Metabolic plasticity and adaptive resistance to metabolic inhibitors in tumor cells

Although tumor metabolism is becoming a major source of inspiration to develop new anticancer drugs, the current number of breakthrough discoveries under clinical evaluation is so far limited. One explanation besides the usual PK and PD issues that stop drug candidates during their development is the metabolic plasticity of tumors. There are indeed numerous examples where tumors were shown to adapt their metabolic preferences according to the (un-)availability of nutrients or the (in-)capacity to transform them into biosynthetic intermediates and ATP. Direct consequences of metabolic adaptation are the development of resistance and the progressive loss of activity of the drug. Such metabolic adaptations can occur through the selection of cancer cell clones bearing a mutation that supports the alternate metabolic route. However, the intricate network of biochemical pathways that govern cell metabolism can also offer mutation-independent modes of adaptation for cells to fulfill their bioenergetic needs through another path than the one blocked by the administered drug. Our study aims to identify the optimal combination of two drugs targeting metabolic pathways in order to prevent a possible escape for cancer cells. To do so, we used 3-bromopyruvate (3-BrPA) as a first metabolism-targeting drug to induce metabolic addictions to given pathways that could be targeted in a second time (synthetic lethality). Our data indicate that a pre-challenge with 3-BrPA leads to metabolic alterations within tumor cells, leading in particular to a strict dependence towards MCT4 expression to support glycolytic flux and cell growth. This metabolic adaptation can be therapeutically targeted. More generally, our study highlights a new strategy to overcome metabolic plasticity and associated therapy resistance by treating tumor cells with two metabolic inhibitors, in a sequential mode, with a synthetic lethality purpose

Hypoxia imaging with the nitroimidazole 18F-FAZA PET tracer: A comparison with Oxylite, EPR Oximetry and 19F-MRI Relaxometry

Background and purpose: Tumor hypoxia has been considered as an important prognosis factor in oncology [1]. Quantitative information and distribution of oxygen concentration would be valuable for the improvement of therapeutic strategies. Given the importance of this aspect, there has been many techniques reported to be useful for hypoxia detection in tumors [2,3]; but a limited number of methods have been implemented into clinical practice. One of techniques currently available for detection of hypoxia in human application is Positron Emission Tomography (PET). The accumulation of hypoxia-specific PET tracers can reflect hypoxic areas inside tumors that are relevant in terms of radiation resistance; however, this is an indirect method that cannot provide absolute values of pO2. Therefore, to evaluate the potential of PET hypoxia images with tracer 18F-fluoroazomycin-arabinoside (18F-FAZA), we compared PET hypoxia imaging to other oximetry techniques: Oxylite, Electron Paramagnetic Resonance (EPR) spectroscopy and Nuclear Magnetic Resonance Imaging by fluorine relaxometry (19F-MRI), respectively. Methods: Male adult WAG/Rij rats grafted with rhabdomyosarcoma in thighs were used for this study. In all comparisons, animals were randomly divided into two groups, breathing either room air or carbogen. PET imaging was performed on a dedicated small-animal PET scanner 3h after intravenous injection of 18.5-29.6 MBq 18F-FAZA. The median pO2 value of each tumor measured by Oxylite was calculated from the measurement at ten independent sites within the tumor. EPR acquisition was carried out 24h after charcoal implantation by using an L-band EPR spectrometer. For MRI oximetry, perfluoro-15-crown-5ether (15C5) was used as oxygen sensitive sensor, the spin-lattice relaxation time (T1) of 19F nuclei was acquired by an 11.7 T system with a tunable 1H/19F surface coil. Linewidth of EPR spectra and T1-value obtained by MRI were then converted to pO2 using the calibration curves. Results: The results from both Oxylite and EPR showed an increase of pO2 in the breathing carbogen group. In accordance with this tendency, the tumor-to-background (T/B) ratio measured by PET under high oxygen condition exhibited about 1.3-fold decrease than that under normoxic condition. The scatter-plots of T/B ratio versus measured pO2 were traced by using data from all individual tumors. There was a good correlation between the results obtained by PET and EPR (R = 0.9306). In the case of Oxylite, although the poorer correlation coefficient was observed (R = 0.5469), the trend for two values to agree was still related to the inverse function theoretically predicted. For the comparison of 18F-FAZA and 15C5 marker, we could not found the significant difference in values T1. This could be explained by the lower sensitivity of 19F-relaxometry compared to EPR and OxyLite. Conclusions: Our present study demonstrated a clear correlation between 18F-FAZA-PET signal intensities and tumor oxygenation. These results suggest that 18F-FAZA is an effective surrogate of hypoxia fractions and support the use of 18F-FAZA PET as an imaging technique to guide cancer therapy

Sirtuin 1 mediates acidosis-induced metabolic reprogramming of tumor cells

Mechanisms underlying cancer progression are strongly influenced by the physico-chemical properties of the tumor microenvironment. In particular, tumor cells must adapt to survive under low pO2 and low pH. Although the impact of hypoxia on tumor metabolism is well described, the influence of extracellular acidosis on the metabolic preferences of cancer cells is largely unknown. Still, tumor acidosis is known to affect important energy-consuming cellular functions including proliferation and invasion. In this study, we have therefore exposed tumor cells (derived from various tissues) to low pH conditions (pH 6.5) for several weeks. After acidic acclimation, we found that tumor cells proliferate at the same rate as parent cells maintained at pH 7.4. More interestingly, we documented that chronic low pH exposure triggers the metabolic reprogramming of tumor cells from the preferential use of glucose towards glutamine metabolism. The use of glutamine as a major fuel of the TCA cycle in low pH-adapted tumor cells was proven using a variety of techniques including measurement of [U13C5]-glutamine incorporation in metabolites (determined by GC-MS) and determination of the oxygen consumption rate (OCR) using a Seahorse microplate analyser. Mechanistic dissection of the low pH-driven phenotype led us to document that the metabolic switch was mediated by SIRT1, a NAD+-dependent protein deacetylase. We actually found that at acidic pH, SIRT1 promotes glutamine metabolism in a HIF2-dependent manner. By contrast, we observed a SIRT-1-mediated decrease in HIF1 signalling and associated glycolysis. These observations were repeated with the exact same conclusions using tumor cell lines of various origins (cervix SiHa, pharynx FaDu and colon HCT116). Also, the 'glutamine-addicted' phenotype was proven to be reversible when acclimated cells were again cultured under physiological pH, thereby excluding a clonal selection of tumor cells in response to the acidic conditions. Finally, pharmacological inhibition of either glutamine metabolism (using the glutaminase inhibitor BPTES) or SIRT1 deacetylase activity preferentially killed low pH-adapted cancer cells in vitro (vs. parent cells) and delayed the growth of corresponding tumor xenografts in vivo. Altogether, these observations indicate that a major metabolic shift from glucose to glutamine metabolism is induced in tumor cells chronically exposed to an acidic environment and importantly makes them particularly suited for dedicated pharmacological treatments

Metabolic adaptation of tumor cells under chronic acidosis: a shift towards reductive glutamine metabolism driven by the SIRT1/HIF2alpha axis

Cancer progression is strongly influenced by the physico-chemical properties of the tumor microenvironment. In particular, tumor cells must adapt to survive under low pO2 and low pH. Although the impact of hypoxia on tumor metabolism is well described, little is known on how tumor cells adapt their metabolism to acidosis. Here, we exposed tumor cells derived from various tissues to low pH conditions (pH 6.5) for several weeks until they ended up proliferating at the same rate as parent cells maintained at pH 7.4. This low pH acclimation triggered the reprogramming of tumor cells from a mainly glycolytic metabolism towards the preferred use of glutamine as documented by tracking the fate of [U-13C] glucose and [U-13C] glutamine by GC-MS analysis of metabolites. The use of glutamine as a major fuel of the TCA cycle in low pH-adapted tumor cells was further associated with an increase in the oxygen consumption rate (OCR) as determined using a Seahorse microplate analyser. The search for the determinants of the low pH-driven phenotype led us to document that the metabolic switch was mediated by SIRT1, a protein deacetylase activated by the increased pool of NAD+ in low pH-adapted cells, through two distinct mechanisms. First, free acetate acted as a counteranion to export excess protons out of the cells via MCT1, maintaining the intracellular pH in a physiological range. Second, SIRT1 stimulated the activity of HIF2α thereby supporting the glutamine metabolism via the upregulation of the glutamine transporter SLC1A5 and enzymes supporting the reductive glutamine metabolism including IDH1. Finally, pharmacological inhibition of either glutamine metabolism with the glutaminase inhibitor BPTES or of SIRT1 deacetylase activity preferentially killed low pH-adapted cancer cells in vitro (vs. parent cells) and delayed the growth of corresponding tumor xenografts in vivo. Altogether, these data indicate that a major metabolic shift from glucose to glutamine metabolism is induced in tumor cells chronically exposed to an acidic environment and importantly makes them particularly suited for dedicated pharmacological treatments