FOXA2-Interacting FOXP2 Prevents Epithelial-Mesenchymal Transition of Breast Cancer Cells by Stimulating E-Cadherin and PHF2 Transcription.

FOXP2, a member of forkhead box transcription factor family, was first identified as a language-related gene that played an important role in language learning and facial movement. In addition, FOXP2 was also suggested regulating the progression of cancer cells. In previous studies, we found that FOXA2 inhibited epithelial-mesenchymal transition (EMT) in breast cancer cells. In this study, by identifying FOXA2-interacting proteins from FOXA2-pull-down cell lysates with Mass Spectrometry Analysis, we found that FOXP2 interacted with FOXA2. After confirming the interaction between FOXP2 and FOXA2 through Co-IP and immunofluorescence assays, we showed a correlated expression of FOXP2 and FOXA2 existing in clinical breast cancer samples. The overexpression of FOXP2 attenuated the mesenchymal phenotype whereas the stable knockdown of FOXP2 promoted EMT in breast cancer cells. Even though FOXP2 was believed to act as a transcriptional repressor in most cases, we found that FOXP2 could activate the expression of tumor suppressor PHF2. Meanwhile, we also found that FOXP2 could endogenously bind to the promoter of E-cadherin and activate its transcription. This transcriptional activity of FOXP2 relied on its interaction with FOXA2. Furthermore, the stable knockdown of FOXP2 enhanced the metastatic capacity of breast cancer cells in vivo. Together, the results suggested that FOXP2 could inhibit EMT by activating the transcription of certain genes, such as E-cadherin and PHF2, in concert with FOXA2 in breast cancer cells.

The cell-penetrating FOXM1 N-terminus (M1-138) demonstrates potent inhibitory effects on cancer cells by targeting FOXM1 and FOXM1-interacting factor SMAD3.

Transcription factor FOXM1 is involved in stimulating cell proliferation, enhancing DNA damage repair, promoting metastasis of cancer cells, and the inhibition of FOXM1 has been shown to prevent the initiation and progression of multiple cancers and FOXM1 is considered to be an effective target for tumor therapeutic drug development. The N-terminus of FOXM1 has been found to prevent transcriptional activities of FOXM1 and to mediate the interaction between FOXM1 and SMAD3. METHODS: A recombinant FOXM1 N-terminal domain (1-138aa) fused with a nine arginine cell-penetrating peptide is produced with an E. coli expression system and named as M1-138. The effects of M1-138 on the proliferation, migration, and tumorigenic ability of cancer cells are analyzed in vitro with cell counting, transwell assays, and colony formation assays. Electrophoretic mobility shift assays (EMSAs) and Luciferase activity assays are used to test the DNA binding ability and transcriptional activity of transcription factors. The levels of mRNAs and proteins are measured by quantitative-PCR, Western blotting or Immunohistochemistry. The interactions among proteins are analyzed with Pull-down and Co-immunoprecipitation (Co-IP) assays. The nude mouse engrafted tumor models are used to test the inhibitory effects of M1-138 in vivo. RESULTS: M1-138 diminishes the proliferation and migration abilities of cancer cells through binding to FOXM1 and FOXM1-interacting factor SMAD3, and consequently attenuating FOXM1 transcriptional activities from both direct and indirect FOXM1-promoter binding mechanisms and interfering with the interaction between FOXM1 and SMAD3. Treatment of M1-138 prevents tumorigenicity of cancer cells and inhibits tumor growth in nude mouse xenograft models with no obvious signs of toxicity. CONCLUSION: M1-138 is a promising drug candidate for the development of anti-cancer therapeutics targeting FOXM1 and SMAD3.

Paris polyphylla 26 triggers G2/M phase arrest and induces apoptosis in HepG2 cells via inhibition of the Akt signaling pathway.

OBJECTIVES: Paris polyphylla 26 (PP-26) is a monomer purified from Paris polyphylla, which has traditionally been used as an antimicrobial, hemostatic, and anticancer agent in China. The anti-proliferation effect and underlying molecular mechanism of PP-26 were investigated in vitro. METHODS: The effects of PP-26 on various tumor cells were detected by MTT assay. PP-26-affected cell cycle and cell cycle-related proteins in HepG2 cells were detected by flow cytometry and western blotting, respectively. Apoptosis in response to PP-26 was assessed by Hoechst 33258 staining and flow cytometry. PP-26-affected apoptosis-related proteins and Akt signaling were detected by western blotting. The inhibitory effect of PP-26 on HepG2 cells, when combined with 5-fluorouracil (5-FU), was also assessed. RESULTS: PP-26 inhibited proliferation of HepG2 cells in a dose-dependent manner by triggering G2/M-phase arrest. Moreover, PP-26 induced apoptosis of HepG2 cells. Expression levels of apoptosis proteins caspase 9, caspase 3, PARP, Bcl-2, Bcl-xL, and Mcl-1 were downregulated, while the expression level of apoptosis protein Bax was upregulated. Expression levels of p-Akt, p-GSK-3ß, and p-Foxo3 were downregulated. Combination with PP-26 enhanced 5-FU inhibition of HepG2 cell proliferation. CONCLUSIONS: PP-26 triggers G2/M-phase arrest and induces apoptosis in HepG2 cells via inhibition of the Akt signaling pathway.

PP-22 promotes autophagy and apoptosis in the nasopharyngeal carcinoma cell line CNE-2 by inducing endoplasmic reticulum stress, downregulating STAT3 signaling, and modulating the MAPK pathway.

Paris polyphylla var. yunnanensis, named Chong Lou, is considered an antitumor substance. In this study, we investigated the effect of PP-22, a monomer purified from P. polyphylla var. yunnanensis, on the nasopharyngeal carcinoma cell line CNE-2 in vitro. The results showed that PP-22 could inhibit the proliferation of CNE-2 cells via the induction of apoptosis, with evidence of the characteristic morphological changes in the apoptosis in the nucleus and an increase in Annexin V-positive cells. In addition, we found that PP-22 could activate the p38 mitogen-activated protein kinase (MAPK) pathway and that this activation was reversed by SB203580, a specific inhibitor of the p38 MAPK pathway. In contrast, PP-22 promoted apoptosis via an intrinsic pathway, including the endoplasmic reticulum stress pathway, in a caspase-dependent manner. A further study showed that PP-22 also induced apoptosis by downregulating the signal transducers and activators of transcription 3 (STAT3) pathway, and the inhibitory effect was also confirmed by STAT3 small interfering RNA. In addition, PP-22 could promote autophagy by inhibiting the extracellular regulated protein kinases (ERK) pathway. And autophagy plays a protective role against apoptosis. Together, these data show that PP-22 promotes autophagy and apoptosis in the nasopharyngeal carcinoma CNE-2 cell line.

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.

SERS assay of telomerase activity at single-cell level and colon cancer tissues via quadratic signal amplification.

As an important biomarker and therapeutic target, telomerase has attracted extensive attention concerning its detection and monitoring. Recently, enzyme-assisted amplification approaches have provided useful platforms for the telomerase activity detection, however, further improvement in sensitivity is still hindered by the single-step signal amplification. Herein, we develop a quadratic signal amplification strategy for ultrasensitive surface-enhanced Raman scattering (SERS) detection of telomerase activity. The central idea of our design is using telomerase-induced silver nanoparticles (AgNPs) assembly and silver ions (Ag(+))-mediated cascade amplification. In our approach, each telomerase-aided DNA sequence extension could trigger the formation of a long double-stranded DNA (dsDNA), making numerous AgNPs assembling along with this long strand through specific Ag-S bond, to form a primary amplification element. For secondary amplification, each conjugated AgNP was dissolved into Ag(+), which can effectively induce the 4-aminobenzenethiol (4-ABT) modified gold nanoparticles (AuNPs@4-ABT) to undergo aggregation to form numerous "hot-spots". Through quadratic amplifications, a limit of detection down to single HeLa cell was achieved. More importantly, this method demonstrated good performance when applied to tissues from colon cancer patients, which exhibits great potential in the practical application of telomerase-based cancer diagnosis in early stages. To demonstrate the potential in screening the telomerase inhibitors and telomerase-targeted drugs, the proposed design is successfully employed to measure the inhibition of telomerase activity by 3'-azido-3'-deoxythymidine.

Ultrasensitive detection of single nucleotide polymorphism in human mitochondrial DNA utilizing ion-mediated cascade surface-enhanced Raman spectroscopy amplification.

Although surface-enhanced Raman spectroscopy (SERS) has been featured by high sensitivity, additional signal enhancement is still necessary for trace amount of biomolecules detection. In this paper, a SERS amplified approach, featuring "ions-mediated cascade amplification (IMCA)", was proposed by utilizing the dissolved silver ions (Ag(+)) from silver nanoparticles (AgNPs). We found that using Ag(+) as linkage agent can effectively control the gaps between neighboring 4-aminobenzenethiol (4-ABT) encoded gold nanoparticles (AuNPs@4-ABT) to form "hot spots" and thus produce SERS signal output, in which the SERS intensity was proportional to the concentration of Ag(+). Inspired by this finding, the IMCA was utilized for ultrasensitive detection of single nucleotide polymorphism in human mitochondrial DNA (16189T → C). Combining with the DNA ligase reaction, each target DNA binding event could successfully cause one AgNP introduction. By detecting the dissolved Ag(+) from AgNPs using IMCA, low to 3.0 × 10(-5) fm/µL targeted DNA can be detected, which corresponds to extractions from 200 nL cell suspension containing carcinoma pancreatic ß-cell lines from diabetes patients. This IMCA approach is expected to be a universal strategy for ultrasensitive detection of analytes and supply valuable information for biomedical research and clinical early diagnosis.

The miR-134 attenuates the expression of transcription factor FOXM1 during pluripotent NT2/D1 embryonal carcinoma cell differentiation.

Transcription factor FOXM1 plays a critical role in maintenance of stem cell pluripotency through stimulating the transcription of pluripotency-related genes in mouse pluripotent stem cells. In this study, we have found that the repression of FOXM1 expression is mediated by FOXM1 3'UTR during retinoic acid-induced differentiation of human pluripotent NT2/D1 embryonal carcinoma cells. FOXM1 3'UTR contains a microRNA response element (MRE) for miR-134, which has been shown to attenuate the expression of pluripotency-related genes post-transcriptionally during mouse embryonic stem cell differentiation. We have determined that miR-134 is induced during RA-induced differentiation of NT2/D1 cells and the overexpression of miR-134 represses the expression of FOXM1 protein but not FOXM1 mRNA. Furthermore, the expression of OCT4 is diminished by FOXM1 knockdown and the OCT4 promoter is regulated directly by FOXM1, suggesting that FOXM1 is required for maintaining the expression of OCT4 in NT2/D1 cells. Together, our results suggest that FOXM1 is essential for human pluripotent stem cells and miR-134 attenuates its expression during differentiation.

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.

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.

Stable expression of FoxA1 promotes pluripotent P19 embryonal carcinoma cells to be neural stem-like cells.

FoxA1 belongs to the fork head/winged-helix transcription factor family and participates in stimulating neuronal differentiation of pluripotent stem cells at early stages. To explore the biological roles of FoxA1 during this process, the stable expression of a GFP-FoxA1 fusion protein was established in P19 pluripotent embryonal carcinoma cells. Although they still express pluripotency-related transcription factors such as Oct4, Nanog, and Sox2, the generated P19 GFPFoxA1 cells exhibited a decreased activity of alkaline phosphatase and an increased expression of SSEA-3 compared with P19 cells. Elevated levels of nestin expression and prominin-1+ populations were observed in P19 GFPFoxA1 cells, implicating that the stable expression of FoxA1 promoted P19 cells to gain partial characteristics of neural stem cells. Furthermore, the promoter of nestin was confirmed to be bound and activated by FoxA1 directly. The expression of neuron-specific marker tubulin betaIII also existed in P19 GFPFoxA1 cells. P19 GFPFoxA1 cells showed an earlier onset of differentiation during RA-induced neuronal differentiation, evidenced by a more rapid change on the Nanog decrease and the tubulin betaIII increase. Thus, overexpression of FoxA1 alone may promote pluripotent P19 cells to become neural stem-like cells.

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.