Norcantharidin promotes cancer radiosensitization through Cullin1 neddylation-mediated CDC6 protein degradation.

Radiotherapy (RT) is a conventional cancer therapeutic modality. However, cancer cells tend to develop radioresistance after a period of treatment. Diagnostic markers and therapeutic targets for radiosensitivity are severely lacking. Our recently published studies demonstrated that the cell division cycle (CDC6) is a critical molecule contributing to radioresistance, and maybe a potential therapeutic target to overcome radioresistance. In the present study, we for the first time reported that Norcantharidin (NCTD), a demethylated form of cantharidin, re-sensitized radioresistant cancer cells to overcome radioresistance, and synergistically promoted irradiation (IR)-induced cell killing and apoptosis by inducing CDC6 protein degradation. Mechanistically, NCTD induced CDC6 protein degradation through the ubiquitin-proteasome pathways. By using small interfering RNA (siRNA) interference or small compound inhibitors, we further determined that NCTD induced CDC6 protein degradation through a neddylation-dependent pathway, but not through Huwe1, Cyclin F, and APC/C-mediated ubiquitin-proteasome pathways. We screened the six most relevant Cullin subunits (CUL1, 2, 3, 4A, 4B, and 5) using siRNAs. The knockdown of Cullin1 but not the other five cullins remarkably elevated CDC6 protein levels. NCTD promoted the binding of Cullin1 to CDC6, thereby promoting CDC6 protein degradation through a Cullin1 neddylation-mediated ubiquitin-proteasome pathway. NCTD can be used in combination with radiotherapy to achieve better anticancer efficacy, or work as a radiosensitizer to overcome cancer radioresistance.

UHRF1 promotes androgen receptor-regulated CDC6 transcription and anti-androgen receptor drug resistance in prostate cancer through KDM4C-Mediated chromatin modifications.

The UHRF1 and CDC6, oncogenes play critical roles in therapeutic resistance. In the present study, we found that UHRF1 mediates androgen receptor (AR)-regulated CDC6 transcription in prostate cancer cells. In prostate cancer tissues and cell lines, levels of UHRF1 and CDC6 were simultaneously upregulated, and this was associated with worse survival. UHRF1 silencing significantly promoted the cytotoxicity and anti-prostate cancer efficacy of bicalutamide in mouse xenografts by inhibiting CDC6 gene expression. UHRF1 promoted AR-regulated CDC6 transcription by binding to the CCAAT motif near the androgen response element (ARE) in the CDC6 promoter. We further found that UHRF1 promoted androgen-dependent chromatin occupancy of AR protein by recruiting the H3K9me2/3-specific demethyltransferase KDM4C and modifying the intense heterochromatin status. Altogether, we found for the first time that UHRF1 promotes AR-regulated CDC6 transcription through a novel chromatin modification mechanism and contributes to anti-AR drug resistance in prostate cancer. Targeting AR and UHRF1 simultaneously may be a novel and promising therapeutic modality for prostate cancer.

USP7 is a novel Deubiquitinase sustaining PLK1 protein stability and regulating chromosome alignment in mitosis.

BACKGROUND: The deubiquitinase USP7 has been identified as an oncogene with key roles in tumorigenesis and therapeutic resistance for a series of cancer types. Recently small molecular inhibitors have been developed to target USP7. However, the anticancer mechanism of USP7 inhibitors is still elusive. METHODS: Cell viability or clonogenicity was tested by violet crystal assay. Cell apoptosis or cell cycle was analyzed by flow cytometry, and chromosome misalignment was observed by a fluorescent microscopy. The protein interaction of PLK1 and USP7 was detected by tandem affinity purification and high throughput proteomics, and further confirmed by co-immunoprecipitation, GST pull-down and protein co-localization. The correlation between USP7 level of tumor tissues and taxane-resistance was evaluated. RESULTS: Pharmacological USP7 inhibition by P5091 retarded cell proliferation and induced cell apoptosis. Further studies showed that P5091 induced cell cycle arrest at G2/M phase, and particularly induced chromosome misalignment, indicating the key roles of USP7 in mitosis. USP7 protein was detected in the PLK1-interacted protein complex. USP7 interacts with PLK1 protein through its PBD domain by catalytic activity. USP7 as a deubiquitinase sustained PLK1 protein stability via the C223 site, and inversely, USP7 inhibition by P5091 promoted the protein degradation of PLK1 through the ubiquitination-proteasome pathway. By overexpressing PLK1, USP7 that had been depleted by RNAi ceased to induce chromosome misalignment in mitosis and again supported cell proliferation and cell survival. Both USP7 and PLK1 were overexpressed in taxane-resistant cancer cells, and negatively correlated with the MP scores in tumor tissues. Either USP7 or PLK1 knockdown by RNAi significantly sensitized taxane-resistant cells to taxane cell killing. CONCLUSION: This is the first report that PLK1 is a novel substrate of USP7 deubiquitinase, and that USP7 sustained the protein stability of PLK1. USP7 inhibition induces cell apoptosis and cell cycle G2/M arrest, and overcomes taxane resistance by inducing the protein degradation of PLK1, resulting in chromosome misalignment in mitosis.

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.

BACKGROUND: The 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. METHODS: Several 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. RESULTS: PCa 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. CONCLUSIONS: Our 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.

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.

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.