Forkhead Box Protein 3 Expression Research Articles

Increased miR-6132 promotes deep vein thrombosis formation by downregulating FOXP3 expression.

Deep vein thrombosis (DVT) is associated with aberrant gene expression that is a common peripheral vascular disease. Here, we aimed to elucidate that the epigenetic modification of forkhead box protein 3 (FOXP3) at the post-transcriptional level, which might be the key trigger leading to the down-regulation of FOXP3 expression in DVT. In order to explore the relationship between microRNAs (miRNAs) and FOXP3, mRNA and microRNA microarray analysis were performed. Dual luciferase reporter assay was used to verify the upstream miRNAs of FOXP3. Quantitative real-time polymerase chain reaction, flow cytometry and Western blot were used to detect the relative expression of miR-6132 and FOXP3. Additionally, DVT models were established to investigate the role of miR-6132 by Murine Doppler Ultrasound and Hematoxylin-Eosin staining. Microarray and flow cytometry results showed that the FOXP3 expression was decreased while miR-6132 level was increased substantially in DVT, and there was significant negative correlation between miR-6132 and FOXP3. Moreover, we discovered that overexpressed miR-6132 reduced FOXP3 expression and aggravated DVT formation, while miR-6132 knockdown increased FOXP3 expression and alleviated DVT formation. Dual luciferase reporter assay validated the direct binding of miR-6132 to FOXP3. Collectively, our data elucidate a new avenue through which up-regulated miR-6132 contributes to the formation and progression of DVT by inhibiting FOXP3 expression.

Identification of FOXP3+ epithelial cells contributing to pancreatic proliferation and angiogenesis.

Forkhead box protein 3 (FOXP3), traditionally recognized as a specific transcription factor for regulatory T cells (Tregs), has also been identified in various tumor epithelial cells (named as cancer-FOXP3, c-FOXP3). However, the natural state and functional role of FOXP3 positive tumor epithelial cells remain unknown. Monoclonal cells expressing varying levels of c-FOXP3 were isolated from established PANC-1 cells using limited dilution. Whole transcriptome sequencing and weighted gene co-expression network analysis (WGCNA) were conducted on these subsets, followed by in vitro and in vivo functional investigations. In addition, we identified c-FOXP3+E-cadherin- epithelial cells in human pancreatic cancer tissues after radical resection by immunofluorescence co-staining. We also investigated the connection between c-FOXP3+E-cadherin- epithelial cells and their clinicopathological features. Our study uncovered a distinct subset of c-FOXP3+ tumor epithelial cells characterized by reduced E-cadherin expression. C-FOXP3+E-cadherin- cells displayed significant proliferation potential and pro-angiogenic effect through the expression of chemokines, including C-X-C motif ligand 1 (CXCL1), C-X-C motif ligand 5 (CXCL5), and C-X-C motif ligand 8 (CXCL8). Notably, higher counts of c-FOXP3+E-Cadherin- cells correlated with poorer prognosis, lower tumor differentiation, lymph node metastasis, and vascular invasion in pancreatic ductal adenocarcinoma (PDAC). In conclusion, this work revealed the stable expression of FOXP3 in tumor epithelial cells, marking a distinct subset. C-FOXP3+E-cadherin- epithelial cells exhibit active proliferation and promote angiogenesis in a vascular endothelial growth factor A (VEGFA) independent manner. These findings provide novel insights into PDAC prognosis and therapeutic avenues.NEW & NOTEWORTHY In this study, we revealed a novel c-FOXP3+ tumor epithelial cell subset marked by diminished E-cadherin and stable FOXP3 expression. These subpopulations not only show robust proliferation and drive angiogenesis via CXCL1, CXCL5, and CXCL8, bypassing VEGFA pathways, but their heightened presence also correlates with adverse PDAC outcomes. By challenging traditional epithelial cell definitions and extending lymphocyte markers to these cells, our findings present innovative targets for PDAC treatment and enrich our understanding of cell biology.

Radiomics Model for Predicting FOXP3 Expression Level and Survival in Clear Cell Renal Carcinoma

We aimed to evaluate the predictive significance of forkhead box protein 3 (FOXP3) expression levels among individuals with clear cell renal carcinoma (ccRCC) and establish a radiomics model for predicting FOXP3 expression. 430 patients with ccRCC were included in the gene-based prognostic analyses; 100 samples were used for radiomics feature generation, model development, and evaluation. A gradient boosting machine was employed to model the selected radiomics features. The developed model generated radiomics scores (RS) that predicted FOXP3 expression. The FOXP3 prognostic model combining imaging features was applied for survival and clinical indicator correlation analyses. FOXP3 was highly expressed in patients with ccRCC and served as an independent predictive marker (hazard ratio [HR]=2.357, 95% CI [confidence interval]: 1.582-3.511, p<0.001). The radiomics model formed by three radiomics characteristics was identified as a strong prognostic indicator of overall survival (OS). The predictive power of the model was commendable (areas under the curve: 0.835 and 0.809 for training and validation sets, respectively). Significant between-group variations in RS distribution were identified, as indicated by gene expression levels (p<0.05). Disparities were observed in pathological stage, pharmaceutical therapy, and neoplasm status between low and high RS cohorts (p<0.001). Kaplan-Meier curves revealed a significant correlation between increased RS and decreased OS (p=0.001), which was also observed in the multivariate analyses (HR=3.411, 95% CI: 1.039-11.196, p=0.043). Prognostic outcome of ccRCC is closely linked to FOXP3 expression level. Computed tomography-based radiomics shows promise for prognostic prediction in ccRCC.

Tregs are involved in VEGFA/ VASH1-related angiogenesis pathway in ovarian cancer

Vasohibin1 (VASH1) is a kind of vasopressor, produced by negative feedback from vascular endothelial growth factor A (VEGFA). Anti-angiogenic therapy targeting VEGFA is currently the first-line treatment for advanced ovarian cancer (OC), but there are still many adverse effects. Regulatory T cells (Tregs) are the main lymphocytes mediating immune escape function in the tumor microenvironment (TME) and have been reported to influence the function of VEGFA. However, whether Tregs are associated with VASH1 and angiogenesis in TME in OC is unclear. We aimed to explore the relationship between angiogenesis and immunosuppression in the TME of OC. We validated the relationship between VEGFA, VASH1, and angiogenesis in ovarian cancer and their prognostic implications. The infiltration level of Tregs and its marker forkhead box protein 3 (FOXP3) were explored in relation to angiogenesis-related molecules. The results showed that VEGFA and VASH1 were associated with clinicopathological stage, microvessel density and poor prognosis of ovarian cancer. Both VEGFA and VASH1 expression were associated with angiogenic pathways and there was a positive correlation between VEGFA and VASH1 expression. Tregs correlated with angiogenesis-related molecules and indicated that high FOXP3 expression is harmful to the prognosis. Gene set enrichment analysis (GSEA) predicted that angiogenesis, IL6/JAK/STAT3 signaling, PI3K/AKT/mTOR signaling, TGF-β signaling, and TNF-α signaling via NF-κB may be common pathways for VEGFA, VASH1, and Tregs to be involved in the development of OC. These findings suggest that Tregs may be involved in the regulation of tumor angiogenesis through VEGFA and VASH1, providing new ideas for synergistic anti-angiogenic therapy and immunotherapy in OC.

Upregulated miR-146a Expression in Peripheral Blood Relates to Th17 and Treg Imbalance in Elder Rheumatoid Arthritis Patients

Background: The expression level of microRNA-146a (miR-146a) increased in peripheral blood and synovialis tissue of rheumatoid arthritis (RA) patient, and it may play an important role in the pathological process of RA. We investigated its possibility as a diagnostic marker and the correlation with T helper 17 (Th17) and Treg cells in elder RA patients. Methods: Blood samples were collected from 38 active RA patients, 38 inactive RA patients, and 40 healthy controls. RNA expression levels of miR-146a were detected from the peripheral blood samples. The proportion of Th17 and Treg cells were analyzed, as well as their cell-specific transcription factor retinoic acid-related orphan receptor variant 2 (RORc) and forkhead box protein 3 (FOXP3). Furthermore, secretion of pre-inflammatory and anti-inflammatory factors was detected. Correlations between miR-146a and these factors were also analyzed. Results: Compared with healthy control, expression levels of miR-146a in inactive and active groups were significantly higher, with the highest level in active group. The expression of miR-146a and the RA severity, Th17 cell ratio, RORc expression, IL-17 level showed a significant positive correlation, while it showed a significantly negative correlation with Treg cell ration, FOXP3 expression, and TGF-β1 secretion. Conclusions: These results suggested that miR-146a may be used as a disease progression marker in the peripheral blood of elder RA patients.

FOXP3‑induced LINC00885 promotes the proliferation and invasion of cervical cancer cells.

Cervical cancer (CC) is the fourth most common type of cancer in women worldwide. LINC00885, a novel oncogenic type of long non-coding RNA, is upregulated in various types of cancer, but its expression in CC remains unknown. The aim of the present study was to investigate the expression of LINC00885 in CC, and its effect on the proliferation and invasion of CC cells in vitro. The expression levels of LINC00885 and forkhead box protein 3 (FOXP3) from The Cancer Genome Atlas (TCGA) were selected to analyze the differences between CC tissues and normal cervical tissues using bioinformatics analyses. Reverse transcription-quantitative (RT-q)PCR was used to detect the relative expression of LINC00885 in CC cell lines, and its expression was knocked down. Cell Counting Kit-8 assays and EdU staining were used to detect the changes in cell proliferative capacity of cells. Transwell experiments were used to examine cell invasion. Dual-luciferase reporter and chromatin immunoprecipitation assays were performed to examine the interactions between LINC00885 and FOXP3. FOXP3 and epithelial-mesenchymal transition (EMT)-related proteins were detected using western blotting. The expression of LINC00885 and FOXP3 in CC tissues and CC cells was significantly higher compared with the normal cervical tissues and normal cervical epithelial cells. FOXP3 could specifically interact with the promoter of LINC00885 and activate LINC00885 transcription. Knockdown of LINC00885 and silencing of FOXP3 significantly inhibited proliferation, invasion and EMT of CC cells. Overexpression of LINC00885 reversed the inhibitory effects of FOXP3 knockdown on the proliferation and invasion of CC cells. Collectively, LINC00885 and FOXP3 may serve as biomarkers for the early diagnosis of CC and as a potential therapeutic target for reversing the malignant phenotype of CC.

FOXP3 and HER-2/ErbB2 in Breast Cancer: Finding Regulatory Links

The transmembrane tyrosine kinase receptor, HER2/ErbB2, has been a subject of many studies owing to itspredictive and prognostic values and its being a target of antibody-mediated treatment. Retrospective evidencestrongly proposes that the over expression of HER2 is related to reduced disease free and overall survival innode positive and perhaps also node negative breast cancers. Prospective attempts showed that antibodiesto HER2 can form tumor responses in advanced breast cancer women which overexpress this molecule.The existence of forkhead box protein 3 (FOXP3) in cells of breast cancer is a subject of debate. We havesystematically analyzed the FOXP3 expression in 20 breast carcinoma samples at transcript levels. Recentprogresses in understanding breast cancer crosstalk, homing processes, tumor cell dormancy, premetastaticniche formation and finally recognition of their micromilieu cytokines, growth factors and chemokinesmightprovide the foundation of developing targeted treatment plans potentially rendering primary carcinomas andtheir metastases more responsive to chemotherapies. The current review has focused on the deep connectionsbetween HER-2 and FoxP3 in context of considering the determination of both of these molecules togetherto rationalize or more personalize the future research directions leading to better treatment for women withbreast carcinoma.

Gene editing using CRISPR enables FOXP3 gene repair in HSPCs and IPEX patient T cells

Background & Aim Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is the prototypical primary immune regulatory disorder caused by mutations in the forkhead box protein 3 (FOXP3) gene, a critical transcription factor required for regulatory T cells (Tregs). Patients typically present with severe early onset autoimmune manifestations that can be fatal within the first year of life. Currently, the only cure for IPEX is allogeneic hematopoietic stem cell transplant (HSCT), but this comes with risk of complications and the survival probability at 15 years is 73.2% (F. Barzaghi et al 2018). Moreover, a suitable donor is not always available. As a monogenic immune disease with limited treatment options, IPEX is an ideal candidate for a gene therapy approach whereby patient hematopoietic stem cells are gene edited and reinfused for autologous transplant. Methods, Results & Conclusion We developed a CRISPR/Cas9 approach combined with AAV delivery of a donor template to restore FOXP3 expression at the endogenous locus, permitting regulated expression of wild-type FOXP3 irrespective of downstream mutations. We were able to edit CD4+ effector T cells (Teff) and Tregs, showing that they maintain characteristic phenotypic markers and are functional in vitro. IPEX patient Tregs edited with the construct had partial restoration of both FOXP3 expression and functional suppression upon co-culture with responder T cells. Gene edited cord blood hematopoietic stem and progenitor Cells (HSPCs) are capable of long-term engraftment in humanized mice and differentiate into multiple hematopoietic subsets (CD13, CD19, CD56, CD3) and partially reconstitute Tregs (CD4+CD25+FOXP3+). These results demonstrate the feasibility of using gene edited HSPCs and hold promise for autologous transplant in genetic autoimmune diseases such as IPEX syndrome.

FOXP3 promotes colorectal carcinoma liver metastases by evaluating MMP9 expression via regulating S-adenosylmethionine metabolism.

BackgroundGrowing evidence has proved that Forkhead box protein 3 (FOXP3), which is a master regulatory gene in the development and function of regulatory T-cells, is expressed in human cancer cells. This expression indicates the crucial role FOXP3 takes up as the disease progresses. However, its role in colorectal cancer (CRC) liver metastasis is still mostly unknown. This study set out to explore the molecular characteristics of FOXP3 in driving the liver metastasis within CRC.MethodsWe downloaded the RNA-seq data from the GSE50760. Weighted gene co-expression network analysis (WGCNA)WGCNA and RNA-Seq analysis were applied to find the key gene network associated with colorectal cancer liver metastasis. Then we performed pathway enrichment analysis on liver metastasis-associated gene set. Immunohistochemistry, in vitro and in vivo studies were conducted to test expression and function of FOXP3 in CRC tissues and liver metastasis tissues. Non-targeted metabolomics analysis was performed to identify the alteration of FOXP3 expression in metabolites of colorectal cancer liver metastasis. Western blot was performed to confirm changes of matrix metalloproteinase 9MMP9 expression were downstream events of S-adenosyl-methionine (SAM).ResultsWe found that FOXP3 and MMP9 exhibited co-expression relationships and affected liver metastasis in CRC. Upregulation of FOXP3 promotes cell migration and invasion in CRC, which suggests a pro-cancer effect. Moreover, metabolomics analysis showed that knockdown of FOXP3 significantly reduced SAM levels, and changes of MMP9 expression were downstream events of SAM, which is concentration-dependent. Besides, The Kyoto Encyclopedia of Genes and Genomes (KEGG) and Western blot analysis confirmed that overexpression of FOXP3 activates the Wnt pathway to promote colon cancer metastasis.ConclusionsOur results altogether suggested that FOXP3 expression inhibited the SAM cycle to reduce SAMe levels, resulting in altered MMP9 expression and helped CRC liver metastasis.

DNA hypermethylation of the promoter attenuates forkhead box protein 3 (FOXP3) expression in hepatocellular carcinoma cells

Background: Forkhead box protein 3 (FOXP3) plays a pivotal role in tumor progression and impacts the methylation status and levels of expression of an associated promoter region. However, the methylation status and how this parameter impacts the FOXP3 promoter in cases of human hepatocellular carcinoma (HCC) remains unclear. This study sought to investigate the relationship between methylation status of transcriptional FOXP3 gene promoter and the levels of expression of FOXP3 in human HCC afflicted cell lines and tissues. Methods: We used quantitative real-time PCR (Real-time qPCR) to detect the levels of expression of FOXP3 mRNA in human HCC afflicted cell lines and tissues. Immunohistochemistry and western blot were used to examine protein expression levels of FOXP3 in human HCC afflicted cell lines and tissues. Reverse transcription PCR was used to detect the levels of expression of the FOXP3 gene promoter in HCC afflicted cell lines. Finally, methylation sequencing was used to examine the influences of epigenetic regulation upon the FOXP3 promoter region in HCC afflicted cell lines. Results: Levels of FOXP3 expression were down-regulated in HCC afflicted tissues compared with adjacent non-tumorous tissues. Levels of expression of FOXP3 in portal vein tumor thrombus (PVTT) were much lower than were levels in HCC afflicted tissues. Further, FOXP3 was abnormally expressed in HCC afflicted cell lines. The FOXP3 promoter was also abnormally expressed among HCC afflicted cell lines. Lastly, the FOXP3 promoter was hypermethylated in HCC afflicted cell lines. Conclusions: Results suggest that levels of expression of the FOXP3 promoter were decreased for measures of both mRNA and protein expression levels in HCC afflicted tissues and cell lines. The FOXP3 gene promoter region when hypermethylated may help inhibit FOXP3 expression and the development and progression of HCC.

DNA hypermethylation of the promoter attenuates forkhead box protein 3 (FOXP3) expression in hepatocellular carcinoma cells.

BackgroundForkhead box protein 3 (FOXP3) plays a pivotal role in tumor progression and impacts the methylation status and levels of expression of an associated promoter region. However, the methylation status and how this parameter impacts the FOXP3 promoter in cases of human hepatocellular carcinoma (HCC) remains unclear. This study sought to investigate the relationship between methylation status of transcriptional FOXP3 gene promoter and the levels of expression of FOXP3 in human HCC afflicted cell lines and tissues.MethodsWe used quantitative real-time PCR (Real-time qPCR) to detect the levels of expression of FOXP3 mRNA in human HCC afflicted cell lines and tissues. Immunohistochemistry and western blot were used to examine protein expression levels of FOXP3 in human HCC afflicted cell lines and tissues. Reverse transcription PCR was used to detect the levels of expression of the FOXP3 gene promoter in HCC afflicted cell lines. Finally, methylation sequencing was used to examine the influences of epigenetic regulation upon the FOXP3 promoter region in HCC afflicted cell lines.ResultsLevels of FOXP3 expression were down-regulated in HCC afflicted tissues compared with adjacent non-tumorous tissues. Levels of expression of FOXP3 in portal vein tumor thrombus (PVTT) were much lower than were levels in HCC afflicted tissues. Further, FOXP3 was abnormally expressed in HCC afflicted cell lines. The FOXP3 promoter was also abnormally expressed among HCC afflicted cell lines. Lastly, the FOXP3 promoter was hypermethylated in HCC afflicted cell lines.ConclusionsResults suggest that levels of expression of the FOXP3 promoter were decreased for measures of both mRNA and protein expression levels in HCC afflicted tissues and cell lines. The FOXP3 gene promoter region when hypermethylated may help inhibit FOXP3 expression and the development and progression of HCC.

Expression of inflammatory molecules cyclooxygenase-2 and forkhead box protein 3 in Wilms’ tumor microenvironment and their clinicopathological significance

Background and purpose The role of inflammation in cancer has been reported in various adult malignant neoplasms. Cyclooxygenase-2 (COX-2) is overexpressed in invasive breast carcinoma, colonic adenocarcinomas, as well as other tumors, and inhibitors of COX-2 have increasingly become therapeutic alternatives in many tumors. Forkhead box protein 3 (FOXP-3) transcription factor is important for the development of Tregs and hence maintenance of immunologic self-tolerance. Activity of Tregs is increased in lung cancer, gastric, and several adult-onset tumors, and they play a role in suppressing the antitumor immune responses. However, the exact role of these inflammatory molecules in pediatric malignancies especially Wilms’ tumor (WT) has not yet been fully addressed. Aim The aim of the present study was to investigate the expression of the inflammatory molecules COX-2 and FOXP-3 in WTs and their possible association with the different clinicopathological parameters. Patients and methods In this study, 21 formalin-fixed paraffin-embedded specimens of WTs were evaluated. Anaplasia [unfavorable histology (UFH)] was found in 31.6% of the studied tumors. Protein expression of COX-2 and FOXP-3 was investigated using immunohistochemistry. The degree of expression of both COX-2 and FOXP-3 expression was semiquantitatively assessed for each of the studied tumors using modified H-scoring. Results The present study revealed that COX-2 was expressed in the cytoplasm of all studied cases (100%). Its expression was higher in anaplastic WT (UFH) (median H-score, 200) compared with tumors with favorable histology (median H-score, 160). However, this difference was not statistically significant (P=0.167). On the contrary, FOXP-3 expression in WT was mostly low. Its expression varied between an H-score of 10 and 130 (median, 20). No significant difference in expression was noted between favorable histology versus UFH tumors. The present study also demonstrated a significant positive association between expression of COX-2 and FOX-P3 in WTs (P=0.033). Conclusion Our results show that COX-2 is strongly expressed in all WTs with a trend toward higher expression in UFH ones. Furthermore, the significant association between COX-2 expression and FOXP-3 expression in the studied cases suggests that inhibition of COX-2 may lead to an associated suppression of FOXP-3 activity and thereby enhancing the antitumor immune responses in these patient, which is an interesting finding that needs to be further explored.

MKL1/miR34a/FOXP3 axis regulates cell proliferation in gastric cancer.

Megakaryoblastic leukemia 1 (MKL1) was closely related to the pathogenesis of various human malignant cancers. MiR34a was reported to be closely related to cancer cell proliferation. Forkhead box protein 3 (FOXP3) was a transcription factor that played a different role in different cancer types. CDK6 was involved in cell cycle progression and was upregulated in several types of cancers. The present study investigated the effects of MKL1/miR34a/FOXP3 axis on cell proliferation in MGC803 gastric cancer cells. Our results demonstrated that overexpression of MKL1 promoted proliferation of MGC80-3 cells, MKL1 directly binding to the promoter of CDK6 to increase its expression. Knockdown of FOXP3 promoted proliferation of MGC80-3 cells and MKL1 inhibited the expression of FOXP3 via miR-34a. The finding can contribute to elucidating the regulatory mechanism involved in the cell cycle progression of gastric cancer cells and may aid in screening potential gene targets for the biological therapy of gastric cancer.

Effect of PD-1, FOXP3 and CSF-1R Protein Expression on the Prognosis of Patients with Hodgkin's Lymphoma

To investigate the effect of the expression of colony-stimulating factor 1 receptor (CSF-1R), forkhead box protein 3 (FOXP3) and programmed death 1 (PD-1) protein on the prognosis of patients with Hodgkin's lymphoma. The clinical features and treatment schemes of 54 patients with Hodgkin's lymphoma treated in our hospital were recorded, the specimens were collected and the relevant micro-environment prognostic factors such as the protein expression of CSF-1R, FOXP3 and PD-1 were detected by immunohistochemical staining, and EB virus and EB virus-encoded RNA (EBER) were detected by in situ hybridization; the correlation between CSF-1R, FOXP3, PD-1 protein expression with prognosis of patients with Hodgkin lymphoma were analyzed; both the unvariate analysis and multivariate analysis (using Cox proportional hazard model) were uased to analyze the influence factors of prognosis. Among the 54 cases of Hodgkin's lymphoma, 22 cases (40.74%) were positive for CSF-1R, 28 cases (51.85%) showed high expression of FOXP3 and 9 cases (16.67%) were positive for PD-1. Unvariate analysis revealed that the international prognostic index (IPI) score, EBER and FOXP3 protein expression were the effecting factors of progression-free survival (PFS) of patients with Hodgkin's lymphoma (all P<0.05); clinical staging (Ann Arbor stage), IPI score, EBER, CSF-1R and FOXP3 protein expression were the effecting factors of overall survival (OS) (all P<0.05). Multivariate analysis (Cox proportional hazard model) results showed that the EBER state was the effecting factors of PFS and OS in patients with Hodgkin's lymphoma (P<0.05); the expression of FOXP3 protein was the effecting factors of PFS (P<0.05); Ann Arbor stage and the expression of CSF-1R protein were the effecting factors for OS (both P<0.05). CSF-1R and FOXP3 closely relate with the prognosis of the patients with Hodgkin's lymphoma, which can provide a basis for targeted therapy of this disease.

FOXP3 expression is modulated by TGF‑β1/NOTCH1 pathway in human melanoma.

Forkhead box protein 3 (FOXP3) transcription factor is expressed by immune cells and several human cancers and is associated with tumor aggressiveness and unfavorable clinical outcomes. NOTCH and transforming growth factor-β (TGF-β) protumorigenic effects are mediated by FOXP3 expression in several cancer models; however, their interaction and role in melanoma is unknown. We investigated TGF-β-induced FOXP3 gene expression during NOTCH1 signaling inactivation. Primary (WM35) and metastatic melanoma (A375 and A2058) cell lines and normal melanocytes (NHEM) were used. FOXP3 subcellular distribution was evaluated by immuno cytochemical analysis. Gene expression levels were assessed by reverse transcription-quantitative polymerase chain reaction. Protein levels were assessed by western blot analysis. The γ-secretase inhibitor (GSI) was used for NOTCH1 inhibition and recombinant human (rh)TGF-β was used for melanoma cell stimulation. Cell proliferation and viability were respectively assessed by MTT and Trypan blue dye assays. FOXP3 mRNA and protein levels were progressively higher in WM35, A375 and A2058 cell lines compared to NHEM and their levels were further increased after stimulation with rh-TGF-β. TGF-β-mediated FOXP3 expression was mediated by NOTCH1 signaling. Inhibition of NOTCH1 with concomitant rh-TGF-β stimulation determined the reduction in gene expression and protein level of FOXP3. Finally, melanoma cell line proliferation and viability were reduced by NOTCH1 inhibition. The results show that nn increase in FOXP3 expression in metastatic melanoma cell lines is a potential marker of tumor aggressiveness and metastasis. NOTCH1 is a central mediator of TGF-β-mediated FOXP3 expression and NOTCH1 inhibition produces a significant reduction of melanoma cell proliferation and viability.

Label-free quantitative proteomic analysis identifies CTNNB1 as a direct target of FOXP3 in gastric cancer cells.

Forkhead box protein 3 (FOXP3) is expressed in numerous types of tumor cell and is associated with tumor progression and prognosis. A previous study reported that FOXP3 inhibited cellular proliferation and induced apoptosis of gastric cancer (GC) cells by activating the apoptosis signaling pathway. In the present study, label-free quantitative proteomic analysis and chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR) was performed to investigate the mechanism by which the anticancer role of FOXP3 was mediated and the proteins that with which it may interact. Label-free quantitative proteomic analysis was used to screen for proteins differentially expressed between FOXP3-overexpressing GC (AF) and vector (ANC) cells. Catenin β1 (CTNNB1) was one of the proteins that exhibited the greatest difference between AF and ANC among 3,313 proteins identified by liquid chromatography with tandem mass spectrometry analysis. The expression of CTNNB1 was evaluated by reverse transcription-quantitative PCR and western blotting. The association between FOXP3 and CTNNB1 was confirmed by ChIP-PCR in AGS cells. The changes in expression of epithelial-mesenchymal transition-associated proteins were analyzed by western blotting. The level of FOXP3 expression was positively associated with CTNNB1 and E-cadherin expression, but not with vimentin and N-cadherin expression. FOXP3 positively regulates CTNNB1 and binds to it directly. Along with the upregulation of glycogen synthase kinase 3β (GSK3β), which was also a protein whose expression was found to change significantly in proteomic analysis and has a key role in the Wnt pathway. This association is an attractive and novel hypothesis for the mechanism by which FOXP3 inhibits the invasion and metastasis of GC cells.

Foxp3 expression in lymph node metastases in patients with head and neck cancer

Introduction: The prevalence and activity of regulatory T cells in patients with cancer correlates with poor prognosis. These cells are characterized by their expression of Forkhead box protein-3 (Foxp3). Squamous cell carcinoma is the most prevalent type of cancer in the head and neck region with overall poor survival rates, also due to early spread of metastatic cells.Material and methods: Primary tumor specimens as well as lymph node specimens harvested during neck dissection of 65 patients with a diagnosis of HNSCC were subjected to immunohistochemical and H-score analysis of Foxp3 expression. Demographics, diagnoses, histopathology and subsequent outcome were analyzed.Results: The primary cancer was squamous cell carcinoma in all patients (male/female 55:10) with the following tumor locations: oral cavity n = 16, oropharynx n = 28, hypopharynx n = 11 and larynx n = 10 (Stage III n = 18; Stage IVA n = 45; Stage IVB n = 2). The H-score for Foxp3 expression in the primary lesion as well as metastatic lymph nodes was significantly higher in advanced stages compared to early stages with differences among tumor locations, which were not significant. High Foxp3 expression was associated with inferior overall survival rates at a mean follow-up of 83.4 months (6–204 months)Conclusions: Foxp3 expression in HNSCC varied from the anatomical site and correlated positively with tumor stage and was associated with poor prognosis. Therefore, Foxp3 expressions in primary lesions as well as lymphogenic metastases appear to predict high-risk HSNCC patients. Novel therapeutic approaches targeting Foxp3+ cells might seem promising for this patient population.

Expression and prognostic significance of microenvironment related prognostic factors in patients with classical Hodgkin's lymphoma

Objective: To observe the expression of three microenvironment related prognostic factors, i. e. programmed death 1 (PD-1), forkhead box protein 3(FOXP3) and colony-stimulating factor 1 receptor(CSF-1R) protein in classical Hodgkin's lymphoma (CHL) patients, and to explore the correlation between the protein expression and the prognosis of the patients. Methods: A total of 45 cases of CHL patients, who had been admitted to the Tianjin Medical University Cancer Institute and Hospital and Chinese PLA General Hospital from February 2005 to August 2010 were analyzed, including clinical features, prognostic factors, and treatment regimens. CHL patients' specimens were collected and the expression of PD-1, FOXP3, and CSF-1R proteins analyzed by immunohistochemical staining. Epstein-Barr virus encoded mRNA (EBER) was detected by in situ hybridization analysis. The relationship between the protein expression of PD-1, FOXP3 and CSF-1R and the patients' outcome was analyzed with clinical and follow-up data. Survival analysis was performed by Kaplan-Meier method, the Cox proportional hazard model was used to perform multivariate analysis. Results: In this cohort of 45 CHL patients, PD-1 positive was found in 7 cases (15.6%), FOXP3 high expression in 23 cases (51.1%), CSF-1R positive in 18 cases (40.0%). In the univariate analysis, the expression of FOXP3 and CSF-1R, International Prognostic Index (IPI) score, Ann Arbor stage and EBER were related with the patients' 5-year overall survival (OS); IPI score, the expression of FOXP3 and EBER were related with the patients' 5-year progress-free survival (PFS). Multivariate analysis indicated that CSF-1R protein expression was the independent prognostic factor affecting the patients' 5-year OS(HR: 8.918, P=0.020), and FOXP3 protein expression was the independent prognostic factor affecting the patients' 5-year PFS (HR: 0.122, P<0.001). And EBV was an independent prognostic factor of PFS and OS in the CHL patients. Conclusion: Microenvironment related prognostic factors FOXP3, CSF-1R and EBV may be independent prognostic factors of CHL and this study may provide novel strategies for targeted therapy of CHL.

Association of nuclear FOXP3 expression with low Ki67 index and better prognosis in patients with breast invasive ductal carcinoma

Recent studies have provided clear evidence that some types of human cancer cells expressed Forkhead Box Protein 3 (FOXP3). However, the presence and role of FOXP3 in breast cancer are still contradictory up to now. In this study, we detected the expression of FOXP3 protein by immunohistochemistry in 123 cases of breast invasive ductal carcinoma. It exhibited that the subcellular localization of FOXP3 expression in breast cancer cells is heterogeneous. In nucleus, FOXP3 expression ratio was 47.97% (59/123) and the nuclear FOXP3 expression was significantly associated with lower Ki67 index (P=0.041), negative vessel tumor embolus (P=0.024). It was also significantly correlated with the molecular subtypes of breast cancer (P=0.002), displaying the highest ratio in the Luminal A subtype (68.18%). Kaplan-Meier analysis indicated that high nuclear FOXP3 expression was associated with better overall survival (OS) (94.92% vs. 82.81%, P=0.022) and disease-free survival (DFS) (91.53% vs. 76.56%, P=0.026). Moreover, nuclear FOXP3 represented an independent prognostic factor for OS (P =0.033) in multivariate analysis. However, in cytoplasm, FOXP3 expression ratio was 63.41% (78/123) and no statistic prognostic significance was found. Thus, our data demonstrated that nuclear FOXP3 expression correlated with low Ki-67 index and better outcome in breast invasive ductal carcinoma, indicating that FOXP3 acted as a potential prognostic marker for breast cancer.

Different Expression Patterns of CXCR4, CCR7, Maspin and FOXP3 in Luminal Breast Cancers and Their Sentinel Node Metastases.

Luminal A breast cancers (BC) represent low-risk tumors conferring better outcome than luminal B and human epidermal growth factor 2 (HER2)-positive or triple-negative tumors. One reason for the heterogeneous outcome among patients with luminal BC is the variation in cell proliferation. As chemokine receptors and tumor suppressors show potential for estimation of infiltration to regional lymph nodes, we aimed to compare differently sized sentinel node metastases with their primary tumors (PT). We compared 29 BCs of luminal subtype A and 23 of subtype B (Ki-67 cut off at 14%) by immunohistochemistry for the chemokine receptors C-X-C chemokine receptor 4 (CXCR4), C-C-chemokine receptor 7 (CCR7), the tumor suppressor Maspin and the regulatory T-cell immunosuppressor forkhead box protein 3 (FOXP3) between PTs and their metastases of different size. Expression of CXCR4 was low in luminal A type tumors, and CCR7 and FOXP3 expression were high in luminal B type cancer. CXCR4 expression significantly positively correlated with CCR7 both in PTs and metastases. Most Maspin-positive PTs became negative in the metastases. The PTs for all Maspin-positive metastases were luminal B type. High CXCR4 expression in PTs was found to be associated with luminal A type tumor, suggesting more favorable outcome. In contrast, CCR7 and FOXP3 expressions in PTs represented luminal B tumors, pointing to more aggressive tumor behavior. Maspin expression did not differ between luminal types.