title: Doxycycline down-regulates DNA-PK and radiosensitizes tumor initiating cells: Implications for more effective radiation therapy.
abstract: DNA-PK is an enzyme that is required for proper DNA-repair and is thought to confer radio-resistance in cancer cells. As a consequence, it is a high-profile validated target for new pharmaceutical development. However, no FDA-approved DNA-PK inhibitors have emerged, despite many years of drug discovery and lead optimization. This is largely because existing DNA-PK inhibitors suffer from poor pharmacokinetics. They are not well absorbed and/or are unstable, with a short plasma half-life. Here, we identified the first FDA-approved DNA-PK inhibitor by "chemical proteomics". In an effort to understand how doxycycline targets cancer stem-like cells (CSCs), we serendipitously discovered that doxycycline reduces DNA-PK protein expression by nearly 15-fold (> 90%). In accordance with these observations, we show that doxycycline functionally radio-sensitizes breast CSCs, by up to 4.5-fold. Moreover, we demonstrate that DNA-PK is highly over-expressed in both MCF7- and T47D-derived mammospheres. Interestingly, genetic or pharmacological inhibition of DNA-PK in MCF7 cells is sufficient to functionally block mammosphere formation. Thus, it appears that active DNA-repair is required for the clonal expansion of CSCs. Mechanistically, doxycycline treatment dramatically reduced the oxidative mitochondrial capacity and the glycolytic activity of cancer cells, consistent with previous studies linking DNA-PK expression to the proper maintenance of mitochondrial DNA integrity and copy number. Using a luciferase-based assay, we observed that doxycycline treatment quantitatively reduces the anti-oxidant response (NRF1/2) and effectively blocks signaling along multiple independent pathways normally associated with stem cells, including STAT1/3, Sonic Hedgehog (Shh), Notch, WNT and TGF-beta signaling. In conclusion, we propose that the efficacy of doxycycline as a DNA-PK inhibitor should be tested in Phase-II clinical trials, in combination with radio-therapy. Doxycycline has excellent pharmacokinetics, with nearly 100% oral absorption and a long serum half-life (18-22 hours), at a standard dose of 200-mg per day. In further support of this idea, we show that doxycycline effectively inhibits the mammosphere-forming activity of primary breast cancer samples, derived from metastatic disease sites (pleural effusions or ascites fluid). Our results also have possible implications for the radio-therapy of brain tumors and/or brain metastases, as doxycycline is known to effectively cross the blood-brain barrier. Further studies will be needed to determine if other tetracycline family members also confer radio-sensitivity.
authors.affiliation: The Breakthrough Breast Cancer Research Unit, Institute of Cancer Sciences, University of Manchester, UK.|The Manchester Centre for Cellular Metabolism (MCCM), Institute of Cancer Sciences, University of Manchester, UK.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Lamb
authors.affiliation: The Breakthrough Breast Cancer Research Unit, Institute of Cancer Sciences, University of Manchester, UK.|The Manchester Centre for Cellular Metabolism (MCCM), Institute of Cancer Sciences, University of Manchester, UK.|The Department of Pharmacy, Health and Nutritional Sciences, The University of Calabria, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Fiorillo
authors.affiliation: The Breakthrough Breast Cancer Research Unit, Institute of Cancer Sciences, University of Manchester, UK.|The Manchester Centre for Cellular Metabolism (MCCM), Institute of Cancer Sciences, University of Manchester, UK.
authors.forename: Amy
authors.initials: A
authors.lastname: Chadwick
authors.affiliation: The Breakthrough Breast Cancer Research Unit, Institute of Cancer Sciences, University of Manchester, UK.|The Manchester Centre for Cellular Metabolism (MCCM), Institute of Cancer Sciences, University of Manchester, UK.
authors.forename: Bela
authors.initials: B
authors.lastname: Ozsvari
authors.affiliation: The Breakthrough Breast Cancer Research Unit, Institute of Cancer Sciences, University of Manchester, UK.|The Manchester Centre for Cellular Metabolism (MCCM), Institute of Cancer Sciences, University of Manchester, UK.
authors.forename: Kimberly J
authors.initials: KJ
authors.lastname: Reeves
authors.affiliation: The Cancer Research UK Manchester Institute, University of Manchester, UK.
authors.forename: Duncan L
authors.initials: DL
authors.lastname: Smith
authors.affiliation: The Breakthrough Breast Cancer Research Unit, Institute of Cancer Sciences, University of Manchester, UK.
authors.forename: Robert B
authors.initials: RB
authors.lastname: Clarke
authors.affiliation: The Breakthrough Breast Cancer Research Unit, Institute of Cancer Sciences, University of Manchester, UK.
authors.forename: Sacha J
authors.initials: SJ
authors.lastname: Howell
authors.affiliation: The Department of Pharmacy, Health and Nutritional Sciences, The University of Calabria, Italy.
authors.forename: Anna Rita
authors.initials: AR
authors.lastname: Cappello
authors.affiliation: The Sidney Kimmel Cancer Center, Philadelphia, PA, USA.
authors.forename: Ubaldo E
authors.initials: UE
authors.lastname: Martinez-Outschoorn
authors.affiliation: The Breakthrough Breast Cancer Research Unit, Institute of Cancer Sciences, University of Manchester, UK.|The Manchester Centre for Cellular Metabolism (MCCM), Institute of Cancer Sciences, University of Manchester, UK.
authors.forename: Maria
authors.initials: M
authors.lastname: Peiris-Pagès
authors.affiliation: The Breakthrough Breast Cancer Research Unit, Institute of Cancer Sciences, University of Manchester, UK.|The Manchester Centre for Cellular Metabolism (MCCM), Institute of Cancer Sciences, University of Manchester, UK.
authors.forename: Federica
authors.initials: F
authors.lastname: Sotgia
authors.affiliation: The Breakthrough Breast Cancer Research Unit, Institute of Cancer Sciences, University of Manchester, UK.|The Manchester Centre for Cellular Metabolism (MCCM), Institute of Cancer Sciences, University of Manchester, UK.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Lisanti
mesh_terms: D001943:Breast Neoplasms / Q000188:drug therapy / Q000473:pathology / Q000532:radiotherapy*; D045744:Cell Line, Tumor; D051747:DNA-Activated Protein Kinase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D004318:Doxycycline / Q000494:pharmacology*; D005260:Female; D006801:Humans; D061986:MCF-7 Cells; D014411:Neoplastic Stem Cells / Q000201:enzymology / Q000473:pathology / Q000528:radiation effects*; D040901:Proteomics / Q000379:methods; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology*
chemical_list: D011838:Radiation-Sensitizing Agents; D051747:DNA-Activated Protein Kinase; D004318:Doxycycline
keywords: DNA-PK; doxycycline; mitochondrial biogenesis; proteomic analysis; radiation resistance