The Role of FAS Receptor Methylation in Osteosarcoma Metastasis.

Osteosarcoma is the most frequent primary malignant bone tumor with an annual incidence of about 400 cases in the United States. Osteosarcoma primarily metastasizes to the lungs, where FAS ligand (FASL) is constitutively expressed. The interaction of FASL and its cell surface receptor, FAS, triggers apoptosis in normal cells; however, this function is altered in cancer cells. DNA methylation has previously been explored as a mechanism for altering FAS expression, but no variability was identified in the CpG island (CGI) overlapping the promoter. Analysis of an expanded region, including CGI shores and shelves, revealed high variability in the methylation of certain CpG sites that correlated significantly with FAS mRNA expression in a negative manner. Bisulfite sequencing revealed additional CpG sites, which were highly methylated in the metastatic LM7 cell line but unmethylated in its parental non-metastatic SaOS-2 cell line. Treatment with the demethylating agent, 5-azacytidine, resulted in a loss of methylation in CpG sites located within the FAS promoter and restored FAS protein expression in LM7 cells, resulting in reduced migration. Orthotopic implantation of 5-azacytidine treated LM7 cells into severe combined immunodeficient mice led to decreased lung metastases. These results suggest that DNA methylation of CGI shore sites may regulate FAS expression and constitute a potential target for osteosarcoma therapy, utilizing demethylating agents currently approved for the treatment of other cancers.

Proteomic analyses reveal distinct chromatin-associated and soluble transcription factor complexes.

The current knowledge on how transcription factors (TFs), the ultimate targets and executors of cellular signalling pathways, are regulated by protein-protein interactions remains limited. Here, we performed proteomics analyses of soluble and chromatin-associated complexes of 56 TFs, including the targets of many signalling pathways involved in development and cancer, and 37 members of the Forkhead box (FOX) TF family. Using tandem affinity purification followed by mass spectrometry (TAP/MS), we performed 214 purifications and identified 2,156 high-confident protein-protein interactions. We found that most TFs form very distinct protein complexes on and off chromatin. Using this data set, we categorized the transcription-related or unrelated regulators for general or specific TFs. Our study offers a valuable resource of protein-protein interaction networks for a large number of TFs and underscores the general principle that TFs form distinct location-specific protein complexes that are associated with the different regulation and diverse functions of these TFs.

Epithelial-mesenchymal transition-derived cells exhibit multilineage differentiation potential similar to mesenchymal stem cells.

The epithelial-to-mesenchymal transition (EMT) is an embryonic process that becomes latent in most normal adult tissues. Recently, we have shown that induction of EMT endows breast epithelial cells with stem cell traits. In this report, we have further characterized the EMT-derived cells and shown that these cells are similar to mesenchymal stem cells (MSCs) with the capacity to differentiate into multiple tissue lineages. For this purpose, we induced EMT by ectopic expression of Twist, Snail, or transforming growth factor-beta in immortalized human mammary epithelial cells. We found that the EMT-derived cells and MSCs share many properties including the antigenic profile typical of MSCs, that is, CD44(+), CD24(-), and CD45(-). Conversely, MSCs express EMT-associated genes, such as Twist, Snail, and mesenchyme forkhead 1 (FOXC2). Interestingly, CD140b (platelet-derived growth factor receptor-beta), a marker for naive MSCs, is exclusively expressed in EMT-derived cells and not in their epithelial counterparts. Moreover, functional analyses revealed that EMT-derived cells but not the control cells can differentiate into alizarin red S-positive mature osteoblasts, oil red O-positive adipocytes and alcian blue-positive chondrocytes similar to MSCs. We also observed that EMT-derived cells but not the control cells invade and migrate towards MDA-MB-231 breast cancer cells similar to MSCs. In vivo wound homing assays in nude mice revealed that the EMT-derived cells home to wound sites similar to MSCs. In conclusion, we have demonstrated that the EMT-derived cells are similar to MSCs in gene expression, multilineage differentiation, and ability to migrate towards tumor cells and wound sites.

Derivation of a cost-saving screening strategy for asymptomatic primary hyperparathyroidism.

BACKGROUND: Our study seeks to find a cost-saving screening strategy in a primary care population for diagnosing primary hyperparathyroidism based on peak serum total calcium level, age, and patient sex. METHODS: Laboratory data resulting from primary care office visits at our institution between January 2016 through December 2017 to evaluate patients who had at least 1 episode of hypercalcemia (≥10.5 mg/dL). For each serum calcium threshold, we calculated the percentage of patients who were found to have an increased parathyroid hormone level (≥65 pg/mL). We determined whether net cost savings could be achieved by screening hypercalcemic patients given their probability of primary hyperparathyroidism and expected cost savings from fracture risk reduction, given their sex and age. RESULTS: From 155,350 unique patients in the study period, a total of 2,271 had a minimum of 1 hypercalcemic lab value. After exclusion criteria, there were 1,326 patients of whom 27.5% had a parathyroid hormone level checked. Cost savings was established at a screening threshold of 10.5 for all patients until age 66 years for men and 69 years for women. For men aged 67-68 y and women aged 70-71 years, the optimal screening threshold was 10.8 mg/dl. CONCLUSION: Cost savings can be achieved by screening hypercalcemic patients with a life expectancy exceeding 16 years, with varying thresholds based on age and sex.

Comparison of algorithms for pre-processing of SELDI-TOF mass spectrometry data.

MOTIVATION: Surface-enhanced laser desorption and ionization (SELDI) time of flight (TOF) is a mass spectrometry technology. The key features in a mass spectrum are its peaks. In order to locate the peaks and quantify their intensities, several pre-processing steps are required. Though different approaches to perform pre-processing have been proposed, there is no systematic study that compares their performance. RESULTS: In this article, we present the results of a systematic comparison of various popular packages for pre-processing of SELDI-TOF data. We evaluate their performance in terms of two of their primary functions: peak detection and peak quantification. Regarding peak quantification, the performance of the algorithms is measured in terms of reproducibility. For peak detection, the comparison is based on sensitivity and false discovery rate. Our results show that for spectra generated with low laser intensity, the software developed by Ciphergen Biosystems (ProteinChip Software 3.1 with the additional tool Biomarker Wizard) produces relatively good results for both peak quantification and detection. On the other hand, for the data produced with either medium or high laser intensity, none of the methods show uniformly better performances under both criteria. Our analysis suggests that an advantageous combination is the use of the packages MassSpecWavelet and PROcess, the former for peak detection and the latter for peak quantification.

Semi-parametric differential expression analysis via partial mixture estimation.

We develop an approach for microarray differential expression analysis, i.e. identifying genes whose expression levels differ between two or more groups. Current approaches to inference rely either on full parametric assumptions or on permutation-based techniques for sampling under the null distribution. In some situations, however, a full parametric model cannot be justified, or the sample size per group is too small for permutation methods to be valid. We propose a semi-parametric framework based on partial mixture estimation which only requires a parametric assumption for the null (equally expressed) distribution and can handle small sample sizes where permutation methods break down. We develop two novel improvements of Scott's minimum integrated square error criterion for partial mixture estimation [Scott, 2004a,b]. As a side benefit, we obtain interpretable and closed-form estimates for the proportion of EE genes. Pseudo-Bayesian and frequentist procedures for controlling the false discovery rate are given. Results from simulations and real datasets indicate that our approach can provide substantial advantages for small sample sizes over the SAM method of Tusher et al. [2001], the empirical Bayes procedure of Efron and Tibshirani [2002], the mixture of normals of Pan et al. [2003] and a t-test with p-value adjustment [Dudoit et al., 2003] to control the FDR [Benjamini and Hochberg, 1995].

A framework for evaluation of deformable image registration spatial accuracy using large landmark point sets.

Expert landmark correspondences are widely reported for evaluating deformable image registration (DIR) spatial accuracy. In this report, we present a framework for objective evaluation of DIR spatial accuracy using large sets of expert-determined landmark point pairs. Large samples (>1100) of pulmonary landmark point pairs were manually generated for five cases. Estimates of inter- and intra-observer variation were determined from repeated registration. Comparative evaluation of DIR spatial accuracy was performed for two algorithms, a gradient-based optical flow algorithm and a landmark-based moving least-squares algorithm. The uncertainty of spatial error estimates was found to be inversely proportional to the square root of the number of landmark point pairs and directly proportional to the standard deviation of the spatial errors. Using the statistical properties of this data, we performed sample size calculations to estimate the average spatial accuracy of each algorithm with 95% confidence intervals within a 0.5 mm range. For the optical flow and moving least-squares algorithms, the required sample sizes were 1050 and 36, respectively. Comparative evaluation based on fewer than the required validation landmarks results in misrepresentation of the relative spatial accuracy. This study demonstrates that landmark pairs can be used to assess DIR spatial accuracy within a narrow uncertainty range.

Pulmonary blood mass dynamics on 4DCT during tidal breathing.

We previously reported that apparent lung mass varies across the phases of 4D computed tomography (4DCT) images. We hypothesize that these variations correspond to the physiologic changes in pulmonary perfusion induced during normal tidal breathing, and should therefore be present in every breathing patient. In this study, we characterize and quantify the respiratory induced variation in pulmonary blood mass (▵PBM) on 89 patients treated with stereotactic body radiotherapy. ▵PBM was computed from the treatment planning helical 4DCT images of each patient. Conversion from Hounsfield Units (HU) to density and mass per voxel was made using the density calibration curve, applied to the lung parenchyma volume within each phase. A difference in the lung mass with breathing was found for all cases, as was a substantial individual variation in lung volume. We found that the ▵PBM increased during inhalation, and decreased during exhalation. A significant correlation between the individual ▵PBM and tidal volume was observed; ▵PBM increased with tidal volume. We further evaluated the anatomic distribution of ▵PBM variation comparing the central versus peripheral lung, cranial versus caudal, dependent versus non-dependent lung. Our observations regarding spatial distribution of the ▵PBM agree with previously reported differences among similar regions for the supine patient. These results show that a variation in pulmonary mass during respiration is apparent on 4DCT and suggest that these variations reflect respiratory induced changes in the pulmonary perfusion. Therefore, the 4DCT derived respiratory induced ▵PBM signal can provide further insight into the pulmonary circulation and advance the overall understanding and diagnosis of human health and disease.

Robust CT ventilation from the integral formulation of the Jacobian.

Computed tomography (CT) derived ventilation algorithms estimate the apparent voxel volume changes within an inhale/exhale CT image pair. Transformation-based methods compute these estimates solely from the spatial transformation acquired by applying a deformable image registration (DIR) algorithm to the image pair. However, approaches based on finite difference approximations of the transformation's Jacobian have been shown to be numerically unstable. As a result, transformation-based CT ventilation is poorly reproducible with respect to both DIR algorithm and CT acquisition method. PURPOSE: We introduce a novel Integrated Jacobian Formulation (IJF) method for estimating voxel volume changes under a DIR-recovered spatial transformation. The method is based on computing volume estimates of DIR mapped subregions using the hit-or-miss sampling algorithm for integral approximation. The novel approach allows for regional volume change estimates that (a) respect the resolution of the digital grid and (b) are based on approximations with quantitatively characterized and controllable levels of uncertainty. As such, the IJF method is designed to be robust to variations in DIR solutions and thus overall more reproducible. METHODS: Numerically, Jacobian estimates are recovered by solving a simple constrained linear least squares problem that guarantees the recovered global volume change is equal to the global volume change obtained from the inhale and exhale lung segmentation masks. Reproducibility of the IJF method with respect to DIR solution was assessed using the expert-determined landmark point pairs and inhale/exhale phases from 10 four-dimensional computed tomographies (4DCTs) available on www.dir-lab.com. Reproducibility with respect to CT acquisition was assessed on the 4DCT and 4D cone beam CT (4DCBCT) images acquired for five lung cancer patients prior to radiotherapy. RESULTS: The ten Dir-Lab 4DCT cases were registered twice with the same DIR algorithm, but with different smoothing parameter. Finite difference Jacobian (FDJ) and IFJ images were computed for both solutions. The average spatial errors (300 landmarks per case) for the two DIR solution methods were 0.98 (1.10) and 1.02 (1.11). The average Pearson correlation between the FDJ images computed from the two DIR solutions was 0.83 (0.03), while for the IJF images it was 1.00 (0.00). For intermodality assessment, the IJF and FDJ images were computed from the 4DCT and 4DCBCT of five patients. The average Pearson correlation of the spatially aligned FDJ images was 0.27 (0.11), while it was 0.77 (0.13) for the IFJ method. CONCLUSION: The mathematical theory underpinning the IJF method allows for the generation of ventilation images that are (a) computed with respect to DIR spatial accuracy on the digital voxel grid and (b) based on DIR-measured subregional volume change estimates acquired with quantifiable and controllable levels of uncertainty. Analyses of the experiments are consistent with the mathematical theory and indicate that IJF ventilation imaging has a higher reproducibility with respect to both DIR algorithm and CT acquisition method, in comparison to the standard finite difference approach.

Determination of respiratory motion for distal esophagus cancer using four-dimensional computed tomography.

PURPOSE: To investigate the motion characteristics of distal esophagus cancer primary tumors using four-dimensional computed tomography (4D CT). METHODS AND MATERIALS: Thirty-one consecutive patients treated for esophagus cancer who received respiratory-gated 4D CT imaging for treatment planning were selected. Deformable image registration was used to map the full expiratory motion gross tumor volume (GTV) to the full-inspiratory CT image, allowing quantitative assessment of each voxel's displacement. These displacements were correlated with patient tumor and respiratory characteristics. RESULTS: The mean (SE) tidal volume was 608 (73) mL. The mean GTV volume was 64.3 (10.7) mL on expiration and 64.1 (10.7) mL on inspiration (no significant difference). The mean tumor motion in the x-direction was 0.13 (0.006) cm (average of absolute values), in the y-direction 0.23 (0.01) cm (anteriorly), and in the z-direction 0.71 (0.02) cm (inferiorly). Tumor motion correlated with tidal volume. Comparison of tumor motion above vs. below the diaphragm was significant for the average net displacement (p = 0.014), motion below the diaphragm was greater than above. From the cumulative distribution 95% of the tumors moved less than 0.80 cm radially and 1.75 cm inferiorly. CONCLUSIONS: Primary esophagus tumor motion was evaluated with 4D CT. According to the results of this study, when 4D CT is not available, a radial margin of 0.8 cm and axial margin of +/-1.8 cm would provide tumor motion coverage for 95% of the cases in our study population.

Impact of hypothermia on implementation of CPAP for neonatal respiratory distress syndrome in a low-resource setting.

BACKGROUND: Neonatal hypothermia is widely associated with increased risks of morbidity and mortality, but remains a pervasive global problem. No studies have examined the impact of hypothermia on outcomes for preterm infants treated with CPAP for respiratory distress syndrome (RDS). METHODS: This retrospective analysis assessed the impact of hypothermia on outcomes of 65 neonates diagnosed with RDS and treated with either nasal oxygen (N = 17) or CPAP (N = 48) in a low-resource setting. A classification tree approach was used to develop a model predicting survival for subjects diagnosed with RDS. FINDINGS: Survival to discharge was accurately predicted based on three variables: mean temperature, treatment modality, and mean respiratory rate. None of the 23 neonates with a mean temperature during treatment below 35.8°C survived to discharge, regardless of treatment modality. Among neonates with a mean temperature exceeding 35.8°C, the survival rate was 100% for the 31 neonates treated with CPAP and 36.4% for the 11 neonates treated with nasal oxygen (p<0.001). For neonates treated with CPAP, outcomes were poor if more than 50% of measured temperatures indicated hypothermia (5.6% survival). In contrast, all 30 neonates treated with CPAP and with more than 50% of temperature measurements above 35.8°C survived to discharge, regardless of initial temperature. CONCLUSION: The results of our study suggest that successful implementation of CPAP to treat RDS in low-resource settings will require aggressive action to prevent persistent hypothermia. However, our results show that even babies who are initially cold can do well on CPAP with proper management of hypothermia.

Reduction of pulmonary compliance found with high-resolution computed tomography in irradiated mice.

PURPOSE: To demonstrate that high-resolution computed tomography (CT) can be used to quantify loss of pulmonary compliance in irradiated mice. METHODS AND MATERIALS: Computed tomography images of three nonirradiated (controls) and three irradiated mice were obtained 200 days after a single dose of 16-Gy Co (60) thoracic irradiation. While intubated, each animal was imaged at static breath-hold pressures of 2, 10, and 18 cm H2O. A deformable image registration algorithm was used to calculate changes in air volume between adjacent-pressure CT image pairs (e.g., 2 and 10 cm H2O), and functional images of pulmonary compliance were generated. The mass-specific compliance was calculated as the change in volume divided by the pressure difference between the 2 image sets and the mass of lung tissue. RESULTS: For the irradiated mice, the lung parenchyma mean CT values ranged from -314 (+/- 11) Hounsfield units (HU) to -378 (+/- 11) HU. For the control mice, the mean CT values ranged from -549 (+/- 11) HU to -633 (+/- 11) HU. Irradiated mice had a 60% (45, 74%; 95% confidence interval) lower mass-specific compliance than did the controls (0.039 [+/- 0.0038] vs. 0.106 [+/- 0.0038] mL air per cm H2O per g lung) from the 2-cm to 10-cm H2O CT image pair. The difference in compliance between groups was less pronounced at the higher distending pressures. CONCLUSION: High-resolution CT was used to quantify a reduction in mass-specific compliance following whole lung irradiation in mice. This small animal radiation injury model and assay may be useful in the study of lung injury.

Reduction of normal lung irradiation in locally advanced non-small-cell lung cancer patients, using ventilation images for functional avoidance.

PURPOSE: To investigate the ability of four-dimensional computed tomography (4D-CT)-derived ventilation images to identify regions of highly functional lung for avoidance in intensity-modulated radiotherapy (IMRT) planning in locally advanced non-small-cell lung cancer (NSCLC). METHODS AND MATERIALS: The treatment-planning records from 21 patients with Stage III NSCLC were selected. Ventilation images were generated from the 4D-CT sets, and each was imported into the treatment-planning system. Ninetieth percentile functional volumes (PFV90), constituting the 10% of the lung volume where the highest ventilation occurs, were generated. Baseline IMRT plans were generated using the lung volume constraint on V20 (<35%), and two additional plans were generated using constraints on the PFV90 without a volume constraint. Dose-volume and dose-function histograms (DVH, DFH) were generated and used to evaluate the planning target volume coverage, lung volume, and functional parameters for comparison of the plans. RESULTS: The mean dose to the PFV90 was reduced by 2.9 Gy, and the DFH at 5 Gy (F5) was reduced by 9.6% (SE = 2.03%). The F5, F10, V5, and V10 were all significantly reduced from the baseline values. We identified a favorable subset of patients for whom there was a further significant improvement in the mean lung dose. CONCLUSIONS: Four-dimensional computed tomography-derived ventilation regions were successfully used as avoidance structures to reduce the DVH and DFH at 5 Gy in all cases. In a subset, there was also a reduction in the F10 and V10 without a change in the V20, suggesting that this technique could be safely used.

Harmonizing the definition of the metabolic syndrome: comparison of the criteria of the Adult Treatment Panel III and the International Diabetes Federation in United States American and European populations.

The 2 most widely used criteria to diagnose the metabolic syndrome (MS) are those developed by the United States Adult Treatment Panel III of the National Cholesterol Education Program (ATP III) and by the International Diabetes Federation (IDF). A major difference between these 2 sets of criteria is that the IDF places more emphasis on waist circumference. We compared the prevalence of MS using the ATP III and the IDF guidelines in 2 American (the Dallas Health Study and National Health and Nutrition Examination Survey) and 1 German (Prospective Cardiovascular Munster study) population samples. When the ATP III criteria were used, the prevalence of MS was higher in the United States than the German samples in both women (37% vs. 18%) and men (30% vs 25%), whereas when the IDF criteria were used, the prevalence of MS was 25% higher in the German than the American sample. Although in the United States both sets of criteria identified mostly the same people (concordance of about 90%), this was less true in Germany (concordance about 80%). To determine which criteria better predicted adverse cardiovascular outcomes, the incidence of coronary events associated with MS, as defined using the ATP III or the IDF criteria, were compared over a 10-year period among the middle-aged men in the German sample (n = 7,152). A total of 3.4% of men without MS developed an event. A much higher percentage of the men with MS defined by the ATP III criteria (10.7%) than the IDF criteria (5.5%) had a cardiovascular event. In conclusion, although the prevalence of MS was higher when the IDF criteria were used in the German sample, the IDF criteria have lower predictive power for coronary events.

Lipoprotein(a) and apolipoprotein(a) isoforms: no association with coronary artery calcification in the Dallas Heart Study.

BACKGROUND: Elevated plasma levels of lipoprotein(a) [Lp(a)] are an independent risk factor for cardiovascular disease in whites. Blacks have 2- to 3-fold higher plasma levels of Lp(a) than whites and yet do not have a correspondingly higher rate of coronary events. It remains unclear whether elevated plasma levels of Lp(a) are an independent risk factor for coronary atherosclerosis in individuals of African descent. METHODS AND RESULTS: The relationship between plasma levels of Lp(a), apolipoprotein(a) isoform sizes, and the presence of coronary calcium was examined in 761 blacks and 527 whites (men aged >40 years, women aged >45 years) from a population-based sample. No relationship was found between plasma levels of Lp(a), apolipoprotein(a) isoform size, or a combination of these 2 variables and coronary artery calcium (CAC) in whites or blacks. No correlation was observed between plasma levels of Lp(a) and coronary calcium scores in any group, although all black men with very high plasma levels of Lp(a) (>300 micromol/L; n=7) were CAC-positive. Whites with high plasma levels of Lp(a) plus elevated plasma levels of LDL cholesterol (men) or reduced levels of HDL cholesterol (men and women) or who smoked (women) had a higher prevalence of CAC. In contrast, no joint effects between plasma levels of Lp(a) and other cardiovascular risk factors on coronary calcium were found in blacks. CONCLUSIONS: No consistent independent relationship between plasma levels of Lp(a) or apolipoprotein(a) isoform size and coronary calcium was found in whites or blacks.

cDNA microarray analysis of endothelial cells subjected to cyclic mechanical strain: importance of motion control.

Microarrays were utilized to determine gene expression of vascular endothelial cells (ECs) subjected to mechanical stretch for insight into the role of strain in vascular pathophysiology. Over 4,000 genes were screened for expression changes resulting from cyclic strain (10%, 1 Hz) of human umbilical vein ECs for 6 and 24 h. Comparison of t-statistics and adjusted P values identified genes having significantly different expression between strained and static cells but not between strained and motion control. Relative to static, 6 h of cyclic stretch upregulated two genes and downregulated two genes, whereas 24 h of cyclic stretch upregulated eight genes but downregulated no genes. However, incorporating the motion control revealed that fluid agitation over the cells, rather than strain, is the primary regulator of differential expression. Furthermore, no gene exceeded a threefold change when comparing cyclic strain to either static or motion control. Quantitative real-time polymerase chain reaction confirmed the dominance of fluid agitation in gene regulation with the exception of heat shock protein 10 at 24 h and plasminogen activator inhibitor 1 at 6 h. Taken together, the small number of differentially expressed genes and their low fold expression levels indicate that cyclic strain is a weak inducer of gene regulation in ECs. However, many of the differentially expressed genes possess antioxidant properties, suggesting that oxidative mechanisms direct EC adaptation to cyclic stretch.

Quantification of regional ventilation from treatment planning CT.

PURPOSE: We describe a method of quantifying regional ventilation from the radiotherapy treatment planning computed tomography (CT) images, with the goal of developing functional images for treatment planning and optimization. METHODS AND MATERIALS: A series of exhalation breath-hold (eBH-CT) and inhalation breath-hold (iBH-CT) CT images obtained using a feedback-guided breath-hold technique for radiotherapy treatment planning was selected. The eBH-CT was mapped on a voxel-by-voxel basis to the iBH-CT using a deformable image registration algorithm. By using the average CT number over a 3 mm(3) region surrounding each pair of mapped voxels, the change in fraction of air per voxel (i.e., regional ventilation) was calculated. This methodology was applied to a series of 22 patients. The calculated total ventilation was compared to the change in contoured lung volumes between the exhalation and inhalation CTs (measured tidal volume). RESULTS: A significant correlation was found between the calculated and measured tidal volumes for the left (R = 0.982) and right (R = 0.985), and for both lungs combined (R = 0.985). In the resulting images, the regional ventilation was highly variable and corresponded with the spatial distribution of differences in the CT values (Hounsfield units) between the eBH-CT and the iBH-CT images. CONCLUSIONS: A method of quantifying regional ventilation from radiotherapy treatment planning CT data sets was demonstrated. The ventilation images can be used in plan optimization to minimize injury to functioning lung.

Sources of variability in genetic association studies: insights from the analysis of hepatic lipase (LIPC).

Genetic association studies have been widely used to identify loci that influence plasma lipoprotein concentrations, but few of the associations reported have proved consistently reproducible across different study populations. This lack of consistency is a widely recognized limitation of association studies, and is often ascribed to inadequate statistical power, population substructure, and population-specific linkage disequilibrium. However, few studies have assessed the causes of variability underlying a given genotype-phenotype association. We have examined two possible sources of variability in the association between the -514 polymorphism in hepatic lipase (LIPC) and plasma HDL-C concentrations. First, we compared the association between this polymorphism and hepatic lipase activity in four populations. A single copy of the -514C allele was associated with a 10 mmol.hr(-1).l(-1) increase in hepatic lipase activity in white American and Turkish men but only approximately 5 mmol.hr(-1).l(-1) in Chinese and African-American men. Second, we tested the effects of a stanozolol-induced increase in hepatic lipase activity on plasma HDL-C concentrations in men with normal (< 150mg/dl) or elevated (150-300mg/dl) levels of plasma triglyceride. The increase in hepatic lipase activity was similar in the two groups, but the resulting decline in HDL-C levels was significantly greater in normolipidemic men. These data suggest that the effect of a polymorphism on gene expression can vary among individuals, and that the resulting phenotype may be further modified by interactions with other factors. Three novel LIPC polymorphisms were identified in the study (-1596insC, -2740A>G, and -2880G>C).

FOXC2 expression links epithelial-mesenchymal transition and stem cell properties in breast cancer.

Resistance to chemotherapy and metastases are the major causes of breast cancer-related mortality. Moreover, cancer stem cells (CSC) play critical roles in cancer progression and treatment resistance. Previously, it was found that CSC-like cells can be generated by aberrant activation of epithelial-mesenchymal transition (EMT), thereby making anti-EMT strategies a novel therapeutic option for treatment of aggressive breast cancers. Here, we report that the transcription factor FOXC2 induced in response to multiple EMT signaling pathways as well as elevated in stem cell-enriched factions is a critical determinant of mesenchymal and stem cell properties, in cells induced to undergo EMT- and CSC-enriched breast cancer cell lines. More specifically, attenuation of FOXC2 expression using lentiviral short hairpin RNA led to inhibition of the mesenchymal phenotype and associated invasive and stem cell properties, which included reduced mammosphere-forming ability and tumor initiation. Whereas, overexpression of FOXC2 was sufficient to induce CSC properties and spontaneous metastasis in transformed human mammary epithelial cells. Furthermore, a FOXC2-induced gene expression signature was enriched in the claudin-low/basal B breast tumor subtype that contains EMT and CSC features. Having identified PDGFR-ß to be regulated by FOXC2, we show that the U.S. Food and Drug Administration-approved PDGFR inhibitor, sunitinib, targets FOXC2-expressing tumor cells leading to reduced CSC and metastatic properties. Thus, FOXC2 or its associated gene expression program may provide an effective target for anti-EMT-based therapies for the treatment of claudin-low/basal B breast tumors or other EMT-/CSC-enriched tumors.

Ganglioside GD2 identifies breast cancer stem cells and promotes tumorigenesis.

Cancer stem cells (CSCs) are a small subpopulation of cancer cells that have increased resistance to conventional therapies and are capable of establishing metastasis. However, only a few biomarkers of CSCs have been identified. Here, we report that ganglioside GD2 (a glycosphingolipid) identifies a small fraction of cells in human breast cancer cell lines and patient samples that are capable of forming mammospheres and initiating tumors with as few as 10 GD2+ cells. In addition, the majority of GD2+ cells are also CD44hiCD24lo, the previously established CSC-associated cell surface phenotype. Gene expression analysis revealed that GD3 synthase (GD3S) is highly expressed in GD2+ as well as in CD44hiCD24lo cells and that interference with GD3S expression, either by shRNA or using a pharmacological inhibitor, reduced the CSC population and CSC-associated properties. GD3S knockdown completely abrogated tumor formation in vivo. Also, induction of epithelial-mesenchymal transition (EMT) in transformed human mammary epithelial cells (HMLER cells) dramatically increased GD2 as well as GD3S expression in these cells, suggesting a role of EMT in the origin of GD2+ breast CSCs. In summary, we identified GD2 as a new CSC-specific cell surface marker and GD3S as a potential therapeutic target for CSCs, with the possibility of improving survival and cure rates in patients with breast cancer.