Abstract

Here, we developed a new synthetic lethal strategy for further optimizing the eradication of cancer stem cells (CSCs). Briefly, we show that chronic treatment with the FDA-approved antibiotic Doxycycline effectively reduces cellular respiration, by targeting mitochondrial protein translation. The expression of four mitochondrial DNA encoded proteins (MT-ND3, MT-CO2, MT-ATP6 and MT-ATP8) is suppressed, by up to 35-fold. This high selection pressure metabolically synchronizes the surviving cancer cell sub-population towards a predominantly glycolytic phenotype, resulting in metabolic inflexibility. We directly validated this Doxycycline-induced glycolytic phenotype, by using metabolic flux analysis and label-free unbiased proteomics.

Next, we identified two natural products (Vitamin C and Berberine) and six clinically-approved drugs, for metabolically targeting the Doxycycline-resistant CSC population (Atovaquone, Irinotecan, Sorafenib, Niclosamide, Chloroquine, and Stiripentol). This new combination strategy allows for the more efficacious eradication of CSCs with Doxycycline, and provides a simple pragmatic solution to the possible development of Doxycycline-resistance in cancer cells. In summary, we propose the combined use of i) Doxycycline (Hit-1: targeting mitochondria) and ii) Vitamin C (Hit-2: targeting glycolysis), which represents a new synthetic-lethal metabolic strategy for eradicating CSCs.

This type of metabolic Achilles’ heel will allow us and others to more effectively “starve” the CSC population.

Conclusions

Numerous functional studies have now directly shown that mitochondria are an important new therapeutic target in cancer cells [3, 5, 8-21, 40-53]. Since Doxycycline, an FDA-approved antibiotic, behaves as an inhibitor of mitochondrial protein translation, it may have therapeutic value in the specific targeting of mitochondria in cancer cells. However, in this paper, we have identified a novel metabolic mechanism by which CSCs successfully escape from the anti-mitochondrial effects of Doxycycline, by assuming a purely glycolytic phenotype. Therefore, DoxyR CSCs are then more susceptible to other metabolic perturbations, because of their metabolic inflexibility, allowing for their eradication with natural products and other FDA-approved drugs. Thus, understanding the metabolic basis of Doxycycline-resistance has ultimately helped us to develop a new synthetic lethal strategy, for more effectively targeting CSCs.

Materials and Methods

Materials

Doxycycline, Ascorbic Acid, 2-Deoxy-D-glucose (2-DG), Irinotecan, Berberine Chloride, Niclosamide, Chloroquine diphosphate, Stiripentol and Atovaquone were all purchased from Sigma Aldrich. Sorafenib was obtained from Generon. All compounds were dissolved in DMSO, except Ascorbic Acid, 2-deoxy-D-glucose (2-DG) and Chloroquine diphosphate, which were dissolved in cell culture medium.

Cell cultures

MCF7 breast cancer cells were obtained from ATCC and cultured in DMEM (Sigma Aldrich). MCF-7 cells resistant to Doxycycline (MCF7 DoxyR) were selected by a stepwise exposure to increasing concentration of Doxycycline. In particular, wild type MCF7 cells were initially exposed to 12.5 µM Doxycycline and the dose gradually increased to 50 µM over a 3-month period. The population of resistant cells, named MCF7 DoxyR, was selected after 3 weeks of treatment with 12.5 µM Doxycycline, followed by 3 weeks of treatment with 25 µM Doxycycline. MCF7 DoxyR cells were routinely maintained in regular medium supplemented with 25 µM Doxycycline.

Mammosphere formation

A single cell suspension of MCF7 or MCF7 DoxyR cells was prepared using enzymatic (1x Trypsin-EDTA, Sigma Aldrich), and manual disaggregation (25 gauge needle) [54]. Cells were then plated at a density of 500 cells/cm2 in mammosphere medium (DMEM-F12/ B27 / 20-ng/ml EGF/PenStrep) in nonadherent conditions, in culture dishes coated with (2-hydroxyethylmethacrylate) (poly-HEMA, Sigma), in the presence of treatments, were required. Cells were grown for 5 days and maintained in a humidified incubator at 37°C at an atmospheric pressure in 5% (v/v) carbon dioxide/air. After 5 days for culture, spheres > 50 μm were counted using an eye piece graticule, and the percentage of cells plated which formed spheres was calculated and is referred to as percentage mammosphere formation. Mammosphere assays were performed in triplicate and repeated three times independently.

Evaluation of mitochondrial mass and function

To measure mitochondrial mass by FACS analysis, cells were stained with MitoTracker Deep Red (Life Technologies), which localizes to mitochondria regardless of mitochondrial membrane potential. Cells were incubated with pre-warmed MitoTracker staining solution (diluted in PBS/CM to a final concentration of 10 nM) for 30-60 min at 37 °C. All subsequent steps were performed in the dark. Cells were washed in PBS, harvested, re-suspended in 300 μL of PBS and then analyzed by flow cytometry (Fortessa, BD Bioscience). Data analysis was performed using FlowJo software. Extracellular acidification rates (ECAR) and real-time oxygen consumption rates (OCR) for MCF7 cells were determined using the Seahorse Extracellular Flux (XFe-96) analyzer (Seahorse Bioscience) [15]. Briefly, 15,000 MCF7 and MCF7 DoxyR cells per well were seeded into XFe-96 well cell culture plates for 24h. Then, cells were washed in pre-warmed XF assay media (or for OCR measurement, XF assay media supplemented with 10mM glucose, 1mM Pyruvate, 2mM L-glutamine and adjusted at 7.4 pH). Cells were then maintained in 175 µL/well of XF assay media at 37C, in a non-CO2 incubator for 1 hour. During the incubation time, 5 µL of 80mM glucose, 9 µM oligomycin, and 1 M 2-deoxyglucose (for ECAR measurement) or 10µM oligomycin, 9 µM FCCP, 10 µM Rotenone, 10 µM antimycin A (for OCR measurement), were loaded in XF assay media into the injection ports in the XFe-96 sensor cartridge. Data set was analyzed by XFe-96 software after the measurements were normalized by protein content (SRB). All experiments were performed three times independently.

ALDEFLUOR assay and separation of the ALDH positive population

ALDH activity was assessed by FACS analysis (Fortessa, BD Bioscence) in MCF7 cells and MCF7 DoxyR cells. The ALDEFLUOR kit (StemCell Technologies) was used to isolate the population with high ALDH enzymatic activity. Briefly, 1 × 105 MCF7 and MCF7 DoxyR cells were incubated in 1ml ALDEFLUOR assay buffer containing ALDH substrate (5 μl/ml) for 40 minutes at 37°C. In each experiment, a sample of cells was stained under identical conditions with 30 μM of diethylaminobenzaldehyde (DEAB), a specific ALDH inhibitor, as a negative control. The ALDEFLUOR-positive population was established in according to the manufacturer’s instructions and was evaluated in 3 × 104 cells. Data analysis was performed using FlowJo software.

Anoikis assay

MCF7 and MCF7 DoxyR cells were seeded on low-attachment plates to enrich for the CSC population [54]. Under these conditions, the non-CSC population undergoes anoikis (a form of apoptosis induced by a lack of cell-substrate attachment) and CSCs are believed to survive. The surviving CSC fraction was analyzed by FACS analysis. Briefly, 1 x 105 MCF7 and MCF7 DoxyR monolayer cells were seeded for 48h in 6-well plates. Then, cells were trypsinized and seeded in low-attachment plates in mammosphere media. After 10h, cells were spun down and incubated with CD24 (IOTest CD24-PE, Beckman Coulter) and CD44 (APC mouse Anti-Human CD44, BD Pharmingen) antibodies for 15 minutes on ice. Cells were rinsed twice and incubated with LIVE/DEAD dye (Fixable Dead Violet reactive dye; Life Technologies) for 10 minutes. Samples were then analyzed by FACS (Fortessa, BD Bioscence). Only the live population, as identified by the LIVE/DEAD dye staining, was analyzed for CD24/CD44 expression. Data were analyzed using FlowJo software.

Label-free semi-quantitative proteomics analysis

Cell lysates were prepared for trypsin digestion by sequential reduction of disulphide bonds with TCEP and alkylation with MMTS. Then, the peptides were extracted and prepared for LC-MS/MS. All LC-MS/MS analyses were performed on an LTQ Orbitrap XL mass spectrometer (Thermo Scientific, San Jose, CA) coupled to an Ultimate 3000 RSLC nano system (Thermo Scientific, formerly Dionex, The Netherlands). Xcalibur raw data files acquired on the LTQ-Orbitrap XL were directly imported into Progenesis LCMS software (Waters Corp., Milford, MA, formerly Non-linear dynamics, Newcastle upon Tyne, UK) for peak detection and alignment. Data were analyzed using the Mascot search engine. Five technical replicates were analyzed for each sample type [8, 12].

Immuno-blot analysis

MCF7 and MCF7 DoxyR cells protein lysates were electrophoresed through a reducing SDS/10% (w/v) polyacrylamide gel, electroblotted onto a nitrocellulose membrane and probed with primary antibodies against phosphorylated AKT (Ser 473) and ATK (Cell Signaling), Phopshorylated ERK 1/2 (E-4), ERK2 (C-14), TOMM20 (F-10) and β-actin (C2), all purchased from Santa Cruz Biotechnology. Proteins were detected by horseradish peroxidase-linked secondary antibodies and revealed using the SuperSignal west pico chemiluminescent substrate (Fisher Scientific).

Click-iT EdU proliferation assay

48h after seeding MCF7 and MCF7 DoxyR were subjected to proliferation assay using Click-iT Plus EdU Pacific Blue Flow Cytometry Assay Kit (Life Technologies), customized for flow cytometry. Briefly, cells were treated with 10 µM EdU for 2 hours and then fixed and permeabilized. EdU was detected after permeabilization by staining cells with Click-iT Plus reaction cocktail containing the Fluorescent dye picolylazide for 30 min at RT. Samples were then washed and analyzed using flow cytometer (Fortessa, BD Bioscence). Background values were estimated by measuring non-EdU labeled, but Click-iT stained cells. Data were analyzed using FlowJo software.

Migration assay

MCF7 and MCF7 DoxyR cells were allowed to grow in regular growth medium until they were 70-80 % confluent. Next, to create a scratch of the cell monolayer, a p200 pipette tip was used. Cells were washed twice with PBS and then incubated at 37° C in regular medium for 24h. The migration assay was evaluated using Incucyte Zoom (Essen Bioscience) [55]. The rate of migration was measured by quantifying the % of wound closure area, determined using the software ImageJ, according to the formula:

% of wound closure = [(At = 0 h - At = Δ h)/At = 0 h] × 100%

Statistical analysis

Data is represented as the mean ± standard error of the mean (SEM), taken over ≥ 3 independent experiments, with ≥ 3 technical replicates per experiment, unless otherwise stated. Statistical significance was measured using the t-test. P ≤ 0.05 was considered significant.

Author contributions

Professor Michael Lisanti and Dr. Federica Sotgia conceived and initiated this collaborative project. All the experiments in this paper were performed by Dr. Ernestina M. De Francesco, with minor technical assistance from other lab members; Dr. Ernestina M. De Francesco analyzed all the data and generated the final figures and tables, and she wrote significant portions of the manuscript. Drs. Michael P. Lisanti, Ernestina M. De Francesco, Gloria Bonuccelli, Marcello Maggiolini and Federica Sotgia all contributed to the writing and the editing of the manuscript. Professor Lisanti generated the schematic summary diagrams.

Acknowledgments

We are grateful to the University of Manchester, which allocated start-up funds and administered a donation, to provide the necessary resources required to start and complete this drug discovery project (to MPL and FS). Dr. Ernestina M. De Francesco was supported by a fellowship from the Associazione Italiana per la Ricerca sul Cancro (AIRC) co-funded by the European Union. The Lisanti and Sotgia Laboratories are currently supported by private donations, and by funds from the Healthy Life Foundation (HLF) and the University of Salford (to MPL and FS). We also wish to thank Dr. Duncan Smith, who performed the proteomics analysis on whole cell lysates, within the CRUK Core Facility. MM was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC, IG 16719).

Conflicts of Interest

MPL and FS hold a minority interest in Lunella, Inc.

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