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.

A mitochondrial based oncology platform for targeting cancer stem cells (CSCs): MITO-ONC-RX

“…For example, Doxycycline behaves as a radio-sensitizer, making CSCs approximately 3 to 5 times more sensitive to radiation treatment [36]. In addition, Doxycycline effectively targets hypoxic CSCs and overcomes Paclitaxel-resistance, under conditions of hypoxia; this may have important implications for achieving more effective anti-angiogenic therapy [37].…”

Chemical Proteomic Approaches Targeting Cancer Stem Cells: A Review of Current Literature

“…To better understand the mechanism of action of doxycycline on CSC metabolism, an unbiased label-free chemical proteomics approach has been used to identify proteins effectively down-regulated by doxycycline. Doxycycline can significantly reduce the expression of many key protein targets functionally associated with mitochondrial metabolism, glycolysis, epithelial to mesenchymal transition (EMT), protein synthesis, DNA damage response, inflammation, and protein degradation in human breast cancer cells (85). In particular, doxycycline can remarkably reduce DNA-PK protein expression.…”

“…Furthermore, DNA-PK is significantly over-expressed in breast cancer mammospheres. Genetic or pharmacological inhibition of DNA-PK can block mammosphere formation (85). Doxycycline also inhibits multiple signaling pathways involved in self-renewal and drug resistance of CSCs such as Sonic Hedgehog, Notch, Wnt, and TGF-β signaling (85).…”

“…Genetic or pharmacological inhibition of DNA-PK can block mammosphere formation (85). Doxycycline also inhibits multiple signaling pathways involved in self-renewal and drug resistance of CSCs such as Sonic Hedgehog, Notch, Wnt, and TGF-β signaling (85). In conclusion, doxycycline may target both mitochondrial ribosomes and DNA-PK, thereby reducing energy metabolism in breast CSCs.…”

Studies on antitumor activity spectrum of doxycycline

“…[6] As a semi-synthetic tetracycline, doxycycline inhibits matrix metalloproteinase (MMP) activation, prevents cell proliferation, and downregulates DNA-dependent protein kinase. [7,8] …”