Folate and vitamin B12 status and predicted neural tube defects risk among nonpregnant women of reproductive age from the Malawi National Micronutrient Survey, 2015-2016.

BACKGROUND: Maternal folate and vitamin B12 deficiency can lead to serious adverse pregnancy outcomes. There are no nationally representative estimates on folate and vitamin B12 status among women of reproductive age (WRA) in Malawi. OBJECTIVE: We assessed folate and vitamin B12 status among nonpregnant WRA in Malawi and predicted the risk of folate-sensitive neural tube defects (NTDs) were they to become pregnant. METHODS: Using data from the cross-sectional, nationally representative 2015-2016 Malawi Micronutrient Survey, we calculated the proportion of folate and vitamin B12 deficiency and insufficiency by demographic characteristics among 778 nonpregnant WRA (15-49 years). We predicted NTD prevalence using red blood cell (RBC) folate distributions and a published Bayesian model of the association between RBC folate and NTD risk. Analyses accounted for complex survey design. RESULTS: Among WRA, 8.5% (95% CI: 6.2, 11.6) and 13.3% (10.0, 17.4) had serum (<7 nmol/L) and RBC folate (<305 nmol/L) deficiency, respectively. The proportion of vitamin B12 deficiency (<148 pmol/L) and insufficiency (≤221 pmol/L) was 11.8% (8.6, 16.0) and 40.6% (34.1, 47.4), respectively. RBC folate insufficiency (<748 nmol/L, defined as the concentration associated with the threshold for elevated NTD risk: >8 cases per 10,000 births) was widespread: 81.4% (75.0, 86.4). The predicted NTD risk nationally was 24.7 cases per 10,000 live births. RBC folate insufficiency and higher predicted NTD risk were more common among WRA living in urban areas or with higher education. CONCLUSIONS: These findings highlight the importance of nutritional and NTD surveillance in Malawi and the opportunity for improving folate and vitamin B12 nutrition among Malawian WRA.

Dihydromyricetin supplementation improves ethanol-induced lipid accumulation and inflammation.

Introduction: Excessive alcohol consumption leads to a myriad of detrimental health effects, including alcohol-associated liver disease (ALD). Unfortunately, no available treatments exist to combat the progression of ALD beyond corticosteroid administration and/or liver transplants. Dihydromyricetin (DHM) is a bioactive polyphenol and flavonoid that has traditionally been used in Chinese herbal medicine for its robust antioxidant and anti-inflammatory properties. It is derived from many plants, including Hovenia dulcis and is found as the active ingredient in a variety of popular hangover remedies. Investigations utilizing DHM have demonstrated its ability to alleviate ethanol-induced disruptions in mitochondrial and lipid metabolism, while demonstrating hepatoprotective activity. Methods: Female c57BL/6J mice (n = 12/group) were treated using the Lieber DeCarli forced-drinking and ethanol (EtOH) containing liquid diet, for 5 weeks. Mice were randomly divided into three groups: (1) No-EtOH, (2) EtOH [5% (v/v)], and (3) EtOH [5% (v/v)] + DHM (6 mg/mL). Mice were exposed to ethanol for 2 weeks to ensure the development of ALD pathology prior to receiving dihydromyricetin supplementation. Statistical analysis included one-way ANOVA along with Bonferroni multiple comparison tests, where p ≤ 0.05 was considered statistically significant. Results: Dihydromyricetin administration significantly improved aminotransferase levels (AST/ALT) and reduced levels of circulating lipids including LDL/VLDL, total cholesterol (free cholesterol), and triglycerides. DHM demonstrated enhanced lipid clearance by way of increased lipophagy activity, shown as the increased interaction and colocalization of p62/SQSTM-1, LC3B, and PLIN-1 proteins. DHM-fed mice had increased hepatocyte-to-hepatocyte lipid droplet (LD) heterogeneity, suggesting increased neutralization and sequestration of free lipids into LDs. DHM administration significantly reduced prominent pro-inflammatory cytokines commonly associated with ALD pathology such as TNF-α, IL-6, and IL-17. Discussion: Dihydromyricetin is commercially available as a dietary supplement. The results of this proof-of-concept study demonstrate its potential utility and functionality as a cost-effective and safe candidate to combat inflammation and the progression of ALD pathology.

Estimating the serum folate concentration that corresponds to the red blood cell folate concentration threshold associated with optimal neural tube defects prevention: A population-based biomarker survey in Southern India.

BACKGROUND: RBC folate concentrations are monitored at the population level, with a recommended threshold for optimal neural tube defect (NTD) prevention. A corresponding threshold for serum folate has not been established. OBJECTIVES: This study aimed to estimate the serum folate insufficiency threshold corresponding to the RBC folate threshold for NTD prevention and examine how this threshold is modified by vitamin B12 status. METHODS: Participants were women (15-40 y; not pregnant or lactating; n = 977) from a population-based biomarker survey in Southern India. RBC folate and serum folate were measured via microbiologic assay. RBC folate deficiency (<305 nmol/L) and insufficiency (<748 nmol/L), serum vitamin B12 deficiency (<148 pmol/L) and vitamin B12 insufficiency (<221 pmol/L), elevated plasma MMA (>0.26 µmol/L), elevated plasma homocysteine (>10.0 µmol/L), and elevated HbA1c (≥6.5%) were evaluated. Bayesian linear models were used to estimate unadjusted and adjusted thresholds. RESULTS: Compared with adequate vitamin B12 status, the estimated serum folate threshold was higher in participants with serum vitamin B12 deficiency (72.5 vs. 28.1 nmol/L) or vitamin B12 insufficiency (48.7 vs. 24.3 nmol/L) and elevated MMA (55.6 vs. 25.9 nmol/L). The threshold was lower in participants with elevated HbA1c (HbA1c ≥6.5% vs. <6.5%; 21.0 vs. 40.5 nmol/L). CONCLUSIONS: The estimated serum folate threshold for optimal NTD prevention was similar to previous reports (24.3 vs. 25.6 nmol/L) among participants with sufficient vitamin B12 status. However, this threshold was more than 2-fold higher in participants with vitamin B12 deficiency and substantially higher across all indicators of insufficient vitamin B12 status (<221 pmol/L, elevated MMA, combined B12, impaired vitamin B12 status), and lower in participants with elevated HbA1c. Findings suggest a serum folate threshold for NTD prevention may be possible in some settings; however, it may not be appropriate in populations with high prevalence of vitamin B12 insufficiency. Am J Clin Nutr 2023;xx:xx-xx. This trial was registered at https://clinicaltrials.gov as NCT04048330.

Transcriptomic characterization of clinical skeletal muscle biopsy from late-onset Pompe patients.

Pompe disease is a rare lysosomal storage disorder arising from recessive mutations in the acid α-glucosidase gene and resulting in the accumulation of glycogen, particularly in the cardiac and skeletal muscle. The current standard of care is administration of enzyme replacement therapy in the form of alglucosidase alfa or the recently approved avalglucosidase alfa. In order to better understand the underlying cellular processes that are disrupted in Pompe disease, we conducted gene expression analysis on skeletal muscle biopsies obtained from late-onset Pompe disease patients (LOPD) prior to treatment and following six months of enzyme replacement with avalglucosidase alfa. The LOPD patients had a distinct transcriptomic signature as compared to control patient samples, largely characterized by perturbations in pathways involved in lysosomal function and energy metabolism. Although patients were highly heterogeneous, they collectively exhibited a strong trend towards attenuation of the dysregulated genes following just six months of treatment. Notably, the enzyme replacement therapy had a strong stabilizing effect on gene expression, with minimal worsening in genes that were initially dysregulated. Many of the cellular process that were altered in LOPD patients were also affected in the more clinically severe infantile-onset (IOPD) patients. Additionally, both LOPD and IOPD patients demonstrated enrichment across several inflammatory pathways, despite a lack of overt immune cell infiltration. This study provides further insight into Pompe disease biology and demonstrates the positive effects of avalglucosidase alfa treatment.

Differential Ly6C Expression after Renal Ischemia-Reperfusion Identifies Unique Macrophage Populations.

Macrophages are a heterogeneous cell type implicated in injury, repair, and fibrosis after AKI, but the macrophage population associated with each phase is unclear. In this study, we used a renal bilateral ischemia-reperfusion injury mouse model to identify unique monocyte/macrophage populations by differential expression of Ly6C in CD11b(+) cells and to define the function of these cells in the pathophysiology of disease on the basis of microarray gene signatures and reduction strategies. Macrophage populations were isolated from kidney homogenates by fluorescence-activated cell sorting for whole genome microarray analysis. The CD11b(+)/Ly6C(high) population associated with the onset of renal injury and increase in proinflammatory cytokines, whereas the CD11b(+)/Ly6C(intermediate) population peaked during kidney repair. The CD11b(+)/Ly6C(low) population emerged with developing renal fibrosis. Principal component and hierarchical cluster analyses identified gene signatures unique to each population. The CD11b(+)/Ly6C(intermediate) population had a distinct phenotype of wound healing, confirmed by results of studies inhibiting the macrophage colony-stimulating factor 1 receptor,whereas the CD11b(+)/Ly6C(low) population had a profibrotic phenotype. All populations, including the CD11b(+)/Ly6C(high) population, carried differential inflammatory signatures. The expression of M2-specific markers was detected in both the CD11b(+)/Ly6C(intermediate) and CD11b(+)/Ly6C(low) populations, suggesting these in vivo populations do not fit into the traditional classifications defined by in vitro systems. Results of this study in a renal ischemia-reperfusion injury model allow phenotype and function to be assigned to CD11b(+)/Ly6C(+) monocyte/macrophage populations in the pathophysiology of disease after AKI.

Noninvasive detection of fetal subchromosomal abnormalities by semiconductor sequencing of maternal plasma DNA.

Noninvasive prenatal testing (NIPT) using sequencing of fetal cell-free DNA from maternal plasma has enabled accurate prenatal diagnosis of aneuploidy and become increasingly accepted in clinical practice. We investigated whether NIPT using semiconductor sequencing platform (SSP) could reliably detect subchromosomal deletions/duplications in women carrying high-risk fetuses. We first showed that increasing concentration of abnormal DNA and sequencing depth improved detection. Subsequently, we analyzed plasma from 1,456 pregnant women to develop a method for estimating fetal DNA concentration based on the size distribution of DNA fragments. Finally, we collected plasma from 1,476 pregnant women with fetal structural abnormalities detected on ultrasound who also underwent an invasive diagnostic procedure. We used SSP of maternal plasma DNA to detect subchromosomal abnormalities and validated our results with array comparative genomic hybridization (aCGH). With 3.5 million reads, SSP detected 56 of 78 (71.8%) subchromosomal abnormalities detected by aCGH. With increased sequencing depth up to 10 million reads and restriction of the size of abnormalities to more than 1 Mb, sensitivity improved to 69 of 73 (94.5%). Of 55 false-positive samples, 35 were caused by deletions/duplications present in maternal DNA, indicating the necessity of a validation test to exclude maternal karyotype abnormalities. This study shows that detection of fetal subchromosomal abnormalities is a viable extension of NIPT based on SSP. Although we focused on the application of cell-free DNA sequencing for NIPT, we believe that this method has broader applications for genetic diagnosis, such as analysis of circulating tumor DNA for detection of cancer.

Folate status and concentrations of serum folate forms in the US population: National Health and Nutrition Examination Survey 2011-2.

Serum and erythrocyte (RBC) total folate are indicators of folate status. No nationally representative population data exist for folate forms. We measured the serum folate forms (5-methyltetrahydrofolate (5-methylTHF), unmetabolised folic acid (UMFA), non-methyl folate (sum of tetrahydrofolate (THF), 5-formyltetrahydrofolate (5-formylTHF), 5,10-methenyltetrahydrofolate (5,10-methenylTHF)) and MeFox (5-methylTHF oxidation product)) by HPLC-MS/MS and RBC total folate by microbiologic assay in US population ≥ 1 year (n approximately 7500) participating in the National Health and Nutrition Examination Survey 2011-2. Data analysis for serum total folate was conducted including and excluding MeFox. Concentrations (geometric mean; detection rate) of 5-methylTHF (37·5 nmol/l; 100 %), UMFA (1·21 nmol/l; 99·9 %), MeFox (1·53 nmol/l; 98·8 %), and THF (1·01 nmol/l; 85·2 %) were mostly detectable. 5-FormylTHF (3·6 %) and 5,10-methenylTHF (4·4 %) were rarely detected. The biggest contributor to serum total folate was 5-methylTHF (86·7 %); UMFA (4·0 %), non-methyl folate (4·7 %) and MeFox (4·5 %) contributed smaller amounts. Age was positively related to MeFox, but showed a U-shaped pattern for other folates. We generally noted sex and race/ethnic biomarker differences and weak (Spearman's r< 0·4) but significant (P< 0·05) correlations with physiological and lifestyle variables. Fasting, kidney function, smoking and alcohol intake showed negative associations. BMI and body surface area showed positive associations with MeFox but negative associations with other folates. All biomarkers showed significantly higher concentrations with recent folic acid-containing dietary supplement use. These first-time population data for serum folate forms generally show similar associations with demographic, physiological and lifestyle variables as serum total folate. Patterns observed for MeFox may suggest altered folate metabolism dependent on biological characteristics.

Blood levels of folate at birth and risk of childhood leukemia.

BACKGROUND: A role for folate in cancer etiology has long been suspected because of folate's function as a cofactor in DNA methylation and maintenance of DNA synthesis. Previous case-control studies examining the association between risk of childhood acute lymphoblastic leukemia (ALL) and mothers' self-reported folate intake and supplementation have been inconclusive. MATERIALS AND METHODS: We used a quantitative microbiologic assay to measure newborn folate concentrations in archived dried bloodspots collected at birth from 313 incident ALL cases, 44 incident acute myeloid leukemia (AML) cases, and 405 matched population-based controls. RESULTS: Overall, we found no difference in hemoglobin-normalized newborn folate concentrations (HbFol, nmol/g) between ALL cases and controls (2.76 vs. 2.77, P = 0.97) or between AML cases and controls (2.93 vs. 2.76, P = 0.32). Null results persisted after stratification by both birth period (1982-94, 1995-98, and 1999-2002) to account for the start of folate fortification of grain products in the United States, and by self-reported maternal prepregnancy supplement use. Similarly, no association was observed for major ALL subgroups. CONCLUSIONS: Our results do not support an association between birth folate concentrations and risk of childhood AML or major ALL subgroups. IMPACT: However, they do not rule out a role for folate through exposures after birth or in early stages of fetal development.

Foxp3 expression is required for the induction of therapeutic tissue tolerance.

CD4(+)Foxp3(+) regulatory T cells (Treg) are essential for immune homeostasis and maintenance of self-tolerance. They are produced in the thymus and also generated de novo in the periphery in a TGF-ß-dependent manner. Foxp3(+) Treg are also required to achieve tolerance to transplanted tissues when induced by coreceptor or costimulation blockade. Using TCR-transgenic mice to avoid issues of autoimmune pathology, we show that Foxp3 expression is both necessary and sufficient for tissue tolerance by coreceptor blockade. Moreover, the known need in tolerance induction for TGF-ß signaling to T cells can wholly be explained by its role in induction of Foxp3, as such signaling proved dispensable for the suppressive process. We analyzed the relative contribution of TGF-ß and Foxp3 to the transcriptome of TGF-ß-induced Treg and showed that TGF-ß elicited a large set of downregulated signature genes. The number of genes uniquely modulated due to the influence of Foxp3 alone was surprisingly limited. Retroviral-mediated conditional nuclear expression of Foxp3 proved sufficient to confer transplant-suppressive potency on CD4(+) T cells and was lost once nuclear Foxp3 expression was extinguished. These data support a dual role for TGF-ß and Foxp3 in induced tolerance, in which TGF-ß stimulates Foxp3 expression, for which sustained expression is then associated with acquisition of tolerance.

Testing of the topoisomerase 1 inhibitor Genz-644282 by the pediatric preclinical testing program.

BACKGROUND: Genz-644282 is a novel non-camptothecin topoisomerase I poison that is in clinical development. PROCEDURES: Genz-644282 was tested against the PPTP in vitro panel (0.1 nM to 1 µM), and in vivo using three times per week × 2 schedule repeated at day 21 at its maximum tolerated dose (MTD) of 4 mg/kg. Subsequently Genz-644282 was tested at 4, 3, 2, and 1 mg/kg in 3 models to assess the dose-response relationship. mRNA gene signatures predictive for Genz-644282 response in vitro were applied to select 15 tumor models that were evaluated prospectively. RESULTS: In vitro, Genz-644282 demonstrated potent cytotoxic activity with a median IC(50) of 1.2 nM (range 0.2-21.9 nM). In vivo, Genz-644282 at its MTD (4 mg/kg) induced maintained complete responses (MCR) in 6/6 evaluable solid tumor models. At 2 mg/kg Genz-644282 induced CR or MCR in 3/3 tumor models relatively insensitive to topotecan, but there were no objective responses at 1 mg/kg. Further testing at 2 mg/kg showed that Genz-644282 induced objective regressions in 7 of 17 (41%) models. There was a significant correlation between predictive response scores based on Affymetrix U133Plus2 baseline tumor expression profiles and the observed in vivo responses to Genz-644282. CONCLUSIONS: Genz-644282 was highly active within a narrow dose range (2-4 mg/kg), typical of other topoisomerase I poisons. As with other topoisomerase I poisons, how accurately these data will translate to clinical activity will depend upon the drug exposures that can be achieved in children treated with this agent.

Alemtuzumab induction of intracellular signaling and apoptosis in malignant B lymphocytes.

The molecular changes induced by alemtuzumab following binding of CD52 on B tumor cells were investigated. Alemtuzumab alone had no detectable impact on cell signaling but cross-linking of alemtuzumab on the surface of B tumor lines with anti-human Fc antibodies induced a transient Ca(2+) flux followed by phosphorylation of several kinases involved in stress and survival pathways, and expression of associated proteins including TNF-α. Cross-linking of alemtuzumab also induced capping and caspase-dependent apoptosis of the tumor lines. When using primary cells from B-CLL patients, alemtuzumab alone was capable of inducing protein phosphorylation and apoptosis through the cross-linking of alemtuzumab by FcγRIIb receptors on B-CLL cells. Apoptosis was prevented by blocking of FcγRIIb receptors with anti-CD32 antibody. Overall, our results indicate that cross-linking of alemtuzumab on B tumor cells can occur naturally through Fc receptor interaction and leads to the activation of specific cellular pathways and induction of apoptosis.

sFLT01: a novel fusion protein with antiangiogenic activity.

sFLT01 is a novel fusion protein that consists of the VEGF/PlGF (placental growth factor) binding domain of human VEGFR1/Flt-1 (hVEGFR1) fused to the Fc portion of human IgG(1) through a polyglycine linker. It binds to both human VEGF (hVEGF) and human PlGF (hPlGF) and to mouse VEGF (mVEGF) and mouse PlGF (mPlGF). In vitro, sFLT01 inhibited the proliferation of human umbilical vein endothelial cells and pericytes stimulated by either hVEGF or hPlGF. In vivo, sFLT01 had robust and significant antitumor activity in numerous preclinical subcutaneous tumor models including H460 non-small cell lung carcinoma, HT29 colon carcinoma, Karpas 299 lymphoma, MOLM-13 AML (acute myeloid leukemia), 786-O, and RENCA renal cell carcinoma (RCC). sFLT01 also increased median survival in the orthotopic RENCA RCC model. sFLT01 had strong antiangiogenic activity and altered intratumoral microvessel density, blood vessel lumen size and perimeter, and vascular and vessel areas in RCC models. sFLT01 treatment resulted in fewer endothelial cells and pericytes within the tumor microenvironment. sFLT01 in combination with cyclophosphamide resulted in greater inhibition of tumor growth than either agent used alone as a monotherapy in the A673 Ewing's sarcoma model. Gene expression profiling indicated that the molecular changes in the A673 sarcoma tumors are similar to changes observed under hypoxic conditions. sFLT01 is an innovative fusion protein that possessed robust antitumor and antiangiogenic activities in preclinical cancer models. It is a dual targeting agent that neutralizes both VEGF and PlGF and, therefore, has potential as a next generation antiangiogenic therapeutic for oncology.

The chemosensitizing activity of inhibitors of glucosylceramide synthase is mediated primarily through modulation of P-gp function.

Glucosylceramide synthase (GCS) is a key enzyme engaged in the biosynthesis of glycosphingolipids and in regulating ceramide metabolism. Studies exploring alterations in GCS activity suggest that the glycolase may have a role in chemosensitizing tumor cells to various cancer drugs. The chemosensitizing effect of inhibitors of GCS (e.g. PDMP and selected analogues) has been observed with a variety of tumor cells leading to the proposal that the sensitizing activity of GCS inhibitors is primarily through increases in intracellular ceramide leading to induction of apoptosis. The current study examined the chemosensitizing activity of the novel GCS inhibitor, Genz-123346 in cell culture. Exposure of cells to Genz-123346 and to other GCS inhibitors at non-toxic concentrations can enhance the killing of tumor cells by cytotoxic anti-cancer agents. This activity was unrelated to lowering intracellular glycosphingolipid levels. Genz-123346 and a few other GCS inhibitors are substrates for multi-drug resistance efflux pumps such as P-gp (ABCB1, gP-170). In cell lines selected to over-express P-gp or which endogenously express P-gp, chemosensitization by Genz-123346 was primarily due to the effects on P-gp function. RNA interference studies using siRNA or shRNA confirmed that lowering GCS expression in tumor cells did not affect their responsiveness to commonly used cytotoxic drugs.

Knockdown of human deubiquitinase PSMD14 induces cell cycle arrest and senescence.

The PSMD14 (POH1, also known as Rpn11/MPR1/S13/CepP1) protein within the 19S complex (19S cap; PA700) is responsible for substrate deubiquitination during proteasomal degradation. The role of PSMD14 in cell proliferation and senescence was explored using siRNA knockdown in carcinoma cell lines. Our results reveal that down-regulation of PSMD14 by siRNA transfection had a considerable impact on cell viability causing cell arrest in the G0-G1 phase, ultimately leading to senescence. The molecular events associated with decreased cell proliferation, cell cycle arrest and senescence include down-regulation of cyclin B1-CDK1-CDC25C, down-regulation of cyclin D1 and up-regulation of p21(/Cip) and p27(/Kip1). Most notably, phosphorylation of the retinoblastoma protein was markedly reduced in PSMD14 knockdown cells. A comparative study with PSMB5, a subunit of the 20S proteasome, revealed that PSMB5 and PSMD14 have different effects on cell cycle, senescence and associated molecular events. These data support the view that the 19S and 20S subunits of the proteasome have distinct biological functions and imply that targeting 19S and 20S would have distinct molecular consequences on tumor cells.

Identification of genes potentially involved in the acquisition of androgen-independent and metastatic tumor growth in an autochthonous genetically engineered mouse prostate cancer model.

BACKGROUND: A major focus of prostate cancer research has been to identify genes that are deregulated during tumor progression, potentially providing diagnostic markers and therapeutic targets. METHODS: We have employed serial analysis of gene expression (SAGE) and microarray hybridization to identify alterations that occur during malignant transformation in the Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model. Many of these alterations were validated by real-time PCR (rtPCR). RESULTS: We identified several hundred mRNAs that were deregulated. Cluster analysis of microarray profiles with samples from various stages of the disease demonstrated that androgen-independent (AI) primary tumors are similar to metastases; 180 transcripts have expression patterns suggesting an involvement in the genesis of late-stage tumors, and our data support a role for phospholipase A2 group IIA in the acquisition of their highly aggressive characteristics. CONCLUSIONS: Our analyses identified well-characterized genes that were previously known to be involved in prostate cancer, validating our study, and also uncovered transcripts that had not previously been implicated in prostate cancer progression.

Protein tyrosine phosphatase PRL-3 in malignant cells and endothelial cells: expression and function.

Protein tyrosine phosphatase PRL-3 mRNA was found highly expressed in colon cancer endothelium and metastases. We sought to associate a function with PRL-3 expression in both endothelial cells and malignant cells using in vitro models. PRL-3 mRNA levels were determined in several normal human endothelial cells exposed or unexposed to the phorbol ester phorbol 12-myristate 13-acetate (PMA) and in 27 human tumor cell lines. In endothelial cells, PRL-3 mRNA expression was increased in human umbilical vascular endothelial cells and human microvascular endothelial cells (HMVEC) exposed to PMA. An oligonucleotide microarray analysis revealed that PRL-3 was among the 10 genes with the largest increase in expression on PMA stimulation. Phenotypically, PMA-treated HMVEC showed increased invasion, tube formation, and growth factor-stimulated proliferation. A flow cytometric analysis of cell surface markers showed that PMA-treated HMVEC retained endothelial characteristics. Infection of HMVEC with an adenovirus expressing PRL-3 resulted in increased tube formation. In tumor cells, PRL-3 mRNA levels varied markedly with high expression in SKNAS neuroblastoma, MCF-7 and BT474 breast carcinoma, Hep3B hepatocellular carcinoma, and HCT116 colon carcinoma. Western blotting analysis of a subset of cell line lysates showed a positive correlation between PRL-3 mRNA and protein levels. PRL-3 was stably transfected into DLD-1 colon cancer cells. PRL-3-overexpressing DLD-1 subclones were assessed for doubling time and invasion. Although doubling time was similar among parental, empty vector, and PRL-3 subclones, invasion was increased in PRL-3-expressing subclones. In models of endogenous expression, we observed that the MCF-7 cell line, which expresses high levels of PRL-3, was more invasive than the SKBR3 cell line, which expresses low levels of PRL-3. However, the MDA-MB-231 cell line was highly invasive with low levels of PRL-3, suggesting that in some models invasion is PRL-3 independent. Transfection of a PRL-3 small interfering RNA into MCF-7 cells inhibited PRL-3 expression and cell invasion. These results indicate that PRL-3 is functional in both endothelial cells and malignant cells and further validate PRL-3 as a potentially important molecular target for anticancer therapy.

Alterations in vascular gene expression in invasive breast carcinoma.

The molecular signature that defines tumor microvasculature will likely provide clues as to how vascular-dependent tumor proliferation is regulated. Using purified endothelial cells, we generated a database of gene expression changes accompanying vascular proliferation in invasive breast cancer. In contrast to normal mammary vasculature, invasive breast cancer vasculature expresses extracellular matrix and surface proteins characteristic of proliferating and migrating endothelial cells. We define and validate the up-regulated expression of VE-cadherin and osteonectin in breast tumor vasculature. In contrast to other tumor types, invasive breast cancer vasculature induced a high expression level of specific transcription factors, including SNAIL1 and HEYL, that may drive gene expression changes necessary for breast tumor neovascularization. We demonstrate the expression of HEYL in tumor endothelial cells and additionally establish the ability of HEYL to both induce proliferation and attenuate programmed cell death of primary endothelial cells in vitro. We also establish that an additional intracellular protein and previously defined metastasis-associated gene, PRL3, appears to be expressed predominately in the vasculature of invasive breast cancers and is able to enhance the migration of endothelial cells in vitro. Together, our results provide unique insights into vascular regulation in breast tumors and suggest specific roles for genes in driving tumor angiogenesis.