Dichloroacetic acid and rapamycin synergistically inhibit tumor progression Enhanced Publication

Dichloroacetic acid and rapamycin synergistically inhibit tumor progression 二氯乙酸联合雷帕霉素协同抑制肿瘤进程 1 3 equal first-author https://orcid.org/0000-0001-7451-0386 2 equal 1 1 2 3 corresp Hai-long PIAO, hpiao@dicp.ac.cn Hai-long PIAO, hpiao@dicp.ac.cn hpiao@dicp.ac.cn https://orcid.org/0000-0001-7451-0386 中国科学院分析化学分离科学重点实验室， 中国科学院大连化学物理研究所， 中国大连市， 116023 中国科学院分析化学分离科学重点实验室， 中国科学院大连化学物理研究所， 中国大连市， 116023 CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China 中国医科大学生命学院， 生命化学与分子生物学系， 中国沈阳市， 110122 中国医科大学生命学院， 生命化学与分子生物学系， 中国沈阳市， 110122 Department of Biochemistry & Molecular Biology, School of Life Sciences, China Medical University, Shenyang 110122, China Department of Biochemistry & Molecular Biology, School of Life Sciences, China Medical University, Shenyang 110122, China 中国科学院大学， 中国北京市， 100049 中国科学院大学， 中国北京市， 100049 University of Chinese Academy of Sciences, Beijing 100049, China University of Chinese Academy of Sciences, Beijing 100049, China Mammalian target of rapamycin (mTOR) controls cellular anabolism, and mTOR signaling is hyperactive in most cancer cells. As a result, inhibition of mTOR signaling benefits cancer patients. Rapamycin is a US Food and Drug Administration (FDA)-approved drug, a specific mTOR complex 1 (mTORC1) inhibitor, for the treatment of several different types of cancer. However, rapamycin is reported to inhibit cancer growth rather than induce apoptosis. Pyruvate dehydrogenase complex (PDHc) is the gatekeeper for mitochondrial pyruvate oxidation. PDHc inactivation has been observed in a number of cancer cells, and this alteration protects cancer cells from senescence and nicotinamide adenine dinucleotide (NAD +‍ ) exhaustion. In this paper, we describe our finding that rapamycin treatment promotes pyruvate dehydrogenase E1 subunit alpha 1 (PDHA1) phosphorylation and leads to PDHc inactivation dependent on mTOR signaling inhibition in cells. This inactivation reduces the sensitivity of cancer cells’ response to rapamycin. As a result, rebooting PDHc activity with dichloroacetic acid (DCA), a pyruvate dehydrogenase kinase (PDK) inhibitor, promotes cancer cells’ susceptibility to rapamycin treatment in vitro and in vivo. 哺乳动物雷帕霉素靶蛋白（mTOR）控制细胞的合成代谢，并且在大多数的肿瘤细胞中mTOR信号通路高度活化，因此抑制mTOR信号通路对癌症患者有益。雷帕霉素是一种美国食品药品监督管理局（FDA）批准的临床一线药物，是mTORC1的特异性抑制剂，用于治疗多种不同类型的癌症。然而，研究发现雷帕霉素仅抑制肿瘤细胞的增殖并不引起细胞的凋亡。丙酮酸脱氢酶复合体（PDHc）在线粒体丙酮酸氧化过程起着决定作用。许多肿瘤细胞中的PDHc处于失活状态，这一变化可以使肿瘤细胞免于衰老和NAD + 耗竭。在本研究中，发现雷帕霉素处理细胞导致依赖mTOR信号通路抑制的丙酸脱氢酶α1（PDHA1）磷酸化水平升高，并导致PDHc酶活降低。PDHc失活直接引起肿瘤细胞对雷帕霉素敏感度下降。因此，在体内和体外的实验中通过使用丙酮酸脱氢酶激酶（PDK）的抑制剂二氯乙酸（DCA）可以重新激活PDHc的活力，进而增加肿瘤细胞对雷帕霉素的敏感性。 二氯乙酸（DCA） 雷帕霉素 丙酸脱氢酶α1（PDHA1） 哺乳动物雷帕霉素靶蛋白（mTOR） Dichloroacetic acid (DCA) Rapamycin Pyruvate dehydrogenase E1 subunit alpha 1 (PDHA1) Mammalian target of rapamycin (mTOR) 1 Introduction 1 Mammalian target of rapamycin (mTOR) controls cell anabolic metabolism, including protein translation and DNA and RNA synthesis ( Beretta et al., 1996 Beretta et al., 1996 ; Robitaille et al., 2013 Robitaille et al., 2013 ; Valvezan et al., 2017 Valvezan et al., 2017 ). This signaling is controlled by two individual TOR complexes: mTORC1 and mTORC2 ( Kim and Guan, 2019 Kim and Guan, 2019 ). In most cancer cells, mTOR signaling is hyperactive as a result of demand for building blocks ( Menon and Manning, 2008 Menon and Manning, 2008 ). mTORC2 promotes metastasis by controlling the actin cytoskeleton ( Jacinto et al., 2004 Jacinto et al., 2004 ; Zhang et al., 2019 Zhang et al., 2019 ). Meanwhile, TOR1 complex is inhibited by rapamycin-‍12-kDa FK506-binding protein (FKBP12) ( Sabatini et al., 1994 Sabatini et al., 1994 ), which prevents substrates from entering the kinase active site ( Yang et al., 2017 Yang et al., 2017 ). Although mTORC2 is insensitive to rapamycin, prolonged treatment with rapamycin can inhibit mTORC2 signaling by dissociating mTORC2 assembly ( Sarbassov et al., 2006 Sarbassov et al., 2006 ; Lamming et al., 2012 Lamming et al., 2012 ). Moreover, rapamycin severely reduces tumor angiogenesis by inhibiting mTOR signaling ( Phung et al., 2006 Phung et al., 2006 ). To date, mTOR inhibition has shown little effect in restricting the growth of cancer cells compared to chemotherapeutics. Inhibition of mTOR induces autophagy, which protects cancer cells from the stress of poor nutrition ( Levy et al., 2017 Levy et al., 2017 ; Bai et al., 2022 Bai et al., 2022 ). In addition, mTOR signaling inhibition limits the cancer-cell growth rate rather than induces apoptosis ( Kim and Guan, 2019 Kim and Guan, 2019 ). As a result, combining mTOR inhibition with other therapies produces significantly better therapeutic effects ( Lorusso, 2016 Lorusso, 2016 ; Zhuang et al., 2016 Zhuang et al., 2016 ). Pyruvate dehydrogenase complex (PDHc) catalyzes pyruvate converting to acetyl-coenzyme A (AcCoA), which is further synthesized to citrate ( Patel et al., 2014 Patel et al., 2014 ). PDHc enzymatic activity is tightly controlled by reversible phosphorylation of pyruvate dehydrogenase E1 subunit alpha 1 (PDHA1), which is mediated by pyruvate dehydrogenase kinase (PDK) and pyruvate dehydrogenase phosphatase 1/2 (PDP1/2) ( Holness and Sugden, 2003 Holness and Sugden, 2003 ). In several cancer cells, PDHc activity is downregulated, and this downregulation promotes nicotinamide adenine dinucleotide (NAD + ) regeneration ( Luengo et al., 2021 Luengo et al., 2021 ) and resistance to senescence ( Kaplon et al., 2013 Kaplon et al., 2013 ). Recent studies have demonstrated that mitochondrial Ras homolog enriched in brain (Rheb) regulates PDHc activity via binding to PDP1/2, independent of mTOR signaling ( Yang et al., 2021 Yang et al., 2021 ). However, we observed that not only rapamycin treatment but also amino acid starvation strongly induces PDHA1 phosphorylation by inhibiting mTOR signaling. Therefore, this study aims to investigate the interplay between mTOR signaling and PDHA1 phosphorylation in cancer cells, and the potential therapeutic implications of dichloroacetic acid (DCA) and rapamycin. 2 Results 1 2.1 PDHA1 phosphorylation induced by rapamycin treatment 2 A previous study demonstrated that Rheb regulates PDHc activity via PDHA1 phosphorylation, independent of mTOR signaling ( Yang et al., 2021 Yang et al., 2021 ). However, rapamycin (a well-known mTORC1 inhibitor) and its clinical derivative compound everolimus significantly promote the phosphorylation of PDHA1 in MDA-MB-231 cells ( Fig. 1 Fig. 1 a). In line with this, we found that rapamycin treatment significantly increased phosphorylation of PDHA1: P-s232 and P-s300 in 4T1 cells; P-s300 in CT26 cells; P-s232 in B16F10 cells; and P-s232 and P-s300 in Hepa1-6 cells. However, it almost completely inhibited phosphorylation of ribosomal protein S6 in several different cell lines ( Figs. 1 Figs. 1 b‍–‍1e). Moreover, even a much lower concentration of rapamycin (2.5 nmol/L) completely inhibited S6 phosphorylation and activated PDHA1 phosphorylation ( Fig. 1 Fig. 1 f). These results indicated that rapamycin treatment induced PDHA1 phosphorylation, and this was a widespread phenomenon in different cell lines. 2.2 mTORC1 involved in PDHA1 regulation 2 Since rapamycin is a specific mTORC1 inhibitor, we speculated that it induces PDHA1 phosphorylation dependent on mTOR signaling. Next, to demonstrate this hypothesis, we starved cells with different amino acids for 6 h to activate mTOR signaling. We observed that arginine and glutamine starvation significantly inhibited S6 phosphorylation; however, leucine starvation did not affect S6 phosphorylation within 6 h ( Fig. 2 Fig. 2 a), so it may require prolonged starvation. Notably, phosphorylation of s293-PDHA1 was upregulated by arginine and glutamine starvation ( Fig. 2 Fig. 2 a), indicating that inhibition of mTOR signaling induces PDHA1 phosphorylation. In addition, S6 phosphorylation was almost completely inhibited by rapamycin within 1 h ( Fig. 2 Fig. 2 b), and three phosphorylation sites of PDHA1 increased in a time-dependent manner. Especially, the s293-PDHA1 responded to rapamycin treatment at an early time point‍—‍30 min ( Fig. 2 Fig. 2 b). This phenomenon was also confirmed with time-dependent glutamine starvation and re-addition (Figs. 2 2 c and 2 2 d). Finally, we found that fetal bovine serum (FBS) and glutamine stimulated S6 phosphorylation and inhibited PDHA1 phosphorylation ( Fig. 2 Fig. 2 e); however this effect was inversed by rapamycin treatment ( Fig. 2 Fig. 2 e). Taken together, rapamycin-inhibited mTOR signaling increases PDHA1 phosphorylation. 2.3 Role of DCA in rapamycin-induced PDHA1 phosphorylation 2 PDKs and PDP1/2 reversibly regulate the phosphorylation of PDHA1 ( Holness and Sugden, 2003 Holness and Sugden, 2003 ). Thus, we wondered whether rapamycin-promoted PDHA1 phosphorylation could be eliminated by PDK inhibition. We therefore treated MDA-MB-231 cells with DCA, a PDK inhibitor ( Tso et al., 2014 Tso et al., 2014 ). We observed that three phosphorylation sites of PDHA1 were greatly decreased in control and rapamycin-treated cells ( Fig. 3 Fig. 3 a). In addition, we observed that DCA treatment did not affect S6 phosphorylation ( Fig. 3 Fig. 3 a), indicating that although mTOR signaling controls PDHc enzymatic activity, the converse is not valid. Similar results were also obtained for B16F10 cells ( Fig. 3 Fig. 3 b). These findings demonstrate that PDK inhibition could overcome rapamycin-induced PDHA1 phosphorylation. 2.4 Synergistic effect of co-treatment with DCA and rapamycin on cancer cell growth in vitro 2 Since mTOR signaling is hyperactive in most human cancers ( Menon and Manning, 2008 Menon and Manning, 2008 ), mTOR signaling inhibition has potential therapeutic benefits in different types of cancer ( Guba et al., 2002 Guba et al., 2002 ; Ghobrial et al., 2016 Ghobrial et al., 2016 ; Rodrik-Outmezguine et al., 2016 Rodrik-Outmezguine et al., 2016 ). DCA significantly decreased the growth of MDA-MB-231 cells under rapamycin or everolimus treatment ( Fig. 4 Fig. 4 a). In addition, we observed that cell growth was significantly inhibited by 1 nmol/L of rapamycin, but this inhibition did not increase with higher concentrations of rapamycin in several cancer cell lines (Figs. 4 4 b and 4 4 c). Importantly, DCA treatment enhanced rapamycin-induced cell-growth inhibition (Figs. 4 4 b and 4 4 c), indicating that the activation of PDHc activity may weaken cancer cells which are resistant to mTOR inhibition. We found that rapamycin treatment reduced the cell colonization of MDA-MB-231 cells ( Fig. 4 Fig. 4 d), although 2 mmol/L DCA treatment had no effect on cell growth in MDA-MB-231 cells. However, DCA combined with rapamycin had higher growth-inhibiting efficacy than rapamycin alone ( Fig. 4 Fig. 4 d). In addition, we observed that rapamycin significantly inhibited cell colonization but did not affect colony numbers in 4T1 cells ( Fig. 4 Fig. 4 e). However, DCA treatment significantly reduced colony numbers in rapamycin-treated 4T1 cells ( Fig. 4 Fig. 4 e). Next, we found that DCA only, or DCA combined with rapamycin, activated the apoptosis protein marker cleaved poly(ADP-ribose) polymerase (PARP); but this phenomenon was not evident for treatment with rapamycin alone ( Fig. 4 Fig. 4 f), indicating that PDHc activation may induce cancer cell apoptosis. It is evident from these results that DCA sensitizes cancer cells to rapamycin-induced growth inhibition. 2.5 Effects of the combined treatment of DCA and rapamycin on cancer cells 2 To further confirm the synergistic efficacy of DCA and rapamycin in vivo, we performed a drug-combination study in vivo ( Fig. 5 Fig. 5 a). We found that rapamycin treatment inhibited tumor growth, and DCA treatment only showed slight effects (Figs. 5 5 b and 5 5 c). However, DCA combined with rapamycin significantly suppressed tumor volume and weight compared to rapamycin treatment alone ( Figs. 5 Figs. 5 b‍–‍5d), indicating that combined treatment with DCA and rapamycin synergistically inhibited tumor growth in vivo. Previous research demonstrated that patients treated with rapamycin had a decreased spleen size due to its immunosuppressive role ( Araújo et al., 2014 Araújo et al., 2014 ). Consistently, we observed that the spleens of vehicle-treated mice were greatly enlarged compared to those treated with rapamycin (Figs. 5 5 e and 5 5 f). In addition, mice that underwent combined DCA and rapamycin treatment exhibited the smallest spleens compared to other groups (Figs. 5 5 e and 5 5 f), arguing that DCA may inhibit cancer metastasis in vivo ( Jacobs et al., 2012 Jacobs et al., 2012 ). These results demonstrate that DCA cooperates with rapamycin to inhibit tumor progression in vivo. 3 Discussion and conclusions 1 mTOR signaling is hyperactive for the anabolic metabolism of rapidly proliferating cancer cells. Thus, mTOR inhibition is a potent therapeutic strategy for cancer patients. Many mTOR inhibitors have been used in clinical trials, including rapamycin and rapamycin derivatives known as rapalogs ( Benjamin et al., 2011 Benjamin et al., 2011 ). However, rapamycin and rapalogs have proved to reduce tumor growth but not induce cell apoptosis, perhaps because the mTORC2-protein kinase B (AKT) complex, a pro-survival regulator, is insensitive to these inhibitors ( Hsieh et al., 2012 Hsieh et al., 2012 ). The second-generation mTOR inhibitors (Torin1, WAY-600, and PP242) are designed to compete for adenosine-triphosphate (ATP) near the catalytic site of the kinase domain. These inhibitors bind directly to the kinase domain of both mTORC1 and mTORC2, resulting in considerable therapeutic effects ( Ghobrial et al., 2016 Ghobrial et al., 2016 ; Fan et al., 2017 Fan et al., 2017 ). Unfortunately, long-term treatment with these inhibitors can reboot the AKT pathway ( Rodrik-Outmezguine et al., 2011 Rodrik-Outmezguine et al., 2011 ), which is the primary limitation of the second-generation inhibitors ( Ghobrial et al., 2016 Ghobrial et al., 2016 ; Rodrik-Outmezguine et al., 2016 Rodrik-Outmezguine et al., 2016 ). In this study, we discovered that mTOR inhibition by rapamycin induces PDHA1 phosphorylation, which is inconsistent with the recently reported result in liver-cancer cells ( Yang et al., 2021 Yang et al., 2021 ). In addition, we observed that amino acid starvation induced PDHA1 phosphorylation followed by mTOR inhibition. These findings strongly indicate that mTOR inhibition reduces PDHc activity. In addition, rapamycin-activated PDHA1 phosphorylation is suppressed by DCA. A previous report demonstrated that PDK4 inactivation switches cells from pro-survival to pro-apoptosis mode under inflammatory stress ( Wu et al., 2018 Wu et al., 2018 ). We also observed that DCA sensitized tumor cells to rapamycin treatment, so we argue that reduced PDHc activity can reduce cell proliferation from mTOR-signaling inhibition-induced growth arrest. PDHc activity reduction promotes NAD + regeneration ( Luengo et al., 2021 Luengo et al., 2021 ) and increases cells’ resistance to senescence ( Kaplon et al., 2013 Kaplon et al., 2013 ). However, we observed that DCA treatment had limited efficacy in inhibiting growth of cancer cells in vitro and in vivo, consistent with previous studies ( Wu et al., 2018 Wu et al., 2018 ; Tataranni and Piccoli, 2019 Tataranni and Piccoli, 2019 ). DCA combined with rapamycin treatment exhibited much smaller tumor lumps compared to DCA or rapamycin alone in a homograft mouse model, confirming that DCA may have better therapeutic benefits in cooperation with other anti-cancer drugs ( Cao et al., 2008 Cao et al., 2008 ; Lucido et al., 2018 Lucido et al., 2018 ; Verma et al., 2019 Verma et al., 2019 ). Hypoxia inducible factor-1α (HIF-1α), a key regulator for metastasis, is downregulated when mTOR signaling is inhibited ( Bernardi et al., 2006 Bernardi et al., 2006 ). We consistently observed that spleen volume was reduced in the mice treated by the combination of DCA and rapamycin. Since DCA also inhibits HIF‍-‍1α expression ( Sutendra et al., 2013 Sutendra et al., 2013 ; Škorja Milić et al., 2021 Škorja Milić et al., 2021 ), we speculate that DCA and rapamycin may synergistically inhibit cancer metastasis by suppressing the HIF-1α signaling pathway. 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Oncotarget , 7 ( 51 ): 84688 - 84694 . https://doi.org/10.18632/oncotarget.12423 https://doi.org/10.18632/oncotarget.12423 MTOR inhibition reversed drug resistance after combination radiation with erlotinib in lung adenocarcinoma Huan CHEN and Hai-long PIAO conceived the project, designed and performed most of the experiments and the data analysis, and wrote the manuscript with input from all other authors. Hai-long PIAO supervised the project. Kunimg LIANG performed the most of experiments. Huan CHEN, Kunimg LIANG, and Cong HOU provided significant intellectual input. All authors have read and approved the final manuscript, and therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data. This work was supported by the National Key Research and Development Program of China (No. 2022YFA0806503), the National Natural Science Foundation of China (No. 81972625), the Dalian Science and Technology Innovation Funding (No. 2019J12SN52), and the Liaoning Revitalization Talents Program (No. XLYC2002035), China. 10.1631/jzus.B2200356 1673-1581（2023）05-0397-09 陈欢,梁昆明,侯聪等.二氯乙酸联合雷帕霉素协同抑制肿瘤进程[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2023,24(05):397-405. 陈欢,梁昆明,侯聪等.二氯乙酸联合雷帕霉素协同抑制肿瘤进程[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2023,24(05):397-405. This work was supported by the National Key Research and Development Program of China (No. 2022YFA0806503), the National Natural Science Foundation of China (No. 81972625), the Dalian Science and Technology Innovation Funding (No. 2019J12SN52), and the Liaoning Revitalization Talents Program (No. XLYC2002035), China. 2022-07-03 2022-12-07 2023-05-15 2023-05-15 2023-03-31 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2022 2023 浙江大学出版社 37538257 Huan CHEN, Kunming LIANG, Cong HOU, and Hai-long PIAO declare that they have no conflict of interest. All work performed with animals was approved by the Medical Animal Care and Use Committee of China Medical University, Shenyang, China (No. KT2020061). Compliance with ethics guidelines Supplementary information 1 Materials and methods 浙江大学学报（英文版）（B辑：生物医学和生物技术） 5 24