Regulation Of FOXM1 Research Articles

The activation of asparagine synthetase by the transcription factor FOXM1 plays a pivotal role in the initiation and progression of ESCC.

This study explores the role of the transcription factor FOXM1 in the initiation and progression of oesophageal squamous cell carcinoma (ESCC). Our findings reveal that FOXM1 is highly expressed in ESCC and correlates with the prognosis of the disease. The relationship between FOXM1 and asparagine synthetase (ASNS) is investigated, and the study demonstrates that FOXM1 activates ASNS, impacting the tumour stemness of ESCC. In this study, we reveal the association between FOXM1 and ESCC development, as well as FOXM1's promotion of migration and proliferation in ESCC cells. The study also highlights FOXM1's regulation of ASNS transcription and the functional role of ASNS in ESCC metastasis and growth. Furthermore, the study explores the impact of FOXM1 and ASNS on ESCC stemness and their potential implications for chemotherapy resistance.

Contribution of CENP-F to FOXM1-Mediated Discordant Centromere and Kinetochore Transcriptional Regulation.

Proper chromosome segregation is required to ensure chromosomal stability. The centromere (CEN) is a unique chromatin domain defined by CENP-A and is responsible for recruiting the kinetochore (KT) during mitosis, ultimately regulating microtubule spindle attachment and mitotic checkpoint function. Upregulation of many CEN/KT genes is commonly observed in cancer. Here, we show that although FOXM1 occupies promoters of many CEN/KT genes with MYBL2, FOXM1 overexpression alone is insufficient to drive the FOXM1-correlated transcriptional program. CENP-F is canonically an outer kinetochore component; however, it functions with FOXM1 to coregulate G2/M transcription and proper chromosome segregation. Loss of CENP-F results in altered chromatin accessibility at G2/M genes and reduced FOXM1-MBB complex formation. We show that coordinated CENP-FFOXM1 transcriptional regulation is a cancer-specific function. We observe a small subset of CEN/KT genes including CENP-C, that are not regulated by FOXM1. Upregulation of CENP-C in the context of CENP-A overexpression leads to increased chromosome missegregation and cell death suggesting that escape of CENP-C from FOXM1 regulation is a cancer survival mechanism. Together, we show that FOXM1 and CENP-F coordinately regulate G2/M genes, and this coordination is specific to a subset of genes to allow for maintenance of chromosome instability levels and subsequent cell survival.

High-risk histological subtype-related FAM83A hijacked FOXM1 transcriptional regulation to promote malignant progression in lung adenocarcinoma.

According to the histopathology, lung adenocarcinoma (LUAD) could be divided into five distinct pathological subtypes, categorized as high-risk (micropapillary and solid) group, intermediate-risk (acinar and papillary) group, and low-risk (lepidic) group. Despite this classification, there is limited knowledge regarding the role of transcription factors (TFs) in the molecular regulation of LUAD histology patterns. Publish data was mined to explore the candidate TFs associated with high-risk histopathology in LUAD, which was validated in tissue samples. Colony formation, CCK8, EdU, transwell, and matrigel assays were performed to determine the biological function of FAM83A in vitro. Subcutaneous tumor-bearing in BALB/c nude mice and xenograft perivitelline injection in zebrafish were utilized to unreal the function of FAM83A in vivo. We also performed chromatin immunoprecipitation (ChIP), dual-luciferase reporter, and rescue assays to uncover the underline mechanism of FAM83A. Immunohistochemistry (IHC) was performed to confirm the oncogenic role of FAM83A in clinical LUAD tissues. Screening the transcriptional expression data from TCGA-LUAD, we focus on the differentially expressed TFs across the divergent pathological subtypes, and identified that the expression of FAM83A is higher in patients with high-risk groups compared with those with intermediate or low-risk groups. The FAM83A expression is positively correlated with worse overall survival, progression-free survival, and advanced stages. Gain- and loss-of-function assays revealed that FAM83A promoted cell proliferation, invasion, and migration of tumor cell lines both in vivo and in vitro. Pathway enrichment analysis shows that FAM83A expression is significantly enriched in cell cycle-related pathways. The ChIP and luciferase reporter assays revealed that FAM83A hijacks the promoter of FOXM1 to progress the malignant LUAD, and the rescue assay uncovered that the function of FAM83A is partly dependent on FOXM1 regulation. Additionally, patients with high FAM83A expression positively correlated with higher IHC scores of Ki-67 and FOXM1, and patients with active FAM83A/FOXM1 axis had poor prognoses in LUAD. Taken together, our study revealed that the high-risk histological subtype-related FAM83A hijacks FOXM1 transcriptional regulation to promote malignant progression in lung adenocarcinoma, which implies targeting FAM83A/FOXM1 is the therapeutic vulnerability.

Transcription factor FoxM1 promotes cyst growth in PKD1 mutant ADPKD.

Autosomal dominant polycystic kidney disease (ADPKD) is driven by mutations in the PKD1 and PKD2 genes, and it is characterized by renal cyst formation, inflammation and fibrosis. Forkhead box protein M1 (FoxM1), a transcription factor of the Forkhead box (Fox) protein super family, has been reported to promote tumor formation, inflammation and fibrosis in many organs. However, the role and mechanism of FoxM1 in regulation of ADPKD progression is still poorly understood. Here, we show that FoxM1 is an important regulator of cyst growth in ADPKD. FoxM1 is upregulated in cyst-lining epithelial cells in Pkd1 mutant mouse kidneys and human ADPKD kidneys. FoxM1 promotes cystic renal epithelial cell proliferation by increasing the expression of Akt and Stat3 and the activation of ERK and Rb. FoxM1 also regulates cystic renal epithelial cell apoptosis through NF-κB signaling pathways. In addition, FoxM1 regulates the recruitment and retention of macrophages in Pkd1 mutant mouse kidneys, a process that is associated with FoxM1-mediated upregulation of monocyte chemotactic protein 1. Targeting FoxM1 with its specific inhibitor, FDI-6, delays cyst growth in rapidly progressing and slowly progressing Pkd1 mutant mouse kidneys. This study suggests that FoxM1 is a central and upstream regulator of ADPKD pathogenesis and provides a rationale for targeting FoxM1 as a therapeutic strategy for ADPKD treatment.

The Synergistic Effects of SHR6390 Combined With Pyrotinib on HER2+/HR+ Breast Cancer.

HER2+/HR+ breast cancer is a special molecular type of breast cancer. Existing treatment methods are prone to resistance; “precision treatment” is necessary. Pyrotinib is a pan-her kinase inhibitor that can be used in HER2-positive tumors, while SHR6390 is a CDK4/6 inhibitor that can inhibit ER+ breast cancer cell cycle progression and cancer cell proliferation. In cancer cells, HER2 and CDK4/6 signaling pathways could be nonredundant; co-inhibition of both pathways by combination of SHR6390 and pyrotinib may have synergistic anticancer activity on HER2+/HR+ breast cancer. In this study, we determined the synergy of the two-drug combination and underlying molecular mechanisms. We showed that the combination of SHR6390 and pyrotinib synergistically inhibited the proliferation, migration, and invasion of HER2+/HR+ breast cancer cells in vitro. The combination of two drugs induced G1/S phase arrest and apoptosis in HER2+/HR+ breast cancer cell lines. The combination of two drugs prolonged the time to tumor recurrence in the xenograft model system. By second-generation RNA sequencing technology and enrichment analysis of the pyrotinib-resistant cell line, we found that FOXM1 was associated with induced resistance to HER2-targeted therapy. In HER2+/HR+ breast cancer cell lines, the combination of the two drugs could further reduce FOXM1 phosphorylation, thereby enhancing the antitumor effect to a certain extent. These findings suggest that SHR6390 combination with pyrotinib suppresses the proliferation, migration, and invasion of HER2+/HR+ breast cancers through regulation of FOXM1.

Alternative splicing regulation of cell-cycle genes by SPF45/SR140/CHERP complex controls cell proliferation.

The regulation of pre-mRNA processing has important consequences for cell division and the control of cancer cell proliferation, but the underlying molecular mechanisms remain poorly understood. We report that three splicing factors, SPF45, SR140, and CHERP, form a tight physical and functionally coherent complex that regulates a variety of alternative splicing events, frequently by repressing short exons flanked by suboptimal 3′ splice sites. These comprise alternative exons embedded in genes with important functions in cell-cycle progression, including the G2/M key regulator FOXM1 and the spindle regulator SPDL1. Knockdown of either of the three factors leads to G2/M arrest and to enhanced apoptosis in HeLa cells. Promoting the changes in FOXM1 or SPDL1 splicing induced by SPF45/SR140/CHERP knockdown partially recapitulates the effects on cell growth, arguing that the complex orchestrates a program of alternative splicing necessary for efficient cell proliferation.

Abstract 123: A CRISPR/Cas9-engineered ARID1A-deficient human gastric cancer organoid model reveals essential and non-essential modes of oncogenic transformation

Abstract Mutations in ARID1A, encoding a subunit of the BAF chromatin remodeling complex, rank amongst the most common molecular aberrations in human cancer. However, oncogenic consequences of ARID1A mutation in human cells remain poorly defined due to lack of forward human genetic model systems. Here, CRISPR/Cas9 knockout of ARID1A in primary TP53-/- human gastric organoids induced morphologic dysplasia, tumorigenicity and mucinous differentiation but impaired Wnt/β-catenin signaling. Genetic Wnt pathway activation rescued mucinous differentiation, but not hyperproliferation, suggesting alternative essential pathways of ARID1A-mediated transformation. ARID1A loss induced transcriptional regulatory modules characteristic of MSI and EBV subtype human gastric cancer, including FOXM1 regulation of multiple mitotic regulatory genes and BIRC5/survivin. Convergently, high-throughput compound screening indicated selective vulnerability of ARID1A-deficient organoids to BIRC5/survivin inhibition, functionally implicating BIRC5/survivin as an essential mediator of proliferation following initial ARID1A loss. However, CRISPR inactivation of ARID1A in established gastric cancer cell lines did not elicit sensitivity to BIRC5/survivin inhibitors, suggesting that this pathway may be selectively required during the early establishment of tumorigenesis. Overall, we define distinct pathways downstream of oncogenic ARID1A mutation, with non-essential Wnt-inhibited mucinous differentiation in parallel with essential transcriptional FOXM1/BIRC5-stimulated proliferation, indicating the general utility of organoid-based forward genetic cancer analysis in human cells. Citation Format: Yuan-Hung Lo, Kevin S. Kolahi, Yuhong Du, Chiung-Ying Chang, Andrey Krokhotin, Ajay Nair, Walter D. Sobba, Kasper Karlsson, Sunny J. Jones, Teri A. Longacre, Amanda T. Mah, Alexandra Sockell, Jose A. Seoane, Jin Chen, Jonathan S. Weissman, Christina Curtis, Andrea Califano, Haian Fu, Gerald R. Crabtree, Calvin J. Kuo. A CRISPR/Cas9-engineered ARID1A-deficient human gastric cancer organoid model reveals essential and non-essential modes of oncogenic transformation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 123.

An Integrated Analysis of the Response of Colorectal Adenocarcinoma Caco-2 Cells to X-Ray Exposure.

Colorectal cancer is among the three top cancer types for incidence and the second in terms of mortality, usually managed with surgery, chemotherapy and radiotherapy. In particular, radiotherapeutic concepts are crucial for the management of advanced rectal cancer, but patients’ survival remains poor, despite advances in treatment modalities. The use of well-characterized in vitro cell culture systems offers an important preclinical strategy to study mechanisms at the basis of cell response to therapeutic agents, including ionizing radiation, possibly leading to a better understanding of the in vivo response to the treatment. In this context, we present an integrated analysis of results obtained in an extensive measurement campaign of radiation effects on Caco-2 cells, derived from human colorectal adenocarcinoma. Cells were exposed to X-rays with doses up to 10 Gy from a radiotherapy accelerator. We measured a variety of endpoints at different post-irradiation times: clonogenic survival after ~ 2 weeks; cell cycle distribution, cell death, frequency of micronucleated cells and atypical mitoses, activation of matrix metalloproteases (MMPs) and of different proteins involved in DNA damage response and cell cycle regulation at earlier time points, up to 48 h post-exposure. Combined techniques of flow cytometry, immunofluorescence microscopy, gelatin zymography and western blotting were used. For selected endpoints, we also addressed the impact of the irradiation protocol, comparing results obtained when cells are plated before irradiation or first-irradiated and then re-plated. Caco-2 resistance to radiation, previously assessed up to 72 h post exposure in terms of cell viability, does not translate into a high clonogenic survival. Survival is not affected by the irradiation protocol, while endpoints measured on a shorter time frame are. Radiation mainly induces a G2-phase arrest, confirmed by associated molecular markers. The activation of death pathways is dose- and time-dependent, and correlates with a dose-dependent inhibition of MMPs. Genomic aberrations are also found to be dose-dependent. The phosphorylated forms of several proteins involved in cell cycle regulation increase following exposure; the key regulator FoxM1 appears to be downregulated, also leading to inhibition of MMP-2. A unified molecular model of the chain of events initiated by radiation is proposed to interpret all experimental results.

Glycogen synthase kinase-3β inhibits tubular regeneration in acute kidney injury by a FoxM1-dependent mechanism.

Acute kidney injury (AKI) is characterized by injury to the tubular epithelium that leads to the sudden loss of renal function. Proper tubular regeneration is essential to prevent progression to chronic kidney disease. In this study, we examined the role of FoxM1, a forkhead box family member transcription factor in tubular repair after AKI. Renal FoxM1 expression increased after renal ischemia/reperfusion (I/R)-induced AKI in mouse kidneys. Treatment with thiostrepton, a FoxM1 inhibitor, reduced FoxM1 regulated pro-proliferative factors and cell proliferation in vitro, and tubular regeneration in mouse kidneys after AKI. Glycogen synthase kinase-3 (GSK3) was found to be an upstream regulator of FoxM1 because GSK3 inhibition or renal tubular GSK3β gene deletion significantly increased FoxM1 expression, and improved tubular repair and renal function. GSK3 inactivation increased β-catenin, Cyclin D1, and c-Myc, and reduced cell cycle inhibitors p21 and p27. Importantly, thiostrepton treatment abolished the improved tubular repair in GSK3β knockout mice following AKI. These results demonstrate that FoxM1 is important for renal tubular regeneration following AKI and that GSK3β suppresses tubular repair by inhibiting FoxM1.

Abstract 3704: MicroRNA-3977 acts as a tumor suppressor and inhibits triple-negative breast cancer tumorigenesis by targeting FoxM1

Abstract Triple negative breast cancer (TNBC) is an aggressive type of breast cancer characterized by the absence of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). TNBC is associated with poor prognosis, high rates of relapse and distant metastasis despite surgery and adjuvant chemotherapy. The lack of effective targeted therapies for TNBC requires new therapeutic approaches. The Forkhead box protein M1 (FoxM1) is a member of forkhead superfamily transcription factors that has been associated with normal cell growth and proliferation through cell cycle transition and mitotic spindle control. FoxM1 is also identified as a master regulator of tumor metastasis. Based on analysis of pooled clinical datasets derived from online accessible databases as Breast Cancer Gene-Expression Miner v4.3 and Kaplan-Meier plotter, the expression and prognostic values of FoxM1 increase in TNBC compare with other breast cancer subtypes. Directly binding to the 3'-untranslated region (3' UTR) of FoxM1 mRNAs suppress its high expression in TNBC that leads to inhibition of cell proliferation, motility, and invasion. These results implied that FoxM1 is an essential poor prognostic factor and promising candidate target in the treatment of TNBC. We identified miR-3977 as a potential regulator of FoxM1 by using in silico prediction algorithms, and we investigated its role in TNBC. In this study, we found miR-3977 expression is significantly suppressed in TNBC cell lines and inversely correlated with FoxM1 levels. We demonstrate that miR-3977 directly bound to the 3′ UTR of FoxM1 mRNA and repressed its expression. We also find reduced miR-3977 expression is associated with shorter patient survival in TNBC based on Kaplan-Meier plotter database. Moreover, in vivo delivery of miR-3977 nanoparticles inhibited FoxM1 expression and tumor growth in TNBC xenograft mouse models. In conclusion, we provide the first evidence that miR-3977 functions as a tumor suppressor and gain of miR-3977 expression leads to decrease in FoxM1 expression and inhibits to cell growth, colony formation, invasion, and progression of TNBC. Taken together, our data suggest that miR-3977-based gene therapy is a potential therapeutic strategy in TNBC. Citation Format: Selda Karamil, Nuray Altintas, Bulent Ozpolat. MicroRNA-3977 acts as a tumor suppressor and inhibits triple-negative breast cancer tumorigenesis by targeting FoxM1 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3704.

Aberrant ASPM expression mediated by transcriptional regulation of FoxM1 promotes the progression of gliomas.

Gliomas are the most common form of malignant tumour in the central nervous system. However, the molecular mechanism of the tumorigenesis and progression of gliomas remains unclear. In this study, we used the GEO database to identify genes differentially expressed in gliomas and predict the prognosis of glioma. We observed that ASPM mRNA was increased obviously in glioma tissue, and higher ASPM mRNA expression predicted worse disease prognosis. ASPM was highly expressed in glioma cell lines U87‐MG and U251, and knockdown of ASPM expression in these cells significantly repressed the proliferation, migration and invasion ability and induced G0/G1 phase arrest. In addition, down‐regulation of ASPM suppressed the growth of glioma in nude mice. Five potential binding sites for transcription factor FoxM1 were predicted in the ASPM promoter. FoxM1 overexpression significantly increased the expression of ASPM and promoted the proliferation and migration of glioma cells, which was abolished by ASPM ablation. ChIP and dual‐luciferase reporter analysis confirmed that FoxM1 bound to the ASPM promoter at −236 to ‐230 bp and −1354 to ‐1348 bp and activated the transcription of ASPM directly. Collectively, our results demonstrated for the first time that aberrant ASPM expression mediated by transcriptional regulation of FoxM1 promotes the malignant properties of glioma cells.

LncRNA MFI2-AS1 promotes HCC progression and metastasis by acting as a competing endogenous RNA of miR-134 to upregulate FOXM1 expression

Hepatocellular carcinoma (HCC) is worldwide accepted most common malignancies, as well as the second major cause of death among Chinese with cancer. There is an increasing evidence that could prove the potential effect of long non-coding RNAs (lncRNAs) to the biological performance of HCC. In present study, with high expression level in The Cancer Genome Atlas (TCGA) HCC samples, lncRNA MFI2 Antisense RNA 1 (MFI2-AS1) was closely related to poor prognosis and advanced stage among patients with HCC. In addition, up-regulation of MFI2-AS1 was further comfirmed in HCC tissues and HCC cell line. Ectopic expression of MFI2-AS1 stimulated the proliferation and metastasis of HCC cells, but knockdown MFI2-AS1 suppressed HCC cell proliferation and metastasis, indicating that MFI2-AS1 exerted oncogenic functions in the tumorigenesis of HCC. Simultaneously, compared with the negative control group, xenograft tumors in MFI2-AS1 group were characterized with poor growth, smaller volumes and less liver metastases. The post-transcriptional regulation of FOXM1 by MFI2-AS1 occured mechanistically, playing a role of competing with endogenous RNA (ceRNA) in HCC to sponge miR-134. Over-expression of MFI2-AS1 increased FOXM1 expression both at mRNA and protein level, whereas it was reducd by miR-134. Meanwhile, knockdown of miR-134 abolished the repression of shMFI2-AS1 on FOXM1 expression. Furthermore, we demonstrated that miR-134 reverses the impact of MFI2-AS1 on HCC proliferation and metastasis through regulation on FOXM1. Collectively, we determined that MFI2-AS1 crucially acted in HCC progression via functioning as miR-134 sponge to upregulating FOXM1 expression, and was conducive to the promotion of better understanding the direct diagnostics and iatreusiology of lncRNA in HCC.

Abstract P5-05-05: Suppression of FOXM1 activities and breast cancer growth in vitro and in vivo by a new class of compounds

Abstract The transcription factor FOXM1 is up-regulated and overexpressed in aggressive, therapy-resistant forms of hormone receptor-positive and triple negative breast cancers, and is associated with less good patient survival. FOXM1 signaling is also a key driver in many other cancers. Here, we identify a new class of compounds effective in suppressing FOXM1 activity in breast cancers, and displaying good potency for antitumor efficacy. The compounds bind directly to FOXM1 and alter its proteolytic sensitivity, reduce the cellular level of FOXM1 protein by a proteasome-dependent process, and suppress breast cancer cell proliferation and cell cycle progression and increase apoptosis. RNA-seq and gene set enrichment analyses indicate that the compounds decrease expression of FOXM1-regulated genes and suppress gene ontologies under FOXM1 regulation. Several compounds have favorable pharmacokinetic properties and show good tumor suppression in preclinical breast tumor models. These compounds may be suitable for further clinical evaluation in targeting aggressive breast cancers driven by FOXM1. (Supported by Breast Cancer Research Foundation grant (BCRF-083 to BSK), The Julius and Mary Landfield Cancer Research Fund (to BSK), NIH grant R01 DK015556 (to JAK), NIH T32 GM070421 Fellowship (to VSG), Bankhead-Coley Foundation grant 09BW04 (to DEA), USDA award ILLU-698-909 and National Center for Supercomputing Applications Faculty Fellowship (to ZME), and UIUC Environmental Toxicology Scholarship (to BPS)). Citation Format: Benita S. Katzenellenbogen, Yvonne Ziegler, Mary J Laws, Valeria S. Guillen, SungHoon Kim, Parama Dey, Brandi P. Smith, Ping Gong, Noah Bindman, Yuechao Zhao, Kathryn Carlson, Mayuri A. Yasuda, Divya Singh, Zhong Li, Dorraya El-Ashry, Zeynep Madak-Erdogan, John A. Katzenellenbogen. Suppression of FOXM1 activities and breast cancer growth in vitro and in vivo by a new class of compounds [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-05-05.

Anticancer Impact and FOXM1 Regulation of Zinc Oxide Nanoparticles on HCT116 Colorectal Carcinoma Cell Line

Anticancer Impact and FOXM1 Regulation of Zinc Oxide Nanoparticles on HCT116 Colorectal Carcinoma Cell Line

Suppression of FOXM1 activities and breast cancer growth in vitro and in vivo by a new class of compounds

The transcription factor FOXM1 is upregulated and overexpressed in aggressive, therapy-resistant forms of hormone receptor-positive and triple negative breast cancers, and is associated with less good patient survival. FOXM1 signaling is also a key driver in many other cancers. Here, we identify a new class of compounds effective in suppressing FOXM1 activity in breast cancers, and displaying good potency for antitumor efficacy. The compounds bind directly to FOXM1 and alter its proteolytic sensitivity, reduce the cellular level of FOXM1 protein by a proteasome- dependent process, and suppress breast cancer cell proliferation and cell cycle progression and increase apoptosis. RNA-seq and gene set enrichment analyses indicate that the compounds decrease expression of FOXM1-regulated genes and suppress gene ontologies under FOXM1 regulation. Several compounds have favorable pharmacokinetic properties and show good tumor suppression in preclinical breast tumor models. These compounds may be suitable for further clinical evaluation in targeting aggressive breast cancers driven by FOXM1.

PBX1 Co-Operates with FOXM1 to Regulate Myeloma Cell Proliferation and to Define an Ultra High-Risk chr1q Gain Myeloma Patient Subgroup

BACKGROUND: The biological basis for the adverse prognosis of chr1q gain/amplification (1q+) present in ~30% of patients with multiple myeloma (MM) remains ill-defined. The transcription factor (TF) Pre-B-cell leukaemia homeobox 1 (PBX1), encoded on chr1q, acts as master regulator of early hematopoiesis and as an oncogene in leukemia and other malignancies. Herein, we hypothesized that PBX1 orchestrates proliferative regulatory networks that underpin the poor prognosis associated with 1q+ in MM. METHODS: We employed qPCR for mRNA quantification, western blotting and immunohistochemistry for protein analysis, lentiviral shRNA-mediated knock-down, Hoechst/Annexin V staining and flow-cytometry for apoptosis and cell cycle analysis, ChIPseq for cistrome and RNAseq after knock-down for transcriptome analysis. Additional data were obtained from MMRF/CoMMpass, Blueprint Consortium and Arkansas datasets. Computational analysis of clinical and "-omics" data was performed using standard bioinformatic work-flows; pathway enrichment analysis using EnrichR and GSEA. RESULTS: Combined genomic (WGS/WES) and transcriptomic (RNAseq) analysis of the CoMMpass dataset identified a subgroup of 1q+ MM patients (60%) characterized by aberrant PBX1 overexpression and amplification (PBX1amp); survival analysis revealed significantly worse outcome of this subgroup compared to 1q+ non-PBX1amp (p<0.05) and 1q-negative (p<0.001) patients. Analysis of the Arkansas dataset validated these findings while immunohistochemistry confirmed PBX1 protein expression in 8/11 1q+ MM patient bone marrow biopsies. Depletion of PBX1 using 2 validated shRNAs was toxic to the 1q+ MM1.S t(14;16) and U266 t(11;14) myeloma cell lines (MMCL) in vitro, revealing a novel myeloma cell addiction to PBX1. Transcriptome analysis of PBX1-depleted cells showed significant de-regulation of genes (MM1.S: 753down/320up; U266: 598 down/288up) highly enriched in cell cycle and proteasome/ER stress response pathways. Flow-cytometric analysis confirmed that PBX1-depleted myeloma cells undergo cell cycle arrest (G0/1 phase) and apoptosis (n=3; p<0.001). Genome-wide binding profiling of PBX1 identified 7,354 significantly enriched peaks shared between MM1.S and U266 MMCL. These peaks were distributed at active promoters (~50%) and active enhancers (~30%), as defined by ChromHMM maps; the PBX1 consensus binding motif was identified as top hit upon motif enrichment analysis. Transcriptome-cistrome integration identified 728 and 282 genes directly activated or repressed, respectively, by PBX1 in MM1.S cells. Again, pathway enrichment analysis of PBX1 direct targets demonstrated significant over-representation of cell cycle and proliferation pathways, as well as positive regulation of various metabolic processes (oxidative phosphorylation, glycolysis, unfolded protein response). Strikingly, enrichment analysis against public ChIPseq datasets revealed significant overlap with gene targets of FOXM1 (200) and E2F (150) TF, suggesting the presence of a united PBX1-FOXM1-E2F regulatory circuitry. In fact, PBX1 binding was detected at an active FOXM1 enhancer and at the E2F1/2 promoters, while FOXM1 and E2F1/2 mRNA levels decreased significantly upon PBX1 depletion in two MMCL. To further explore the PBX1-FOXM1 interaction, we overlaid the FOXM1-depleted transcriptome with the PBX1 network in MM.1S cells. This identified 160 genes to be co-regulated by PBX1 and FOXM1, including genes previously linked to myeloma cell drug resistance (NEK2, TOP2A, AURKA); however, PBX1 expression was not altered in FOXM1-depleted cells, suggesting that PBX1 is functionally upstream of FOXM1. Additional analysis across 814 primary MM patient CoMMpass transcriptomes confirmed significant co-expression of PBX1 with FOXM1, E2F1/2 and another 67 PBX1-FOXM1 highly expressed, co-regulated gene targets, conferring poor OS (p<0.0001). CONCLUSIONS: We found that genetic amplification and overexpression of PBX1 defines an ultra high-risk subgroup of 1q+ MM with proliferative features. Myeloma cells are addicted to aberrant PBX1 expression. As well as essential metabolic pathways, PBX1 regulates cell cycle progression by direct transcriptional regulation of FOXM1 and of its target genes. These data reveal PBX1 as a critical biological link between chr1q gain/amplification and poor prognosis in a subset of MM patients. Disclosures Auner: Karyopharm: Consultancy; Amgen: Other: Consultancy and Research Funding; Takeda: Consultancy. Caputo:GSK: Research Funding. Karadimitris:GSK: Research Funding.

Transcriptional regulation of FoxM1 by HIF‑1α mediates hypoxia‑induced EMT in prostate cancer.

Hypoxia is a tumorigenesis-related microenvironment change which usually occurs in the earliest stage of prostate cancer (PCa) development. Accumulating evidence has demonstrated that hypoxia/hypoxia-inducing factor (HIF) is involved in the induction of epithelial-mesenchymal transition (EMT) and increased metastatic potential in PCa. However, the mechanism by which hypoxia/HIF regulates EMT remains unclear. In the present study, we demonstrated the molecular mechanisms of hypoxia-induced EMT in PCa, focusing on HIF-1α/Forkhead box M1 (FoxM1) signaling pathway. PCa PC3 and DU145 cell lines were used as the model system in vitro. Our data revealed that hypoxia induced EMT in PCa cells. Bioinformatics analysis identified the possible association between HIF-1α and FoxM1. Additionally, FoxM1 was significantly associated with PCa development and Gleason scores of PCa. Exposure to hypoxia resulted in the increased expression of HIF-1α and FoxM1. Genetic knockdown FoxM1 abolished hypoxia-induced EMT in PCa, while exogenous overexpression of FoxM1 facilitated hypoxia-induced EMT. Furthermore, the increase of FoxM1 during hypoxia was due to the transcriptional regulation on the FoxM1 promoter by HIF-1α. We also confirmed the binding site of HIF-1α on the FoxM1 promoter by different lengths promoter sequences. These findings provide new insights into how EMT is regulated in PCa under hypoxic stress. It is worthwhile to investigate in future that inhibition of FoxM1 as a potential target may be an effective therapeutic strategy against PCa.

An order-to-disorder structural switch activates the FoxM1 transcription factor.

Intrinsically disordered transcription factor transactivation domains (TADs) function through structural plasticity, adopting ordered conformations when bound to transcriptional co-regulators. Many transcription factors contain a negative regulatory domain (NRD) that suppresses recruitment of transcriptional machinery through autoregulation of the TAD. We report the solution structure of an autoinhibited NRD-TAD complex within FoxM1, a critical activator of mitotic gene expression. We observe that while both the FoxM1 NRD and TAD are primarily intrinsically disordered domains, they associate and adopt a structured conformation. We identify how Plk1 and Cdk kinases cooperate to phosphorylate FoxM1, which releases the TAD into a disordered conformation that then associates with the TAZ2 or KIX domains of the transcriptional co-activator CBP. Our results support a mechanism of FoxM1 regulation in which the TAD undergoes switching between disordered and different ordered structures.

Naringin Ameliorates Streptozotocin-Induced Diabetes through Forkhead Box M1-Mediated Beta Cell Proliferation

Background: Adult pancreatic beta cells, though quiescent, can proliferate in response to physiological need. This inherent character is used in exploring the possibilities of expanding the beta cell mass in the treatment of diabetes. Forkhead box M1 (FoxM1) transcription factor is an important regulator in the proliferation and survival of adult beta cell mass. Naringin, a flavanone glycoside, is reported to have antidiabetic activity and exhibited an increase in insulin levels in diabetic animals. Objectives: The present study tried to evaluate the role of naringin in the regulation of FoxM1 in the pancreas of diabetic rats and to reascertain its antilipidemic and antioxidant properties. Methods: Diabetes was induced in male rats using streptozotocin and treated with naringin (100 mg/kg) orally for 4 and 8 weeks. Serum biochemical parameters, insulin, gene and protein expression of FoxM1, and antioxidant markers in rat pancreas were analyzed. Results: Naringin administration reduced the blood sugar, urea, creatinine, and cholesterol values and improved the pancreatic antioxidant status in diabetic rats. Naringin-treated diabetic rats showed a significant increase in mRNA and protein expression of FoxM1 compared to the diabetic control rats, indicating regeneration of cells. It also increased the insulin immunopositive cells, indicating functional beta cells. Conclusion: Naringin was found to upregulate the FoxM1 transcription factor in diabetic animals, which influenced the proliferation and functional status of beta cells.

Pan-Cancer Analyses Reveal Genomic Features of FOXM1 Overexpression in Cancer.

FOXM1 is frequently overexpressed in cancer, but this has not been studied in a comprehensive manner. We utilized genotype-tissue expression (GTEx) normal and The Cancer Genome Atlas (TCGA) tumor data to define FOXM1 expression, including its isoforms, and to determine the genetic alterations that promote FOXM1 expression in cancer. Additionally, we used human fallopian tube epithelial (FTE) cells to dissect the role of Retinoblastoma (Rb)-E2F and Cyclin E1 in FOXM1 regulation, and a novel human embryonic kidney cell (HEK293T) CRISPR FOXM1 knockout model to define isoform-specific transcriptional programs. FOXM1 expression, at the mRNA and protein level, was significantly elevated in tumors with FOXM1 amplification, p53 inactivation, and Rb-E2F deregulation. FOXM1 expression was remarkably high in testicular germ cell tumors (TGCT), high-grade serous ovarian cancer (HGSC), and basal breast cancer (BBC). FOXM1 expression in cancer was associated with genomic instability, as measured using aneuploidy signatures. FTE models confirmed a role for Rb-E2F signaling in FOXM1 regulation and in particular identified Cyclin E1 as a novel inducer of FOXM1 expression. Among the three FOXM1 isoforms, FOXM1c showed the highest expression in normal and tumor tissues and cancer cell lines. The CRISPR knockout model demonstrated that FOXM1b and FOXM1c are transcriptionally active, while FOXM1a is not. Finally, we were unable to confirm the existence of a FOXM1 auto-regulatory loop. This study provides significant and novel information regarding the frequency, causes, and consequences of elevated FOXM1 expression in human cancer.