Invasion of ovarian cancer cells is induced by PITX2-mediated activation of TGF-β and Activin-A

Background:Most ovarian cancers are highly invasive in nature and the high burden of metastatic disease make them a leading cause of mortality among all gynaecological malignancies. The homeodomain transcription factor, PITX2 is associated with cancer in different tissues. Our previous studies demonstrated increased PITX2 expression in human ovarian tumours. Growing evidence linking activation of TGF-β pathway by homeodomain proteins prompted us to look for the possible involvement of this signalling pathway in PITX2-mediated progression of ovarian cancer. Methods: The status of TGF-β signalling in human ovarian tissues was assessed by immunohistochemistry. The expression level of TGFB/INHBA and other invasion-associated genes was measured by quantitative-PCR (Q-PCR) and Western Blot after transfection/treatments with clones/reagents in normal/cancer cells. The physiological effect of PITX2 on invasion/motility was checked by matrigel invasion and wound healing assay. The PITX2- and activin-induced epithelial-mesenchymal transition (EMT) was evaluated by Q-PCR of respective markers and confocal/phase-contrast imaging of cells. Results: Human ovarian tumours showed enhanced TGF-β signalling. Our study uncovers the PITX2-induced expression of TGFB1/2/3 as well as INHBA genes (p < 0.01) followed by SMAD2/3-dependent TGF-β signalling pathway. PITX2-induced TGF-β pathway regulated the expression of invasion-associated genes, SNAI1, CDH1 and MMP9 (p < 0.01) that accounted for enhanced motility/invasion of ovarian cancers. Snail and MMP9 acted as important mediators of PITX2-induced invasiveness of ovarian cancer cells. PITX2 over-expression resulted in loss of epithelial markers (p < 0.01) and gain of mesenchymal markers (p < 0.01) that contributed significantly to ovarian oncogenesis. PITX2-induced INHBA expression (p < 0.01) contributed to EMT in both normal and ovarian cancer cells. Conclusions: Overall, our findings suggest a significant contributory role of PITX2 in promoting invasive behaviour of ovarian cancer cells through up-regulation of TGFB/INHBA. We have also identified the previously unknown involvement of activin-A in promoting EMT. Our work provides novel mechanistic insights into the invasive behavior of ovarian cancer cells. The extension of this study have the potential for therapeutic applications in future

Relationship between Tumor DNA Methylation Status and Patient Characteristics in African-American and European-American Women with Breast Cancer

Aberrant DNA methylation is critical for development and progression of breast cancer. We investigated the association of CpG island methylation in candidate genes and clinicopathological features in 65 African-American (AA) and European-American (EA) breast cancer patients. Quantitative methylation analysis was carried out on bisulfite modified genomic DNA and sequencing (pyrosequencing) for promoter CpG islands of p16, ESR1, RASSF1A, RARβ2, CDH13, HIN1, SFRP1 genes and the LINE1 repetitive element using matched paired non-cancerous and breast tumor specimen (32 AA and 33 EA women). Five of the genes, all known tumor suppressor genes (RASSF1A, RARβ2, CDH13, HIN1 and SFRP1), were found to be frequently hypermethylated in breast tumor tissues but not in the adjacent non-cancerous tissues. Significant differences in the CDH13 methylation status were observed by comparing DNA methylation between AA and EA patients, with more obvious CDH13 methylation differences between the two patient groups in the ER- disease and among young patients (age<50). In addition, we observed associations between CDH13, SFRP1, and RASSF1A methylation and breast cancer subtypes and between SFRP1 methylation and patient's age. Furthermore, tumors that received neoadjuvant therapy tended to have reduced RASSF1A methylation when compared with chemotherapy naïve tumors. Finally, Kaplan Meier survival analysis showed a significant association between methylation at 3 loci (RASSF1A, RARβ2 and CDH13) and reduced overall disease survival. In conclusion, the DNA methylation status of breast tumors was found to be significantly associated with clinicopathological features and race/ethnicity of the patients

Abnormal accumulation of autophagic vesicles correlates with axonal and synaptic pathology in young Alzheimer’s mice hippocampus

Dystrophic neurites associated with amyloid plaques precede neuronal death and manifest early in Alzheimer’s disease (AD). In this work we have characterized the plaque-associated neuritic pathology in the hippocampus of young (4- to 6-month-old) PS1M146L/APP751SL mice model, as the initial degenerative process underlying functional disturbance prior to neuronal loss. Neuritic plaques accounted for almost all fibrillar deposits and an axonal origin of the dystrophies was demonstrated. The early induction of autophagy pathology was evidenced by increased protein levels of the autophagosome marker LC3 that was localized in the axonal dystrophies, and by electron microscopic identification of numerous autophagic vesicles filling and causing the axonal swellings. Early neuritic cytoskeletal defects determined by the presence of phosphorylated tau (AT8-positive) and actin–cofilin rods along with decreased levels of kinesin-1 and dynein motor proteins could be responsible for this extensive vesicle accumulation within dystrophic neurites. Although microsomal Aβ oligomers were identified, the presence of A11-immunopositive Aβ plaques also suggested a direct role of plaque-associated Aβ oligomers in defective axonal transport and disease progression. Most importantly, presynaptic terminals morphologically disrupted by abnormal autophagic vesicle buildup were identified ultrastructurally and further supported by synaptosome isolation. Finally, these early abnormalities in axonal and presynaptic structures might represent the morphological substrate of hippocampal dysfunction preceding synaptic and neuronal loss and could significantly contribute to AD pathology in the preclinical stages

Defects in Mitochondrial Dynamics and Metabolomic Signatures of Evolving Energetic Stress in Mouse Models of Familial Alzheimer's Disease

The identification of early mechanisms underlying Alzheimer's Disease (AD) and associated biomarkers could advance development of new therapies and improve monitoring and predicting of AD progression. Mitochondrial dysfunction has been suggested to underlie AD pathophysiology, however, no comprehensive study exists that evaluates the effect of different familial AD (FAD) mutations on mitochondrial function, dynamics, and brain energetics.We characterized early mitochondrial dysfunction and metabolomic signatures of energetic stress in three commonly used transgenic mouse models of FAD. Assessment of mitochondrial motility, distribution, dynamics, morphology, and metabolomic profiling revealed the specific effect of each FAD mutation on the development of mitochondrial stress and dysfunction. Inhibition of mitochondrial trafficking was characteristic for embryonic neurons from mice expressing mutant human presenilin 1, PS1(M146L) and the double mutation of human amyloid precursor protein APP(Tg2576) and PS1(M146L) contributing to the increased susceptibility of neurons to excitotoxic cell death. Significant changes in mitochondrial morphology were detected in APP and APP/PS1 mice. All three FAD models demonstrated a loss of the integrity of synaptic mitochondria and energy production. Metabolomic profiling revealed mutation-specific changes in the levels of metabolites reflecting altered energy metabolism and mitochondrial dysfunction in brains of FAD mice. Metabolic biomarkers adequately reflected gender differences similar to that reported for AD patients and correlated well with the biomarkers currently used for diagnosis in humans.Mutation-specific alterations in mitochondrial dynamics, morphology and function in FAD mice occurred prior to the onset of memory and neurological phenotype and before the formation of amyloid deposits. Metabolomic signatures of mitochondrial stress and altered energy metabolism indicated alterations in nucleotide, Krebs cycle, energy transfer, carbohydrate, neurotransmitter, and amino acid metabolic pathways. Mitochondrial dysfunction, therefore, is an underlying event in AD progression, and FAD mouse models provide valuable tools to study early molecular mechanisms implicated in AD

New therapeutic targets in Alzheimer's disease: brain deregulation of calcium and zinc

The molecular determinants of Alzheimer's (AD) disease are still not completely known; however, in the past two decades, a large body of evidence has indicated that an important contributing factor for the disease is the development of an unbalanced homeostasis of two signaling cations: calcium (Ca2+) and zinc (Zn2+). Both ions serve a critical role in the physiological functioning of the central nervous system, but their brain deregulation promotes amyloid-β dysmetabolism as well as tau phosphorylation. AD is also characterized by an altered glutamatergic activation, and glutamate can promote both Ca2+ and Zn2+ dyshomeostasis. The two cations can operate synergistically to promote the generation of free radicals that further intracellular Ca2+ and Zn2+ rises and set the stage for a self-perpetuating harmful loop. These phenomena can be the initial steps in the pathogenic cascade leading to AD, therefore, therapeutic interventions aiming at preventing Ca2+ and Zn2+ dyshomeostasis may offer a great opportunity for disease-modifying strategies

Neurodegeneration of the retina in mouse models of Alzheimer’s disease: what can we learn from the retina?

Alzheimer’s disease (AD) is an age-related progressive neurodegenerative disease commonly found among elderly. In addition to cognitive and behavioral deficits, vision abnormalities are prevalent in AD patients. Recent studies investigating retinal changes in AD double-transgenic mice have shown altered processing of amyloid precursor protein and accumulation of β-amyloid peptides in neurons of retinal ganglion cell layer (RGCL) and inner nuclear layer (INL). Apoptotic cells were also detected in the RGCL. Thus, the pathophysiological changes of retinas in AD patients are possibly resembled by AD transgenic models. The retina is a simple model of the brain in the sense that some pathological changes and therapeutic strategies from the retina may be observed or applicable to the brain. Furthermore, it is also possible to advance our understanding of pathological mechanisms in other retinal degenerative diseases. Therefore, studying AD-related retinal degeneration is a promising way for the investigation on (1) AD pathologies and therapies that would eventually benefit the brain and (2) cellular mechanisms in other retinal degenerations such as glaucoma and age-related macular degeneration. This review will highlight the efforts on retinal degenerative research using AD transgenic mouse models

DNA hypermethylation of PITX2 is a marker of poor prognosis in untreated lymph node-negative hormone receptor-positive breast cancer patients.

BACKGROUND: In this study, we evaluated if PITX2 DNA methylation is a marker for disease recurrence in lymph node-negative (LNN), steroid hormone receptor-positive (HR+) breast cancer patients. In addition, we studied the association between PITX2 DNA methylation and PITX2 gene expression. PATIENTS AND METHODS: PITX2 DNA-methylation was measured in tumor tissue from 412 LNN/HR+ breast cancer patients who had not received any adjuvant systemic treatment. In addition, PITX2 DNA-methylation and mRNA expression was evaluated in 32 breast cancer cell lines. RESULTS: In univariate Cox regression analysis, DNA-methylation of PITX2 as a continuous variable was associated with early distant metastasis (HR = 1.71; P < 0.01) and poor overall survival (HR = 1.71; P < 0.01). In multivariate analysis together with the established prognostic factors age, tumor size and tumor grade, and steroid hormone receptor levels, both associations retained their significance (for MFS, HR = 1.74; P < 0.01; for OS, HR = 1.46; P = 0.02). In the breast cancer cell lines, PITX2 DNA methylation was inversely association with PITX2A and PITX2B mRNA expression (P < 0.01). CONCLUSIONS: Hypermethylation of PITX2 is, in cell lines, negatively associated with PITX2 mRNA expression and, in clinical specimens, positively associated with breast cancer disease progression