Nada Al-Saffar1, Jasmin Sidhu1, Udai Banerji1, Yuen-Li Chung1, and Martin O. Leach1
1The Institute of Cancer Research and The Royal Marsden NHS Foundation Trust, London, United Kingdom
Synopsis
Previous
reports showed that activation of the PI3K/mTOR pathway is associated with
chemoresistance in ovarian cancer cells, and this can be reversed by combining chemotherapy with the mTORC1/2
inhibitor vistusertib (AZD2014). This combination is now being pursued clinically.
In this study, we used NMR spectroscopy to establish biomarkers for this
combination in A2780Cis
human ovarian carcinoma cells. We report that this combination resulted in
decreases in lactate, PC, GPC and tCho and was associated with inhibition of
glycolysis- and choline-regulatory enzymes. This indicates that choline metabolites and lactate may provide
potential non-invasive biomarkers of tumour response to this combination
therapy.
INTRODUCTION
Activation of the PI3K/mTOR signalling pathway has been shown to
contribute to resistance in ascitic cells from ovarian cancer patients showing
chemoresistance to cisplatin.1 Additive apoptosis was seen when
vistusertib (AZD2014, a dual mTORC1/2 inhibitor) was added to paclitaxel in
preclinical models, and this combination is now being pursued clinically.2
Our aim was to assess whether this combination results in metabolic changes
that are detectable by NMR spectroscopy providing potential non-invasive
biomarkers for monitoring response to
AZD2014 and paclitaxel combination during early stage clinical trials.
METHODS
The A2780Cis human cisplatin-resistant ovarian carcinoma cell line was
treated with AZD2014 (AstraZeneca; 2.5xGI50) and paclitaxel (2.5xGI50)
both individually and in combination (2.5xGI50 +2.5xGI50)
for 24hr. Treatment effects were studied at molecular and cellular levels.
Metabolic changes were evaluated by NMR spectroscopy at 25°C with a 500 MHz
Bruker spectrometer. Metabolite contents were determined by integration and
normalised relative to the peak integral of an internal reference and the
number of cells extracted per sample.RESULTS & DISCUSSION
Treatment of A2780Cis cells with paclitaxel reduced cell number (down to
67±5, P=0.001) relative to their controls. AZD2014 also resulted in a decrease
in the cell number (down to 78±6, P=0.008) of treated cells compared to
controls. Combination of paclitaxel with AZD2014 resulted in a further decrease
in treated cell number (down to 56±8, P=0.003) compared to controls. Consistent
with the mechanism of action, immunoblotting analysis of cells treated with
AZD2014 showed an inhibition of PI3K/mTOR signalling observed as a
decrease in the levels of phosphorylated AKT(Ser473) and RPS6(Ser240/244) in treated cells compared to their
controls, while paclitaxel induced apoptosis as detected by cleaved PARP. Combination
treatment of paclitaxel with AZD2014 resulted in combined molecular effects of
both agents including inhibition of PI3K/mTOR signalling and induction of
apoptosis. 1H-NMR analysis showed that treatment with AZD2014
induced a significant (P≤0.05) decrease in the levels of lactate,
phosphocholine (PC), glycerophosphocholine (GPC) and total choline (tCho; Fig.
1a&b). In contrast, paclitaxel resulted in increased levels of tCho (P≤0.05; Fig. 1a&b). Interestingly,
combination treatment of paclitaxel with AZD2014 resulted in metabolic changes similar
to those caused by single treatment with AZD2014 including a decrease in the
levels of lactate, PC and tCho (P≤0.05; Fig. 1a&b). These metabolic changes in cells support our
previous findings where similar changes in choline-containing metabolites were
observed both in vivo and ex vivo.3 To explore potential mechanisms
underlying the detected metabolic changes, immunoblotting was used to
assess treatment effects on protein levels of enzymes involved in glycolysis
and choline metabolism. Our results showed that the decrease in levels of PC observed
following treatment with AZD2014 alone and in combination with paclitaxel was
associated with a decrease in the protein levels of choline kinase α (CHKA),
the enzyme responsible for choline phosphorylation into PC (Fig. 1c). A previous
report showed that the mTORC1 inhibitor Everolimus resulted in a decrease in
the hyperpolarized lactate-to-pyruvate ratio and that was associated with a
drop in expression of LDHA (catalyses the conversion of pyruvate to lactate).4
In line with this study, a decrease in the protein expression level of LDHA was
detected following AZD2014 treatment and the combination (Fig. 1c). In
addition, a reduction in the glycolytic enzyme HK2 (phosphorylates glucose into
glucose-6-phosphate) was observed (Fig. 1c), providing further evidence for the
inhibition of glycolysis as a mechanism for the NMR-detected depletion of
lactate.CONCLUSION
Our results show that NMR spectroscopy can detect metabolic changes
including changes in lactate and choline metabolites following combination
treatment of paclitaxel with the mTORC1/2 inhibitor AZD2014. Our findings
correlate with in vivo NMR-detected biomarkers and indicate that MRS or
other imaging modalities (e.g. PET and DNP) may have the potential to be used
to monitor response to this combination therapy during early stage clinical
trials.Acknowledgements
CRUK and EPSRC support to the Cancer Imaging Centre at ICR and RMH in association with MRC and Department of Health C1060/A10334, C1060/A16464 and NHS funding to the NIHR Biomedical Research Centre and the Clinical Research Facility in Imaging.References
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