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.
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.
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.
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.
Subscribe to Journal
Get full journal access for 1 year
only $20.79 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Carbone, M., Adusumilli, P. S., Alexander, H. R., Baas, P., Bardelli, F., Bononi, A. et al. Mesothelioma: scientific clues for prevention, diagnosis, and therapy. CA Cancer J Clin. https://doi.org/10.3322/caac.21572 (2019).
Vogelzang, N. J., Rusthoven, J. J., Symanowski, J., Denham, C., Kaukel, E., Ruffie, P. et al. Phase III study of pemetrexed in combination with cisplatin versus cisplatin alone in patients with malignant pleural mesothelioma. J. Clin. Oncol. 21, 2636–2644 (2003).
Ceresoli, G. L., Zucali, P. A., Favaretto, A. G., Grossi, F., Bidoli, P., Del Conte, G. et al. Phase II study of pemetrexed plus carboplatin in malignant pleural mesothelioma. J. Clin. Oncol. 24, 1443–1448 (2006).
Sugarbaker, P. H. Update on the management of malignant peritoneal mesothelioma. Transl. Lung Cancer Res. 7, 599–608 (2018).
Righi, L., Papotti, M. G., Ceppi, P., Billè, A., Bacillo, E., Molinaro, L. et al. Thymidylate synthase but not excision repair cross-complementation group 1 tumor expression predicts outcome in patients with malignant pleural mesothelioma treated with pemetrexed-based chemotherapy. J. Clin. Oncol. 28, 1534–1539 (2010).
Zucali, P. A., Giovannetti, E., Destro, A., Mencoboni, M., Ceresoli, G. L., Gianoncelli, L. et al. Thymidylate synthase and excision repair cross-complementing group-1 as predictors of responsiveness in mesothelioma patients treated with pemetrexed/carboplatin. Clin. Cancer Res. 17, 2581–2590 (2011).
Lustgarten, D. E. S., Deshpande, C., Aggarwal, C., Wang, L.-C., Saloura, V., Vachani, A. et al. Thymidylate synthase and folyl-polyglutamate synthase are not clinically useful markers of response to pemetrexed in patients with malignant pleural mesothelioma. J. Thorac. Oncol. 8, 469–477 (2013).
Mairinger, F., Vollbrecht, C., Halbwedl, I., Hatz, M., Stacher, E., Gülly, C. et al. Reduced folate carrier and folylpolyglutamate synthetase, but not thymidylate synthase predict survival in pemetrexed-treated patients suffering from malignant pleural mesothelioma. J. Thorac. Oncol. 8, 644–653 (2013).
Matherly, L. H., Hou, Z. & Gangjee, A. The promise and challenges of exploiting the proton-coupled folate transporter for selective therapeutic targeting of cancer. Cancer Chemother. Pharm. 81, 1–15 (2018).
Cherian, C., Kugel Desmoulin, S., Wang, L., Polin, L., White, K., Kushner, J. et al. Therapeutic targeting malignant mesothelioma with a novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate via its selective uptake by the proton-coupled folate transporter. Cancer Chemother. Pharm. 71, 999–1011 (2013).
Zhao, R. & Goldman, I. D. The molecular identity and characterization of a proton-coupled folate transporter–PCFT; biological ramifications and impact on the activity of pemetrexed. Cancer Metastasis Rev. 26, 129–139 (2007).
Gonen, N., Bram, E. E. & Assaraf, Y. G. PCFT/SLC46A1 promoter methylation and restoration of gene expression in human leukemia cells. Biochem. Biophys. Res. Commun. 376, 787–792 (2008).
Giovannetti, E., Zucali, P. A., Assaraf, Y. G., Funel, N., Gemelli, M., Stark, M. et al. Role of proton-coupled folate transporter in pemetrexed resistance of mesothelioma: clinical evidence and new pharmacological tools. Ann. Oncol. 28, 2725–2732 (2017).
Raz, S., Sheban, D., Gonen, N., Stark, M., Berman, B. & Assaraf, Y. G. Severe hypoxia induces complete antifolate resistance in carcinoma cells due to cell cycle arrest. Cell Death Dis. 5, e1067 (2014).
Peters, G. J., Smitskamp-Wilms, E., Smid, K., Pinedo, H. M. & Jansen, G. Determinants of activity of the antifolate thymidylate synthase inhibitors Tomudex (ZD1694) and GW1843U89 against mono- and multilayered colon cancer cell lines under folate-restricted conditions. Cancer Res. 59, 5529–5535 (1999).
Klabatsa, A., Sheaff, M. T., Steele, J. P. C., Evans, M. T., Rudd, R. M. & Fennell, D. A. Expression and prognostic significance of hypoxia-inducible factor 1α (HIF-1α) in malignant pleural mesothelioma (MPM). Lung Cancer 51, 53–59 (2006).
Maftouh, M., Avan, A., Sciarrillo, R., Granchi, C., Leon, L. G., Rani, R. et al. Synergistic interaction of novel lactate dehydrogenase inhibitors with gemcitabine against pancreatic cancer cells in hypoxia. Br. J. Cancer 110, 172–182 (2014).
Giovannetti, E., Leon, L. G., Gómez, V. E., Zucali, P. A., Minutolo, F. & Peters, G. J. A specific inhibitor of lactate dehydrogenase overcame the resistance toward gemcitabine in hypoxic mesothelioma cells, and modulated the expression of the human equilibrative transporter-1. Nucleosides Nucleotides Nucleic Acids 35, 643–651 (2016).
Simon, G. R., Verschraegen, C. F., Jänne, P. A., Langer, C. J., Dowlati, A., Gadgeel, S. M. et al. Pemetrexed plus gemcitabine as first-line chemotherapy for patients with peritoneal mesothelioma: final report of a phase II trial. J. Clin. Oncol. 26, 3567–3572 (2008).
Burt, B. M., Richards, W. G., Lee, H.-S., Bartel, S., Dasilva, M. C., Gill, R. R. et al. A phase I trial of surgical resection and intraoperative hyperthermic cisplatin and gemcitabine for pleural mesothelioma. J. Thorac. Oncol. 13, 1400–1409 (2018).
Capkova, L., Koubkova, L. & Kodet, R. Expression of carbonic anhydrase IX (CAIX) in malignant mesothelioma. An immunohistochemical and immunocytochemical study. Neoplasma 61, 161–169 (2014).
Nabavi, N., Bennewith, K. L., Churg, A., Wang, Y., Collins, C. C. & Mutti, L. Switching off malignant mesothelioma: exploiting the hypoxic microenvironment. Genes Cancer 7, 340–354 (2016).
Avan, A., Caretti, V., Funel, N., Galvani, E., Maftouh, M., Honeywell, R. J. et al. Crizotinib inhibits metabolic inactivation of gemcitabine in c-Met-driven pancreatic carcinoma. Cancer Res. 73, 6745–6756 (2013).
Sciarrillo, R., Wojtuszkiewicz, A., El Hassouni, B., Funel, N., Gandellini, P., Lagerweij, T. et al. Splicing modulation as novel therapeutic strategy against diffuse malignant peritoneal mesothelioma. EBioMedicine 39, 215–225 (2019).
Koukourakis, M. I., Giatromanolaki, A., Sivridis, E., Gatter, K. C. & Harris, A. L., Tumour Angiogenesis Research Group. Lactate dehydrogenase 5 expression in operable colorectal cancer: strong association with survival and activated vascular endothelial growth factor pathway-a report of the Tumour Angiogenesis Research Group. J. Clin. Oncol. 24, 4301–4308 (2006).
Giovannetti, E., Zucali, P. A., Assaraf, Y. G., Leon, L. G., Smid, K., Alecci, C. et al. Preclinical emergence of vandetanib as a potent antitumour agent in mesothelioma: molecular mechanisms underlying its synergistic interaction with pemetrexed and carboplatin. Br. J. Cancer 105, 1542–1553 (2011).
Calvaresi, E. C., Granchi, C., Tuccinardi, T., Di Bussolo, V., Huigens, R. W., Lee, H. Y. et al. Dual targeting of the Warburg effect with a glucose-conjugated lactate dehydrogenase inhibitor. Chembiochem 14, 2263–2267 (2013).
Barbone, D., Ryan, J. A., Kolhatkar, N., Chacko, A. D., Jablons, D. M., Sugarbaker, D. J. et al. The Bcl-2 repertoire of mesothelioma spheroids underlies acquired apoptotic multicellular resistance. Cell Death Dis. 2, e174 (2011).
Massihnia, D., Avan, A., Funel, N., Maftouh, M., van Krieken, A., Granchi, C. et al. Phospho-Akt overexpression is prognostic and can be used to tailor the synergistic interaction of Akt inhibitors with gemcitabine in pancreatic cancer. J. Hematol. Oncol. 10, 9 (2017).
van der Wilt, C. L., Backus, H. H., Smid, K., Comijn, L., Veerman, G., Wouters, D. et al. Modulation of both endogenous folates and thymidine enhance the therapeutic efficacy of thymidylate synthase inhibitors. Cancer Res. 61, 3675–3681 (2001).
Cavazzoni, A., La Monica, S., Alfieri, R., Ravelli, A., Van Der Steen, N., Sciarrillo, R. et al. Enhanced efficacy of AKT and FAK kinase combined inhibition in squamous cell lung carcinomas with stable reduction in PTEN. Oncotarget 8, 53068–53083 (2017).
Grasso, C., Jansen, G. & Giovannetti, E. Drug resistance in pancreatic cancer: impact of altered energy metabolism. Crit. Rev. Oncol. Hematol. 114, 139–152 (2017).
Matherly, L. H., Wilson, M. R. & Hou, Z. The major facilitative folate transporters solute carrier 19A1 and solute carrier 46A1: biology and role in antifolate chemotherapy of cancer. Drug Metab. Dispos. 42, 632–649 (2014).
Leon, L. G., Gemelli, M., Sciarrillo, R., Avan, A., Funel, N. & Giovannetti, E. Synergistic activity of the c-Met and tubulin inhibitor tivantinib (ARQ197) with pemetrexed in mesothelioma cells. Curr. Drug Targets 15, 1331–1340 (2014).
Yang, H., Bocchetta, M., Kroczynska, B., Elmishad, A. G., Chen, Y., Liu, Z. et al. TNF-alpha inhibits asbestos-induced cytotoxicity via a NF-kappaB-dependent pathway, a possible mechanism for asbestos-induced oncogenesis. Proc. Natl Acad. Sci. USA 103, 10397–10402 (2006).
Goparaju, C. M., Blasberg, J. D., Volinia, S., Palatini, J., Ivanov, S., Donington, J. S. et al. Onconase mediated NFKβ down-regulation in malignant pleural mesothelioma. Oncogene 30, 2767–2777 (2011).
Servais, E. L., Colovos, C., Rodriguez, L., Bograd, A. J., Nitadori, J., Sima, C. et al. Mesothelin overexpression promotes mesothelioma cell invasion and MMP-9 secretion in an orthotopic mouse model and in epithelioid pleural mesothelioma patients. Clin. Cancer Res. 18, 2478–2489 (2012).
Gonen, N. & Assaraf, Y. G. The obligatory intestinal folate transporter PCFT (SLC46A1) is regulated by nuclear respiratory factor 1. J. Biol. Chem. 285, 33602–33613 (2010).
Firuzi, O., Che, P. P., El Hassouni, B., Buijs, M., Coppola, S., Löhr, M. et al. Role of c-MET inhibitors in overcoming drug resistance in spheroid models of primary human pancreatic cancer and stellate cells. Cancers 11, 638 (2019).
El Hassouni, B., Sciarrillo, R., Gómez, V., Maftouh, M., Mantini, G., Vonk, C. et al. Targeting hypoxic pancreatic cancer cells with glucose conjugated lactate dehydrogenase inhibitor NHI-Glc-2. Abstract 3082, Cancer Res. 3082–3082 (2019).
Koch, A., Ebert, E. V., Seitz, T., Dietrich, P., Berneburg, M., Bosserhoff, A. et al. Characterization of glycolysis-related gene expression in malignant melanoma. Pathol. Res. Pr. 216, 152752 (2020).
Zhao, R., Najmi, M., Aluri, S., Spray, D. C. & Goldman, I. D. Concentrative transport of antifolates mediated by the proton-coupled folate transporter (SLC46A1); augmentation by a HEPES buffer. Mol. Pharmacol. 93, 208–215 (2018).
Zhao, R., Qiu, A., Tsai, E., Jansen, M., Akabas, M. H. & Goldman, I. D. The proton-coupled folate transporter: impact on pemetrexed transport and on antifolates activities compared with the reduced folate carrier. Mol. Pharmacol. 74, 854–862 (2008).
Wang, D., Zhang, J., Lu, Y., Luo, Q. & Zhu, L. Nuclear respiratory factor-1 (NRF-1) regulated hypoxia-inducible factor-1α (HIF-1α) under hypoxia in HEK293T. IUBMB Life 68, 748–755 (2016).
El Hassouni, B., Granchi, C., Vallés-Martí, A., Supadmanaba, I. G. P., Bononi, G., Tuccinardi, T. et al. The dichotomous role of the glycolytic metabolism pathway in cancer metastasis: interplay with the complex tumor microenvironment and novel therapeutic strategies. Semin Cancer Biol. https://doi.org/10.1016/j.semcancer.2019.08.025 (2019).
Gonlugur, T. E. & Gonlugur, U. Pleural fluid findings as prognostic factors for malignant pleural mesothelioma. J. Clin. Lab Anal. 22, 334–336 (2008).
Singhal, S., Wiewrodt, R., Malden, L. D., Amin, K. M., Matzie, K., Friedberg, J. et al. Gene expression profiling of malignant mesothelioma. Clin. Cancer Res. 9, 3080–3097 (2003).
Zhao, D., Zou, S.-W., Liu, Y., Zhou, X., Mo, Y., Wang, P. et al. Lysine-5 acetylation negatively regulates lactate dehydrogenase A and is decreased in pancreatic cancer. Cancer Cell 23, 464–476 (2013).
Sciarrillo, R., Minutolo, F., Peters, G. J. & Giovannetti, E. Lactate dehydrogenase acetylation adds another piece to the puzzle of metabolic reprogramming in pancreatic cancer. Transl. Gastrointest. Cancer 3, https://doi.org/10.3978/j.issn.2224-4778.2014.04.02 (2014).
Gorlach, A., Bolling, B., Holtermann, G., Schwachofer, J., Carlsson, J. & Acker, H. Changes in growth, po(2) and ph after exposure to oxamate - studies of 2 human tumor-cell lines growing as multicellular spheroids. Int J. Oncol. 7, 831–839 (1995).
Oehl, K., Kresoja-Rakic, J., Opitz, I., Vrugt, B., Weder, W., Stahel, R. et al. Live-cell mesothelioma biobank to explore mechanisms of tumor progression. Front. Oncol. 8, https://doi.org/10.3389/fonc.2018.00040 (2018).
Wu, L., Allo, G., John, T., Li, M., Tagawa, T., Opitz, I. et al. Patient-derived xenograft establishment from human malignant pleural mesothelioma. Clin. Cancer Res. 23, 1060–1067 (2017).
Smitskamp-Wilms, E., Pinedo, H. M., Veerman, G., Ruiz van Haperen, V. W. & Peters, G. J. Postconfluent multilayered cell line cultures for selective screening of gemcitabine. Eur. J. Cancer 34, 921–926 (1998).
Levin, M., Stark, M., Berman, B. & Assaraf, Y. G. Surmounting Cytarabine-resistance in acute myeloblastic leukemia cells and specimens with a synergistic combination of hydroxyurea and azidothymidine. Cell Death Dis. 10, 1–14 (2019).
Degwert., N., Latuske, E., Vohwinkel., G., Stamm, H., Klokow, M., Bokemeyer, C. et al. Deoxycytidine kinase is downregulated under hypoxic conditions and confers resistance against cytarabine in acute myeloid leukaemia. Eur. J. Haematol. 97, 239–244 (2016).
Wilson, W. R. & Hay, M. P. Targeting hypoxia in cancer therapy. Nat. Rev. Cancer 11, 393–410 (2011).
Zhuo, Y., Lin, L., Wei, S. & Zhang, M. Pretreatment elevated serum lactate dehydrogenase as a significant prognostic factor in malignant mesothelioma: a meta-analysis. Medicine 95, e5706 (2016).
Pergolini, I., Morales-Oyarvide, V., Mino-Kenudson, M., Honselmann, K. C., Rosenbaum, M. W., Nahar, S. et al. Tumor engraftment in patient-derived xenografts of pancreatic ductal adenocarcinoma is associated with adverse clinicopathological features and poor survival. PLoS ONE 12, e0182855 (2017).
Bronte, G., Incorvaia, L., Rizzo, S., Passiglia, F., Galvano, A., Rizzo, F. et al. The resistance related to targeted therapy in malignant pleural mesothelioma: why has not the target been hit yet? Crit. Rev. Oncol. Hematol. 107, 20–32 (2016).
Zucali, P. A. Target therapy: new drugs or new combinations of drugs in malignant pleural mesothelioma. J. Thorac. Dis. 10, S311–S321 (2018).
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.
Ethics approval and consent to participate
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.
Consent to publish
Consent to publish has been obtained from all the participants (or legal parent or guardian for children) to report individual patient data.
All data generated or analysed during this study are included in this published article (and in supplementary information files).
The authors declare no competing interests.
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).
Note This work is published under the standard license to publish agreement. After 12 months the work will become freely available and the license terms will switch to a Creative Commons Attribution 4.0 International (CC BY 4.0).
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
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