Expression of FOXA1 Is Associated with the Tumor-Infiltrating M2 Macrophage, Cytotoxic T Lymphocyte, and Effect of Chemotherapy in Bladder Cancer

Purpose: Cisplatin-containing combination chemotherapy has been the standard of care since the late 1980s, but the response rate is <50%. Studies have shown that the efficiency of chemotherapy differs among molecular subtypes of bladder cancer. In this study, we aimed to correlate FOXA1, a marker for differentiation of the basal and luminal subtypes, with tumor immune cell infiltration and the effect of chemotherapy in bladder cancer. Materials and Methods: Eighty-three patients with bladder cancer treated with chemotherapy were reviewed. Clinicopathological variables for each case were recorded. FOXA1, M2 tumor-associated macrophage (TAM), dendritic cell (DC), and cytotoxic T lymphocyte (CTL) were examined by immunohistochemistry. The relationship between FOXA1, immune cell infiltration, and clinical response to chemotherapy was assessed. Results: The overall objective response rate was 34%. The objective response rate for tumors with lower FOXA1 expression was 58% and for tumors with higher FOXA1 expression was 12%. Tumors with infiltrated M2 TAM proportion <3% had a higher objective response rate compared with infiltrated M2 TAM proportion >3% tumors (46% vs. 21%, p = 0.02). Tumors with infiltrated CTL proportion >5% had a higher objective response rate compared with infiltrated CTL proportion <5% tumors (50% vs. 17%, p = 0.002). DCs showed no significant differences. We found that the objective response rate for tumors with lower FOXA1 expression, proportion <3% M2 TAM infiltration, and proportion >5% CTL infiltration is 82%. Lower FOXA1 expression was associated with low M2 TAM infiltration and high CTL infiltration. Conclusions: Thus, we showed that in patients with bladder cancer who received chemotherapy, the higher clinical response rate is associated with low FOXA1 expression, low M2 TAM infiltration, and high CTL infiltration.

Investigating the Role of Chromatin Remodeler FOXA1 in Ferroptotic Cell Death

Ferroptosis is a lipid peroxidation-dependent mechanism of regulated cell death known to suppress tumor proliferation and progression. Although several genetic and protein hallmarks have been identified in ferroptotic cell death, it remains challenging to fully characterize ferroptosis signaling pathways and to find suitable biomarkers. Moreover, changes taking place in the epigenome of ferroptotic cells remain poorly studied. In this context, we aimed to investigate the role of chromatin remodeler forkhead box protein A1 (FOXA1) in RSL3-treated multiple myeloma cells because, similar to ferroptosis, this transcription factor has been associated with changes in the lipid metabolism, DNA damage, and epithelial-to-mesenchymal transition (EMT). RNA sequencing and Western blot analysis revealed that FOXA1 expression is consistently upregulated upon ferroptosis induction in different in vitro and in vivo disease models. In silico motif analysis and transcription factor enrichment analysis further suggested that ferroptosis-mediated FOXA1 expression is orchestrated by specificity protein 1 (Sp1), a transcription factor known to be influenced by lipid peroxidation. Remarkably, FOXA1 upregulation in ferroptotic myeloma cells did not alter hormone signaling or EMT, two key downstream signaling pathways of FOXA1. CUT&RUN genome-wide transcriptional binding site profiling showed that GPX4-inhibition by RSL3 triggered loss of binding of FOXA1 to pericentromeric regions in multiple myeloma cells, suggesting that this transcription factor is possibly involved in genomic instability, DNA damage, or cellular senescence under ferroptotic conditions.

Systematic Pan-Cancer Analysis and Experimental Verification Identify FOXA1 As An Immunological and Prognostic Biomarker In Epithelial Ovarian Cancer

Background: Epithelial ovarian cancer (EOC) has the lowest survival rate among female reproductive cancers present with symptoms of aggressive malignancies, poor prognosis, drug resistance and postoperative recurrence. The majority of patients with EOC are diagnosed at an advanced stage due to the therapeutic challenges including lack of early diagnosis and effective therapeutic targets for EOC.Methods: Pan-cancer analyses were performed to explore the features of Forkhead box (FOX) A1 (FOXA1) using data from TCGA and GTEx database. R package “clusterprofiler” was used to perform the enrichment analysis of FOXA1 in EOC. Data downloaded from Drug Sensitivity in Cancer (GDSC) database were used to evaluate the association between FOXA1 and anti-tumor drug sensitivity. In experimental verification, FOXA1 expression was detected using qRT-PCR and Western blot assays. Western blot, immunofluorescence staining and transwell assays were used to assess the influence of FOXA1 silencing on epithelial-mesenchymal transition (EMT) of EOC cellsResults: We found that FOXA1 was highly expressed in EOC and predicted poorer survival of EOC patients. We observed that FOXA1 expression was positively correlated EMT related pathways. Through experimental verification, we found the underlying function of FOXA1 to promote EMT in ovarian cancers. Results from western blot, immunofluorescence staining and transwell assays showed that FOXA1 silencing impeded the progression of EMT and invasiveness of the cancer cells. Furthermore, CCK-8 and invasion assays suggested that siRNA-FOXA1 attenuated the ability of cancer cells to metastasize and proliferate. Dual-luciferase reporter assays confirmed the binding activity of FOXA1 to the promoter of connective tissue growth factor (CTGF). In addition, we found that FOXA1 was closely correlated immunosuppressive microenvironment of EOC. High FOXA1 expression may contribute to resistance of many anti-cancer drugs.Conclusions: Our results predict and validate the function of FOXA1 in promoting EMT and the progression of disease in EOC. Targeting FOXA1 may improve the sensitivity of EOC treatment.

FOXA1 and adaptive response determinants to HER2 targeted therapy in TBCRC 036

Inhibition of the HER2/ERBB2 receptor is a keystone to treating HER2-positive malignancies, particularly breast cancer, but a significant fraction of HER2-positive (HER2+) breast cancers recur or fail to respond. Anti-HER2 monoclonal antibodies, like trastuzumab or pertuzumab, and ATP active site inhibitors like lapatinib, commonly lack durability because of adaptive changes in the tumor leading to resistance. HER2+ cell line responses to inhibition with lapatinib were analyzed by RNAseq and ChIPseq to characterize transcriptional and epigenetic changes. Motif analysis of lapatinib-responsive genomic regions implicated the pioneer transcription factor FOXA1 as a mediator of adaptive responses. Lapatinib in combination with FOXA1 depletion led to dysregulation of enhancers, impaired adaptive upregulation of HER3, and decreased proliferation. HER2-directed therapy using clinically relevant drugs (trastuzumab with or without lapatinib or pertuzumab) in a 7-day clinical trial designed to examine early pharmacodynamic response to antibody-based anti-HER2 therapy showed reduced FOXA1 expression was coincident with decreased HER2 and HER3 levels, decreased proliferation gene signatures, and increased immune gene signatures. This highlights the importance of the immune response to anti-HER2 antibodies and suggests that inhibiting FOXA1-mediated adaptive responses in combination with HER2 targeting is a potential therapeutic strategy.

Knockdown of Forkhead box A1 suppresses the tumorigenesis and progression of human colon cancer cells through regulating the phosphatase and tensin homolog/Akt pathway

Background This study aimed to evaluate the role and the underlying mechanisms of Forkhead box A1 (encoded by FOXA1) in colon cancer. Methods We analyzed FOXA1 mRNA and protein expression in colon cancer tissues and cell lines. We also silenced FOXA1 expression in HCT116 and SW480 cells to evaluate the effects on cell proliferation, cell cycle, migration, and invasion by using MTT, colony formation, flow cytometry, and the Transwell assay, respectively. Results FOXA1 immunostaining was higher in colon cancer tissues than adjacent healthy tissues. FOXA1 mRNA and protein expression was significantly increased in human colon cancer cells compared with a normal colonic cell line. FOXA1 expression was also significantly higher in colorectal cancer tissues from TCGA data sets and was associated with worse prognosis in the R2 database. FOXA1 expression was negatively correlated with the extent of its methylation, and its knockdown reduced proliferation, migration, and invasion, and induced G2/M phase arrest in HCT116 and SW480 cells by suppressing the phosphatase and tensin homolog/Akt signaling pathway and inhibiting epithelial–mesenchymal transition. Conclusion FOXA1 may act as an oncogene in colon cancer tumorigenesis and development.

The Role and Mechanism of Human Papillomavirus16 E7 (HPV16 E7) in the Proliferation and Invasion of Cervical Cancer Cells Through Regulating Forkhead Box Protein A1

High-risk HPV16 is an important factor for cervical cancer. HPV16 E7 can promote the malignant transformation of cervical epithelial cells. Forkhead box protein A1 (FOXA1) is abnormally expressed in several tumors. Our study assessed HPV16 E7's effect on cervical cancer cells. Hela cells were divided into control group; HPV16 E7 group; and siFOXA1+ HPV16 E7 group followed by analysis of HPV16 E7 and FOXA1 expression by Real-time PCR and Western blot, cell proliferation by MTT assay, Caspase 3 activity, Bax and Bcl-2 expression by Real-time PCR as well as cell invasion by Transwell assay. In HPV16 E7 group, HPV16 E7 and HOXA1 expression was significantly increased, cell proliferation was promoted, invasive ability was increased, Caspase 3 activity and Bax expression was decreased, and Bcl-2 expression was increased compared to control group (P < 0 05). Conversely, inhibition of FOXA1 expression in Hela cells overexpressing HPV16 E7 can significantly inhibit cell proliferation and invasion, and promote apoptosis (P < 0 05). HPV16 E7 protein can up-regulate FOXA1 in host cells, and promote cervical cancer cell growth, proliferation and invasion, indicating that it is one of the key factors contributing to cervical cancer.

FOXA1 expression in Iraqi women with ER+ breast cancer: A case control study

Background: Breast cancer is a heterogeneous disease that can be classified into many subtypes according to histopathological and molecular characteristics. Forkhead box protein A1 (FOXA1) is a pioneer factor of estrogen receptor (α-ER)-chromatin binding and function. FOXA1 expression is related to luminal breast cancer with a good prognosis. Objectives: The present study is sought to determine the gene expression of FOXA1 in Iraqi women with ER+ breast cancer from fresh tissue. Methods: Forty-eight fresh malignant breast tissues analyzed by immunohistochemistry assay to choose ER+ sample submitted to RT-qPCR to evaluate FOXA1 gene expression. Results: ER-positive was 72.91% of the total samples, luminal A was the most common molecular subtype with 56.25%. Conclusions: Highly significant FOXA1 gene expression in Iraqi women with breast cancer makes it eligible to be a good predictor or a biomarker for breast cancer.

FOXA1 Expression in Nasopharyngeal Carcinoma: Association with Clinicopathological Characteristics and EMT Markers

The forkhead box (FOXA) family of transcription factors regulates gene expression and chromatin structure during tumorigenesis and embryonic development. Until now, the relationship between FOXA1 and the nasopharyngeal carcinoma (NPC) has not yet been reported. Therefore, our purpose is to analyze the expression of FOXA1 in 56 NPC patients compared to 10 normal nasopharyngeal mucosae and to correlate the expression with the clinicopathological features. Besides, we investigated the association between FOXA1 and LMP1 gene expression, as well as the EMT markers namely the E-cadherin and Twist1. Among 56 NPC tissues, 34 (60.7%) cases were positive for FOXA1. Furthermore, we noticed that FOXA1 expression correlated with TNM (p=0.037), and age at diagnosis (p=0.05). Moreover, positive expression of FOXA1 is likely to be associated with prolonged disease-free survival and overall survival rates. On the other hand, we observed a positive association between the expression of E-cadherin and FOXA1 (p=0.0051) whereas Twist1 correlated negatively with FOXA1 (p=0.004). Furthermore, knowing that LMP1 plays a key role in the pathogenesis of NPC, we explored the association of FOXA1 with the LMP1 gene expression in both NPC cell lines and tissues. We found that, in the C666-1 which displays low levels of LMP1, the expression of FOXA1 is high, and inversely in the C15 cell line that expresses a high level of LMP1, the level of FOXA1 is low. Besides, in accordance to our results, we found that in NPC tissues there is a negative association between LMP1 and FOXA1. In conclusion, our results suggest that the overexpression of FOXA1 is associated with a nonaggressive behavior and favorable prognosis in NPC patients. FOXA1 could contribute in the EMT process through key factors as E-cadherin, Twist1, and LMP1.