1,721,053 research outputs found
Enhanced lineage reprogramming towards induced neural stem cells with re-engineered SOX17
No full textForced expression of transcription factors (TFs) can program cellular fate changes. The direct transdifferentiation of induced neural stem cells (iNSCs) from somatic cells with defined TFs has been reported in several studies. iNSCs have great potentials in regenerative medicine and as authentic models for age-associated neurological diseases. Compared to cells derived from induced pluripotent stem cells (iPSCs), iNSCs could be a shortcut for the generation of cell models, avoid oncogenic cell states and circumvent the rejuvenation confounding authentic models of age-associated diseases. However, low efficiency, slow speed, poor reproducibility and an unresolved mechanism of iNSC reprogramming impede their routine applications in research and for clinical studies.
In this study, to enhance and dissect iNSC generation, we aimed to identify artificially evolved and enhanced TFs (eTFs) to boost iNSC reprogramming based on a Brn4, Sox2, Klf4 and c-Myc (BSKM) cocktail. We identified mutant variants of the endoderm factor Sox17 that can effectively produce iNSCs, whereas wild-type Sox2 and Sox17 fail. An engineered eSox17 with three point mutations can drive efficient and rapid generation of iNSCs from fetal, adult and old mouse fibroblasts. These iNSCs maintain long-term self-renewal, express neural stem cell markers and are capable of differentiating into neurons, astrocytes and oligodendrocytes.
By scaling down the number of TFs in the reprogramming cocktail, we found exogenous Brn4 and c-Myc factors are dispensable. eSox17 and Klf4 are necessary and sufficient to generate iNSCs. This two-factor cocktail drives direct reprogramming to neural lineage without passing a pluripotent state, demonstrated by the lack of pluripotency gene activation using an Oct4-GFP reporter and Nanog-CreER lineage tracing system. In addition, metabolic assays revealed that Klf4 induces glycolysis essential for iNSC reprogramming.
To uncover the molecular mechanism underlying the unique ability of eSox17 to generate iNSCs, we investigated transcriptional dynamic and chromatin accessibility during neural reprogramming. Global gene expression and chromatin opening are similar at the early stage of reprogramming with different Sox factors.
Compared with Sox2 and Sox17, eSox17 differentially targets genomic loci with canonical Sox:Oct DNA elements and activates a small subset of genes to mediate cell fate change during neural reprogramming. Importantly, exogenous TF silencing is required to mature reprogramming cells into iNSCs at the late stage.
In summary, this study shows eSox17 enables robust reprogramming of somatic cells into iNSCs. The translation of these findings to human iNSC generation could lead to readily available, authentic and personalized cell models for neurodegenerative diseases that bypass the shortcomings associated with iPSC-based strategies. The application of eTFs to generate desired cell types could provide promising cell sources for research and regenerative medicine.published_or_final_versionBiomedical SciencesDoctoralDoctor of Philosoph
Molecular basis and cellular events of brachydactyly type A1 pathogenesis
No full textThe synovial joints connect articulating elements and provide mobility, while proper
embryonic joint development is crucial to ensure postnatal function. Interzones form as
joint progenitors appear, differentiate and cavitate to form joint components. Amongst,
cavitation is poorly understood but a defective process likely contributes to joint ablating
condition. Here, programmed cell death (PCD) is detected from early interzone till end of
cavitation in phalangeal joint and corresponds to specific joint developmental stage.
Inhibiting caspases result in poor cavitation and inability to maintain interzone molecular
signature, resulting in anlage fusion. Therefore, the controversial PCD is essential to initiate
or progress joint cavitation, forming the basis of dominant Indian hedgehog (Ihh)E95K
mutation-causing brachydactyly type A1 (BDA1) pathogenesis.
In human and homozygous mouse model, BDA1 is characterized as short middle phalanges
in digit II to V, with an absent distal phalangeal joint in digit V. Despite extended IhhE95K
range and attenuated signalling capacity are responsible for short anlage, the disease
mechanism for ablated joint remains elusive. Here, TUNEL quantification revealed BDA1
joints have significantly less PCD, with a more severe reduction in distal joints. Indeed, the
ablated joint developed a delayed interzone-like structure but no PCD event, suggesting
inability to cavitate. Single cell transcriptomics revealed a decrease in pro-apoptotic
pathway in BDA1 joint where B cell lymphoma (Bcl-2) family of intrinsic apoptosis regulators is tipped towards anti-apoptosis. Henceforth, when apoptosis is introduced specifically to
developing interzone of ex vivo BDA1 paw culture, the absent joint is partially rescued with
signs of cavitation.
The extended IhhE95K signalling field is likely the driving mechanism for the cellular changes.
Recent discoveries on growth arrest specific-1 (Gas1) and cell adhesion
associated/oncogene regulated (Cdo) Hh co-receptors demonstrated their pro-apoptotic
and Hh potentiating property. They express in developing interzone, muscle and periinterzone
cells. In Gas1 genetically overexpressed joint, anlage has extended Ihh range but
lower Ihh expression, leading to BDA1-like deranged growth plate with reduction in
hypertrophy and ossification. This molecular and phenotypic pattern is opposite of Cdo-/-
mutants. These suggest Gas1 and Cdo function in developing limb as long range Ihh
promoter to inhibit growth plate differentiation. Therefore, the 2-fold increase in Cdoexpressing
cells and strong Gas1 and Cdo staining in BDA1 joints likely further joint
chondrogenicity and enhance BDA1 growth plate phenotype.
Cdo-/- mutants demonstrated Cdo pro-apoptotic property in IhhE95K/+ interzones but opposite
in WT interzones. Meanwhile, Gas1 overexpression promotes PCD in developing joint. The
reciprocal expression nature between joint Gas1 and Cdo indicates the final Cdo/Gas1/Ihh
output in regulating PCD occurrence. The absence of Gas1 and Cdo expression in BDA1
ablated joint in conjunction with increased pro-survival Ihh contributed to insufficient PCD,
resulting in cavitation failure and an absent joint.published_or_final_versionBiomedical SciencesMasterMaster of Philosoph
Induction of cells with osteo-chondrogenic potential by transcription factor-mediated reprogramming process
No full textSkeletal system plays a crucial role in our life. Skeletal diseases and disorders unlike cancer, are not fatal, but affect the quality of our life. Cell-based therapeutic strategies to generate targeted desired cell types for repair or replacement of damaged skeletal tissues are ideal regenerative medicines. Because of the heterogeneous cell types generated from embryonic and mesenchymal stem cells, the ability of progenitor population to differentiate into a target cell type appear to be a better alternative for tissue regeneration. Osteo-chondroprogenitors uniquely co-expressing Sox9 and Runx2 with dual differentiation potential to become chondrocytes and osteoblasts is a progenitor cell which is suitable for cell based therapy of bone disease. Therefore, developing effective strategies to generate sufficient quantities of osteo-chondroprogenitors are essential.
Toward this, we took advantage of two lineage conversion approaches. The first strategy was to interrogate the ability of osteoblasts to be reprogrammed into induced pluripotent stem (iPS) cells and another one was to use defined transcription factors to induce chondrocyte lineage from skin fibroblasts. The selection of osteoblasts is based on the fact that it is originally derived from osteo-chondroprogenitor lineage and the stochastic events of iPS induction might revert osteoblasts first to their progenitor state before becoming pluripotent. The second approach is based on a previous report using three transcription factors (Sox9, Klf4 and c-Myc) to reprogramme skin fibroblasts into chondrocyte lineage. Our aim is to examine whether osteo-chondroprogenitors would be formed during the two reprogramming processes using Sox9-EGFP knock-in mice as a reporter.
We reasoned that osteoblasts can be reprogrammed into iPS cells by four Yamanaka’s factors with pluripotency as shown by their ability to form teratomas and contribute to chimeric embryos. However base on the limitation of selector marker of osteo-chondroprogenitor we still cannot capture this progenitor during iPS reprogramming. And because of the pluripotency potential, pluripotent reprogramming approach also brings high risk of teratoma formation. Therefore our second objective was performed to examine whether osteo-chondroprogenitors would be formed during lineage reprogramming. Transient appearance of Sox9-EGFP/Runx2+ve cells was observed in the intermediate stage of over 14 days of chondrocyte lineage induction from skin fibroblasts by Sox9, klf4 and c-Myc. Cells expressing Sox9-EGFP/Runx2+ve showed typical molecular markers of osteo-chondroprogenitors. In vitro and in vivo differentiation assays demonstrated that Sox9-EGFP/Runx2+ve cells can differentiate predominantly into osteoblasts and chondrocytes. Taken together our data indicate that cells with osteo-chondrogenic potential could be generated by defined transcription factors-mediated reprogramming processes.published_or_final_versionBiochemistryDoctoralDoctor of Philosoph
DLC1 exerts oncogenic role through association with FOXK1 to synergistically activate MMP9 expression in metastatic melanoma
No full textMelanoma is considered as the most aggressive form of skin cancer, accounting for 72% of skin cancer-related deaths globally. Over 50% of melanoma patients harbor the BRAF mutation, which leads to constitutively activated oncogenic signaling pathways, resulting in the neoplastic transformation of melanocytes into metastatic melanomas. Despite improved overall survival of patients with metastatic melanoma treated with BRAF inhibitors, the cancer can rapidly acquire resistance to the treatment that limits its long-term efficacy. Therefore, there is an urgent need to identify new factors in governing melanoma colonization as alternative therapeutic targets.
Deleted in liver cancer 1 (DLC1), a RhoGTPase-activating (RhoGAP) protein, has been characterized as a tumor suppressor, as its expression was frequently found to be downregulated in various cancer types, and restoration of its expression inhibited tumorigenic growth. DLC1 negatively regulates the activity of RHOA, RHOB, RHOC, and to a lesser extent CDC42 by promoting the hydrolysis of active GTP-bound state into the inactive GDP-bound form. Most of the studies focused on the inhibitory effects of DLC1 on RHOA activity, which is involved in cell proliferation and actin cytoskeletal reorganization for cell migration. A previous report demonstrated that DLC1 inhibited TGF--induced expression of parathyroid hormone-like hormone through suppression of RHO-ROCK signaling to prevent breast cancer bone metastasis. In contrast, studies in other cellular contexts showed that interaction of DLC1 with partner factors contributed to the anti-tumorigenic functions via a RhoGAP-independent mechanism, implying a context-dependent role of DLC1. Apart from its cytoplasmic role in regulating RHO signaling, DLC1 can be localized in the nucleus where it was shown to be less efficient in exerting tumor suppressor activity in hepatocellular carcinoma. Similarly, DLC1 was also found to be localized in both the cytoplasm and nuclei of metastatic melanomas but how this subcellular localization of DLC1 regulates melanoma progression is largely unknown.
I
n the current study, I found that DLC1 expression was upregulated in the majority of melanoma tissues where it was localized in both the nuclei and the cytoplasm. Functional assays showed that nuclear DLC1 promoted growth and invasion of melanoma cells in a RhoGAP-independent manner. By proteomic analysis, I identified Forkhead box transcription factor, FOXK1, was crucial for DLC1 nuclear translocation and retention to orchestrate oncogenic programs. RNA-sequencing identified Matrix metalloproteinase 9 (MMP9) was commonly downregulated in both FOXK1 and DLC1 knockdown cells. Mechanistically, DLC1 was essential for FOXK1 occupancy to the promoter of MMP9, and both DLC1 and FOXK1 synergistically activate MMP9 expression for melanoma invasion and metastasis. My results provide a new paradigm of the underlying mechanism by which a well-known tumor suppressor DLC1 functions in the nucleus as an oncogene in melanoma.published_or_final_versionBiomedical SciencesDoctoralDoctor of Philosoph
DEPDC1B promotes melanoma angiogenesis and metastasis through sequestration of ubiquitin ligase CDC16 to stabilize secreted SCUBE3
No full textAngiogenesis, the formation of new blood vessels from pre-existing vasculature, is pivotal to melanoma progression and metastasis that contributes to melanoma lethality. Triggered by various oncogenic factors, angiogenesis can be inhibited by targeting secreted molecules and their specific signaling transduction pathways, which have been clinically proven as effective therapeutic interventions with cancer metastasis. However, the single-targeted anti-vascular endothelial growth factor (VEGF) therapy has limited efficacy on melanoma and induces rapid resistance, suggesting the existence of other key drivers governing the blood vessel formation in melanoma. Therefore, identification of the mechanisms underlying melanoma angiogenesis for the development of novel druggable targets is in urgent need.
Here, I found that a SRY-related HMG-box 10 (SOX10)-regulated oncogenic DEP domain-containing protein 1B (DEPDC1B) is critical in melanoma growth and metastasis through promoting angiogenesis. Skin cutaneous melanoma (SKCM) patient samples revealed higher expression of DEPDC1B is associated with poor survival. Tissue microarray immunostaining demonstrates a positive correlation between DEPDC1B and SOX10 protein expression in different stages of melanoma. As a result, depletion of DEPDC1B leads to a significant reduction of melanoma cell proliferation, colony formation, invasion, xenograft tumorigenesis and lung metastasis. Conversely, overexpression (OE) of DEPDC1B promotes the corresponding phenotypes. In addition, the dual-luciferase reporter assay, chromatin immunoprecipitation (ChIP) assay and epistasis analysis validated DEPDC1B as a direct downstream target of SOX10 to partly mediate its oncogenic activity. Furthermore, I have observed extensive vascular distribution in mouse lung nodules induced by DEPDC1B OE and enhanced formation of human umbilical vein endothelial cell (HUVEC) tubular network upon treatment of DEPDC1B OE conditioned medium (CM), indicating that DEPDC1B is sufficient and required for promoting angiogenesis. Importantly, mass spectrometry identifies a secreted protein SCUBE3 in the CM and cycloheximide chase analysis suggested SCUBD3 can be stabilized by DEPDC1B. Functional studies showed that SCUBE3 exhibits oncogenic properties in melanoma and is pro-angiogenic both in vitro and in vivo.
Mechanistically, DEPDC1B regulates SCUBE3 protein stability through the competitive association with ubiquitin ligase CDC16 to inhibit the interaction of SCUBE3 with CDC16, thereby preventing SCUBE3 from undergoing degradation via the ubiquitin-proteasome pathway. Notably, expression of SOX10, DEPDC1B, and SCUBE3 are positively correlated with higher microvessel density in the advanced stage of metastatic melanomas in tissue microarray. In conclusion, I revealed a SOX10-DEPDC1B-SCUBE3 regulatory axis that promotes melanoma angiogenesis and metastasis, which suggests targeting secreted SCUBE3 can be a therapeutic strategy against metastatic melanoma.published_or_final_versionBiomedical SciencesDoctoralDoctor of Philosoph
IHH and binding partners regulating progression of joint formation
No full textFunctional synovial joints require proper joint development. Phalangeal joint forms by segmentation of cartilage template. This process starts with de-differentiation of cells at the future joint site (interzone), that progresses to cavitation. Indian hedgehog (IHH), an important morphogen in developmental patterning, is necessary for joint formation as mutations in this gene give rise to a rare congenital disease, Brachydactyly type A1 (BDA1) where joint formation is affected. A common molecular outcome of IHH mutations causing BDA1 is reduced binding affinity with its receptors (PTCH1) and regulators (HIP1), leading to reducing signaling capacity but increase in the signaling range resulting in more IHH in the developing interzone. However, the molecular and cellular impact for this increased concentration of IHH leading to abnormal joint formation in not known. Single cell transcriptomics analysis of WT and BDA1 developing mouse phalangeal joint interzones at E14.5 showed cellular apoptosis was affected, and identified two differentially expressed genes, Cdon and Gli3 to be associated with BDA1. Interestingly, Cdon, has been demonstrated to have dependence receptor function linked to apoptosis. Previously, we showed that apoptosis is required for phalangeal joint interzone cavitation. Indeed, inactivation of Cdon in mice and in a mouse model of BDA1 previously generated can modify apoptosis in phalangeal joint formation and progression to cavitation. Importantly, the ability for CDON to induce apoptosis is suppressed when IHH is in excess. Further preliminary findings of apoptosis from overexpression studies in ATDC5 cells and assessments using the developing chick neural tube as a model system, support the proposed the apoptosis relationship between IHH and CDON, and a potential interplay between CDON and GAS1 in regulating apoptosis. Thus, we proposed a model by which a precise level of IHH regulated apoptosis that is needed for interzone cavitation in phalangeal joint formation, and this is achieved through the interaction with CDON and/or GAS1. While further investigations are needed, our findings have provided further novel insights into the role of the hedgehog morphogenic gradient in regulating organogenesis in developmental process.published_or_final_versionBiomedical SciencesMasterMaster of Philosoph
A dual role for DLC1 in regulating avian cranial neural crest cell fate commitment and delamination
No full textIn response to neural crest inductive signals, cells in the open neural plate border
region are bestowed with competence to give rise to neural crest cells. Upon neural
tube closure, prospective neural crest cells undergo epithelial-mesenchymal
transition (EMT) to delaminate from the dorsal neuroepithelium, migrate and
differentiate into various cell types throughout the embryos. These sequential steps
of neural crest ontogeny are strictly governed by a gene regulatory network (GRN)
comprising of an array of transcription factors. However, the molecule mechanisms
leading to the emergence and segregation of definitive neural crest cells from other
lineages have remained elusive. Here, this study revealed that a RhoGTPase
activating (RhoGAP) protein, deleted in liver cancer 1 (DLC1) exhibits both
nuclear and cytoplasmic localization in the prospective avian cranial neural crest
cells. Intriguingly, forced expression and knockdown of DLC1 led to loss of neural
crest specifier genes, SOX9, SNAIL2 and FOXD3 expression and the expansion of
SOX2-positive neural progenitor domain into the neural crest forming territory while PAX7-positive neural plate border region remains unaltered, suggesting that
appropriate level of DLC1 expression in the nucleus is required for the segregation
of neural crest lineage through regulation of their specifier genes expression. This
is accomplished by an essential requirement of DLC1 to recruit PAX7 binding to
the cis-regulatory elements of NC specifier genes together with transcription
elongation factor B polypeptide 1 (TCEB1) for transcriptional regulation.
In addition, this study further showed that cytoplasmic DLC1 recruits a novel
partner, Serine-Threonine Kinase Receptor-Associated Protein (STRAP) which is
a WD-containing protein through its amino acid 69-322 region. The cooperation of
DLC1 and STRAP prevented SNAIL2 from degradation via inhibition of GSK3β
activity in an RHOA-independent manner, resulting in repression of its nuclear
target Cad6B for the proper onset of neural crest delamination. Together, these
findings unravel unprecedented subcellular roles of DLC1 in regulating both avian
cranial neural crest cell fate commitment and delamination, providing a new
paradigm for our understanding of the neural crest-GRN and also on future neural
crest reprograming studies.published_or_final_versionBiomedical SciencesDoctoralDoctor of Philosoph
The role of EphA4/ephrin-A1 signaling in enteric neural crest interaction
No full textEnteric neural crest cells (ENCCs) form the enteric ganglia and neuronal network during enteric nervous system (ENS) development. Ret and p75 are well-known ENCC markers that are important in ENCC survival and cell fate commitment. Apart from premature differentiation and reduction of progenitor cell pool, failure of ENCCs to completely colonize the gut during ENS development can contribute to aganglionosis in Hirschsprung’s disease (HSCR). Eph/ephrin is a family of cell surface receptor and ligand molecules that mediate forward and reverse signaling. Cranial and trunk neural crests express Ephs and ephrins that regulate cell migration through repulsion or attraction. Protein expression of Ephs and ephrins in mouse developing gut has been recently discovered. However, the role and mechanism of Eph/ephrin signaling in ENCC interaction and migration were not well-understood. Mutation in EFNA1, EPHA5 and EPHB2 were also reported in human HSCR patients, suggesting that Ephs and ephrins are required in ENS development. In this project, I aimed to elucidate the role of EphA4/ephrin-A1 signaling in ENCC interaction by analyzing ENCC RNA-seq datasets, studying protein interaction of wildtype and mutant ephrin-A1 with Ret and p75 and investigating the functional role of EphA4/ephrin-A1 signaling in vitro.
Bioinformatic analysis of Wnt1Cre ENCC bulk RNA-seq dataset and Sox10Cre ENCC single cell RNA-seq dataset of E12.5 embryonic guts showed that Eph and ephrin genes were expressed in subpopulations of ENCCs. From cell lineage plot, EphA4 expression was widespread in gliogenic, neurogenic and bi-potential populations of ENCCs. Ephrin-A1 was expressed in gliogenic and bi-potential populations with high expression level, but it was completely absent in the neurogenic population. Ephrin-A1 co-expressed with ENCC markers Ret and p75. Ephrin-A1 expression was particularly correlated with the expression of p75 visualized in correlation matrix.
GPI-linked Ephrin-A ligands recruit co-receptors to exert intracellular signals upon binding to EphA receptors. Co-receptors identified includes Ret and p75. Protein-protein interaction experiments were done to study the link between ephrin-A1, Ret and p75. Results showed that ephrin-A1 could interact with Ret and p75 in HEK293T cells. These protein interactions suggested that Ret and p75 could be involved in ENCC interaction and migration by partnering with ephrin-A1. Interactions between ephrin-A1-H135Q mimicking human HSCR patient mutation and Ret and p75 were not altered.
Furthermore, cell segregation assay suggested that EphA4/ephrin-A1 interaction mediated forward and reverse repulsive signaling. Ephrin-A1 mutation seemingly exaggerated the repulsion between ephrin-A1- and EphA4-expressing cells. Preliminary results showed that ephrin-A1 mutation could also alter the phosphorylation level of Ret, p75 and FAK. As FAK affects cell-cell interaction through integrin signaling, ephrin-A1 mutation could be causing defect in ENCC migration by dysregulating ENCC cell-cell interaction through FAK.
Taken together, I highlighted the novelty of Eph and ephrin expression in ENCCs and protein interaction of ephrin-A1 with ENCC markers, Ret and p75. My study also suggested the possible mechanism of EphA4/ephrin-A1 repulsive signaling in affecting ENCC interaction and migration.published_or_final_versionBiomedical SciencesMasterMaster of Philosoph
Functional characterization, clinical relevance and therapeutic implication of follistatin-like 1 (FSTL1) in hepatocellular carcinoma
No full textHepatocellular carcinoma (HCC) ranks the third most common cause of cancerrelated mortality in Hong Kong. Despite advances in diagnosis and treatment strategies, the prognosis of HCC remains dismal. Thus, novel therapeutic options are still urgently awaited. Over 80% of HCC cases are presented with a background of fibrosis and/or cirrhosis, a pathological state where chronic inflammation and hepatocellular regeneration in the presence of enriched stromal myofibroblasts are commonly observed. Throughout the past decade, much emphasis has been placed on the role of tumor microenvironment (TME) in cancer development. Numerous studies conducted have proved that cancer cells are regulated by signals generated from fibroblasts in the TME. Thus, it is logical to speculate that targeting signaling factors within the TME may shed light to novel therapeutic options. Follistatin-like 1 (FSTL1) has been widely reported as a pro-inflammatory mediator in different fibrosis-related and inflammatory diseases. Here, we found FSTL1 to be up-regulated during liver regeneration and HCC development in our partial hepatectomy and HCC mice models. Moreover, we found FSTL1 to either be absent or expressed at very low levels in human HCC cells, but preferentially secreted from the myofibroblasts of peri-tumor liver and HCC tissue. By immunofluorescence and Person correlation analysis performed in human HCC clinical samples, fibrosis/HCC-related mouse models and expression data of an online HCC cohort (TCGA), we demonstrated FSTL1 to co-localize and positively correlate with α-SMA, a marker of myofibroblasts, confirming the source of FSTL1 as liver myofibroblasts including activated fibroblasts in both tumor and peri-tumor compartments. Clinically, high FSTL1 in FAP+ fibroblasts was significantly correlated with more advanced pathological TNM staging in HCC patients. As a secretory protein, it shows potential prognostic value to be adopted as a novel serological biomarker marker for HCC screening and/or diagnosis. Subsequent in vitro functional analyses found FSTL1 to induce HCC cell proliferation, metastasis and sorafenib resistance; while FSTL1 attenuation by a FSTL1 specific neutralizing antibody (nAb) elicited the opposite effects. Such oncogenic proliferative and metastatic effects were also observed in HCC patient-derived organoid cultures. Further, FSTL1 was also found to enhance HCC cell tumor formation and metastasis in in vivo models; while the administration of FSTL1 nAb exhibited opposing effects, thus suggesting a therapeutic efficacy of neutralizing FSTL1. RNA-sequencing was subsequently performed in hope to elucidate the dysregulated downstream molecular mechanism mediated by FSTL1. By Gene Set Enrichment Analysis (GSEA), we found the myc pathway to be activated in FSTL1-treated HCC cells. Western Blot analyses subsequently demonstrated enhanced phosphorylation of two key upstream players of the myc pathway, namely TAK-1 and p38 MAPK, which further substantiated the GSEA results. To conclude, myofibroblast-secreted FSTL1 mediates HCC progression and metastasis via a deregulated TAK-1/ p38 MAPK/ c-myc signaling cascade and anti-FSTL1 nAb holds therapeutic potential for HCC patients. We believe that FSTL1 could potentially serve as both a novel diagnostic/prognostic biomarker and therapeutic target in HCC.published_or_final_versionBiomedical SciencesMasterMaster of Philosoph
Revealing determinants for SARS-CoV-2 spike-mediated syncytia formation
No full textMultinucleated syncytial pneumonocytes, induced by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), are commonly observed in the postmortem lung tissues of severe COVID-19 cases. The syncytia formation is primarily determined by the fusogenicity of the spike protein of SARS-CoV-2. Infected cells express spikes on their cell surface, which leads to cell-cell fusion with neighboring cells, forming syncytia. Syncytia formation potentially contributes to pathology by facilitating viral transmission, immune evasion, inflammatory response, and cytopathicity. Since its emergence, SARS-CoV-2 has been continuously evolving. Mutations in spike protein result in numerous variants with different degrees of infectivity and pathogenicity. Functionally annotating the syncytia-forming potential of spike variants could help identify variants that may cause severe pathological consequences and should be tracked in the future. Additionally, the in-depth investigation of the host factors crucial for syncytia formation may facilitate the development of novel therapeutic strategies.
In this study, we conducted deep mutational scanning on the cytoplasmic tail of the SARS-CoV-2 spike in the context of full-length spike protein under human cell environments. The K1255F mutation was identified and validated to enhance the spike’s fusogenicity and ACE2 binding capacity. Mechanistically, it was demonstrated that the K1255F substitution creates a diaromatic FF motif, which improves endoplasmic reticulum (ER) exit and thus increases spike’s availability at the cell surface for triggering fusion with neighboring cells and forming syncytia.
In addition, we developed a size-exclusion selection-based strategy and combined it with the genome-wide CRISPR knockout screen to systematically identify host factors crucial for SARS-CoV-2 spike-mediated syncytia formation. Apart from the known receptor ACE2, two key regulators of clathrin-mediated endocytosis (CME), AP2M1 and FCHO2, were identified as crucial factors for SARS-CoV-2 spike-mediated syncytia formation. It was validated that AP2M1 or FCHO2 knockout in receiver cells significantly reduced the syncytia formation mediated by both the D614G and Omicron spikes. Furthermore, the involvement of CME machinery in driving syncytia formation was confirmed by CHC knockdown and the treatment with CME inhibitors. Moreover, it was demonstrated that the treatment of CME-inhibiting drugs, Chlorpromazine and Fluvoxamine, significantly inhibits SARS-CoV-2 replication and reduces the presence of syncytium-like multinucleated cells in the lung tissues of hamsters infected with SARS-CoV-2.
Taken together, these findings demonstrate the crucial role of the CME machinery in driving SARS-CoV-2-induced syncytia formation, providing support for the repurposing of Chlorpromazine and Fluvoxamine to alleviate COVID-19 severity in patients.published_or_final_versionBiomedical SciencesDoctoralDoctor of Philosoph
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