Targeting EMT in Cancer with Repurposed Metabolic Inhibitors
Authors: Vignesh Ramesh, Thomas Brabletz, Paolo Ceppi
Journal: Trends in cancer (2020)

Epithelial-to-mesenchymal transition (EMT), an embryonic phenotypic plasticity program, in cancer confers invasiveness, dissemination, and chemo/immunotherapy resistance.EMT drives through dynamic intermediate states (partial EMT), which possess high metastasis-initiating potential. Genomic and functional analyses have determined the existence of EMT/partial EMT features in multiple human cancer types.EMT is associated with significant metabolic rewiring, and some metabolic pathways promote and maintain EMT.Repurposing of antimetabolism drugs in EMT-driven cancers could reduce the incidence of metastasis and improve patients' response to treatments.A better understanding of the interdependence between EMT and metabolism could accelerate the discovery of novel treatments for aggressive cancers. Epithelial-to-mesenchymal transition (EMT) determines the most lethal features of cancer, metastasis formation and chemoresistance, and therefore represents an attractive target in oncology. However, direct targeting of EMT effector molecules is, in most cases, pharmacologically challenging. Since emerging research has highlighted the distinct metabolic circuits involved in EMT, we propose the use of metabolism-specific inhibitors, FDA approved or under clinical trials, as a drug repurposing approach to target EMT in cancer. Metabolism-inhibiting drugs could be coupled with standard chemo- or immunotherapy to combat EMT-driven resistant and aggressive cancers. Epithelial-to-mesenchymal transition (EMT) determines the most lethal features of cancer, metastasis formation and chemoresistance, and therefore represents an attractive target in oncology. However, direct targeting of EMT effector molecules is, in most cases, pharmacologically challenging. Since emerging research has highlighted the distinct metabolic circuits involved in EMT, we propose the use of metabolism-specific inhibitors, FDA approved or under clinical trials, as a drug repurposing approach to target EMT in cancer. Metabolism-inhibiting drugs could be coupled with standard chemo- or immunotherapy to combat EMT-driven resistant and aggressive cancers. EMT, a classical developmental phenotypic plasticity program, is governed by the switching in the epithelial phenotype of a cell to mesenchymal form conferring various biological processes such as in gastrulation, neural crest migration, and wound healing [1.Kalluri R. Weinberg R.A. The basics of epithelial–mesenchymal transition.J. Clin. Invest. 2009; 119: 1420-1428Crossref PubMed Scopus (6986) Google Scholar]. Cancer being a highly heterogeneous disease known for its accumulations of various abnormalities in the cell, often recapitulates and manipulates the developmental EMT program in a partial and transient fashion to acquire advantageous features for its survival and propagation [2.Hanahan D. Weinberg R.A. Hallmarks of cancer: the next generation.Cell. 2011; 144: 646-674Abstract Full Text Full Text PDF PubMed Scopus (39909) Google Scholar]. The EMT phenomenon is mediated through multiple transcription factors, aiding enhanced invasive ability, dissemination to distant sites, metastatic colonization, cancer stemness, and chemoresistance [3.Williams E.D. et al.Controversies around epithelial–mesenchymal plasticity in cancer metastasis.Nat. Rev. Cancer. 2019; 19: 716-732Crossref PubMed Scopus (184) Google Scholar,4.Brabletz T. et al.EMT in cancer.Nat. Rev. Cancer. 2018; 18: 128-134Crossref PubMed Scopus (973) Google Scholar]. In some tissues, the EMT process has also been observed in the fundamental early steps of tumorigenesis [5.Sato M. et al.Human lung epithelial cells progressed to malignancy through specific oncogenic manipulations.Mol. Cancer Res. 2013; 11: 638-650Crossref PubMed Scopus (138) Google Scholar,6.Vaz M. et al.Chronic cigarette smoke-induced epigenomic changes precede sensitization of bronchial epithelial cells to single-step transformation by KRAS mutations.Cancer Cell. 2017; 32 (e6): 360-376Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar]. Recent evidence highlighted that cancer cells that have undergone full EMT possess less metastatic potential, while cells in the partial or hybrid state inherently contain a high degree of plasticity and metastasis-initiating ability [7.Beerling E. et al.Plasticity between epithelial and mesenchymal states unlinks EMT from metastasis-enhancing stem cell capacity.Cell Rep. 2016; 14: 2281-2288Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 8.Pastushenko I. et al.Identification of the tumour transition states occurring during EMT.Nature. 2018; 556: 463-468Crossref PubMed Scopus (643) Google Scholar, 9.Kröger C. et al.Acquisition of a hybrid E/M state is essential for tumorigenicity of basal breast cancer cells.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 7353-7362Crossref PubMed Scopus (192) Google Scholar]. Dissemination of tumor cells as clusters by collective migration has also been proposed to be regulated through EMT-dependent mechanisms [10.Lambert A.W. et al.Emerging biological principles of metastasis.Cell. 2017; 168: 670-691Abstract Full Text Full Text PDF PubMed Scopus (1400) Google Scholar]. Several studies took a logical approach to quantitatively discern the EMT states in clinical cancer samples by using gene expression-based signature metrics, and identified a correlation with poor patient survival [11.Taube J.H. et al.Core epithelial-to-mesenchymal transition interactome gene-expression signature is associated with claudin-low and metaplastic breast cancer subtypes.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 15449-15454Crossref PubMed Scopus (755) Google Scholar, 12.Tan T.Z. et al.Epithelial–mesenchymal transition spectrum quantification and its efficacy in deciphering survival and drug responses of cancer patients.EMBO Mol. Med. 2014; 6: 1279-1293Crossref PubMed Scopus (388) Google Scholar, 13.George J.T. et al.Survival outcomes in cancer patients predicted by a partial EMT gene expression scoring metric.Cancer Res. 2017; 77: 6415-6428Crossref PubMed Scopus (122) Google Scholar]. Importantly, a pan-cancer EMT signature derived from patients across multiple cancer types revealed a strong association with various immune checkpoint targets [14.Mak M.P. et al.A patient-derived, pan-cancer EMT signature identifies global molecular alterations and immune target enrichment following epithelial-to-mesenchymal transition.Clin. Cancer Res. 2016; 22: 609-620Crossref PubMed Scopus (260) Google Scholar]. Recently, phenotypic plasticity of EMT, including partial EMT, has also been observed in patient biopsies from several advanced metastatic carcinomas [15.Navas T. et al.Clinical evolution of epithelial–mesenchymal transition in human carcinomas.Cancer Res. 2020; 80: 304-318PubMed Google Scholar]. These together unveil a central role for EMT in tumor progression with clinical relevance. Moreover, EMT contributes to chemoresistance [16.Fischer K.R. et al.Epithelial-to-mesenchymal transition is not required for lung metastasis but contributes to chemoresistance.Nature. 2015; 527: 472-476Crossref PubMed Scopus (1203) Google Scholar, 17.Zheng X. et al.Epithelial-to-mesenchymal transition is dispensable for metastasis but induces chemoresistance in pancreatic cancer.Nature. 2015; 527: 525-530Crossref PubMed Scopus (1356) Google Scholar, 18.Shibue T. Weinberg R.A. EMT, CSCs, and drug resistance: the mechanistic link and clinical implications.Nat. Rev. Clin. Oncol. 2017; 14: 611-629Crossref PubMed Scopus (1198) Google Scholar] and resistance to immune checkpoint blockade [19.Horn L.A. et al.Tumor plasticity and resistance to immunotherapy.Trends Cancer. 2020; 6: 432-441Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar]. EMT promotes chemotherapeutic resistance in multiple cancers, including cisplatin resistance in non-small cell lung cancer (NSCLC) [20.Kurokawa M. et al.Cisplatin influences acquisition of resistance to molecular-targeted agents through epithelial–mesenchymal transition-like changes.Cancer Sci. 2013; 104: 904-911Crossref PubMed Scopus (40) Google Scholar] and gastric cancer [21.Huang D. et al.Cisplatin resistance in gastric cancer cells is associated with HER2 upregulation-induced epithelial–mesenchymal transition.Sci. Rep. 2016; 6: 20502Crossref PubMed Scopus (65) Google Scholar] and docetaxel resistance in prostate cancer with high relapse [22.Marín-Aguilera M. et al.Epithelial-to-mesenchymal transition mediates docetaxel resistance and high risk of relapse in prostate cancer.Mol. Cancer Ther. 2014; 13: 1270-1284Crossref PubMed Scopus (110) Google Scholar]. In addition, occurrence of chemoresistant cancer stem cells (CSCs) bearing a partial EMT state (CSC+/pEMT+) with a poor clinical outcome was observed in metastatic breast cancer patients receiving chemotherapy [23.Papadaki M.A. et al.Circulating tumor cells with stemness and epithelial-to-mesenchymal transition features are chemoresistant and predictive of poor outcome in metastatic breast cancer.Mol. Cancer Ther. 2019; 18: 437-447Crossref PubMed Scopus (84) Google Scholar]. All of these observations indicate a primary requisite for the development of targeted treatment modalities suppressing EMT and metastatic dissemination, which could also be combined with standard chemotherapy to overcome drug resistance. However, targeting EMT at the molecular level by blocking key transcription factors is pharmacologically challenging [24.Dang C.V. et al.Drugging the 'undruggable' cancer targets.Nat. Rev. Cancer. 2017; 17: 502-508Crossref PubMed Scopus (377) Google Scholar]. We describe how metabolic inhibitory drugs, approved by the FDA or under clinical trials, can target EMT-associated metabolic processes. Cellular metabolism regulates various molecular and biochemical processes and is indispensable in response to the changes in the internal and external milieu of a cell [25.Mouchiroud L. et al.Transcriptional coregulators: fine-tuning metabolism.Cell Metab. 2014; 20: 26-40Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar]. Cancer cells, in particular during EMT, fine-tune their metabolic circuitry to meet the increased bioenergetic demands of encountering complex challenges during the cellular transit [26.Campbell S.L. Wellen K.E. Metabolic signaling to the nucleus in cancer.Mol. Cell. 2018; 71: 398-408Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar,27.Halldorsson S. et al.Metabolic re-wiring of isogenic breast epithelial cell lines following epithelial to mesenchymal transition.Cancer Lett. 2017; 396: 117-129Crossref PubMed Scopus (37) Google Scholar]. Therefore, understanding the switch in metabolic pathways during EMT would unravel key vulnerable therapeutic targets. Recent studies have reported significant metabolic reprogramming events during EMT. Unsupervised clustering of metabolism-specific gene expression from a comprehensive collection of cancer cell lines identified a mesenchyme-specific subgroup with high dihydropyrimidine (DHP) dehydrogenase (DPYD) enzyme-driven production of DHPs [28.Shaul Y.D. et al.Dihydropyrimidine accumulation is required for the epithelial–mesenchymal transition.Cell. 2014; 158: 1094-1109Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar]. A further study revealed that compounds specifically targeting mesenchyme-like cells largely inhibited glutathione peroxidase 4 (GPX4), which is involved in the lipid peroxidase pathway to evade ferroptosis, a nonapoptotic form of cell death [29.Viswanathan V.S. et al.Dependency of a therapy-resistant state of cancer cells on a lipid peroxidase pathway.Nature. 2017; 547: 453-457Crossref PubMed Scopus (624) Google Scholar]. Metabolic rewiring can be overtly regulated by EMT-associated transcription factors (EMT-TFs). For instance, ZEB1, a strong transcriptional EMT regulator, aids the mesenchymal state of the cell by promoting glycosphingolipid metabolism [30.Mathow D. et al.Zeb1 affects epithelial cell adhesion by diverting glycosphingolipid metabolism.EMBO Rep. 2015; 16: 321-331Crossref PubMed Scopus (35) Google Scholar]. Similarly, SNAI1 mediates the repression of fructose-1,6-bisphosphatase 1 (FBP1) to activate glycolysis in breast cancer [31.Dong C. et al.Loss of FBP1 by Snail-mediated repression provides metabolic advantages in basal-like breast cancer.Cancer Cell. 2013; 23: 316-331Abstract Full Text Full Text PDF PubMed Scopus (517) Google Scholar]. Metabolic reprogramming is therefore an inevitable component of the EMT phenotypic switching of cancer cells. However, metabolism has also been described as an upstream regulator of cellular plasticity. For instance, when the TCA cycle enzyme fumarate hydratase (FH) is ablated in renal cancer, it causes fumarate accumulation in the cell, which in turn epigenetically silences the EMT-suppressing miRNA miR-200 [32.Ceppi P. Peter M.E. MicroRNAs regulate both epithelial-to-mesenchymal transition and cancer stem cells.Oncogene. 2014; 33: 269-278Crossref PubMed Scopus (86) Google Scholar] and activates EMT-TFs [33.Sciacovelli M. et al.Fumarate is an epigenetic modifier that elicits epithelial-to-mesenchymal transition.Nature. 2016; 537: 544-547Crossref PubMed Scopus (302) Google Scholar]. Similarly, the nucleotide metabolic enzyme thymidylate synthase (TS), typically known for its involvement in cell proliferation, has been shown to regulate the EMT phenotype and breast cancer de-differentiation with a requisite of DPYD-dependent pyrimidine catabolism [34.Siddiqui A. et al.Thymidylate synthase maintains the de-differentiated state of triple negative breast cancers.Cell Death Differ. 2019; 26: 2223-2236Crossref PubMed Scopus (17) Google Scholar]. In addition, an EMT-focused transcriptomic analysis identified elevated levels of AKR1B1 in mesenchymal cells contributing to EMT and stemness. The polyol pathway, a two-step metabolic pathway mediated by aldo-keto-reductase-1 B1 (AKR1B1) and sorbitol dehydrogenase (SORD), converts excess glucose to fructose in cells. When activated by excess glucose, the polyol pathway can induce EMT through autocrine TGF-β stimulation [35.Schwab A. et al.Polyol pathway links glucose metabolism to the aggressiveness of cancer cells.Cancer Res. 2018; 78: 1604-1618Crossref PubMed Scopus (49) Google Scholar], implicating a strong connection between glucose metabolism and EMT. D-2-Hydroxyglutarate, an oncometabolite produced by glutamine anaplerosis, induced ZEB1-mediated EMT [36.Colvin H. et al.Oncometabolite D-2-hydroxyglurate directly induces epithelial-mesenchymal transition and is associated with distant metastasis in colorectal cancer.Sci. Rep. 2016; 6: 36289Crossref PubMed Scopus (67) Google Scholar]. Similarly, branched-chain amino acid metabolism, through its counterpart enzyme branched-chain α-keto acid dehydrogenase kinase (BCKDK), promotes EMT and metastasis in colorectal cancer [37.Tian Q. et al.Phosphorylation of BCKDK of BCAA catabolism at Y246 by Src promotes metastasis of colorectal cancer.Oncogene. 2020; 39: 3980-3996Crossref PubMed Scopus (12) Google Scholar]. Collectively these findings show that reprogramming of metabolism and EMT are interdependent events and, with the priming of therapeutic exploitation at the metabolic level, EMT can be effectively targeted. Here, we discuss some promising pharmacological inhibitors of metabolic enzymes or pathways (summarized in Table 1, Key Table), which could be repurposed to revert or reduce EMT phenotypes.Table 1Key Table. List of Metabolism-Inhibiting Drugs, FDA Approved or Under Clinical Trials, with EMT-Inhibiting FeatureDrugTargetFDA approval/treatment forClinical trial forTrial IDSimvastatinHMG-CoAFDA approved for hypolipidemiaAdvanced rectal cancerNCT02161822Pancreatic cancerNCT00944463Small cell lung cancerNCT01441349Brain metastasisNCT02104193OlaparibPARPFDA approved for cancerMetastatic renal cell carcinomaNCT03786796DisulfiramALDH1FDA approved for alcoholismMetastatic breast cancerNCT03323346CTC/EMT metastatic breast cancerNCT04265274EtodolacCOX-2FDA approved for pain and inflammationColorectal metastasisNCT03919461Metastatic pancreatic cancerNCT03838029SuraminHeparanaseTreatment for trypanosomiasisAutismNCT02508259Prostate cancerNCT00002881Advanced solid tumorsNCT00003038NSCLCNCT00066768RolipramPDE4Treatment for depressionMultiple sclerosisNCT00011375L-NAMEiNOSCardiovascularNCT03684213CardiovascularNCT03679780L-NMMAMultiple cancersNCT03236935Metastatic TNBCNCT028344032-DGGlycolysisAdvanced tumorsNCT00096707AscorbateVitamin CResectable or metastaticNCT03146962Pancreatic cancerNCT02905578Malignant tumorsNCT04033107 Open table in a new tab Below we highlight the importance of repositioning various FDA-approved or under-trial drugs for the inhibition of metabolism, which in turn mitigate EMT features (Figure 1). Given the inhibitory potential of these drugs in EMT, an inclusion criterion for EMT-guided patient selection in clinical trials would be an additional benefit to the therapeutic outcome. The tabulated list of drugs with repurposing ability (Table 1) demonstrates that metabolism-centered targeting could represent a valuable resource for future clinical research in EMT-driven aggressive cancers. Activation of signaling pathways such as TGF-β, Wnt, and Notch can strongly induce EMT [38.Thiery J.P. Sleeman J.P. Complex networks orchestrate epithelial–mesenchymal transitions.Nat. Rev. Mol. Cell Biol. 2006; 7: 131-142Crossref PubMed Scopus (3152) Google Scholar] and rewire the metabolic circuitry in cells to mediate the process. For instance, TGF-β promotes EMT by increasing the levels of phosphodiesterase 4 (PDE4), an enzyme involved in the conversion of cAMP to AMP to maintain the cAMP pool in the cells [39.Kolosionek E. et al.Expression and activity of phosphodiesterase isoforms during epithelial mesenchymal transition: the role of phosphodiesterase 4.Mol. Biol. Cell. 2009; 20: 4751-4765Crossref PubMed Scopus (77) Google Scholar]. Rolipram, a selective inhibitor of PDE4 with anticancer and antipsychotic effects [40.Goldhoff P. et al.Targeted inhibition of cyclic AMP phosphodiesterase-4 promotes brain tumor regression.Clin. Cancer Res. 2008; 14: 7717-7725Crossref PubMed Scopus (103) Google Scholar,41.Wiescholleck V. Manahan-Vaughan D. PDE4 inhibition enhances hippocampal synaptic plasticity in vivo and rescues MK801-induced impairment of long-term potentiation and object recognition memory in an animal model of psychosis.Transl. Psychiatry. 2012; 2e89Crossref PubMed Scopus (45) Google Scholar], attenuated the TGF-β-induced PDE4-mediated EMT process in a SMAD-independent manner with inhibition of reactive oxygen species (ROS) production [39.Kolosionek E. et al.Expression and activity of phosphodiesterase isoforms during epithelial mesenchymal transition: the role of phosphodiesterase 4.Mol. Biol. Cell. 2009; 20: 4751-4765Crossref PubMed Scopus (77) Google Scholar]. Pretreatment of lung cancer cells with rolipram followed by treatment with TGF-β1 showed a significant increase in epithelial markers (E-cadherin and cytokeratin-18) and a decrease in a mesenchymal marker (fibronectin) at both the transcript and the protein level. Interestingly, a chemopreventive effect of rolipram was observed in an in vivo lung cancer model in preventing lung tumorigenesis with the inhibition of EMT [42.Yeo C.D. et al.Chemopreventive effect of phosphodieasterase-4 inhibition in benzo(a)pyrene-induced murine lung cancer model.Exp. Lung Res. 2014; 40: 500-506Crossref PubMed Scopus (16) Google Scholar]. Similar to rolipram, TGF-β-induced EMT was abrogated on treatment with simvastatin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and a FDA-approved drug used for the treatment of hyperlipidemia [43.Yang T. et al.Simvastatin attenuates TGF-β1-induced epithelial–mesenchymal transition in human alveolar epithelial cells.Cell. Physiol. Biochem. 2013; 31: 863-874Crossref PubMed Scopus (65) Google Scholar,44.Wang G. et al.Simvastatin induces cell cycle arrest and inhibits proliferation of bladder cancer cells via PPARγ signalling pathway.Sci. Rep. 2016; 6: 35783Crossref PubMed Scopus (72) Google Scholar]. In bladder cancer, simvastatin inhibited EMT with distinct downregulation of the bladder cancer biomarker vimentin and upregulation of the epithelial marker E-cadherin [44.Wang G. et al.Simvastatin induces cell cycle arrest and inhibits proliferation of bladder cancer cells via PPARγ signalling pathway.Sci. Rep. 2016; 6: 35783Crossref PubMed Scopus (72) Google Scholar]. In addition, downregulation of mesenchyme-associated proteins including β-catenin and reduced migration or invasion were observed on treatment with simvastatin in multiple bladder cancer cell lines. Interestingly, a study observed that pancreatic ductal adenocarcinoma (PDAC) patients given statin medication before undergoing surgery were found to have lower levels of CXCR4, c-Met, and vimentin in PDAC tissues compared with patients without statin medication [45.Yin Y. et al.Simvastatin inhibits sonic hedgehog signaling and stemness features of pancreatic cancer.Cancer Lett. 2018; 426: 14-24Crossref PubMed Scopus (20) Google Scholar]. Further, simvastatin was found to have inhibitory effect in vivo on metastasis and improved the overall survival outcome in patients with high-grade serous ovarian cancer [46.Kato S. et al.Simvastatin interferes with cancer 'stem-cell' plasticity reducing metastasis in ovarian cancer.Endocr. Relat. Cancer. 2018; 25: 821-836Crossref PubMed Scopus (27) Google Scholar]. Various Phase II clinical trials in advanced stages of carcinoma are ongoing using simvastatin as a standalone or combinatorial drug. Interactions between tumor-associated stroma and cancer cells play a vital role in triggering the EMT program [10.Lambert A.W. et al.Emerging biological principles of metastasis.Cell. 2017; 168: 670-691Abstract Full Text Full Text PDF PubMed Scopus (1400) Google Scholar]. For example, the polysaccharide heparan sulphate, on cleavage and processing by heparanase, remodels the extracellular matrix for the bioavailability of growth factors, such as TGF-β, to induce EMT for cancer progression [47.Masola V. et al.Heparanase is a key player in renal fibrosis by regulating TGF-β expression and activity.Biochim. Biophys. Acta. 2014; 1843: 2122-2128Crossref PubMed Scopus (50) Google Scholar]. Selective inhibition of heparanase with suramin mitigated EMT in gastric signet-ring cell adenocarcinoma with downregulation of the expression of TGF-β1 and mesenchymal markers (Slug, vimentin, and α-SMA) along with upregulation of E-cadherin levels [48.Shah S. et al.The close relationship between heparanase and epithelial mesenchymal transition in gastric signet-ring cell adenocarcinoma.Oncotarget. 2018; 9: 33778-33787Crossref PubMed Scopus (11) Google Scholar]. Suramin is a widely used drug for the treatment of African trypanosomiasis, and a randomized clinical trial showed a promising approach for its use in autism spectrum disorder [49.Naviaux R.K. et al.Low-dose suramin in autism spectrum disorder: a small, Phase I/II, randomized clinical trial.Ann. Clin. Transl. Neurol. 2017; 4: 491-505Crossref PubMed Google Scholar]. A Phase I/II trial in metastatic breast cancer patients given prior treatment with taxanes showed a well-tolerated efficacy when treated with non-cytotoxic doses of suramin in combination with weekly paclitaxel [50.Lustberg M.B. et al.Phase I/II trial of non-cytotoxic suramin in combination with weekly paclitaxel in metastatic breast cancer treated with prior taxanes.Cancer Chemother. Pharmacol. 2012; 70: 49-56Crossref PubMed Scopus (22) Google Scholar]. Nitric oxide functions as both a pro- and an antitumorigenic factor, produced by the enzyme nitric oxide synthase (NOS) from L-arginine [51.Burke A.J. et al.The yin and yang of nitric oxide in cancer progression.Carcinogenesis. 2013; 34: 503-512Crossref PubMed Scopus (247) Google Scholar]. In triple-negative breast cancer (TNBC), inhibition of endogenous inducible NOS (iNOS) with a potent iNOS-specific inhibitor (1400W) and pan-NOS inhibitors (L-NAME and L-NMMA) showed downregulation of EMT-TFs, including Snail, Slug, and Twist, by the inhibition of multiple deregulated interconnected pathways of HIF-1α, ER stress, and the TGF-β/ATF3/ATF4 signaling axis. These inhibitors possess activity in suppressing the tumor-initiating capacity of CSCs and lung metastasis [52.Granados-Principal S. et al.Inhibition of iNOS as a novel effective targeted therapy against triple-negative breast cancer.Breast Cancer Res. 2015; 17: 25Crossref PubMed Scopus (128) Google Scholar]. While clinically L-NMMA could cause acute blood pressure elevation, administration of L-NMMA with amlodipine and docetaxel as a combinatorial drug regimen in orthotopic mouse models of TNBC was convincingly shown to reduce the blood pressure elevation by L-NMMA. Importantly, such combinatorial treatment also reduced tumor growth, with improved survival compared with the docetaxel-treated group. In accordance with this, L-NMMA improved the chemotherapeutic sensitivity to docetaxel in patient-derived xenograft (PDX) models of TNBC [53.Dávila-González D. et al.Pharmacological inhibition of NOS activates ASK1/JNK pathway augmenting docetaxel-mediated apoptosis in triple-negative breast cancer.Clin. Cancer Res. 2018; 24: 1152-1162Crossref PubMed Scopus (45) Google Scholar]. Several clinical trials are ongoing to test the therapeutic efficacy of L-NAME and L-NMMA in treating cardiovascular diseases and multiple cancers, respectively (Table 1). Vitamin metabolism has been shown to possess anticancer activity, with ascorbate having high selectivity towards cancer cells compared with normal [54.Ma Y. et al.High-dose parenteral ascorbate enhanced chemosensitivity of ovarian cancer and reduced toxicity of chemotherapy.Sci. Transl. Med. 2014; 6222ra18Crossref PubMed Scopus (273) Google Scholar]. Intravenous ascorbate has a potentially greater pharmacological effect than its oral counterpart, with an improved therapeutic response when used in combination with standard chemotherapy or immunotherapy [55.Alexander M.S. et al.Pharmacologic ascorbate reduces radiation-induced normal tissue toxicity and enhances tumor radiosensitization in pancreatic cancer.Cancer Res. 2018; 78: 6838-6851Crossref PubMed Scopus (47) Google Scholar, 56.Allen B.G. et al.First-in-human Phase I clinical trial of pharmacologic ascorbate combined with radiation and temozolomide for newly diagnosed glioblastoma.Clin. Cancer Res. 2019; 25: 6590-6597Crossref PubMed Scopus (29) Google Scholar, 57.Magrì A. et al.High-dose vitamin C enhances cancer immunotherapy.Sci. Transl. Med. 2020; 12eaay8707Crossref PubMed Scopus (72) Google Scholar]. The use of high-dose intravenous ascorbate has been found to be able to inhibit EMT along with metastasis as a standalone agent compared with as a combinatorial agent with gemcitabine in mice bearing orthotopic pancreatic xenografts. Pharmacological-level concentrations of ascorbate were able to deplete cellular levels of NAD+ selectively in cancer cells, leading to cell death with depletion of ATP content. A simultaneous increase in the acetylation of α-tubulin occurs with the depletion of NAD+ content, contributing to the disruption of microtubule dynamics and thereby inhibiting the motility of cancer cells. However, the molecular mechanistic mode of ascorbate in inhibiting EMT still needs future investigations. A Phase I/IIa trial with ascorbate showed improved overall survival in combination with gemcitabine in pancreatic cancer patients [58.Polireddy K. et al.High dose parenteral ascorbate inhibited pancreatic cancer growth and metastasis: mechanisms and a Phase I/IIa study.Sci. Rep. 2017; 7: 17188Crossref PubMed Scopus (71) Google Scholar]. Several other enzyme-related processes also contribute to EMT, and their inhibition showed an effect in treating tumors or drug-resistant cancers. For example, the poly-ADP-ribose (PAR) polymerase (PARP) enzyme catalyzes the synthesis of PAR from NAD+. Targeting of PARP with its specific inhibitor, olaparib, has been shown to impede TGF-β-induced EMT in mouse mammary gland cells, although the precise molecular mechanism of olaparib action on EMT has to be uncovered [59.Schacke M. et al.PARP-1/2 inhibitor olaparib prevents or partially reverts EMT induced by TGF-β in NMuMG cells.Int. J. Mol. Sci. 2019; 20: 518Crossref Scopus (19) Google Scholar]. Olaparib is an FDA-approved oral drug for the treatment of breast cancer and is currently in a Phase II study in metastatic renal cell carcinoma patients (ClinicalTrials.gov: NCT03786796). The aldehyde dehydrogenase 1A1 (ALDH1A1) enzyme, involved in alcohol metabolism, was recently shown to contribute to the development of erlotinib resistance with the acquisition of EMT/CSC features in lung cancer, and this was reverted on treatment with disulfiram, an ALDH1-specific inhibitor [60.Lei H.M. et al.Aldehyde dehydrogenase 1A1 confers erlotinib resistance via facilitating the reactive oxygen species-reactive carbonyl species metabolic pathway in lung adenocarcinomas.Theranostics. 2019; 9: 7122-7139Crossref PubMed Scopus (21) Google Scholar]. In lung adenocarcinoma xenografts, continuous treatment with erlotinib showed erlotinib resistance in the tumors and combinatorial treatment with disulfiram increased the sensitivity of tumor growth inhibition. In addition, disulfiram blocked brain metastasis with no measurable toxicity. Inhibition of ALDH1A1 by disulfiram in vivo effectively overcame erlotinib resistance-induced tumor dissemination and prolonged the survival outcome. Acquisition of ALDH1A1-induced erlotinib resistance is dependent on the ROS and reactive carbonyl species (RCS) metabolic enzymes SOD2 and GPX4, respectively, and disulfiram inhibited ALDH1A1 down