ROS Promote Hypoxia-Induced Keratinocyte Epithelial-Mesenchymal Transition by Inducing SOX2 Expression and Subsequent Activation of Wnt/β-Catenin.
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ABSTRACT: We previously showed that wound-induced hypoxia is related to keratinocyte migration. The ability of keratinocytes within wound healing to undergo epithelial to mesenchymal transition (EMT) contributes significantly to the acquisition of migratory properties. However, the effect of hypoxia on keratinocyte EMT on wound healing and the potential mechanism are poorly documented. This study first demonstrated that reactive oxygen species (ROS) appear to be an essential signalling mediator in keratinocytes with increased EMT and migration subjected to hypoxic conditions. Next, we showed that the expression of sex-determining region Y-box 2 (SOX2), a stemness-associated molecule, is ROS-dependent under hypoxia and that SOX2 inhibition in keratinocytes dramatically prevented hypoxia-induced EMT and migration. In addition, β-catenin was found to be a potential molecular target of SOX2, and the activation of Wnt/β-catenin was required for hypoxia-induced EMT and migration. Using an in vitro skin culture model and an in vivo skin wound model, our study further reinforced the critical role of ROS in inducing EMT through SOX2 expression and subsequent activation of Wnt/β-catenin, allowing for rapid reepithelialization of the wound area. Taken together, our findings reveal a previously unknown mechanism by which hypoxia promotes wound healing by promoting reepithelialization through the production of ROS, inducing keratinocyte EMT and migration via the enhancement of SOX2 and activation of Wnt/β-catenin.
Project description:Progenitor cells in the central nervous system must leave the cell cycle to become neurons and glia, but the signals that coordinate this transition remain largely unknown. We previously found that Wnt signaling, acting through Sox2, promotes neural competence in the Xenopus retina by activating proneural gene expression. We now report that Wnt and Sox2 inhibit neural differentiation through Notch activation. Independently of Sox2, Wnt stimulates retinal progenitor proliferation and this, when combined with the block on differentiation, maintains retinal progenitor fates. Feedback inhibition by Sox2 on Wnt signaling and by the proneural transcription factors on Sox2 mean that each element of the core pathway activates the next element and inhibits the previous one, providing a directional network that ensures retinal cells make the transition from progenitors to neurons and glia.
Project description:BackgroundOsteosarcoma (OS) patients with lung metastasis have poor prognoses, and effective therapeutic strategies for delaying or inhibiting the spread of lung metastasis from the primary OS site are lacking. Hence, it is critical to elucidate the underlying mechanisms of OS metastasis and to identify additional new effective treatment strategies for patients.MethodsDifferential expression and functional analyses were performed to identify key genes and relevant signaling pathways associated with OS lung metastasis. The expression of CCR9 in OS cell lines and tissues was measured by RT-qPCR, western blotting and immunohistochemistry. Cell migration and invasion were assessed by wound healing and Transwell Matrigel invasion assays, respectively. The regulatory relationship between CCR9 and the Wnt/β-catenin signaling pathway was further evaluated by rescue experiments.ResultsThe expression of CCR9 was elevated in OS cell lines and patients with lung metastasis. CCR9 promoted MG63 and HOS cell migration and invasion by activating the Wnt/β-catenin signaling pathway. Furthermore, knockdown of CCR9 repressed epithelial-mesenchymal transition (EMT) by downregulating mesenchymal markers (N-cadherin and Vimentin) and EMT-associated transcription factors (twist and snail) and upregulating an epithelial marker (E-cadherin).ConclusionsOur findings suggest that CCR9 promotes EMT by activating Wnt/β-catenin pathways to promote OS metastasis. CCR9 may be a promising therapeutic target to inhibit lung metastasis and serve as a novel prognostic marker for OS.
Project description:Ubiquitin-specific protease 5 (USP5) is a deubiquitinating enzyme that functions as an oncoprotein in a variety of human cancers. However, the expression and role of USP5 in the metastasis of non-small cell lung cancer (NSCLC) have not been addressed. In this study, we examined the expression and prognostic significance of USP5 in NSCLC. The results revealed that USP5 was overexpressed and correlated with metastasis and overall survival in NSCLC tissues. A further in vitro study revealed that the levels of USP5 protein in NSCLC cells were associated with epithelial-mesenchymal transition (EMT) markers. Furthermore, USP5 overexpression significantly enhanced, whereas USP5 silencing significantly decreased the expression of EMT proteins and migration and invasion of NSCLC cells. In addition, the results from western blotting demonstrated that USP5 regulated EMT via the Wnt/?-catenin signaling pathway. Further immunohistochemical analysis revealed that USP5 was significantly associated with the expression of ?-catenin and EMT markers in NSCLC tissues. Overall, USP5 upregulation is associated with tumor metastasis and poor prognosis in patients with NSCLC. USP5 promotes EMT and the invasion and migration of NSCLC cells. Therefore, USP5 may serve as a novel prognostic biomarker and provide a potential target for the treatment of metastasis in NSCLC.
Project description:Disrupted mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) generation are often associated with macrophage pyroptosis. It remains unclear how these forms of mitochondrial dysfunction relate to inflammasome activation and gasdermin-D (Gsdmd) cleavage, two central steps of the pyroptotic process. Here, we also found MMP collapse and ROS generation induced by Nlrp3 inflammasome activation as previous studies reported. The elimination of ROS alleviated the cleavage of Gsdmd, suggesting that Gsdmd cleavage occurs downstream of ROS release. Consistent with this result, hydrogen peroxide treatment augmented the cleavage of Gsdmd by caspase-1. Indeed, four amino acid residues of Gsdmd were oxidized under oxidative stress in macrophages. The efficiency of Gsdmd cleavage by inflammatory caspase-1 was dramatically reduced when oxidative modification was blocked by mutation of these amino acid residues. These results demonstrate that Gsdmd oxidation serves as a de novo mechanism by which mitochondrial ROS promote Nlrp3 inflammasome-dependent pyroptotic cell death.
Project description:BNIP3 is an atypical BH3-only member of the Bcl-2 family with pro-death, pro-autophagic, and cytoprotective functions, depending on the type of stress and cellular context. Recently, we demonstrated that BNIP3 stimulates the migration of epidermal keratinocytes under hypoxia. In the present study found that autophagy and BNIP3 expression were concomitantly elevated in the migrating epidermis during wound healing in a hypoxia-dependent manner. Inhibition of autophagy through lysosome-specific chemicals (CQ and BafA1) or Atg5-targeted small-interfering RNAs greatly attenuated the hypoxia-induced cell migration, and knockdown of BNIP3 in keratinocytes significantly suppressed hypoxia-induced autophagy activation and cell migration, suggesting a positive role of BNIP3-induced autophagy in keratinocyte migration. Furthermore, these results indicated that the accumulation of reactive oxygen species (ROS) by hypoxia triggered the activation of p38 and JNK mitogen-activated protein kinase (MAPK) in human immortalized keratinocyte HaCaT cells. In turn, activated p38 and JNK MAPK mediated the activation of BNIP3-induced autophagy and the enhancement of keratinocyte migration. These data revealed a previously unknown mechanism that BNIP3-induced autophagy occurs through hypoxia-induced ROS-mediated p38 and JNK MAPK activation and supports the migration of epidermal keratinocytes during wound healing.
Project description:This study explored the role of fibulin-3 in osteosarcoma progression and the possible signaling pathway involved. Fibulin-3 mRNA and protein expression in normal tissue, benign fibrous dysplasia, osteosarcoma, osteosarcoma cell lines (HOS and U-2OS), the normal osteoblastic cell line hFOB, and different invasive subclones was evaluated by immunohistochemistry (IHC) or immunocytochemistry (ICC) and real time reverse transcriptase-polymerase chain reaction (real time qRT-PCR). To assess the role of fibulin-3 in the invasion and metastasis of osteosarcoma cells, lentiviral vectors with fibulin-3 small hairpin RNA (shRNA) and pLVX-fibulin-3 were constructed and used to infect the highly invasive and low invasive subclones. The effects of fibulin-3 knockdown and upregulation on the biological behavior of osteosarcoma cells were investigated by functional in vitro and in vivo assays. The results revealed that fibulin-3 expression was upregulated in osteosarcoma, and was positively correlated with low differentiation, lymph node metastasis, and poor prognosis. Fibulin-3 could promote osteosarcoma cell invasion and metastasis by inducing EMT and activating the Wnt/β-catenin signaling pathway. Collectively, our findings demonstrate that fibulin-3 is a promoter of osteosarcoma development and progression, and suggest a novel therapeutic target for future studies.
Project description:Fusobacterium nucleatum, a Gram-negative oral anaerobe, is a significant contributor to colorectal cancer. Using an in vitro cancer progression model, we discover that F. nucleatum stimulates the growth of colorectal cancer cells without affecting the pre-cancerous adenoma cells. Annexin A1, a previously unrecognized modulator of Wnt/?-catenin signaling, is a key component through which F. nucleatum exerts its stimulatory effect. It is specifically expressed in proliferating colorectal cancer cells and involved in activation of cyclin D1. Annexin A1 expression level in colon cancer is a predictor of poor prognosis independent of cancer stage, grade, age and sex. The FadA adhesin from F. nucleatum up-regulates Annexin A1 expression through E-cadherin. A positive feedback loop between FadA and Annexin A1 is identified in the cancerous cells, absent in the non-cancerous cells. We therefore propose a "two-hit" model in colorectal carcinogenesis, with somatic mutation(s) as the first hit, and F. nucleatum as the second hit exacerbating cancer progression after benign cells become cancerous. This model extends the "adenoma-carcinoma" model and identifies microbes such as F. nucleatum as cancer "facilitators".
Project description:Using whole genome sequencing, we identified gene amplification of CREPT in colorectal cancer (CRC). In this study, we aim to clarify its clinical significance, biological effects, and mechanism in CRC. CREPT was upregulated in CRC cell lines and in 47.37% (72/152) of primary CRC tumors. Amplification of CREPT was detected in 48.28% (56/116) of primary CRC tumors, which was positively correlated with its overexpression (P?<?0.001). Multivariate analysis showed that CRC patients with CREPT protein overexpression were significantly associated with poor disease-free survival (P?<?0.05). CREPT significantly accelerated CRC cell proliferation and metastasis both in vitro and in vivo. RNA-sequencing (seq) analysis uncovered that the tumor-promoting effect by CREPT was attributed to enhancing Wnt/?-catenin signaling. Using co-immunoprecipitation coupled with mass spectroscopy, we identified p300 protein was a novel CREPT interacting partner. CREPT greatly increased the interaction between p300 and ?-catenin, thus promoting p300-mediated ?-catenin acetylation and stabilization. Moreover, CREPT cooperated with p300, leading to elevated active histone acetylation markers H3K27ac and H4Ac and decreased repressive histone marker H3K9me3 at the promoters of Wnt downstream targets. In summary, CREPT plays a pivotal oncogenic role in colorectal carcinogenesis through promoting Wnt/?-catenin pathway via cooperating with p300. CREPT may serve as a prognostic biomarker of patients with CRC.
Project description:Wnt/?-catenin signaling is active in small subpopulations of Ewing sarcoma cells, and these cells display a more metastatic phenotype, in part due to antagonism of EWS-FLI1-dependent transcriptional activity. Importantly, these ?-catenin-activated Ewing sarcoma cells also alter secretion of extracellular matrix (ECM) proteins. We thus hypothesized that, in addition to cell-autonomous mechanisms, Wnt/?-catenin-active tumor cells might contribute to disease progression by altering the tumor microenvironment (TME). Analysis of transcriptomic data from primary patient biopsies and from ?-catenin-active versus -nonactive tumor cells identified angiogenic switch genes as being highly and reproducibly upregulated in the context of ?-catenin activation. In addition, in silico and in vitro analyses, along with chorioallantoic membrane assays, demonstrated that ?-catenin-activated Ewing cells secreted factors that promote angiogenesis. In particular, activation of canonical Wnt signaling leads Ewing sarcoma cells to upregulate expression and secretion of proangiogenic ECM proteins, collectively termed the angiomatrix. Significantly, our data show that induction of the angiomatrix by Wnt-responsive tumor cells is indirect and is mediated by TGF-?. Mechanistically, Wnt/?-catenin signaling antagonizes EWS-FLI1-dependent repression of TGF-? receptor type 2, thereby sensitizing tumor cells to TGF-? ligands. Together, these findings suggest that Wnt/?-catenin-active tumor cells can contribute to Ewing sarcoma progression by promoting angiogenesis in the local TME.
Project description:The Wnt-β-catenin signal transduction pathway is essential for embryonic development and adult tissue homeostasis. Wnt signaling converts TCF from a transcriptional repressor to an activator in a process facilitated by the E3 ligase XIAP. XIAP-mediated monoubiquitylation of the transcriptional corepressor Groucho (also known as TLE) decreases its affinity for TCF, thereby allowing the transcriptional coactivator β-catenin to displace it on TCF. Through a genome-scale screen in cultured Drosophila melanogaster cells, we identified the deubiquitylase USP47 as a positive regulator of Wnt signaling. We found that USP47 was required for Wnt signaling during Drosophila and Xenopus laevis development, as well as in human cells, indicating evolutionary conservation. In human cells, knockdown of USP47 inhibited Wnt reporter activity, and USP47 acted downstream of the β-catenin destruction complex. USP47 interacted with TLE3 and XIAP but did not alter their amounts; however, knockdown of USP47 enhanced XIAP-mediated ubiquitylation of TLE3. USP47 inhibited ubiquitylation of TLE3 by XIAP in vitro in a dose-dependent manner, suggesting that USP47 is the deubiquitylase that counteracts the E3 ligase activity of XIAP on TLE. Our data suggest a mechanism by which regulated ubiquitylation and deubiquitylation of TLE enhance the ability of β-catenin to cycle on and off TCF, thereby helping to ensure that the expression of Wnt target genes continues only as long as the upstream signal is present.