The circadian cryptochrome, CRY1, is a pro-tumorigenic factor that rhythmically modulates DNA repair

AbstractMechanisms regulating DNA repair processes remain incompletely defined. Here, the circadian factor CRY1, an evolutionally conserved transcriptional coregulator, is identified as a tumor specific regulator of DNA repair. Key findings demonstrate that CRY1 expression is androgen-responsive and associates with poor outcome in prostate cancer. Functional studies and first-in-field mapping of the CRY1 cistrome and transcriptome reveal that CRY1 regulates DNA repair and the G2/M transition. DNA damage stabilizes CRY1 in cancer (in vitro, in vivo, and human tumors ex vivo), which proves critical for efficient DNA repair. Further mechanistic investigation shows that stabilized CRY1 temporally regulates expression of genes required for homologous recombination. Collectively, these findings reveal that CRY1 is hormone-induced in tumors, is further stabilized by genomic insult, and promotes DNA repair and cell survival through temporal transcriptional regulation. These studies identify the circadian factor CRY1 as pro-tumorigenic and nominate CRY1 as a new therapeutic target.

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Conditionally Reprogrammed Cells from Patient-Derived Xenograft to Model Neuroendocrine Prostate Cancer Development

Cells ◽

10.3390/cells9061398 ◽

2020 ◽

Vol 9 (6) ◽

pp. 1398 ◽

Cited By ~ 2

Author(s):

Xinpei Ci ◽

Jun Hao ◽

Xin Dong ◽

Hui Xue ◽

Rebecca Wu ◽

...

Keyword(s):

Prostate Cancer ◽

Ex Vivo ◽

Cancer Cell Line ◽

Marker Genes ◽

Patient Derived Xenograft ◽

Pdx Model ◽

Neuroendocrine Prostate Cancer ◽

Parental Tumor

Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer. It develops mainly via NE transdifferentiation of prostate adenocarcinoma in response to androgen receptor (AR)-inhibition therapy. The study of NEPC development has been hampered by a lack of clinically relevant models. We previously established a unique and first-in-field patient-derived xenograft (PDX) model of adenocarcinoma (LTL331)-to-NEPC (LTL331R) transdifferentiation. In this study, we applied conditional reprogramming (CR) culture to establish a LTL331 PDX-derived cancer cell line named LTL331_CR_Cell. These cells retain the same genomic mutations as the LTL331 parental tumor. They can be continuously propagated in vitro and can be genetically manipulated. Androgen deprivation treatment on LTL331_CR_Cells had no effect on cell proliferation. Transcriptomic analyses comparing the LTL331_CR_Cell to its parental tumor revealed a profound downregulation of the androgen response pathway and an upregulation of stem and basal cell marker genes. The transcriptome of LTL331_CR_Cells partially resembles that of post-castrated LTL331 xenografts in mice. Notably, when grafted under the renal capsules of male NOD/SCID mice, LTL331_CR_Cells spontaneously gave rise to NEPC tumors. This is evidenced by the histological expression of the NE marker CD56 and the loss of adenocarcinoma markers such as PSA. Transcriptomic analyses of the newly developed NEPC tumors further demonstrate marked enrichment of NEPC signature genes and loss of AR signaling genes. This study provides a novel research tool derived from a unique PDX model. It allows for the investigation of mechanisms underlying NEPC development by enabling gene manipulations ex vivo and subsequent functional evaluations in vivo.

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Acquired temozolomide resistance in MGMT-deficient glioblastoma cells is associated with regulation of DNA repair by DHC2

Brain ◽

10.1093/brain/awz202 ◽

2019 ◽

Vol 142 (8) ◽

pp. 2352-2366 ◽

Cited By ~ 15

Author(s):

Guo-zhong Yi ◽

Guanglong Huang ◽

Manlan Guo ◽

Xi’an Zhang ◽

Hai Wang ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Dna Methyltransferase ◽

Molecular Mechanisms ◽

Progression Free Survival ◽

Recurrent Glioblastoma ◽

Dna Lesions ◽

Dna Repair Proteins

Abstract The acquisition of temozolomide resistance is a major clinical challenge for glioblastoma treatment. Chemoresistance in glioblastoma is largely attributed to repair of temozolomide-induced DNA lesions by O6-methylguanine-DNA methyltransferase (MGMT). However, some MGMT-deficient glioblastomas are still resistant to temozolomide, and the underlying molecular mechanisms remain unclear. We found that DYNC2H1 (DHC2) was expressed more in MGMT-deficient recurrent glioblastoma specimens and its expression strongly correlated to poor progression-free survival in MGMT promotor methylated glioblastoma patients. Furthermore, silencing DHC2, both in vitro and in vivo, enhanced temozolomide-induced DNA damage and significantly improved the efficiency of temozolomide treatment in MGMT-deficient glioblastoma. Using a combination of subcellular proteomics and in vitro analyses, we showed that DHC2 was involved in nuclear localization of the DNA repair proteins, namely XPC and CBX5, and knockdown of either XPC or CBX5 resulted in increased temozolomide-induced DNA damage. In summary, we identified the nuclear transportation of DNA repair proteins by DHC2 as a critical regulator of acquired temozolomide resistance in MGMT-deficient glioblastoma. Our study offers novel insights for improving therapeutic management of MGMT-deficient glioblastoma.

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Simultaneous targeting of DNA replication and homologous recombination in glioblastoma with a polyether ionophore

Neuro-Oncology ◽

10.1093/neuonc/noz159 ◽

2019 ◽

Cited By ~ 1

Author(s):

Yi Chieh Lim ◽

Kathleen S Ensbey ◽

Carolin Offenhäuser ◽

Rochelle C J D’souza ◽

Jason K Cullen ◽

...

Keyword(s):

Dna Repair ◽

Homologous Recombination ◽

First Generation ◽

Ex Vivo ◽

Drug Efficacy ◽

Tumor Formation ◽

Dna Lesions ◽

Dual Functions

Abstract Background Despite significant endeavor having been applied to identify effective therapies to treat glioblastoma (GBM), survival outcomes remain intractable. The greatest nonsurgical benefit arises from radiotherapy, though tumors typically recur due to robust DNA repair. Patients could therefore benefit from therapies with the potential to prevent DNA repair and synergize with radiotherapy. In this work, we investigated the potential of salinomycin to enhance radiotherapy and further uncover novel dual functions of this ionophore to induce DNA damage and prevent repair. Methods In vitro primary GBM models and ex vivo GBM patient explants were used to determine the mechanism of action of salinomycin by immunoblot, flow cytometry, immunofluorescence, immunohistochemistry, and mass spectrometry. In vivo efficacy studies were performed using orthotopic GBM animal xenograft models. Salinomycin derivatives were synthesized to increase drug efficacy and explore structure-activity relationships. Results Here we report novel dual functions of salinomycin. Salinomycin induces toxic DNA lesions and prevents subsequent recovery by targeting homologous recombination (HR) repair. Salinomycin appears to target the more radioresistant GBM stem cell–like population and synergizes with radiotherapy to significantly delay tumor formation in vivo. We further developed salinomycin derivatives which display greater efficacy in vivo while retaining the same beneficial mechanisms of action. Conclusion Our findings highlight the potential of salinomycin to induce DNA lesions and inhibit HR to greatly enhance the effect of radiotherapy. Importantly, first-generation salinomycin derivatives display greater efficacy and may pave the way for clinical testing of these agents.

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Drug-induced DNA damage and tumor chemosensitivity.

Journal of Clinical Oncology ◽

10.1200/jco.1990.8.12.2062 ◽

1990 ◽

Vol 8 (12) ◽

pp. 2062-2084 ◽

Cited By ~ 65

Author(s):

R J Epstein

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Tumor Cell ◽

Malignant Disease ◽

Dna Lesions ◽

Drug Induced ◽

Cell Subpopulations ◽

Therapeutic Cell

Cytotoxic drugs act principally by damaging tumor-cell DNA. Quantitative analysis of this interaction provides a basis for understanding the biology of therapeutic cell kill as well as a rational strategy for optimizing and predicting tumor response. Recent advances have made it possible to correlate assayed DNA lesions with cytotoxicity in tumor cell lines, in animal models, and in patients with malignant disease. In addition, many of the complex interrelationships between DNA damage, DNA repair, and alterations of gene expression in response to DNA damage have been defined. Techniques for modulating DNA damage and cytotoxicity using schedule-specific cytotoxic combinations, DNA repair inhibitors, cell-cycle manipulations, and adjunctive noncytotoxic drug therapy are being developed, and critical therapeutic targets have been identified within tumor-cell subpopulations and genomic DNA alike. Most importantly, methods for predicting clinical response to cytotoxic therapy using both in vitro markers of tumor-cell sensitivity and in vivo measurements of drug-induced DNA damage are now becoming a reality. These advances can be expected to provide a strong foundation for the development of innovative cytotoxic drug strategies over the next decade.

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EZH2 inhibition reduces cartilage loss and functional impairment related to osteoarthritis

Scientific Reports ◽

10.1038/s41598-020-76724-9 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Lyess Allas ◽

Sybille Brochard ◽

Quitterie Rochoux ◽

Jules Ribet ◽

Cleo Dujarrier ◽

...

Keyword(s):

Functional Disability ◽

Ex Vivo ◽

Cartilage Degradation ◽

Motor Functions ◽

Methyl Transferase ◽

Expression Of Genes ◽

Medial Meniscectomy ◽

The Impact

Abstract Histone methyltransferase EZH2 is upregulated during osteoarthritis (OA), which is the most widespread rheumatic disease worldwide, and a leading cause of disability. This study aimed to assess the impact of EZH2 inhibition on cartilage degradation, inflammation and functional disability. In vitro, gain and loss of EZH2 function were performed in human articular OA chondrocytes stimulated with IL-1β. In vivo, the effects of EZH2 inhibition were investigated on medial meniscectomy (MMX) OA mouse model. The tissue alterations were assayed by histology and the functional disabilities of the mice by actimetry and running wheel. In vitro, EZH2 overexpression exacerbated the action of IL-1β in chondrocytes increasing the expression of genes involved in inflammation, pain (NO, PGE2, IL6, NGF) and catabolism (MMPs), whereas EZH2 inhibition by a pharmacological inhibitor, EPZ-6438, reduced IL-1β effects. Ex vivo, EZH2 inhibition decreased IL-1β-induced degradation of cartilage. In vivo, intra-articular injections of the EZH2 inhibitor reduced cartilage degradation and improved motor functions of OA mice. This study demonstrates that the pharmacological inhibition of the histone methyl-transferase EZH2 slows the progression of osteoarthritis and improves motor functions in an experimental OA model, suggesting that EZH2 could be an effective target for the treatment of OA by reducing catabolism, inflammation and pain.

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Mouse prostate tumor inhibition via disruption of murine telomerase: In vivo and in vitro data for a new preclinical cancer model

Journal of Clinical Oncology ◽

10.1200/jco.2009.27.15_suppl.e14631 ◽

2009 ◽

Vol 27 (15_suppl) ◽

pp. e14631-e14631

Author(s):

T. Xu ◽

Y. Xu ◽

R. Lao ◽

K. He ◽

L. Xue ◽

...

Keyword(s):

Prostate Cancer ◽

Dna Damage ◽

Cancer Cells ◽

Cancer Cell Line ◽

Telomerase Activity ◽

Telomerase Rna ◽

Prostate Cancer Cells ◽

Mouse Prostate

e14631 Background: Telomerase-interference (TI), a novel therapeutic strategy, exploits the high telomerase activity in prostate cancer by introducing a mutated telomerase RNA (MT-Ter) that encodes toxic telomeres. Until now, TI has been tested by targeting human telomerase in tumor cells xenografted into immuno-deficient mice, an inadequate model for predicting efficacy and toxicity. We designed and validated 2 new TI gene constructs that specifically target murine telomerase RNA (mTER), enabling the study of TI in preclinical mouse models that are immuno-competent and that develop endogenous prostate tumors. Methods: We designed 2 constructs and cloned them into a lentiviral delivery system: MT-mTER and siRNA against wild type mTer (α-mTer-siRNA). Using a mouse prostate cancer cell line, E4, we tested the 2 constructs for expression (RT-PCR), telomerase activity (TRAP), and biologic activity (53bp1 DNA damage staining, MTS growth assay, TUNEL and caspase apoptosis assays), as well as in vivo efficacy (NOD-SCID allografts). Results: We confirmed MT-mTER expression (∼50-fold) and showed that α-mTer-siRNA specifically depleted WT-mTER (80% reduction) but not MT-mTER when the 2 constructs are co-expressed; thus, the 2 constructs in combination effectively substituted MT-mTer for WT-mTer in the mouse prostate cancer cells. MT-mTER caused mutant telomeric repeats (TTTGGG instead of TTAGGG) to be added to the ends of telomeres, resulting in rapid telomeric uncapping marked by 53bp1 DNA damage foci (an average 7.5 foci/cell vs. 1.4 foci/cell in vector control). This, in turn, led to rapid and significant apoptosis (>90% TUNEL and caspase +) and growth inhibition in vitro (90% reduction by MTS) and in vivo (75% reduction in tumor allograft size). Conclusions: We successfully designed and validated MT-mTer and α-mTer-siRNA, 2 novel gene constructs that specifically target and co-opt murine telomerase activity within mouse prostate cancer cells. These constructs offer a significant advantage, as they can be used to investigate TI in immuno-competent mice that develop prostate cancer, thereby modeling actual human disease and testing TI-based therapies in a much more informative and authentic manner. No significant financial relationships to disclose.

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Differential response of prostate cancer cells to ionizing radiation: The RB status.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.5_suppl.106 ◽

2012 ◽

Vol 30 (5_suppl) ◽

pp. 106-106

Author(s):

Robert Benjamin Den ◽

Steve Ciment ◽

Ankur Sharma ◽

Hestia Mellert ◽

Steven McMahon ◽

...

Keyword(s):

Prostate Cancer ◽

Cell Cycle ◽

Dna Damage ◽

Dna Repair ◽

Ionizing Radiation ◽

Cell Survival ◽

Hormone Sensitive ◽

Clonogenic Cell ◽

Castrate Resistant

106 Background: Prostate cancer is the most frequently diagnosed malignancy and the second leading cause of cancer death in U.S. men. The retinoblastoma tumor suppressor protein, RB, plays a critical role in cell cycle regulation and loss of RB has been observed in 25-30% of prostate cancers. We have previously shown that RB loss results in a castrate resistant phenotype, however the consequences of RB status with regard to radiation response are unknown. We hypothesized that RB loss would downregulate the G1-S cell cycle checkpoint arrest normally induced by irradiation, inhibit DNA repair, and subsequently sensitize cells to ionizing radiation. Methods: Experimental work was performed with multiple isogenic prostate cancer cell lines (hormone sensitive: LNCaP and LAP-C4 cells and hormone resistant C42, 22Rv1 cells; stable knockdown of RB using shRNA). Gamma H2AX assays were used to quantitate DNA damage and PARP cleavage and Caspase 3 were used to quantitate apoptosis. FACS analysis with BrdU was used to assess the cell cycle. Cell survival was measured using the clonogenic cell survival assay. In vivo work was performed in nude mice with tumor xenografts. Results: We observed that loss of RB increased radioresponsiveness in both transient and clonogenic cell survival assays in both hormone sensitive and castrate resistant cell lines (p<0.05). Cell death was not mediated through increased apoptosis nor was perturbations in cell cycle noted. However, loss of RB effected DNA repair as measured by gamma H2AX staining as well as cellular senescence. In vivo xenografts of the RB deficient tumors exhibited diminished tumor mass, lower PSA kinetics and decreased tumor growth after treatment with single fraction of ionizing radiation in comparison to RB intact tumors (p<0.05). Conclusions: Loss of RB results in a differential response to ionizing radiation. Isogenic cells with RB knockdown are more sensitive to DNA damage and result in reduced cell survival. The underlying mechanism appears to be related to DNA damage repair and cellular senescence.

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Leveraging RB status to define therapy for castrate-resistant prostate cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2014.32.4_suppl.96 ◽

2014 ◽

Vol 32 (4_suppl) ◽

pp. 96-96

Author(s):

Renee de Leeuw ◽

Clay de Comstock ◽

Daniela de Pollutri ◽

Matthew Joseph Schiewer ◽

Stephen J Ciment ◽

...

Keyword(s):

Prostate Cancer ◽

Kinase Inhibitors ◽

Ex Vivo ◽

Tissue Samples ◽

Ki67 Staining ◽

Cell Architecture ◽

Castrate Resistant Prostate Cancer ◽

Castrate Resistant

96 Background: Loss of retinoblastoma (RB) tumor suppressor is overrepresented in castrate-resistant prostate cancer (CRPC) compared to primary PCa. We previously showed using analyses of human tissue and in vitro and in vivo modeling that RB constrains androgen receptor (AR) function, and that loss of RB is sufficient promote resistance to castration and AR antagonists. Thus, novel strategies are needed to treat RB-deficient tumor. By contrast, in tumors retaining RB, suppressing enhancing RB activity would be of therapeutic advantage, and may be accomplished through next-generation Cdk4/6 inhibitors. Methods: Stable isogenic pairs of prostate cancer cell lines either retaining RB or RB depleted (by shRNA) were assessed in vitro and in xenografts for response to Cdk4/6 kinase inhibitors or the cabazitaxel. In addition, using an ex vivo explant assay, fresh tumor tissue samples from radical prostatectomy were exposed to the Cdk4/6 inhibitor or cabazitaxel for up to 7 days, and evaluated by IHC for Ki67, Caspase-3, and AR. Results: Cdk4/6 inhibition blocks tumor cell proliferation dependent on RB status. This was further confirmed ex vivo, as evidenced by a marked reduction in Ki67 staining in Cdk4/6 inhibitor treated explant tissue from two prostate cancer patients. Conversely, in vitro studies revealed a modest sensitization of RB-depleted tumors to cabazitaxel that was dramatically enhanced in vivo and after castration. Cabazitaxel, like docetaxel, targets the cell architecture and induces cell death, but also induces a distinct gene expression profile that may partially explain efficacy in docetaxel-resistant tumors. Neither taxane showed affects on AR nuclear localization using in vivoor explant studies. Conclusions: These results strongly support our hypothesis that RB status can be used as a metric to define therapeutic response to cabazitaxel, as such that loss of RB function induces sensitization taxanes, whereas RB proficient tumors give an enhanced response to Cdk4/6 kinase inhibitors.

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MINDIN secretion by prostate tumors induces premetastatic changes in bone via β-catenin

Endocrine Related Cancer ◽

10.1530/erc-20-0116 ◽

2020 ◽

Vol 27 (7) ◽

pp. 441-456

Author(s):

Juan A Ardura ◽

Luis Álvarez-Carrión ◽

Irene Gutiérrez-Rojas ◽

Peter A Friedman ◽

Arancha R Gortázar ◽

...

Keyword(s):

Gene Expression ◽

Prostate Cancer ◽

Tumor Cell ◽

Ex Vivo ◽

Prostate Tumor ◽

Tumor Cell Adhesion ◽

Prostate Tumors ◽

Cell Homing

Bone metastases are common in advanced prostate cancer patients, but mechanisms by which specific pro-metastatic skeletal niches are formed before tumor cell homing are unclear. We aimed to analyze the effects of proteins secreted by primary prostate tumors on the bone microenvironment before the settlement and propagation of metastases. Here, using an in vivo pre-metastatic prostate cancer model based on the implantation of prostate adenocarcinoma TRAMP-C1 cells in immunocompetent C57BL/6 mice, we identify MINDIN as a prostate tumor secreted protein that induces bone microstructural and bone remodeling gene expression changes before tumor cell homing. Associated with these changes, increased tumor cell adhesion to the endosteum ex vivo and to osteoblasts in vitro was observed. Furthermore, MINDIN promoted osteoblast proliferation and mineralization and monocyte expression of osteoclast markers. β-catenin signaling pathway revealed to mediate MINDIN actions on osteoblast gene expression but failed to affect MINDIN-induced adhesion to prostate tumor cells or monocyte differentiation to osteoclasts. Our study evidences that MINDIN secretion by primary prostate tumors creates a favorable bone environment for tumor cell homing before metastatic spread.

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MicroRNA regulation of the MRN complex impacts DNA damage, cellular senescence and angiogenic signaling

10.1101/132258 ◽

2017 ◽

Author(s):

Cristina Espinosa-Diez ◽

RaeAnna Wilson ◽

Namita Chatterjee ◽

Clayton Hudson ◽

Rebecca Ruhl ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Targeted Delivery ◽

Genotoxic Stress ◽

Telomerase Activity ◽

Loss Of Function ◽

Mrn Complex ◽

Vegf Signaling

AbstractMicroRNAs contribute to biological robustness by buffering cellular processes from external perturbations. Here we report an unexpected link between DNA damage response and angiogenic signaling that is buffered by two distinct microRNAs. We demonstrate that genotoxic stress-induced miR-494 and miR-99b inhibit the DNA repair machinery by targeting the MRE11a-RAD50-NBN (MRN) complex. Functionally, gain and loss of function experiments show that miR-494 and miR-99b affect telomerase activity, activate p21 and Rb pathways and diminish angiogenic sproutingin vitroandin vivo. Genetic and pharmacological disruption of VEGFR-2 signaling and the MRN complex reveal a surprising co-dependency of these pathways in regulating endothelial senescence and proliferation. Vascular-targeted delivery of miR-494 decreases both growth factor-induced and tumor angiogenesis in mouse models. Mechanistically, disruption of the MRN complex induced CD44, a known driver of senescence and regulator of VEGF signaling in addition to suppressing IL-13 a stimulator of VEGF signaling. Our work identifies a putative miR-facilitated mechanism by which endothelial cells can be insulated against VEGF signaling to facilitate the onset of senescence and highlight the potential of targeting DNA repair to disrupt pathological angiogenesis.

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