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1.
Nature ; 590(7846): 504-508, 2021 02.
Article in English | MEDLINE | ID: mdl-33536620

ABSTRACT

Amplification of chromosomal region 8p11-12 is a common genetic alteration that has been implicated in the aetiology of lung squamous cell carcinoma (LUSC)1-3. The FGFR1 gene is the main candidate driver of tumorigenesis within this region4. However, clinical trials evaluating FGFR1 inhibition as a targeted therapy have been unsuccessful5. Here we identify the histone H3 lysine 36 (H3K36) methyltransferase NSD3, the gene for which is located in the 8p11-12 amplicon, as a key regulator of LUSC tumorigenesis. In contrast to other 8p11-12 candidate LUSC drivers, increased expression of NSD3 correlated strongly with its gene amplification. Ablation of NSD3, but not of FGFR1, attenuated tumour growth and extended survival in a mouse model of LUSC. We identify an LUSC-associated variant NSD3(T1232A) that shows increased catalytic activity for dimethylation of H3K36 (H3K36me2) in vitro and in vivo. Structural dynamic analyses revealed that the T1232A substitution elicited localized mobility changes throughout the catalytic domain of NSD3 to relieve auto-inhibition and to increase accessibility of the H3 substrate. Expression of NSD3(T1232A) in vivo accelerated tumorigenesis and decreased overall survival in mouse models of LUSC. Pathological generation of H3K36me2 by NSD3(T1232A) reprograms the chromatin landscape to promote oncogenic gene expression signatures. Furthermore, NSD3, in a manner dependent on its catalytic activity, promoted transformation in human tracheobronchial cells and growth of xenografted human LUSC cell lines with amplification of 8p11-12. Depletion of NSD3 in patient-derived xenografts from primary LUSCs containing NSD3 amplification or the NSD3(T1232A)-encoding variant attenuated neoplastic growth in mice. Finally, NSD3-regulated LUSC-derived xenografts were hypersensitive to bromodomain inhibition. Thus, our work identifies NSD3 as a principal 8p11-12 amplicon-associated oncogenic driver in LUSC, and suggests that NSD3-dependency renders LUSC therapeutically vulnerable to bromodomain inhibition.


Subject(s)
Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/pathology , Histone-Lysine N-Methyltransferase/metabolism , Histones/chemistry , Histones/metabolism , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Nuclear Proteins/metabolism , Animals , Biocatalysis , Carcinogenesis/genetics , Carcinoma, Squamous Cell/genetics , Female , Histone-Lysine N-Methyltransferase/deficiency , Histone-Lysine N-Methyltransferase/genetics , Humans , Lung Neoplasms/genetics , Male , Methylation , Mice , Models, Molecular , Mutation , Nuclear Proteins/deficiency , Nuclear Proteins/genetics , Receptor, Fibroblast Growth Factor, Type 1/deficiency , Receptor, Fibroblast Growth Factor, Type 1/genetics , Receptor, Fibroblast Growth Factor, Type 1/metabolism , Xenograft Model Antitumor Assays
2.
Gastroenterology ; 2024 Aug 02.
Article in English | MEDLINE | ID: mdl-39097198

ABSTRACT

BACKGROUND & AIMS: Metastases from gastric adenocarcinoma (GAC) lead to high morbidity and mortality. Developing innovative and effective therapies requires a comprehensive understanding of the tumor and immune biology of advanced GAC. Yet, collecting matched specimens from advanced, treatment-naïve patients with GAC poses a significant challenge, limiting the scope of current research, which has focused predominantly on localized tumors. This gap hinders deeper insight into the metastatic dynamics of GAC. METHODS: We performed in-depth single-cell transcriptome and immune profiling on 68 paired, treatment-naïve, primary metastatic tumors to delineate alterations in cancer cells and their tumor microenvironment during metastatic progression. To validate our observations, we conducted comprehensive functional studies both in vitro and in vivo, using cell lines and multiple patient-derived xenograft and novel mouse models of GAC. RESULTS: Liver and peritoneal metastases exhibited distinct properties in cancer cells and dynamics of tumor microenvironment phenotypes, supporting the notion that cancer cells and their local tumor microenvironments co-evolve at metastatic sites. Our study also revealed differential activation of cancer meta-programs across metastases. We observed evasion of cancer cell ferroptosis via GPX4 up-regulation during GAC progression. Conditional depletion of Gpx4 or pharmacologic inhibition of ferroptosis resistance significantly attenuated tumor growth and metastatic progression. In addition, ferroptosis-resensitizing treatments augmented the efficacy of chimeric antigen receptor T-cell therapy. CONCLUSIONS: This study represents the largest single-cell dataset of metastatic GACs to date. High-resolution mapping of the molecular and cellular dynamics of GAC metastasis has revealed a rationale for targeting ferroptosis defense in combination with chimeric antigen receptor T-cell therapy as a novel therapeutic strategy with potential immense clinical implications.

3.
Gut ; 72(9): 1651-1663, 2023 09.
Article in English | MEDLINE | ID: mdl-36918265

ABSTRACT

OBJECTIVE: Gastric cancer (GC) is a leading cause of cancer mortality, with ARID1A being the second most frequently mutated driver gene in GC. We sought to decipher ARID1A-specific GC regulatory networks and examine therapeutic vulnerabilities arising from ARID1A loss. DESIGN: Genomic profiling of GC patients including a Singapore cohort (>200 patients) was performed to derive mutational signatures of ARID1A inactivation across molecular subtypes. Single-cell transcriptomic profiles of ARID1A-mutated GCs were analysed to examine tumour microenvironmental changes arising from ARID1A loss. Genome-wide ARID1A binding and chromatin profiles (H3K27ac, H3K4me3, H3K4me1, ATAC-seq) were generated to identify gastric-specific epigenetic landscapes regulated by ARID1A. Distinct cancer hallmarks of ARID1A-mutated GCs were converged at the genomic, single-cell and epigenomic level, and targeted by pharmacological inhibition. RESULTS: We observed prevalent ARID1A inactivation across GC molecular subtypes, with distinct mutational signatures and linked to a NFKB-driven proinflammatory tumour microenvironment. ARID1A-depletion caused loss of H3K27ac activation signals at ARID1A-occupied distal enhancers, but unexpectedly gain of H3K27ac at ARID1A-occupied promoters in genes such as NFKB1 and NFKB2. Promoter activation in ARID1A-mutated GCs was associated with enhanced gene expression, increased BRD4 binding, and reduced HDAC1 and CTCF occupancy. Combined targeting of promoter activation and tumour inflammation via bromodomain and NFKB inhibitors confirmed therapeutic synergy specific to ARID1A-genomic status. CONCLUSION: Our results suggest a therapeutic strategy for ARID1A-mutated GCs targeting both tumour-intrinsic (BRD4-assocatiated promoter activation) and extrinsic (NFKB immunomodulation) cancer phenotypes.


Subject(s)
Stomach Neoplasms , Transcription Factors , Humans , Transcription Factors/genetics , Transcription Factors/metabolism , Stomach Neoplasms/genetics , Stomach Neoplasms/therapy , Stomach Neoplasms/pathology , Nuclear Proteins/genetics , Epigenomics , Mutation , Tumor Microenvironment/genetics , DNA-Binding Proteins/genetics , Cell Cycle Proteins/genetics
4.
J Biol Chem ; 293(50): 19317-19329, 2018 12 14.
Article in English | MEDLINE | ID: mdl-30337365

ABSTRACT

Human pluripotent stem cells hold great promise for improving regenerative medicine. However, a risk for tumor formation and difficulties in generating large amounts of subtype derivatives remain the major obstacles for clinical applications of stem cells. Here, we discovered that zinc finger E-box-binding homeobox 1 (ZEB1) is highly expressed upon differentiation of human embryonic stem cells (hESCs) into neuronal precursors. CRISPR/Cas9-mediated ZEB1 depletion did not impede neural fate commitment, but prevented hESC-derived neural precursors from differentiating into neurons, indicating that ZEB1 is required for neuronal differentiation. ZEB1 overexpression not only expedited neural differentiation and neuronal maturation, which ensured safer neural cell transplantation, but also facilitated the generation of excitatory cortical neurons, which were valuable for managing certain neurological disorders, such as Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS). Our study provides useful information on how human neural cells are generated, which may help in forming strategies for developing and improving replacement therapies for treating patients with neurological diseases.


Subject(s)
Cell Differentiation , Human Embryonic Stem Cells/cytology , Zinc Finger E-box-Binding Homeobox 1/metabolism , Animals , Brain/cytology , Gene Expression Regulation , Gene Knockout Techniques , Humans , Mice , Neurons/cytology , Zinc Finger E-box-Binding Homeobox 1/deficiency , Zinc Finger E-box-Binding Homeobox 1/genetics
5.
J Biol Chem ; 292(31): 12959-12970, 2017 08 04.
Article in English | MEDLINE | ID: mdl-28500132

ABSTRACT

The zinc finger E-box-binding transcription factor Zeb1 plays a pivotal role in the epithelial-mesenchymal transition. Numerous studies have focused on the molecular mechanisms by which Zeb1 contributes to this process. However, the functions of Zeb1 beyond the epithelial-mesenchymal transition remain largely elusive. Using a transdifferentiation system to convert mouse embryonic fibroblasts (MEFs) into functional neurons via the neuronal transcription factors achaete-scute family bHLH (basic helix-loop-helix) transcription factor1 (Ascl1), POU class 3 homeobox 2 (POU3F2/Brn2), and neurogenin 2 (Neurog2, Ngn2) (ABN), we found that Zeb1 was up-regulated during the early stages of transdifferentiation. Knocking down Zeb1 dramatically attenuated the transdifferentiation efficiency, whereas Zeb1 overexpression obviously increased the efficiency of transdifferentiation from MEFs to neurons. Interestingly, Zeb1 improved the transdifferentiation efficiency induced by even a single transcription factor (e.g. Asc1 or Ngn2). Zeb1 also rapidly promoted the maturation of induced neuron cells to functional neurons and improved the formation of neuronal patterns and electrophysiological characteristics. Induced neuron cells could form functional synapse in vivo after transplantation. Genome-wide RNA arrays showed that Zeb1 overexpression up-regulated the expression of neuron-specific genes and down-regulated the expression of epithelial-specific genes during conversion. Taken together, our results reveal a new role for Zeb1 in the transdifferentiation of MEFs into neurons.


Subject(s)
Cell Transdifferentiation , Fibroblasts/metabolism , Gene Expression Regulation, Developmental , Nerve Tissue Proteins/metabolism , Neurogenesis , Neurons/metabolism , Zinc Finger E-box-Binding Homeobox 1/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cells, Cultured , Embryo, Mammalian/cytology , Fibroblasts/cytology , Gene Expression Profiling , Germ-Free Life , Hippocampus , Mice, Inbred C57BL , Mice, Inbred ICR , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/genetics , Neurons/cytology , Neurons/transplantation , POU Domain Factors/genetics , POU Domain Factors/metabolism , RNA Interference , Recombinant Proteins/metabolism , Zinc Finger E-box-Binding Homeobox 1/antagonists & inhibitors , Zinc Finger E-box-Binding Homeobox 1/genetics
6.
Reproduction ; 2016 Oct 14.
Article in English | MEDLINE | ID: mdl-27742864

ABSTRACT

The syncytiotrophoblast (STB) plays a key role in maintaining the function of the placenta during human pregnancy. However, the molecular network that orchestrates STB development remains elusive. The aim of this study was to obtain broad and deep insight into human STB formation via transcriptomics. We adopted RNA sequencing (RNA-Seq) to investigate genes and isoforms involved in forskolin (FSK)-induced fusion of BeWo cells. BeWo cells were treated with 50 µM FSK or dimethylsulfoxide (DMSO) as a vehicle control for 24 and 48 h, and the mRNAs at 0, 24 and 48 h was sequenced. We detected 28,633 expressed genes and identified 1,902 differentially expressed genes (DEGs) after FSK treatment for 24 and 48 h. Among the 1,902 DEGs, 461 were increased and 395 were decreased at 24 h, while 879 were up-regulated and 763 were down-regulated at 48 h. When the 856 DEGs identified at 24 h were traced individually at 48 h, they separated into 6 dynamic patterns via a K-means algorithm, and most were enriched in down-even and up-even patterns. Moreover, the Gene Ontology (GO) terms syncytium formation, cell junction assembly, cell fate commitment, calcium ion transport, regulation of epithelial cell differentiation and cell morphogenesis involved in differentiation were clustered, and the MAPK pathway was most significantly regulated. Analyses of alternative splicing isoforms detected 123,200 isoforms, of which 1,376 were differentially expressed. The present deep analysis of the RNA-Seq data of BeWo cell fusion provides important clues for understanding the mechanisms underlying human STB formation.

7.
Reproduction ; 149(4): 339-46, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25614620

ABSTRACT

The placenta has numerous functions, such as transporting oxygen and nutrients and building the immune tolerance of the fetus. Cell fusion is an essential process for placental development and maturation. In human placental development, mononucleated cytotrophoblast (CTB) cells can fuse to form a multinucleated syncytiotrophoblast (STB), which is the outermost layer of the placenta. Nephrin is a transmembrane protein that belongs to the Ig superfamily. Previous studies have shown that nephrin contributes to the fusion of myoblasts into myotubes in zebrafish and mice, presenting a functional conservation with its Drosophila ortholog sticks and stones. However, whether nephrin is involved in trophoblast syncytialization remains unclear. In this study, we report that nephrin was localized predominantly in the CTB cells and STB of human placenta villi from first trimester to term pregnancy. Using a spontaneous fusion model of primary CTB cells, the expression of nephrin was found to be increased during trophoblast cell fusion. Moreover, the spontaneous syncytialization and the expression of syncytin 2, connexin 43, and human chorionic gonadotropin beta were significantly inhibited by nephrin-specific siRNAs. The above results demonstrate that nephrin plays an important role in trophoblast syncytialization.


Subject(s)
Cell Differentiation , Giant Cells/cytology , Membrane Proteins/metabolism , Placenta/cytology , Trophoblasts/cytology , Animals , Blotting, Western , Cell Fusion , Cells, Cultured , Chorionic Gonadotropin, beta Subunit, Human/genetics , Chorionic Gonadotropin, beta Subunit, Human/metabolism , Connexin 43/genetics , Connexin 43/metabolism , Female , Giant Cells/metabolism , Humans , Immunoenzyme Techniques , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , Mice , Placenta/metabolism , Pregnancy , Pregnancy Proteins/genetics , Pregnancy Proteins/metabolism , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Trophoblasts/metabolism
8.
Biol Reprod ; 90(6): 117, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24740598

ABSTRACT

Human trophoblast syncytialization, a process of cell-cell fusion, is one of the most important yet least understood events during placental development. Investigating the fusion process in a placenta in vivo is very challenging given the complexity of this process. Application of primary cultured cytotrophoblast cells isolated from term placentas and BeWo cells derived from human choriocarcinoma formulates a biphasic strategy to achieve the mechanism of trophoblast cell fusion, as the former can spontaneously fuse to form the multinucleated syncytium and the latter is capable of fusing under the treatment of forskolin (FSK). Live-cell imaging is a powerful tool that is widely used to investigate many physiological or pathological processes in various animal models or humans; however, to our knowledge, the mechanism of trophoblast cell fusion has not been reported using a live- cell imaging manner. In this study, a live-cell imaging system was used to delineate the fusion process of primary term cytotrophoblast cells and BeWo cells. By using live staining with Hoechst 33342 or cytoplasmic dyes or by stably transfecting enhanced green fluorescent protein (EGFP) and DsRed2-Nuc reporter plasmids, we observed finger-like protrusions on the cell membranes of fusion partners before fusion and the exchange of cytoplasmic contents during fusion. In summary, this study provides the first video recording of the process of trophoblast syncytialization. Furthermore, the various live-cell imaging systems used in this study will help to yield molecular insights into the syncytialization process during placental development.


Subject(s)
Membrane Microdomains/physiology , Microscopy, Fluorescence/methods , Microscopy, Video/methods , Placentation/physiology , Trophoblasts/cytology , Adjuvants, Immunologic/pharmacology , Benzimidazoles/pharmacology , Cell Fusion/methods , Cell Line, Tumor , Choriocarcinoma/pathology , Colforsin/pharmacology , Cytoplasm/physiology , Female , Fluorescent Dyes/pharmacology , Humans , Pregnancy , Primary Cell Culture , Trophoblasts/drug effects , Uterine Neoplasms/pathology
9.
Reproduction ; 148(4): 343-52, 2014 Oct.
Article in English | MEDLINE | ID: mdl-24989904

ABSTRACT

Placenta-specific protein 1 (PLAC1), a placenta-specific gene, is known to be involved in the development of placenta in both humans and mice. However, the precise role of PLAC1 in placental trophoblast function remains unclear. In this study, the localization of PLAC1 in human placental tissues and its physiological significance in trophoblast invasion and migration are investigated by technical studies including real-time RT-PCR, in situ hybridization, immunohistochemistry, and functional studies by utilizing cell invasion and migration assays in the trophoblast cell line HTR8/SVneo as well as the primary inducing extravillous trophoblasts (EVTs). The results show that PLAC1 is mainly detected in the trophoblast columns and syncytiotrophoblast of the first-trimester human placental villi, as well as in the EVTs that invade into the maternal decidua. Knockdown of PLAC1 by RNA interference significantly suppresses the invasion and migration of HTR8/SVneo cells and shortens the distance of the outgrowth of the induced EVTs from the cytotrophoblast column of the explants. All the above data suggests that PLAC1 plays an important role in human placental trophoblast invasion and migration.


Subject(s)
Cell Movement , Pregnancy Proteins/metabolism , Trophoblasts/metabolism , Cell Line , Female , Gene Expression Regulation, Developmental , Gestational Age , Humans , Pregnancy , Pregnancy Proteins/genetics , Pregnancy Trimesters , RNA Interference , RNA, Messenger/metabolism , Signal Transduction , Tissue Culture Techniques , Transfection
10.
Sci Rep ; 14(1): 8510, 2024 04 12.
Article in English | MEDLINE | ID: mdl-38609433

ABSTRACT

Pancreatic neuroendocrine tumors (PanNETs) are a heterogeneous group of tumors that exhibit an unpredictable and broad spectrum of clinical presentations and biological aggressiveness. Surgical resection is still the only curative therapeutic option for localized PanNET, but the majority of patients are diagnosed at an advanced and metastatic stage with limited therapeutic options. Key factors limiting the development of new therapeutics are the extensive heterogeneity of PanNETs and the lack of appropriate clinically relevant models. In that context, genomic sequencing of human PanNETs revealed recurrent mutations and structural alterations in several tumor suppressors. Here, we demonstrated that combined loss of MEN1, ATRX, and PTEN, tumor suppressors commonly mutated in human PanNETs, triggers the development of high-grade pancreatic neuroendocrine tumors in mice. Histopathological evaluation and gene expression analyses of the developed tumors confirm the presence of PanNET hallmarks and significant overlap in gene expression patterns found in human disease. Thus, we postulate that the presented novel genetically defined mouse model is the first clinically relevant immunocompetent high-grade PanNET mouse model.


Subject(s)
Neuroendocrine Tumors , Pancreatic Neoplasms , Animals , Humans , Mice , Chromosome Mapping , Disease Models, Animal , Neuroendocrine Tumors/genetics , Pancreatic Neoplasms/genetics , PTEN Phosphohydrolase/genetics , X-linked Nuclear Protein/genetics
11.
Cell Discov ; 10(1): 12, 2024 Jan 31.
Article in English | MEDLINE | ID: mdl-38296970

ABSTRACT

Malignant forms of breast cancer refractory to existing therapies remain a major unmet health issue, primarily due to metastatic spread. A better understanding of the mechanisms at play will provide better insights for alternative treatments to prevent breast cancer cell dispersion. Here, we identify the lysine methyltransferase SMYD2 as a clinically actionable master regulator of breast cancer metastasis. While SMYD2 is overexpressed in aggressive breast cancers, we notice that it is not required for primary tumor growth. However, mammary-epithelium specific SMYD2 ablation increases mouse overall survival by blocking the primary tumor cell ability to metastasize. Mechanistically, we identify BCAR3 as a genuine physiological substrate of SMYD2 in breast cancer cells. BCAR3 monomethylated at lysine K334 (K334me1) is recognized by a novel methyl-binding domain present in FMNLs proteins. These actin cytoskeleton regulators are recruited at the cell edges by the SMYD2 methylation signaling and modulate lamellipodia properties. Breast cancer cells with impaired BCAR3 methylation lose migration and invasiveness capacity in vitro and are ineffective in promoting metastases in vivo. Remarkably, SMYD2 pharmacologic inhibition efficiently impairs the metastatic spread of breast cancer cells, PDX and aggressive mammary tumors from genetically engineered mice. This study provides a rationale for innovative therapeutic prevention of malignant breast cancer metastatic progression by targeting the SMYD2-BCAR3-FMNL axis.

12.
Nat Biomed Eng ; 7(9): 1170-1187, 2023 09.
Article in English | MEDLINE | ID: mdl-37652986

ABSTRACT

The design of chimeric antigen receptor (CAR) T cells would benefit from knowledge of the fate of the cells in vivo. This requires the permanent labelling of CAR T cell products and their pooling in the same microenvironment. Here, we report a cell-barcoding method for the multiplexed longitudinal profiling of cells in vivo using single-cell RNA sequencing (scRNA-seq). The method, which we named shielded-small-nucleotide-based scRNA-seq (SSN-seq), is compatible with both 3' and 5' single-cell profiling, and enables the recording of cell identity, from cell infusion to isolation, by leveraging the ubiquitous Pol III U6 promoters to robustly express small-RNA barcodes modified with direct-capture sequences. By using SSN-seq to track the dynamics of the states of CAR T cells in a tumour-rechallenge mouse model of leukaemia, we found that a combination of cytokines and small-molecule inhibitors that are used in the ex vivo manufacturing of CAR T cells promotes the in vivo expansion of persistent populations of CD4+ memory T cells. By facilitating the probing of cell-state dynamics in vivo, SSN-seq may aid the development of adoptive cell therapies.


Subject(s)
CD4-Positive T-Lymphocytes , Transcriptome , Humans , Animals , Mice , Cell- and Tissue-Based Therapy , Cytokines , Nucleotides
13.
JSLS ; 27(4)2023.
Article in English | MEDLINE | ID: mdl-38226337

ABSTRACT

Study Objective: To determine if a pre-operative morphine/bupivacaine spinal injection prior to laparoscopic hysterectomy reduced postoperative pain and resulted in less opioid consumption during the hospital stay. Methods: A retrospective cohort study (Canadian Task Force Classification II-2) was conducted at a single institution regional referral center (community hospital) in North Carolina. Three hundred nineteen patients met criteria for inclusion: 192 received spinal anesthesia and 127 did not. Baseline demographics were similar between the two groups. Median pain scores were significantly lower in the treatment than the control group on day of surgery (DOS) (2 vs. 6; P < 0.001) and postoperative day 1 (POD1) (2 vs. 4; P < 0.001). Results: Primary outcomes were pain scores on DOS and POD1 and inpatient opioid use. Pain scores were obtained using the 0 to 10 Numerical Rating Scale. Opioids were converted to oral morphine milliequivalents (OME). Median opioid use was also significantly lower in the treatment than the control group on DOS (0 vs. 15.00 OME; P < 0.001) and POD1 (0 vs. 7.5 OME; P < 0.001). Median length of stay between the groups was not significantly different. Conclusion: Pre-operative morphine spinal injection for laparoscopic hysterectomy led to significantly lower pain scores and inpatient opioid consumption. Pre-operative spinal anesthesia for benign laparoscopic hysterectomy appears helpful for enhancing the postoperative experience.


Subject(s)
Anesthesia, Spinal , Laparoscopy , Female , Humans , Analgesics, Opioid/therapeutic use , Retrospective Studies , Pain, Postoperative/drug therapy , Pain, Postoperative/prevention & control , Bupivacaine , Hysterectomy/methods , Laparoscopy/methods , Morphine Derivatives , Morphine
14.
Cell Discov ; 9(1): 30, 2023 Mar 17.
Article in English | MEDLINE | ID: mdl-36928215

ABSTRACT

The placenta is one of the most important yet least understood organs. Due to the limitations of conventional research approaches, we are still far from a comprehensive understanding of mouse placentation, especially regarding the differentiation of trophoblast lineages at the early developmental stage. To decipher cell compositions and developmental processes, we systematically profile the single-cell transcriptomes of trophoblast cells from extraembryonic tissues (embryonic day 7.5 (E7.5) and E8.5) and placentae (E9.5-E14.5) at one-day intervals. We identify distinct trophoblast cell types during mouse placentation, including unreported progenitor cells and intermediate precursor cells. An updated differentiation roadmap of mouse trophoblast lineages is presented following systematic transcriptome analyses. Based on transcriptomic regulatory network inference, we specify transcription factors responsible for the regulation of dynamic developmental processes during lineage diversification. We map lineage differentiation trajectories and find that sinusoid trophoblast giant cells arise from the subpopulation of ectoplacental cone cells. We provide a comprehensive single-cell data resource to shed light on future mechanistic studies of the gene regulatory networks governing hemochorial placentation.

15.
bioRxiv ; 2023 Sep 18.
Article in English | MEDLINE | ID: mdl-37790557

ABSTRACT

Malignant forms of breast cancer refractory to existing therapies remain a major unmet health issue, primarily due to metastatic spread. A better understanding of the mechanisms at play will provide better insights for alternative treatments to prevent breast cancer cells dispersion. Here, we identify the lysine methyltransferase SMYD2 as a clinically actionable master regulator of breast cancer metastasis. While SMYD2 is overexpressed in aggressive breast cancers, we notice that it is not required for primary tumor growth. However, mammary-epithelium specific SMYD2 ablation increases mouse overall survival by blocking the primary tumor cells ability to metastasize. Mechanistically, we identify BCAR3 as a genuine physiological substrate of SMYD2 in breast cancer cells. BCAR3 monomethylated at lysine K334 (K334me1) is recognized by a novel methyl-binding domain present in FMNLs proteins. These actin cytoskeleton regulators are recruited at the cell edges by the SMYD2 methylation signaling and modulates lamellipodia properties. Breast cancer cells with impaired BCAR3 methylation loose migration and invasiveness capacity in vitro and are ineffective in promoting metastases in vivo . Remarkably, SMYD2 pharmacologic inhibition efficiently impairs the metastatic spread of breast cancer cells, PDX and aggressive mammary tumors from genetically engineered mice. This study provides a rationale for innovative therapeutic prevention of malignant breast cancer metastatic progression by targeting the SMYD2-BCAR3-FMNL axis.

16.
J Mol Cell Biol ; 11(11): 967-978, 2019 12 23.
Article in English | MEDLINE | ID: mdl-31408157

ABSTRACT

Human trophoblast syncytialization is one of the most important yet least understood events during placental development. In this study, we found that detyrosinated α-tubulin (detyr-α-tub), which is negatively regulated by tubulin tyrosine ligase (TTL), was elevated during human placental cytotrophoblast fusion. Correspondingly, relatively high expression of TTL protein was observed in first-trimester human placental cytotrophoblast cells, but fusing trophoblast cells exhibited much lower levels of TTL. Notably, fusion of preeclamptic cytotrophoblast cells was compromised but could be partially rescued by knockdown of TTL levels. Mechanistically, chronic downregulation of TTL in trophoblast cells resulted in significantly elevated expression of detyr-α-tub. Restoration of detyr-α-tub thus contributed to the cell surface localization of the fusogenic protein Syncytin-2 and the gap junction protein Connexin 43 (Cx43), which in turn promoted successful fusion between trophoblast cells. Taken together, the results suggest that tubulin detyrosination plays an essential role in human trophoblast fusogenic protein aggregation and syncytialization. Insufficient tubulin detyrosination leads to defects in syncytialization and potentially to the onset of preeclampsia.


Subject(s)
Giant Cells/metabolism , Trophoblasts/metabolism , Tubulin/metabolism , Biomarkers , Female , Gene Expression , Gene Products, env/metabolism , Humans , Models, Biological , Placenta/metabolism , Pregnancy , Pregnancy Proteins/metabolism
19.
Cell Res ; 28(8): 819-832, 2018 08.
Article in English | MEDLINE | ID: mdl-30042384

ABSTRACT

The placenta is crucial for a successful pregnancy and the health of both the fetus and the pregnant woman. However, how the human trophoblast lineage is regulated, including the categorization of the placental cell subtypes is poorly understood. Here we performed single-cell RNA sequencing (RNA-seq) on sorted placental cells from first- and second-trimester human placentas. New subtypes of cells of the known cytotrophoblast cells (CTBs), extravillous trophoblast cells (EVTs), Hofbauer cells, and mesenchymal stromal cells were identified and cell-type-specific gene signatures were defined. Functionally, this study revealed many previously unknown functions of the human placenta. Notably, 102 polypeptide hormone genes were found to be expressed by various subtypes of placental cells, which suggests a complex and significant role of these hormones in regulating fetal growth and adaptations of maternal physiology to pregnancy. These results document human placental trophoblast differentiation at single-cell resolution and thus advance our understanding of human placentation during the early stage of pregnancy.


Subject(s)
Peptide Hormones/genetics , Placenta/cytology , Placentation/genetics , Trophoblasts/metabolism , Base Sequence , Cell Differentiation , Female , Gene Expression , Humans , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Pregnancy , Pregnancy Trimester, First/genetics , Pregnancy Trimester, First/metabolism , Pregnancy Trimester, Second/genetics , Pregnancy Trimester, Second/metabolism , Sequence Analysis, RNA/methods , Single-Cell Analysis/methods
20.
Cell Rep ; 21(5): 1150-1159, 2017 Oct 31.
Article in English | MEDLINE | ID: mdl-29091755

ABSTRACT

Many types of multinucleated cells (syncytia) generated by cell-cell fusion are post-mitotic, but it remains unclear how this state is maintained and why. Here, we utilized the fluorescent ubiquitination-based cell-cycle indicator (Fucci) reporter system to show that human placental trophoblast cells were all in the G0 phase before they fuse. Expression of the fusogenic protein (fusogen) Syncytin-2 was confined to G0 cells. Overexpression of Syncytin-2 in cycling cells overrode the cell-cycle restriction and enabled fusion of cells in the S/G2/M phases but resulted in the unstable syncytia retaining mitotic features. The Syncytin-2-induced syncytia were functionally compromised with respect to pathogen defense and hormone secretion. We found that, during trophoblast fusion, the cell-cycle inhibitor p21 interacted with the GCM1 transcription factor, and this complex bound to the promoter of Syncytin-2 and promoted its transcription. These findings demonstrate that G0-restricted Syncytin-2 expression is a prerequisite for development of functional post-mitotic syncytia.


Subject(s)
Giant Cells/metabolism , Placenta/metabolism , Pregnancy Proteins/metabolism , CRISPR-Cas Systems/genetics , Cadherins/metabolism , Cell Fusion , Cell Line , Colforsin/metabolism , Cyclin-Dependent Kinase Inhibitor p21/deficiency , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , DNA-Binding Proteins , Female , Giant Cells/cytology , Humans , Microscopy, Fluorescence , Mitosis , Nuclear Proteins/metabolism , Pregnancy , Pregnancy Proteins/genetics , Promoter Regions, Genetic , Resting Phase, Cell Cycle , Time-Lapse Imaging , Transcription Factors/metabolism , Transcription, Genetic , Trophoblasts/cytology , Trophoblasts/metabolism
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