Hadassa Degani

Phosphocholine as a biomarker of breast cancer: Molecular and biochemical studies

The discovery of metabolic and molecular markers that help improving the detection and diagnosis of breast cancer is an important goal to be achieved. A high composite‐choline signal in magnetic resonance spectra of breast lesions has been demonstrated to improve the accuracy of breast cancer diagnosis. In the present study we revealed the principal molecular and biochemical steps associated with the induction of choline metabolism and phosphocholine accumulation in human breast cancer cell‐lines in comparison with normal human mammary epithelial cells. We found upregulation of the expression levels of specific choline transporters: organic cation transporter‐2 and choline high affinity transporter‐1, as well as of the enzyme choline kinase α in the cancerous cells in comparison with that in the normal mammary epithelial cells. The expression levels of choline transporter like‐1, organic cation transporter‐1 and choline kinase β were similar in normal and cancerous cells. We further showed that choline transport rates and choline kinase activity indeed increased by several fold in the cancer cells leading to the elevation of phosphocholine. The results strongly suggest that phosphocholine can serve as a biomarker of breast cancer reflecting upregulation of specific choline transporters and choline kinase genes.

Triple-negative breast cancer: Present challenges and new perspectives

Triple‐negative breast cancers (TNBC), characterized by absence of estrogen receptor (ER), progesterone receptor (PR) and lack of overexpression of human epidermal growth factor receptor 2 (HER2), are typically associated with poor prognosis, due to aggressive tumor phenotype(s), only partial response to chemotherapy and present lack of clinically established targeted therapies. Advances in the design of individualized strategies for treatment of TNBC patients require further elucidation, by combined ‘omics’ approaches, of the molecular mechanisms underlying TNBC phenotypic heterogeneity, and the still poorly understood association of TNBC with BRCA1 mutations. An overview is here presented on TNBC profiling in terms of expression signatures, within the functional genomic breast tumor classification, and ongoing efforts toward identification of new therapy targets and bioimaging markers. Due to the complexity of aberrant molecular patterns involved in expression, pathological progression and biological/clinical heterogeneity, the search for novel TNBC biomarkers and therapy targets requires collection of multi‐dimensional data sets, use of robust multivariate data analysis techniques and development of innovative systems biology approaches.

Inhibition of Tumor Growth and Elimination of Multiple Metastases in Human Prostate and Breast Xenografts by Systemic Inoculation of a Host Defense–Like Lytic Peptide

We report on a short host defense-like peptide that targets and arrests the growth of aggressive and hormone-resistant primary human prostate and breast tumors and prevents their experimental and spontaneous metastases, respectively, when systemically inoculated to immunodeficient mice. These effects are correlated with increased necrosis of the tumor cells and a significant decrease in the overall tumor microvessel density, as well as newly formed capillary tubes and prostate-specific antigen secretion (in prostate tumors). Growth inhibition of orthotopic tumors derived from stably transfected highly fluorescent human breast cancer cells and prevention of their naturally occurring metastases were visualized in real time by using noninvasive whole-body optical imaging. The exclusive selectivity of the peptide towards cancer derives from its specific binding to surface phosphatidylserine and the killing of the cancer cells via cytoplasmic membrane depolarization. These data indicate that membrane disruption can provide a therapeutic means of inhibiting tumor growth and preventing metastases of various cancers.

Magnetic resonance imaging reveals functional diversity of the vasculature in benign and malignant breast lesions

Tumor perfusion through the microvascular network can be imaged noninvasively by dynamic contrast‐enhanced magnetic resonance imaging (DCE‐MRI). The objective of the current study was to quantify the microvascular perfusion parameters in various human breast lesions and to determine whether they varied between benign lesions and malignancy and whether they were altered with increased invasiveness.

Noninvasive Magnetic Resonance Imaging of Transport and Interstitial Fluid Pressure in Ectopic Human Lung Tumors

Tumor response to blood borne drugs is critically dependent on the efficiency of vascular delivery and transcapillary transfer. However, increased tumor interstitial fluid pressure (IFP) forms a barrier to transcapillary transfer, leading to resistance to drug delivery. We present here a new, noninvasive method which estimates IFP and its spatial distribution in vivo using contrast-enhanced magnetic resonance imaging (MRI). This method was tested in ectopic human non-small-cell lung cancer which exhibited a high IFP of approximately 28 mm Hg and, for comparison, in orthotopic MCF7 human breast tumors which exhibited a lower IFP of approximately 14 mm Hg, both implanted in nude mice. The MRI protocol consisted of slow infusion of the contrast agent [gadolinium-diethylenetriaminepentaacetic acid (GdDTPA)] into the blood for approximately 2 hours, sequential acquisition of images before and during the infusion, and measurements of T1 relaxation rates before infusion and after blood and tumor GdDTPA concentration reached a steady state. Image analysis yielded parametric images of steady-state tissue GdDTPA concentration with high values of this concentration outside the tumor boundaries, approximately 1 mmol/L, declining in the tumor periphery to approximately 0.5 mmol/L, and then steeply decreasing to low or null values. The distribution of steady-state tissue GdDTPA concentration reflected the distribution of IFP, showing an increase from the rim inward, with a high IFP plateau inside the tumor. The changes outside the borders of the tumors with high IFP were indicative of convective transport through the interstitium. This work presents a noninvasive method for assessing the spatial distribution of tumor IFP and mapping barriers to drug delivery and transport.

Glycolysis and glucose transporter 1 as markers of response to hormonal therapy in breast cancer.

Estrogen plays a key role in the development and progression of breast cancer; hence, antiestrogens, such as tamoxifen, have a marked impact on the treatment and outcome of breast cancer patients. Estrogen‐induced growth requires continuous replenishment of energy, predominantly generated by glycolysis. Previous work from this laboratory demonstrated estrogen induction and tamoxifen inhibition of glycolysis in MCF7 human breast cancer cells in vitro (Furman et al., J Steroid Biochem Mol Biol 1992;43:189–95). We present here studies of estrogen vs. tamoxifen regulation of glycolysis in orthotopic MCF7 human breast cancer xenografts in vivo. In addition we investigated mediation of this metabolic regulation through glucose transporter 1, in the same cells, in vitro, as well as in 2 other hormone‐responsive human breast cancer cells. Tumor response and glycolysis were monitored noninvasively by means of magnetic resonance imaging and 13C spectroscopy, respectively. During estrogen‐stimulated tumor growth (from ≈0.5 to ≈1.3 cm3 in 10 days), the rate of glucose metabolism through glycolysis in vivo was high at 40 ± 4 μmole/g/min. However, treatment for 10 days with tamoxifen induced growth arrest and a concomitant decrease of 2‐fold in the rate of glycolysis. In congruence, glucose transporter 1 expression was stimulated by estrogen, reaching after 72 hr a 2‐ to 3‐fold higher level of expression relative to that in tamoxifen‐treated cells. Thus, estrogen‐induced changes in glycolysis appeared to be mediated via its regulation of glucose transporter 1 expression. The in vivo monitoring of glycolysis may serve as a tool to expose hormonal regulation of glucose transporter 1 expression in breast cancer tumors, as well as to assess response to hormonal therapy.

Ionic permeabilities of membranes

Ion transport across membranal barriers plays a fundamental role in cellular activity; the generation of nerve impulses and their transfer to the muscles, the renal activity and activated transport of various metabolites are only a few examples. Thus, studies of the permeability characteristics of membranes are essential for the understanding of many physiological processes. Several methods have been employed to study ion permeability of vesicular membranes. The method mostly used is the tracers technique which employes radioactive isotopes. This method in general is limited to relatively slow transport rates corresponding to lifetimes longer than several seconds. Also its application requires the destruction of the sample. In this communication we present a method in which the transport process can be directly monitored using the nuclear magnetic resonance (NMR) of the transporting species. It makes use of paramagnetic relaxation reagents (PRR) and is similar to the method used [ 1,2] to study water transport. Here we extend the method by using an anionic PRR for positively charged transporting species for which the usual PRRs are not suitable. Specifically we consider the transport of alkali metal ions (sodium and lithium) through phospholipid membranes of vesicles using 23Na and ‘Li NMR and employ Gd(EDTA)as a relaxation reagent for cations [3]. The method is nondestructive and can be applied for both very slow kinetics (halflives longer than several minutes) as well as very fast rates (halflives shorter than 10m3 s).

Parametric diffusion tensor imaging of the breast

Objectives:To investigate the ability of parametric diffusion tensor imaging (DTI), applied at 3 Tesla, to dissect breast tissue architecture and evaluate breast lesions. Materials and Methods:All protocols were approved and a signed informed consent was obtained from all subjects. The study included 21 healthy women, 26 women with 33 malignant lesions, and 14 women with 20 benign lesions. Images were recorded at 3 Tesla with a protocol optimized for breast DTI at a spatial resolution of 1.9 × 1.9 × (2–2.5) mm3. Image processing algorithms and software, applied at pixel resolution, yielded vector maps of prime diffusion direction and parametric maps of the 3 orthogonal diffusion coefficients and of the fractional anisotropy and maximal anisotropy. Results:The DTI-derived vector maps and parametric maps revealed the architecture of the entire mammary fibroglandular tissue and allowed a reliable detection of malignant lesions. Cancer lesions exhibited significantly lower values of the orthogonal diffusion coefficients, &lgr;1, &lgr;2, &lgr;3, and of the maximal anisotropy index &lgr;1-&lgr;3 as compared with normal breast tissue (P < 0.0001) and to benign breast lesions (P < 0.0009 and 0.004, respectively). Maps of &lgr;1 exhibited the highest contrast-to-noise ratio enabling delineation of the cancer lesions. These maps also provided high sensitivity/specificity of 95.6%/97.7% for differentiating cancers from benign lesions, which were similar to the sensitivity/specificity of dynamic contrast-enhanced magnetic resonance imaging of 94.8%/92.9%. Maps of &lgr;1-&lgr;3 provided a secondary independent diagnostic parameter with high sensitivity of 92.3%, but low specificity of 69.5% for differentiating cancers from benign lesions. Conclusion:Mapping the diffusion tensor parameters at high spatial resolution provides a potential novel means for dissecting breast architecture. Parametric maps of &lgr;1 and &lgr;1-&lgr;3 facilitate the detection and diagnosis of breast cancer.

Critical role of spatial resolution in dynamic contrast-enhanced breast MRI†

The spatial resolution of three‐dimensional (3D) gradient‐echo T1‐weighted images, from 40 women with 25 malignant and 23 benign lesions, was purposely degraded to determine the role of spatial resolution in recording, analysis, and diagnosis of dynamic contrast‐enhanced breast MRI. Images were recorded and analyzed at pixel resolution according to the 3TP method (Degani et al., Nat Med 1997;3:780–782). Reduction in spatial resolution degraded the appearance of foci with fast wash‐in and fast washout dynamics. This resulted in an increase in false‐negative diagnoses. The sensitivity for differentiating between malignant and benign lesions, using threshold criteria defined by the 3TP analysis, of 76% decreased to 60% and 24% for a 2‐ and 4‐fold reduction in spatial resolution, respectively, without affecting significantly the high specificity (96–100%). In order to minimize false‐negative diagnoses of contrast‐enhanced breast MRI and maintain high specificity, it is essential to record and analyze the dynamic behavior at high spatial resolution. J. Magn. Reson. Imaging 2001;13:862–867.

Lipid metabolism in T47D human breast cancer cells:31P and13C-NMR studies of choline and ethanolamine uptake

31P and 13C-NMR were used to determine the kinetics of choline and ethanolamine incorporation in T47D clone 11 human breast cancer cells grown as small (150 microns) spheroids. Spheroids were perfused inside the spectrometer with 1,2-13C-labeled choline or 1,2-13C-labeled ethanolamine (0.028 mM) and the buildup of labeled phosphoryl-choline (PC) or phosphorylethanolamine (PE) was monitored. Alternatively the PC and GPC pools were prelabeled with 13C and the reduction of label was monitored. 31P spectra were recorded from which the overall energetic status as well as total pool sizes could be determined. The ATP content was 8 +/- 1 fmol/cell, and the total PC and PE pool sizes were 16 and 14 fmol/cell, respectively. PC either increased by 50% over 24 h or remained constant, while PE remained constant in medium without added ethanolamine but increased 2-fold within 30 h in medium containing ethanolamine, indicating a dependence on precursor concentration in the medium. The 31P and 13C data yielded similar kinetic results: the rate of the enzymes phosphocholine kinase and phosphoethanolamine kinase were both on the order of 1.0 fmol/cell per h, and the rate constants for CTP:phosphocholine cytidyltransferase and CTP:phosphoethanolamine kinase were 0.06 h-1 for both enzymes. The kinetics of choline incorporation did not alter in the presence of 0.028 mM ethanolamine indicating that they have non-competing pathways.