The miR-27a/FOXJ3 Axis Dysregulates Mitochondrial Homeostasis in Colorectal Cancer Cells

miR-27a plays a driver role in rewiring tumor cell metabolism. We searched for new miR-27a targets that could affect mitochondria and identified FOXJ3, an apical factor of mitochondrial biogenesis. We analyzed FOXJ3 levels in an in vitro cell model system that was genetically modified for miR-27a expression and validated it as an miR-27a target. We showed that the miR-27a/FOXJ3 axis down-modulates mitochondrial biogenesis and other key members of the pathway, implying multiple levels of control. As assessed by specific markers, the miR-27a/FOXJ3 axis also dysregulates mitochondrial dynamics, resulting in fewer, short, and punctate organelles. Consistently, in high miR-27a-/low FOXJ3-expressing cells, mitochondria are functionally characterized by lower superoxide production, respiration capacity, and membrane potential, as evaluated by OCR assays and confocal microscopy. The analysis of a mouse xenograft model confirmed FOXJ3 as a target and suggested that the miR-27a/FOXJ3 axis affects mitochondrial abundance in vivo. A survey of the TCGA-COADREAD dataset supported the inverse relationship of FOXJ3 with miR-27a and reinforced cellular component organization or biogenesis as the most affected pathway. The miR-27a/FOXJ3 axis acts as a central hub in regulating mitochondrial homeostasis. Its discovery paves the way for new therapeutic strategies aimed at restraining tumor growth by targeting mitochondrial activities.

Incorporation of unnatural amino acid for the tagging of cannabinoid receptors 1 and 2 reveals receptor roles in regulating cAMP levels

The role of CB1/CB2 co-expression in cell signaling remains elusive. We established a simplified mammalian cell model system in which expression of CB1 or CB2 can be easily monitored under a confocal microscope. For this, we applied amber codon suppression in live cells to incorporate a single trans-cyclooctene (TCO) bearing amino acid in one of the extracellular loops of CB1 or CB2, followed by fluorescent labeling via click chemistry. We employed genetically encoded biosensors to measure the roles of CB1 and/or CB2 in regulating intracellular calcium ([Ca2+]i) and cAMP ([cAMP]i) levels. We show that the agonist-mediated activation of tagged-CB1 or -CB2 can transiently elevate [Ca2+]i levels. However, when the two receptors were co-expressed in the same cell, CB2 no longer signaled through calcium although CB1-mediated transient elevation of [Ca2+]i levels was unaffected. Because of the existence of crosstalk between calcium and cAMP signaling, we measured the effects of CB1 and/or CB2 in regulating adenylate cyclase activity. We found that the expression of CB1 increased forskolin-induced [cAMP]i levels compared to non-transfected cells. Conversely, CB2 expression decreased stimulated [cAMP]i levels under the same conditions. Finally, co-expressed CB1 and CB2 receptors showed additive yet opposing effects on stimulated [cAMP]i levels. These observations suggest that co-expressed CB1/CB2 act locally as a pair in regulating cell excitability by modulating stimulated [cAMP]i levels.

Using 3D epigenomic maps of primary olfactory neuronal cells from living individuals to understand gene regulation

As part of PsychENCODE, we developed a three-dimensional (3D) epigenomic map of primary cultured neuronal cells derived from olfactory neuroepithelium (CNON). We mapped topologically associating domains and high-resolution chromatin interactions using Hi-C and identified regulatory elements using chromatin immunoprecipitation and nucleosome positioning assays. Using epigenomic datasets from biopsies of 63 living individuals, we found that epigenetic marks at distal regulatory elements are more variable than marks at proximal regulatory elements. By integrating genotype and metadata, we identified enhancers that have different levels corresponding to differences in genetic variation, gender, smoking, and schizophrenia. Motif searches revealed that many CNON enhancers are bound by neuronal-related transcription factors. Last, we combined 3D epigenomic maps and gene expression profiles to predict enhancer-target gene interactions on a genome-wide scale. This study not only provides a framework for understanding individual epigenetic variation using a primary cell model system but also contributes valuable data resources for epigenomic studies of neuronal epithelium.

Inducible alpha-synuclein overexpression affects human Neural Stem Cells behavior

Converging evidence suggest that levels of alpha-Synuclein (aSyn) expression play a critical role in Parkinson’s disease (PD). Several mutations of the SNCA gene, encoding for aSyn have been associated to either the familial or the sporadic forms of PD. Nonetheless, the mechanism underlying wild type aSyn-mediated neurotoxicity in neuronal cells as well as its specific driving role in PD pathogenesis has yet to be fully clarified. In this view, the development of proper in vitro cellular systems is a crucial step.Here we present a novel human Tet-on hNSC cell line, in which aSyn timing and level of expression can be tightly experimentally tuned. Induction of aSyn in self-renewing hNSCs leads to progressive formation of aSyn aggregates and impairs their proliferation and cell survival. Furthermore, aSyn induction during the neuronal differentiation process results in impaired neurogenic potential due to enhanced refractoriness to exit self-renewal and to increase of gliogenic vs neurogenic competence. Finally, acute aSyn induction in hNSC-derived dopaminergic neuronal cultures results in cell toxicity.This novel conditional in vitro cell model system may be a valuable tool for dissecting of aSyn pathogenic effects in hNSCs and neurons and in developing new potential therapeutic strategies.

A functional drug re-purposing screening identifies carfilzomib as a drug preventing 17β-estradiol: ERα signaling and cell proliferation in breast cancer cells

Most cases of breast cancer (BC) are estrogen receptor α-positive (ERα+) at diagnosis. The presence of ERα drives the therapeutic approach for this disease, which often consists of endocrine therapy (ET). 4OH-Tamoxifen and faslodex (i.e., fulvestrant - ICI182,780) are two ETs that render tumor cells insensitive to 17β-estradiol (E2)-dependent proliferative stimuli and prevent BC progression. However, ET has limitations and serious failures in different tissues and organs. Thus, there is an urgent need to identify novel drugs to fight BC in the clinic. Re-positioning of old drugs for new clinical purposes is an attractive alternative for drug discovery. For this analysis, we focused on the modulation of intracellular ERα levels in BC cells as target for the screening of about 900 Food and Drug Administration (FDA) approved compounds that would hinder E2:ERα signaling and inhibit BC cell proliferation. We found that carfilzomib induces ERα degradation and prevents E2 signaling and cell proliferation in two ERα+ BC cell lines. Remarkably, the analysis of carfilzomib effects on a cell model system with an acquired resistance to 4OH-tamoxifen revealed that this drug has an antiproliferative effect superior to faslodex in BC cells. Therefore, our results identify carfilzomib as a drug preventing E2:ERα signaling and cell proliferation in BC cells and suggest its possible re-position for the treatment of ERα+ BC as well as for those diseases that have acquired resistance to 4OH-tamoxifen.