Publications by authors named "Changxian Shen"

25 Publications

  • Page 1 of 1

Wee1 Kinase Inhibitor AZD1775 Effectively Sensitizes Esophageal Cancer to Radiotherapy.

Clin Cancer Res 2020 Jul 27;26(14):3740-3750. Epub 2020 Mar 27.

The Ohio State University Medical Center, Arthur G. James Comprehensive Cancer Center and Richard J. Solove Research Institute, Columbus, Ohio.

Purpose: Esophageal cancer is a deadly malignancy with a 5-year survival rate of only 5% to 20%, which has remained unchanged for decades. Esophageal cancer possesses a high frequency of mutations leading to dysfunctional G cell-cycle checkpoint, which likely makes esophageal cancer cells highly reliant upon G-M checkpoint for adaptation to DNA replication stress and DNA damage after radiation. We aim to explore whether targeting Wee1 kinase to abolish G-M checkpoint sensitizes esophageal cancer cells to radiotherapy.

Experimental Design: Cell viability was assessed by cytotoxicity and colony-forming assays, cell-cycle distribution was analyzed by flow cytometry, and mitotic catastrophe was assessed by immunofluorescence staining. Human esophageal cancer xenografts were generated to explore the radiosensitizing effect of AZD1775 .

Results: The IC concentrations of AZD1775 on esophageal cancer cell lines were between 300 and 600 nmol/L. AZD1775 (100 nmol/L) as monotherapy did not alter the viability of esophageal cancer cells, but significantly radiosensitized esophageal cancer cells. AZD1775 significantly abrogated radiation-induced G-M phase arrest and attenuation of p-CDK1-Y15. Moreover, AZD1775 increased radiation-induced mitotic catastrophe, which was accompanied by increased γH2AX levels, and subsequently reduced survival after radiation. Importantly, AZD1775 in combination with radiotherapy resulted in marked tumor regression of esophageal cancer tumor xenografts.

Conclusions: Abrogation of G-M checkpoint by targeting Wee1 kinase with AZD1775 sensitizes esophageal cancer cells to radiotherapy and in mouse xenografts. Our findings suggest that inhibition of Wee1 by AZD1775 is an effective strategy for radiosensitization in esophageal cancer and warrants clinical testing.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-3373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7367716PMC
July 2020

EGFL7 Antagonizes NOTCH Signaling and Represents a Novel Therapeutic Target in Acute Myeloid Leukemia.

Clin Cancer Res 2020 02 31;26(3):669-678. Epub 2019 Oct 31.

The Ohio State University Comprehensive Cancer Center, Columbus, Ohio.

Purpose: EGF-like domain 7 (EGFL7) is a secreted protein and recently has been shown to play an important role in acute myeloid leukemia (AML); however, the underlying mechanism by which EGFL7 promotes leukemogenesis is largely unknown.

Experimental Design: Using an antibody interaction array, we measured the ability of EGFL7 to bind directly approximately 400 proteins expressed by primary AML blasts. Primary patient samples were stimulated with recombinant EGFL7 (rEGFL7) or anti-EGFL7 blocking antibody to assess alterations in downstream signaling and the ability to effect blast differentiation and survival. We treated three independent AML models with anti-EGFL7 or IgG1 control to determine whether anti-EGFL7 could prolong survival .

Results: We found EGFL7 significantly binds several signaling proteins important for normal and malignant hematopoiesis including NOTCH. Stimulation of AML blasts with rEGFL7 reduced NOTCH intracellular domain and NOTCH target gene expression while treatment with an anti-EGFL7 blocking antibody resulted in reactivation of NOTCH signaling, increased differentiation, and apoptosis. Competitive ligand-binding assays showed rEGFL7 inhibits DELTA-like (DLL) 4-mediated NOTCH activation while anti-EGFL7 combined with DLL4 significantly increased NOTCH activation and induced apoptosis. Using three different AML mouse models, we demonstrated that treatment with anti-EGFL7 alone results in increased survival.

Conclusions: Our data demonstrate that EGFL7 contributes to NOTCH silencing in AML by antagonizing canonical NOTCH ligand binding. Reactivation of NOTCH signaling using anti-EGFL7 results in prolonged survival of leukemic mice, supporting the use of EGFL7 as a novel therapeutic target in AML.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-2479DOI Listing
February 2020

mTOR Signaling Upregulates CDC6 via Suppressing miR-3178 and Promotes the Loading of DNA Replication Helicase.

Sci Rep 2019 07 8;9(1):9805. Epub 2019 Jul 8.

Department of Thoracic Surgery, Sir Run Run Shaw Hospital, College of Medicine Zhejiang University, Hangzhou, 310016, China.

mTOR signaling pathway is deregulated in most cancers and uncontrolled cell cycle progression is a hallmark of cancer cell. However, the precise molecular mechanisms of the regulation of DNA replication and chromatin metabolism by mTOR signaling are largely unknown. We herein report that mTOR signaling promotes the loading of MCM2-7 helicase onto chromatin and upregulates DNA replication licensing factor CDC6. Pharmacological inhibition of mTOR kinase resulted in CHK1 checkpoint activation and decreased MCM2-7 replication helicase and PCNA associated with chromatins. Further pharmacological and genetic studies demonstrated CDC6 is positively controlled by mTORC1-S6K1 and mTORC2 signaling. miRNA screening revealed mTOR signaling suppresses miR-3178 thereby upregulating CDC6. Analysis of TCGA data found that CDC6 is overexpressed in most cancers and associates with the poor survival of cancer patients. Our findings suggest that mTOR signaling may control DNA replication origin licensing and replisome stability thereby cell cycle progression through CDC6 regulation.
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http://dx.doi.org/10.1038/s41598-019-46052-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6614418PMC
July 2019

Inhibiting BRAF Oncogene-Mediated Radioresistance Effectively Radiosensitizes BRAF-Mutant Thyroid Cancer Cells by Constraining DNA Double-Strand Break Repair.

Clin Cancer Res 2019 08 16;25(15):4749-4760. Epub 2019 May 16.

Arthur G. James Comprehensive Cancer Center and Richard J. Solove Research Institute, The Ohio State University Medical Center, Columbus, Ohio.

Purpose: Activating BRAF mutations, most commonly BRAF, are a major oncogenic driver of many cancers. We explored whether BRAF promotes radiation resistance and whether selectively targeting BRAF with a BRAF inhibitor (vemurafenib, BRAFi) sensitizes BRAF thyroid cancer cells to radiotherapy.

Experimental Design: Immunoblotting, neutral comet, immunocytochemistry, functional reporter, and clonogenic assays were used to analyze the outcome and molecular characteristics following radiotherapy with or without BRAF or vemurafenib in thyroid cancer cells.

Results: BRAF thyroid cancer cell lines were associated with resistance to ionizing radiation (IR), and expression of BRAF into wild-type BRAF thyroid cancer cells led to IR resistance. BRAFi inhibited ERK signaling in BRAF mutants, but not BRAF wild-type thyroid cancer cell lines. BRAFi selectively radiosensitized and delayed resolution of IR-induced γH2AX nuclear foci in BRAF cells. Moreover, BRAFi impaired global DNA repair and altered the resolution of 53BP1 and RAD51 nuclear foci in BRAF cells following IR. BRAF mutants displayed enhanced nonhomologous end-joining (NHEJ) repair activity, which was abolished by BRAFi. Intriguingly, BRAF mutation led to upregulation of XLF, a component of NHEJ, which was prevented by BRAFi. Importantly, BRAFi in combination with radiotherapy resulted in marked and sustained tumor regression of BRAF thyroid tumor xenografts.

Conclusions: BRAF mutation promotes NHEJ activity leading to radioresistance and BRAFi selectively radiosensitizes BRAF thyroid cancer cells through inhibiting NHEJ. Our findings suggest that combining BRAFi and radiation may improve the therapeutic outcome of patients with BRAF-mutant thyroid cancer.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-3625DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6677585PMC
August 2019

LCL161, a SMAC-mimetic, Preferentially Radiosensitizes Human Papillomavirus-negative Head and Neck Squamous Cell Carcinoma.

Mol Cancer Ther 2019 06 23;18(6):1025-1035. Epub 2019 Apr 23.

The Ohio State University Medical Center, Arthur G. James Comprehensive Cancer Center and Richard J. Solove Research Institute, Columbus, Ohio.

Targeting inhibitor of apoptosis proteins (IAP) with second mitochondria-derived activator of caspase (SMAC) mimetics may promote cancer cell death. We tested whether cIAP1 predicts poor prognosis in head and neck squamous cell carcinoma (HNSCC) and whether a novel Smac-mimetic, LCL161, could radiosensitize human papillomavirus-positive (HPV) and -negative (HPV) HNSCC. The association of (encoding cIAP1) mRNA level with HPV status in HNSCC was analyzed using The Cancer Genome Atlas (TCGA) database. cIAP1 was assessed by IHC on an HNSCC tissue microarray (TMA, = 84) followed by correlation analysis with HPV status and patient outcomes. Human cell culture and animal models of HNSCC were used to analyze the outcome and molecular characteristics following radiotherapy in combination with LCL161. cIAP1 expression is increased in HPV compared with HPVHNSCC tumors in the TCGA database. In our TMA, cIAP1 was overexpressed in HNSCC compared with normal tissues ( = 0.0003) and associated with a poor overall survival ( = 0.0402). cIAP1 levels were higher in HPV than that in HPVHNSCC tumors ( = 0.004) and patients with cIAP1/HPV HNSCC had the worst survival. LCL161 effectively radiosensitized HPV HNSCC cells, which was accompanied with enhanced apoptosis, but not HPV HNSCC cells. Importantly, LCL161 in combination with radiotherapy led to dramatic tumor regression of HPV HNSCC tumor xenografts, accompanied by cIAP1 degradation and apoptosis activation. These results reveal that cIAP1 is a prognostic and a potential therapeutic biomarker for HNSCC, and targeting cIAP1 with LCL161 preferentially radiosensitizes HPV HNSCC, providing justification for clinical testing of LCL161 in combination with radiation for patients with HPV HNSCC.
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http://dx.doi.org/10.1158/1535-7163.MCT-18-1157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6548673PMC
June 2019

Prognostic and biological significance of the proangiogenic factor EGFL7 in acute myeloid leukemia.

Proc Natl Acad Sci U S A 2017 06 22;114(23):E4641-E4647. Epub 2017 May 22.

The Ohio State University Comprehensive Cancer Center, Columbus, OH 43210;

Epithelial growth factor-like 7 (EGFL7) is a protein that is secreted by endothelial cells and plays an important role in angiogenesis. Although EGFL7 is aberrantly overexpressed in solid tumors, its role in leukemia has not been evaluated. Here, we report that levels of both mRNA and EGFL7 protein are increased in blasts of patients with acute myeloid leukemia (AML) compared with normal bone marrow cells. High mRNA expression associates with lower complete remission rates, and shorter event-free and overall survival in older (age ≥60 y) and younger (age <60 y) patients with cytogenetically normal AML. We further show that AML blasts secrete EGFL7 protein and that higher levels of EGFL7 protein are found in the sera from AML patients than in sera from healthy controls. Treatment of patient AML blasts with recombinant EGFL7 in vitro leads to increases in leukemic blast cell growth and levels of phosphorylated AKT. EGFL7 blockade with an anti-EGFL7 antibody reduced the growth potential and viability of AML cells. Our findings demonstrate that increased EGFL7 expression and secretion is an autocrine mechanism supporting growth of leukemic blasts in patients with AML.
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http://dx.doi.org/10.1073/pnas.1703142114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5468639PMC
June 2017

P53 suppresses ribonucleotide reductase via inhibiting mTORC1.

Oncotarget 2017 Jun;8(25):41422-41431

Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, USA.

Balanced deoxyribonucleotides pools are essential for cell survival and genome stability. Ribonucleotide reductase is the rate-limiting enzyme for the production of deoxyribonucleotides. We report here that p53 suppresses ribonucleotide reductase subunit 1 (RRM1) and 2 (RRM2) via inhibiting mammalian target of rapamycin complex 1 (mTORC1). In vitro, cancer cell lines and mouse embryonic fibroblast cells were treated with different concentrations of pharmacological inhibitors for different times. In vivo, rhabdomyosarcoma Rh30 cell tumor-bearing mice were treated with rapamycin or AZD8055. Protein levels and phosphorylation status were assessed by immunoblotting and mRNA levels were determined by real time RT-PCR. Pharmacological inhibition of mTORC1 with rapamycin, mTOR kinase with AZD8055 or protein kinase B with MK2206 resulted in decrease of RRM1 and RRM2 in Rh30 cells both in vitro and in mouse tumor xenografts. Moreover, eukaryotic translational initiation factor 4E-binding proteins 1 and 2 double knockout mouse embryonic fibroblast cells demonstrated an elevation of RRM1 and RRM2. Furthermore, down-regulation of mTOR-protein kinase B signaling or cyclin dependent kinase 4 led to decrease of RRM1 and RRM2 mRNAs. In addition, TP53 mutant cancer cells had elevation of RRM1 and RRM2, which was reduced by rapamycin. Importantly, human double minute 2 inhibitor nutlin-3 decreased RRM1 and RRM2 in TP53 wild type rhabdomyosarcoma Rh18 but not in TP53 mutated Rh30 cells. Our data demonstrated that mTOR enhances the cap-dependent protein translation and gene transcription of RRM1 and RRM2. Our findings might provide an additional mechanism by which p53 maintains genome stability.
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http://dx.doi.org/10.18632/oncotarget.17440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5522210PMC
June 2017

Regulation of CHK1 by mTOR contributes to the evasion of DNA damage barrier of cancer cells.

Sci Rep 2017 05 8;7(1):1535. Epub 2017 May 8.

Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, USA.

Oncogenic transformation leads to dysregulated cell proliferation, nutrient deficiency, and hypoxia resulting in metabolic stress and increased DNA damage. In normal cells, such metabolic stress leads to inhibition of signaling through the mammalian Target of Rapamycin Complex 1 (mTORC1), reduction of protein translation, cell cycle arrest, and conservation of energy. In contrast, negative regulation of mTORC1 signaling by DNA damage is abrogated in many cancer cells, thus mTORC1 signaling remains active under microenvironmental conditions that potentially promote endogenous DNA damage. Here we report that mTORC1 signaling suppresses endogenous DNA damage and replication stress. Pharmacological inhibition of mTOR signaling resulted in phosphorylation of H2AX concomitant with the decrease of CHK1 levels both in cell culture and mouse rhadomyosarcoma xenografts. Further results demonstrated that mTORC1-S6K1 signaling controls transcription of CHK1 via Rb-E2F by upregulating cyclin D and E. Consistent with these results, downregulation of CHK1 by inhibition of mTOR kinase resulted in defects in the slow S phase progression following DNA damage. These results indicate that, under stressful conditions, maintained mTORC1 signaling in cancer cells promotes survival by suppressing endogenous DNA damage, and may control cell fate through the regulation of CHK1.
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http://dx.doi.org/10.1038/s41598-017-01729-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5431544PMC
May 2017

Noncatalytic PTEN missense mutation predisposes to organ-selective cancer development in vivo.

Genes Dev 2015 Aug;29(16):1707-20

Solid Tumor Biology Program, James Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, USA; Department of Molecular Genetics, College of Arts and Sciences, The Ohio State University, Columbus, Ohio 43210, USA; Department of Molecular Virology, Immunology, and Medical Genetics, College of Medicine, The Ohio State University, Columbus, Ohio 43210, USA;

Inactivation of phosphatase and tensin homology deleted on chromosome 10 (PTEN) is linked to increased PI3K-AKT signaling, enhanced organismal growth, and cancer development. Here we generated and analyzed Pten knock-in mice harboring a C2 domain missense mutation at phenylalanine 341 (Pten(FV)), found in human cancer. Despite having reduced levels of PTEN protein, homozygous Pten(FV/FV) embryos have intact AKT signaling, develop normally, and are carried to term. Heterozygous Pten(FV/+) mice develop carcinoma in the thymus, stomach, adrenal medulla, and mammary gland but not in other organs typically sensitive to Pten deficiency, including the thyroid, prostate, and uterus. Progression to carcinoma in sensitive organs ensues in the absence of overt AKT activation. Carcinoma in the uterus, a cancer-resistant organ, requires a second clonal event associated with the spontaneous activation of AKT and downstream signaling. In summary, this PTEN noncatalytic missense mutation exposes a core tumor suppressor function distinct from inhibition of canonical AKT signaling that predisposes to organ-selective cancer development in vivo.
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http://dx.doi.org/10.1101/gad.262568.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4561480PMC
August 2015

Inhibition of MEK confers hypersensitivity to X-radiation in the context of BRAF mutation in a model of childhood astrocytoma.

Pediatr Blood Cancer 2015 Oct 15;62(10):1768-74. Epub 2015 May 15.

Center for Childhood Cancer and Blood Diseases, Nationwide Children's Hospital, Columbus, Ohio.

Purpose: Curative therapy for childhood glioma presents challenges when complete resection is not possible. Patients with recurrent low-grade tumors or anaplastic astrocytoma may receive radiation treatment; however, the long-term sequellae from radiation treatment can be severe. As many childhood gliomas are associated with activation of BRAF, we have explored the combination of ionizing radiation with MEK inhibition in a model of BRAF-mutant anaplastic astrocytoma.

Experimental Design: The regulation of TORC1 signaling by BRAF was examined in BT-40 (BRAF mutant) and BT-35 (BRAF wild type) xenografts, in a cell line derived from the BT-40 xenograft and two adult BRAF mutant glioblastoma cell lines. The effect of MEK inhibition (selumetinib), XRT (total dose 10 Gy as 2 Gy daily fractions), or the combination of selumetinib and XRT was evaluated in subcutaneous BT-40 xenografts.

Results: Inhibition of MEK signaling by selumetinib suppressed TORC1 signaling only in the context of the BRAF-mutant both in vitro and in vivo. Inhibition of MEK signaling in BT-40 cells or in xenografts lead to a complete suppression of FANCD2 and conferred hypersensitivity to XRT in BT-40 xenografts without increasing local skin toxicity.

Conclusions: Selumetinib suppressed TORC1 signaling in the context of BRAF mutation. Selumetinib caused a rapid downregulation of FANCD2 and markedly potentiated the effect of XRT. These data suggest the possibility of potentiating the effect of XRT selectively in tumor cells by MEK inhibition in the context of mutant BRAF or maintaining tumor control at lower doses of XRT that would decrease long-term sequelae.
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http://dx.doi.org/10.1002/pbc.25579DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4561855PMC
October 2015

Targeting FANCD2 for therapy sensitization.

Oncotarget 2014 Jun;5(11):3426-7

Center for Childhood Cancer and Blood Diseases, Nationwide Children's Hospital, Columbus, OH.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4116492PMC
http://dx.doi.org/10.18632/oncotarget.2070DOI Listing
June 2014

FANCD2 is a potential therapeutic target and biomarker in alveolar rhabdomyosarcoma harboring the PAX3-FOXO1 fusion gene.

Clin Cancer Res 2014 Jul 30;20(14):3884-95. Epub 2014 Apr 30.

Nationwide Children's Hospital, Columbus, OH

Purpose: Alveolar rhabdomyosarcoma that harbors the PAX3-FOXO1 fusion gene (t-ARMS) is a common and lethal subtype of this childhood malignancy. Improvement in clinical outcomes in this disease is predicated upon the identification of novel therapeutic targets.

Experimental Design: Robust mouse models were used for in vivo analysis, and molecular studies were performed on xenografts treated in parallel. Two independent patient sets (n = 101 and 124) of clinically annotated tumor specimens were used for analysis of FANCD2 levels and its association with clinical and molecular characteristics and outcomes.

Results: Our xenograft studies reveal a selective suppression of FANCD2 by m-TOR kinase inhibition and radiosensitization of the t-ARMS line only. In the initial patient set, we show that FANCD2 transcript levels are prognostic in univariate analysis, and are significantly associated with metastatic disease and that the copresence of the translocation and high expression of FANCD2 is independently prognostic. We also demonstrate a significant and nonrandom enrichment of mTOR-associated genes that correlate with FANCD2 gene expression within the t-ARMS samples, but not within other cases. In the second patient set, we show that on a protein level, FANCD2 expression correlates with PAX3-FOXO1 fusion gene and is strongly associated with phospho-P70S6K expression in cases with the fusion gene.

Conclusions: Our data demonstrate that FANCD2 may have a significant role in the radiation resistance and virulence of t-ARMS. Indirectly targeting this DNA repair protein, through mTOR inhibition, may represent a novel and selective treatment strategy.
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http://dx.doi.org/10.1158/1078-0432.CCR-13-0556DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4102646PMC
July 2014

The mTOR pathway negatively controls ATM by up-regulating miRNAs.

Proc Natl Acad Sci U S A 2013 Jul 1;110(29):11869-74. Epub 2013 Jul 1.

Center for Childhood Cancer and Blood Diseases, Nationwide Children's Hospital, Columbus, OH 43205, USA.

The ataxia telangiectasia mutated (ATM) checkpoint is the central surveillance system that maintains genome integrity. We found that in the context of childhood sarcoma, mammalian target of rapamycin (mTOR) signaling suppresses ATM by up-regulating miRNAs targeting ATM. Pharmacological inhibition or genetic down-regulation of the mTOR pathway resulted in increase of ATM mRNA and protein both in mouse sarcoma xenografts and cultured cells. mTOR Complex 1 (mTORC1) suppresses ATM via S6K1/2 signaling pathways. microRNA-18a and microRNA-421, both of which target ATM, are positively controlled by mTOR signaling. Our findings have identified a negative feedback loop for the signaling between ATM and mTOR pathways and suggest that oncogenic growth signals may promote tumorigenesis by dampening the ATM checkpoint.
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http://dx.doi.org/10.1073/pnas.1220898110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3718104PMC
July 2013

Regulation of FANCD2 by the mTOR pathway contributes to the resistance of cancer cells to DNA double-strand breaks.

Cancer Res 2013 Jun 30;73(11):3393-401. Epub 2013 Apr 30.

Center for Childhood Cancer & Blood Diseases, The Research Institute, Nationwide Children's Hospital, 700 Children's Drive,ResearchBuilding II,Columbus,OH 43205, USA.

Deregulation of the mTOR pathway is closely associated with tumorigenesis. Accordingly, mTOR inhibitors such as rapamycin and mTOR-selective kinase inhibitors have been tested as cancer therapeutic agents. Inhibition of mTOR results in sensitization to DNA-damaging agents; however, the molecular mechanism is not well understood. We found that an mTOR-selective kinase inhibitor, AZD8055, significantly enhanced sensitivity of a pediatric rhabdomyosarcoma xenograft to radiotherapy and sensitized rhabdomyosarcoma cells to the DNA interstrand cross-linker (ICL) melphalan. Sensitization correlated with drug-induced downregulation of a key component of the Fanconi anemia pathway, FANCD2 through mTOR regulation of FANCD2 gene transcripts via mTORC1-S6K1. Importantly, we show that FANCD2 is required for the proper activation of ATM-Chk2 checkpoint in response to ICL and that mTOR signaling promotes ICL-induced ATM-Chk2 checkpoint activation by sustaining FANCD2. In FANCD2-deficient lymphoblasts, FANCD2 is essential to suppress endogenous and induced DNA damage, and FANCD2-deficient cells showed impaired ATM-Chk2 and ATR-Chk1 activation, which was rescued by reintroduction of wild-type FANCD2. Pharmacologic inhibition of PI3K-mTOR-AKT pathway in Rh30 rhabdomyosarcoma cells attenuated ICL-induced activation of ATM, accompanied with the decrease of FANCD2. These data suggest that the mTOR pathway may promote the repair of DNA double-strand breaks by sustaining FANCD2 and provide a novel mechanism of how the Fanconi anemia pathway modulates DNA damage response and repair.
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http://dx.doi.org/10.1158/0008-5472.CAN-12-4282DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3674187PMC
June 2013

Pro-apoptosis and anti-proliferation effects of a recombinant dominant-negative survivin-T34A in human cancer cells.

Anticancer Res 2009 Apr;29(4):1423-8

Department of Nuclear Medicine, University of Ulm, Ulm, Germany.

Background: Survivin is an attractive target for anti-cancer drug development; however targeting it by small molecules or antibodies is difficult, as survivin is neither a kinase nor a cell surface protein. Protein transduction domain (PTD)-mediated macromolecular therapeutics provides an alternative avenue for targeting survivin.

Materials And Methods: A plasmid expressing a dominant-negative survivin-T34A fused with the immunodeficiency virus protein transduction domain TAT was constructed. The fusion protein was expressed and purified from E. coli. The inhibition of proliferation and induction of apoptosis was tested in human lung carcinoma cell line A549 by directly adding survivin-T34A to the cell culture medium.

Results: Recombinant survivin-T34A was efficiently expressed and purified by affinity chromatography. It induced cell apoptosis as demonstrated by induction of caspase 3 activation and higher percentage of Annexin V staining, and inhibited cell proliferation as determined by cell number counting.

Conclusion: This functional recombinant protein is promising for development of macromolecular therapeutics targeting survivin.
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April 2009

Molecular imaging of proliferation in vivo: positron emission tomography with [18F]fluorothymidine.

Methods 2009 Jun 24;48(2):205-15. Epub 2009 Mar 24.

Nuklearmedizinische Klinik und Poliklinik, Klinikum rechts der Isar der Technischen Universität München, Ismaninger Strasse 22, D-81675 Munich, Germany.

Deregulated cell cycle progression is a hallmark of cancer. Accordingly, a major part of therapeutic drugs has been designed to inhibit cell proliferation and tumor growth. Metabolic imaging with positron emission tomography (PET) and the glucose analog 2'-[(18)F]fluoro-2'-deoxyglucose (FDG) has been demonstrated to sensitively detect malignant tumors and to identify responding tumors early in the course of anticancer treatment. However, tumoral uptake of FDG reflects proliferation only in part and is associated with false positive findings due to unspecific tracer retention in inflammatory processes. Most recent advances in cancer treatment have come from the development of disease specific, molecular agents, many of which induce cell cycle arrest (cytostatic effect) instead of tumor cell death (cytotoxic effect). Thus, evaluating alterations in DNA metabolism may reflect response to treatment better than alterations in glucose utilization. PET with the thymidine analog 3'-deoxy-3'-[(18)F]fluorothymidine (FLT) enables non-invasive imaging and quantification of the proliferation fraction of tumors. Furthermore, FLT has been suggested as surrogate marker for assessment of response to treatment, especially when targeted drugs are utilized. This article reports on metabolic pathways of radionucleosides in proliferating cells. Methods for in vivo assessment of the proliferative activity in preclinical and clinical studies are described with a focus on early monitoring response to therapy.
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http://dx.doi.org/10.1016/j.ymeth.2009.03.009DOI Listing
June 2009

PTEN mutation: many birds with one stone in tumorigenesis.

Anticancer Res 2008 Nov-Dec;28(6A):3613-9

Department of Life Sciences, Huaihua University, Huaihua, Peoples Republic of China.

The PTEN (phosphatase and tensin homolog deleted on chromosome ten) tumor suppressor gene is mutated in a wide range of malignancies and recent studies have demonstrated that PTEN prevents tumorigenesis through multiple mechanisms. PTEN functions as a plasma-membrane lipid phosphatase that antagonizes the PI3K (phosphoinositide 3 kinase)-AKT pathway. PTEN physically and genetically interacts with the central genome guardian p53. PTEN also associates with the centromeric protein CENP-C to maintain centromere integrity and suppresses chromosomal instability from DNA double-strand breaks (DSBs) through transcriptional regulation of Rad51 (radiosensitive yeast mutant 51). Moreover PTEN controls the growth and proliferation of haematopoietic stem cells (HSC) and restrains cells from leukemia in an mTOR (mammalian target of rapamycin) dependent manner. Thus, restoring PTEN functions in cancer cells directly or indirectly holds great promise for cancer therapy.
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March 2009

Chromosome instability and tumor lethality suppression in carcinogenesis.

J Cell Biochem 2008 Dec;105(6):1327-41

Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.

The maintenance and survival of each organism depends on its genome integrity. Alterations of essential genes, or aberrant chromosome number and structure lead to cell death. Paradoxically, cancer cells, especially in solid tumors, contain somatic gene mutations and are chromosome instability (CIN), suggesting a mechanism that cancer cells have acquired to suppress the lethal mutations and/or CIN. Herein we will discuss a tumor lethality suppression concept based on the studies of yeast genetic interactions and transgenic mice. During the early stages of the multistep process of tumorigenesis, incipient cancer cells probably have adopted genetic and epigenetic alterations to tolerate the lethal mutations of other genes that ensue, and to a larger extent CIN. In turn, CIN mediated massive gain and loss of genes provides a wider buffer for further genetic reshuffling, resulting in cancer cell heterogeneity, drug resistance and evasion of oncogene addiction, thus CIN may be both the effector and inducer of tumorigenesis. Accordingly, interfering with tumor lethality suppression could lead to cancer cell death or growth defects. Further validation of the tumor lethality suppression concept would help to elucidate the role of CIN in tumorigenesis, the relationship between CIN and somatic gene mutations, and would impact the design of anticancer drug development.
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http://dx.doi.org/10.1002/jcb.21937DOI Listing
December 2008

TOR signaling is a determinant of cell survival in response to DNA damage.

Mol Cell Biol 2007 Oct 13;27(20):7007-17. Epub 2007 Aug 13.

Department of Molecular Pharmacology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105, USA.

The conserved TOR (target of rapamycin) kinase is part of a TORC1 complex that regulates cellular responses to environmental stress, such as amino acid starvation and hypoxia. Dysregulation of Akt-TOR signaling has also been linked to the genesis of cancer, and thus, this pathway presents potential targets for cancer chemotherapeutics. Here we report that rapamycin-sensitive TORC1 signaling is required for the S-phase progression and viability of yeast cells in response to genotoxic stress. In the presence of the DNA-damaging agent methyl methanesulfonate (MMS), TOR-dependent cell survival required a functional S-phase checkpoint. Rapamycin inhibition of TORC1 signaling suppressed the Rad53 checkpoint-mediated induction of ribonucleotide reductase subunits Rnr1 and Rnr3, thereby abrogating MMS-induced mutagenesis and enhancing cell lethality. Moreover, cells deleted for RNR3 were hypersensitive to rapamycin plus MMS, providing the first demonstration that Rnr3 contributes to the survival of cells exposed to DNA damage. Our findings support a model whereby TORC1 acts as a survival pathway in response to genotoxic stress by maintaining the deoxynucleoside triphosphate pools necessary for error-prone translesion DNA polymerases. Thus, TOR-dependent cell survival in response to DNA-damaging agents coincides with increased mutation rates, which may contribute to the acquisition of chemotherapeutic drug resistance.
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http://dx.doi.org/10.1128/MCB.00290-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2168917PMC
October 2007

Adenovirus-delivered siRNA.

Methods Mol Biol 2004 ;252:523-32

Department of Nuclear Medicine, University Hospital of Ulm, Germany.

RNA interference is the process that double-stranded RNA (dsRNA) induces the homology-dependent degradation of cognate mRNA mediated by 21- to 23-nt small interfering RNA (siRNA). Successful application of RNAi in functional genomics and proteomics, cancer gene therapy, and virus protection depends on the efficient delivery of siRNA into mammalian cells. The availability of high virus titer, infection of a broad spectrum of cell types, and independence on active cell division makes adenovirus the vector of choice for siRNA delivery. To this end, we developed a new adenovirus shuttle vector designated as pShuttle-H1 to host H1-RNA promoter and unique BglII and HindIII sites for insertion of oligos for expression of siRNA. In this chapter, we describe an adenovirus system that uses a commercially available adenovirus system and pShuttle-H1 to deliver siRNA-expressing cassette into cells to silence a specific gene in mammalian cells.
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http://dx.doi.org/10.1385/1-59259-746-7:523DOI Listing
June 2004

Triplex-forming oligodeoxynucleotides targeting survivin inhibit proliferation and induce apoptosis of human lung carcinoma cells.

Cancer Gene Ther 2003 May;10(5):403-10

Department of Nuclear Medicine University of Ulm, Ulm, Germany.

Survivin is expressed in most cancers but is undetectable in differentiated adult cells, and plays an important role both in the suppression of apoptosis and mitotic spindle checkpoint; thus it has attracted great interest as a potential drug target. In this study, we investigated the antigene and antiproliferative effects of triplex-forming oligodeoxynucleotides (TFO) targeting survivin in human lung carcinoma A549 cells. Survivin-specific TFOs form stable triplexes under physiological conditions as tested by electrophoretic mobility shift assays. Treatment of A549 cells with survivin-specific but not control TFOs at a concentration of 400 nM in the presence of uptake-enhancing liposome significantly reduced survivin protein level, inhibited cell proliferation, and induced cell apoptosis as demonstrated by immunoblot, cell number counting, and Annexin V-staining. Moreover, we found that the triplex-forming potential of TFOs measured in vitro does not necessarily correlate with the ability of TFOs to affect expression of a targeted gene in vivo. Our results indicate that targeting survivin is a promising alternative strategy for the development of novel anticancer therapeutics.
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http://dx.doi.org/10.1038/sj.cgt.7700581DOI Listing
May 2003

Targeting bcl-2 by triplex-forming oligonucleotide--a promising carrier for gene-radiotherapy.

Cancer Biother Radiopharm 2003 Feb;18(1):17-26

Department of Nuclear Medicine, University of Ulm, Germany.

Triplex forming oligonucleotides (TFO) provide a promising tool for gene therapy. DNA damaging agents have been successfully coupled to TFOs and induce site-directed DNA damages. Here, we attempted to apply this antigen strategy using a TFO incorporated with a Conversion-electron-emitter, (99m)technetium, to target bcl-2 gene, the prototypical inhibitor of apoptosis. In the bcl-2 promoter region, we found two TFO binding sites which bind corresponding TFOs with very high specificity and affinity. Both partially and completely phosphorothioated TFOs form stable triplexes and significantly inhibit gene transcription in vitro. We also found that purine motif TFO with a thymidine opposite a thymidine interruption at the polypurine strand can form a stable triplex. In addition, (99m)technetium-conjugated TFOs were found to form a stable triplex and to inhibit bcl-2 gene transcription in vitro. Our results suggest a promising application of this triplex-forming oligonucleotide based Conversion-electron-emitter mediated gene radiotherapy in diseases related to bcl-2 overexpression.
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http://dx.doi.org/10.1089/108497803321269296DOI Listing
February 2003

Gene silencing by adenovirus-delivered siRNA.

FEBS Lett 2003 Mar;539(1-3):111-4

Department of Nuclear Medicine, University of Ulm, Robert-Koch-Strasse 8, D-89070 Ulm, Germany.

RNA interference is the process that double-stranded RNA induces the homology-dependent degradation of cognate mRNA mediated by 21-23 nucleotide short interfering RNA (siRNA). Here, we describe a simple virus vector for efficient delivery of siRNA into mammalian cells utilizing the well-defined H1-RNA promoter and conventional adenovirus. In this pilot study, p53 was targeted by this vector. Our results demonstrate efficient and specific knock-down of p53 in breast cancer MCF-7 and lung carcinoma A549 cells and indicate a prospective application of this siRNA expressing recombinant adenovirus system in functional genomics, cancer gene therapy and virus inhibition.
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http://dx.doi.org/10.1016/s0014-5793(03)00209-6DOI Listing
March 2003

FDG uptake in breast cancer: correlation with biological and clinical prognostic parameters.

Eur J Nucl Med Mol Imaging 2002 Oct 26;29(10):1317-23. Epub 2002 Jul 26.

Department of Nuclear Medicine, University of Ulm, Robert-Koch-Strasse 8, 89081 Ulm, Germany.

The aim of this study was to evaluate the possible correlation between preoperative FDG-PET results in human breast cancer and the prognostic markers Ki-67, c- erb B2, p53, oestrogen/progesterone receptor status, axillary lymph node status, tumour size and tumour grading. Seventy-five female patients with breast cancer were included in this prospective study. Patient selection was independent of tumour size and the suspected clinical stage of disease. A high-resolution full-ring scanner (Siemens ECAT HR+) was used for PET imaging. The FDG uptake of breast tumours was calculated as the tumour to background ratio (TBR). In resected cancer tissue specimens, the proliferative fraction was evaluated by Ki-67 immunostaining. Additionally, immunostaining of the prognostic markers c-erb B2, p53, and progesterone and oestrogen receptors was performed. Haematoxylin and eosin-stained sections were used for tumour grading. Correlations between FDG uptake and prognostic markers were assumed to be significant at P<0.05 using the Mann-Whitney U test. In ductal breast cancer, mean TBR was 17.3 (median 7.7, range 1.6-122.7), while in lobular cancer it was 6.5 (median 3.7, range 1.4-22.7). Mean proliferative fraction (% Ki-67 positive tumour cells) was 15%+/-13.8% (median 10%, range 0%-60%). Twenty-three carcinomas showed <5% Ki-67 positive tumour cells. Statistical analysis indicated a positive correlation between FDG uptake and proliferative index in ductal breast cancer ( P<0.0001, r=0.63). By contrast, there was no correlation between FDG uptake and c- erb B2 ( P=0.79), p53 ( P=0.92), tumour grading ( P=0.09), oestrogen receptor status ( P=0.41), progesterone receptor status ( P=0.34), axillary lymph node status ( P=0.90) and tumour size ( P=0.3). It is concluded that FDG uptake is significantly higher in ductal breast cancer than in lobular cancer ( P<0.05). FDG uptake correlates with proliferative activity assessed by Ki-67 immunostaining ( P<0.05). A significant correlation with the other prognostic markers, however, could not be demonstrated.
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http://dx.doi.org/10.1007/s00259-002-0880-8DOI Listing
October 2002

Liposomal delivery of antisense oligonucleotides for efficient downregulation of Bcl-2 and induction of apoptosis.

Cancer Biother Radiopharm 2002 Jun;17(3):281-9

Department of Nuclear Medicine, University of Ulm Robert-Koch-Str. 8, D-89070 Ulm.

Aim: The aim of this study was to enhance the delivery and thus anti-tumoral efficiency of antisense bcl-2 oligonucleotides (ODN's).

Methods: Bcl-2 overexpressing DoHH2 lymphoma and HeLa-cells were transfected with ODN's using a polycationic liposome preparation. Specific hybridization of antisense ODN's was demonstrated by gel-shift assays and in vitro transcription/translation studies. Cellular uptake of oligonucleotides was evaluated by fluorescence microscopy. Inhibition of bcl-2 translation was demonstrated by quantitative RT-PCR and Western Blot. TUNEL assay, ANNEXIN V-binding and Apo-2.7 expression were performed to evaluate induction of apoptosis.

Results: Using polycationic liposomes, a ODN transfection rate of 95% in HeLa and 45% in DoHH2 cells were demonstrated by fluorescence microscopy. 24 hours after transfection quantitative RT-PCR detected a 56% decrease of bcl-2 mRNA in antisense and a 7% decrease in sense transfected DoHH2 cells (p < 0.05). In HeLa-cells, bcl-2 expression was almost completely inhibited 72 hours after antisense ODN transfection. Antisense treated cells also showed significant induction of apoptosis.

Conclusions: Polycationic liposome-mediated transfection of bcl-2 antisense ODN's causes enhanced cellular uptake and efficient bcl-2 downregulation in bcl-2 overexpressing cell lines. This delivery strategy may explain why significant induction of apoptosis was achieved at low oligonucleotide concentrations (approximately 200 pmol/5 x 10(5) tumor cells).
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http://dx.doi.org/10.1089/10849780260179242DOI Listing
June 2002