results from the xenograft models, tissue samples from triple negative breast carcinomas had higher GPC/PCho ratio than samples from ER+/PgR+ carcinomas, suggesting that the choline metabolism in the experimental models is representative for luminal-like and basal-like human breast cancer.Conclusions: The differences in choline metabolite concentrations corresponded well with differences in gene expression, demonstrating distinct metabolic profiles in the xenograft models representing basal-like and luminal-like breast cancer. The same characteristics of choline metabolite profiles were also observed in patient material from ER+/PgR+ and triple-negative breast cancer, suggesting that the xenografts are relevant model systems for studies of choline metabolism in luminal-like and basal-like breast cancer. [ABSTRACT FROM AUTHOR]