FOXA1 Expression Research Articles

Loss of miR-634 contributes to the formation FOXA1-positive triple negative breast cancer subtype

BackgroundTriple-negative breast cancer (TNBC), the most aggressive subtype of breast cancer, lacks targeted therapies, posing a substantial challenge for treatment. Therefore, investigating its pathogenesis is a crucial research focus. FOXA1 and miR-634 are involved in tumorigenesis. However, the molecular mechanisms underlying the aberrant upregulation of FOXA1 expression in TNBC remain unclear. Therefore, we explore the role of miR-634 in the FOXA1-positive TNBC subtype.MethodsQuantitative reverse transcription polymerase chain reaction was used to detect miR-634 expression in breast cancer tissues and cell lines. Aberrantly activated signaling pathways and related genes in TNBC were analyzed using The Cancer Genome Atlas. The potential target of miR-634 was predicted by TargetScan, the TNBC cell proliferation rate was detected using an MTT assay, the in vitro metastatic capacity was determined by transwell assay and the cell cycle distribution was tested using flow cytometry. Western blotting was performed to measure the expression of proteins involved in the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway.ResultsThe expression of miR-634 was significantly down-regulated in both TNBC tissues and cells, compared with adjacent non-cancerous tissues and MCF10A, respectively. Ectopic expression of miR-634 inhibits breast cancer cell proliferation and in vitro metastasis. The TCGA-based expression profile analysis of TNBC revealed that aberrantly activated PI3K/AKT signaling may contribute to its malignant phenotype. FOXA1, the top hit of aberrantly upregulated genes in TNBC, was a direct target of miR-643. Moreover, forced expression of miR-643 drastically suppressed FOXA1 expression by the inactivation PI3K/AKT signaling.ConclusionMiR-634 suppresses FOXA1 to inhibit the proliferation and metastasis of TNBC cells by inactivating the PI3K/AKT signaling pathway.

Immunohistochemical expression of FOXA1 and Androgen receptor in breast carcinoma in Upper Egypt

Immunohistochemical expression of FOXA1 and Androgen receptor in breast carcinoma in Upper Egypt

TGF-β1 facilitates gallbladder carcinoma metastasis by regulating FOXA1 translation efficiency through m6A modification

TGF-β1 plays a pivotal role in the metastatic cascade of malignant neoplasms. N6-methyladenosine (m6A) stands as one of the most abundant modifications on the mRNA transcriptome. However, in the metastasis of gallbladder carcinoma (GBC), the effect of TGF-β1 with mRNA m6A modification, especially the effect of mRNA translation efficiency associated with m6A modification, remains poorly elucidated. Here we demonstrated a negative correlation between FOXA1 and TGF-β1 expression in GBC. Overexpression of FOXA1 inhibited TGF-β1-induced migration and epithelial-mesenchymal transition (EMT) in GBC cells. Mechanistically, we confirmed that TGF-β1 suppressed the translation efficiency of FOXA1 mRNA through polysome profiling analysis. Importantly, both in vivo and in vitro experiments showed that TGF-β1 promoted m6A modification on the coding sequence (CDS) region of FOXA1 mRNA, which was responsible for the inhibition of FOXA1 mRNA translation by TGF-β1. We demonstrated through MeRIP and RIP assays, dual-luciferase reporter assays and site-directed mutagenesis that ALKBH5 promoted FOXA1 protein expression by inhibiting m6A modification on the CDS region of FOXA1 mRNA. Moreover, TGF-β1 inhibited the binding capacity of ALKBH5 to the FOXA1 CDS region. Lastly, our study confirmed that overexpression of FOXA1 suppressed lung metastasis and EMT in a nude mice lung metastasis model. In summary, our research findings underscore the role of TGF-β1 in regulating TGF-β1/FOXA1-induced GBC EMT and metastasis by inhibiting FOXA1 translation efficiency through m6A modification.

LINC01133 contributes to the malignant phenotypes of non-small cell lung cancer by targeting miR-30b-5p/FOXA1 pathway.

This study aimed to explore the mechanism of action of LINC01133 in non-small cell lung cancer. LINC01133 expression in NSCLC patient tissues and cells was detected by qRT-PCR. After transfecting siRNA-LINC01133 in NSCLC cells, the proliferation and invasive migration ability of the cells were assessed via CCK-8 and Transwell assay, respectively. The sublocalization of LINC01133 in NSCLC cells was analyzed by bioinformatics prediction and nucleoplasm separation assay and RNA-FISH assay. Analysis of the binding relationship between LINC01133, FOXA1 and miR-30b-5p was all through bioinformatics website analysis, dual-luciferase reporter and RNA Pulldown assay. Functional rescue experiments confirmed the character of miR-30b-5p and FOXA1 in LINC01133 regulating the NSCLC cells biological behavior. LINC01133 high expressions were found in NSCLC tissues and cells. siRNA-LINC01133 treatment inhibited NSCLC cells malignant behavior. Mechanistically: LINC01133 promoted FOXA1 expression through adsorption binding of miR-30b-5p. Knocking down miR-30b-5p expression or up-regulating FOXA1 expression was able to reverse siRNA-LINC01133 inhibitory effect of tumor cell malignant behavior. LINC01133 promoted FOX1 expression by competitively binding miR-30b-5p, which attenuated the targeting inhibitory effect of miR-30b-5p on FOXA1 and ultimately promoted proliferation and invasive migration of NSCLC cells.

Abstract PO4-16-03: Ectopic expression of lineage-specific transcription factors in Breast Cancer

Abstract Background: Lineage-specific transcription factors (LTFs) are purported to define cellular lineages and their expression could be used to identify subtypes of cancer. In many cancers, tuft cell differentiation appears to have prognostic impact. However, such differentiation has not been documented in breast cancer. Methods: We sought to study the fidelity of LTFs (MYOG, POU2F3, FOXA1, FOXA2, FOXA3, FOXJ1, HNF4A, HNF4G, SPIB, SOX8, GRHL1, GRHL2, and GRHL3) for their presence and prognostic impact in breast cancer. Single cell RNA data from GSE 161529, which contains 421,761 single cells, was downloaded and analyzed for the expression and co-expression of LTFs. The data was first subdivided based on ER+ and TNBC status. Analysis was performed using cells from 10ER+ samples and 4 TNBC samples. Cell clustering using Louvain’s method was run using all genes. Cell type assignment was then performed using well-known markers for both tumor and lymphocyte marker from existing publications. Further analysis was restricted to keratin expressing cells and to study the presence and co-expression of LTFs in the epithelial cells. KM-Plotter was employed to determine the prognostic value of these LTFs. The analysis was further stratified by expression of estrogen receptor positive and negative status. The endpoint of relapse free survival was used, as it provided data on the largest number of patients. Results: Single-cell analysis pipeline successfully identified distinct populations of tumor epithelial cells accompanied by various lymphocytes, including B cells, CD8+ T cells, CD4+ T cells, dendritic cells, and macrophages, in both ER+ tumors and TNBC tumor samples. We further confirmed the expression of the LTFs in the epithelial (keratin+) compartment. Expression of all LTFs (except MYOG, and FOXA2) was scattered in the different epithelial clusters. Further analysis of single LTFs showed differentiational co-expression based on ER+ status. POU2F3+/ER+ cells also co-expressed FOXA1 and GRHL1 while POU2F3+/ER- cells were GRHL1+ and (weak) SPIB+. The analysis of LTFs using KM-Plotter tool revealed their expression documenting multiple cell lineages in breast cancer; this was noted in ER+, HER2+ cancers and TNBCs. The directionality of the outcome (good or bad prognosis) varied with the specific TFs and subtype of breast cancer. High expression of POU2F3, and FOXA1, and SPIB were associated with better relapse-free survival (RFS) in ER+ breast cancer. POU2F3, FOXA2, FOXA3 were associated with worse RFS, whereas SPIB was with prognosis in ER- breast cancers. Conclusions: We for the first time document the expression of LTFs in breast cancer and their prognostic relevance based on ER+ and ERneg status. Expression of POU2F3, a marker of tuft cells which are known to regulate immune response, was noted to be associated with improved outcomes in ER+ tumors. Further characterization of the relevance of LTFs and their co-expression with respect to therapeutic response is ongoing. Citation Format: Sunil Badve, Jian Hu, Yu Zhang, Yesim Gokmen-Polar. Ectopic expression of lineage-specific transcription factors in Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-16-03.

Knockdown of KBTBD7 attenuates septic lung injury by inhibiting ferroptosis and improving mitochondrial dysfunction

Lung injury in sepsis is caused by an excessive inflammatory response caused by the entry of pathogenic microorganisms into the body. It is also accompanied by the production of large amounts of ROS. Ferroptosis and mitochondrial dysfunction have also been shown to be related to sepsis. Finding suitable sepsis therapeutic targets is crucial for sepsis research. BTB domain-containing protein 7 (KBTBD7) is involved in regulating inflammatory responses, but its role and mechanism in the treatment of septic lung injury are still unclear. In this study, we evaluated the role and related mechanisms of KBTBD7 in septic lung injury. In in vitro studies, we established an in vitro model by inducing human alveolar epithelial cells with lipopolysaccharide (LPS) and found that KBTBD7 was highly expressed in the in vitro model. KBTBD7 knockdown could reduce the inflammatory response by inhibiting the secretion of pro-inflammatory factors and inhibit the production of ROS, ferroptosis and mitochondrial dysfunction. Mechanistic studies show that KBTBD7 interacts with FOXA1, promotes FOXA1 expression, and indirectly inhibits SLC7A11 transcription. In vivo studies have shown that knocking down KBTBD7 improves lung tissue damage in septic lung injury mice, inhibits inflammatory factors, ROS production and ferroptosis. Taken together, knockdown of KBTBD7 shows an alleviating effect on septic lung injury in vitro and in vivo, providing a potential therapeutic target for the treatment of septic lung injury.

FOXA1 regulates ribosomal RNA transcription in prostate cancer.

Ribosome biogenesis is excessively activated in tumor cells, yet it is little known whether oncogenic transcription factors (TFs) are involved in the ribosomal RNA (rRNA) transactivation. Nucleolar proteomics data and large-scale immunofluorescence were re-analyzed to jointly identify the proteins localized at nucleolus. RNA-Seq data of five prostate cancer (PCa) cohorts were combined and integrated with multi-dimensional data to define the upregulated nucleolar TFs in PCa tissues. Then, ChIP-Seq data of PCa cell lines and two PCa clinical cohorts were re-analyzed to reveal the TF binding patterns at ribosomal DNA (rDNA) repeats. The TF binding at rDNA was validated by ChIP-qPCR. The effect of the TF on rRNA transcription was determined by rDNA luciferase reporter, nascent RNA synthesis, and global protein translation assays. In this study, we reveal the role of oncogenic TF FOXA1 in regulating rRNA transcription within nucleolar organization regions. By analyzing human TFs in prostate cancer clinical datasets and nucleolar proteomics data, we identified that FOXA1 is partially localized in the nucleolus and correlated with global protein translation. Our extensive FOXA1 ChIP-Seq analysis provides robust evidence of FOXA1 binding across rDNA repeats in prostate cancer cell lines, primary tumors, and castration-resistant variants. Notably, FOXA1 occupancy at rDNA repeats correlates with histone modifications associated with active transcription, namely H3K27ac and H3K4me3. Reducing FOXA1 expression results in decreased transactivation at rDNA, subsequently diminishing global protein synthesis. Our results suggest FOXA1 regulates aberrant ribosome biogenesis downstream of oncogenic signaling in prostate cancer.

Abstract 6432: Co-expression of HERV-R, CCND1, BRAF, FOXA1, TMPRSS2, ATF4 and low expression of BAZ1B and KMT2D is associated with early detection and poor prognosis of prostate cancer in African Black men from Nigeria

Abstract Prostate cancer (CaP) is a heterogeneous disease characterized by features of non-aggressive, slow-growing disorders to fast-growing disease. Men of African ancestry have disproportionately higher incidence and mortality rates for CaP than men of European ancestry. While there is evidence for a higher genetic predisposition for incidence of CaP in men of African ancestry compared to men of European ancestry, there is paucity of molecular biology and transcriptomic studies on the role of Human endogenous retroviruses (HERVs) in relation to CaP-associated genes in CaP pathogenesis and prognosis. In this study, we performed bulk RNA sequencing (RNA-seq) on formalin-fixed paraffin-embeded (FFPE) CaP samples from Nigerian CaP patients to investigate the expression significance of HERV-R in relation to FOXA1, TMPRSS2, BAZ1B, KMT2D, ATF4 and BRAF genes. Based on this findings, we used immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA) and immunofluorescence (IF) to confirm the expression pattern of the HERV-R and FOXA1, TMPRSS2, BAZ1B, KMT2D, ATF4 and BRAF proteins on 45 FFPE CaP and blood plasma samples, and 5 CaP-free samples from patients in Zaria, Nigeria. Most of these patients were longitudinally followed from 2017 to date. The DESeq2 procedure was used for data processing, analysis, and differential expression analysis of the aforementioned genes. We found a significant expression of HERV-R in relation with FOXA1, TMPRSS2, BAZ1B, KMT2D, ATF4, CCND1 and BRAF in the CaP from rural Africans in Nigeria. HERV-R expression was found to be associated with increased PSA level, higher Gleason grade and poor prognosis of CaP. A CaP sample from an 80-year-old patient that was histologically diagnosed to have no malignancy seen despite high PSA level of >100 and poor disease outcomes shows a significant cytoplasmic expression of HERV-R, indicating that HERV-R may serve as an important biomarker for early detection. Unfortunately, we lost the 80-year-old patient to late detection before revisiting his case for proper histological diagnosis and management. In addition, some histologically diagnosed BPH samples that are apparently reported to be CaP-free show stromal cells expression of HERV-R and in some cases show expression of HERV-R in the prostatic glandular areas. In contrast, stromal cells of the CaP samples show no expression of HERV-R, but strong expression in the glandular areas. This may implicate a gradual transition from benign prostatic lesion to malignant prostatic disease. HERV-R expression in CaP may have a relationship with BRAF, CCND1, FOXA1, TMPRSS2 and ATF4 genes. These findings combined together, underscore the importance of the expanded concept of HERV-R expression and CaP associated genes in early detection of CaP and prognosis. Citation Format: Faruk Mohammed, Konrad Stawiski, Halimatu Sadiya Musa, Sani Kamarudeen Owolabi, Sani Abubakar, Solomon O. Rotimi, Adoke Kasimu Umar, Yawale Iliyasu, Ahmad Bello, Folakemi T. Odedina, Clayton C. Yates, Kevin Petrie, Franklin W. Huang, Sani Ibrahim. Co-expression of HERV-R, CCND1, BRAF, FOXA1, TMPRSS2, ATF4 and low expression of BAZ1B and KMT2D is associated with early detection and poor prognosis of prostate cancer in African Black men from Nigeria [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6432.

Abstract A076: The role of PARP-1-mediated FoxA1 ADP-ribosylation in breast cancer biology

Abstract Breast cancer is the most common cancer diagnosed in women. At the time of diagnosis, approximately 70% of breast cancers express Estrogen Receptor alpha (ERα), a key signature of luminal subtype tumors. Equally important in these cases is the expression of FoxA1, a subtype-specific transcription factor (TF) essential for ERα-mediated cell proliferation. FoxA1 is a key pioneer TF that assists ERα with the assembly of enhancers and the transcription of estrogen (E2)-regulated target genes. Together, the FoxA1-ERα axis is a major driver of cancer cell growth. Our recently published work implicates poly(ADP-ribose) polymerase 1 (PARP-1) and ADP-ribosylation (ADPRylation) in the regulation of E2-dependent transcriptional responses in ERα+ breast cancer cells. To determine the underlying mechanisms, we identified FoxA1 as a substrate of PARP-1-mediated ADPRylation. We hypothesize that (1) PARP-1 elicits its effects on E2 signaling and ERα enhancer function, in part, by ADPRylating FoxA1 at specific residues and (2) Loss of these regulatory ADPRylation events alters FoxA1 activity, which in turn can alter the assembly of ERα enhancers and transform the breast cancer cell transcriptome. Indeed, the sites of FoxA1 ADPRylation are recurrently mutated in breast cancers and are highly enriched in metastatic cases. Using an integrated set of biochemical, molecular, cell based, genomic and proteomic approaches, we have validated the ADPRylation of FoxA1 by PARP-1 in vitro and in vivo. We selected two FoxA1 ADPRylation sites, D226 and E255, for further analysis due to their positions in a mutation ‘hot spot’ region corresponding to the C-terminal forkhead DNA-binding domain. Mutation of these sites blocks FoxA1 ADPRylation and enhances cell proliferation in MCF-7 human breast cancer cells. Additionally, preliminary ChIP-seq analysis indicates that the binding of both ERα and FoxA1 are globally reduced at ERα enhancers in MCF-7 cells expressing the FoxA1 ADPRylation site mutants compared to wild-type but may also increase both at selected enhancers. While there is a global reduction in mutant FoxA1 binding near the canonical estrogen-regulated genes, interestingly, we observe an overall enhanced binding of FoxA1 D226N compared to wild-type outside of this canonical program. Collectively, these studies will (1) reveal new molecular endpoints modulated by PARP-1, providing new insights about the use of PARPi to treat ERα+ breast cancers, and (2) suggest new therapeutic strategies to disrupt the aberrant signaling pathways associated with mutant FoxA1 luminal breast cancers, and more importantly metastatic cases, which is of utmost clinical significance. Citation Format: Cristel V. Camacho, Aishwarya Sundaresan, Tulip Nandu, W. Lee Kraus. The role of PARP-1-mediated FoxA1 ADP-ribosylation in breast cancer biology [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A076.

Abstract A059: Heterogeneous cell populations in organoid models derived from breast preinvasive and invasive carcinomas after endocrine therapy

Abstract Approximately 80% of ductal carcinoma in situ (DCIS) and invasive breast cancers express hormone receptors (HR), and are typically treated with selective estrogen receptor modulators (SERMs) or aromatase inhibitors, or with additional HR-targeting agents in the research setting. However, the development of resistance poses a significant challenge. To better understand the mechanisms behind endocrine resistance and explore new treatment strategies, it is essential to have a reliable model system that accurately reflects the heterogeneous nature of breast lesions. In this study, we developed a protocol to generate patient-derived organoid cultures of pre-invasive and malignant breast lesions from patients with persistent or residual disease in the active surveillance or neoadjuvant setting after treatment with endocrine therapy. We generated n = 11 HR+ DCIS and n = 18 HR+ breast cancer organoids and used single-cell RNA-sequencing to characterize cell heterogeneity within individual organoid cultures. We observed mixed non-cancerous and malignant cells present within some cultures, and optimized protocols to minimize non-cancerous cell types. To better define endocrine sensitivity versus resistance, we also assessed responsiveness to estrogen and anti-estrogen therapy in individual cultures by monitoring organoid growth through live cell imaging and evaluating estrogen-response markers at the protein and RNA levels in response to treatment in vitro. Next, we applied our model system to evaluate endocrine resistance in heterogeneous tumor populations. We compared changes in cellular composition and signaling pathways in endocrine-resistant and endocrine-sensitive breast organoids based on in vitro metrics and/or correlation with clinical variables. In addition, within single cultures heterogeneous cell populations were identified with differential expression of ESR1 and HR target genes, generally falling into three cluster groups that could be defined based on different patterns of FOXA1, GATA3, TFF1, and XBP1 expression, among others. These populations also differed with evidence of co-expressed genes and signaling pathways related to breast tumorigenesis and differentiation (estrogen response, androgen response, mTOR signaling, metabolism) in putative HR-active cell populations in both pre-invasive and invasive breast cases. In summary, our study demonstrates that patient-derived breast and breast cancer organoids can be used to model estrogen responsiveness and endocrine resistance. We have established a platform that enables us to investigate the mechanisms underlying endocrine resistance at different premalignant and tumor stages to evaluate new treatment approaches. The ultimate goal of this work is to contribute to the development of new targeted therapies for endocrine-resistant breast lesions. Citation Format: Nadine Goldhammer, Shruti Warhadpande, Liana Beld, Cristian K. Maldonado Rodas, Allison Lam, Tam Binh Bui, Shoko Emily Abe, Michael Alvarado, Cheryl Ewing, Karen Goodwin, Rita Mukhtar, Jasmine Wong, Lamorna Brown Swigart, Gillian Hirst, Christopher Schwartz, Laura van't Veer, Laura Esserman, Jo Chien, Jennifer M. Rosenbluth. Heterogeneous cell populations in organoid models derived from breast preinvasive and invasive carcinomas after endocrine therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A059.

Deciphering the Immune Microenvironment at the Forefront of Tumor Aggressiveness by Constructing a Regulatory Network with Single-Cell and Spatial Transcriptomic Data.

The heterogeneity and intricate cellular architecture of complex cellular ecosystems play a crucial role in the progression and therapeutic response of cancer. Understanding the regulatory relationships of malignant cells at the invasive front of the tumor microenvironment (TME) is important to explore the heterogeneity of the TME and its role in disease progression. In this study, we inferred malignant cells at the invasion front by analyzing single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST) data of ER-positive (ER+) breast cancer patients. In addition, we developed a software pipeline for constructing intercellular gene regulatory networks (IGRNs), which help to reduce errors generated by single-cell communication analysis and increase the confidence of selected cell communication signals. Based on the constructed IGRN between malignant cells at the invasive front of the TME and the immune cells of ER+ breast cancer patients, we found that a high expression of the transcription factors FOXA1 and EZH2 played a key role in driving tumor progression. Meanwhile, elevated levels of their downstream target genes (ESR1 and CDKN1A) were associated with poor prognosis of breast cancer patients. This study demonstrates a bioinformatics workflow of combining scRNA-seq and ST data; in addition, the study provides the software pipelines for constructing IGRNs automatically (cIGRN). This strategy will help decipher cancer progression by revealing bidirectional signaling between invasive frontline malignant tumor cells and immune cells, and the selected signaling molecules in the regulatory network may serve as biomarkers for mechanism studies or therapeutic targets.

Expression of BCL2, TP53, FOXA1, and GATA3 in pTa bladder cancer recurrence.

In this study, we analyzed pTa bladder cancer (BC) for molecular markers BCL2, TP53, FOXA1, and GATA3 in relation to cancer recurrence. We analyzed samples of 79 patients with the pTa stage of BC using areal-time polymerase chain reaction (real-time PCR) between September 2018 and September 2020. The expression levels of BCL2, TP53, FOXA1, and GATA3 were compared with homologous non-tumor bladder tissue. Expression of FOXA1, GATA3, and TP53 was significantly higher (p<0.01) in NMIBC samples compared to homologous non-tumor tissue. The expression of TP53 and FOXA1 in pTa was significantly lower (p<0.01) in the high-grade (HG) tumor when compared to the low-grade (LG) tumor. In contrast, the relative quantification (RQ) of GATA3 was significantly higher (p<0.01) in HG pTa. Patients with recurrence (pTa=33) had significantly higher expression of TP53, and GATA3 (p<0.01), and the gene of FOXA1 (p<0.01) had asignificantly lower expression when compared to pTa tumors without recurrence. The expression of Bcl-2 was not statistically significant. Our results, indicate, that comparing expression levels of these genes in cancer and cancer-free tissue could provide valuable data, as patients with pTa BC recurrence within up to 54 months of follow-up had asignificantly higher RQ of TP53, GATA3, and FOXA1 when compared to pTa BC patients without recurrence (Tab. 2, Fig. 8, Ref. 54). Text in PDF www.elis.sk Keywords: bladder cancer, gene expression, recurrence, GATA3, FOXA1, TP53, BCL2.

FOXA1, induced by RC48, regulates HER2 transcription to enhance the tumorigenic capacity of lung cancer through PI3K/AKT pathway.

Lung cancer remains the tumor with the highest global incidence and mortality rates. Current primary treatment modalities encompass targeted therapy, immunotherapy, and chemotherapy; however, a subset of patients derives no benefit from these interventions. Recently, the HER2-targeting antibody drug Disitamab vedotin (RC48) was approved and introduced primarily for gastric and bladder cancers, with minimal investigation in the field of lung cancer. This study demonstrates that FOXA1 directly binds to the promoter region of HER2, influencing the HER2/PI3K/AKT signaling pathway, which consequently modulates factors that foster lung cancer proliferation and impede apoptosis. Unlike FOXA1, HER2 does not influence the expression of FOXA1. Intriguingly, in lung cancer cells, RC48 not only impacts the HER2/PI3K/AKT pathway but also affects the FOXA1/HER2/PI3K/AKT pathway, thereby exerting a robust antitumor effect. In clinical specimens, heightened expressions of FOXA1 and HER2 correlate positively with clinical progression and poorer prognosis. These findings suggest that FOXA1 may serve as a potential biomarker or therapeutic target in future non-small cell lung cancer (NSCLC) treatments, and ongoing research may position RC48 as a transformative agent in lung cancer therapy.

In Silico Analysis Uncovers FOXA1 as a Potential Biomarker for Predicting Neoadjuvant Chemotherapy Response in Fine-Needle Aspiration Biopsies.

Background: The preoperative identification of neoadjuvant chemotherapy (NAC) treatment responsiveness in breast cancer (BC) patients is advantageous for tailoring treatment regimens. There is a relative scarcity in the current research exploring NAC treatment responsive biomarkers using bulk sequencing data obtained from fine-needle aspiration (FNA). Materials and Methods: Limma was employed for the selection of differentially expressed genes. Additionally, WGCNA, machine learning, and Genetic Perturbation Similarity Analysis (GPSA) were utilized to identify key genes associated with NAC treatment response. ConsensusClusterPlus was employed for unsupervised clustering. Rt-qPCR and WB were conducted to assess gene expression and protein levels in clinical tissues and cell lines. The Seahorse XF96 Extracellular Flux Analyzer was utilized to evaluate Extracellular Acidification Rate (ECAR) and Oxygen Consumption Rate (OCR). The "pRRophetic" package was used for drug sensitivity prediction, while CB-Dock2 was applied for molecular docking and optimal pose presentation. Spatial transcriptomic analysis was based on the CROST database. Results: Eleven biomarkers were identified associated with NAC treatment response in BC patients, with FOXA1 identified as a pivotal hub gene among them. The expression levels of FOXA1 showed a significant positive correlation with genomic stability and a marked negative correlation with the homologous recombination deficiency (HRD) score. Downregulation of the FOXA1 gene resulted in reduced glycolysis in MCF-7 cells.Additionally, FOXA1 were found to serve as a biomarker for both NAC and PARP inhibitor treatment sensitivity in BC patients. Spatial transcriptomic analysis indicates significantly elevated infiltration of T follicular helper (T-FH) cells and mast cells surrounding tumors exhibiting high FOXA1 expression. Conclusion: In summary, our study involved the analysis of diverse sequencing datasets derived from various FNA samples to identify biomarkers sensitive to NAC, thereby offering novel insights into resources for future personalized clinical treatment strategies.

THU479 FOXA1 Loss Remodels The Tumor Immune Microenvironment In Late-stage Prostate Cancer

Abstract Disclosure: L.T. Brea: None. H. Shi: None. V. Keo: None. W. Xie: None. X. Lu: None. G. Gritsina: None. X. Wang: None. S.H. Park: None. J. Zhao: None. J. Yu: None. Background: Castration resistant prostate cancer (CRPC) has shown a poor response to immune checkpoint inhibitors due to its immunosuppressive nature. CRPC is infiltrated by tumor-associated macrophages (TAMs) and T regulatory cells (Tregs), which induce an immunosuppressive tumor immune microenvironment (TIME) and promote tumor progression. A deeper understanding of tumor intrinsic mechanisms shaping the TIME in prostate cancer (PCa) is needed to harness immunotherapies for PCa patients. Previous studies have found that epithelial transcription factor FOXA1 is downregulated in CRPC. In accordance, we have reported that FOXA1 loss induces tumor cell invasion and macrophage recruitment in vitro through HIF1A-CCL2 signaling. However, the extent to which FOXA1 loss regulates the TIME in PCa remains unclear, largely due to the lack of immune-proficient mouse models for the study of FOXA1 function in the context of PCa. Methods: To examine how FOXA1 loss regulates the PCa TIME, we generated a novel genetically engineered mouse model (GEMM) with prostate-specific deletion of Pten and Foxa1 (PbCre4:Ptenf/fFoxa1f/f) to compare with the established Pten-null PCa mouse model (PbCre4:Ptenf/f). We performed immunohistochemistry analyses on the GEMM prostate tumor tissues to characterize histopathology changes and assess tumor immune infiltration. We also performed spatial transcriptomics and scRNA-seq analyses of the GEMMs to comprehensively evaluate the effect of Foxa1 loss on downstream pathways and the surrounding TIME. Finally, we performed bioinformatics analysis of PCa patient datasets to validate the clinical relevance. Results: We found PbCre4:Ptenf/fFoxa1f/f mouse prostate tumors exhibited a more aggressive histopathologic tumor phenotype compared to age-matched PbCre4:Ptenf/f. Notably, immunohistochemistry staining and spatial transcriptomic analysis of the GEMMs revealed striking remodeling of the TIME upon Foxa1 loss, with marked increases in tumor infiltration by Tregs and TAMs. Moreover, CIBERSORTx analysis of patient datasets revealed FOXA1 expression negatively correlates with Treg and M2-macrophage infiltration. We also found pro-tumorigenic inflammatory cytokine signaling genes, linked to the recruitment of immunosuppressive cells, were upregulated in PbCre4:Ptenf/fFoxa1f/f prostate tumors. Furthermore, we confirmed inflammatory pathways were upregulated among FOXA1-low patient samples. Additional studies are ongoing to further elucidate the mechanisms by which FOXA1 regulates inflammatory cytokine signaling and the TIME in PCa.Conclusion: This study presents a novel GEMM for the study of FOXA1 function in an immunocompetent setting. Our data supports FOXA1 as a critical tumor intrinsic regulator of the TIME. This will have important implications on the design of immunotherapeutic approaches for late-stage PCa often with FOXA1 loss. Presentation: Thursday, June 15, 2023

Endometrial expression of FOX proteins (FOXA1 and FOXA2) in women of reproductive age with different endometrial thickness: prospective cohort comparative study

Aim. To evaluate the expression of FOX proteins (FOXA1 and FOXA2) in the endometrium during the implantation window in women with a history of reproductive dysfunctions with different thickness of the endometrium.&#x0D; Materials and methods. The prospective cohort comparative study was conducted. The main group included patients with "thin" endometrium (7 mm according to ultrasound on preovulatory days; n=52), the comparison group consisted of women with normal endometrial thickness (7 mm according to ultrasound; n=62; women of both groups with reproductive dysfunctions of unknown reason), the control group included 16 healthy fertile women. Aspiration biopsy of the endometrium was performed on the 68 days after ovulation, as well as venipuncture to obtain a sample of peripheral blood to determine the levels of sex steroids (estradiol E2 and progesterone P). A combined histological and immunohistochemical study of endometrial biopsies was performed.&#x0D; Results. All women had ovulatory values of progesterone P16.1 nmol/l (68 days after ovulation) and normoestrogenemia (E2, pmol/l) in the blood. E2/P was similar in all groups (p0, 05). A pronounced expression of FOXA1 was noted in women with thin endometrium significantly more often (p0.05) with various hormone-receptor characteristics of the endometrium (42% n=22 out of 52) compared with healthy participants (0%; n=0). Reduced FOXA2 expression in the uterine mucosa was significantly more often detected on 68 days after ovulation in women with "thin" endometrium (56% n=29 of 52) than in women with normal endometrial thickness, both in women from the comparison group and in healthy women from the control group (p0.05). In a generalized analysis of the expression of FOX proteins in the endometrium on days 68 after ovulation, it was generally found that every second (50%; n=57 out of 114) women with a history of reproductive disorders (with a reduced and normal M-echo value) expression of proteomic markers differed from healthy women. In the case of "thin" endometrium, more than two thirds of patients (71%; n=37 out of 52) showed differences in endometrial expression of FOX proteins compared with women without a burdened reproductive history.&#x0D; Conclusion. In the majority of women (71%) with a thin endometrium and a history of reproductive dysfunctions, the expression of FOX proteins in the endometrium differed from the control group. Overall, endometrial expression of FOX proteins, which is likely to be different from healthy women, in patients with reproductive dysfunctions of unknown origin is a significant predictor of reproductive failure. At the same time, such an isolated indicator as M-echo value 7 mm according to ultrasound data is not an absolute prognostic marker of endometrial receptivity disorders.

Tamoxifen-predictive value of gene expression signatures in premenopausal breast cancer: data from the randomized SBII:2 trial

BackgroundGene expression (GEX) signatures in breast cancer provide prognostic information, but little is known about their predictive value for tamoxifen treatment. We examined the tamoxifen-predictive value and prognostic effects of different GEX signatures in premenopausal women with early breast cancer.MethodsRNA from formalin-fixed paraffin-embedded tumor tissue from premenopausal women randomized between two years of tamoxifen treatment and no systemic treatment was extracted and successfully subjected to GEX profiling (n = 437, NanoString Breast Cancer 360™ panel). The median follow-up periods for a recurrence-free interval (RFi) and overall survival (OS) were 28 and 33 years, respectively. Associations between GEX signatures and tamoxifen effect were assessed in patients with estrogen receptor-positive/human epidermal growth factor receptor 2-negative (ER+ /HER2−) tumors using Kaplan–Meier estimates and Cox regression. The prognostic effects of GEX signatures were studied in the entire cohort. False discovery rate adjustments (q-values) were applied to account for multiple hypothesis testing.ResultsIn patients with ER+/HER2− tumors, FOXA1 expression below the median was associated with an improved effect of tamoxifen after 10 years with regard to RFi (hazard ratio [HR]FOXA1(high) = 1.04, 95% CI = 0.61–1.76, HRFOXA1(low) = 0.30, 95% CI = 0.14–0.67, qinteraction = 0.0013), and a resembling trend was observed for AR (HRAR(high) = 1.15, 95% CI = 0.60–2.20, HRAR(low) = 0.42, 95% CI = 0.24–0.75, qinteraction = 0.87). Similar patterns were observed for OS. Tamoxifen was in the same subgroup most beneficial for RFi in patients with low ESR1 expression (HRRFi ESR1(high) = 0.76, 95% CI = 0.43–1.35, HRRFi, ESR1(low) = 0.56, 95% CI = 0.29–1.06, qinteraction = 0.37). Irrespective of molecular subtype, higher levels of ESR1, Mast cells, and PGR on a continuous scale were correlated with improved 10 years RFi (HRESR1 = 0.80, 95% CI = 0.69–0.92, q = 0.005; HRMast cells = 0.74, 95% CI = 0.65–0.85, q < 0.0001; and HRPGR = 0.78, 95% CI = 0.68–0.89, q = 0.002). For BC proliferation and Hypoxia, higher scores associated with worse outcomes (HRBCproliferation = 1.54, 95% CI = 1.33–1.79, q < 0.0001; HRHypoxia = 1.38, 95% CI = 1.20–1.58, q < 0.0001). The results were similar for OS.ConclusionsExpression of FOXA1 is a promising predictive biomarker for tamoxifen effect in ER+/HER2− premenopausal breast cancer. In addition, each of the signatures BC proliferation, Hypoxia, Mast cells, and the GEX of AR, ESR1, and PGR had prognostic value, also after adjusting for established prognostic factors.Trial registration This trial was retrospectively registered in the ISRCTN database the 6th of December 2019, trial ID: https://clinicaltrials.gov/ct2/show/ISRCTN12474687.

Hepatocyte-specific HDAC3 ablation promotes hepatocellular carcinoma in females by suppressing Foxa1/2

BackgroundHepatocellular carcinoma (HCC), the most common primary liver cancer, prevails mainly in males and has long been attributed to androgens and higher circumstantial levels of interleukin-6 (IL-6) produced by resident hepatic macrophages.MethodsConstitutively hepatocyte-specific histone deacetylase 3 (HDAC3)-deficient (HDAC3LCKO) mice and constitutively hepatocyte-specific HDAC3 knockout and systemic IL-6 simultaneously ablated (HDAC3LCKO& IL-6−/−) mice were used in our study to explore the causes of sex differences in HCC. Additionally, we performed human HCC tissues with an IHC score. Correlation analysis and linear regression plots were constructed to reveal the association between HDAC3 and its candidate genes. To further elucidate that HDAC3 controls the expression of Foxa1/2, we knocked down HDAC3 in HUH7 liver cancer cells.ResultsWe observed a contrary sex disparity, with an earlier onset and higher incidence of HCC in female mice when HDAC3 was selectively ablated in the liver. Loss of HDAC3 led to constant liver injury and the spontaneous development of HCC. Unlike the significant elevation of IL-6 in male mice at a very early age, female mice exhibit stable IL-6 levels, and IL-6 ablation did not eliminate the sex disparity in hepatocarcinogenesis in HDAC3-deficient mice. Oestrogen often protects the liver when combined with oestrogen receptor alpha (ERα); however, ovariectomy in HDAC3-ablated female mice significantly delayed tumourigenesis. The oestrogen-ERα axis can also play a role in tumour promotion in the absence of Foxa1 and Foxa2 in the receptor complex. Loss of HDAC3 profoundly reduced the expression of both Foxa1 and Foxa2 and impaired the binding between Foxa1/2 and ERα. Furthermore, a more frequent HDAC3 decrease accompanied by the simultaneous Foxa1/2 decline was found in female HCC compared to that in male HCC.ConclusionIn summary, we reported that loss of HDAC3 reduces Foxa1/2 and thus promotes HCC development in females in an oestrogen-dependent manner.

Lysosome-dependent FOXA1 ubiquitination contributes to luminal lineage of advanced prostate cancer.

Changes in FOXA1 (forkhead box protein A1) protein levels are well associated with prostate cancer (PCa) progression. Unfortunately, direct targeting of FOXA1 in progressive PCa remains challenging due to variations in FOXA1 protein levels, increased FOXA1 mutations at different stages of PCa, and elusive post-translational FOXA1 regulating mechanisms. Here, we show that SKP2 (S-phase kinase-associated protein 2) catalyzes K6- and K29-linked polyubiquitination of FOXA1 for lysosomal-dependent degradation. Our data indicate increased SKP2:FOXA1 protein ratios in stage IV human PCa compared to stages I-III, together with a strong inverse correlation (r = -0.9659) between SKP2 and FOXA1 levels, suggesting that SKP2-FOXA1 protein interactions play a significant role in PCa progression. Prostate tumors of Pten/Trp53 mice displayed increased Skp2-Foxa1-Pcna signaling and colocalization, whereas disruption of the Skp2-Foxa1 interplay in Pten/Trp53/Skp2 triple-null mice demonstrated decreased Pcna levels and increased expression of Foxa1 and luminal positive cells. Treatment of xenograft mice with the SKP2 inhibitor SZL P1-41 decreased tumor proliferation, SKP2:FOXA1 ratios, and colocalization. Thus, our results highlight the significance of the SKP2-FOXA1 interplay on the luminal lineage in PCa and the potential of therapeutically targeting FOXA1 through SKP2 to improve PCa control.

Prognostic value of FOXA1 in patients with Т1–2N0M0 breast cancer

Background. New biomarkers of breast cancer are actively being studied to used them for breast cancer prognosis and develop optimal approaches to therapy. FOXA1 is one of these factors whose prognostic value for breast cancer has been analyzed in several studies.Aim. To evaluate the prognostic value of FOXA1 in patients with T1–2N0M0 breast cancer.Materials and methods. We measured FOXA1 expression in tumor cells of patients with hormone-dependent and hormone-independent breast cancer using the method developed by us (patent No. RU2748716C1 dated 03.06.2020). We performed immunohistochemical examination of tumor tissue using specific antibodies for staining. The Allred scale was used for results interpretation (to assess the number of cells with stained nuclei and the intensity of their staining). Then the total Allred score was calculated and used as a prognostic parameter. Patients with the Allred score between 4 and 8 had a favorable prognosis. We followed up patients in the groups and evaluated their survival. A univariate analysis was used to assess the risk of lethal outcome depending on the level of FOXA1 expression.Results. In 75 % of patients with hormone-dependent and 82 % of patients with hormone-independent breast cancer (stages T1–2, N0 in both groups), the prognostic parameter measured using the Allred scale ranged between 4 and 8 points; their 10-year overall survival was 93 % and 92 %, respectively.Conclusion. Our findings suggest high accuracy of our method for breast cancer prognosis. It can be used for breast cancer prognosis in early stages (T1–2, N0) and expands the range of techniques for breast cancer prognosis.