Urine Of Breast Cancer Patients Research Articles

Establishing pteridine metabolism in a progressive isogenic breast cancer cell model.

Pteridines include folate-derived metabolites that have been putatively associated with certain cancers in clinical studies. However, their biological significance in cancer metabolism and role in cancer development and progression remains poorly understood. The purpose of this study was to examine the effects of tumorigenicity on pteridine metabolism by studying a panel of 15 pteridine derivatives using a progressive breast cancer cell line model with and without folic acid dosing. The MCF10A progressive breast cancer model, including sequentially derived MCF10A (benign), MCF10AT (premalignant), and MCF10CA1a (malignant) cell lines were dosed with 0, 100, and 250 mg/L folic acid. Pteridines were analyzed in both intracellular and extracellular contexts using an improved high-performance liquid chromatography-tandem mass spectrometry method. Pteridines were located predominately in the extracellular media. Folic acid dosing increased extracellular levels of pterin, 6-hydroxylumazine, xanthopterin, 6-hydroxymethylpterin, and 6-carboxypterin in a dose-dependent manner. In particular, pterin and 6-hydroxylumazine levels were positively correlated with tumorigenicity upon folate dosing. Folic acid is a primary driver for pteridine metabolism in human breast cell. Higher folate levels contribute to increased formation and excretion of pteridine derivatives to the extracellular media. In breast cancer, this metabolic pathway becomes dysregulated, resulting in the excretion of certain pteridine derivatives and providing in vitro evidence for the observation of elevated pteridines in the urine of breast cancer patients. Finally, this study reports a novel use of the MCF10A progressive breast cancer model for metabolomics applications that may readily be applied to other metabolites of interest.

The expression and significance of 8-hydroxydeoxyguanosine in breast cancer patients’ blood, urine and cancer tissue

Objective: To explore the expression and clinic significance of 8-OHdG in breast cancer. Methods: Pre-operative serum 8-OHdG levels were detected with an enzyme-linked immunosorbent assay in a well-defined series of 173 breast cancer patients. 8-OHdG expression in cancer cells from 150 of these patients was examined by immunohistochemistry. The HPLC-ECD method is used to determine 8-OHdG concentration in urine. Results: The serum 8-OHdG levels and immunohistochemical 8-OHdG expression were in concordance with each other (P < 0.05, r = 0.163). Breast cancer patients with negative 8-OHdG immunostaining show lower survival rate according to the multivariate analysis (P < 0.01). This observation was even more remarkable in ductal carcinomas (n = 140) patients (P < 0.001). A low serum 8-OHdG level was associated statistically significantly with lymphatic vessel invasion and a positive lymph node status. Comparison of 8-OHdG concentration in urine of breast cancer patients and healthy women was statistical significance (P < 0.01). Conclusion: Low serum 8-OHdG levels and a low immunohistochemical 8-OHdG expression were associated with an aggressive breast cancer phenotype. In addition, negative 8-OHdG immunostaining was an independent prognostic factor for breast cancer-specific death in breast carcinoma patients. Using 8-OHdG concentration in urine to predict DNA damage resulting from breast cancer can provide good biological indicators for detecting harm in early breast cancer.

Biochemical Background in Mitochondria Affects 2HG Production by IDH2 and ADHFE1 in Breast Carcinoma.

Mitochondrial production of 2-hydroxyglutarate (2HG) can be catalyzed by wild-type isocitrate dehydrogenase 2 (IDH2) and alcohol dehydrogenase, iron-containing 1 (ADHFE1). We investigated whether biochemical background and substrate concentration in breast cancer cells promote 2HG production. To estimate its role in 2HG production, we quantified 2HG levels and its enantiomers in breast cancer cells using analytical approaches for metabolomics. By manipulation of mitochondrial substrate fluxes using genetic and pharmacological approaches, we demonstrated the existence of active competition between 2HG producing enzymes, i.e., IDH2 and ADHFE1. Moreover, we showed that distinct fractions of IDH2 enzyme molecules operate in distinct oxido-reductive modes, providing NADPH and producing 2HG simultaneously. We have also detected 2HG release in the urine of breast cancer patients undergoing adjuvant therapy and detected a correlation with stages of breast carcinoma development. In summary, we provide a background for vital mitochondrial production of 2HG in breast cancer cells with outcomes towards cancer biology and possible future diagnosis of breast carcinoma.

Implementing a central composite design for the optimization of solid phase microextraction to establish the urinary volatomic expression: a first approach for breast cancer.

Breast cancer (BC) is positioned as the second among all cancers remaining at the top of women´s diseases worldwide followed by colorectum, lung, cervix, and thyroid cancers. The main drawback of most the screening/diagnostic methods is their low sensitivity/specificity and in some cases the invasive procedure required to obtain the samples. On the present investigation, we report a statistical design was to evaluate by central composite design the influence towards the optimization of the most significant variables of solid-phase microextraction (SPME) procedure for the isolation of volatile organic metabolites (VOMs) from urine of BC patients (N = 31) and healthy individuals (CTL; N = 40). The establishment of the urinary volatomic composition, through gas chromatography-mass spectrometry (GC-MS) analysis, can boost the identification of volatile organic metabolites (VOMs) potential BC biomarkers useful to be used together or to complement the current BC diagnostics tools. Better early detection methods are needed to improve the outcomes of patients with BC. Several combinations of experiments were considered with a central composite design (CCD) of response surface methodology (RSM) for the urinary volatomic pattern. Three-level three-factor CCD was employed assessing the most important extraction-influencing variables-fiber coating, NaCl amount, extraction time and temperature. The optimal conditions were achieved using a carboxen/polydimethylsiloxane fiber with 15% (w/v) NaCl during 75min at 50°C. A total of ten VOMs belonging to sulfur compounds, terpenoids and carbonyl compounds presented the highest contribution towards discrimination of BC patients from CTL (variable importance in projection (VIP) > 1, p < 0.05). The discrimination efficiency and accuracy of urinary metabolites was ascertained by receiver operating characteristic (ROC) curve analysis that allowed the identification of some metabolites with highest sensitivity and specificity to discriminate the groups. The results obtained with this approach suggest the possibility to identify endogenous metabolites as a platform to discovery potential BC biomarkers and paves a way to explore the related metabolomic pathways in order to improve BC diagnostic tools.

Urinary metallomics as a novel biomarker discovery platform: Breast cancer as a case study

BackgroundUrinary metallomics is presented here as a new “omics” approach that aims to facilitate personalized cancer screening and prevention by improving our understanding of urinary metals in disease. MethodsTwenty-two urinary metals were examined with inductively-coupled plasma-mass spectrometry in 138 women newly diagnosed with breast cancer and benign conditions. Urinary metals from spot urine samples were adjusted to renal dilution using urine specific gravity. ResultsTwo urinary metals, copper (P-value=0.036) and lead (P-value=0.003), were significantly increased in the urine of breast cancer patients. A multivariate model that comprised copper, lead, and patient age afforded encouraging discriminatory power (AUC: 0.728, P-value<0.0005), while univariate models of copper (61.7% sensitivity, 50.0% specificity) and lead (76.6% sensitivity, 51.2% specificity) at optimized cutoff thresholds compared favorably with other breast cancer diagnostic modalities such as mammography. Correlations found among various metals suggested potential geographic and dietary influences on the urine metallome that warrant further investigation. ConclusionsThis proof-of-concept work introduces urinary metallomics as a noninvasive, potentially transformative “omics” approach to early cancer detection. Urinary copper and lead have also been preliminarily identified as potential breast cancer biomarkers.

Urinary concentrations of ADAM 12 from breast cancer patients pre- and post-surgery vs. cancer-free controls: a clinical study for biomarker validation

BackgroundThe ADAMs (A Disintegrin and Metalloproteinases) are a family of multi-domain, zinc-dependent metalloproteinase enzymes. ADAM 12 has been previously associated with the onset and progression of breast cancer, and elevated levels of ADAM 12 have been previously found in the urine of breast cancer patients. Aims of the current study are: 1) establish the viability of urinary ADAM 12 as a diagnostic marker for breast cancer, and 2) explore the effects of surgical tumor removal on the levels of urinary ADAM 12.MethodsA total of 96 patients have been recruited for this study, including 50 patients diagnosed with cancer, and 46 age-matched controls. Commercially available ELISA kits for ADAM 12 were used to quantify the presence and concentration of this enzyme in the urine from cancer patients with ductal carcinoma in situ (DCIS) and invasive breast cancer (IBC) both prior to any treatment and approximately two weeks following surgery, as well as from controls.ResultsWe find no statistically significant differences between the concentrations of ADAM 12 in the urine of breast cancer patients prior to treatment and that of their age-matched controls; however the concentration of ADAM 12, both alone and as a function of urine total protein, are significantly elevated following surgery (p < 0.0001). Patients who underwent a mastectomy have significantly higher urinary ADAM 12 concentrations than those who underwent a lumpectomy (significant at p = 0.0271).ConclusionsThese findings suggest that urinary ADAM 12 may not correlate directly with the status and stage of breast cancer as previously thought; rather these increases may be a result of tissue injury and inflammation from biopsy and surgical resection. Results of this study may suggest a need for biomarkers to be evaluated carefully in the context of tissue damage.

Potential of fluorescent metalloproteinase substrates for cancer detection

Objectives MMP-2, MMP-9, their complexes and ADAM12 are detected in the urine of breast cancer patients and predict disease status. We assessed the use of FRET-based substrates in an assay to distinguish breast cancer patients from controls. Design and methods Substrates with varying specificities for MMP-9 and MMP-2 and several ADAMs were screened. Flsub21 and Flsub13, substrates for ADAM12 and ADAM8 respectively, were studied. Results Flsub21 and Flsub13 cleavage activities were detected in the urine of patients with invasive and metastatic breast cancers at significantly higher frequencies compared to controls. Our model predicted probabilities of 90% when both Flsub21 and Flsub13 were positive, 65% when Flsub21 alone was positive, 55% when Flsub13 alone was positive and 20% when both substrates were negative. Conclusions These data suggest the potential utility of FRET substrates to non-invasively identify invasive and/or metastatic breast cancer.

ADAM12 induces estrogen-independence in breast cancer cells

Antiestrogen therapy has been used successfully to prolong disease-free and overall survival of ER positive breast cancer patients. However, 50% of patients with ER+tumors fail to respond to such therapy or eventually acquire resistance to endocrine therapy, resulting in tumor progression and mortality. It is imperative, therefore, to understand the mechanisms that lead to hormone refractory breast cancer in order to develop therapeutics that can modulate the resistance to antiestrogen therapy. The protease, ADAM12, can be detected in the urine of breast cancer patients and its levels correlate with disease status, stage, and cancer risk. Within the context of this study, the authors have investigated the role of the two distinct isoforms of ADAM12 in breast tumor cell proliferation and as potential mediators of endocrine resistance. Using stable clones of ADAM12-overexpressing MCF-7 cells, the authors analyzed proliferation rates of these ER+breast tumor cells both in estrogen-depleted medium and in the presence of the antiestrogens, tamoxifen, and ICI 182,780. Acquired estrogen resistance in these cells was analyzed using phospho-RTK analysis. Upregulation and phosphorylation of proteins were detected via immunoprecipitation and immunoblotting. EGFR and MAPK inhibitors were used to explore the mechanism of acquired estrogen resistance in breast tumor cells. It was observed that overexpression of the two isoforms, transmembrane ADAM12-L, and secreted ADAM12-S, in breast tumor cells promoted estrogen-independent proliferation. In ADAM12-L-expressing cells, estrogen-independence was a direct result of increased EGFR expression and MAPK activation, whereas, the mechanism in ADAM12-S-expressing cells may be enhanced IGF-1R signaling. The importance of the EGFR signaling pathway in the estrogen-independent growth of ADAM12-L expressing cells was highlighted by the effect of EGFR inhibitors AG1478 and PD15035 or MAPK inhibitor U0126, each of which abolished the antiestrogen resistance in these cells. Taken together, these results demonstrate that ADAM12 isoforms confer a proliferative advantage to MCF-7 cells in the absence of estrogen stimulation, and suggest that downregulation of ADAM12 in combination with endocrine therapy may represent a useful pharmacological approach to breast cancer therapy.

Quantitative analysis of urinary phospholipids found in patients with breast cancer by nanoflow liquid chromatography–tandem mass spectrometry: II. Negative ion mode analysis of four phospholipid classes

Analysis was performed on four different categories of phospholipids (phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), and phosphatidic acid (PA)) from urine in patients with breast cancer. This quantitative analysis was conducted using nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS-MS). This study shows the profiling of the phospholipids (PLs) that can be identified by the negative ion mode of MS. A previous study (Kim et al. Anal. Bioanal. Chem. 393:1649, 21) focused on only two PL classes: phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) and were identified by positive ion mode. PLs were extracted by lyophilization of 1 mL of urine from both healthy normal females and breast cancer patients before and after surgery. Separation of PLs was performed by nLC followed by structural identification of PLs using data-dependent collision-induced dissociation. A total of 34 urinary PL molecules (12 PSs, 12 PIs, four PGs, and six PAs) were quantitatively examined. Among the four PL categories examined in this study, most PL classes showed an increase in the total amounts in the cancer patients, yet PIs exhibited some decreases. The present study suggests that the lipid composition found in the urine of breast cancer patients can be utilized for the possible development of disease markers, when the analysis is performed with negative ion mode of nLC-ESI-MS-MS.

TNF receptor p55 and IL-872 and IL-877 isoforms: blood and urine levels in breast cancer patients

In the preliminary study reported here, 37 patients with breast cancer and 10 healthy volunteers were analyzed for soluble TNF-R p55 and two variants of IL-8 consisting of 72 and 77 amino acid residues (IL-872 and IL-877, respectively) in their blood and urine with novel ELISA test systems. The clinical/prognostic values of determining these inflammatory cytokines at different stages of the cancer process appeared to depend on the treatment course being evaluated. In contrast to expectations, it was noted that there was a stabile tendency for decreased TNF-R p55 and IL-872 levels in the plasma and urine of breast cancer patients as compared with levels observed with healthy controls. Moreover, patients that underwent polychemotherapy treatments were notable for significant decreases in IL-872 and TNF-R p55 levels in their blood plasma; these findings contrasted with significant increases in these parameters in these patients’ urine. Interestingly, the IL-877 isoform that now appeared both in the urine and plasma of patients was not detectable before initiation of the polychemotherapy. In spite of all these findings, individual fluctuations among these parameters still do not allow us to establish, at this time, any strong correlations between these values with any particular breast cancer stage or a type of treatment. Nonetheless, while the results here are preliminary, they demonstrate that testing for TNF-R, along with IL-8 isoforms, in the blood plasma and urine could potentially present a valid means for monitoring of the overall immune and disease progress/remission status in breast cancer patients. Ongoing studies with larger patient sample sizes, as well as collecting and analyzing samples at multiple timepoints—to minimize the potential influence of any inherent variability in cytokine levels in humans—will hopefully allow us to specify what these preliminary results reported here suggest, i.e., the potential utility of this experimental approach for determining disease progression or efficacy of treatment in cancer patients.

Autoantibody to Tumor Antigen, Alpha 2-HS Glycoprotein: A Novel Biomarker of Breast Cancer Screening and Diagnosis

We sought to identify a new serum biomarker for breast cancer screening and diagnosis using stepwise proteomic analysis of sera from breast cancer patients to detect the presence of autoantibodies that react with urinary protein. Two-dimensional immunoblotting was done for screening autoimmunogenic tumor antigens in the urine of breast cancer patients. Reactive spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Among urinary proteins separated by two-dimensional electrophoresis, 13 spots showed strong reactivity with pooled sera from breast cancer patients or control sera. By mass spectrometry, we identified alpha 2-HS glycoprotein (AHSG) as a tumor antigen. Peripheral blood was obtained from 81 women diagnosed with breast cancer before surgery and 73 female donors without evidence of any malignancy for the individual analysis. In one-dimensional Western blot analysis, AHSG autoantibody was detected in 64 of 81 breast cancer patients (79.1%) and in 7 of 73 controls (9.6%). The sensitivity of this test in breast cancer patients was 79.0%. Our results suggest that AHSG and anti-AHSG autoantibody may be useful serum biomarkers for breast cancer screening and diagnosis.

Urinary Metalloproteinases: Noninvasive Biomarkers for Breast Cancer Risk Assessment

Matrix metalloproteinases (MMP) and a disintegrin and metalloprotease 12 (ADAM 12) can be detected in the urine of breast cancer patients and provide independent prediction of disease status. To evaluate the potential of urinary metalloproteinases as biomarkers to predict breast cancer risk status, urine samples from women with known risk marker lesions, atypical hyperplasia and lobular carcinoma in situ (LCIS), were analyzed. Urine samples were obtained from 148 women: 44 women with atypical hyperplasia, 24 women with LCIS, and 80 healthy controls. MMP analysis was done using gelatin zymography and ADAM 12 analysis was done via immunoblotting with monospecific antibodies and subsequent densitometric measurement. Positive urinary MMP-9 levels indicated a 5-fold risk of atypical hyperplasia and >13-fold risk of LCIS compared with normal controls. Urinary ADAM 12 levels were significantly elevated in women with atypical hyperplasia and LCIS from normal controls, with receiver operating characteristic curve analysis showing an area under the curve of 0.914 and 0.950, respectively. To assess clinical applicability, a predictive index was developed using ADAM 12 in conjunction with Gail risk scores for women with atypia. Scores above 2.8 on this ADAM 12-Gail risk prediction index score are predictive of atypical hyperplasia (sensitivity, 0.976; specificity, 0.977). Our data suggest that the noninvasive detection and analysis of urinary ADAM 12 and MMP-9 provide important clinical information for use as biomarkers in the identification of women at increased risk of developing breast cancer.

Targeted inhibition of EG-1 blocks breast tumor growth

EG-1 is a gene product that is significantly elevated in human breast cancer tissues. Previously, we have shown that EG-1 overexpression stimulates cellular proliferation both in vitro and in vivo. Here, we ask whether this molecule can be targeted for experimental therapeutic purpose. siRNA lentivirus and polyclonal antibody were designed to suppress EG-1 expression. These agents were then used in cell culture proliferation assays and breast tumor xenograft models. Serum and urine from breast cancer patients were also analyzed for the presence of EG-1 peptide. We report here for the first time that endogenous EG-1 can be targeted to inhibit breast tumor growth. This inhibition, whether delivered via siRNA lentivirus or polyclonal antibody, resulted in decreased cellular proliferation in culture and smaller xenografts in mice. The effects were shown in both ER (estrogen receptor)-positive human breast cancer MCF-7 cells, as well as in ER-negative MDA-MB-231 cells. Furthermore, we detected soluble EG-1 in serum and urine of breast cancer patients. These observations demonstrate that EG-1 is relevant to human breast cancer, and is a molecular target worthy of translational efforts into effective breast cancer therapy.

The Matrix Metalloproteinase-9/Neutrophil Gelatinase-Associated Lipocalin Complex Plays a Role in Breast Tumor Growth and Is Present in the Urine of Breast Cancer Patients

Having previously shown that the binding of neutrophil gelatinase-associated lipocalin (NGAL) to matrix metalloproteinase-9 (MMP-9) protects this extracellular matrix remodeling enzyme from autodegradation, we hypothesized that the addition of NGAL to breast cancer cells, which do not express this protein but do express MMP-9, might result in a more aggressive phenotype in vivo. Based on our previous reports that MMPs can be detected in the urine of cancer patients, we also asked whether MMP-9/NGAL could be detected in the urine of breast cancer patients and whether it might be predictive of disease status. Clones of MCF-7 human breast cancer cells differentially expressing NGAL were generated by stable transfection with human NGAL expression constructs. The established clones were then implanted s.c. in immunodeficient mice and tumor growth was monitored. In addition, we analyzed the urine of individuals with breast cancer and age-matched, sex-matched controls using gelatin zymography for the presence of MMP-9/NGAL. Increased NGAL expression resulted in significant stimulation of tumor growth. Immunohistochemical analysis of MCF-7 tumors revealed that the NGAL-overexpressing ones exhibited increased growth rates that were accompanied by increased levels of MMP-9, increased angiogenesis, and an increase in the tumor cell proliferative fraction. In addition, MMP-9/NGAL complex was detected in 86.36% of the urine samples from breast cancer patients but not in those from healthy age and sex-matched controls. These findings suggest, for the first time, that NGAL may play an important role in breast cancer in vivo by protecting MMP-9 from degradation thereby enhancing its enzymatic activity and facilitating angiogenesis and tumor growth. Clinically, these data suggest that the urinary detection of MMP-9/NGAL may be useful in noninvasively predicting disease status of breast cancer patients.

ADAM 12 Cleaves Extracellular Matrix Proteins and Correlates with Cancer Status and Stage

ADAM 12 is a member of a family of disintegrin-containing metalloproteases that have been implicated in a variety of diseases including Alzheimer's disease, arthritis, and cancer. We purified ADAM 12 from the urine of breast cancer patients via Q-Sepharose anion exchange and gelatin-Sepharose affinity chromatography followed by protein identification by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Four peptides were identified that spanned the amino acid sequence of ADAM 12. Immunoblot analysis using ADAM 12-specific antibodies detected an approximately 68-kDa band identified as the mature form of ADAM 12. To characterize catalytic properties of ADAM 12, full-length ADAM 12-S was expressed in COS-7 cells and purified. Substrate specificity studies demonstrated that ADAM 12-S degrades gelatin, type IV collagen, and fibronectin but not type I collagen or casein. Gelatinase activity of ADAM 12 was completely abrogated by zinc chelators 1,10-phenanthroline and EDTA and was partially inhibited by the hydroxamate inhibitor Marimastat. Endogenous matrix metalloprotease inhibitor TIMP-3 inhibited activity. To validate our initial identification of this enzyme in human urine, 117 urine samples from breast cancer patients and controls were analyzed by immunoblot. The majority of samples from cancer patients were positive for ADAM 12 (67 of 71, sensitivity 0.94) compared with urine from controls in which ADAM 12 was detected with significantly lower frequency. Densitometric analyses of immunoblots demonstrated that ADAM 12 protein levels were higher in urine from breast cancer patients than in control urine. In addition, median levels of ADAM 12 in urine significantly increased with disease progression. These data demonstrate for the first time that ADAM 12 is a gelatinase, that it can be detected in breast cancer patient urine, and that increased urinary levels of this protein correlate with breast cancer progression. They further support the possibility that detection of urinary ADAM 12 may prove useful in the development of noninvasive diagnostic and prognostic tests for breast and perhaps other cancers.

Can 8-oxo-dG be used as a predictor for individual radiosensitivity?

Purpose: To develop predictive tests for individual radiosensitivity of tumor patients. Methods and Materials: Acute skin reactions were clinically scored among 40 women after 46 Gy, given with 2 Gy fractions to breast and regional lymph nodes, adjuvant after surgery. The acute skin reactions were compared to the excretion of 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dG) in urine, determined by high-performance liquid chromatography (HPLC) with electrochemical detector. Specimens of urine were collected before and during postoperative radiation treatment at given intervals. We compared a group of 9 patients with the most pronounced skin reactions with another group of 8 patients with almost no skin reactions at 46 Gy. Results: The level of 8-oxo-dG excreted in urine during 8 h was measured. After normalizing the excretion to irradiated volumes, dose per volume and excretion before irradiation, the 8-oxo-dG level in urine was significantly ( p < 0.001) lower for the patients with pronounced skin reactions as compared to patients with minor skin reactions, at an accumulated dose of 12 Gy. In addition, the background level of 8-oxo-dG excreted before treatment started, was significantly ( p = 0.043) lower for patients with minor skin reactions as compared to patients with pronounced skin reactions. The background level of 8-oxo-dG was corrected for body weight and normalized to BMI. Conclusion: We suggest that the excretion of 8-oxo-dG into urine of breast cancer patients is a possible marker for acute radiosensitivity.

Concentration of epidermal growth factor (EGF) receptor ligands in the urine of breast cancer patients & control subjects

Concentration of epidermal growth factor (EGF) receptor ligands in the urine of breast cancer patients & control subjects