Yongjun Tan

Foxm1 transcription factor is required for maintenance of pluripotency of P19 embryonal carcinoma cells

Transcription factor Foxm1 plays a critical role during embryonic development and its expression is repressed during retinoic acid (RA)-induced differentiation of pluripotent P19 embryonal carcinoma cells at the early stage, correlated with downregulation of expression of pluripotency markers. To study whether Foxm1 participates in the maintenance of pluripotency of stem cells, we knock down Foxm1 expression in P19 cells and identify that Oct4 are regulated directly by Foxm1. Knockdown of Foxm1 also results in spontaneous differentiation of P19 cells to mesodermal derivatives, such as muscle and adipose tissues. Maintaining Foxm1 expression prevents the downregulation of pluripotency-related transcription factors such as Oct4 and Nanog during P19 cell differentiation. Furthermore, overexpression of FOXM1 alone in RA-differentiated P19 cells (4 days) or human newborn fibroblasts restarts the expression of pluripotent genes Oct4, Nanog and Sox2. Together, our results suggest a critical involvement of Foxm1 in maintenance of stem cell pluripotency.

FOXM1 promotes the epithelial to mesenchymal transition by stimulating the transcription of Slug in human breast cancer.

The Forkhead Box M1 (FOXM1) transcription factor is involved in tumorigenesis and tumor progression in multiple human carcinomas. In this study, we found that FOXM1 promoted the epithelial to mesenchymal transition (EMT) in human breast cancer. We observed a strong correlation between the expression levels of FOXM1 and the mesenchymal phenotype. Knockdown of FOXM1 inhibited the mesenchymal phenotype, whereas stable overexpression of FOXM1 induced EMT in breast cancer cells. FOXM1 was found to endogenously bind to and stimulate the promoter of Slug that is crucial for EMT progression. The knockdown of Slug abolished the EMT-inducing function of FOXM1. The stable overexpression of FOXM1 promoted metastasis of breast cancer cells in vivo. This study confirmed that FOXM1 promoted EMT in breast cancer cells by stimulating the transcription of EMT-related genes such as Slug.

Inhibition of FOXM1 transcription factor suppresses cell proliferation and tumor growth of breast cancer.

The forkhead box M1 (FOXM1) transcription factor regulates the expression of genes essential for cell proliferation and transformation and is implicated in tumorigenesis and tumor progression. FOXM1 has been considered as a potential target for the prevention and/or therapeutic intervention in human carcinomas. In this study, we observed a strong expression of FOXM1 in clinical tissue specimens and cell lines of human breast cancer and a correlation between FOXM1 levels and the proliferation ability in the tested MCF-7, MDA-MB-231 and ZR-75-30 cells. By using an adenovirus vector (named AdFOXM1shRNA) that expresses a short hairpin RNA (shRNA) to downregulate FOXM1 expression specifically, we found that the knockdown of FOXM1 expression diminished the proliferation and anchorage-independent growth of the breast cancer cells. The FOXM1 silencing in ZR-75-30 cells dramatically prevented the tumorigenicity of the AdFOXM1shRNA-treated cells in vitro and in vivo. Furthermore, the efficacy of AdFOXM1shRNA for tumor gene therapy was assessed with the breast cancer xenograft mouse model and the tumor growth was significantly suppressed when inoculated mice were injected with AdFOXM1shRNA in the tumors. Together, our results suggest that FOXM1 is a potential therapeutic target for breast cancer and AdFOXM1shRNA may be an additional gene therapeutic intervention for breast cancer treatment.

Adenovirus-mediated RNA interference targeting FOXM1 transcription factor suppresses cell proliferation and tumor growth of nasopharyngeal carcinoma

The Forkhead Box M1 (FOXM1) transcription factor, which regulates the expression of genes essential for cell proliferation and transformation, is implicated in tumorigenesis and tumor progression. FOXM1 has attracted much attention as a potential target for the prevention and/or therapeutic intervention in human carcinomas.

Two-fold elevation of expression of FoxM1 transcription factor in mouse embryonic fibroblasts enhances cell cycle checkpoint activity by stimulating p21 and Chk1 transcription

Objectives:  Forkhead Box M1 (FoxM1) transcription factor regulates expression of cell cycle effective genes and is stabilized by checkpoint kinase 2 (Chk2) to stimulate expression of DNA repair enzymes in response to DNA damage. This study intended to test whether FoxM1 is involved in cell cycle checkpoint pathways.

FOXA2 attenuates the epithelial to mesenchymal transition by regulating the transcription of E-cadherin and ZEB2 in human breast cancer

The Forkhead Box A2 (FOXA2) transcription factor is required for embryonic development and for normal functions of multiple adult tissues, in which the maintained expression of FOXA2 is usually related to preventing the progression of malignant transformation. In this study, we found that FOXA2 prevented the epithelial to mesenchymal transition (EMT) in human breast cancer. We observed a strong correlation between the expression levels of FOXA2 and the epithelial phenotype. Knockdown of FOXA2 promoted the mesenchymal phenotype, whereas stable overexpression of FOXA2 attenuated EMT in breast cancer cells. FOXA2 was found to endogenously bind to and stimulate the promoter of E-cadherin that is crucial for epithelial phenotype of the tumor cells. Meanwhile, FOXA2 prevented EMT of breast cancer cells by repressing the expression of EMT-related transcription factor ZEB2 through recruiting a transcriptional corepressor TLE3 to the ZEB2 promoter. The stable overexpression of FOXA2 abolished metastasis of breast cancer cells in vivo. This study confirmed that FOXA2 inhibited EMT in breast cancer cells by regulating the transcription of EMT-related genes such as E-cadherin and ZEB2.

Increased levels of FoxA1 transcription factor in pluripotent P19 embryonal carcinoma cells stimulate neural differentiation.

Transcription factor FoxA1 plays a critical role during embryonic development and is activated during retinoic acid (RA)-induced neural differentiation of pluripotent P19 embryonal carcinoma cells at the early stage, which is marked by decreased expression of Nanog and increased expression of neural stem cell marker Nestin. To further understand how FoxA1 mediates neural differentiation, we have overexpressed FoxA1 through an adenovirus vector in P19 cells and identified that early neurogenesis-related sonic hedgehog (Shh) gene is activated directly by FoxA1. Knockdown of FoxA1 expression during P19 cell neural differentiation results in prevention of Shh and Nestin induction. FoxA1 binds to Shh promoter at -486 to -462 bp region and activates the promoter in cotransfection assays. Furthermore, overexpression of FoxA1 alone in P19 cells stimulates expression of Nestin and results in decreased protein levels of Nanog. During RA-induced P19 cell differentiation, elevated levels of FoxA1 increase the population of neurons, evidenced by stimulated expression of neuron-specific Neurofilament-1 and Tubulin betaIII. Together, our results suggest a critical involvement of FoxA1 in stimulating neural differentiation of pluripotent stem cells at early stages.

Foxm1 mediates LIF/Stat3-dependent self-renewal in mouse embryonic stem cells and is essential for the generation of induced pluripotent stem cells.

Activation of signal transducer and activator of transcription 3 (Stat3) by leukemia inhibitory factor (LIF) is required for maintaining self-renewal and pluripotency of mouse embryonic stem cells (mESCs). Here, we have confirmed transcription factor Forkhead Box m1 (Foxm1) as a LIF/Stat3 downstream target that mediates LIF/Stat3-dependent mESC self-renewal. The expression of Foxm1 relies on LIF signaling and is stimulated by Stat3 directly in mESCs. The knockdown of Foxm1 results in the loss of mESC pluripotency in the presence of LIF, and the overexpression of Foxm1 alone maintains mESC pluripotency in the absence of LIF and feeder layers, indicating that Foxm1 is a mediator of LIF/Stat3-dependent maintenance of pluripotency in mESCs. Furthermore, the inhibition of Foxm1 expression prevents the reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells (iPSCs), suggesting that Foxm1 is essential for the reprogramming of somatic cells into iPSCs. Our results reveal an essential function of Foxm1 in the LIF/Stat3-mediated mESC self-renewal and the generation of iPSCs.

Silencing of FOXM1 transcription factor expression by adenovirus-mediated RNA interference inhibits human hepatocellular carcinoma growth

The Forkhead Box M1 (FOXM1) transcription factor has been considered as a potential target for the prevention and/or therapeutic intervention in human carcinomas because of its roles in tumorigenesis and tumor progression through regulating the expression of genes relevant to cell proliferation and transformation. In this study, FOXM1 was found to express strongly in both clinical tissue specimens and human hepatocellular carcinoma (HCC) cell lines such as Huh-6, Huh-7 and HepG2. The knockdown of FOXM1 expression through an adenovirus vector (named AdFOXM1shRNA), which expresses a short hairpin RNA to downregulate FOXM1 expression specifically, diminished the proliferation of Huh-7 and HepG2 cells and anchorage-independent growth of Huh-7 cells. Furthermore, we assessed the efficacy of AdFOXM1shRNA for tumor gene therapy with the Huh-7 cell xenograft mouse model and found that the tumor growth was significantly suppressed when inoculated mice were injected with AdFOXM1shRNA in the tumors. Together, our results suggest that FOXM1 is a potential therapeutic target for HCC and AdFOXM1shRNA may be an additional gene therapeutic intervention for HCC treatment.

Suppression of FOXM1 Transcriptional Activities via a Single-Stranded DNA Aptamer Generated by SELEX

The transcription factor FOXM1 binds to its consensus sequence at promoters through its DNA binding domain (DBD) and activates proliferation-associated genes. The aberrant overexpression of FOXM1 correlates with tumorigenesis and progression of many cancers. Inhibiting FOXM1 transcriptional activities is proposed as a potential therapeutic strategy for cancer treatment. In this study, we obtained a FOXM1-specific single stranded DNA aptamer (FOXM1 Apt) by SELEX with a recombinant FOXM1 DBD protein as the target of selection. The binding of FOXM1 Apt to FOXM1 proteins were confirmed with electrophoretic mobility shift assays (EMSAs) and fluorescence polarization (FP) assays. Phosphorthioate-modified FOXM1 Apt (M-FOXM1 Apt) bound to FOXM1 as wild type FOXM1 Apt, and co-localized with FOXM1 in nucleus. M-FOXM1-Apt abolished the binding of FOXM1 on its consensus binding sites and suppressed FOXM1 transcriptional activities. Compared with the RNA interference of FOXM1 in cancer cells, M-FOXM1 Apt repressed cell proliferation and the expression of FOXM1 target genes without changing FOXM1 levels. Our results suggest that the obtained FOXM1 Apt could be used as a probe for FOXM1 detection and an inhibitor of FOXM1 transcriptional functions in cancer cells at the same time, providing a potential reagent for cancer diagnosis and treatment in the future.