Angio-Associated Migratory Cell Protein (AAMP) Regulates the Hippo/YAP Pathway and Mitochondrial Functionality to Drive Osteosarcoma Metastasis

Background: Osteosarcoma (OS) is the prevalent form of primary bone cancer among adolescents, but the 5-year overall survival rate for patients with a metastatic or recurrent OS is under 20%. Angio-associated migratory cell protein (AAMP) is known to be a key regulator of cellular migration, yet its role in the context of OS metastasis has yet to be firmly established.Methods: Bioinformatics analyses were used to explore the association between AAMP and YAP expression and the prognosis of OS patients, and to evaluate differences in AAMP expression in patients with primary OS, recurrent OS, and pulmonary metastatic OS. Immunohistochemical (IHC) staining was additionally performed to compare AAMP levels in primary OS and pulmonary metastatic OS patient samples. Lentiviral transduction was further used to establish OS cell lines in which AAMP or YAP had been stably knocked down or overexpressed. OS cell migration and invasion were assessed using wound healing and Transwell assays. Proteins associated with the mitochondria, the epithelial-mesenchymal transition (EMT), YAP, and its target proteins were assessed in OS cell lines via Western blotting. OS cell lamellipodia were detected via phalloidin staining. Mitochondrial morphological characteristics were assessed via transmission electron microscopy following the knockdown of AAMP. An ATP kit was employed to measure ATP levels in OS cells in which AAMP had been knocked down. Animal model studies were used to confirm indices associated with OS cell lung metastasis following AAMP knockdown. Results: Patients with metastatic OS exhibit higher levels of AAMP expression that are correlated with poorer patient prognosis. Knocking down AAMP suppressed the migratory, invasive, and EMT activity of analyzed OS cell lines. AAMP was found to regulate CFL1 and thereby control OS cell protrusion. AAMP knockdown was further found to promote OS cell mitochondrial dysfunction and decreased intracellular ATP production, with these AAMP knockdown cells exhibiting impaired migratory and invasive activity as a consequence of YAP inhibition. Consistently, the knockdown of AAMP suppressed the in vivo metastasis of OS cells. Conclusions: Together, these data highlight a model wherein AAMP can promote OS cell migratory and invasive activity by regulating YAP and mitochondrial functionality. The AAMP/CFL1/YAP signaling pathway may thus represent a viable therapeutic target for efforts aimed at suppressing the metastatic progression of OS.

MO433IDENTIFICATION AND VALIDATION OF PEPTIDIC FEATURES IN CKD PATIENTS AND UNRAVELLING OF A POTENTIAL INFLAMMATION INDUCER

Background and Aims Chronic Kidney Disease (CKD) is causing serious cardiovascular diseases. Creatinine quantification and eGFR estimation are suboptimal approaches for the diagnosis of CKD, especially at early stages. Therefore, there is a strong need for identification of mediators for CKD diagnosis and prediction of disease progression. In this study we follow a cohort of renal healthy patients (controls) and CKD patients (cases) for two years, defining three time points (baseline, after 12 months and after 24 months), with the aim of identifying and characterizing mediators of disease which could be an indication for the development and progression of CKD and its outcome. Method By the employment of liquid chromatography-mass spectrometry (LC-MS) we analyzed the plasma samples from the patients and identified the mediators1 : lysine (K), an angio-associated migratory cell protein (AAMP) peptide and an amiloride-sensitive oxidase (AOC1) peptide, which were consistently and differentially expressed in cases and controls at all time points. Correlation analyses between the mediators and clinical markers were performed using the software R-Studio (RStudio Team (2020). RStudio: Integrated Development for R. RStudio, PBC, Boston, MA URL http://www.rstudio.com/). The AAMP peptide was subsequently tested in a fibroblasts cells culture to investigate whether it was an inflammation inducer, its action was investigated at four different concentrations (0.1nM, 1nM, 100nM, 1000nM). Cells were stimulated for 48h and relative expression of two inflammation markers (CCL2 and IL6) was measured through PCR. Results Correlation analyses revealed that the AAMP peptide showed from modest to strong relations with clinical markers such as creatinine, hemoglobin, blood urea nitrogen, homocysteine, fibrinogen and parathyroid hormone. Results showed that the peptide after 48h of stimulation did not cause an increase in the expression of gene CCL2 at any concentration, but caused a strong increase of gene IL6 (interleukin-6), a cytokine promoting inflammation and B cells maturation. Conclusion In conclusion, angio-associated migratory cell peptide, might be involved in CKD by inducing inflammation and driving the development of cardiovascular consequences such as atherosclerosis. Acknowledgments This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 764474.

The nucleus acts as a ruler tailoring cell responses to spatial constraints

The microscopic environment inside a metazoan organism is highly crowded. Whether individual cells can tailor their behavior to the limited space remains unclear. In this study, we found that cells measure the degree of spatial confinement by using their largest and stiffest organelle, the nucleus. Cell confinement below a resting nucleus size deforms the nucleus, which expands and stretches its envelope. This activates signaling to the actomyosin cortex via nuclear envelope stretch-sensitive proteins, up-regulating cell contractility. We established that the tailored contractile response constitutes a nuclear ruler–based signaling pathway involved in migratory cell behaviors. Cells rely on the nuclear ruler to modulate the motive force that enables their passage through restrictive pores in complex three-dimensional environments, a process relevant to cancer cell invasion, immune responses, and embryonic development.

The heart of the neural crest: cardiac neural crest cells in development and regeneration

ABSTRACTCardiac neural crest cells (cNCCs) are a migratory cell population that stem from the cranial portion of the neural tube. They undergo epithelial-to-mesenchymal transition and migrate through the developing embryo to give rise to portions of the outflow tract, the valves and the arteries of the heart. Recent lineage-tracing experiments in chick and zebrafish embryos have shown that cNCCs can also give rise to mature cardiomyocytes. These cNCC-derived cardiomyocytes appear to be required for the successful repair and regeneration of injured zebrafish hearts. In addition, recent work examining the response to cardiac injury in the mammalian heart has suggested that cNCC-derived cardiomyocytes are involved in the repair/regeneration mechanism. However, the molecular signature of the adult cardiomyocytes involved in this repair is unclear. In this Review, we examine the origin, migration and fates of cNCCs. We also review the contribution of cNCCs to mature cardiomyocytes in fish, chick and mice, as well as their role in the regeneration of the adult heart.

The nucleus acts as a ruler tailoring cell responses to spatial constraints

The microscopic environment inside a metazoan organism is highly crowded. Whether individual cells can tailor their behavior to the limited space remains unclear. Here, we found that cells measure the degree of spatial confinement using their largest and stiffest organelle, the nucleus. Cell confinement below a resting nucleus size deforms the nucleus, which expands and stretches its envelope. This activates signaling to the actomyosin cortex via nuclear envelope stretch-sensitive proteins, upregulating cell contractility. We established that the tailored contractile response constitutes a nuclear ruler-based signaling pathway involved in migratory cell behaviors. Cells rely on the nuclear ruler to modulate the motive force enabling their passage through restrictive pores in complex three-dimensional (3D) environments, a process relevant to cancer cell invasion, immune responses and embryonic development.One Sentence SummaryNuclear envelope expansion above a threshold triggers a contractile cell response and thus acts as a ruler for the degree of cell deformation.