Metformin alleviates benzo[a]pyrene-induced alveolar injury by inhibiting necroptosis and protecting AT2 cells.

Exposure to benzo[a]pyrene (B[a]P) has been linked to lung injury and carcinogenesis. Airway epithelial cells express the B[a]P receptor AHR, so B[a]P is considered to mainly target airway epithelial cells, whereas its potential impact on alveolar cells remains inadequately explored. Metformin, a first-line drug for diabetes, has been shown to exert anti-inflammatory and tissue repair-promoting effects under various injurious conditions. Here, we explored the effect of chronic B[a]P exposure on alveolar cells and the impact of metformin on B[a]P-induced lung injury by examining the various parameters including lung histopathology, inflammation, fibrosis, and related signal pathway activation. MLKL knockout (Mlkl-/-) and AT2-lineage tracing mice (SftpcCre-ERT2;LSL-tdTomatoflox+/-) were used to delineate the role of necroptosis in B[a]P-induced alveolar epithelial injury and repair. Mice receiving weekly administration of B[a]P for 6 weeks developed a significant alveolar damaging phenotype associated with pulmonary inflammation, fibrosis, and activation of the necroptotic cell death pathway. These effects were significantly relieved in MLKL null mice. Furthermore, metformin treatment, which were found to promote AMPK phosphorylation and inhibit RIPK3, as well as MLKL phosphorylation, also significantly alleviated B[a]P-induced necroptosis and lung injury phenotype. However, the protective efficacy of metformin was rendered much less effective in Mlkl null mice or by blocking the necroptotic pathway with RIPK3 inhibitor. Our findings unravel a potential protective efficacy of metformin in mitigating the detrimental effects of B[a]P exposure on lung health by inhibiting necroptosis and protecting AT2 cells.

SPAUTIN-1 alleviates LPS-induced acute lung injury by inhibiting NF-κB pathway in neutrophils.

BACKGROUND: Acute lung injury (ALI) is an inflammatory condition characterized by acute damage to lung tissue. SPAUTIN-1, recognized as a small molecule drug targeting autophagy and USP10/13, has been reported for its potential to inhibit oxidative stress damage in various tissue injuries. However, the role and mechanism of SPAUTIN-1 in ALI remain unclear. This study aims to elucidate the protective effects of SPAUTIN-1 on ALI, with a particular focus on its role and mechanism in pulmonary inflammatory responses. METHODS: Lipopolysaccharides (LPS) were employed to induce inflammation-mediated ALI. Bleomycin was used to induce non-inflammation-mediated ALI. The mechanism of SPAUTIN-1 action was identified through RNA-Sequencing and subsequently validated in mouse primary cells. Tert-butyl hydroperoxide (TBHP) was utilized to create an in vitro model of lung epithelial cell oxidative stress with MLE-12 cells. RESULTS: SPAUTIN-1 significantly mitigated LPS-induced lung injury and inflammatory responses, attenuated necroptosis and apoptosis in lung epithelial cells, and inhibited autophagy in leukocytes and epithelial cells. However, SPAUTIN-1 exhibited no significant effect on bleomycin-induced lung injury. RNA-sequencing results demonstrated that SPAUTIN-1 significantly inhibited the NF-κB signaling pathway in leukocytes, a finding consistently confirmed by mouse primary cell assays. In vitro experiments further revealed that SPAUTIN-1 effectively mitigated oxidative stress injury in MLE-12 cells induced by TBHP. CONCLUSION: SPAUTIN-1 alleviated LPS-induced inflammatory injury by inhibiting the NF-κB pathway in leukocytes and protected epithelial cells from oxidative damage, positioning it as a potential therapeutic candidate for ALI.

ESE1/AGR2 axis antagonizes TGF-ß-induced epithelial-mesenchymal transition in low-grade pancreatic cancer.

Epithelium-specific ETS transcription factor 1 (ESE1) has been implicated in epithelial homeostasis, inflammation, as well as tumorigenesis, and cancer progression. However, numerous studies have reported contradictory roles-as an oncogene or a tumor suppressor of ESE1 in different cancers, and its function in the development and progression of pancreatic ductal adenocarcinoma (PDAC) has remained largely unexplored. Herein, we report that ESE1 was found upregulated in primary PDAC compared to normal pancreatic tissue, but high expression of ESE1 correlated to better relapse-free survival in patients with PDAC. Interestingly, ESE1 was found to exhibit dual roles in regulation of malignant properties of PDAC cells in that its overexpression promoted cell proliferation, whereas its downregulation enhanced epithelial-mesenchymal transition (EMT) phenotype. In the context of TGF-ß-induced EMT, ESE1 is markedly downregulated at post-transcriptional level, and reconstituted ESE1 expression partially reversed TGF-ß-induced EMT marker expression. Furthermore, we identify AGR2 as a novel transcriptional target of ESE1 that participates in TGF-ß-induced EMT in PDAC. Collectively, our findings reveal an ESE1/AGR2 axis that interacts with TGF-ß signaling to modulate EMT phenotype in PDAC.

Enhanced nuclear localization of YAP1-2 contributes to EGF-induced EMT in NSCLC.

YAP1, a key mediator of the Hippo pathway, plays an important role in tumorigenesis. Alternative splicing of human YAP1 mRNA results in two major isoforms: YAP1-1, which contains a single WW domain, and YAP1-2, which contains two WW domains, respectively. We here investigated the functions and the underlying regulatory mechanisms of the two YAP1 isoforms in the context of EGF-induced epithelial-mesenchymal transition (EMT) in non-small cell lung cancer (NSCLC). Human NSCLC cell lines express both YAP1-1 and YAP1-2 isoforms-although when compared to YAP1-1, YAP1-2 mRNA levels are higher while its protein expression levels are lower. EGF treatment significantly promoted YAP1 expression as well as EMT process in NSCLCs, whereas EGF-induced EMT phenotype was significantly alleviated upon YAP1 knockdown. Under normal culture condition, YAP1-1 stable expression cells exhibited a stronger migration ability than YAP1-2 expressing cells. However, upon EGF treatment, YAP1-2 stable cells showed more robust migration than YAP1-1 expressing cells. The protein stability and nuclear localization of YAP1-2 were preferentially enhanced with EGF treatment. Moreover, EGF-induced EMT and YAP1-2 activity were suppressed by inhibitor of AKT. Our results suggest that YAP1-2 is the main isoform that is functionally relevant in promoting EGF-induced EMT and ultimately NSCLC progression.

Yap1-2 Isoform Is the Primary Mediator in TGF-ß1 Induced EMT in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is the most aggressive human malignancy and intrinsically resistant to conventional therapies. YAP1, as a key downstream effector of the Hippo pathway, plays an important role in tumorigenesis including PDAC. Alternative mRNA splicing of YAP1 results in at least 8 protein isoforms, which are divided into two subgroups (YAP1-1 and YAP1-2) based on the presence of either a single or double WW domains. We investigated the functions and regulatory mechanisms of YAP1-1 and YAP1-2 in PDAC cells induced by TGF-ß to undergo epithelial-to-mesenchymal transition (EMT). CRISPR-Cas9 and shRNA were used to silence YAP1 expression in pancreatic cancer cells. Re-constituted lentivirus mediated overexpression of each single YAP1 isoform was generated in the parental knockout L3.6 cells. EMT was induced by treatment with TGF-ß, EGF and bFGF in parental and the constructed stable cell lines. Western blot and qPCR were used to detect the expression of EMT markers. Scratch wound healing and transwell assays were used to detect cell migration. The stability and subcellular localization of YAP1 proteins were determined by Western blot analysis, immunofluorescence, as well as ubiquitination assays. We showed that TGF-ß, EGF and bFGF all significantly promoted EMT in PDAC cells, which was inhibited by knockdown of YAP1 expression. Interestingly, YAP1-1 stable cells exhibited a stronger migratory ability than YAP1-2 cells under normal culture condition. However, upon TGF-ß treatment, L3.6-YAP1-2 cells exhibited a stronger migratory ability than L3.6-YAP1-1 cells. Mechanistically, TGF-ß treatment preferentially stabilizes YAP1-2 and enhances its nuclear localization. Furthermore, TGF-ß-induced EMT and YAP1-2 activity were both blocked by inhibition of AKT signaling. Our results showed that both YAP1-1 and YAP1-2 isoforms are important mediators in the EMT process of pancreatic cancer. However, YAP1-2 is more important in mediating TGF-ß-induced EMT, which requires AKT signaling.

Corrigendum: An FGFR/AKT/SOX2 Signaling Axis Controls Pancreatic Cancer Stemness.

[This corrects the article DOI: 10.3389/fcell.2020.00287.].

An FGFR/AKT/SOX2 Signaling Axis Controls Pancreatic Cancer Stemness.

Cancer stemness is associated with high malignancy and low differentiation, as well as therapeutic resistance of tumors including pancreatic ductal adenocarcinoma (PDAC). Fibroblast growth factors (FGFs) exert pleiotropic effects on a variety of cellular processes and functions including embryonic stem cell pluripotency and cancer cell stemness via the activation of four tyrosine kinase FGF receptors (FGFRs). FGF ligands have been a major component of the cocktail of growth factors contained in the cancer stemness-inducing (CSI) and organoid culture medium. Although FGF/FGFR signaling has been hypothesized to maintain cancer stemness, its function in this process is still unclear. We report that inhibition of FGF/FGFR signaling impairs sphere-forming ability of PDAC in vitro, and knocking down FGFR1 and FGFR2 decreased their tumorigenesis abilities in vivo. Mechanistically, we demonstrated that SOX2 is down-regulated upon loss of FGFR signaling. The overexpression of SOX2 in SOX2-negative cells, which normally do not display stemness capabilities, is sufficient to induce spheroid formation. Additionally, we found that AKT phosphorylation was reduced upon FGFR signaling inhibition. The inhibition of AKT using specific pharmacological inhibitors in the context of CSI medium leads to the loss of spheroid formation associated with loss of SOX2 nuclear expression and increased degradation. We demonstrate that an FGFR/AKT/SOX2 axis controls cancer stemness in PDAC and therefore may represent an important therapeutic target in the fight against this very aggressive form of cancer.

Endogenous Nodal promotes melanoma undergoing epithelial-mesenchymal transition via Snail and Slug in vitro and in vivo.

Nodal, an important embryonic morphogen, has been reported to modulate tumorigenesis. Epithelial-mesenchymal transition (EMT) plays an important role in cancer metastasis. We have previously reported that recombinant Nodal treatment can promote melanoma undergoing EMT, but the effects of endogenous Nodal on EMT are still unknown. Here we generated both Nodal-overexpression and -knockdown stable cell lines to investigate the in vitro and in vivo characteristics of Nodal-induced EMT in murine melanoma cells. Nodal-overexpression cells displayed increased migration ability, accompanied by typical phenotype changes of EMT. In contrast, Nodal-knockdown stable cells repressed the EMT phenotype as well as reduced cell motility. Results of animal experiments confirmed that overexpression of Nodal can promote the metastasis of melanoma tumor in vivo. Mechanistically, we found that Nodal-induced expression of Snail and Slug involves its activation of ALK/Smads and PI3k/AKT pathways, which is an important process in the Nodal-induced EMT. However, we also found that the EMT phenotype was not completely inhibited by blocking the paracrine activity of Nodal in Nodal overexpression cell line suggesting the presence of additional mechanism(s) in the Nodal-induced EMT. This study provides a better understanding of Nodal function in melanoma, and suggests targeting Nodal as a potential strategy for melanoma therapey.