Cellular and Molecular Biology

Impact of hypoxia on chemoresistance of mesothelioma mediated by the proton-coupled folate transporter, and preclinical activity of new anti-LDH-A compounds

Abstract

Background

Expression of proton-coupled folate transporter (PCFT) is associated with survival of mesothelioma patients treated with pemetrexed, and is reduced by hypoxia, prompting studies to elucidate their correlation.

Methods

Modulation of glycolytic gene expression was evaluated by PCR arrays in tumour cells and primary cultures growing under hypoxia, in spheroids and after PCFT silencing. Inhibitors of lactate dehydrogenase (LDH-A) were tested in vitro and in vivo. LDH-A expression was determined in tissue microarrays of radically resected malignant pleural mesothelioma (MPM, N = 33) and diffuse peritoneal mesothelioma (DMPM, N = 56) patients.

Results

Overexpression of hypoxia marker CAIX was associated with low PCFT expression and decreased MPM cell growth inhibition by pemetrexed. Through integration of PCR arrays in hypoxic cells and spheroids and following PCFT silencing, we identified the upregulation of LDH-A, which correlated with shorter survival of MPM and DMPM patients. Novel LDH-A inhibitors enhanced spheroid disintegration and displayed synergistic effects with pemetrexed in MPM and gemcitabine in DMPM cells. Studies with bioluminescent hypoxic orthotopic and subcutaneous DMPM athymic-mice models revealed the marked antitumour activity of the LDH-A inhibitor NHI-Glc-2, alone or combined with gemcitabine.

Conclusions

This study provides novel insights into hypoxia/PCFT-dependent chemoresistance, unravelling the potential prognostic value of LDH-A, and demonstrating the preclinical activity of LDH-A inhibitors.

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Fig. 1: Hypoxia affects PCFT expression and pemetrexed activity.
Fig. 2: Correlation of hypoxia and PCFT silencing with LDH-A expression.
Fig. 3: Cytotoxic effect of anti-LDH-A compounds alone and in combination with pemetrexed or gemcitabine on MPM and DMPM monolayers and spheroids.
Fig. 4: In vivo activity of the new anti-LDH-A compound NHI-Glc-2 on orthotopic and subcutaneous DMPM models.
Fig. 5: High expression of LDH-A correlates with significantly shorter overall survival (OS) and progression-free survival (PFS) in both MPM and DMPM patients.

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Acknowledgements

The authors would like to thank Naomi Petersen (VUmc, Amsterdam, The Netherlands), Claudio Ricci and Luca Pollina (AOUP, Pisa, Italy) for their work on the mouse models and TMA stainings. We extend our gratitude to Maria Gemelli (Cancer Center Humanitas, Milano, Italy) for her work on the clinical database, Paolo Gandellini (Molecular Pharmacology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy) for his work on the microarray data, Leticia G. Leon (Cancer Pharmacology Lab, Pisa, Italy) for preliminary in vitro studies and to Zhanjun Hou (Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI, USA) who provided the anti-PCFT antibody.

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E.G., F.M. and G.J.P. designed and coordinated the study. G.L.P., B.E.H. and R.S. were the principal investigators and take primary responsibility for the experiments; N.F., E.A.Z., A.A., T.L., A.B., K.S. and G.M. performed additional experimental work and prepared some of the figures; N.F. and E.G. performed the studies on TMA; S.C., N.Z., B.P., M.D., C.G., L.H.M. and P.A.Z. provided essential material; N.Z., J.C., G.J., Y.G.A., P.D. and R.T.S. participated in the research design; G.L.P. and E.G. wrote the paper; G.L.P., G.J.P., Y.G.A. and E.G. edited the paper. All authors read and approved the final paper.

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Correspondence to Elisa Giovannetti.

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This study was approved by the Local Ethics Committee of the Humanitas Cancer Center (Rozzano, Milano, Italy) and National Cancer Center (Milano, Italy). Patients who provided tissues and primary cell cultures have provided a written informed consent. The study was performed in accordance with the Declaration of Helsinki.

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Consent to publish has been obtained from all the participants (or legal parent or guardian for children) to report individual patient data.

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All data generated or analysed during this study are included in this published article (and in supplementary information files).

Competing interests

The authors declare no competing interests.

Funding information

This work was partially supported in the collections and analysis of data by the following grants: “the Law Offices of Peter G. Angelos Grant” from the Mesothelioma Applied Research Foundation (MARF), United States (Elisa Giovannetti, Paolo A Zucali, Filippo Minutolo and Godefridus J. Peters), Fondazione Humanitas, Milano, Italy (Elisa Giovannetti, Paolo A Zucali), CCA Foundation 2012 (Elisa Giovannetti, Godefridus J Peters and Amir Avan), 2013 (Jacqueline Cloos, Elisa Giovannetti) 2015 and 2016 (Elisa Giovannetti) grants, KWF Dutch Cancer Society grants (KWF project# 10401 and # 11957, Elisa Giovannetti), Polish National Science Center project 2018/31/B/NZ7/02909 (Godefridus J. Peters, Elisa Giovannetti, Filippo Minutolo and Ryszard T. Smolenski), University of Pisa (Intramural Funds, Carlotta Granchi, Elisa Giovannetti and Filippo MInutolo), Italian Association for Cancer Research AIRC/Start-Up grant and (Elisa Giovannetti), R01 CA53535 (Larry Matherly) from the National Institutes of Health and the Eunice and Milton Ring Endowed Chair for Cancer Research (Larry Matherly).

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Li Petri, G., El Hassouni, B., Sciarrillo, R. et al. Impact of hypoxia on chemoresistance of mesothelioma mediated by the proton-coupled folate transporter, and preclinical activity of new anti-LDH-A compounds. Br J Cancer 123, 644–656 (2020). https://doi.org/10.1038/s41416-020-0912-9

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