Discovery of a novel hybrid coumarin-hydroxamate conjugate targeting the HDAC1-Sp1-FOSL2 signaling axis for breast cancer therapy.

BACKGROUND: Breast cancer is one of the most lethal cancers in women. Despite significant advances in the diagnosis and treatment of breast cancer, many patients still succumb to this disease, and thus, novel effective treatments are urgently needed. Natural product coumarin has been broadly investigated since it reveals various biological properties in the medicinal field. Accumulating evidence indicates that histone deacetylase inhibitors (HDACIs) are promising novel anti-breast cancer agents. However, most current HDACIs exhibit only moderate effects against solid tumors and are associated with severe side effects. Thus, to develop more effective HDACIs for breast cancer therapy, hydroxamate of HDACIs was linked to coumarin core, and coumarin-hydroxamate hybrids were designed and synthesized. METHODS: A substituted coumarin moiety was incorporated into the classic hydroxamate HDACIs by the pharmacophore fusion strategy. ZN444B was identified by using the HDACI screening kit and cell viability assay. Molecular docking was performed to explore the binding mode of ZN444B with HDAC1. Western blot, immunofluorescent staining, cell viability, colony formation and cell migration and flow cytometry assays were used to analyze the anti-breast cancer effects of ZN444B in vitro. Orthotopic studies in mouse models were applied for preclinical evaluation of efficacy and toxicity in vivo. Proteomic analysis, dual-luciferase reporter assay, chromatin immunoprecipitation, co-immunoprecipitation, immunofluorescent staining assays along with immunohistochemical (IHC) analysis were used to elucidate the molecular basis of the actions of ZN444B. RESULTS: We synthesized and identified a novel coumarin-hydroxamate conjugate, ZN444B which possesses promising anti-breast cancer activity both in vitro and in vivo. A molecular docking model showed that ZN444B binds to HDAC1 with high affinity. Further mechanistic studies revealed that ZN444B specifically decreases FOS-like antigen 2 (FOSL2) mRNA levels by inhibiting the deacetylase activity of HDAC1 on Sp1 at K703 and abrogates the binding ability of Sp1 to the FOSL2 promoter. Furthermore, FOSL2 expression positively correlates with breast cancer progression and metastasis. Silencing FOSL2 expression decreases the sensitivity of breast cancer cells to ZN444B treatment. In addition, ZN444B shows no systemic toxicity in mice. CONCLUSIONS: Our findings highlight the potential of FOSL2 as a new biomarker and therapeutic target for breast cancer and that targeting the HDAC1-Sp1-FOSL2 signaling axis with ZN444B may be a promising therapeutic strategy for breast cancer.

FOSL2-mediated transcription of ISG20 induces M2 polarization of macrophages and enhances tumorigenic ability of glioblastoma cells.

BACKGROUND: Interferon stimulated exonuclease gene 20 (ISG20) has been reported to be correlated with macrophage infiltration in glioblastoma (GBM) in previous bioinformatics-based studies. This study explores the exact effect of ISG20 on macrophage polarization in GBM. METHODS: ISG20 expression in GBM tissues and cells was determined by RT-qPCR and/or immunohistochemistry. GBM cells were co-cultured with M0 macrophages (PMA-stimulated THP-1 cells) in vitro, followed by flow cytometry and ELISA to analyze the M2 polarization of macrophages. Fluorescence-contained GBM cells were intracranially injected into nude mice along with M0 macrophages to generate orthotopic xenograft tumor models. Upstream regulator of ISG20 was predicted using bioinformatics. Loss- or gain-of-function assays of Fos like 2 (FOSL2) and ISG20 were performed in GBM cells. DNA methylation level of FOSL2 was analyzed by bisulfite sequencing analysis. RESULTS: ISG20 was found highly expressed in GBM tissues and cells. ISG20 silencing in GBM cells decreased CD206 and CD163 levels in the co-cultured macrophages and reduced secretion of IL-10 and TGF-ß. It also enhanced survival of nude mice bearing xenograft tumors, blocked tumor growth, and suppressed M2 polarization of macrophages in vivo. FOSL2, highly expressed in GBM, bound to the ISG20 promoter to activate its transcription. FOSL2 silencing similarly blocked M2 polarization of macrophages, which was negated by ISG20 overexpression. The high FOSL2 expression in GBM was attributed to DNA hypomethylation. CONCLUSION: This study demonstrates that FOSL2 is highly expressed in GBM due to DNA hypomethylation. It activates transcription of ISG20, thus promoting M2 polarization of macrophages and GBM development.

SOX2 dosage sustains tumor-promoting inflammation to drive disease aggressiveness by modulating the FOSL2/IL6 axis.

BACKGROUND: Inflammation is undoubtedly a hallmark of cancer development. Its maintenance within tumors and the consequences on disease aggressiveness are insufficiently understood. METHODS: Data of 27 tumor entities (about 5000 samples) were downloaded from the TCGA and GEO databases. Multi-omic analyses were performed on these and in-house data to investigate molecular determinants of tumor aggressiveness. Using molecular loss-of-function data, the mechanistic underpinnings of inflammation-induced tumor aggressiveness were addressed. Patient specimens and in vivo disease models were subsequently used to validate findings. RESULTS: There was significant association between somatic copy number alterations (sCNAs) and tumor aggressiveness. SOX2 amplification was the most important feature among novel and known aggressiveness-associated alterations. Mechanistically, SOX2 regulates a group of genes, in particular the AP1 transcription factor FOSL2, to sustain pro-inflammatory signaling pathways, such as IL6-JAK-STAT3, TNFA and IL17. FOSL2 was found overexpressed in tumor sections of specifically aggressive cancers. In consequence, prolonged inflammation induces immunosuppression and activates cytidine deamination and thus DNA damage as evidenced by related mutational signatures in aggressive tumors. The DNA damage affects tumor suppressor genes such as TP53, which is the most mutated gene in aggressive tumors compared to less aggressive ones (38% vs 14%), thereby releasing cell cycle control. These results were confirmed by analyzing tissues from various tumor types and in vivo studies. CONCLUSION: Our data demonstrate the implication of SOX2 in promoting DNA damage and genome instability by sustaining inflammation via FOSL2/IL6, resulting in tumor aggressiveness.

Long non-coding RNA MIR17HG impedes FOSL2-mediated transcription activation of HIC1 to maintain a pro-inflammatory phenotype of microglia during intracerebral haemorrhage.

Activation and polarization of microglia play decisive roles in the progression of intracerebral haemorrhage (ICH), and lactate exposure correlates with microglia polarization. This study explores molecules influencing lactate production and microglia phenotype alteration following ICH. A murine model of ICH was induced by intracerebral injection of collagenase. The mice experienced autonomous neurological function recovery, haematoma resolution and rapid lactate production, along with a gradual increase in angiogenesis activity, neuronal recovery and an M1-to-M2 phenotype change of microglia. Galloflavin, a lactate dehydrogenase antagonist, suppressed this phenotype change and the functional recovery in mice. FOS like 2 (FOSL2) was significantly upregulated in the brain tissues from day 7 post-ICH. Overexpression of FOSL2 induced an M1-to-M2 phenotype shift in microglia and accelerated lactate production in vivo and in haemoglobin-treated microglia in vitro. Long non-coding RNA MIR17HG impeded FOSL2-mediated transcription activation of hypermethylated in cancer 1 (HIC1). MIR17HG overexpression induced pro-inflammatory activation of microglia in mice, which was blocked by further HIC1 overexpression. Overall, this study demonstrates that MIR17HG maintains a pro-inflammatory phenotype of microglia during ICH progression by negating FOSL2-mediated transcription activation of HIC1. Specific inhibition of MIR17HG or upregulation of FOSL2 or HIC1 may favour inflammation inhibition and haematoma resolution in ICH.

The transcription factors Junb and Fosl2 cooperate to regulate Cdh3 expression in 15P-1 Sertoli cells.

In Sertoli cells of the testis, cadherins (Cdh) are important cell-to-cell interaction proteins and contribute to the formation of the blood-testis barrier being essential for germ cells' protection. P-cadherin or Cdh3 is only expressed in Sertoli cells from embryonic to prepubertal development. Interestingly, the expression profile of Cdh3 correlates with that of activating protein-1 (AP-1) transcription factors during Sertoli cells development. To assess their potential implications in the regulation of Cdh3, different AP-1 transcription factors were overexpressed in 15P-1 Sertoli cells. We found that the overexpressions of Junb and Fosl2 activated Cdh3 promoter. ChIP-qPCR assay and luciferase reporter assay with 5' promoter deletions and site-directed mutagenesis confirmed the recruitment of Junb and Fosl2 to an AP-1 regulatory element at -47 bp in the proximal region of Cdh3 promoter in 15P-1 cells. These findings were further supported by histone modification markers and chromatin accessibility surrounding Cdh3 promoter in mouse testis. Moreover, the knockdowns of Junb and/or Fosl2 by siRNA decreased Cdh3 protein levels. Taken together, these data suggest that in 15P-1 Sertoli cells, the AP-1 family members Junb and Fosl2 are responsible for the regulation of Cdh3 expression, which requires the recruitment of both factors to the proximal region of the Cdh3 promoter.

Circ_0005615 Regulates the Progression of Colorectal Cancer Through the miR-873-5p/FOSL2 Signaling Pathway.

To determine the effects of circ_0005615 in CRC development and underneath mechanism. The expression levels of circ_0005615, microRNA-873-5p (miR-873-5p) and FOS-like antigen 2 (FOSL2) mRNA were determined by quantitative real-time polymerase chain reaction (qRT-PCR). The protein levels of exosome makers, proliferation-related makers and FOSL2 were detected by western blot or immunohistochemistry assay. Cell proliferation was evaluated by cell counting kit-8 (CCK-8) and cell colony formation assays. Cell migration and invasion were demonstrated by a transwell assay. Cell apoptosis was investigated by flow cytometry analysis. The binding relationship between miR-873-5p and circ_0005615 or FOSL2 was predicted by circular RNA interactome and targetscan online databases, respectively, and identified by dual-luciferase reporter assay. The impacts of circ_0005615 silencing on tumor formation were determined by in vivo tumor formation assay. Circ_0005615 expression was dramatically upregulated in serum exosomes of CRC patients compared with the control group. The CRC patients with a high circ_0005615 expression had a poor survival rate. Circ_0005615 and FOSL2 expressions were apparently increased, while miR-873-5p was decreased in CRC tissues or cells relative to control groups. Circ_0005615 knockdown inhibited cell proliferation, migration, and invasion, whereas promoted cell apoptosis in CRC; however, miR-873-5p inhibitor attenuated these impacts. Additionally, circ_0005615 acted as a sponge of miR-873-5p and miR-873-5p bound to FOSL2. FOSL2 overexpression restrained the effects of miR-873-5p mimic on CRC progression. Furthermore, circ_0005615 knockdown suppressed tumor growth in vivo. Circ_0005615 modulated CRC malignant progression by controlling FOSL2 expression through sponging miR-873-5p. This finding lays a foundation for the study on circRNA-mediated CRC therapy.

FOSL2 truncating variants in the last exon cause a neurodevelopmental disorder with scalp and enamel defects.

PURPOSE: We aimed to investigate the molecular basis of a novel recognizable neurodevelopmental syndrome with scalp and enamel anomalies caused by truncating variants in the last exon of the gene FOSL2, encoding a subunit of the AP-1 complex. METHODS: Exome sequencing was used to identify genetic variants in all cases, recruited through Matchmaker exchange. Gene expression in blood was analyzed using reverse transcription polymerase chain reaction. In vitro coimmunoprecipitation and proteasome inhibition assays in transfected HEK293 cells were performed to explore protein and AP-1 complex stability. RESULTS: We identified 11 individuals from 10 families with mostly de novo truncating FOSL2 variants sharing a strikingly similar phenotype characterized by prenatal growth retardation, localized cutis scalp aplasia with or without skull defects, neurodevelopmental delay with autism spectrum disorder, enamel hypoplasia, and congenital cataracts. Mutant FOSL2 messenger RNAs escaped nonsense-mediated messenger RNA decay. Truncated FOSL2 interacts with c-JUN, thus mutated AP-1 complexes could be formed. CONCLUSION: Truncating variants in the last exon of FOSL2 associate a distinct clinical phenotype by altering the regulatory degradation of the AP-1 complex. These findings reveal a new role for FOSL2 in human pathology.

Circ-FAT1 Up-Regulates FOSL2 Expression by Sponging miR-619-5p to Facilitate Colorectal Cancer Progression.

Circular RNA FAT atypical cadherin 1 (circ-FAT1) has been reported to play roles in colorectal cancer (CRC) development. Here, the purpose of this study was to investigate the function and mechanism of circ-FAT1 in CRC tumorigenesis and its potential value in the clinic. Levels of genes and proteins were examined by quantitative real-time polymerase chain reaction and Western blot. In vitro assays were conducted using cell counting kit-8 assay, 5-Ethynyl-2'-deoxyuridine assay, flow cytometry, transwell assay, and tube formation assay, respectively. The target relationship between miR-619-5p and circ-FAT1 or FOS-like antigen 2 (FOSL2) was verified by dual-luciferase reporter and RNA immunoprecipitation assays. In vivo assay was performed using a mouse subcutaneous xenograft model. Circ-FAT1 and FOSL2 were highly expressed in CRC tissues and cells. Functionally, knockdown of circ-FAT1 or FOSL2 suppressed CRC cell apoptosis, migration, invasion, and angiogenesis, but induced cell apoptosis in vitro. Mechanistically, circ-FAT1 acted as a sponge for miR-619-5p to up-regulate the expression of FOSL2, which was confirmed to be a target of miR-619-5p. A series of rescue experiments demonstrated that miR-619-5p inhibition or FOSL2 overexpression reversed the inhibitory action of circ-FAT1 silencing on CRC cell malignant phenotypes mentioned above. Pre-clinically, lentivirus-mediated circ-FAT1 knockdown inhibited the tumorigenesis of CRC xenografts in nude mice via regulating miR-619-5p and FOSL2. Circ-FAT1 knockdown repressed FOSL2 expression by sponging miR-619-5p to suppress CRC tumorigenesis, providing a potential approach for CRC therapeutics.

Circ_0091581 Promotes the Progression of Hepatocellular Carcinoma Through Targeting miR-591/FOSL2 Axis.

BACKGROUND: Circular RNAs (circRNAs) have shown crucial regulatory roles in cancer biology. We aimed to uncover the role and underlying mechanism of circ_0091581 in hepatocellular carcinoma (HCC) progression. METHODS: The abundance of circ_0091581, microRNA-591 (miR-591) and FOS like 2, AP-1 transcription factor subunit (FOSL2) was measured by quantitative real-time polymerase chain reaction. Cell viability, colony formation ability, and invasion ability were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony formation assay, and transwell invasion assay. The migration ability was analyzed by transwell migration assay and wound healing assay. Flow cytometry was used to evaluate the cell cycle and apoptosis of HCC cells. The interaction between miR-591 and circ_0091581 or FOSL2 was predicted by Circular RNA Interactome database or TargetScan database and confirmed by dual-luciferase reporter assay and RNA immune co-precipitation assay. FOSL2 protein expression was measured by Western blot assay. Xenograft tumor assay was conducted to analyze the role of circ_0091581 in HCC tumor growth in vivo. RESULTS: Circ_0091581 was highly expressed in HCC tissue samples and cell lines in contrast to that in adjacent normal tissue samples and THLE-2 cell line. Circ_0091581 accelerated the viability, colony formation, metastasis, and cell cycle, while it impeded the apoptosis of HCC cells. MiR-591 bound to circ_0091581, and circ_0091581 knockdown-mediated effects in HCC cells were largely overturned by miR-591 silencing. FOSL2 was a target of miR-591, and FOSL2 overexpression largely reversed miR-591 accumulation-induced influences in HCC cells. FOSL2 protein expression was down-regulated by circ_0091581 silencing, and the addition of miR-591 inhibitor partly recovered the expression of FOSL2 in HCC cells. Circ_0091581 interference notably suppressed HCC tumor growth in vivo. CONCLUSION: Circ_0091581 acted as an oncogene to enhance the viability, colony formation, metastasis and cell cycle and inhibit the apoptosis of HCC cells through targeting miR-591/FOSL2 axis.

Long-lasting transcription in hippocampal area CA1 after contextual fear conditioning.

A fundamental question is how memory is stored for several weeks and even longer. A long-lasting increase in gene transcription has been suggested to mediate such long-term memory storage. Here, we used contextual fear conditioning in mice to search for lasting transcription that may contribute to long-term memory storage. Our study focussed on hippocampal area CA1, which has been suggested to have a role for at least one week in contextual fear memory. Using an unbiased microarray analysis followed by confirmatory quantitative real-time PCR, we identified an upregulation of two transcription factors, Fosl2 and Nfil3, which lasted for seven days after conditioning. To our knowledge these are the longest transcriptional changes ever detected in the hippocampus after contextual fear conditioning. Thus, our findings suggest novel transcriptional candidates for long-term memory storage.

The AP-1 transcription factor component Fosl2 potentiates the rate of myocardial differentiation from the zebrafish second heart field.

The vertebrate heart forms through successive phases of cardiomyocyte differentiation. Initially, cardiomyocytes derived from first heart field (FHF) progenitors assemble the linear heart tube. Thereafter, second heart field (SHF) progenitors differentiate into cardiomyocytes that are accreted to the poles of the heart tube over a well-defined developmental window. Although heart tube elongation deficiencies lead to life-threatening congenital heart defects, the variables controlling the initiation, rate and duration of myocardial accretion remain obscure. Here, we demonstrate that the AP-1 transcription factor, Fos-like antigen 2 (Fosl2), potentiates the rate of myocardial accretion from the zebrafish SHF. fosl2 mutants initiate accretion appropriately, but cardiomyocyte production is sluggish, resulting in a ventricular deficit coupled with an accumulation of SHF progenitors. Surprisingly, mutant embryos eventually correct the myocardial deficit by extending the accretion window. Overexpression of Fosl2 also compromises production of SHF-derived ventricular cardiomyocytes, a phenotype that is consistent with precocious depletion of the progenitor pool. Our data implicate Fosl2 in promoting the progenitor to cardiomyocyte transition and uncover the existence of regulatory mechanisms to ensure appropriate SHF-mediated cardiomyocyte contribution irrespective of embryonic stage.

Fos-like antigen 2 (FOSL2) promotes metastasis in colon cancer.

Among different cancers, incidence and mortality of colorectal cancer (CRC) is one of the highest. KRAS mutation is one of the underlying features in the pathogenesis of CRC with CRC tumors harboring mutant KRAS exhibiting a more aggressive behavior compared to CRC tumors with wild type KRAS. We had earlier shown that the microRNA-143 (miR-143) replenishment not only chemosensitizers CRC cell line with mutant KRAS instead of wild-type KRAS gene, to paclitaxel-mediated cytotoxicity, but also inhibits cell migration and invasion ability. Hence, the study aimed to determine how miR-143 replenishment is inhibiting pre-metastatic behavior in CRC cells with mutant KRAS. Top ten mRNA targets of miR-143 as predicted by TargetScan were evaluated by qRT-PCR in LoVo cells which were performed mock transfection or miR-143 mimic transfection. Evaluation of the changes in cognate mRNA target(s) was done in 30 paired CRC tissue and tumor adjacent normal tissue specimens and in LoVo cells by western blot. Effect of the mRNA target on pro-metastatic behavior was assayed by gain- and loss-of-function studies using a combination of western blotting and in vitro cell proliferation and transwell migration/invasion assay in LoVo cells and in the normal colonic epithelium cell line FHC. In vivo effect of the cognate mRNA target on CRC metastasis was assayed by xenograft assay. Of the 10 predicted mRNA targets, FOSL2 (P < 0.05) and IGFBP5 (P > 0.05) was down regulated in LoVo cells transfected with the miR-143 mimic. FOSL2 mRNA levels were significantly downregulated in CRC tissue specimens compared with adjacent normal tissue (P < 0.05). Immunoblot analysis showed that FOSL2, but not IGFBP5, protein expression is down regulated in LoVo cells after the miR-143 mimic transfection. FOSL2 overexpression in the normal colonic epithelial cell line FHC or siRNA-mediated silencing in LoVo cells induced and repressed, respectively, pro-mesenchymal cell features. Whereas manipulation of FOSL2 expression did not have any effect on cell proliferation rates, silencing its expression inhibited cell migration and invasion ability in vitro. In addition, silencing of FOSL2 expression in the LoVo cells can significantly inhibited invasion of hepatic, while no effect was found for tumorigenic potential. Our results suggest that FOSL2 is a critical regulator of CRC metastasis and might be an important marker for prognostic in CRC patients.

Osteoblast-specific expression of Fra-2/AP-1 controls adiponectin and osteocalcin expression and affects metabolism.

Recent studies have established that the skeleton functions as an endocrine organ affecting metabolism through the osteoblast-derived hormone osteocalcin (Ocn). However, it is not fully understood how many transcription factors expressed in osteoblasts regulate the endocrine function. Here, we show that mice with osteoblast-specific deletion of Fra-2 (Fosl2) have low bone mass but increased body weight. In contrast, transgenic expression of Fra-2 in osteoblasts leads to increased bone mass and decreased body weight accompanied by reduced serum glucose and insulin levels, improved glucose tolerance and insulin sensitivity. In addition, mice lacking Fra-2 have reduced levels of circulating Ocn, but high adiponectin (Adipoq), whereas Fra-2 transgenic mice exhibit high Ocn and low Adipoq levels. Moreover, we found that Adipoq was expressed in osteoblasts and that this expression was transcriptionally repressed by Fra-2. These results demonstrate that Fra-2 expression in osteoblasts represents a novel paradigm for a transcription factor controlling the endocrine function of the skeleton.

BACH1 transcriptionally upregulates FOSL2 to induce M2 macrophages phenotype by activating TGFß/SMAD signaling to promote the transformation of lung fibroblasts into myofibroblasts.

Pulmonary fibrosis is a chronic and progressive lung disorder. The pro-fibrosis factors induced by M2 macrophage phenotype promote the differentiation of fibroblasts into myofibroblasts, which is essential for pulmonary fibrosis. We aimed to explore the role and mechanism of BTB domain and CNC homology 1 (BACH1) in pulmonary fibrosis. BACH1 was knocked down in THP-1 polarized M2 macrophages with or without FOS-like antigen 2 (FOSL2) overexpression, the expression of M2 macrophage markers was detected. Cell viability, migration, invasion and extracellular matrix (ECM) accumulation were estimated by CCK-8, wound healing, transwell, western bot and immunofluorescence staining. Luciferase reporter and chromatin immunoprecipitation assays were used to verify the binding of BACH1 to FOSL2 promotor region. In vivo, a bleomycin (BLM)-induced pulmonary fibrosis mice model was established to evaluate the effect of BACH1 silencing on the histopathological changes, M2 macrophage phenotype and extracellular matrix (ECM) deposition. Expression of proteins was assessed with western blot. Results indicated that BACH1 expression was upregulated in M2 macrophages polarized from THP-1 cells. BACH1 deficiency inhibited the polarization of THP-1 to the M2 macrophage phenotype to promote the transformation of lung fibroblasts into myofibroblasts. Additionally, BACH1 could transcriptionally activate FOSL2 expression in THP-1-derived macrophages to upregulate TGFß/SMAD signaling in HFL-1 cells. The animal experiments indicated that BACH1 knockdown alleviated BLM-induced pulmonary fibrosis, M2 macrophage polarization and inactivated FOSL2/TGFß/SMAD signaling in mice lung tissues. Together, this finding suggests BACH1/FOSL2 may be useful therapeutic targets for the treatment of pulmonary fibrosis.

HTB50-2 Inhibits Growth and Migration of Triple-negative Breast Cancer via FOSL2/FOXC1 Signaling Axis and Subsequent Ferroptosis.

BACKGROUND: The manipulation of ferroptosis in cancer cells is a possible therapeutic technique that has been investigated for use in the treatment of cancer. Consequently, ferroptosis-inducing medications have recently received increased interest in cancer therapy. In this research, we assessed the anticancer efficacy of 14ß-hydroxy- 3ß-(ß-D-Glucopyranosyloxy)-5α-bufa-20,22-dienolide (HTB50-2), a natural product derived from the plant Helleborus thibetanus Franch, in Triple-Negative Breast Cancer (TNBC). Moreover, we also studied its potential mechanisms. METHODS: The biological effects of HTB50-2 in a series of breast cancer cell lines were analyzed using sulforhodamine B (SRB) and other methods. The migration ability was analyzed using three methods: wound healing assay, transwell assay, and Western blot. Meanwhile, the potential therapeutic value of HTB50-2 was evaluated in BALB/c mice by orthotopic transplantation. Transcriptome sequencing was conducted to explore the FOS-like antigen 2 (FOSL2) gene, and its role in ferroptosis was verified by Western blot and immunohistochemistry. The association of FOSL2 and ferroptosis-related genes was analyzed using NetworkAnalyst databases, and a TF-Gene interaction network was constructed. RESULTS: Ferroptosis was found to be induced in TNBC cells by HTB50-2. Furthermore, HTB50-2 inhibited tumor development by inducing ferroptosis in TNBC in vivo. Mechanistically, we demonstrated that a transcription factor FOSL2 mediated ferroptosis by HTB50-2. Additionally, it was found that Forkhead box C1 (FOXC1) was regulated by FOSL2 and correlated with ferroptosis. CONCLUSION: Our data suggest that HTB50-2 exerts its anti-cancer properties by ferroptosis via FOSL2/FOXC1 signaling pathway. Hence, HTB50-2 has an important application potential in the treatment of TNBC.

FasL impacts Tgfb signaling in osteoblastic cells.

Fas ligand (FasL, CD178) belongs to classical apoptotic molecules, however, recent evidence expands the spectrum of FasL functions into non-apoptotic processes which also applies for the bone. Tgfb subfamily members (Tgfb1, Tgfb2, Tgfb3) represent major components in osteogenic pathways and extracellular matrix. Their possible association with FasL has not yet been investigated but can be postulated. To test such a hypothesis, FasL deficient (gld) calvaria-derived cells were examined with a focus on the expression of Tgfb receptor ligands. The qPCR analysis revealed significantly increased expression of Tgfb1, Tgfb2 and Tgfb3 in gld cells. To check the vice versa effect, the gld cells were stimulated by soluble FasL. As a consequence, a dramatic decrease in expression levels of all three ligands was observed. This phenomenon was also confirmed in IDG-SW3 (osteoblastic cells of endochondral origin). TFLink gateway identified Fosl2 as an exclusive candidate of FasL capable to impact expression of all three Tgfb ligands. However, Fosl2 siRNA did not cause any significant changes in expression of Tgfb ligands. Therefore, the upregulation of the three ligands is likely to occur separately. In this respect, we tested the only exclusive candidate transcription factor for Tgfb3, Prrx1. Additionally, an overlapping candidate for Tgfb1 and Tgfb2, Mef2c capable to modulate expression of sclerostin, was examined. Prrx1 as well as Mef2c were found upregulated in gld samples and their expression decreased after addition of FasL. The same effect of FasL treatment was observed in the IDG-SW3 model. Taken together, FasL deficiency causes an increase in the expression of Tgfb ligands and stimulation by FasL reduces Tgfb expression in osteoblastic cells. The candidates mediating the effect comprise Prrx1 for Tgfb3 and Mef2c for Tgfb1/2. These results indicate FasL as a novel cytokine interfering with Tgfb signaling and thus the complex osteogenic network. The emerging non-apoptotic functions of FasL in bone development and maintenance should also be considered in treatment strategies such as the anti-osteoporotic factor.

Genome-Wide Characterization of TAZ Binding Sites in Mammary Epithelial Cells.

The transcriptional co-activator with PDZ binding motif (TAZ) is a key effector of the Hippo signaling pathway. We and others previously reported that high expression levels of TAZ are positively associated with decreased survival rates and shorter times to relapse in basal-like breast cancer (BLBC) patients. The oncogenic activity of TAZ involves the regulation of diverse signal transduction pathways that direct processes such as cell proliferation, migration, and resistance to apoptosis, albeit through poorly characterized gene expression programs. Here, using a tet-inducible system in mammary epithelial MCF10A cells, we have characterized the TAZ-regulated transcription program using RNA sequencing in a temporal and spatial manner. We further identified global TAZ binding sites at different TAZ activation time points by chromatin immunoprecipitation (ChIP) sequencing analysis. We found that the vast majority of TAZ was rapidly localized in enhancer regions at the early TAZ activation time point and then gradually spread to promoter regions. TAZ bound to enhancer regions following a switch in potential TEAD and FOSL2 transcription factor motifs. Furthermore, the ATAC sequencing analysis indicated that TAZ activation led to chromatin structural alterations. Together, our results have revealed the landscape of genome-wide TAZ binding sites and may lead to improvements in the current understanding of how TAZ regulates the gene expression program that contributes to the development of breast cancer.

FOSL2 promotes intertumoral infiltration of T cells and increases pathological complete response rates in locally advanced rectal cancer patients.

The outcome of neoadjuvant chemoradiotherapy (nCRT) remains highly unpredictable for individuals with locally advanced rectal cancer (LARC). We set out to characterize effective biomarkers that promote a pathological complete response (pCR). We quantified the abundances of 6483 high-confidence proteins in pre-nCRT biopsies of 58 LARC patients from two hospitals with pressure cycling technology (PCT)-assisted pulse data-independent acquisition (PulseDIA) mass spectrometry. Compared with non-pCR patients, pCR patients achieved long-term disease-free survival (DFS) and had higher tumor immune infiltration, especially CD8+ T cell infiltration, before nCRT. FOSL2 was selected as the candidate biomarker for predicting pCR and was found to be significantly upregulated in pCR patients, which was verified in another 54 pre-nCRT biopsies of LARC patients by immunohistochemistry. FOSL2 expression was able to predict pCR by multiple reaction monitoring (MRM) with high efficiency (Area under curve (AUC) = 0.939, specificity = 1.000, sensitivity = 0.850), and high FOSL2 expression was associated with long-term DFS (p = 0.044). When treated with simulated nCRT, FOSL2 sufficiency resulted in more significant inhibition of cell proliferation, and more significant promotion of cell cycle arrest and cell apoptosis. Moreover, CXCL10 secretion with abnormal cytosolic dsDNA accumulation was found in FOSL2-wildtype (FOSL2-WT) tumor cells over nCRT, which might elevate CD8+ T-cell infiltration and CD8+ T-cell-mediated cytotoxicity to promote nCRT-induced antitumor immunity. Our study revealed proteomic profiles in LARC patients before nCRT and highlighted immune activation in the tumors of patients who achieved pCR. We identified FOSL2 as a promising biomarker to predict pCR and promote long-term DFS by contributing to CD8+ T-cell infiltration.

Inhibition of FOSL2 aggravates the apoptosis of ovarian cancer cells by promoting the formation of inflammasomes.

BACKGROUND: Ovarian cancer is a common gynecological malignancy among female patients and poses a serious threat to women's health. Although it has been established that Fos-like antigen 2 (FOSL2) is linked to ovarian cancer (OC), its exact role in the development of OC remains unknown. OBJECTIVE: This article aims to investigate the role of FOSL2 in ovarian cancer development. METHODS: FOSL2 expression in ovarian carcinoma and adjacent tissues was assessed using real-time fluorescent quantitative PCR and western blot. We constructed OE/sh-FOSL2 plasmids and Caspase-1 specific inhibitors (Yvad-CMK) and transfected A 2780 cells with them to identify the relevant cell functions. Furthermore, we used western blot assay to determine the changes in expression of apoptosis-associated speck-like protein containing a CARD (ASC), cysteine aspartate-specific proteasezymogen procaspase 1 (pro-caspase-1), cysteinyl aspartate-specific proteinase-1 (caspase-1), interleukin-1ß precursor (pro-IL-1ß), interleukin-1ß (IL-1ß), interleukin-18 precursor (pro-IL-18), and interleukin-18 (IL-18). In addition, we measured the concentration of IL-1ß and IL-18 using an enzyme-linked immunosorbent assay (ELISA). Moreover, Tthe level of lactate dehydrogenase (LDH) in the cell supernatant was measured by LDH release assay kit. RESULTS: The expression of FOSL2 was significantly higher compared with the surrounding tissues. The proliferation, migration, and invasion of A2780 cells were enhanced after transfection with OE-FOSL2 plasmids; however, the cell apoptosis was significantly decreased. When FOSL2 was overexpressed, the inflammasome-associated proteins such as ASC, caspase-1, IL-1ß, and IL-18 were downregulated. Furthermore, FOSL2 induced apoptosis and activated the production of inflammasomes in A2780 cells. Co-therapy with Yvad-CMK and substantially inhibited apoptosis and activation of inflammasomes. CONCLUSIONS: Inhibition of FOSL2 promotes the apoptosis of OC cells by mediating the formation of an inflammasome.

FOSL2 deficiency delays nonalcoholic steatohepatitis progression by regulating LY6D-mediated NLRP3 activation.

Lymphocyte antigen 6 family member D (LY6D) was enhanced specifically in senescent cells, while its effects on pyroptosis, a programmed cell death, remains unknown. The goal of this study was to assess the role of LY6D in the mediation of pyroptosis during nonalcoholic steatohepatitis (NASH). After screening out LY6D as a specific liver fibrosis-associated gene using the GSE55747 dataset from the GEO database, we established a NASH mouse model using methionine and choline deficient-diet feeding and an in vitro model using lipopolysaccharide (LPS)-treated hepatocytes. LY6D was overexpressed in NASH livers as well as in LPS-treated hepatocytes. Silencing of LY6D inhibited NASH-associated hepatocyte pyroptosis. With the aid of bioinformatics analysis, promoter-luciferase reporter and ChIP-qPCR assays, we identified FOSL2 as an upstream transcription factor of LY6D. FOSL2, which was highly expressed in NASH, promoted LY6D transcription by binding to the promoter of LY6D. Depletion of FOSL2 significantly inhibited NASH-associated hepatocyte pyroptosis, which was significantly reversed after overexpression of LY6D. Moreover, the promotion of hepatocyte pyroptosis by the FOSL2/LY6D axis was significantly attenuated by specific inhibition of NLRP3. These findings suggesting that FOSL2/LY6D axis may be a key molecular axis and a potential target for NASH therapeutics.