Objective: The transcription factor forkhead box protein O1 (FOXO1) is critical for regulating cytokine and chemokine secretion. However, its function in the tumor microenvironment (TME) remains largely unexplored. In this study, we characterized the prognostic value of FOXO1 and the interaction between tumor-derived FOXO1 and M2 macrophages in esophageal squamous cell carcinoma (ESCC). Methods: FOXO1 expression and macrophage infiltration in clinical samples and mouse models were quantified using quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry staining. Western blotting, qRT-PCR, and enzyme-linked immunosorbent assay were used to evaluate chemokine ligand 20 (CCL20) and colony stimulating factor 1 (CSF-1) expression in FOXO1(+) and FOXO1(-) tumor cells. Macrophage phenotypes were determined using qRT-PCR, flow cytometry, and RNA sequencing. Transcriptional activity was measured using chromatin immunoprecipitation (ChIP)-qPCR. Tumor viability was investigated using XTT proliferation and foci formation assays. Results: FOXO1 upregulation in tumor tissues was found to drive the polarization of M0 macrophages and infiltration of M2 macrophages into the TME, resulting in worse prognosis in ESCC patients. CSF-1, a vital factor inducing M0-to-M2 polarization, was upregulated via a FOXO1-mediated mechanism. RNA sequencing results corroborated that the FOXO1-induced macrophages exhibited similar molecular signatures to the IL4-stimulated M2 macrophages. The transwell assays showed that FOXO1 promoted the migration of M2 macrophages via CCL20 secretion, which could be inhibited using an anti-CCL20 antibody. FOXO1(+) tumor-induced M2 macrophages promoted tumor proliferation via the FAK-PI3K-AKT pathway and the PI3K inhibitor could effectively impede the oncogenical process. Conclusions: FOXO1 facilitated M0-to-M2 polarization and the recruitment of M2 macrophages in the TME via the transcriptional modulation of CCL20 and CSF-1. Our data deciphered the FOXO1-dependent mechanism in M2 macrophage infiltration in the TME of ESCC, which has implications for the development of novel prognostic and therapeutic targets to optimize the current treatment against ESCC.

Long noncoding RNAs (LncRNAs), including MALAT1, are critical regulators of tumor development. However, the roles and molecular mechanisms of LncRNAs in cutaneous squamous cell carcinoma (cSCC) remain underexplored. In this study, functional studies using in vitro cellular and in vivo xenograft models confirmed the pro-carcinogenic roles of MALAT1 in cSCC. Further, MALAT1 was identified to regulate epidermal growth factor receptor (EGFR) protein expression but did not affect EGFR mRNA expression. Transcriptomic sequencing identified kinectin 1 (KTN1) as the key mediator for MALAT1 regulation of EGFR. Mechanistic study revealed that MALAT1 interacts with c-MYC to form a complex and directly binds to the promoter region of KTN1 gene and enhances its transactivation to positively regulate EGFR protein expression. Our findings, therefore, establish a novel c-MYC-assisted MALAT1-KTN1-EGFR axis, which contributes to cSCC development and may serve as novel target for therapeutic intervention.

The importance of immunosuppressive myeloid-derived suppressor cells (MDSCs) bearing monocyte markers in tumor metastasis has been well established. Recently, it was reported that these cells possess phenotypic plasticity and differentiate into fibrocytes, very distinct cells that are precursors of tumorigenic myofibroblasts. However, the importance of this transdifferentiation in tumor metastasis has not been explored. Here, we describe the role of MDSC-derived fibrocytes in tumor metastasis that is regulated by Kruppel-like factor 4 (KLF4), a transcription factor that is critical to monocyte differentiation and to promotion of cancer development. Using mouse metastasis models of melanoma and breast cancer, we found that KLF4 knockout was associated with significantly reduced pulmonary metastasis, which was accompanied by decreased populations of MDSCs, fibrocytes and myofibroblasts in the lung. Cause-effect studies by adoptive transfer revealed that KLF4 deficiency in MDSCs led to significantly reduced lung metastasis that was associated with fewer MDSC-derived fibrocytes and myofibroblasts. Mechanistically, KLF4 deficiency significantly compromised the generation of fibrocytes from MDSCs in vitro. During this process, KLF4 expression levels were tightly linked with those of fibroblast-specific protein-1 (FSP-1), deficiency of which resulted in no metastasis in mice as has been previously reported. In addition, KLF4 bound directly to the FSP-1 promoter as determined by chromatin immunoprecipitation and overexpression of KLF4 increased the FSP-1 promoter activities. Taken together, our results suggest that MDSCs not only execute their immunosuppressive function to promote metastatic seeding as reported before, but also boost metastatic tumor growth after they adopt a fibrocyte fate. Therefore, KLF4-mediated fibrocyte generation from MDSCs may represent a novel mechanism of MDSCs contributing to tumor metastasis and supports the feasibility of inhibiting KLF4 or FSP-1 to prevent tumor metastasis.

Gibberellic acid-stimulated Arabidopsis (GASA) proteins, as cysteine-rich peptides (CRPs), play roles in development and reproduction and biotic and abiotic stresses. Although the GASA gene family has been identified in plants, the knowledge about GASAs in Populus euphratica, the woody model plant for studying abiotic stress, remains limited. Here, we referenced the well-sequenced Populus trichocarpa genome, and identified the GASAs in the whole genome of P. euphratica and P. trichocarpa. 21 candidate genes in P. trichocarpa and 19 candidate genes in P. euphratica were identified and categorized into three subfamilies by phylogenetic analysis. Most GASAs with signal peptides were located extracellularly. The GASA genes in Populus have experienced multiple gene duplication events, especially in the subfamily A. The evolution of the subfamily A, with the largest number of members, can be attributed to whole-genome duplication (WGD) and tandem duplication (TD). Collinearity analysis showed that WGD genes played a leading role in the evolution of GASA genes subfamily B. The expression patterns of P. trichocarpa and P. euphratica were investigated using the PlantGenIE database and the real-time quantitative PCR (qRT-PCR), respectively. GASA genes in P. trichocarpa and P. euphratica were mainly expressed in young tissues and organs, and almost rarely expressed in mature leaves. GASA genes in P. euphratica leaves were also widely involved in hormone responses and drought stress responses. GUS activity assay showed that PeuGASA15 was widely present in various organs of the plant, especially in vascular bundles, and was induced by auxin and inhibited by mannitol dramatically. In summary, this present study provides a theoretical foundation for further research on the function of GASA genes in P. euphratica.

Seven polymethoxyflavones (PMFs) were isolated from the dried peels of hallabong, the hybrid Citrus , by a repeated column chromatography. The structures of PMFs were identified as 5,6,7,3',4'-pentamethoxyflavone (1), 6,7,8,3',4'-pentamethoxyflavone (2), 3-hydroxy-5,6,7,4'-tetramethoxyflavone (3), 5,6,7,8,3',4'-hexamethoxyflavone (4), 3,6,7,4'-tetramethoxyflavone (5), 3,5,6,7,8,3',4'-heptamethoxyflavone (6), and 5,6,7,8,4'-pentamethoxyflavone (7) using 1H and 13C NMR in combination with mass spectrometry. Among these compounds, 5 was isolated for the first time from nature. The content of PMFs 1-7 in hallabong was determined by HPLC-UV. The major PMFs of hallabong are 5 in the dried peels (15.4 mg/g) and 7 in the dried leaves (12.2 mg/g).

Bovine mastitis is an inflammatory condition of the mammary gland often caused by (Staphylococcus aureus) S. aureus infection. The aim of this study was to identify mastitis-related miRNAs and their downstream target genes, and therefore elucidate the regulatory mechanisms involved in disease progression and resistance. Three healthy and three mastitic cows were identified on the basis of the somatic cell count and bacterial culture of their milk, and the histological examination of udder tissues. High-throughput RNA sequencing and bioinformatic analyses revealed that 48 differentially expressed miRNAs (DEMs) in the mastitic udder tissues relative to the healthy tissues. Among 48 DEMs, the expression level of bta-miR-223 was the most up-regulated. Overexpression of the bta-miR-223 in Mac-T cells mitigated the inflammatory pathways induced by S. aureus-derived lipoteichoic acid (LTA). The Cbl proto-oncogene B (CBLB) was identified as the target gene of bta-miR-223, and the direct binding of the miRNA to the CBLB promoter was confirmed by dual luciferase reporter assay using wild-type and mutant 3'-UTR constructs. Furthermore, overexpression of CBLB in the LTA-stimulated Mac-T cells significantly upregulated PI3K, AKT, and phosphorylated NF-κB p65, whereas CBLB knockdown had the opposite effect. Consistent with the in vitro findings, the mammary glands of mice infected with 10 8 CFU/100 µL S. aureus showed high levels of CBLB, PI3K, AKT, and p-NF-κB p65 48 h after infection. Taken together, bta-miR-223 is a predominant miRNA involved in mastitis, and bta-miR-223 likely mitigates the inflammatory progression by targeting CBLB and inhibiting the downstream PI3K/AKT/NF-κB pathway.

The current study focused on how dihydrotestosterone (DHT) regulates synaptic plasticity in the hippocampus of mild cognitive impairment male senescence-accelerated mouse prone 8 (SAMP8) mice. Five-month-old SAMP8 mice were divided into the control, castrated and castrated-DHT groups, in which the mice were castrated and treated with physiological doses of DHT for a period of 2 months. To determine the regulatory mechanisms of DHT in the cognitive capacity, the effects of DHT on the morphology of the synapse and the expression of synaptic marker proteins in the hippocampus were investigated using immunohistochemistry, qPCR and western blot analysis. The results showed that the expression of cAMP-response element binding protein (CREB), postsynaptic density protein 95 (PSD95), synaptophysin (SYN) and developmentally regulated brain protein (Drebrin) was reduced in the castrated group compared to the control group. However, DHT promoted the expression of CREB, PSD95, SYN and Drebrin in the hippocampus of the castrated-DHT group. Thus, androgen depletion impaired the synaptic plasticity in the hippocampus of SAMP8 and accelerated the development of Alzheimer's disease (AD)-like neuropathology, suggesting that a similar mechanism may underlie the increased risk for AD in men with low testosterone. In addition, DHT regulated synaptic plasticity in the hippocampus of mild cognitive impairment (MCI) SAMP8 mice and delayed the progression of disease to Alzheimer's dementia. In conclusion, androgen-based hormone therapy is a potentially useful strategy for preventing the progression of MCI in aging men. Androgens enhance synaptic markers (SYN, PSD95, and Drebrin), activate CREB, modulate the fundamental biology of synaptic structure, and lead to the structural changes of plasticity in the hippocampus, all of which result in improved cognitive function.

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