Shotgun proteomics coupled to nanoparticle-based biomarker enrichment reveals a novel panel of extracellular matrix proteins as candidate serum protein biomarkers for early-stage breast cancer detection.

BACKGROUND: The lack of specificity and high degree of false positive and false negative rates when using mammographic screening for detecting early-stage breast cancer is a critical issue. Blood-based molecular assays that could be used in adjunct with mammography for increased specificity and sensitivity could have profound clinical impact. Our objective was to discover and independently verify a panel of candidate blood-based biomarkers that could identify the earliest stages of breast cancer and complement current mammographic screening approaches. METHODS: We used affinity hydrogel nanoparticles coupled with LC-MS/MS analysis to enrich and analyze low-abundance proteins in serum samples from 20 patients with invasive ductal carcinoma (IDC) breast cancer and 20 female control individuals with positive mammograms and benign pathology at biopsy. We compared these results to those obtained from five cohorts of individuals diagnosed with cancer in organs other than breast (ovarian, lung, prostate, and colon cancer, as well as melanoma) to establish IDC-specific protein signatures. Twenty-four IDC candidate biomarkers were then verified by multiple reaction monitoring (LC-MRM) in an independent validation cohort of 60 serum samples specifically including earliest-stage breast cancer and benign controls (19 early-stage (T1a) IDC and 41 controls). RESULTS: In our discovery set, 56 proteins were increased in the serum samples from IDC patients, and 32 of these proteins were specific to IDC. Verification of a subset of these proteins in an independent cohort of early-stage T1a breast cancer yielded a panel of 4 proteins, ITGA2B (integrin subunit alpha IIb), FLNA (Filamin A), RAP1A (Ras-associated protein-1A), and TLN-1 (Talin-1), which classified breast cancer patients with 100% sensitivity and 85% specificity (AUC of 0.93). CONCLUSIONS: Using a nanoparticle-based protein enrichment technology, we identified and verified a highly specific and sensitive protein signature indicative of early-stage breast cancer with no false positives when assessing benign and inflammatory controls. These markers have been previously reported in cell-ECM interaction and tumor microenvironment biology. Further studies with larger cohorts are needed to evaluate whether this biomarker panel improves the positive predictive value of mammography for breast cancer detection.

Urine lipoarabinomannan glycan in HIV-negative patients with pulmonary tuberculosis correlates with disease severity.

An accurate urine test for pulmonary tuberculosis (TB), affecting 9.6 million patients worldwide, is critically needed for surveillance and treatment management. Past attempts failed to reliably detect the mycobacterial glycan antigen lipoarabinomannan (LAM), a marker of active TB, in HIV-negative, pulmonary TB-infected patients' urine (85% of 9.6 million patients). We apply a copper complex dye within a hydrogel nanocage that captures LAM with very high affinity, displacing interfering urine proteins. The technology was applied to study pretreatment urine from 48 Peruvian patients, all negative for HIV, with microbiologically confirmed active pulmonary TB. LAM was quantitatively measured in the urine with a sensitivity of >95% and a specificity of >80% (n = 101) in a concentration range of 14 to 2000 picograms per milliliter, as compared to non-TB, healthy and diseased, age-matched controls (evaluated by receiver operating characteristic analysis; area under the curve, 0.95; 95% confidence interval, 0.9005 to 0.9957). Urinary LAM was elevated in patients with a higher mycobacterial burden (n = 42), a higher proportion of weight loss (n = 37), or cough (n = 50). The technology can be configured in a variety of formats to detect a panel of previously undetectable very-low-abundance TB urinary analytes. Eight of nine patients who were smear-negative and culture-positive for TB tested positive for urinary LAM. This technology has broad implications for pulmonary TB screening, transmission control, and treatment management for HIV-negative patients.

Phosphatidylcholine-specific phospholipase C inhibition reduces HER2-overexpression, cell proliferation and in vivo tumor growth in a highly tumorigenic ovarian cancer model.

Antagonizing the oncogenic effects of human epidermal growth factor receptor 2 (HER2) with current anti-HER2 agents has not yet yielded major progress in the treatment of advanced HER2-positive epithelial ovarian cancer (EOC). Using preclinical models to explore alternative molecular mechanisms affecting HER2 overexpression and oncogenicity may lead to new strategies for EOC patient treatment. We previously reported that phosphatidylcholine-specific phospholipase C (PC-PLC) exerts a pivotal role in regulating HER2 overexpression in breast cancer cells. The present study, conducted on two human HER2-overexpressing EOC cell lines - SKOV3 and its in vivo-passaged SKOV3.ip cell variant characterized by enhanced in vivo tumorigenicity - and on SKOV3.ip xenografts implanted in SCID mice, showed: a) about 2-fold higher PC-PLC and HER2 protein expression levels in SKOV3.ip compared to SKOV3 cells; b) physical association of PC-PLC with HER2 in non-raft domains; c) HER2 internalization and ca. 50% reduction of HER2 mRNA and protein expression levels in SKOV3.ip cells exposed to the PC-PLC inhibitor tricyclodecan-9-yl-potassium xanthate (D609); d) differential effects of D609 and trastuzumab on HER2 protein expression and cell proliferation; e) decreased in vivo tumor growth in SKOV3.ip xenografts during in vivo treatment with D609; f) potential use of in vivo magnetic resonance spectroscopy (MRS) and imaging (MRI) parameters as biomarkers of EOC response to PC-PLC inhibition. Overall, these findings support the view that PC-PLC inhibition may represent an effective means to target the tumorigenic effects of HER2 overexpression in EOC and that in vivo MR approaches can efficiently monitor its effects.

Activation of Phosphatidylcholine-Specific Phospholipase C in Breast and Ovarian Cancer: Impact on MRS-Detected Choline Metabolic Profile and Perspectives for Targeted Therapy.

Elucidation of molecular mechanisms underlying the aberrant phosphatidylcholine cycle in cancer cells plays in favor of the use of metabolic imaging in oncology and opens the way for designing new targeted therapies. The anomalous choline metabolic profile detected in cancer by magnetic resonance spectroscopy and spectroscopic imaging provides molecular signatures of tumor progression and response to therapy. The increased level of intracellular phosphocholine (PCho) typically detected in cancer cells is mainly attributed to upregulation of choline kinase, responsible for choline phosphorylation in the biosynthetic Kennedy pathway, but can also be partly produced by activation of phosphatidylcholine-specific phospholipase C (PC-PLC). This hydrolytic enzyme, known for implications in bacterial infection and in plant survival to hostile environmental conditions, is reported to be activated in mitogen- and oncogene-induced phosphatidylcholine cycles in mammalian cells, with effects on cell signaling, cell cycle regulation, and cell proliferation. Recent investigations showed that PC-PLC activation could account for 20-50% of the intracellular PCho production in ovarian and breast cancer cells of different subtypes. Enzyme activation was associated with PC-PLC protein overexpression and subcellular redistribution in these cancer cells compared with non-tumoral counterparts. Moreover, PC-PLC coimmunoprecipitated with the human epidermal growth factor receptor-2 (HER2) and EGFR in HER2-overexpressing breast and ovarian cancer cells, while pharmacological PC-PLC inhibition resulted into long-lasting HER2 downregulation, retarded receptor re-expression on plasma membrane and antiproliferative effects. This body of evidence points to PC-PLC as a potential target for newly designed therapies, whose effects can be preclinically and clinically monitored by metabolic imaging methods.

Immune surveillance properties of human NK cell-derived exosomes.

Exosomes are nanovesicles released by normal and tumor cells, which are detectable in cell culture supernatant and human biological fluids, such as plasma. Functions of exosomes released by "normal" cells are not well understood. In fact, several studies have been carried out on exosomes derived from hematopoietic cells, but very little is known about NK cell exosomes, despite the importance of these cells in innate and adaptive immunity. In this paper, we report that resting and activated NK cells, freshly isolated from blood of healthy donors, release exosomes expressing typical protein markers of NK cells and containing killer proteins (i.e., Fas ligand and perforin molecules). These nanovesicles display cytotoxic activity against several tumor cell lines and activated, but not resting, immune cells. We also show that NK-derived exosomes undergo uptake by tumor target cells but not by resting PBMC. Exosomes purified from plasma of healthy donors express NK cell markers, including CD56+ and perforin, and exert cytotoxic activity against different human tumor target cells and activated immune cells as well. The results of this study propose an important role of NK cell-derived exosomes in immune surveillance and homeostasis. Moreover, this study supports the use of exosomes as an almost perfect example of biomimetic nanovesicles possibly useful in future therapeutic approaches against various diseases, including tumors.

Inhibition of phosphatidylcholine-specific phospholipase C results in loss of mesenchymal traits in metastatic breast cancer cells.

INTRODUCTION: Acquisition of mesenchymal characteristics confers to breast cancer (BC) cells the capability of invading tissues different from primary tumor site, allowing cell migration and metastasis. Regulators of the mesenchymal-epithelial transition (MET) may represent targets for anticancer agents. Accruing evidence supports functional implications of choline phospholipid metabolism in oncogene-activated cell signaling and differentiation. We investigated the effects of D609, a xanthate inhibiting phosphatidylcholine-specific phospholipase C (PC-PLC) and sphingomyelin synthase (SMS), as a candidate regulator of cell differentiation and MET in the highly metastatic BC cell line MDA-MB-231. METHODS: PC-PLC expression and activity were investigated using confocal laser scanning microscopy (CLSM), immunoblotting and enzymatic assay on human MDA-MB-231 compared with MCF-7 and SKBr3 BC cells and a nontumoral immortalized counterpart (MCF-10A). The effects of D609 on PC-PLC and SMS activity, loss of mesenchymal markers and changes in migration and invasion potential were monitored in MDA-MB-231 cells by enzymatic assays, CLSM, immunoblotting and transwell chamber invasion combined with scanning electron microscopy examinations. Cell proliferation, formation and composition of lipid bodies and cell morphology were investigated in D609-treated BC cells by cell count, CLSM, flow-cytometry of BODIPY-stained cells, nuclear magnetic resonance and thin-layer chromatography. RESULTS: PC-PLC (but not phospholipase D) showed 2- to 6-fold activation in BC compared with nontumoral cells, the highest activity (up to 0.4 pmol/µg protein/min) being detected in the poorly-differentiated MDA-MB-231 cells. Exposure of the latter cells to D609 (50 µg/mL, 24-72 h) resulted into 60-80% PC-PLC inhibition, while SMS was transiently inhibited by a maximum of 21%. These features were associated with progressive decreases of mesenchymal traits such as vimentin and N-cadherin expression, reduced galectin-3 and milk fat globule EGF-factor 8 levels, ß-casein formation and decreased in vitro cell migration and invasion. Moreover, proliferation arrest, changes in cell morphology and formation of cytosolic lipid bodies typical of cell differentiation were induced by D609 in all investigated BC cells. CONCLUSIONS: These results support a critical involvement of PC-PLC in controlling molecular pathways responsible for maintaining a mesenchymal-like phenotype in metastatic BC cells and suggests PC-PLC deactivation as a means to promote BC cell differentiation and possibly enhance the effectiveness of antitumor treatments.

Characterisation of in vivo ovarian cancer models by quantitative 1H magnetic resonance spectroscopy and diffusion-weighted imaging.

Magnetic resonance imaging (MRI) and spectroscopy (MRS) offer powerful approaches for detecting physiological and metabolic alterations in malignancies and help investigate underlying molecular mechanisms. Research on epithelial ovarian carcinoma (EOC), the gynaecological malignancy with the highest death rate characterised by frequent relapse and onset of drug resistance, could benefit from application of these molecular imaging approaches. In this study, MRI/MRS were used to characterise solid tumour models obtained by subcutaneous (s.c.) or intraperitoneal (i.p.) implantation of human SKOV3.ip cells in severe combined immunodeficiency (SCID) mice. In vivo MRI/MRS, ex vivo magic-angle-spinning (MAS), and in vitro (1)H-NMR measurements were carried out at 4.7 T, 9.4 T, and 9.4/16.5 T, respectively. MRI evaluation was performed by T1-, T2-, and diffusion-weighted (DW) multislice spin-echo imaging. The in vivo (1)H spectra of all tumour models showed a prominent resonance of total choline-containing metabolites (tCho). Quantitative in vivo MRS of both i.p. and s.c. SKOV3.ip xenografts showed that the mean tCho content was in the 2.9-4.5 mM range, with a mean PCho/tCho ratio of 0.99 ± 0.01 [23 examinations, 14-34 days post injection (dpi)], in good agreement with ex vivo and in vitro analyses. Myo-inositol ranged between 11.7 and 17.0 mM, with a trend towards higher values in i.p. xenografts at 14-16 dpi. The average apparent diffusion coefficient (ADC) values of SKOV3.ip xenografts [1.64 ± 0.11 (n = 9, i.p.) and 1.58 ± 0.03 x10(-3) mm(2)/s (n = 7, s.c.)] were in agreement with values reported for tumours from patients with EOC, while the mean vascular signal fraction (VSF) was lower (≤ 4%), probably due to the more rapid growth of preclinical models. Both s.c. and i.p. xenografts are valuable preclinical models for monitoring biochemical and physiopathological changes associated with in vivo EOC tumour growth and response to therapy, which may serve as the basis for further clinical development of noninvasive MR approaches.

Inhibition of phosphatidylcholine-specific phospholipase C downregulates HER2 overexpression on plasma membrane of breast cancer cells.

INTRODUCTION: Overexpression on plasma membrane of human epidermal growth factor receptor 2 (HER2) is reported in 25% to 30% of breast cancers. Heterodimer formation with cognate members of the epidermal growth factor receptor (EGFR) family, such as HER3 and EGFR, activates abnormal cell-signalling cascades responsible for tumorigenesis and further transcriptional HER2 gene upregulation. Targeting the molecular mechanisms controlling HER2 overexpression and recycling may effectively deactivate this feedback-amplification loop. We recently showed that inactivation of phosphatidylcholine-specific phospholipase C (PC-PLC) may exert a pivotal role in selectively modulating the expression on the membrane of specific receptors or proteins relevant to cell function. In the present study, we investigated the capability of PC-PLC inhibition to target the molecular mechanisms controlling HER2 overexpression on the membrane of breast cancer cells by altering the rates of its endocytosis and lysosomal degradation. METHODS: Localization on the membrane and interaction of PC-PLC with HER2, EGFR, and HER3 were investigated on HER2-overexpressing and HER2-low breast cancer cell lines, by using confocal laser scanning microscopy, flow cytometry, cell-surface biotinylation, isolation of lipid rafts, and immunoprecipitation experiments. The effects of the PC-PLC inhibitor tricyclodecan-9-yl-potassium xanthate (D609) on HER2 expression on the membrane and on the levels of overall HER2, HER2-HER3, and HER2-EGFR contents were monitored in the HER2-overexpressing SKBr3 cells, after either transient or continuous receptor engagement with anti-HER2 monoclonal antibodies, including trastuzumab. Changes of HER2 expression and cell proliferation were examined in SKBr3, BT-474, and MDA-MB-453 cells continuously exposed to D609 alone or combined with trastuzumab. RESULTS: PC-PLC selectively accumulates on the plasma membrane of HER2-overexpressing cells, where it colocalizes and associates with HER2 in raft domains. PC-PLC inhibition resulted in enhanced HER2 internalization and lysosomal degradation, inducing downmodulation of HER2 expression on the membrane. Moreover, PC-PLC inhibition resulted in strong retardation of HER2 reexpression on the membrane and a decrease in the overall cellular contents of HER2, HER2-HER3, and HER2-EGFR heterodimers. The PC-PLC inhibitor also induced antiproliferative effects, especially in trastuzumab-resistant cells. CONCLUSIONS: The results pointed to PC-PLC inhibition as a potential means to counteract the tumorigenic effects of HER2 amplification and complement the effectiveness of current HER2-targeting therapies.