The cargo protein MAP17 (PDZK1IP1) regulates the cancer stem cell pool activating the Notch pathway by abducting NUMB

Purpose: Cancer stem cells (CSC) are self-renewing tumor cells, with the ability to generate diverse differentiated tumor cell subpopulations. They differ from normal stem cells in the deregulation of the mechanisms that normally control stem cell physiology. CSCs are the origin of metastasis and highly resistant to therapy. Therefore, the understanding of the CSC origin and deregulated pathways is important for tumor control. Experimental Design: We have included experiments in vitro, in cell lines and tumors of different origins. We have used patient-derived xenografts (PDX) and public transcriptomic databases of human tumors. Results: MAP17 (PDZKIP1), a small cargo protein overexpressed in tumors, interacts with NUMB through the PDZ-binding domain activating the Notch pathway, leading to an increase in stem cell factors and cancer-initiating–like cells. Identical behavior was mimicked by inhibiting NUMB. Conversely, MAP17 downregulation in a tumor cell line constitutively expressing this gene led to Notch pathway inactivation and a marked reduction of stemness. In PDX models, MAP17 levels directly correlated with tumorsphere formation capability. Finally, in human colon, breast, or lung there is a strong correlation of MAP17 expression with a signature of Notch and stem cell genes. Conclusions: MAP17 overexpression activates Notch pathway by sequestering NUMB. High levels of MAP17 correlated with tumorsphere formation and Notch and Stem gene transcription. Its direct modification causes direct alteration of tumorsphere number and Notch and Stem pathway transcription. This defines a new mechanism of Notch pathway activation and Stem cell pool increase that may be active in a large percentage of tumors.Ministerio de Economía y Competitividad PI15/00045, CTS-1848Junta de Andalucía PI-00-96-2014, PI-0306-201

Ofd1, a human disease gene, regulates the length and distal structure of centrioles

SUMMARYCentrosomes and their component centrioles represent the principal microtubule organizing centers of animal cells. Here we show that the gene underlying Orofaciodigital Syndrome 1, Ofd1, is a component of the distal centriole that controls centriole length. In the absence of Ofd1, distal regions of centrioles, but not procentrioles, elongate abnormally. These long centrioles are structurally similar to normal centrioles, but contain destabilized microtubules with abnormal post-translational modifications. Ofd1 is also important for centriole distal appendage formation and centriolar recruitment of the intraflagellar transport protein Ift88. To model OFD1 Syndrome in embryonic stem cells, we replaced the Ofd1 gene with missense alleles from human OFD1 patients. Distinct disease-associated mutations cause different degrees of excessive or decreased centriole elongation, all of which are associated with diminished ciliogenesis. Our results indicate that Ofd1 acts at the distal centriole to build distal appendages, recruit Ift88, and stabilize centriolar microtubules at a defined length

Coexistence of Wolbachia with Buchnera aphidicola and a secondary symbiont in the aphid Cinara cedri.

Intracellular symbiosis is very common in the insect world. For the aphid Cinara cedri, we have identified by electron microscopy three symbiotic bacteria that can be characterized by their different sizes, morphologies, and electrodensities. PCR amplification and sequencing of the 16S ribosomal DNA (rDNA) genes showed that, in addition to harboring Buchnera aphidicola, the primary endosymbiont of aphids, C. cedri harbors a secondary symbiont (S symbiont) that was previously found to be associated with aphids (PASS, or R type) and an alpha-proteobacterium that belongs to the Wolbachia genus. Using in situ hybridization with specific bacterial probes designed for symbiont 16S rDNA sequences, we have shown that Wolbachia was represented by only a few minute bacteria surrounding the S symbionts. Moreover, the observed B. aphidicola and the S symbionts had similar sizes and were housed in separate specific bacterial cells, the bacteriocytes. Interestingly, in contrast to the case for all aphids examined thus far, the S symbionts were shown to occupy a similarly sized or even larger bacteriocyte space than B. aphidicola. These findings, along with the facts that C. cedri harbors the B. aphidicola strain with the smallest bacterial genome and that the S symbionts infect all Cinara spp. analyzed so far, suggest the possibility of bacterial replacement in these species

Oxidative stress and mitochondrial dysfunction in Kindler syndrome

This is an Open Access article distributed under the terms of the Creative Commons Attribution License.-- et al.[Background]: Kindler Syndrome (KS) is an autosomal recessive skin disorder characterized by skin blistering, photosensitivity, premature aging, and propensity to skin cancer. In spite of the knowledge underlying cause of this disease involving mutations of FERMT1 (fermitin family member 1), and efforts to characterize genotype-phenotype correlations, the clinical variability of this genodermatosis is still poorly understood. In addition, several pathognomonic features of KS, not related to skin fragility such as aging, inflammation and cancer predisposition have been strongly associated with oxidative stress. Alterations of the cellular redox status have not been previously studied in KS. Here we explored the role of oxidative stress in the pathogenesis of this rare cutaneous disease. [Methods]: Patient-derived keratinocytes and their respective controls were cultured and classified according to their different mutations by PCR and western blot, the oxidative stress biomarkers were analyzed by spectrophotometry and qPCR and additionally redox biosensors experiments were also performed. The mitochondrial structure and functionality were analyzed by confocal microscopy and electron microscopy.[Results]: Patient-derived keratinocytes showed altered levels of several oxidative stress biomarkers including MDA (malondialdehyde), GSSG/GSH ratio (oxidized and reduced glutathione) and GCL (gamma-glutamyl cysteine ligase) subunits. Electron microscopy analysis of both, KS skin biopsies and keratinocytes showed marked morphological mitochondrial abnormalities. Consistently, confocal microscopy studies of mitochondrial fluorescent probes confirmed the mitochondrial derangement. Imbalance of oxidative stress biomarkers together with abnormalities in the mitochondrial network and function are consistent with a pro-oxidant state. [Conclusions]: This is the first study to describe mitochondrial dysfunction and oxidative stress involvement in KS.FL was supported by grants from Instituto de Salud Carlos III (PI11/01225) and Comunidad de Madrid (S2010/BMD- 2359; SKINMODEL). MDR was supported by grants from the Science and Innovation Ministry of Spain SAF2010-16976), Comunidad de Madrid (S2010/BMD-2420; CELLCAM), GENEGRAFT - contract N° HEALTH-F2-2011-261392 and CIBERER ACCI 13-714/172.04. MG is supported in part by ERA-NET grant: E-Rare-2 (SpliceEB). EZ was in part supported by a fellowship from CIBERER and SAF2010-16976.Peer reviewe

Ependymoma associated protein Zfta is expressed in immature ependymal cells but is not essential for ependymal development in mice

The fusion protein of uncharacterised zinc finger translocation associated (ZFTA) and effector transcription factor of tumorigenic NF‑κB signalling, RELA (ZFTA‑RELA), is expressed in more than two‑thirds of supratentorial ependymoma (ST‑EPN‑RELA), but ZFTA¿s expression profile and functional analysis in multiciliated ependymal (E1) cells have not been examined. Here, we showed the mRNA expression of mouse Zfta peaks on embryonic day (E) 17.5 in the wholemount of the lateral walls of the lateral ventricle. Zfta was expressed in the nuclei of FoxJ1‑positive immature E1 (pre‑E1) cells in E18.5 mouse embryonic brain. Interestingly, the transcription factors promoting ciliogenesis (ciliary TFs) (e.g., multicilin) and ZFTA‑RELA upregulated luciferase activity using a 5′ upstream sequence of ZFTA in cultured cells. Zftatm1/tm1 knock‑in mice did not show developmental defects or abnormal fertility. In the Zftatm1/tm1 E1 cells, morphology, gene expression, ciliary beating frequency and ependymal flow were unaffected. These results suggest that Zfta is expressed in pre‑E1 cells, possibly under the control of ciliary TFs, but is not essential for ependymal development or flow. This study sheds light on the mechanism of the ZFTA‑RELA expression in the pathogenesis of ST‑EPNRELA: Ciliary TFs initiate ZFTA‑RELA expression in pre‑E1 cells, and ZFTA‑RELA enhances its own expression using positive feedback

Endoderm development requires centrioles to restrain p53-mediated apoptosis in the absence of ERK activity

Centrioles comprise the heart of centrosomes, microtubule-organizing centers. To study the function of centrioles in lung and gut development, we genetically disrupted centrioles throughout the mouse endoderm. Surprisingly, removing centrioles from the endoderm did not disrupt intestinal growth or development but blocked lung branching. In the lung, acentriolar SOX2-expressing airway epithelial cells apoptosed. Loss of centrioles activated p53, and removing p53 restored survival of SOX2-expressing cells, lung branching, and mouse viability. To investigate how endodermal p53 activation specifically killed acentriolar SOX2-expressing cells, we assessed ERK, aprosurvival cue.ERKwasactivethroughouttheintestine and inthedistal lung buds, correlating with tolerance to centriole loss. Pharmacologically inhibiting ERK activated apoptosis in acentriolar cells, revealing that ERK activity protects acentriolar cells from apoptosis. Therefore, centrioles are largely dispensable for endodermal growth and the spatial distribution of ERK activity in the endoderm shapes the developmental consequences of centriolar defects and p53 activation

The generation of oligodendroglial cells is preserved in the rostral migratory stream during aging.

The subventricular zone (SVZ) is the largest source of newly generated cells in the adult mammalian brain. SVZ-derived neuroblasts migrate via the rostral migratory stream (RMS) to the olfactory bulb (OB), where they differentiate into mature neurons. Additionally, a small proportion of SVZ-derived cells contribute to the generation of myelinating oligodendrocytes. The production of new cells in the SVZ decreases during aging, affecting the incorporation of new neurons into the OB. However, the age-related changes that occur across the RMS are not fully understood. In this study we evaluate how aging affects the cellular organization of migrating neuroblast chains, the proliferation, and the fate of the newly generated cells in the SVZ-OB system. By using electron microscopy and immunostaining, we found that the RMS path becomes discontinuous and its cytoarchitecture is disorganized in aged mice (24-month-old mice). Subsequently, OB neurogenesis was impaired in the aged brain while the production of oligodendrocytes was not compromised. These findings provide new insight into oligodendrocyte preservation throughout life. Further exploration of this matter could help the development of new strategies to prevent neurological disorders associated with senescence

Adult neurogenesis in the telencephalon of the lizard Podarcis liolepis

In adult lizards, new neurons are generated from neural stem cells in the ventricular zone of the lateral ventricles. These new neurons migrate and integrate into the main telencephalic subdivisions. In this work we have studied adult neurogenesis in the lizard Podarcis liolepis (formerly Podarcis hispanica) by administering [3H]-thymidine and bromodeoxyuridine as proliferation markers and euthanizing the animals at different survival times to determine the identity of progenitor cells and to study their lineage derivatives. After short survival times, only type B cells are labeled, suggesting that they are neural stem cells. Three days after administration, some type A cells are labeled, corresponding to recently formed neuroblasts. Type A cells migrate to their final destinations, where they differentiate into mature neurons and integrate into functional circuits. Our results after long survival periods suggest that, in addition to actively dividing type B cells, there is also a type B subpopulation with low proliferative activity. We also found that new neurons incorporated into the olfactory bulb are generated both in situ, in the walls of the anterior extension of the lateral ventricle of the olfactory bulbs, but also at more caudal levels, most likely in anterior levels of the sulcus ventralis/terminalis. These cells follow a tangential migration toward the olfactory bulbs where they integrate. We hypothesized that at least part of the newly generated neurons would undergo a specialization process over time. In support of this prediction, we found two neuronal populations in the cellular layer of the medial cortex, which we named type I and II neurons. At intermediate survival times (1 month) only type II neurons were labeled with [3H]-thymidine, while at longer survival times (3, 6, or 12 months) both type I and type II neurons were labeled. This study sheds light on the ultrastructural characteristics of the ventricular zone of P. liolepis as a neurogenic niche, and adds to our knowledge of the processes whereby newly generated neurons in the adult brain migrate and integrate into their final destinations

Numb-like (NumbL) downregulation increases tumorigenicity, cancer stem cell-like properties and resistance to chemotherapy

NumbL, or Numb-like, is a close homologue of Numb, and is part of an evolutionary conserved protein family implicated in some important cellular processes. Numb is a protein involved in cell development, in cell adhesion and migration, in asymmetric cell division, and in targeting proteins for endocytosis and ubiquitination. NumbL exhibits some overlapping functions with Numb, but its role in tumorigenesis is not fully known. Here we showed that the downregulation of NumbL alone is sufficient to increase NICD nuclear translocation and induce Notch pathway activation. Furthermore, NumbL downregulation increases epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC)-related gene transcripts and CSC-like phenotypes, including an increase in the CSC-like pool. These data suggest that NumbL can act independently as a tumor suppressor gene. Furthermore, an absence of NumbL induces chemoresistance in tumor cells. An analysis of human tumors indicates that NumbL is downregulated in a variable percentage of human tumors, with lower levels of this gene correlated with worse prognosis in colon, breast and lung tumors. Therefore, NumbL can act as an independent tumor suppressor inhibiting the Notch pathway and regulating the cancer stem cell pool.España, Ministerio de Economía y Competitividad PI12/00137, PI15/00045España, Consejería de Ciencia e Innovación CTS-6844España, Consejería de Ciencia e Innovación CTS-1848España, Junta de Andalucía, Consejería de Salud PI-0306-2012España, Junta de Andalucía, Consejería de Salud PI-0096-201

Spontaneous Cardiomyocyte Differentiation From Adipose Tissue Stroma Cells

Cardiomyocyte regeneration is limited in adult life. Thus, the identification of a putative source of cardiomyocyte progenitors is of great interest to provide a usable model in vitro and new perspective in regenerative therapy. As adipose tissues were recently demonstrated to contain pluripotent stem cells, the emergence of cardiomyocyte phenotype from adipose-derived cells was investigated. We demonstrated that rare beating cells with cardiomyocyte features could be identified after culture of adipose stroma cells without addition of 5-azacytidine. The cardiomyocyte phenotype was first identified by morphological observation, confirmed with expression of specific cardiac markers, immunocytochemistry staining, and ultrastructural analysis, revealing the presence of ventricle- and atrial-like cells. Electrophysiological studies performed on early culture revealed a pacemaker activity of the cells. Finally, functional studies showed that adrenergic agonist stimulated the beating rate whereas cholinergic agonist decreased it. Taken together, this study demonstrated that functional cardiomyocyte- like cells could be directly obtained from adipose tissue. According to the large amount of this tissue in adult mammal, it could represent a useful source of cardiomyocyte progenitors.Garcia Verdugo, Jose Manuel, [email protected]