Youhong Liu

Activation of the p38 MAPK/Akt/ERK1/2 signal pathways is required for the protein stabilization of CDC6 and cyclin D1 in low‐dose arsenite‐induced cell proliferation

Arsenic trioxide (ATO) is a first‐line anti‐cancer agent for acute promyelocytic leukemia, and induces apoptosis in other solid cancer cell lines including breast cancer cells. However, as with arsenites found in drinking water and used as raw materials for wood preservatives, insecticides, and herbicides, low doses of ATO can induce carcinogenesis after long‐term exposure. At 24 h after exposure, ATO (0.01–1 µM) significantly increased cell proliferation and promoted cell cycle progression from the G1 to S/G2 phases in the non‐tumorigenic MCF10A breast epithelial cell line. The expression of 14 out of 96 cell‐cycle‐associated genes significantly increased, and seven of these genes including cell division cycle 6 (CDC6) and cyclin D1 (CCND1) were closely related to cell cycle progression from G1 to S phase. Low‐dose ATO steadily increased gene transcript and protein levels of both CDC6 and cyclin D1 in a dose‐ and time‐dependent manner. Low‐dose ATO produced reactive oxygen species (ROS), and activated the p38 MAPK, Akt, and ERK1/2 pathways at different time points within 60 min. Small molecular inhibitors and siRNAs inhibiting the activation of p38 MAPK, Akt, and ERK1/2 decreased the ATO‐increased expression of CDC6 protein. Inhibiting the activation of Akt and ERK1/2, but not p38 MAPK, decreased the ATO‐induced expression of cyclin D1 protein. This study reports for the first time that p38 MAPK/Akt/ERK1/2 activation is required for the protein stabilization of CDC6 in addition to cyclin D1 in ATO‐induced cell proliferation and cell cycle modulation from G1 to S phase. J. Cell. Biochem. 111: 1546–1555, 2010.

Novel Interactions between FOXM1 and CDC25A Regulate the Cell Cycle

FOXM1 is a critical regulator of the G1/S and G2/M cell cycle transitions, as well as of the mitotic spindle assembly. Previous studies have suggested that FOXM1 regulates CDC25A gene transcription, but the mechanism remains unknown. Here, we provide evidence that FOXM1 directly regulates CDC25A gene transcription via direct promoter binding and indirect activation of E2F-dependent pathways. Prior literature reported that CDC25B and CDC25C activate CDK1/cyclinB complexes in order to enable phosphorylation of FOXM1. It was unknown if CDC25A functions in a similar manner. We report that FOXM1 transcriptional activity is synergistically enhanced when co-expressed with CDC25A. The increase is dependent upon CDK1 phosphorylation of FOXM1 at T600, T611 and T620 residues. We also report a novel protein interaction between FOXM1 and CDC25A via the C-terminus of FOXM1. We demonstrate that the phosphorylation of Thr 600 and Thr 611 residues of FOXM1 enhanced this interaction, and that the interaction is dependent upon CDC25A phosphatase activity. Our work provides novel insight into the underlying mechanisms by which FOXM1 controls the cell cycle through its association with CDC25A.

Aberrant overexpression of FOXM1 transcription factor plays a critical role in lung carcinogenesis induced by low doses of arsenic

Environmental or occupational exposure to low doses of arsenic induces a series of health problems including cancer. The molecular events in arsenic‐induced carcinogenicity remain to be defined. In the NuLi‐1 immortalized human lung epithelial cell line with p53 and pRb deficiency, exposure to low doses of arsenic trioxide for 72 h promoted cell proliferation and upregulated the gene transcription levels of FOXM1, CDC6, CDC25A, and cyclin D1, which are both critical cell cycle regulatory genes and proto‐oncogenes. Continuous in vitro exposure to 1 µM arsenic trioxide for 34 wks induced malignant cell transformation, as evidenced by enhanced anchorage‐independent cell growth. The expression of FOXM1, CDC6, CDC25A, and Cyclin D1 was dynamically elevated at the gene transcription and protein levels in the process of cell transformation. The carcinogenic ability of transformed cell colonies coincides with the expression levels of FOXM1 in in vitro anchorage‐independent growth assays and in vivo tumor xenograft formation assays. In reverse, the knockdown of FOXM1 in lung adenocarcinoma A549 cells or arsenic‐transformed NuLi‐1 cells significantly decreased anchorage‐independent cell growth and tumor xenograft formation. The transformed NuLi‐1 cells showed genomic instability in the form of copy number variation (CNV) at chromosome 1, 5, 6, 18, and 20, but not loss of heterozygosity (LOH). These results showed for the first time that chronic exposure to low doses of arsenic trioxide promoted lung carcinogenicity, in part by aberrantly upregulating FOXM1 and its associated oncogenes, when the tumor suppressor genes p53 and pRb were inactivated.

The FOXM1-ABCC5 axis contributes to paclitaxel resistance in nasopharyngeal carcinoma cells.

Paclitaxel is clinically used as a first-line chemotherapeutic regimen for several cancer types, including head and neck cancers. However, acquired drug resistance results in the failure of therapy, metastasis and relapse. The drug efflux mediated by ATP-binding cassette (ABC) transporters and the survival signals activated by forkhead box (FOX) molecules are critical in the development of paclitaxel drug resistance. Whether FOX molecules promote paclitaxel resistance through drug efflux remains unknown. In this study, we developed several types of paclitaxel-resistant (TR) nasopharyngeal carcinoma (NPC) cells. These TR NPC cells acquired cancer stem cell (CSC) phenotypes and underwent epithelial to mesenchymal transition (EMT), and developed multidrug resistance. TR cells exhibited stronger drug efflux than parental NPC cells, leading to the reduction of intracellular drug concentrations and drug insensitivity. After screening the gene expression of ABC transporters and FOX molecules, we found that FOXM1 and ABCC5 were consistently overexpressed in the TR NPC cells and in patient tumor tissues. Further studies demonstrated that FOXM1 regulated abcc5 gene transcription by binding to the FHK consensus motifs at the promoter. The depletion of FOXM1 or ABCC5 with siRNA significantly blocked drug efflux and increased the intracellular concentrations of paclitaxel, thereby promoting paclitaxel-induced cell death. Siomycin A, a FOXM1 inhibitor, significantly enhanced in vitro cell killing by paclitaxel in drug-resistant NPC cells. This study is the first to identify the roles of FOXM1 in drug efflux and paclitaxel resistance by regulating the gene transcription of abcc5, one of the ABC transporters. Small molecular inhibitors of FOXM1 or ABCC5 have the potential to overcome paclitaxel chemoresistance in NPC patients.

FOXM1 promotes the progression of prostate cancer by regulating PSA gene transcription

Androgen/AR is the primary contributor to prostate cancer (PCa) progression by regulating Prostate Specific Antigen (PSA) gene transcription. The disease inevitably evolves to androgen-independent (AI) status. Other mechanisms by which PSA is regulated and develops to AI have not yet been fully determined. FOXM1 is a cell proliferation-specific transcription factor highly expressed in PCa cells compared to non-malignant prostate epithelial cells, suggesting that the aberrant overexpression of FOXM1 contributes to PCa development. In addition to regulating AR gene transcription and cell cycle-regulatory genes, FOXM1 selectively regulates the gene transcription of KLK2 and PSA, typical androgen responsive genes. Screening the potential FOXM1-binding sites by ChIP-PCR, we found that FOXM1 directly binds to the FHK binding motifs in the PSA promoter/enhancer regions. AI C4-2 cells have more FOXM1 binding sites than androgen dependent LNCaP cells. The depletion of FOXM1 by small molecular inhibitors significantly improves the suppression of PSA gene transcription by the anti-AR agent Cadosax. This is the first report showing that FOXM1 promotes PCa progression by regulating PSA gene transcription, particularly in AI PCa cells. The combination of anti-AR agents and FOXM1 inhibitors has the potential to greatly improve therapy for late-stage PCa patients by suppressing PSA levels.

Docetaxel increases antitumor efficacy of oncolytic prostate‐restricted replicative adenovirus by enhancing cell killing and virus distribution

We explored multiple molecular mechanisms of the combination of docetaxel and an oncolytic prostate‐restricted replication competent adenovirus (Ad) (PRRA) in advanced prostate cancer (PCa) models. The combinational therapy has potential to overcome the therapeutic limitations of poor virus distribution inside solid tumors.

FOXM1 contributes to taxane resistance by regulating UHRF1-controlled cancer cell stemness

Therapy-induced expansion of cancer stem cells (CSCs) has been identified as one of the most critical factors contributing to therapeutic resistance, but the mechanisms of this adaptation are not fully understood. UHRF1 is a key epigenetic regulator responsible for therapeutic resistance, and controls the self-renewal of stem cells. In the present study, taxane-resistant cancer cells were established and stem-like cancer cells were expanded. UHRF1 was overexpressed in the taxane-resistant cancer cells, which maintained CSC characteristics. UHRF1 depletion overcame taxane resistance in vitro and in vivo. Additionally, FOXM1 has been reported to play a role in therapeutic resistance and the self-renewal of CSCs. FOXM1 and UHRF1 are highly correlated in prostate cancer tissues and cells, FOXM1 regulates CSCs by regulating uhrf1 gene transcription in an E2F-independent manner, and FOXM1 protein directly binds to the FKH motifs at the uhrf1 gene promoter. This present study clarified a novel mechanism by which FOXM1 controls CSCs and taxane resistance through a UHRF1-mediated signaling pathway, and validated FOXM1 and UHRF1 as two potential therapeutic targets to overcome taxane resistance.

Radiation-promoted CDC6 protein stability contributes to radioresistance by regulating senescence and epithelial to mesenchymal transition

Ionizing radiation (IR) is a conventional cancer therapeutic, to which cancer cells develop radioresistance with exposure. The residual cancer cells after radiation treatment also have increased metastatic potential. The mechanisms by which cancer cells develop radioresistance and gain metastatic potential are still unknown. In this study acute IR exposure induced cancer cell senescence and apoptosis, but after long-term IR exposure, cancer cells exhibited radioresistance. The proliferation of radioresistant cells was retarded, and most cells were arrested in G0/G1 phase. The radioresistant cells simultaneously showed resistance to further IR-induced apoptosis, premature senescence, and epithelial to mesenchymal transformation (EMT). Acute IR exposure steadily elevated CDC6 protein levels due to the attenuation of ubiquitination, while CDC6 overexpression was observed in the radioresistant cells because the insufficiency of CDC6 phosphorylation blocked protein translocation from nucleus to cytoplasm, resulting in subcellular protein accumulation when the cells were arrested in G0/G1 phase. CDC6 ectopic overexpression in CNE2 cells resulted in apoptosis resistance, G0/G1 cell cycle arrest, premature senescence, and EMT, similar to the characteristics of radioresistant CNE2-R cells. Targeting CDC6 with siRNA promoted IR-induced senescence, sensitized cancer cells to IR-induced apoptosis, and reversed EMT. Furthermore, CDC6 depletion synergistically repressed the growth of CNE2-R xenografts when combined with IR. The study describes for the first time cell models for IR-induced senescence, apoptosis resistance, and EMT, three major mechanisms by which radioresistance develops. CDC6 is a novel radioresistance switch regulating senescence, apoptosis, and EMT. These studies suggest that CDC6highKI67low represents a new diagnostic marker of radiosensitivity, and CDC6 represents a new therapeutic target for cancer radiosensitization.

PARP inhibitor veliparib and HDAC inhibitor SAHA synergistically co-target the UHRF1/BRCA1 DNA damage repair complex in prostate cancer cells

BackgroundThe poly ADP ribose polymerase (PARP) inhibitor olaparib has been approved for treating prostate cancer (PCa) with BRCA mutations, and veliparib, another PARP inhibitor, is being tested in clinical trials. However, veliparib only showed a moderate anticancer effect, and combination therapy is required for PCa patients. Histone deacetylase (HDAC) inhibitors have been tested to improve the anticancer efficacy of PARP inhibitors for PCa cells, but the exact mechanisms are still elusive.MethodsSeveral types of PCa cells and prostate epithelial cell line RWPE-1 were treated with veliparib or SAHA alone or in combination. Cell viability or clonogenicity was tested with violet crystal assay; cell apoptosis was detected with Annexin V-FITC/PI staining and flow cytometry, and the cleaved PARP was tested with western blot; DNA damage was evaluated by staining the cells with γH2AX antibody, and the DNA damage foci were observed with a fluorescent microscopy, and the level of γH2AX was tested with western blot; the protein levels of UHRF1 and BRCA1 were measured with western blot or cell immunofluorescent staining, and the interaction of UHRF1 and BRCA1 proteins was detected with co-immunoprecipitation when cells were treated with drugs. The antitumor effect of combinational therapy was validated in DU145 xenograft models.ResultsPCa cells showed different sensitivity to veliparib or SAHA. Co-administration of both drugs synergistically decreased cell viability and clonogenicity, and synergistically induced cell apoptosis and DNA damage, while had no detectable toxicity to normal prostate epithelial cells. Mechanistically, veliparib or SAHA alone reduced BRCA1 or UHRF1 protein levels, co-treatment with veliparib and SAHA synergistically reduced BRCA1 protein levels by targeting the UHRF1/BRCA1 protein complex, the depletion of UHRF1 resulted in the degradation of BRCA1 protein, while the elevation of UHRF1 impaired co-treatment-reduced BRCA1 protein levels. Co-administration of both drugs synergistically decreased the growth of xenografts.ConclusionsOur studies revealed that the synergistic lethality of HDAC and PARP inhibitors resulted from promoting DNA damage and inhibiting HR DNA damage repair pathways, in particular targeting the UHRF1/BRCA1 protein complex. The synergistic lethality of veliparib and SAHA shows great potential for future PCa clinical trials.

AB033. Novel roles of mitochondrial outer membrane proteins in the maintenance of androgen receptor protein stability, resistance to anti-androgen receptor agents and progression of prostate cancer

Background Anti-androgen/androgen receptor (AR) agents, as the first-line hormone therapy, have been commonly administrated in prostate cancer (PCa) patients. However, the response of patients to these agents is temporary, the diseases inevitably developed to the androgen-refractory stage, and lost the response to these agents. In this current study, we investigated the novel roles of mitochondrial outer membrane (TOM) proteins in the maintenance of AR protein stability, and the development of resistance to anti-AR agents through mitochondria-mediated cell stress, and the activation of cell survival signaling pathways. Methods The co-localization of AR and TOM proteins was observed by confocal microscopy and biochemical assays. The physical interaction between AR and TOM proteins and the interactive domains were identified by the co-immunoprecipitation. TOM genes were interfered by RNAi, and AR protein stability was assessed by confocal microscopy and immunoblot, and the AR transcription activity was tested by RT-PCR and luciferase reporter assay. The mitochondrial functions and intracellular metabolism were evaluated by a Seahorse Bioscience XFe analyzer, and ROS was tested by flow cytometry. The phenotype transition was identified by the cell morphology and specific biomarkers. The sensitivity of anti-AR agents was tested by the MTS assay and cell colony assays. Results In AR-positive PCa cells, AR protein is located in mitochondria, and particularly interacts with TOM proteins through the LLXXL motifs at the C-terminal. Interfering one protein induced the degradation of another protein. The knockdown of TOM protein impaired the AR protein stability, nuclear translocation and the transcription activity. Alternatively, the knockdown of AR or anti-AR agent Casodex impaired the stability of TOM proteins. Furthermore, the interference of TOM proteins drastically altered intracellular metabolism, especially in direction to weaken cell respiration and enhance aerobic glycolysis, so called the “Warburg effect”. The consequence of the metabolic change contributed to the ROS elevation and the activation of PI3K/AKT pathway, cell survival and phenotype transition of PCa cells, and hence promoted cancer progression. Conclusions In addition to HSP proteins, we, at the first time, reported that TOM proteins are alternative novel machine to maintain AR protein stability. Importantly, anti-AR agents obtained the therapeutic effects on PCa by blocking androgen/AR, while activated alternative cell survival pathway through mitochondrial outer membrane proteins-mediated metabolism alternation and ROS elevation. These results implicated a novel mechanism in which PCa patients develop the resistance to anti-AR agents, and develop toward the androgen refractory stage.