Drug repurposing in cancer

Accepted Manuscript Title: Drug repurposing in cancer Authors: Linda Sleire, Hilde Elisabeth Førde, Inger Anne Netland, Lina Leiss, Per Øyvind Enger PII: DOI: Reference:

S1043-6618(17)30853-8 http://dx.doi.org/doi:10.1016/j.phrs.2017.07.013 YPHRS 3644

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Pharmacological Research

Please cite this article as: Sleire Linda, Førde Hilde Elisabeth, Netland Inger Anne, Leiss Lina, Enger Per Øyvind.Drug repurposing in cancer.Pharmacological Research http://dx.doi.org/10.1016/j.phrs.2017.07.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Drug repurposing in cancer Linda Sleire1, Hilde Elisabeth Førde1, Inger Anne Netland1, Lina Leiss1 and Per Øyvind Enger1, 2 1Oncomatrix Research Group, Department of Biomedicine, University of Bergen, Jonas Lies vei 91, 5009 Bergen, Norway. 2 Department of Neurosurgery, Haukeland University Hospital, Jonas Lies vei 71, 5021 Bergen, Norway.

corresponding author Prof Per Øyvind Enger Department of Biomedicine, University of Bergen, Jonas Lies vei 91, 5009 Bergen, Norway. tel: +47 55586347 e-mail: [email protected]

Graphical abstract

Abstract Cancer is a major health issue worldwide, and the global burden of cancer is expected to increase in the coming years. Whereas the limited success with current therapies has driven huge investments into drug development, the average number of FDA approvals per year has declined since the 1990s. This unmet need for more effective anti-cancer drugs has sparked a growing interest for drug repurposing, i.e. using drugs already approved for other indications to treat cancer. As such, data both from pre-

clinical experiments, clinical trials and observational studies have demonstrated antitumor efficacy for compounds within a wide range of drug classes other than cancer. Whereas some of them induce cancer cell death or suppress various aspects of cancer cell behavior in established tumors, others may prevent cancer development. Here, we provide an overview of promising candidates for drug repurposing in cancer, as well as studies describing the biological mechanisms underlying their anti-neoplastic effects.

Keywords: cancer, aspirin, metformin, thalidominde, drug repurposing, tumour associated inflammation

Introduction In 2012, more than 14 million people were diagnosed with cancer and there were 8.2 million estimated cancer deaths (1). Demographic trends including population growth, increasing life expectancy and more affluent life styles due to economic development will be key drivers of a continued increase in cancer burden. According to global statistics, more than 20 million people will be diagnosed with cancer in 2025. Importantly, malignancies such as colorectal, prostate and breast cancer, that are often incurable in advanced stages with current treatments, will contribute significantly to this increase (1). Thus, addressing these present and future challenges requires more effective cancer drugs. Traditionally, cancer drug discovery and development involves identification and optimization of lead compounds, followed by pre-clinical and clinical studies to extensively test and characterize their pharmacological properties, anti-neoplastic effects and toxicity. Since the human genome project produced the first rough draft of the human genome, spending in research and development have increased by almost 50 % the OECD countries (2). Despite these efforts however, successful cancer drug development has proven difficult. The average time span from initial experiments to completed regulatory review vary between 11.4 to 13.5 years (3). Moreover, estimates of capitalized costs vary, ranging from 161-1800 million dollar per drug (4). Currently, there are more than 10'000 clinical trials investigating drug interventions in cancer registered at www.clinicaltrials.gov. However, only a limited number of drug candidates progress to the next phase in clinical trials (5), and the approval rate of cancer drugs entering phase I trials has previously been as low as 5 % (6). Notably,

the U.S. Food and Drug Administration (FDA) approvals in the past years are fewer compared to the 1990’s. In 2016, FDA approved 22 new drugs compared to 45 in 2015 (7). In oncology, the agency approved 4 new drugs in 2016, compared to 14 in 2015, 9 in 2014 and 9 in 2013 (8). Also the European Medicines Agency (EMA) approved fewer drugs in 2016 than previous years. For oncology drugs that receive marketing approval, prices have skyrocketed in recent years, increasing significantly the burden on health economies worldwide. Whereas basic drug discovery for a large part receive public funding and financial support from nonprofit organizations, late stage development is mostly driven by the pharmaceutical industry and venture capital. Given the profit-based incentives of this funding model, drugs that ultimately receive clinical approval are highly priced to cover overall investments – both for failed and successful drug candidates. As such, the costs for a combination targeted therapy for metastatic melanoma, involving the monoclonal antibodies ipilimumab and nivolumab, has been estimated to $400 000 US per responder (9, 10). Collectively, these challenges have boosted the search for alternative ways to improve success rates, shorten processing time and cut costs in cancer drug development. Drug repurposing or repositioning refers to the application of a drug for another indication than it was originally approved for and has received increasing interest as an alternative strategy to de novo drug synthesis (11). A major advantage is that extensive data are often available, reducing the need for additional studies to investigate pharmacokinetic properties and toxicity. Repositioning of drugs for a new indication may nevertheless be accompanied by side effects not previously encountered and will require validation in a new clinical trial. However, the safety profile is likely to resemble that of the original indication, thus increasing the likelihood of the drug making it through the trial. Some of the drugs discussed in this review, like metformin used to treat diabetes type 2, have been discovered to have anti-cancer effect by coincidences. In the case of metformin, some epidemiological studies with diabetes patients showed that there could be a protective effect of this drug on tumor development. More recently, progress in bioinformatics has enabled drugs to be assessed for repurposing by making use of available experimental data, a method referred to as in silico drug repurposing. In such screenings, the identification of new drug candidates requires

additional experimental testing, but is still faster and easier than traditional drug development. In this review we will discuss a number of licensed drug that has the potential to be repurposed as anti-cancer drugs both in cancer prevention and therapy and highlight some of the data regarding signaling pathways and cellular mechanisms that are involved.

Drug repurposing in cancer prevention Aspirin Aspirin is a non-steroidal anti-inflammatory drug (NSAID) and widely used due to its analgesic, and antipyretic properties (12, 13). It has also however, a documented protective effect against cardiovascular disease, as daily intake of aspirin in low doses cause anti-platelet and anti-thrombosis effects through inhibition of COX-1, which again is involved in synthesis of Thromboxane A2, a key factor in the aggregation of platelets (14). Thus Aspirin is also currently used for prevention of thromboembolism in patients with manifest or increased risk for cardiovascular disease (15). Notably, accumulating data from in vitro and in vivo experiments, observational studies and prospective trials have firmly established that Aspirin has anti-neoplastic effects (16-19). Furthermore, numerous experimental and observational studies have established a close link between inflammation and cancer, suggesting that its antiinflammatory properties underly its cancer protective effects. For certain cancer types including colrectal cancer and hepatocarcinoma the inflammatory process is considered a main driver of carcinogenesis. Notably, Rothwell and colleagues reported that regular use of aspirin significantly reduced the incidence of colorectal, esophageal, gastric, biliary and breast cancer after systematically comparing randomized trials with cohort and case-control studies (20). In addition, a reduced risk of metastasis was observed (21-23). Similar effects have also been reported for prostate and lung cancer, although less striking (24) On a mechanistic level, the anti-neoplastic effect of Aspirin has been attributed to its inhibition of cyclooxygenase (COX) enzymes (25) that promote carcinogeneis through synthesis of PGE2. These enzymes represent the rate-limiting step in the synthesis of various prostaglandins, including PGE2 that exert pro-inflammatory and

immunosuppressive effects. PGE2 is present at increased levels in tumour tissue from various cancer types (26) and has been shown to facilitate malignant transformation (27). The effects of PGE2 are mediated through E prostanid (EP) receptor signalling impacting on G-protein coupled receptors. This pathway may promote proliferation, invasion and secretion of angiogenic factors as well as inhibit apoptosis (28). Apart from inhibiting the synthesis of PGE2, Aspirin has also been linked to increased expression of 15-hydroxyprostaglandin dehydrogenase (15-PDGH) wich inactivates PGE2 (29). Furthermore, Aspirin reportedly block PGE2-induced secretion of CCL2 and thereby recruitment of myelo-derived suppressor cells. excert pro-inflammatory activities and contributies to immune supression (28). Aspirin also modulate immune function by increasing COX-dependent production an epimer of Resolvin D1 that resolves inflammation and has a longer half-life, 17(R)-resolvin-D1(30). Aspirins’ ability to inhibit platelets may aditionally regulate immune function since activated platelets supress NK cell-mediated lysis of tumor cells (31). The anti-cancer effect of Aspirin has also been linked to the phosphatidylinositol-3-kinase (PI3K) signalling pathway, as the risk of cancer seems most strongly reduced in colorectal cancers harboring mutations in PIK3CA (32), a catalytic subunit of PI3K. Apart from its inhibition of COX enzymes, acetylation by Aspirin impacts the funcion of multiple protein targets, and has been shown to inhibit activation of the transcription factor NFκB which regulates the expression of numerous genes involved in apoptosis and malignant progression. Aspirin also disrupts the Ras-Raf-MEK-ERK signaling cascade by inhibiting interaction between several components within this pathway (33). The various effects of aspirin is summarized in figure 1. Although prophylactic aspirin is promising in cancer prevention, the overall benefit from long-term use may be compromised by well-known side effects such as intracerebral and gastrointestinal bleeding (34-36). The recommendations for population-wide use of aspirin remain unclear (24). A benefit-harm analysis performed recently by Stegemann et al, suggest that the population at age 40-85 would have greater benefit than harm when using aspirin as a primary cancer prevention tool (37). Another recommendation for daily intake of aspirin for patients above 40 years with increased risk cardiovascular disease and colorectal cancer with low risk of developing side-effects, was recently published by U.S Preventive Services Task Force Recommendation Statement (USPSTF) (38).

Statins Statins inhibit the rate-limiting enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) and thereby the mevalonate pathway that constitutes the initial step in cholesterol biosynthesis. They are among the most commonly prescribed drugs used to treat lipid disorders, due to their effectiveness in preventing development of cardiovascular diseases. Importantly, data suggest that inhibition of the HMG-CoA may also be a mechanism for a protective effect against cancer, since the mevalonate pathway provides Geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP). These compounds are used for prenylation of proteins, a process critical for directing these proteins to the cell membrane. Interference with this process may therefore be disruptive to cell cycle progression and cell proliferation (39, 40), thereby mediating anti-neoplastic effects (41, 42). Several observational studies and meta-analyses have investigated the relationship between cancer incidence and statin use. Both studies of all cancer types as well as specific cancers are inconsistent, although several studies suggest a positive correlation with reduced incidence of gastrointestinal cancers. One meta-analyses including 7611 patients with gastric cancer in 26 randomised controlled trials (RCTs) and 8 observational found a 27% risk reduction associated with statin use (43). Similarly, meta-analyses have reported that statin use is associated with a reduced risk for esophageal cancer (44) and hepatocarcinoma (45). For other prevalent cancer types including breast (46) and colorectal cancer (47) however, several cohort and case-control studies as well as meta-analyses have reported only weak or no significant correlation between reduced cancer risk and statin use. In summary, studies published up to this point have reported conflicting results, and are overall inconclusive regarding the association between statin use and cancer incidence. It should be emphasized however, that most RCTs that have been analyzed have had endpoints relating to cardiovascular disease. A possible role for statins in cancer prevention can only be determined through carefully designed RCTs with a sufficiently long follow-up and cancer incidence as a primary endpoint.

Metformin

Metformin is a first-line oral drug for treatment of type 2 diabetes, and generally well tolerated. Diabetic patients have an increased risk of developing several cancer types. A meta-analysis of 20 studies from 2007 showed that diabetic women have a 20% higher risk of developing breast cancer, and that the risk of cancer-related death is increased (48). Furthermore, several other studies have linked diabetes, in particular type II diabetes, to development of non-Hodgkin’s lymphoma (49, 50), pancreatic (51), colorectal (52), bladder (53) and endometrial (54) cancer. In general, any measure to improve glycemic may therefore be expected to protect against cancer development. A more detailed description of the mechanisms underlying the anti-cancer effect of metformin is provided in the section addressing the use of metformin in cancer treatment (55, 56). Several systematic reviews suggest that diabetic patients using metformin both has reduced incidence and mortality compared to patients on other treatment regimes. Furthermore, in site-specific analysis, the authors found that in particular colorectal, hepatocellular, pancreas, stomach, liver, esophagus and lung cancer occurred at a lower incidence among metformin users (57, 58). In a larger meta-analysis of several anti-diabetic drugs, including 66 articles with metformin, it was found that patients using metformin had an overall decreased risk of developing cancer and a decreased mortality by 14% and 30% respectively. Other anti-diabetic drugs, like insulin, were on the other hand associated with both increased risk and mortality of cancer (59). Despite these findings, no randomized controlled trial has been published that investigates the impact of metformin use on cancer incidence. However, a recent multicenter doubleblind, placebo-controlled, randomized phase 3 trial was recently published investigating the effect of low dose metformin, using the occurrence of colorectal adenomas as a biomarker for cancer as primary endpoint at 1 year after intervention. Notably, colonoscopy showed that both the occurrence and number of adenomas/polyps were reduced in the patients using metformin compared to the control group (60).

SERMs – selective estrogen receptor modulators Selective estrogen receptor modulators (SERMs) are drugs that act on the estrogen receptor (ER) and mediate different effects depending on the organs or tissue. This way, SERMs may simultaneously stimulate the ER pathway in some tissues and inhibit the pathway in others. Tamoxifen, originally developed as a fertility drug, has

been known to have anti-cancer properties since the 1970s, and in 1998 FDA approved tamoxifen as a preventive drug against breast cancer recurrence (61). Another drug belonging to this class is Raloxifene, a benzothiaphene type of SERM, which mimics the action of estrogen in bone tissue, and was initially approved for treatment of osteoporosis in menopausal and post-menopausal women. Raloxifene acts directly on the bone cells, and regulates both the proliferation and activity of osteoclasts and osteoblasts (62). Furthermore it modulates the expression of several matrix proteins important for osteogenesis, as well as collagen and IL-6, the latter important for osteoclast differentiation. In breast and endometrium however, it has anti-estrogen effects through its binding of the ER’s ligand binding domain. This region includes the AF-2 region that serves as a binding site for coactivators and corepresssors. These factors regulate the binding of the Estrogen Receptor to specific DNA sequences called Estrogen responsive elements and thereby regulate transcription of target genes as well. Upon its binding to raloxifene, the ER undergoes a conformational change preventing the AF-2 region from binding coactivators. As a consequence transcription that is normally activated by binding of the estrogen-bound ER to the estrogen response element is instead blocked. In a large multicenter study involving 180 clinical centers in 25 countries, the MORE trial, 7705 postmenopausal women with osteoporosis were evaluated for the risk of breast cancer development. Initial clinical studies showed that postmenopausal women taking raloxifene had increased bone mineral density and a decreased risk of vertebral fractures compared to the placebo group (63). However, occurrence of breast cancer was evaluated as a secondary end point. Risk of invasive breast cancer was reduced by 76% and 72% after 3 and 4 years respectively in the raloxifene group compared to controls, and specifically the risk of estrogen receptor-positive invasive breast cancer was reduced by 90% and 86% after 3 and 4 years (64, 65). The study also investigated the effect of treatment with raloxifene on the endometrium and did not find an increase in the risk of endometrial cancer, which has been reported for tamoxifen. In a follow-up of this study, the CORE trial, the effect of raloxifene was further analyzed after eight years with patients from the MORE trial. The CORE trial found that the breast cancer protection of raloxifene persisted, and that the risk of invasive breast cancer and estrogen receptor-positive breast cancer after eight years was reduced by 66% and 76%, respectively, in the raloxifene compared to the placebo

group (66). Further clinical investigation of raloxifene in breast cancer prevention concluded that raloxifene was as efficient as tamoxifen in risk reduction and that the use of raloxifene was associated with a decreased risk of endometrial cancer as well as less side effects such as DVT, pulmonary embolism and cataracts compared to tamoxifen (67, 68). Raloxifene received approval in the US for breast cancer prevention in potmenopausal women in 2007 and guidelines for the use of SERMs have been issued. In many countries the use of these agents are still limited.

Drug repurposing in cancer therapy Cardiovascular drugs Cardiac glycosides such as digoxin and digitoxin are compounds found in plants and animals that are used to treat different cardiac conditions (69). They inhibit the Na+/K+-ATPase, an ion pump critical for maintaining membrane potential and osmotic stability (70), and increases sodium ion concentrations (71). As a compensatory mechanism, activation of the Na+/Ca+ pump increases Ca+ influx and thereby induces contractility of the myocardial cells (69, 71). Already in 1967 Shiratori reported anti-proliferative effects on HeLa cells by cardiac glycosides (72), and growth inhibitory effects have subsequently been described on cancer cells from pulmonary tumors (73), prostate cancer (74) and breast cancer (75). Anti-proliferative effects were found to be mediated by the release of cytochrome c from the mitochondria, followed by activation of caspases (74) and interference with DNA replication, mainly by inhibiting DNA topoisomerases (75). In addition cardiac drugs also induce the expression of p21Cip1, an inhibitor of cyclin-dependent kinases, resulting in growth arrest (76). Finally, data also suggest that cardiac glycosides can eradicate established xenograft tumors by suppressing HIF-1α and HIF-2α expression (77). These transcription factors are critical regulators of a large number of genes essential for tumors’ adaption to hypoxic environments. Expression of HIFs are often increased in tumors, and are associated with a worse prognosis (78). The number of clinical studies validating cardiac glycosides as cancer drugs is low. A recent clinical trial on ovarian cancer concludes that patients on digoxin at the time of cancer treatment with surgery and platinum-based chemotherapy did not have any survival benefit compared to patients not on digoxin (79). Furthermore, several observations question the clinical feasibility of using cardiac glycosides in cancer

treatment as digitoxin inhibits general protein synthesis in both malignant and nonmalignant cells (80). In addition, mouse cells was found to tolerate much higher doses of the drugs compared to human cells, questioning the relevance of previously conducted pre-clinical studies to a clinical setting. Importantly, these glycosides are also associated with significant toxicity and unwanted drug-drug interactions (81, 82).

Aspirin is used in cardiovascular disease to treat and prevent myocardial infarctions, and it targets the cyclooxygenase (COX) enzymes 1 and 2. COX1 is responsible for the production of thromboxane A2 in platelets, which subsequently leads to platelet aggregation and platelet adherence to cells such as tumor cells. Platelet coverage of tumor cells prevents recognition of immune cells, and thus favors the metastatic process. COX2 is responsible for the production of prostaglandin E2, which can work directly on tumors cells by promoting proliferation (83). Some studies have investigated the role of aspirin as a therapeutic agent in established or chemically induced tumors (19, 84), and several clinical studies indicate that post diagnosis use of aspirin is associated with a favorable survival in patients with colorectal cancer (85-87). However, as most studies are retrospective and the patient selection is heterogeneous, there is a large degree of conflicting findings. Prospective studies are needed to establish the role of aspirin as a therapeutic cancer agent. Several clinical trials are currently ongoing, most investigating the role of aspirin in prevention of disease recurrence.

Beta-blockers are widely used against a range of cardiac conditions, and its main target is beta-adrenoreceptors. These receptors are activated by catecholamines, and their activation is thought to promote tumorigenesis. Catecholamines are increased under chronic stress, and in addition to increased proliferation, the cellular effects include up-regulation of angiogenesis (88, 89) and metastasis (90). Blocking beta-adrenoreceptors can induce anti-proliferative and anti-migratory effects and increase survival in experimental animal models of pancreas (91) and colorectal cancer (92, 93). Furthermore, use of beta-blockers in hypertensive women has in one study been linked to prevention of disease progression of breast cancer; however, the study subject number was low (n=43) (94). By stratifying for breast cancer subtypes, Melhem-Bertrandt and colleagues found that relapse-free survival was better in

women with triple negative breast cancer compared to the other groups when treated with beta-blockers (95).

Metformin is a biguanide compound commonly used to treat diabetes type II. Its mechanism is thought to be inhibition of oxidative phosphorylation, causing energetic stress and inhibition of gluconeogenesis, especially in liver cells (96). Several epidemiological studies have linked metformin intake to reduced cancer risk (discussed above in the cancer prevention chapter). Notably, metformin use among diabetes patients with established cancer has been associated with a favorable response to therapy and increased survival in cancers such as hepatocellular carcinoma (97), colorectal (98), prostate (99), HER2+ breast cancer (100), ovarian (101), pancreas (102), esophageal (103) and rectal cancer (104). In some cancers, the expressions of insulin receptors are elevated, resulting in increased PI3K-mTOR signaling that promotes cellular processes such as proliferation. Thus, some of the anti-neoplastic effects of metformin are likely mediated through interference with insulin signaling (fig. 2) (96). However, metformin may induce energetic stress directly on the mitochondria of tumor cells (105, 106). This is mediated through interference with mitochondrial respiration, decreasing activity of the citric acid cycle and overall production of ATP (106). Cancer cells may counteract this effect by compensatory increasing ATP production through elevated aerobic glycolysis. Thus, the anti-neoplastic effect of metformin depends on the cancer cells ability to activate glycolysis-mediated ATP production and the availability of glucose in the tissue. In addition, cancer cells with mutations in respiratory complex I genes, and thus inability to up-regulate oxidative phosphorylation respond better to metformin (107). Metformin also activates AMP-activated protein kinase (AMPK) by phosphorylation (108, 109). AMPK has several downstream effects, among them inhibition of anabolic pathways and decreased protein synthesis (110, 111), the latter is associated with mTOR down-regulation (112). Indirectly, metformin also activates AMPK through the tumor suppressor liver kinase B1 (LKB1) (113). Mechanistically, mTOR inhibition by metformin seems to occur at the level of translation, and metformin treatment leads to suppression of mRNA translation in general, but also specifically in a set of cancer-promoting genes (114). In breast cancer cells, growth inhibition upon metformin treatment is mediated through AMPK

activation (115) and altered mRNA translation by inhibition of translational proteins such as ribosomal S6 kinase and the eIF4E-binding protein 1 (114, 116). Furthermore, inhibition of the factors 4E-BP1 and EF2, also important for translation, is regulated downstream of AMPK activation by metformin (117). In leukemic cells AMPK also inhibits the unfolded protein response (UPR), which is normally activated to eliminate proteins with incorrect folding in the endoplasmatic reticulum. Thus metformin treatment may cause an accumulation of ER stress by unfolded proteins that are not removed due do defect UPR, resulting in apoptosis (118). A similar mechanism is reported in a study of prostate cancer, in which apoptosis is induced by ER-stress caused by metformin (119). At anti-diabetic doses of metformin, a phase II randomized controlled trial treating patients with advanced pancreatic cancer concluded that there was no survival benefit for the patients by addition of metformin to the standard chemotherapeutic regime (120). However, several clinical studies on breast and endometrial cancer have indicated that at anti-diabetic doses, metformin is able to reduce the number of proliferating cells (121-123). Some studies also suggest that tumors respond to metformin depending on their molecular subtype. In diabetic patients with HER2+ breast tumors, metformin use was associated with a favorable survival outcome compared non-users (100). Clinical findings also suggest that metformin may impact on the disease course in castration-resistant prostate cancer, measured by a reduction in PSA levels, a marker used to evaluate disease status (124).

Antipsychotic and antidepressant drugs Antipsychotic drugs are commonly used to treat conditions such as psychosis, schizophrenia and bipolar disorder, whereas population based studies have indicated that some of these drugs may also reduce the risk of certain cancers (125).

Chlorpromazine (CPZ), which is a derivative of phenothiazine, has traditionally been one of the most important anti-psychotics for treatment of schizophrenia and psychosis, and emerged already in the late sixties and early seventies as an attractive candidate for repurposing (126, 127). A study from the early nineties indicated that schizophrenic men using chlorpromazine might have a decreased risk of developing prostate cancer (128). Chlorpromazine, as well as other phenothiazines, has been

found to inhibit tumor growth in several cancers, including hepatocellular carcinoma (129), glioma (130, 131), leukemia (132) and melanoma (133). Numerous studies have consistently showed that Chlorpromazine exerts antiproliferative and pro-apoptotic effects. The mechanisms underlying these effects have mainly been linked to altered expression of several cell cycle related proteins or interactions with these proteins, as well as interference with mitochondrial processes. In glioma cells, CPZ increases expression of the cyclin-dependent kinase inhibitor p21, causing cell cycle arrest in the G2/M-phase. (130). In colorectal cancer cells, chlorpromazine induces apoptosis via up-regulation of p53 (134). Activation of p53 was dependent on signaling through JNK, a member of the mitogen-activated protein kinase (MAPK) family. CPZ also inhibits the mitotic kinesin KSP/Eg-5, leading to ineffective centrosome separation and consequently mitotic arrest (135). Moreover, CPZ reduces the association of the oncogene K-Ras with the plasma membrane leading to increased KRas in the cytoplasm and internal membranes, accompanied by both growth arrest and apoptosis (136). Notably, Chlorpromazine also reduces mitochondrial ATP production and DNA polymerase activity in the mitochondria of leukemic cells, leading to apoptosis at concentrations that does not affect viability of normal lymphocytes (132). In glioma cells, CPZ was found to induce autophagy through inhibition of the Akt/mTOR pathway (131). In a study of the effect of the phenothiazine derivative trifluoperazine on metastasis, it was found that the potential for cancer cell angiogenesis and invasion is reduced upon drug treatment, an effect mediated through interference with βcatenin signaling (137).

Penfluridol (PF) is a first generation antipsychotic drug used to treat schizophrenia that exerts anti-proliferative effects, presumably by interference with integrin signaling. Anti-tumor effects have been demonstrated in vivo, both in localized disease and in an experimental model of breast cancer metastasis to the brain (138). In pancreatic xenograft tumors, penfluridol slowed tumor growth by inducing apoptosis (139). Contrary to several other anti-psychotic drugs, this study also established that continuous use of penfluridol was not associated with major toxicity.

Fluspirilene is an anti-psychotic drug used to treat schizophrenia and belongs to the

class of diphenylbutylpiperidines. It was identified as a cancer drug candidate through in silico screening studies by altering expression levels of several cell-cycle proteins, such as CDK2, cyclin E and Rb, similar to CPZ. Through a computational approach, fluspirilene was also identified as a drug that could bind to MDM2, possibly blocking the interaction between p53 and MDM2. Importantly, experimental validation has confirmed that fluspirilene possesses strong anti-proliferative effects in hepatic carcinoma cells, due to reduced expression and phosphorylation of CDK2, leading to accumulation of cells in the G1 phase of the cell cycle (140). Fluspirilene also inhibited hepatocellular carcinoma xenografts in mice. Notably, experimental studies showed that fluspirilene inhibits proliferation in various cancer cell lines in a p53 dependent manner consistent with the hypothesis that it interacts with MDM2 in the p53-binding pocket, making p53 available for activation and consequently induction of apoptosis (141).

Antidepressants Tricyclic antidepressants are a class of drugs containing three fused rings in their structure and are used to treat clinical depression and other mood disorders. Although the exact mechanism is not fully elucidated, their effects are partly mediated by inhibiting reuptake of amine neurotransmitters, such as serotonin and norepinephrine transporters. It has also been suggested that these drugs change neuronal plasticity in response to atrophy and cell death seen in depression (142). Notably, several studies have reported antidepressants to have antineoplastic effects (143). Mechanistically it has been shown that tricyclic agents such as clomipramine exerts its antineoplastic effect by inhibition of mitochondrial complex III, leading to decreased oxygen consumption and consequently induction of apoptosis via caspase activation (144). In addition to caspase activation, signaling through c-jun and release of cytochrome c from mitochondria is proposed to be involved in induction of apoptosis for glioma and neuroblastoma cells (145). Similar findings have also been reported for leukemic cells (146-148). Amitriptyline, another tricyclic drug, is suggested for use as an oxidation therapy agent, i.e. a drug that has the ability to both increase levels of reactive oxygen species, as well as lowering antioxidant levels (149).

Lithium (LiCl) has traditionally been used for treatment of depression. Its effects on cancer cells have largely been attributed to the inhibition of glycogen synthase kinase 3, which again impacts on multiple cellular functions (150). GSK3 phosphorylates and inactivates glycogen synthase, an important molecule in glycogen synthesis, and a negative regulator of WNT signaling. (151). However, lithium also changes the release of neurotransmitters, modulates the activity of several phosphoproteins, and directly inhibits inositol monophosphatase (IMPase), important for signaling through G-protein coupled receptors (152). Sun et al showed growth inhibitory effects of LiCl through GSK3 inhibition in prostate cancer cell lines and tumor xenografts, due to reduced interaction between the transcription factor E2F and DNA that induce S-phase gene expression (153). LiCl has also been shown to increase the effect of doxorubicin and etoposide, which act directly on the cell cycle, in prostate cancer cell lines (154, 155). Recent findings on colon cancer cells suggest that although lithium did not inhibit growth, it can prevent metastasis through inhibition of lymphangiogenesis. Inactivation of GSK-3 by lithium down-regulated Smad3, which again reduced expression levels of transforming growth factor beta-induced protein (TGFBIp), a key mediator of lymphangiogenesis in colon cancer (156). Long-term use of lithium has been associated with nephropathy and some indications have been made linking development of renal cancers to chronic use of lithium (157, 158). However, other reports have not supported these observations (159). In a Danish cohort, overall colorectal carcinoma risk was not affected by the use of lithium, although a slight overall risk for distal colon tumors were seen (160). A clinical study of lithium for use in low-grade neuroendocrine tumors concluded that despite promising pre-clinical results, the drug did not result in any anti-tumor effects at doses used for bipolar disorders and depression (161).

Microbiological agents Artemisinins were discovered as part of a drug discovery project for the treatment of malaria infections. Artemisinins are extracted from plants and have been used in traditional Chinese medicine (162). When given as treatment for malaria, the artemisinins induce formation of reactive oxygen species within the infected red blood cells thereby killing the parasite (163). The parasite affects the hemoglobin of

the red blood cells, and some of the effects of artemisinins have been attributed to the reaction of their endoperoxide groups with the iron-containing heme molecules in parasite-infected RBCs (162). Artesunate is the most studied artemisinin for drug repurposing in cancer. It has been linked to anti-angiogenic effects, production of ROS and modulation of drug resistance in various cancer cell types in vitro and in vivo (164). Anti-proliferative and pro-apoptotic effects of artesunate have been found in lymphoma and myeloma cells (165), as well as in hepatocellular carcinoma (HCC) (166, 167). The effects are mediated by up-regulation of pro-apoptotic proteins such as caspase-3, a decrease in MYC oncogene and down-regulation of several anti-apoptotic proteins. In hepatocellular carcinoma the anti-neoplastic effect of artesunate is increased in combination with sorafenib (166) and gemcitabine (167). Furthermore, antiangiogenic effects have been reported both for renal cancer and HCC with reduced tumor growth in vivo, with decreased the number of vessels and down-regulation of vascular endothelial growth factor (168). Dihydroartemisinin (DHA) is another derivative of artemisinin, and shows growthinhibitory effects on leukemia cells through ROS-dependent autophagy and subsequent apoptosis (169). The DHA effect was mediated in part by down-regulation of the protein transferrin receptor 1 (TfR1) and cell cycle growth arrest. In hepatoma and breast cancer cells, DHA treatment resulted in a depletion of iron due to the reduced expression of TfR1 that has a critical role in iron uptake (170). Decreased expression of TfR1 has also been observed in leukemic cells (171). Sensitivity to DHA was seen in a number of papilloma virus infected cells and cervical cancer cells, and found to be dependent on TfR1 expression. Tumor growth was furthermore inhibited in an in vivo papilloma model with DHA treatment (172).

Mebendazole (MZ) is commonly used to treat parasitic worm infections. The antiparasitic effect is mediated through inhibition of tubulin polymerization (173). Some case reports with administration of mebendazole to cancer patients suggest it can also have anti-tumor efficacy in a clinical setting (174, 175). One of the first reports on anti-tumor effect of Mebendazole came in 2002 from Mukhopadhyay and colleagues (176). MZ inhibited the growth of lung cancer cells in vitro, whereas normal endothelial cells and fibroblasts were not affected. In mice, MZ treatment resulted in smaller tumors compared to control groups, and angiogenesis

was inhibited. Moreover, the number of metastases was reduced in the treatment group (176). Other studies have since shown growth inhibition in vitro and in vivo in cell lines from other cancer types including lung cancer (177), colon cancer (178), melanoma (179, 180), glioblastoma (181) and medulloblastoma (182). In melanoma, data suggest that the effect of mebendazole is mediated through induction of apoptosis, specifically by up-regulation of caspases and the pro-apoptotic Bcl-2 and by inhibition of the negative regulator of apoptosis; X-linked inhibitor of apoptosis (XIAP) (179, 180). In colon cancer cell lines, MZ was also showed to have high binding affinities and thus potentially be an inhibitor of several kinases and oncogenes such as ABL and BRAF (178), whereas it acted as a Hedgehog inhibitor in medulloblastoma (183). Mebendazole can also be combined with temozolomide that is routinely used for treatment of malignant gliomas. This combined treatment inhibited tumor growth both in a xenograft and a syngenic model of glioma more than temozolomide alone (181). Notably, case reports suggest that mebendazole exhibit strong anti-tumor effects in some patients. In a patient with metastatic colon cancer, mebendazole was administered after relapse from first- and second-line treatment. Apart from an increase in levels of liver enzymes, after which the dose was lowered, the patient did not experience any adverse events, and mebendazole treatment effectively eradicated almost all metastasis in the lung and lymph nodes, as well as gave a partial remission of liver metastases (174). In a patient with adrenocortical carcinoma, mebendazole was administered upon failure of several chemotherapeutic drugs. No adverse events were seen, the size of metastases decreased and the disease remained stable for 19 months (175).

Itraconazole is an anti-fungal drug that inhibits 14-α-lanosterol demethylase (14LDM), an enzyme important for cholesterol synthesis. In addition, Itraconazole inhibits angiogenesis by decreasing endothelial cell proliferation (184), and inhibits both expression and various downstream targets of VEGFR2 in endothelial cells (185). Treatment of human vascular endothelial cells (HUVECs) with Itraconazole decreased phosphorylation of growth factor receptors, inhibiting migration and tube formation (186). In the same study, Itraconazole treatment decreased angiogenesis and regression of non-small cell lung cancer xenografts. In addition to the anti-angiogenic effects, the anti-tumor efficacy of this compound

may involve other mechanisms: In glioblastoma cells the removal of cholesterol from the plasma membrane lead to decreased AKT1 activity, and subsequently inhibition of its downstream target mTOR, leading to induction of apoptosis and decreased proliferation (187). Itracondazole has been proposed as a Hedgehog inhibitor through Smo, and the in vivo growth of two hedgehog-dependent tumor models, a medulloblastoma and a basal cell carcinoma of the skin, was reduced in animals receiving the drug (188). Similar findings were also reported in a study of pleural mesothelioma cells (189). In a randomized phase II trial with metastatic castration-resistant prostate cancer, daily administration of Itraconazole was moderately effective, with a prolonged PSA progression free survival, reduction in circulating tumor cells and inhibition of Hedgehog signaling (190). In a small phase II study of basal cell carcinoma, 19 patients were treated with Itraconazole (191). Drug treatment resulted in reduced hedgehog signaling accompanied by decreased proliferation and regression of the tumor area. Combining Itraconazole with standard chemotherapy for lung cancer patients in a small phase II study showed that the combination increased both progression free as well as overall survival, and the authors speculate that the effect may be attributed to the anti-angiogenic properties of the drug (192). Some contraindications for the use of Itraconazole exist. There is some evidence that the use of antifungal drugs may interfere with the actions of other anticancer agents, in particular molecular antibodies such as rituximab (193).

Anti-viral drugs Incidences of some HIV-related cancers, like Kaposi’s sarcoma and lymphoma, have decreased after the introduction of protease inhibitors against HIV. Although, the evidence for preventive effects of these drugs still is scarce (194), there are accumulating preclinical data suggesting antineoplastic effects.

Ritonavir is widely used in HIV treatment to increase the potency of other protease inhibitors. A substantial body of data obtained from various cancer types shows that ritonavir can inhibit cell cycle progression, induce apoptosis and alter metabolism through multiple mechanisms. Down-regulation of Rb, cyclin dependent kinases and anti-apoptotic proteins, in addition to up-regulation of p53, result in inhibition of cell cycle progression and increased apoptosis in ovarian, pancreatic and breast cancer

cells (195-197). Ritonavir also inhibits Akt phosphorylation, in breast cancer cells mediated through binding of Hsp90 (197). Levels of the glucose transporter GLUT4 are decreased upon ritonavir treatment (198). As many tumors rely on increased glucose metabolism, treatment with ritonavir may decrease viability. Glucose metabolism was investigated in samples from chronic lymphocytic leukemia patients, and some were found to be sensitive to ritonavir treatment (199). Resistant cells could be sensitized by co-treatment with metformin, which is an inhibitor of oxidative phosphorylation in the mitochondria that may provide a possible compensatory mechanism upon targeting of glycolysis. The same group reported similar findings in a xenograft myeloma animal model (200). Notably, several studies suggest that ritonavir potentiates the efficacy of other drugs. Ritonavir reportedly increases the anti-tumor efficacy of temozolomide in glioma cell lines, an effect that is further increased by adding the antiemetic drug aprepitant (201). In leukemic cells, ritonavir increased the effect of the chemotherapeutic drug ATRA, both in sensitive and resistant cells, and promoted cancer cell differentiation (202). Ritonavir has been investigated as a co-treatment with the proteasome inhibitor bortezomib. In contrast to hematological malignancies, bortezomib has limited efficacy in solid tumors. However, ritonavir induced an unfolded protein response in sarcoma cells, sensitizing otherwise bortezomib-resistant cells to bortezomibdependent apoptosis (203). Similar findings on synergistic effects of bortesomib and ritonavir were reported in a study of renal cancer, where ritonavir was also found to induce histone acetylation by inhibition of histone deacetylases (204). In a subsequent study, the authors also showed that ritonavir works synergistically with panobinostat, a non-selective histone deacetylase inhibitor used for multiple myeloma, in renal cancer cells (205). In combination with lopinavir, ritonavir was administered to patients with high-grade glioma in a phase II study enrolling 19 patients. One patient experienced complete remission, but there was no improvement in progression free survival compared to standard therapeutic regimes for these patients. The authors suggested that restricted delivery to the brain might explain the limited efficacy (206).

Nelfinavir is another protease inhibitor effective against both HIV-1 and HIV-2. As for ritonavir, the drug has anti-cancer effects, with inhibition of Akt-signalling and induction of ER stress proposed as the main mechanisms. Nelfinavir treatment can

reduce phosphorylation of both STAT3 and Akt, and regression of prostate xenograft tumors has been seen upon nelfinavir treatment (207). In multiple myeloma, treatment results in decreased signaling through Akt, STAT3 and Erk1/2 via inhibition of the 26S proteasome (208). Nelfinavir-mediated Akt-inhibiton also results in decreased expression and secretion of VEGF in head and neck squamous cell carcinoma and lung carcinoma, in addition to reduced levels of HIF-1α upon treatment in hypoxia. Furthermore, nelfinavir inhibits angiogenesis in animal models, and intra-tumoral oxygen levels were increased in the treatment group, leading to an increased effect of radiation in the nelfinavir-treated animals (209). Nelfinavir is a potent inducer of endoplasmatic reticulum (ER) stress and subsequent inducer of an unfolded protein response (UPR) in non-small cell lung cancer (210), ovarian cancer (211), liposarcoma (212), and breast cancer cells (213). In liposarcoma cells nelfinavir treatment results in increased levels of sterol regulatory element binding protein-1 (SREBP-1) and activating transcription factor 6 (ATF6), both transcription factors located in ER membranes. These levels increase as a consequence of nelfinavir inhibiting the enzyme site-2-protease (S2P), which normally cleaves the membrane bound transcription factors to participate in protein folding (212). Similar blocking of intramembrane proteolysis has been seen in castration-resistant prostate cancer cells (214, 215). Induction of ER-stress by nelfinavir and COX-2 inhibitors was found to enhance the effect of chemotherapeutic agents in breast cancer cell lines and their chemoresistant derivatives (213). The nelfinavir/COX-2 inhibition combined with drugs inhibiting autophagy could enhance cytotoxic effects in triple negative breast cancer both in vitro and in vivo (216). Pro-apoptotic effects of nelfinavir treatment are mediated by activation of CHOP and caspase-4, as well as an up-regulation of death-receptor 5 (DR5) (217, 218). Studying cervical cancer cells, Xiang et al found that the induction of apoptosis in these cells was mediated by the production of reactive oxygen species in the mitochondria upon nelfinavir treatment. Nelfinavir also reduced the levels of MnSOD, an enzyme responsible for neutralizing mitochondrial ROS (219). As for ritonavir, nelfinavir also works synergistically with bortezomib. In a study of non-small cell lung cancer and multiple myeloma, combination of nelfinavir and bortezomib enhanced ER stress and effectively caused growth inhibition both in vitro and in vivo. Mechanistically, nelfinavir treatment leads to accumulation of ubiquitin (Ub)-proteins while degradation by the proteasome is inhibited by bortezomib,

resulting in proteotoxic stress and cell death (220). Furthermore, multiple myeloma cells can be sensitized to bortezomib by nelfinavir treatment. Nelfinavir inhibits the β2 activity of the proteasome, to which bortezomib is not effective, and it is suggested that this together with inhibition of Akt-phosphorylation is the main mechanisms for the antineoplastic effects of nelfinavir in multiple myeloma cells (221). In a phase I study of liposarcoma, administration of nelfinavir was associated with limited toxicity and plasma levels of the drug was in the range of the anti-proliferative doses seen in pre-clinical experiments. Some patients observed a clinical benefit with partial response or stable disease (222). Hoover et al conducted a phase II trial with nelfinavir against adenoid cystic carcinomas. Several patients experienced adverse events, and the authors concluded that the use of nelfinavir as monotherapy for this cancer had limited efficacy, but that the drug might be effective in combination with other agents (223). Combination of nelfinavir with bortezomib in a phase I trial of hematological cancers showed that out of 10 patients, 3 had partial response and 2 had stable disease. Patients that previously failed bortezomib treatment could be sensitized to bortezomib treatment again when administered together with nelfinavir. The trial concludes that the combination of nelfinavir and bortezomib seems promising with regards to efficacy and that the observed toxicity profile was acceptable (224).

Antibiotics Doxycycline is a tetracyclic antibiotic, effective against a range of infectious diseases. Some tetracyclines were in the early nineties shown to be effective in inhibition of angiogenesis (225), and it was later found that doxycycline had growth inhibitory effects in osteosarcoma, prostate cancer and mesothelioma cells (226-228). Furthermore, pro-apoptotic effects of doxycycline have been observed in pancreatic cells (229, 230), and leukemic cells (231). Tetracyclines are associated with inhibition of matrix metalloproteinases (MMPs) (232). Doxycycline treatment down-regulates the expression of MMP-2 and MMP-9 in leukemic (233) and colorectal cancer cells (234), followed by an inhibition of invasion (234). As MMPs are thought to be important for invasion and metastasis, doxycycline has also been investigated in experimental studies of cancer metastasis. In an animal study of bone metastasis from breast cancer, doxycycline was found to decrease the volume of tumors in the bone and also in the soft tissue surrounding the bones (235). The same authors also showed that the metastatic disease could be

further inhibited in combination with zoledronic acid, a drug used to prevent bone fractures in patients with bone metastasis or osteoporosis (236). Decreased metastasis through MMP-2/9 inhibition has also been reported in animal studies of prostate cancer (237) and oral squamous cell carcinoma (238). Furthermore, treatment with doxycycline can decrease expression of EMT-markers in lung cancer and hepatocellular carcinoma cells, resulting in a reversal of a pro-metastatic phenotype (239, 240). Hepatocellular carcinoma cells enriched with stem-like features had a decrease in colony formation ability and decreased expression of stem cell markers upon doxycycline treatment. In addition, the treatment also reversed EMT and regressed tumor growth of xenograft tumors (241). In a phase II clinical trial of metastatic renal cell carcinoma doxycycline was combined with interferon-alpha therapy. A possible anti-angiogenic effect was monitored by measurements of VEGF levels. Although some initial regression of VEGF levels were seen in some patients, the combination treatment was not effective in patients with metastatic renal cell carcinoma (242). A recent phase II trial in women with metastatic breast cancer has investigated the effect of doxycycline treatment in combination with bone-targeting drugs. The endpoints included evaluation of pain as well as markers relevant for bone metastasis. Doxycycline was found to have no effect in this study, but was instead associated with significant toxicity (243).

NSAIDs NSAIDs (Nonsteroidal anti-inflammatory drugs) comprise a drug class with analgesic, antipyretic and anti-inflammatory effects (244), such as ibuprofen, acetyl salicylic acid, naproxen and diclofenac. In many countries, these are available without prescription (245). Several epidemiological studies demonstrate that the use of NSAIDs is cancer preventive, and recent data also suggest that it may be effective in the treatment of established tumors (246). NSAIDs act by repressing the formation of eicosanoids, mainly through inhibition of the two enzymes cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), both of which catalyze the formation of eicosanoids from arachidonic acid (247). Whereas COX-1 is constitutively expressed and regulates normal physiological processes, COX-2 is expressed in an inflammatory setting (248) and is a well-known tumor

promoter (249). Common side effects of NSAIDs, such as gastrointestinal bleeding, due to COX-1 inhibition, while COX-2 inhibition has been linked to many of the desired effects of NSAIDs (249). This led to the development of selective COX-2 inhibiting drugs. Interetingsly, Celecoxib, a selective COX-2 inhibitor first used to treat pain related to arthritis, was approved by the FDA for treatment of familial adenomatous polyposis (FAP) in 1999 (250). Although the anti-cancer effect of COX-inhibition by NSAIDs is established, COXindependent pathways also exist. As such, it has been shown that all common NSAIDs were able to suppress NF-kappa-B-activation (251). Furthermore, NSAIDs reduce growth of cancer cells by induction of the gene MDA-7 (IL-24) (252), a member of the interleukin-10-family (253).

Ibuprofen is a non-selective COX-inhibitor. In cancer it has been shown to inhibit the growth of prostate cancer cells (254). It also displays a radiosensitizing effect in vitro, yet at higher concentrations than those reported to inhibit eicosanoid synthesis, indicating alternative mechanisms (255). In vitro studies on adenocarcinoma gastric cells indicate anti-tumor effects by anti-angiogenesis, induction of apoptosis, and reduction of cell proliferation, accompanied by altered expression of the cancerassociated genes Akt, P53, PCNA, Bax and Bcl2 (256). In a study of TNF-α mediated migration of metastatic melanoma cell lines, it was seen that administration of ibuprofen both as salt and in a hydrogel could revert migration. Although the mechanism is not fully elucidated, some of the effects were mediated by induction of apoptosis (257). Ibuprofen can also modulate chemosensitivity. In lung cancer cells, treatment with ibuprofen decreased the levels of Hsp70, a heat shock protein associated with resistance to apoptosis. Blocking of Hsp70 and subsequent induction of apoptosis increased the sensitivity to the chemotherapeutic drug cisplatin upon ibuprofen treatment (258).

Naproxen is a non-selective COX-inhibitor classified as a propionic-acid derivative, and has been shown to inhibit cell proliferation, trigger apoptosis and suppress metastasis. Antineoplastic properties have been demonstrated both in vitro and in vivo in leukemic, breast, colon, bladder and osteosarcoma cell lines. Through PI3K targeting, naproxen induces cell-cycle arrest and apoptosis in human urinary bladder cancer cell lines in vitro (259). In vivo, in an animal model of induced colon cancer,

the combination of atorvastatin and naproxen significantly inhibited colonic adenocarcinomas (260). In a phase II trial of recurrent prostate cancer, the combination of calcitriol and naproxen was assessed. The combination was well tolerated, and the prostate specific antigen (PSA) doubling time (PSADT) decreased for 19% of the enrolled patients, while 67% of the patients experienced a prolongation of PSADT compared to baseline (261).

Diclofenac is a derivative of acetic acid with a moderate selectivity for the COX2isoenzyme. Antineoplastic effects have been reported in several tumor types, including fibrosarcoma, hepatoma, colon, ovarian and pancreatic cancer. These studies have involved the effect of topical diclofenac 3% and calcitriol 3 mug/g on superficial basal cell carcinoma cancer types, including ovarian, breast, brain, colon, pancreatic, lung, liver and leukemic cancer cells (262, 263). Moreover, the growth rates and the levels of vascularization were substantially reduced in rat models of fibrosarcoma and hepatoma (264). Diclofenac was later found to have potent antiproliferative effect in human colon cancer cell lines (265). Diclofenac has also demonstrated tumor inhibition in a murine pancreatic cancer model (266), as well as for ovarian cancer (267). In vitro data suggest that induction of apoptosis upon diclofenac treatment is mediated through inhibition of the antioxidant SOD2, leading to increased levels of reactive oxygen species (268). Despite the growing body of evidence for antineoplastic effect of diclofenac, there are currently no ongoing clinical trials assessing diclofenac as treatment for any cancer type. However, a phase II clinical trial of basal cell carcinoma was recently completed. Diclofenac was assessed either as single therapy or in combination with calcitriol. The study concluded that topical application of diclofenac was more effective than combination treatment, and complete tumor regression was seen in 64% of the cases (269).

Other drugs Leflunomide is an immunomodulatory drug commonly used to treat rheumatoid disorders such as rheumatoid arthritis and psoriasisarthritis. Several pre-clinical studies have showed that leflunomide inhibits growth, disrupts cell cycle regulation

and induces apoptosis in thyroid (270), neuroblastoma (271), chronic lymphocytic leukemia (272) and glioma cells (273). Mechanistically, Leflunimode inhibits kinase activity of growth factor receptors such as EGFR and interferes with pyrimidine synthesis both in normal and cancer cells (274-276). A771726, the active metabolite of leflunomide, inhibits the enzyme dihydroorotate dehydrogenase (DHODH), an important factor in pyrimidine synthesis (277). Furthermore, A771726 inhibits phosphorylation of kinases, and modulates tumor-stroma interactions in multiple myeloma cells. In addition to DHODHinhibition, leflunomide also inhibits the Aryl Hydrocarbon Receptor (AhR), suppressing growth in melanoma cells (278). Leflunomide is also able to modulate the chemosensitivity of several cancer cell lines. In chronic lymphocytic leukemia, resistance to the chemotherapeutic drug fludarabine is common, partly mediated by increased CD40/IL-4 signaling and subsequent STATsignaling. This is counteracted by leflunomide treatment which decreases STAT3/6 phosphorylation and induce apoptosis through inhibition of NF-κB and anti-apoptotic proteins (279). Leflunomide may also inhibit angiogenesis, mediated by decreased VEGF expression as well as a decrease in microvessel density (280). However conflicting results exist, as low doses of leflunomide was found to activate the PI3K/Akt pathway, as well as counteract the induction of apoptosis by cytotoxic drugs such as etoposide (281). Thus, the potential of leflunomide for use in cancer therapy is uncertain.

Disufiram has been used as an alcohol deterrent for more than 60 years (282). The compound was initially used in the process of rubber vulcanization, and its first possible medical use was discovered when factory workers, regularly exposed to disulfiram, reported flu-like symptoms shortly after alcohol intake (283). Disulfiram inhibits the enzyme acetaldehyde dehydrogenase, which breaks down the acetaldehyde produced from enzymatic degradation of alcohol (284). Thus, disulfiram increased the acetaldehyde concentration of the blood, leading to severe “hangover symptoms” after alcohol ingestion. Disulfiram has received attention for its antineoplastic effects both as a single agent and in combination with other therapies. Some of the cytotoxic effects ascribed to disulfiram have been attributed to its binding of divalent cations, which interferes with copper- and zinc-dependent processes, such as angiogenesis and apoptosis (285-

287). In ovarian cancer cells, disulfiram treatment has led to apoptosis by a copperdependent induction of heat-shock proteins (288). Disulfiram has further been shown to stabilize IκB, the inhibitor of NFκB, which is deregulated in many cancers. This IκB stabilization has been found to re-sensitize treatment resistant breast and colon cancer cells with enhanced NFκB-signaling towards gemcitabine (289). In one study utilizing breast cancer cells, disulfiram demonstrated effects on spatial disorganization of late endosomes and lysosomes (290). Disulfiram has also re-sensitized treatment resistant glioblastoma cell lines to alkylating treatment by its potent inhibition of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) (291). Based on promising preclinical studies that suggested disulfiram-mediated decrease in toxicity and increase in efficacy upon cisplatin-treatment, a randomized phase II clinical study enrolling patients with known cisplatin-sensitive cancers compared the effects of cisplatin alone, or in combination with disulfiram. However, there was no statistically significant difference in response rate, median survival, or time to progression between the two groups (292). Moreover, a clinical dose escalation trial of disulfiram in patients with non-metastatic recurrent prostate cancer did not suggest any clinical benefits (293).

Auranofin is a gold complex that has been used to treat arthritis (294). Even before auranofin received FDA approval for its current use, the compound’s anti-cancer properties by induction of apoptosis were observed in a wide range of cancers (295297). The antineoplastic activity of auranofin has largely been attributed to two main mechanisms. First, it functions as a potent inhibitor of the reduction/oxidation (redox) enzyme thioredoxin reductase (298), an enzyme essential in controlling the levels of reactive oxygen species (ROS). Many cancer cells overexpress this enzyme, thereby sensitizing them to auranofin. Inhibition of redox enzymes leads to high levels of ROS, causing oxidative stress and cell death. The second main mechanism involves the inhibition of the ubiquitin-proteasome pathway (299). This pathway is required for targeted degradation of proteins within cells, and is upregulated in many cancers, including chronic myeloid leukemia (CML) and hepatocellular carcinoma (300, 301). Marzano et al. reported that auranofin decreases cell viability more effectively than cisplatin in ovarian cancer cells (298), and Pessetto et al. found that auranofin induces apoptosis in gastrointestinal stromal tumour (GIST) cells, even when they are resistant to imatinib mesylate (302). Antineoplastic effects by auranofin have also been

demonstrated for both non-small-cell lung cancer and leukemia (303-305).

Thalidomide is a derivative of glutamic acid (306), and was originally launched as a sedative in 1957 (307), but was also used as an antiemetic in pregnancy. However, the drug was withdrawn from the market in 1961 because of its severe teratogenic effects (308). Thalidomide was since demonstrated to be effective against erythema nodosum leprosum (ENL), a cutaneous variant of leprosy, and FDA (The U.S. Food and Drug Administration) approved thalidomide for treatment of this condition in 1998 (309). Based on its beneficial effects in ENL, and also on its anti-angiogenic effect, the drug has since been used for the treatment of skin disorders, infectious diseases, immunologic and rheumatologic disorders, haematologic diseases, as well as for various cancer types (310-312). In 2006, the FDA approved thalidomide for treatment of newly diagnosed multiple myeloma together with dexamethasone. Thalidomide modulates numerous signaling pathways commonly deregulated in cancer cells (fig. 3) (313). Thalidomide inhibits tumor necrosis factor-α (TNF-α), which is deregulated in many cancers (314). Furthermore, deregulation of the protein complex NF-κB, which controls DNA transcription, cytokine production and cell survival, is linked to cancer progression. Thalidomide inhibits NF-κB activation, through inhibition of IκB kinase (IKK) (315). In addition, thalidomide inhibits interleukin (IL)-1β, IL-6 and IL-12, granulocyte macrophage colony stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and interferon (IFN)-γ (316, 317). In solid organ malignancies, the results have so far not been as good as anticipated. Modest response has been observed in some cancers, such as AIDS-related Kaposi’s sarcoma, renal cell carcinoma and gliomas (318-320). Thalidomide may be a promising agent in treatment of hormone-dependent prostate cancer. In a phase III clinical trial, thalidomide reduced time to prostate specific antigen (PSA)-based progression in patients receiving androgen deprivation therapy (321). Many clinical trials of thalidomide as cancer treatment are however hampered by toxicity issues (322-324), which have resulted in development of several thalidomide analogues with less toxicity and similar anti-cancer effects. Concluding remarks

Drug repurposing has received increasing attention both from the pharmaceutical industry and the public sector/academia as a faster and cheaper strategy for expanding the arsenal of approved cancer drugs. The drugs discussed in this review have all been shown to target one or more of the cancer hallmarks (fig. 4). Searches in the clinicaltrial.gov database using the generic names of these drugs and the word “cancer” yielded 414 recruiting trials (Table 1). Thalidomide and Raloxifene have already been approved for treatment of myeloma and breast cancer, respectively. Other drugs like metformin, aspirin and statins are already widely used on other indications, which should increase the likelihood of obtaining conclusive results regarding their role in cancer treatment. Hopefully, as high-throughput screening technologies become increasingly available and computational methods for analysis are improved, more existing drugs will emerge as candidates for drug repositioning.

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Figure Legends

Figure 1. A key function of Aspirin is its inhibition of COX-1 and -2 enzymes that produce PGH2, a precursor for several prostaglandins that regulate the inflammatory process. Platelets use PGH2 to produce thromboxane A2 (TXA2) that promotes platelet activation and aggregation. Additionally, platelets participate in initiation of the inflammatory process. PGE2 is the most central derivative of PGH2, and its signaling via the EP-receptors drives survival, proliferation, angiogenesis and invasion, but also inhibits NK cells. PGE2 also stimulates the release of cytokines that recruit immunosuppressive MDSCs, which inhibit cytotoxic CD8 T cells. Further immunosuppression is achieved by PGE2-mediated FOXP3 T regulatory cells, which inhibit cytotoxic T cells and NK cells. Importantly, inhibition of COX enzymes shunts its substrate arachidonic acid (AA) to LOX, which promotes resolution of inflammation through production of lipoxin.

Figure 2. In addition to lowering blood insulin and glucose, which in itself may decrease cancer susceptibility, metformin causes energetic stress. Interference with oxidative phosphorylation affects gluconeogenesis and the TCA cycle. Pathways involving stress sensors LKB1 and AMPK are activated upon lowered ATP and ADP levels and increasing AMP levels. Several pro-neoplastic processes required for cellular growth are in turn inhibited, such as glucose, lipid and protein synthesis as well as mTOR activity. Lastly, inhibition of the unfolded protein response by AMPK leads to accumulation of unfolded proteins in the ER, promoting apoptosis.

Figure 3. Thalidomide may inhibit cancer growth by inhibition of angiogenesis as well as its impact on several aspects of the immune system. Central is inhibition of TNF-α, likely by promoting TNF-α mRNA degradation. Cascades downstream of TNF-α mediated by NF-κB are affected, thereby inhibiting cell adhesion, progression through the cell cycle and JAK/STAT and MAPK signaling. Furthermore, Thalidomide inhibits secretion of the angiogenic factors VEGF and bFGF. Thalidomide also acts co-stimulatory on T cells, leading to T cell secretion of IL-2, IL-12, INF-α and INF-γ, important for T cell proliferation and NK cell activation. Moreover, levels of several immune modulatory cytokines such as IL-1β, GM-CSF and IL-10 are altered by Thalidomide.

Figure 4. Drug candidates for repurposing and the hallmarks of cancer that they target.

Table 1: Repurposed drugs currently in clinical trials for oncology Drug

Trade name

Original indication

Clinical trial*

Acetylsalisylic acid

Aspirin

Pain, fever, inflammation

31

Artesunate

Artesunate, ansunate, artex, plasmotrim

Malaria

3

Auranofin

Ridaura

Rheumatoid arthritis

2

Digoxin

Lanoxin

Heart conditions

4

Disulfiram

Antabuse

Alcholism

5

Doxycycline

Doxycylin, doxylin, doxyhexal

Bacterial infections

7

Itraconazole

Sporanox

Fungal infections

14

Leflunomide

Arava, arabloc, lunava, repso, elafra

Rheumatism

1

Lithium

Lithium Carbonate ER, lithobid, eskalith

Depression

3

Mebendazole

Vermox

Worm infections

3

Metformin

Glucophage

Diabetes II

94

Naproxen

Aleve, Naprosyn

NSAID, anti-inflammatory

2

Nelfinavir

Viracept

HIV infection

7

Ritonavir

Norvir

HIV infection

6

High cholesterol

37

Sedative, anti-nausea

195

Statins Thalidomide

Immunoprin

*=Currently recruiting in www.clinicaltrials.gov, July-17. Trials identified by search term “cancer” and the name of the drug.