The association between bronchopulmonary dysplasia grade and risks of adverse neurodevelopmental outcomes among preterm infants born at less than 30 weeks of gestation.

BACKGROUND: Bronchopulmonary dysplasia (BPD) is a multifactorial disease with neurodevelopmental implications. This study aims to quantify the risks of adverse neurodevelopmental outcomes for each BPD grade among preterm infants born at less than 30 weeks' gestation. METHODS: We retrospectively studied infants who received care in our institution until at least 36 weeks postmenstrual age and had a formal neurodevelopmental assessment in our infant follow-up clinic using the Bayley Scales for Infant and Toddler Development (BSID). We assessed the association between BPD grade and adverse neurodevelopmental outcomes using descriptive statistics and regression models. RESULTS: Two hundred and fifty infants, including 89 (35.6%), 87 (34.8%), 65 (20.6%), and 9 (3.6%) with No BPD, Grade 1, Grade 2, and Grade 3 BPD, were included in the study. Small for gestational age, late pulmonary hypertension, dexamethasone administration, and adverse neurodevelopmental outcomes were more common as BPD grade increased. In a logistic regression analysis, Grades 2 and 3, but not Grade 1, BPD were associated with increased odds of a composite adverse neurodevelopmental outcome by 2.7 and 7.2 folds, respectively. A BSID domain-specific analysis showed that higher grades were associated with lower scores in the cognitive, gross motor, and fine motor domains. CONCLUSIONS: Grades 2 and 3 BPD, but not Grade 1, correlate with risks of adverse neurodevelopmental outcomes at a grade-dependent manner in our single-center cohort retrospective study. Further validation using a multi-center large cohort is warranted.

Oxygenation index in the first three weeks of life is a predictor of bronchopulmonary dysplasia grade in very preterm infants.

BACKGROUND: The new bronchopulmonary dysplasia (BPD) grading system was developed based on its correlation with long-term respiratory and neurodevelopmental outcomes and may provide better personalized prognostication. Identifying early-life predictors for accurate BPD grade prediction may allow interventions to be tailored to individual needs. This study aimed to assess whether oxygenation index (OI) dynamics in the first three weeks of life are a predictor of BPD grade. METHODS: A single-center retrospective study was performed. Generalized additive mixed modeling was used to model OI trajectories for each BPD grade subgroup. A multinomial regression model was then developed to quantify the association between OI dynamics and BPD grade. RESULTS: Two hundred fifty-four infants were identified for inclusion in the trajectory modeling. A total of 6,243 OI data points were available for modeling. OI trajectory estimates showed distinct patterns in the three groups, most prominent during the third week of life. The average daily OI change was -0.33 ± 0.52 (n = 85) in the No-BPD group, -0.04 ± 0.75 (n = 82) in the Low-Grade BPD group, and 0.22 ± 0.65 (n = 75) in the High-Grade BPD group (p < 0.001). A multinomial regression analysis showed the initial OI value and the average daily OI change both independently correlated with BPD grade outcomes after adjusting for birth gestation, birth weight z-score, sex, and the duration of invasive ventilation. CONCLUSION: Early-life OI dynamics may be a useful independent marker for BPD grade prediction. Prospective studies may be warranted to further validate the findings.

Machine learning for prediction of bronchopulmonary dysplasia-free survival among very preterm infants.

BACKGROUND: Bronchopulmonary dysplasia (BPD) is one of the most common and serious sequelae of prematurity. Prompt diagnosis using prediction tools is crucial for early intervention and prevention of further adverse effects. This study aims to develop a BPD-free survival prediction tool based on the concept of the developmental origin of BPD with machine learning. METHODS: Datasets comprising perinatal factors and early postnatal respiratory support were used for initial model development, followed by combining the two models into a final ensemble model using logistic regression. Simulation of clinical scenarios was performed. RESULTS: Data from 689 infants were included in the study. We randomly selected data from 80% of infants for model development and used the remaining 20% for validation. The performance of the final model was assessed by receiver operating characteristics which showed 0.921 (95% CI: 0.899-0.943) and 0.899 (95% CI: 0.848-0.949) for the training and the validation datasets, respectively. Simulation data suggests that extubating to CPAP is superior to NIPPV in BPD-free survival. Additionally, successful extubation may be defined as no reintubation for 9 days following initial extubation. CONCLUSIONS: Machine learning-based BPD prediction based on perinatal features and respiratory data may have clinical applicability to promote early targeted intervention in high-risk infants.

Developing a machine learning-based tool to extend the usability of the NICHD BPD Outcome Estimator to the Asian population.

The NICHD BPD Outcome Estimator uses clinical and demographic data to stratify respiratory outcomes of extremely preterm infants by risk. However, the Estimator does not have an option in its pull-down menu for infants of Asian descent. We hypothesize that respiratory outcomes in extreme prematurity among various racial/ethnic groups are interconnected and therefore the Estimator can still be used to predict outcomes in infants of Asian descent. Our goal was to apply a machine learning approach to assess whether outcome prediction for infants of Asian descent is possible with information hidden in the prediction results using White, Black, and Hispanic racial/ethnic groups as surrogates. We used the three racial/ethnic options in the Estimator to obtain the probabilities of BPD outcomes for each severity category. We then combined the probability results and developed three respiratory outcome prediction models at various postmenstrual age (PMA) by a random forest algorithm. We showed satisfactory model performance, with receiver operating characteristics area under the curve of 0.934, 0.850, and 0.757 for respiratory outcomes at PMA 36, 37, and 40 weeks, respectively, in the testing data set. This study suggested an interrelationship among racial/ethnic groups for respiratory outcomes among extremely preterm infants and showed the feasibility of extending the use of the Estimator to the Asian population.

In-Hospital Outcomes in Fontan Completion Surgery According to Age.

Actual timing of the Fontan operation is variable. Our aim was to evaluate the impact of age at the time of Fontan operation on mortality and clinical outcome and characterize patients with worse outcomes. We conducted a retrospective, cross-sectional study on the Fontan operation using nationally representative databases from 2003 to 2016 and categorized the patient into 1 of 5 groups according to their age at the time of surgery: <2, 2, 3, 4, and ≥5 years. Survey-weighted logistic regression models were used to compare the outcomes of the different age groups. A total of 6,647 children underwent the Fontan completion procedure during the study period with median age 3 (interquartile range 2 to 4) years. The in-hospital mortality was 2.1%. In logistic regression models, in-hospital mortality, respiratory failure, acute kidney injury, chylothorax, arrhythmias, and sudden cardiac arrest were similar among the 5 age groups. Compared with children >2 years, those <2 years were less likely to be admitted for surgery on an elective basis (73.5% vs 90.4%, p <0.001), more likely to have chromosomal anomalies (2.7% vs 1.7%), and more likely to have repair of atrioventricular valves (8.5% vs 6.0%, p = 0.027). Mortality was higher in those with an underlying atrioventricular septal defect (AVSD) adjusted odds ratio 4.3 (2.4 to 7.9, p <0.001). Repair of AV valves was more common in the AVSD group compared with those in non-AVSD (14.3% vs 5.5%, p <0.001). In conclusion, age at Fontan completion does not adversely affect the in-hospital outcomes. Our focus should be on optimizing essential factors that are crucial for successful Fontan completion.

The SLC25A42 Transcript Is a Biomarker for Fetal Reprogramming in Response to Placental Insufficiency in Preterm Newborns Under 32 Weeks Gestation-A Pilot Study.

Introduction: Timing of medical delivery of preterm newborns exposed to placental insufficiency is largely determined by umbilical artery blood flow and maternal clinical manifestations. There is a lack of tools to properly assess fetal body response to placental insufficiency before or upon delivery. Yet, short- and long-term comorbidities associated with placental insufficiency and the consequential intrauterine growth restriction may be a result of fetal response following prolonged stress. This study aims to establish a procedure to investigate fetal/neonatal transcriptional response to placental insufficiency as part of an initiative to identify cost-effective biomarkers for assessing fetal response to placental insufficiency. Methods: A prospective pilot study involving newborns with birth gestation <32 weeks was conducted to compare gene expression profiles in whole blood collected at birth among three clinically distinct groups - preeclampsia without placental insufficiency (PE), placental insufficiency (PI), and non-PE/PI groups. Results: Whole blood from 11, 3, and 6 newborns in the non-PE/PI, PE, and PI groups were obtained. A transcriptome analysis found that the majority of the genes were downregulated in the PI group, suggesting global transcriptional inactivation. Intriguingly, SLC25A42, which encodes a mitochondrial transporter for coenzyme A and adenosine-3',5'-diphosphate, was significantly upregulated in the PI group. Conclusion: Transcriptional biomarkers for assessing fetal response to placental insufficiency may provide a useful tool to better understand the pathophysiology of fetal reprogramming in response to placental insufficiency. The validity and the role of SLC25A42, as well as its correlation with short- and long-term neonatal outcomes, warrants further investigation.

Live-cell Migration Assays to Study Motility of Neural andGlial (Oligodendrocyte) Progenitor Cells.

Cell motility has been extensively studied in in vitro models using fibroblasts and keratocytes, but the cell type-specific mechanisms underlying migration of lineage- or disease-specific cells, such as neural and glial progenitor cells, remain an active field for investigation. The migrating neural and glial progenitor cells contribute to the development, tissue repair and tumor invasion in the central nervous system (CNS). Cell migration is a highly dynamic process which relies on membranous protrusions to assemble, extend, disassemble and retract. In the CNS, the motility of neural and glial progenitor cells is affected by various cell-autonomous and non-cell-autonomous mechanisms such as signaling molecules, actin and microtubule interactions, and environmental cues. Here, we described a live-cell migration assay for use in the assessment of neural and glial progenitor cell migration. We first will demonstrate the procedures for isolating and culturing neural and glial progenitor cells. Next, we will demonstrate the acquisition of time-lapse images using phase contrast microscopy, the methods for quantification and the analyses of various motility parameters including speed, velocity, straightness and leading-edge dynamics. This method allows researchers to dissect the mechanisms of cell motility in response to different environmental cues, such as chemoattractive and repulsive signals, matrix adhesiveness and stiffness. This assay also allows researchers to study migration of pharmacologically and genetically manipulated cells.

Improved multilineage human hematopoietic reconstitution and function in NSGS mice.

Genetic manipulation of NOD/SCID (NS) mice has yielded numerous sub-strains with specific traits useful for the study of human hematopoietic xenografts, each with unique characteristics. Here, we have compared the engraftment and output of umbilical cord blood (UCB) CD34+ cells in four immune-deficient strains: NS, NS with additional IL2RG knockout (NSG), NS with transgenic expression of human myeloid promoting cytokines SCF, GM-CSF, and IL-3 (NSS), and NS with both IL2RG knockout and transgenic cytokine expression (NSGS). Overall engraftment of human hematopoietic cells was highest in the IL2RG knockout strains (NSG and NSGS), while myeloid cell output was notably enhanced in the two strains with transgenic cytokine expression (NSS and NSGS). In further comparisons of NSG and NSGS mice, several additional differences were noted. NSGS mice were found to have a more rapid reconstitution of T cells, improved B cell differentiation, increased levels of NK cells, reduced platelets, and reduced maintenance of primitive CD34+ cells in the bone marrow. NSGS were superior hosts for secondary engraftment and both strains were equally suitable for experiments of graft versus host disease. Increased levels of human cytokines as well as human IgG and IgM were detected in the serum of humanized NSGS mice. Furthermore, immunization of humanized NSGS mice provided evidence of a functional response to repeated antigen exposure, implying a more complete hematopoietic graft was generated in these mice. These results highlight the important role that myeloid cells and myeloid-supportive cytokines play in the formation of a more functional xenograft immune system in humanized mice.

Efficacy of prophylactic dexmedetomidine in preventing postoperative junctional ectopic tachycardia in pediatric cardiac surgery patients: A systematic review and meta-analysis.

BACKGROUND: Junctional ectopic tachycardia is a serious tachyarrhythmic complication following pediatric cardiac surgery. It is difficult to manage and is associated with significant morbidity and mortality. Conventional nonpharmacological and pharmacological measures have shown limited effects. Dexmedetomidine is an α2 agonist which has recently been shown in multiple studies to be effective. AIMS: The aim of this systematic review with meta-analysis was to evaluate the efficacy of prophylactic dexmedetomidine administration in the prevention of junctional ectopic tachycardia in pediatric patients following cardiac surgeries. METHODS: We searched MEDLINE, EMBASE, Cochrane, Web of Science, and relevant references published in English before December 20, 2017 and performed meta-analysis on the selected studies, with one group receiving prophylactic perioperative dexmedetomidine administration and another group receiving placebo. The primary outcome was the incidence of junctional ectopic tachycardia, secondary outcomes included bradycardia, hypotension, intensive care unit stay, total hospital stay, inotropic scores, and total mechanical ventilation time. Odds ratio or mean difference with 95% confidence intervals were calculated using a random effect model. RESULTS: Seven studies (5 prospective randomized studies and 2 retrospective case-controlled studies) with a total of 1616 patients were analyzed. The incidence of junctional ectopic tachycardia in the dexmedetomidine group was significantly reduced compared to placebo. Similarly, intensive care unit stay, inotropic scores, and total mechanical ventilation time were also significantly decreased in the dexmedetomidine group. No significant increases in adverse events were found. Mortality was low in both groups. CONCLUSION: Prophylactic dexmedetomidine is effective in reducing the incidence of postoperative junctional ectopic tachycardia without significant increases in adverse events in pediatric patients undergoing surgery for congenital heart diseases.

Neuronal Precursor Migration in Ex Vivo Brain Slice Culture.

Neuronal migration during fetal brain development is a well-coordinated process between the migrating neurons and their substrates, the basal processes of the radial glial cells (RGCs). The progeny-progenitor relationship between the migrating neurons and the RGCs in the developing fetal brain may make interpretations of the results difficult, because the variable in question may affect both the RGCs and the migrating neurons in different ways. A transplantation assay combining migrating cells and the scaffolding tissue from two different sources may circumvent this issue. We developed an ex vivo brain slice transplantation assay that allows recording of migrating neurons in real time.

Immortalization of human AE pre-leukemia cells by hTERT allows leukemic transformation.

Human CD34+ hematopoietic stem and progenitor cells (HSPC) expressing fusion protein AML1-ETO (AE), generated by the t(8;21)(q22;q22) rearrangement, manifest enhanced self-renewal and dysregulated differentiation without leukemic transformation, representing a pre-leukemia stage. Enabling replicative immortalization via telomerase reactivation is a crucial step in cancer development. However, AE expression alone is not sufficient to maintain high telomerase activity to immortalize human HSPC cells, which may hamper transformation. Here, we investigated the cooperativity of telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, and AE in disease progression. Enforced expression of hTERT immortalized human AE pre-leukemia cells in a telomere-lengthening independent manner, and improved the pre-leukemia stem cell function by enhancing cell proliferation and survival. AE-hTERT cells retained cytokine dependency and multi-lineage differentiation potential similar to parental AE clones. Over the short-term, AE-hTERT cells did not show features of stepwise transformation, with no leukemogenecity evident upon initial injection into immunodeficient mice. Strikingly, after extended culture, we observed full transformation of one AE-hTERT clone, which recapitulated the disease evolution process in patients and emphasizes the importance of acquiring cooperating mutations in t(8;21) AML leukemogenesis. In summary, achieving unlimited proliferative potential via hTERT activation, and thereby allowing for acquisition of additional mutations, is a critical link for transition from pre-leukemia to overt disease in human cells. AE-hTERT cells represent a tractable model to study cooperating genetic lesions important for t(8;21) AML disease progression.

AML cells are differentially sensitive to chemotherapy treatment in a human xenograft model.

As acute myeloid leukemia (AML) xenograft models improve, the potential for using them to evaluate novel therapeutic strategies becomes more appealing. Currently, there is little information on using standard chemotherapy regimens in AML xenografts. Here we have characterized the immunodeficient mouse response to combined Ara-C (cytarabine) and doxorubicin treatment. We observed significant toxicity associated with doxorubicin that required optimization of the route of injection as well as the maximum-tolerated dose for immunodeficient strains. Mice treated with an optimized 5-day induction protocol showed transient weight loss, short-term reduction of peripheral blood cell and platelet counts, and slight anemia. Considerable cytotoxicity was observed in the bone marrow (BM), with primitive LSK cells having a significant survival advantage relative to more mature cells, consistent with the idea of chemotherapy targeting actively growing cells. Treated leukemic mice demonstrated reduced disease burden and increased survival, demonstrating efficacy. AML cells showed significantly increased sensitivity to doxorubicin-containing therapy compared with murine BM cells. Although early treatment could result in some cures, mice with significant leukemia grafts were not cured by using induction therapy alone. Overall, the data show that this model system is useful for the evaluation of novel chemotherapies in combination with standard induction therapy.

The thrombopoietin/MPL/Bcl-xL pathway is essential for survival and self-renewal in human preleukemia induced by AML1-ETO.

AML1-ETO (AE) is a fusion product of translocation (8;21) that accounts for 40% of M2 type acute myeloid leukemia (AML). In addition to its role in promoting preleukemic hematopoietic cell self-renewal, AE represses DNA repair genes, which leads to DNA damage and increased mutation frequency. Although this latter function may promote leukemogenesis, concurrent p53 activation also leads to an increased baseline apoptotic rate. It is unclear how AE expression is able to counterbalance this intrinsic apoptotic conditioning by p53 to promote survival and self-renewal. In this report, we show that Bcl-xL is up-regulated in AE cells and plays an essential role in their survival and self-renewal. Further investigation revealed that Bcl-xL expression is regulated by thrombopoietin (THPO)/MPL-signaling induced by AE expression. THPO/MPL-signaling also controls cell cycle reentry and mediates AE-induced self-renewal. Analysis of primary AML patient samples revealed a correlation between MPL and Bcl-xL expression specifically in t(8;21) blasts. Taken together, we propose that survival signaling through Bcl-xL is a critical and intrinsic component of a broader self-renewal signaling pathway downstream of AML1-ETO-induced MPL.

Cdc42 regulates extracellular matrix remodeling in three dimensions.

Extracellular matrix (ECM) actively participates in normal cell regulation and in the process of tumor progression. The Rho GTPase Cdc42 has been shown to regulate cell-ECM interaction in conventional two-dimensional culture conditions by using dominant mutants of Cdc42 in immortalized cell lines that may introduce nonspecific effects. Here, we employ three-dimensional culture systems for conditional gene targeted primary mouse embryonic fibroblasts that better simulate the reciprocal and adaptive interactions between cells and surrounding matrix to define the role of Cdc42 signaling pathways in ECM organization. Cdc42 deficiency leads to a defect in global cell-matrix interactions reflected by a decrease in collagen gel contraction. The defect is associated with an altered cell-matrix interaction that is evident by morphologic changes and reduced focal adhesion complex formation. The matrix defect is also associated with a reduction in synthesis and activation of matrix metalloproteinase 9 (MMP9) and altered fibronectin deposition patterning. A Cdc42 mutant rescue experiment found that downstream of Cdc42, p21-activated kinase (PAK), but not Par6 or WASP, may be involved in regulating collagen gel contraction and fibronectin organization. Thus, in addition to the previously implicated roles in intracellular regulation of actin organization, proliferation, and vesicle trafficking, Cdc42 is essential in ECM remodeling in three dimensions.

Inhibition of Rac GTPase signaling and downstream prosurvival Bcl-2 proteins as combination targeted therapy in MLL-AF9 leukemia.

The Rac family of small Rho GTPases coordinates diverse cellular functions in hematopoietic cells including adhesion, migration, cytoskeleton rearrangements, gene transcription, proliferation, and survival. The integrity of Rac signaling has also been found to critically regulate cellular functions in the initiation and maintenance of hematopoietic malignancies. Using an in vivo gene targeting approach, we demonstrate that Rac2, but not Rac1, is critical to the initiation of acute myeloid leukemia in a retroviral expression model of MLL-AF9 leukemogenesis. However, loss of either Rac1 or Rac2 is sufficient to impair survival and growth of the transformed MLL-AF9 leukemia. Rac2 is known to positively regulate expression of Bcl-2 family proteins toward a prosurvival balance. We demonstrate that disruption of downstream survival signaling through antiapoptotic Bcl-2 proteins is implicated in mediating the effects of Rac2 deficiency in MLL-AF9 leukemia. Indeed, overexpression of Bcl-xL is able to rescue the effects of Rac2 deficiency and MLL-AF9 cells are exquisitely sensitive to direct inhibition of Bcl-2 family proteins by the BH3-mimetic, ABT-737. Furthermore, concurrent exposure to NSC23766, a small-molecule inhibitor of Rac activation, increases the apoptotic effect of ABT-737, indicating the Rac/Bcl-2 survival pathway may be targeted synergistically.

The thrombopoietin/MPL pathway in hematopoiesis and leukemogenesis.

Hematopoietic stem cells (HSC) comprise a small percentage of total hematopoietic cells. Their ability to self-renewal is key to the continuous replenishment of the hematopoietic system with newly formed functional blood cell types while maintaining their multipotential capacity. Understanding the extrinsic signals that are essential to HSC maintenance will provide insights into the regulation of hematopoiesis at its most primitive stage, and with the knowledge applied, will potentially lead to improved clinical transplantation outcomes. In this review, we will summarize the current understanding of the role of the thrombopoietin/MPL signaling pathway in HSC maintenance during adult and fetal hematopoiesis. We will also speculate on the downstream key players in the pathway based on published data, and summarize the role of this pathway in leukemia.

N-Ras(G12D) induces features of stepwise transformation in preleukemic human umbilical cord blood cultures expressing the AML1-ETO fusion gene.

AML1-ETO (AE) is a fusion product of t(8;21) observed in 40% French-American-British M2 type of acute myeloid leukemia (AML). Clinical data suggest that Ras mutation is a frequent cooperating event in t(8;21) AML. Whether constitutively active Ras promotes leukemogenesis on the t(8;21) background has not been demonstrated experimentally. Here, we retrovirally expressed N-Ras(G12D) in AE-expressing human hematopoietic cells to investigate cooperativity. The AE/N-Ras(G12D) cultures were cytokine-independent, enriched for CD34 positivity, and possessed increased colony-forming and replating abilities. N-Ras(G12D) expression led to Bcl-2 up-regulation and reduced apoptosis. Ectopic Bcl-2 expression also resulted in enhanced colony-forming and replating abilities but was insufficient to sustain cytokine independence. AE/N-Ras(G12D) cells were more sensitive to Bcl-2 inhibition with ABT-737 than parent AE cells. Enhanced engraftment of AE/N-Ras(G12D) cells was observed on intrafemoral injection into immunodeficient mice, presumably because of improved survival in the bone marrow microenvironment. N-Ras(G12D) promotes progression toward transformation in AE-expressing cells, partially through up-regulating Bcl-2.

Core binding factor at the crossroads: determining the fate of the HSC.

Hematopoietic development requires coordinated actions from a variety of transcription factors. The core binding factor (CBF), consisting of a Runx protein and the CBFbeta protein, is a transcription factor complex that is essential for emergence of the hematopoietic stem cell (HSC) from an endothelial cell stage. The hematopoietic defects observed in either Runx1 or CBFbeta knockout mice underscore the necessity of this complex for definitive hematopoiesis. Despite the requirement for CBF in establishing definitive hematopoiesis, Runx1 loss has minimal impact on maintaining the HSC state postnatally, while CBFbeta may continue to be essential. Lineage commitment, on the other hand, is significantly affected upon CBF loss in the adult, indicating a primary role for this complex in modulating differentiation. Given the impact of normal CBF function in the hematopoietic system, the severe consequences of disrupting CBF activity, either through point mutations or generation of fusion genes, are obvious. The physiologic role of CBF in differentiation is subverted to an active process of self-renewal maintenance by the genetic aberrations, through several possible mechanisms, contributing to the development of hematopoietic malignancies including myelodysplastic syndrome and leukemia. The major impact of CBF on the hematopoietic system in both development and disease highlights the need for understanding the intricate functions of this complex and reiterate the necessity of continued efforts to identify potential points of therapeutic intervention for CBF-related diseases.

Thiazolidinediones downregulate Wnt/beta-catenin signaling via multiple mechanisms in breast cancer cells.

BACKGROUND: Thiazolidinediones (TZDs) have been demonstrated to possess antitumor effects in breast cancer cells, although the mechanisms are not well established. We sought to better define TZDs' antitumor effects and molecular mechanisms to permit rational utilization of these agents. MATERIALS AND METHODS: We studied the effects of TZDs on DNA synthesis (BrdU enzyme-linked immunosorbent assay), gene expression (microarray, quantitative reverse transcription-polymerase chain reaction, and immunoblot), serine phosphorylation and localization of beta-catenin (nuclear/cytoplasmic fractionation and immunoblot), transactivation activity of beta-catenin/T cell factor 4 (TCF4) (luciferase assay with wild-type and mutant TCF4 responsive element), and beta-catenin/TCF4 complex (immunoprecipitation) in human breast cancer cells MDA-MB-231 and T47D. RESULTS: Troglitazone (TG) down-regulated DNA synthesis in MDA-MB-231 and T47D and decreased mRNA expression of the Wnt co-receptors frizzled-1 and low-density lipoprotein-related protein 6 (LRP6) as well as protein level of LRP6. TG also targets downstream Wnt signaling molecules in T47D cells. TG down-regulates p-beta-catenin (S33/S37/T41) and promotes translocation of beta-catenin into the nucleus. However, TG inhibits beta-catenin-mediated transactivation by down-regulating TCF4 protein levels, thereby inhibiting beta-catenin/TCF4 complex formation. Finally, we found that inhibition of Akt augments TG-mediated down-regulation of DNA synthesis and this result was accompanied with the decreased protein levels of Wnt signaling molecules: LRP6, beta-catenin, and TCF4. CONCLUSION: These results suggest that the Wnt/beta-catenin signaling pathway, which plays an important role in breast cancer pathogenesis, may be a target for TZD treatment. Moreover, a combination of TZDs and a specific Akt inhibitor may serve as a new approach to target Wnt/beta-catenin directly and via PI3K/Akt action on glycogen synthase-3beta.

Troglitazone inhibits cell migration, adhesion, and spreading by modulating cytoskeletal rearrangement in human breast cancer cells.

Metastatic tumors are the primary cause of death in patients with breast cancer. Recent data indicate that the peroxisome proliferator-activated receptor gamma (PPARgamma) ligands, thiazolidinediones (TZDs), possess anti-invasive activities on human breast cancer cells. However, the effects of TZDs on other metastatic properties of breast cancer cells such as adhesion, spreading, and migration are not well established. In this study, we show that troglitazone (TG), a member of the TZD family, inhibits lamellipodia formation or membrane ruffling as well as actin polymerization at these structures in MDA-MB-231 and T47D breast cancer cells. In addition, TG reduces migration, adhesion, and spreading on fibronectin (FN)-coated plates. These phenomena were associated with the dramatic decrease of Tyr397 and Tyr576 phosphorylation of focal adhesion kinase (FAK) and the detergent-insoluble Rac1. We also found that TG upregulates Tyr416 phosphorylation of Src, but downregulates the Src-FAK complex. Moreover, we use a PPARgamma-inactive derivative of TG (STG28) and a PPARgamma antagonist (GW9662) to eliminate PPARgamma-mediated effects. We found that treatment with STG28 or GW9662 plus TG showed similar effects compared to TG treatment alone on tyrosine phosphorylation of FAK and Src, indicating that these effects are not the result of PPARgamma activation. Interestingly, we found that TG upregulates actin filament assembly at the point of cell-cell contact in T47D cells, indicating that TG may also upregulate cell-cell adhesion in breast cancer cells which express E-cadherin. These results suggested that TG should be investigated further for its therapeutic potential in metastatic breast cancer.