Upgrade of an old drug: Auranofin in innovative cancer therapies to overcome drug resistance and to increase drug effectiveness

Abstract Auranofin is an oral gold(I) compound, initially developed for the treatment of rheumatoid arthritis. Currently, Auranofin is under investigation for oncological application within a drug repurposing plan due to the relevant antineoplastic activity observed both in vitro and in vivo tumor models. In this review, we analysed studies in which Auranofin was used as a single drug or in combination with other molecules to enhance their anticancer activity or to overcome chemoresistance. The analysis of different targets/pathways affected by this drug in different cancer types has allowed us to highlight several interesting targets and effects of Auranofin besides the already well‐known inhibition of thioredoxin reductase. Among these targets, inhibitory‐κB kinase, deubiquitinates, protein kinase C iota have been frequently suggested. To rationalize the effects of Auranofin by a system biology‐like approach, we exploited transcriptomic data obtained from a wide range of cell models, extrapolating the data deposited in the Connectivity Maps website and we attempted to provide a general conclusion and discussed the major points that need further investigation.

interesting targets and effects of Auranofin besides the already well-known inhibition of thioredoxin reductase. Among these targets, inhibitory-κB kinase, deubiquitinates, protein kinase C iota have been frequently suggested. To rationalize the effects of Auranofin by a system biology-like approach, we exploited transcriptomic data obtained from a wide range of cell models, extrapolating the data deposited in the Connectivity Maps website and we attempted to provide a general conclusion and discussed the major points that need further investigation.

K E Y W O R D S
Auranofin, cancer, drug resistance, NF-kB inhibition, proteasome inhibition, thioredoxin reductase inhibition 1 | INTRODUCTION Auranofin (AF) is a linear gold(I) complex bearing triethylphosphine and thioglucose tetraacetate as ligands ( Figure 1). AF has been adopted in clinical use since 1985 for the treatment of rheumatoid arthritis. It is an orally administered drug and is considered safe for human use due to the well-known toxicity profile. After oral administration, 15%-25% of the drug can be detected in plasma and the serum albumin has been found to carry about 80%-95% of gold circulating in the blood. 1,2 The pharmacokinetics of AF has been extensively described by Chaffman et al. 3 The mean pick of AF concentration, following a single 6 mg dose, was attained after 102-120 min and it ranged between of 0.066 and 0.23 µg/ml (corresponding to a molar concentration of 0.1-0.34 µM). Steadystate plasma gold concentrations following at least 12 weeks of 2-9 mg daily dose of AF were 0.20-1.0 µg/ml, respectively corresponding to 0.3-1.5 µM. More recently in a Phase I human study, where AF was used as an antiparasitic agent at the dose of 7 mg daily (dose recommended in rheumatoid arthritis therapy), gold level in plasma of treated subjects for over a week reached 1-1.5 µM. 4 The triethyl-phosphine ligand is lipophilic, conferring membrane solubility to the complex. Once the compound has entered into the cells, the thiol ligand reacts with thiol and selenol groups, with which it forms stable and irreversible adducts. 5 In mammals most of the effects caused by this drug are thought to be related to its inhibitory activity against the thioredoxin reductase enzymes: TrxR1 and TrxR2. [6][7][8][9][10] The thioredoxin reductase enzymes play an important role in multiple intracellular processes including DNA synthesis, transcriptional regulation, cell growth and resistance to oxidative agents that induce oxidative stress and apoptosis. An increase of the thioredoxin reductase/thioredoxin (TrxR/Trx) system has been reported in many tumors compared to normal tissues and the ability of certain cancer cells to maintain a highly reduced intracellular environment is correlated with tumor aggressiveness and drug resistance. Furthermore, high expression level of TrxR/Trx system directly correlates with poor prognosis in a variety of cancers including lung F I G U R E 1 Chemical structure of Auranofin cancer 11 and breast cancer. 12 Indeed, this system plays a pivotal role in maintaining the reduced and active state of several proteins including phosphatase and tensin homolog (PTEN) that negatively regulates phosphatidylinositol-3kinase/AKT Serine/Threonine Kinase 1 (PI3K/Akt) pathway which is positively involved cell survival. 13 Several elegant reviews have already discussed the role of TrxR/Trx system under physiological and pathological conditions. 14,15 Other areas of research for the antiproliferative activity of AF focus on the inhibition of signal transducer and activator of transcription 3 (STAT3), [16][17][18] nuclear factor-kappa B (NF-κB) 18,19 and protein kinase C iota (PKCiota) 20,21 signaling. Furthermore, proteasome inhibition and activation of forkhead box O3 (FOXO3) were also reported as possible mechanisms involved in AF cytotoxicity. 22 Based on this evidence, AF is today object of important drug repurposing plans in anticancer therapy with encouraging results, as summarized in this review. In this context, results obtained by research studies have already been translated into clinical trials to develop new alternative therapeutic strategies. Indeed, despite enormous advances in cancer research and treatment, there are still some types of cancer for which there is no specific therapy and hence, chemotherapy and radiation therapy remain pillars of cancer treatment. Furthermore, in these cases patients often respond only partially to therapy and develop resistance over long-term treatment. Hence, the development of combination therapies may be useful to eradicate cancer cells that are unresponsive to monotherapy. Up to now, Phase I and II studies on chronic lymphocytic leukemia (NCT01419691) and a pilot study on ovarian cancer (NCT01747798) have been completed, while other clinical trials with AF as monotherapy or in combination with other drugs are ongoing on glioblastoma (NCT02770378), on lung cancer (NCT01737502), on ovarian cancer (NCT03456700). AF is also under investigation for use in infective diseases (NCT02736968 for Giardia protozoa, NCT02961829 for HIV, and NCT02968927 for tuberculosis).
In this review we have reviewed articles that use AF (alone or in combination with other molecules) as an anticancer drug. We focused first on the effect of AF on different types of cancer cells and/or animal models and then we analysed and integrated the results by a system biology-like approach, with the data available on Connectivity Map website (https://clue.io/cmap). Finally, we attempted to extrapolate general conclusion and discussed the major points that need further investigation. In Table 1 we reported AF effect and mechanism of action on different cancer models (cell lines and/or animal model). Most of the articles are commented on throughout the text.
The bibliographic research was limited to studies published from 1980 to 2020 and found in PubMed and Google Scholar databases. Keywords used to retrieve documents were "Auranofin and cancer." In PubMed the option "Best match" was applied. The word "auranofin" had to be cited in the title, in the abstract or among keywords. Studies performed with auranofin analogues, but not auranofin, were not included. Oxidative stress (Trx system)  References include also studies not specifically described in the text. lenalidomide). [24][25][26] However, due to acquired drug resistance and the heterogeneity of myeloma cells, MM remains incurable. Therefore, new therapeutic approaches are needed to overcome drug resistance and improve clinical outcomes in the treatment of MM.
The first study dealing with the potential use of AF to treat MM was carried out by Nakaya et al. 18 They selected this gold(I) compound based on its well-known anti-inflammatory and immunosuppressive properties through the inactivation of NF-κB signaling. 19,27 It has been reported that chemoresistance in MM is associated with constitutive activation of NF-κB and tyrosine-protein kinase Janus Kinase 2 (JAK2)/STAT3 signaling. 28 32 It is known that hypoxia is a key factor in the acquisition of bortezomib resistance even if the molecular mechanisms involved are still to be elucidated. 33,34 The authors first showed, by using the gene expression data set (GSE4581), that higher TrxR1 levels were associated to acquired drug resistance and to decreased myeloma patient survival. Second, they found an overexpression of TrxR1 in hypoxic myeloma cells RPMI8226 and U266 resistant to bortezomib, suggesting a possible involvement of this enzyme in the hypoxiainduced bortezomib resistance. Thus, they used AF (1-4 µM) as a selective TrxR1 inhibitor in hypoxic myeloma cells.
The results pointed out that TrxR1 inhibition overcame hypoxia-induced bortezomib resistance. Accordingly, they demonstrated that hypoxia increased NF-κB subunit p65 nuclear protein levels and that AF-induced TrxR1 inhibition, decreased both NF-κB p65 protein and messenger RNA (mRNA) levels as well as mRNA levels of downstream NF-κB regulated genes, Survivin and Cyclin D1. Based on these data, the authors proposed TrxR1 inhibitors, such as AF, as single drug or in combination therapy to bypass bortezomib resistance and to increase myeloma patient survival. In the same year Raninga and colleagues investigated a possible crosstalk between TrxR and heme oxygenase-1 (HO-1) in multiple myeloma cell lines RPMI8226, U266, and OPM2, aiming to demonstrate the need to target multiple antioxidant systems. 35 HO-1 participates in hemoglobin degradation and catalyzes the conversion of intracellular heme into biliverdin, free iron, and carbon monoxide. 36 In turn, biliverdin is reduced by biliverdin reductase to bilirubin which has anti-inflammatory, antioxidative, and antiapoptosis properties. 37 HO-1 expression and activity has been found increased in several types of cancer implying its potential involvement in chemoresistance. Moreover, HO-1 expression is regulated by many transcription factors including NF-κB 38 and nuclear factor erythroid 2-related factor 2 (Nrf2) 39

| Leukemia
In Fiskus et al. attempted to provide rationale to AF repurposing for chronic lymphocytic leukemia (CLL) therapy. 53 Standard therapy is based on myelotoxic chemoimmunotherapy. 54 However, in case of relapse a different treatment is required as standard therapy is no longer effective. This type of cancer was suitable for AF repurposing because CLL cells have intrinsically higher levels of ROS. 55 The authors demonstrated for the first time thatAF 1 µM triggered lethal oxidative stress through TrxR inhibition and ER stress response in cultured and patient derived primary CLL cells, including those with genetic features associated with poor clinical outcome. AF anticancer activity was also proved to be efficient in TCL-1 transgenic mice, an in vivo model of CCL. 56 Chen et al. 57 evaluated AF on chronic myelogenous leukemia (CML) cell and animal models, as an alternative therapy to overcome acquired resistance to Imatinib mesylate (IM), a drug resistance mainly due to mutation in Bcr-Abl. 58 2]Cl decreased Bcr-Abl and proto-oncogene c-myc protein expression leading to apoptosis also in IM resistant CML cells.
The obtained results highlighted the effectiveness of TrxR targeting in overcoming IM resistance as well as the crosstalk between the TrxR system and Bcr-Abl signaling pathway.
Acute lymphoblastic leukemia (AAL) is the most frequent neoplasm in childhood 61 and, as various other cancers, it is characterized by an unbalanced redox homeostasis. 62 For this reason, Haß et al. 63 selected different ROS inducers to sensitize ALL cells to therapeutic treatment based on the second mitochondria-derived activator of caspases (Smac) mimetic LCL161 that antagonizes inhibitor of Apoptosis (IAP) proteins. They used AF alone or in combination with LCL161 to target TrxR, Erastin to inhibit the cystine/glutamate transporter XCT required for GSH production and buthionine sulfoximine (BSO) as specific inhibitor of γ-glutamylcysteine ligase (γ-GCL). 64 The results pointed out the effectiveness of this approach in being able to prime ALL cells for LCL161-induced cell death.

| Breast cancer
Breast cancer is the most common female cancer and the second most common cause of cancer death in women.
Up to 20% of breast cancers are "triple negative" tumors (TNBC) as the three most common types of receptors known to fuel most breast cancer growth-estrogen, progesterone, and the receptor tyrosine-protein kinase   80 Using a high-throughput drug screening, they revealed that AF was a potential inhibitor of the expression of NONO, suggesting a new way in which AF could carry out its antitumour activity.

| Colon cancer and gastric cancer
Colon cancer is one of main cancer types that lead to cancer-related death worldwide. 81 Chemotherapy is an important treatment approach for this pathology; however, patients have few treatment options after two cycles of therapy. Hence, the discovery of new treatments for colon cancer care is mandatory. AF (0.5 µM) was combined with 5Z-7-oxozeaenol (5 µM), a molecule able to inhibit the transforming growth factor βactivated kinase 1 (TAK1), leading to colon carcinoma cell death both in vivo and in vitro. 82 The inhibition of TrxR activity and the increased Trx oxidation were proposed to be responsible for the enhancement of sensitization of cancer cells to 5Z-7-oxozeaenol. Moreover, AF has been used in combination with the antiinflammatory drug celecoxib (CE) in different colon carcinoma cell lines. 83 The findings show that AF/CE combination greatly enhanced AF therapeutic efficacy both in vitro and in vivo. Drug mixture generated oxidative stress that inhibited hexokinase, induced TrxR oxidation and modifies mitochondrial redox state.
Particularly in mitochondria, AF/CE promoted the oxidation of TrxR2 and the degradation of the cytochrome c oxidase subunit II, a component of complex IV of the electron transport chain. As consequence, mitochondrial respiration and ATP production resulted inhibited.
5-FU is widely used in chemotherapy for colon cancer, but drug resistance limits its clinical effectiveness. In a recent study, Liu et al. found that nuclear FOXO3 was decreased in 5-FU-resistant SW620 and HCT-8 (SW620/ 5-FU and HCT-8/5-FU) cells, and that overexpressing FOXO3 could significantly reverse 5-FU resistance. 84 FOXO3 is a transcription factor with key role in cell cycle, apoptosis, aging regulation, and tumor suppression.
Several protein kinases (IkB kinase, Akt, MAP) phosphorylate FOXO3, promoting its translocation from the nucleus to the cytosol where it is degraded via the ubiquitin-proteasome pathway. 85 Its inhibition leads to tumor development and progression, suggesting that FOXO3 reactivation could be a promising strategy to develop anticancer therapeutic drugs. The authors demonstrated that TrxR1 was the key protein participating in the regulation of FOXO3-mediated 5-FU resistance. Thus, they used AF as FOXO3 agonist and TrxR1 inhibitor to overcome 5-FU resistance both in vitro and in vivo models.
The possible beneficial effect of AF during radiation treatment has been analysed in subcutaneous CT26 colon tumors, in normal gastrointestinal epithelium of mice and in human organoids formed using malignant and nonmalignant tissues from the same patient. 86 Indeed, one of the disadvantages in the use of radiotherapy in colon carcinoma therapy is the elevated radio sensitivity of normal intestinal epithelium. The results showed that AF pretreatment prevented radiation toxicity in normal cells in all these models. In fact, AF caused cell cycle arrest and p53/p21 pathway activation, leading to reduction of the irradiation-induced DNA damages and increasing cell survival. On the other hand, in malignant cells AF inhibited proteasome activity and induced endoplasmic reticulum stress/unfolded protein response, activating apoptosis.
Concerning gastric cancer, Peng et al. demonstrated that in gastric cell lines, the natural compound piperlongumine (PL), known to induce ROS production, enhanced AF cytotoxicity. 87 In particular, 4 µM AF induced ROS increase, ER stress and mitochondrial dysfunction leading to apoptosis. The combination of AF and PL resulted in an increase of cell death in vitro and in a reduction of tumor size in vivo.

| Lung cancer
Lung cancer is the leading cause of cancer-related deaths in men and women. 88 In particular, non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancers and has a poor prognosis with a 5-year survival rate of 17%. Currently common therapies for NSCLC include platinum-based chemotherapy with concurrent radiotherapy but cancer cell resistance limits treatment utility.  Furthermore, in H1993 xenograft nude mice, treatment with MK2206 or AF as single drugs had no effect on tumor growth, but their combination significantly inhibited tumor growth and significantly prolonged animal survival.
The same research group also investigated the cytotoxicity of AF alone in 10 different NSCLC cell lines and they found that AF sensitivity ranged from less than 1 µM to up to 2 µM. 93 They demonstrated that sensitive cells  94 Hou et al. investigated the effect of AF on cancer stem cell side-population (SP). 95 SP represents a subpopulation of stem-like cancer cells that have a pivotal role in drug resistance due to their high expression of the ATP-binding cassette transporter ABCG2 involved in drug export. The authors found that A549 cell line, contained a considerable amount of SP and AF could effectively deplete SP cells by increasing ROS generation. Interestingly, they proved that AF inhibited the glycolytic enzyme hexokinase leading to ATP depletion and decreasing the activity of ABCG2 pump. Furthermore, the synergistic effect of AF and the chemotherapeutic agent adriamycin was proved both in vivo and in vitro.
Trx/TrxR and glutathione/glutathione-disulfide reductase (GSH/GSR) are the most relevant cellular antioxidant systems and functional deficiency in one of these systems renders cells dependent on the other system for survival. 96 Thus, it has been hypothesized that cells harboring a compromised GSH/GSR system could be more sensitive to TrxR inhibition. Yan et al. found that GSR gene is delete in 6% of lung adenocarcinoma. To investigate whether this deletion could sensitize cells to AF treatment, the authors evaluated AF sensitivity in several cell lines with different expression of genes involved in GSH homeostasis and in AF resistant A459 cell line in which GSR gene was knocked out or silenced. In all these conditions the authors demonstrated that the alteration of GSH homeostasis was correlated with increased sensitivity to AF treatment. 97 Moreover, they also demonstrated in vivo activity of AF in two lung cancer patient-derived xenograft models with relatively low levels of GSR and glutamatecysteine ligase catalytic subunit expression.
Activating EGF mutations represent a driven force in lung tumor progression and limit the therapy based on EGFR inhibitors. It is interesting to note that AF (0.25 µM) enhanced ibrutinib activity in EGFR mutant non-small cell lung cancer cells by inhibiting the expression or phosphorylation of multiple key nodes in Akt/mTOR and MEK/ERK pathways. 98 Ito et al. proved that AF in combination with IPA-3, an inhibitor of p21 activate kinase (PAK1) which is in turn regulated by PKCiota, had a highly synergistic effect in EGFR or KRAS mutant adenocarcinoma and squamous cell carcinoma cell lines and decreased tumor volume in mice models. 99 In these cell lines the combination of IPA-3 and AF abrogated the expression and/or phosphorylation of serine/threonine-protein kinase PAK1, PKCiota, ERK 1/2, mTOR, Akt, and transcriptional coactivator YAP1 proteins.
Liu et al. developed a high-throughput drug screen strategy to identify new drugs that can synergistically enhance antitumour efficiency of cisplatin in chemoresistant small cell lung carcinoma (SCLC) cells. 100 Among the FDA-approved drug library of 1092 compounds they found that AF was able to overcome cisplatin resistance. In this study the authors demonstrated that AF synergized with cisplatin resulting in ROS overproduction, which in turn led to mitochondrial dysfunction and DNA damage in SCLC. The combination resulted in significant reduction of tumor growth also in vivo.

| Ovarian cancer (OC)
OC is the most lethal gynecological disease and is characterized by heterogeneity, high risk of relapse and development of platinum resistance that result in poor prognosis. The cornerstone of treatment is maximal-effort surgical cytoreduction combined with cytotoxic chemotherapy. An estimated 80%-85% of patients with OC who achieve full remission following first-line platinum-based therapy will develop recurrent disease and median survival for these patients ranges from 12 months to 24 months. 101 Except for bevacizumab and poly ADP-ribose polymerase inhibitors, approved for BRCA1/2 mutated patients and for platinum-sensitive patients without BRCA 1/2 mutation, there are few other options for women with platinum-resistant OC.
The first articles that report a cytotoxic activity of AF on ovarian cancer cells were published by Marzano 102 and Rigobello. 103 In these studies, the authors demonstrated that AF could overcome cisplatin resistance in the

| Other cancers
The literature contains several other studies concerning the use of AF as a cytotoxic and potentially antitumor agent in other cancer types. Taken together they confirm the possible broad spectrum of application of this drug.
Concerning hepatoma, Kim et al. reported that 2 μM AF was able to inhibit IL-6 activation of JAK1-STAT3 pathway in a ROS-independent fashion. 16 Huang et al. found that AF (0.2 μM) in combination with Disulfiram (an aldehyde dehydrogenase, in clinical use to treat alcoholism) synergistically induced proteasome-associated DUBs inhibition, reticulum endoplasmic (RE) stress and apoptosis in HepG2 and SMMC-7721 cell lines. 115 They also proved that this mechanism was ROS-independent since scavenging of ROS by 100 µM Vitamin C failed to block cell death. As already shown for other cancer types, also in hepatocellular carcinoma TrxR1 expression positively correlated with advanced clinical staging and poorer patient survival. 116  The high-throughput screen of FDA approved drugs was used to discover new therapies for osteosarcoma and Ewing sarcoma. 130,131 In both cases AF emerged as a possible alternative to traditional treatments both as a single drug and in combination with rapamycin or vorinostat (for osteosarcoma) and with ganetespib, an HSP90 inhibitor (for Ewing sarcoma). Lastly, AF-induced cell death via oxidative stress also in HeLa cervical cancer cells and was used in combination with 2-DG and BSO (inhibitors of glycolysis and GSH synthesis, respectively) to treat highly glycolytic cervical cancer cell lines that were resistant to standard therapy with cisplatin plus pelvic irradiation. 134,135

| DOSE-DEPENDENT EFFECTS OF AURANOFIN THROUGH A SYSTEM BIOLOGY-LIKE APPROACH
In the first part of this review, we reported an update on AF effects according to clinical applications in different cancers.
Reporting the results obtained from different cell models, with different concentrations and exposition times, generates a fragmented vision of the AF mechanism of action. Thus, in this section we have summarized the major effects of AF in cancer cells by revisiting a large data set of transcriptomic data obtained with a wide range of cell models exposed to AF.
The data are deposited in the connectivity maps (CMAP) website (https://clue.io/cmap) 136 that, using transcriptional expression data (mRNA transcript abundance of 978 "landmark" genes), allows to probe the relationships between drugs and many cellular models of diseases. In practice, changes in gene expression in response to a genetic perturbation (knockdown or overexpression of a gene) or treatments with small molecules (perturbagenes) are compared by similarity to all perturbation signatures of the database (more than 1 million of available profiles) obtaining a "tau score" which allows to predict the effects of a perturbagene. CMAP is equipped with all the bioinformatic tools and transcriptomic data sets to compare the effects of different drug dosages and exposition times in many cancer cell lines.
Searching for the connectivities of AF with other "perturbagenes," CMAP performs its prediction using the gene expression profiles of nine cell lines (A375, A549, HCC515, HEPG2, HT29, MCF7, PC3, HA1E, and VCAP) exposed for 6 h at 10 µM of AF which is generally considered a high dose. The highest AF connection is with the NF-κB inhibition ( Figure 2A)  specific genetic background could also play a key role in determining the primary AF targets. This is confirmed by the comparison of the transcript expression profiles between different AF concentrations in two cancer cell lines and the effects of TrxR1 silencing, available in the CMAP data set. For example, A549 cells (lung cancer) exposed to 1 µM AF generate effects very similar to TrxR silencing whereas, MCF7 cells (breast cancer) exposed to different concentration of AF (1 nM to 10 µM) show low correlations with TrxR1 silencing ( Figure 2B). Furthermore, considering the hierarchical clustering of the CMAP transcriptomic profiles of 45 cancer cell lines exposed to 10 µM AF for 6 h, three major clusters can be distinguished ( Figure 2C).

| CONCLUSION AND PERSPECTIVES
Based on the studies performed on different cancer models, several mechanisms have been proposed to explain how AF alone or in combination with other drugs can exert its anticancer activity. These mechanisms can be partially overlapping and tumor and/or AF dosage dependent. As summarized in Table 1, AF EC50 after 24-48 h ranges between 0.5 and 2 µM in most of the cells. Most of the experiments were performed using AF in this range concentration for 12-24 h h. In some studies, AF was used at higher concentration (6-10 µM) for a shorter amount of time (2-6 h). These different approaches could lead to different pathway activations and complicate overall data interpretation as discussed in the previous paragraph. Since in humans, the plasma steady-state physiological range of AF is 1-1.5 µM, 3,4 it is reasonable to hypothesize that in vivo, targets inhibited by low concentrations of AF have greater relevance.
In conclusion the main direct and indirect targets of AF are shown in Figure 3 and, in particularly, a direct interaction has been shown for the following proteins: • TrxR inhibition The direct inhibition of TrxR was demonstrated for the first time in 1998 and up to date it represents the most proposed mechanism. 6 In most articles, the inhibition of TrxR1 and the consequent increase of ROS has been proposed as the primary way by which AF induces cytotoxicity. In this context, it should be also mentioned that TrxR1 inhibition can influence the redox state of several proteins controlled by the TrxR/Trx system and therefore the ultimate effector of AF action may result difficult to identify. 13,27,57 Another point to be considered is that not all cancers have the same expression level of this enzyme and thus, AF sensitivity may depend on this feature. In fact, several studies have shown that cell lines where TrxR1 expression levels were higher were also more resistant to AF cytotoxic activity. 31 • IκB kinase IκB kinase was proposed as direct AF target by Jeon et al. in LPS-stimulated macrophages. The same authors also demonstrated that AF binds Cys179 of IκB kinase, thus blocking its activity. 19,27 IκB kinase activity is strictly related to activation of NF-κB. Indeed, IκB kinase phosphorylates IκB protein that binds NF-κB and sequestrates it in the cytosol in an inactive state. Once phosphorylated, IκB dissociates from NF-κB, that migrates in the nucleus where it carries out its transcriptional activity. 139 In this review we have described several studies pointing out a reduction of NF-κB activity upon AF treatment. This can impact on cancer progression, since NF-κB regulates the expression of a plethora of genes involved in inflammation, oxidative stress and cell survival. In particular, since the correlation between tumor aggressiveness and inflammation has been extensively proved, the anti-inflammatory properties of AF could be relevant if used in combination with other antitumour drugs. 140 Interestingly, IκB is not the only substrate of IκB kinase, indeed Hu et al. found that it also phosphorylates the tumor suppressor FOXO3 that once phosphorylated, translocates to the cytosol and becomes inactive. 109 Thus, IκB kinase inhibition by AF can evoke various responses whose importance for the cytotoxicity of the drug could depend on the specific cell type and, in vivo, on the tumor microenvironment.
• PKCiota PKCiota was indicated as AF target in 2013 by Wang et al. 20 A possible direct interaction has been proposed since two analogues of AF, namely aurothiomalate and aurothioglucose, inhibit the enzyme, by binding a critical cysteine residue. 108 Amplification of the PKCiota gene and elevated PKCiota mRNA level are observed in several cancers, that is, human primary lung squamous cell carcinomas, serous epithelial ovarian cancer, and cervical cancer. 141 Furthermore, high PKCiota expression level is associated with decreased survival in patients with NSCLC, ovarian, bile duct, and prostate tumors, suggesting its possible role as prognostic marker. 142 In consideration of all these aspects the AF inhibitory role on this kinase needs further investigation especially in cancer with high expression of this enzyme.
• Hexokinase The study of Hou et al. demonstrated that the purified enzyme hexokinase was inhibited by AF at 4-6 μM and that in vivo cellular glucose uptake, lactate and ATP production were also significantly suppressed by the same dose. 95 Most cancer cells express high levels of hexokinase and this is implicated in the accelerated glycolytic flux. 143 Furthermore, hexokinase (in particular the isoform II) can bind to mitochondria, where it plays additional functions such as cell death inhibition and autophagy regulation. 144 Thus, targeting hexokinase is considered a promising antitumour strategy, but further evidence is needed to understand the possible involvement of AF especially if used at more physiological doses.
• Proteasome-associated DUBs In 2014 Liu et al. demonstrated that the proteasome associated deubiquitinases UCHL5 and USP14 of the 16S regulatory subunit are inhibited by AF both in vitro and in vivo and that this inhibition played a key role in AF cytotoxicity. 76,125 As discussed in previous paragraph, Zhang et al. considered DUBs an off-target effect occurring at high dose of AF. 138 Further studies are needed to better understand dose-dependent and/or cell type dependent inhibition of these targets.
All the other proteins summarized in Figure 3 resulted affected by AF treatment but, as fa as we know, no evidence was provided regarding a direct interaction. It is reasonable to assume that their modulation is related to the inhibition of the direct targets previously described, but in several cases the signaling cascade has not been clarified. Furthermore, more than one mechanism can contribute to the ultimate effect of the drug.
Overall, the studies described in this review support the use of AF in combination with the drugs normally used in the therapy of specific cancer, as a valuable aid to overcome resistance to chemotherapy and/or enhance the effectiveness of conventional therapies. Further efforts are needed to understand the effective mechanism through which AF performs its action on different types of cancer in relation to specific genome background and protein pattern expression. The studies reported in this review confirm the growing interest in AF drug repurposing and provide the basis for further insights that can support the translation of research in this field into clinical trials.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available in PubMed.