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Vitamin C and Doxycycline: A synthetic lethal combination therapy targeting metabolic flexibility in cancer stem cells (CSCs)

PDF |  HTML |  Order a ReprintOncotarget. 2017; 8:67269-67286. https://doi.org/10.18632/oncotarget.18428

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Ernestina Marianna De Francesco^1,2, Gloria Bonuccelli³, Marcello Maggiolini¹, Federica Sotgia³ and Michael P. Lisanti³

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.

INTRODUCTION

Cancer stem cells (CSCs) are thought to be the “root cause” of tumor recurrence, distant metastasis and therapy-resistance, driving poor clinical outcome in advanced cancer patients [1–3]. Therefore, new therapeutic strategies are necessary to identify and eradicate CSCs [4–7]. As such, this goal remains an unmet medical need. 

Recently, we identified that high mitochondrial mass is a new common and characteristic feature of CSCs, based on high-resolution proteomics analysis [8–10]. Importantly, high mitochondrial mass is a surrogate marker for increased mitochondrial biogenesis and/or elevated mitochondrial protein translation. Thus, this simple metabolic observation provides a new means for both i) CSC identification [9–13] and ii) CSC eradication [9, 10, 14–19]. 

Specifically, we showed that a mitochondrial fluorescent dye (MitoTracker) could be effectively used for the enrichment and purification of CSC activity from a heterogeneous population of living cells [11–13]. In this context, cancer cells with the highest mitochondrial mass had the strongest functional ability to undergo anchorage-independent growth, a characteristic normally associated with metastatic potential [11–13]. The ‘Mito-high’ cell sub-population also had the highest tumor-initiating activity in vivo, as shown using pre-clinical models. High mitochondrial mass was strictly correlated with i) increased hTERT activity and ii) the ability to undergo cell proliferation, which was sensitive to CDK4/6 inhibitors, such as palbociclib [16]. Complementary results were obtained with other fluorescent mitochondrial probes for ROS and hydrogen peroxide, as well as NADH auto-fluorescence, an established marker of mitochondrial “power”/high OXPHOS activity [13].

Moreover, we demonstrated that several classes of non-toxic antibiotics could be used to halt CSC propagation [14–19]. Because of the conserved evolutionary similarities between aerobic bacteria and mitochondria, certain classes of antibiotics inhibit mitochondrial protein translation, as an off-target side-effect [14]. One such group of antibiotics is the tetracyclines, the prototypic family member being Doxycycline. 

Through this analysis, it became apparent that tetracycline antibiotics, such as Doxycycline, could be re-purposed to eradicate CSCs, in multiple cancer types [14, 20, 21]. These eight distinct cancer types included: DCIS, breast (ER(+) and ER(-)), ovarian, prostate, lung, and pancreatic carcinomas, as well as melanoma and glioblastoma. Doxycycline was also effective in halting the propagation of primary cultures of CSCs from breast cancer patients, with advanced metastatic disease (isolated from ascites fluid and/or pleural effusions) [20]. 

Remarkably, Doxycycline behaves as a strong radio-sensitizer, successfully overcoming radio-resistance in breast CSCs [20]. This has important clinical implications, as the majority of ER(+) breast cancer patients are currently treated with breast-conserving surgery (lumpectomy) plus radiation therapy and hormonal therapy with an anti-estrogen. 

Doxycycline is an FDA-approved drug, which first became available in 1967, ~50 years ago now. It has excellent pharmacokinetic properties, with absorption of nearly 100% and a half-life of 18 to 24 hours. However, as with any new potential therapy, there is always a concern regarding the possible development of drug-resistance. 

Here, we show that cancer cells can indeed escape the effects of Doxycycline, by reverting to a purely glycolytic phenotype. Fortunately, the metabolic inflexibility conferred by this escape mechanism allows Doxycycline-resistant (DoxyR) CSCs to be more effectively targeted with many other metabolic inhibitors, including Vitamin C, which functionally blocks aerobic glycolysis. 

Interestingly, previous studies have shown that Vitamin C inhibits GAPDH (a glycolytic enzyme) and depletes the cellular pool of glutathione, resulting in high ROS production and oxidative stress [22]. We show here that DoxyR CSCs are between 4- to 10-fold more susceptible to the effects of Vitamin C, inhibiting their propagation in the range of 100 to 250 µM. Therefore, Doxycycline and Vitamin C may represent a new synthetic lethal drug combination for eradicating CSCs, by ultimately targeting both mitochondrial and glycolytic metabolism. 

RESULTS

Metabolic flexibility is the intrinsic ability of a cell to change from one carbon fuel source to another; conversely, metabolic inflexibility is the exact opposite: the lack of ability (or dramatically reduced ability) to change fuel sources. It is believed that metabolic flexibility in cancer cells allows them to escape therapeutic eradication, leading to chemo- and radio-resistance. Here, we used doxycycline to pharmacologically induce metabolic inflexibility in CSCs, by chronically inhibiting mitochondrial biogenesis. This treatment resulted in a purely glycolytic population of surviving cancer cells. Then, we identified six other clinically-approved therapeutics, two natural products and one experimental drug, that all successfully eradicate the remaining glycolytic CSCs. Therefore, Doxycycline-induced metabolic inflexibility may be a practical solution to avoiding treatment failure, in a variety of cancer types.