Investigating the Correlation Between Long-Term Response in Patients with Metastatic HER2+ Breast Cancer and the Activity of Regulatory T Cells: A Retrospective Study.

Background: Trastuzumab is commonly utilized in the management of metastatic HER2-positive breast cancer. Our main goal was to examine the clinical outcomes and immune markers of patients who received trastuzumab and chemotherapy treatment. Methods: Between 1995 and 2012, a total of 98 patients diagnosed with metastatic HER2-positive breast cancer were retrospectively analyzed at Ege University Hospital and Tepecik Training and Research Hospital. The clinicopathological characteristics and clinical outcomes of the patients were assessed, and the associations between response rates, survival and the immune profiles of tumor infiltrating lymphocytes were statistically evaluated. Results: The average age of patients at the time of diagnosis was 50.1±10.3 (ranging from 30 to 79) years. The mean follow-up period for all patients was 97.9±53.8 months. Among the patients, complete response was observed in 24.5%, partial response in 61.2%, and stable disease in 8.2% of cases. The average progression-free survival was 50.3±26.9 months (ranging from 1 to 163 months), and the average overall survival was 88.8±59.4 months (ranging from 12 to 272 months). After analyzing all cases, it was found that patients who were younger (p=0.006), exhibited higher CD3-positivity (p=0.041), presented with higher FOXP3-positivity (p=0.025), showed complete or at least partial response to treatment (p=0.008), and experienced a long-term response to trastuzumab (and chemotherapy) treatment had longer survival (p=0.001). Conclusion: Patients with HER2-positive breast cancer, who initially respond positively to palliative trastuzumab and chemotherapy treatment, can achieve long-term tumor remission lasting for several years.

Blockade of forkhead box protein O1 signaling alleviates primary sclerosing cholangitis-induced sarcopenia in mice model.

AIMS: Primary sclerosing cholangitis (PSC) is a cholestatic liver disease that affects the hepatic bile ducts, leading to hepatic inflammation and fibrosis. PSC can also impact skeletal muscle through the muscle-liver axis, resulting in sarcopenia, a complication characterized by a generalized loss of muscle mass and strength. The underlying mechanisms and therapy of PSC-induced sarcopenia are not well understood, but one potential regulator is the transcription factor forkhead box protein O1 (FOXO1), which is involved in the ubiquitin proteasome system. Thus, the aim of this study is to assess the pharmacological potential of FOXO1 inhibition for treating PSC-induced sarcopenia. MATERIALS AND METHODS: To establish diet-induced PSC model, we provided mice with a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet for 4 weeks. Mice were intramuscularly injected with AS1842856 (AS), a FOXO1 inhibitor, at a dose of 3.5 mg/kg twice a week for last two weeks. C2C12 myotubes with cholic acid (CA) or deoxycholic acid (DCA) were treated with AS. KEY FINDINGS: We observed a decrease in muscle size and performance in DDC-fed mice with upregulated expression of FOXO1 and E3 ligases such as ATROGIN1 and MuRF1. We found that myotube diameter and MyHC protein level were decreased by CA or DCA in C2C12 myotubes, but treatment of AS reversed these reductions. We observed that intramuscular injection of AS effectively mitigates DDC diet-induced sarcopenia in a rodent PSC model. SIGNIFICANCE: Our study suggests that a FOXO1 inhibitor could be a potential leading therapeutic drug for relieving PSC-induced sarcopenia.

Conserved linear motif within the immediate early protein ORF45 promotes its engagement with KSHV lytic cycle-promoting forkhead transcription factors, FOXK1 and FOXK2.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that can cause several cancers, such as Kaposi sarcoma and primary effusion lymphoma (PEL). We and others have recently demonstrated that Forkhead box (FOX) transcription factors can be dysregulated by KSHV, and they can affect KSHV infection. Herein, we focus on dissecting the role of two FOXK subfamily members, FOXK1 and FOXK2, in the KSHV life cycle. FOXK proteins are key host regulators of cellular functions, yet their role in KSHV infection remains unknown. Here, we demonstrated that both FOXK proteins are essential for efficient KSHV lytic reactivation in PEL cells. FOXK1 and FOXK2 are unique as they are the only FOX proteins that contain a Forkhead-associated (FHA) domain. The FHA domain is a specialized protein binding domain that recognizes a short linear serine/threonine-rich (S/T) motif. Through an unbiased motif survey, we found that KSHV viral protein ORF45 and its gammaherpesvirus homologs contain a putative FHA-binding motif. ORF45 is an immediate early tegument protein, vital for lytic reactivation and virus production. We demonstrated that ORF45 uses its novel conserved motif to interact with the FHA domain containing FOXK factors in the nucleus of infected cells. A single-point mutation of the conserved threonine residue in the motif within ORF45 abolished the ORF45-FOXK1/2 interaction. Our data indicates that FOXK proteins interact with ORF45 homologs encoded by murine gammaherpesvirus 68 (MHV68) and Rhesus macaque rhadinovirus (RRV), and that the FHA domains of FOXK proteins are sufficient for their interactions, highlighting a conserved mechanism.IMPORTANCEThe dysregulation of Forkhead transcription factors contributes to many different human diseases, including cancers, but their impact on herpesvirus lifecycle and pathogenesis is less understood. Our study uncovers a critical pro-lytic function of the FOXK subfamily and its requirement for KSHV lytic reactivation in PEL. We found that FOXK proteins bind to a key immediate early KSHV protein ORF45 using its novel short linear S/T motif. Notably, even though ORF45 homologs in gammaherpesviruses are highly diverse, we identified a similar S/T short linear motif in ORF45 homologs and also showed an evolutionary conserved interaction between FOXK proteins and ORF45 homologs of MHV68 and RRV. Our study provides a basis for future studies in animal models to evaluate the role of FOXK proteins and the impact of their interactions with ORF45 in gammaherpesvirus infection and pathogenesis. Targeting these interactions could allow a novel way to limit gammaherpesvirus infections.

MicroRNAs as Epigenetic Regulators of Obesity.

In obesity, the process of adipogenesis largely determines the number of adipocytes in body fat depots. Adipogenesis is regulated by several adipocyte-selective micro-ribonucleic acids (miRNAs) and transcription factors that modulate adipocyte proliferation and differentiation. However, some miRNAs block the expression of master regulators of adipogenesis. Since the specific miRNAs display different expressions during adipogenesis, in mature adipocytes and permanent obesity, their use as biomarkers or therapeutic targets is feasible. Upregulated miRNAs in persistent obesity are downregulated during adipogenesis. Moreover, some of the downregulated miRNAs in obese individuals are upregulated in mature adipocytes. Induction of adipocyte stress and hypertrophy leads to the release of adipocyte-derived exosomes (AdEXs) that contain the cargo molecules, miRNAs. miRNAs are important messengers for intercellular communication involved in metabolic responses and have very specific signatures that direct the metabolic activity of target cells. While each miRNA targets multiple messenger RNAs (mRNAs), which may coordinate or antagonize each other's functions, several miRNAs are dysregulated in other tissues during obesity-related comorbidities. Deletion of the miRNA-processing enzyme DICER in pro-opiomelanocortin-expressing cells results in obesity, which is characterized by hyperphagia, increased adiposity, hyperleptinemia, defective glucose metabolism, and alterations in the pituitary-adrenal axis. In recent years, RNA-based therapeutical approaches have entered clinical trials as novel therapies against overweight and its complications. Development of lipid droplets, macrophage accumulation, macrophage polarization, tumor necrosis factor receptor-associated factor 6 activity, lipolysis, lipotoxicity, and insulin resistance are effectively controlled by miRNAs. Thereby, miRNAs as epigenetic regulators are used to determine the new gene transcripts and therapeutic targets.

METTL14 inhibits the proliferation, migration and invasion of prostate cancer cells by increasing m6A methylation of CDK4.

Background: Methyltransferase-like (METTL) plays an important role in various biological processes, but its role in prostate cancer (PCa) is still unclear. This study aimed to explore the mechanism by which methyltransferase-like 14 (METTL14) inhibits the physiological activity of PCa cells by increasing the N6-methyladenosine (m6A) modification of cyclin-dependent kinase 4 (CDK4). Methods: Clinical samples were collected for bioinformatics analysis. A PCa mouse model was constructed. Cell counting kit-8 (CCK-8), flow cytometry, colony formation assays, scratch assays, Transwell assays, real-time quantitative polymerase chain reaction (RT-qPCR), immunofluorescence and western blotting were used to detect the corresponding indicators. Results: METTL14 was found to be beneficial to inhibit the proliferation, invasion, and migration of PCa cells. When the m6A RNA increased, the half-life of CDK4 mRNA decreased after oe-METTL14 (overexpression of METTL14). Overexpression of CDK4 reversed the effect of oe-METTL14. Coimmunoprecipitation experiments revealed there were interactions between CDK4 and forkhead box M1 (FOXM1). Transfection of si-CDK4 was similar to transfection of oe-METTL14. After transfection with oe-FOXM1, the invasion and migration ability of cells increased, and cell apoptosis decreased. After transfection with si-FOXM1 alone, autophagy related 7 (ATG7) expression was significantly downregulated, and autophagy levels were reduced. The overexpression of ATG7 reversed the effect of si-FOXM1. The tumor volume and weight of the oe-METTL14 group mice were significantly reduced, and tumor proliferation was decreased in comparison to untreated tumor-bearing mice. Conclusions: METTL14 inhibits the invasion and migration of PCa cells and induces cell apoptosis by inhibiting CDK4 stability and FOXM1/ATG7-mediated autophagy.

Inhibition of FoxO1 ameliorates hepatic steatosis and hepatitis in nonalcoholic steatohepatitis mice through regulation of gut microbiota.

OBJECTIVE: We aimed to investigate the role of forkhead box O1 (FoxO1) inhibitor AS1842856 (AS) in nonalcoholic steatohepatitis (NASH) mice and the potential mechanisms. METHODS: Mice were given methionine-choline-sufficient (MCS), or methionine- and choline-deficient (MCD) diet for 5 weeks, along with AS (60 mg/kg) or vehicle gavage treatment (0.2 mL/day). Body and liver weight, serum triglyceride (TG), low-density lipoprotein-cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C), alanine aminotransferase (ALT), aspartate aminotransferase (AST), fasting glucose and insulin levels were measured. Liver macrophage infiltration and ileal ZO-1 protein expression were also detected. Interleukin (IL)-6, IL-1ß, and tumor necrosis factor (TNF)-α, sterol regulatory element binding protein (SREBP)-1c, phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (G6Pase), α-smooth muscle actin (SMA), recombinant collagen type III α1 (Col3a1), and connective tissue growth factor (Ctgf) expressions were measured. Stool samples were collected for 16S rDNA sequencing. RESULTS: Compared to the MCD group, AS attenuated liver weight, reduced serum TG, ALT, and AST levels, increased HDL-C levels, mitigated hepatic steatosis, decreased macrophage infiltration, and augmented ileal ZO-1 proteins in NASH mice. It also reduced the levels of IL-6, IL-1ß, and TNF-α, alongside with the Srebp-1c mRNA expression. However, no significant effects on Pepck, G6Pase, α-SMA, Col3a1, or Ctgf were observed. Furthermore, AS promoted diversity and altered gut microbiota composition in NASH mice, causing increased beneficial bacteria like Akkermansia muciniphila, Parabacteroides distasonis, and Prevotellamassilia, which were associated with metabolic functions. CONCLUSION: FoxO1 inhibitor AS ameliorated hepatic steatosis, inflammation, and intestinal dysbiosis in NASH mice, making it a potentially promising treatment for NASH.

FOXO1-NCOA4 Axis Contributes to Cisplatin-Induced Cochlea Spiral Ganglion Neuron Ferroptosis via Ferritinophagy.

Mammalian cochlea spiral ganglion neurons (SGNs) are crucial for sound transmission, they can be damaged by chemotherapy drug cisplatin and lead to irreversible sensorineural hearing loss (SNHL), while such damage can also render cochlear implants ineffective. However, the mechanisms underlying cisplatin-induced SGNs damage and subsequent SNHL are still under debate and there is no currently effective clinical treatment. Here, this study demonstrates that ferroptosis is triggered in SGNs following exposure to cisplatin. Inhibiting ferroptosis protects against cisplatin-induced SGNs damage and hearing loss, while inducing ferroptosis intensifies these effects. Furthermore, cisplatin prompts nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy in SGNs, while knocking down NCOA4 mitigates cisplatin-induced ferroptosis and hearing loss. Notably, the upstream regulator of NCOA4 is identified and transcription factor forkhead box O1 (FOXO1) is shown to directly suppress NCOA4 expression in SGNs. The knocking down of FOXO1 amplifies NCOA4-mediated ferritinophagy, increases ferroptosis and lipid peroxidation, while disrupting the interaction between FOXO1 and NCOA4 in NCOA4 knock out mice prevents the cisplatin-induced SGN ferroptosis and hearing loss. Collectively, this study highlights the critical role of the FOXO1-NCOA4 axis in regulating ferritinophagy and ferroptosis in cisplatin-induced SGNs damage, offering promising therapeutic targets for SNHL mitigation.

Autocrine IL-8 Contributes to Propionibacterium Acnes-induced Proliferation and Differentiation of HaCaT Cells via AKT/FOXO1/ Autophagy.

OBJECTIVE: Proprionibacterium acnes (P. acnes)-induced inflammatory responses, proliferation and differentiation of keratinocytes contribute to the progression of acne vulgaris (AV). P. acnes was found to enhance the production of interleukin-8 (IL-8) by keratinocytes. This study aimed to investigate the role of IL-8 in P. acnes-induced proliferation and differentiation of keratinocytes and the underlying mechanism. METHODS: The P. acnes-stimulated HaCaT cell (a human keratinocyte cell line) model was established. Western blotting and immunofluorescence were performed to detect the expression of the IL-8 receptors C-X-C motif chemokine receptor 1 (CXCR1) and C-X-C motif chemokine receptor 2 (CXCR2) on HaCaT cells. Cell counting kit-8 (CCK-8) assay, 5-ethynyl-20-deoxyuridine (EdU) assay and Western blotting were performed to examine the effects of IL-8/CXCR2 axis on the proliferation and differentiation of HaCaT cells treated with P. acnes, the IL-8 neutralizing antibody, the CXCR2 antagonist (SB225002), or the CXCR1/CXCR2 antagonist (G31P). Western blotting, nuclear and cytoplasmic separation, CCK-8 assay, and EdU assay were employed to determine the downstream pathway of CXCR2 after P. acnes-stimulated HaCaT cells were treated with the CXCR2 antagonist, the protein kinase B (AKT) antagonist (AZD5363), or the constitutively active forkhead box O1 (FOXO1) mutant. Finally, autophagy markers were measured in HaCaT cells following the transfection of the FOXO1 mutant or treatment with the autophagy inhibitor 3-methyladenine (3-MA). RESULTS: The expression levels of CXCR1 and CXCR2 were significantly increased on the membrane of HaCaT cells following P. acnes stimulation. The IL-8/CXCR2 axis predominantly promoted the proliferation and differentiation of P. acnes-induced HaCaT cells by activating AKT/FOXO1/autophagy signaling. In brief, IL-8 bound to its receptor CXCR2 on the membrane of keratinocytes to activate the AKT/FOXO1 axis. Subsequently, phosphorylated FOXO1 facilitated autophagy to promote the proliferation and differentiation of P. acnes-induced keratinocytes. CONCLUSION: This study demonstrated the novel autocrine effect of IL-8 on the proliferation and differentiation of P. acnes-induced keratinocytes, suggesting a potential therapeutic target for AV.

Molecular expression of Forkhead Box C2 gene (FOXC2) and Prospero homeobox gene (PROX-1) in oral squamous carcinoma and their correlation with clinicopathological parameters: A prospective cohort study.

Background: Forkhead box C2 gene (FOXC2) acts as an epithelial-mesenchymal transition (EMT) inducer while Prospero homeobox 1 gene (PROX-1) function as a regulator of lymphangiogenesis and angiogenesis in oral squamous cell carcinoma (OSCC). It is presumed that PROX-1 has both tumour-suppressive and oncogenic effects. The main aim of this study is to evaluate the role of PROX-1 and FOXC2 in the invasion and progression of OSCC cases and to correlate their expression with various histopathological parameters. Materials and Methods: A prospective cohort study was conducted in a total sample size of 52 OSCC tissues and histologically tumour-free margins of 20. mRNA expression and protein levels of FOXC2 and PROX-1 were evaluated using real-time PCR and sandwich enzyme-linked immunosorbent assay techniques. Chi-square analysis and correlation analysis were done. Kaplan-Meier analysis evaluated the survival rate. Results: Mean Ct values of FOXC2 were 1.915 ± 0.519 and PROX-1 was 0.061 ± 0.173. There was a significant 2-fold increase in the FOXC2 expression and a 0.5-fold decrease in the PROX-1 expression in OSCC tissue. Increased levels of FOXC2 protein and decreased levels of PROX-1 with a mean difference of 1.64 ± 0.73 ng/ml and 1.27 ± 0.33 ng/ml were observed in OSCC compared to histologically tumour-free margins. A significant positive correlation was found between the FOXC2 expression and clinicopathological parameters such as staging, perineural invasion (PNI) and lymphovascular invasion (LVI) whereas PROX-1 showed a significant negative correlation with histopathological parameters such as staging, PNI, LVI and tumour staging. There was a significant positive correlation between the PROX-1 and histologically tumour-free margins in disease-free survival patients (P-value = 0.03). Conclusion: FOXC2 and PROX-1 expressions were correlated with lymphovascular invasion, OSCC tumour staging and PNI. Thus, FOXC2 and PROX-1 could be possible therapeutic targets in the treatment of OSCC that can inhibit the EMT in OSCC and thereby favouring a better prognosis.

Advances in Developing Small Molecule Drugs for Alzheimer's Disease.

Alzheimer's disease (AD) is the most common type of dementia among middle-aged and elderly individuals. Accelerating the prevention and treatment of AD has become an urgent problem. New technology including Computer-aided drug design (CADD) can effectively reduce the medication cost for patients with AD, reduce the cost of living, and improve the quality of life of patients, providing new ideas for treating AD. This paper reviews the pathogenesis of AD, the latest developments in CADD and other small-molecule docking technologies for drug discovery and development; the current research status of small-molecule compounds for AD at home and abroad from the perspective of drug action targets; and the development trend of new drug development for AD in the future.