Increasing opportunities of drug repurposing for treating breast cancer by the integration of molecular, histological, and systemic approaches


List of abbreviations

2-DG

2-Deoxyglucose

2-FDG

2-Fludeoxyglucose

3D

Three direct-acting antivirals

ABC7

Adjuvant for breast cancer treatment using seven repurposed drugs

ABCG2

ATP-binding cassette superfamily G member 2

ADH

Alcohol dehydrogenase

ADRB

Adrenoreceptor beta

ALDH

Aldehyde dehydrogenase

AMP

Adenosine monophosphate

AMPK

AMP-activated protein kinase

ART

Artemisinin

ATP

Adenosine triphosphate

BCRP

Breast cancer–resistance protein

BCSCs

Breast cancer stem cells

BLBC

Basal-like breast cancer

BRCA-1

Breast cancer-1

CDDP

Cisplatin

CDK2

Cyclin-dependent kinase-2

COX-2

Cyclooxygenase-2

CSCs

Cancer stem cells

CuGlu

Copper gluconate

CYP

Cytochrome p

DCA

Dichloroacetate

DCIS

Ductal carcinoma in situ

DDI

Drug–drug interaction

DDTC

Diethyldithiocarbamate

DeCoST

Drug Repurposing from Control System Theory

DMBA

Dimethylbenz[a]anthracene

DNA

Deoxyribonucleic acid

DNMT

DNA methyltransferase

DOX

Doxycycline

DSF

Disulfiram

EGF

Epidermal growth factor

EGFP

Enhanced green fluorescent protein

EGFR

Epidermal growth factor receptor

EMT

Epithelial–mesenchymal transition

ENO1

Enolase-1

ER

Estrogen receptor

ERK

Extracellular receptor kinase

FDA

Food and Drug Administration

FLN

Flunarizine

GBM

Glioblastoma

GLP-1

Glucagon like peptide-1

GSTP-1

Glutathione S-transferase P1

HCV

Hepatitis C virus

HER2

Human epidermal growth factor receptor

HIV

Human immunodeficiency virus

HK-2

Hexokinase-2

HR

Hazard ratio

hTERT

Human telomerase reverse transcriptase

IBC

Inflammatory breast cancer

IGF-1

Insulin-like growth factor-1

IGFBP-3

Insulin-like growth factor binding protein-3

IL-6

Interleukin 6

IV

Intravenous

JNK

c-Jun N-terminal kinase

LC3

Light chain-3

LDHA

Lactate dehydrogenase A

Lipo-DS

Liposome-encapsulated disulfiram

LP-1

Lily polysaccharide-1

MAPK

Mitogen-activated protein kinase

MATE

Multidrug and toxin extrusion protein

MBC

Metastatic breast cancer

MBZ

Mebendazole

MGMT

Methylguanine DNA methyltransferase

mM

Millimolar

MMP

Matrix metalloproteinases

mRNA

Mitochondrial ribonucleic acid

MSCs

Mesenchymal stem cells

MTD

Maximum tolerated dose

MTF

Metformin

mTOR

Mammalian target of rapamycin

NAC

N-acetylcysteine

NF-kB

Nuclear factor kappa-light-chain-enhancer of activated B cells

NIH

National Institutes of Health

nM

Nanomolar

NS5B

Nonstructural protein 5B

OATP

Organic anion transporting polypeptide

OCT

Organic cation transporter

OSR

Oxidative stress response

P-gp

P-glycoprotein

PAC

Paclitaxel

PBPK

Physiologically based pharmacokinetics

pCR

Pathological complete response

PDK-1

Phosphoinositide-dependent kinase-1

PEG

Polyethylene glycol

PET

Positron emission tomography

PI3K

Phosphoinositide 3-kinase

PLGA

Poly-lactic-c-glycolic acid

PLK-1

Polo like kinase-1

PMAT

Plasma membrane monoamine transporter

PR

Progesterone receptor

PTEN

Phosphatase and tensin homolog

qPCR

Quantitative polymerase chain reaction

RANKL

Receptor activator of nuclear factor kappa-beta ligand

RECK

Reversion inducing cysteine rich protein with kazal motifs

ROS

Reactive oxygen species

RPFNA

Random periareolar fine-needle aspiration

SIL

Silibinin

SOD

Superoxide dismutase

TCGA

The Cancer Genome Atlas

TGF-b

Transforming growth factor beta

Th2

T helper type 2

TLR

Toll-like receptor

TNBC

Triple-negative breast cancer

Trp53

Transformation related protein 53

TTM

Tetrathiomolybdate

TUNEL

Terminal deoxynucleotidyl transferase dUTP nick end labeling

UGT

Uridine diphosphate glucuronosyltransferase

UPS

Ubiquitin proteasome system

VEGF

Vascular endothelial growth factor

μM

Micromolar

Introduction

Breast cancer is the most common type of cancer affecting women ( https://www.cancer.gov/types/common-cancers ; https://www.who.int/health-topics/cancer#tab=tab_1 ; https://www.wcrf.org/dietandcancer/cancer-trends/skin-cancer-statistics ). The tremendous impact of breast cancer calls for any and every method possible to be used in its therapy and treatment. Drug repurposing (also known as repositioning, reprofiling, redirecting, or rediscovering [ ]) means developing new uses for a drug beyond its original use or initially approved indication. Drug repurposing has attracted increasing attention in recent years as potentially inexpensive alternatives are needed urgently to compensate for the high costs and disappointing success rate associated with the drug discovery pipeline [ ]. Repurposing can help identify new therapies for diseases at a lower cost and in a shorter time, particularly in those cases where preclinical safety studies have already been completed [ ]. Repurposing an old drug for a new use, such as cancer therapy, is an attractive and exciting field [ , ]. During recent years, several reviews on drug repurposing have been published [ , ].

Drug repurposing is a possible alternative to the current therapies in breast cancer treatment. Multiple drugs have shown great promise in this aspect, from inhibition of breast cancer cell proliferation in general to blocking of pathways that specifically cause breast cancer. This chapter will focus on multiple old or currently used drugs that have shown potential promise in the therapy of breast cancer, by summarizing first their original uses or purposes and then their mechanisms of action responsible for the antibreast cancer effects. This chapter will then comprehensively review both preclinical and clinical (as available) studiesusing these repurposed drugs and their multitude of effects on various subtypes of breast cancer. It will end by discussing the future of drug repurposing—both as a field in general and specifically in the therapy of breast cancer.

Metformin

Metformin (MTF; Fig. 5.1 ) is a well-acknowledged biguanide and a multiaction drug advertised under the name Glucophage as well as others. MTF was approved by the FDA in 1995 as an oral hypoglycemic drug in the management of diabetes mellitus and is the first-line medication for type 2 diabetes mellitus treatment [ ] ( Table 5.1 ). MTF could effectively reduce gluconeogenesis in the liver and improve insulin sensitivity by inducing peripheral glucose uptake and lowering the basal and postprandial plasma glucose [ ]. It was reported that MTF has antiinflammatory [ ], antiapoptotic, anticancer, hepatoprotective, cardioprotective [ ], renoprotective [ ], otoprotective [ ], radioprotective, radiosensitizing, and antioxidant activities [ ] ( Table 5.1 ). There has been a recent development in MTF repositioning for anticancer therapy. It exhibited a great potential to change the metabolic reprogramming and acts as a candidate for cancer management [ ]. In 2005, MTF was used for breast cancer treatment [ ].

Figure 5.1, Chemical structures of repurposed drugs in breast cancer treatment.

Table 5.1
Summary of repurposed drugs in breast cancer.
Drug History and use Original mechanisms of action Proposed anticancer mechanisms
MTF FDA approved in 1995 as an oral hypoglycemic drug in the management of diabetes mellitus Inhibition of mitochondrial complex I and ATP production
Inhibition of gluconeogenesis and lipogenesis
Inhibition of fructose-1,6-bisphosphatase
Reduction in CCL11 and proinflammatory cytokines
Suppression of monocyte differentiation in macrophages
Increased GLP-1 secretion in the gut and increased glucose utilization
Reduction in the carcinoma cell proliferation via insulin/IGF-1 pathway, inhibition of NF-kB, AMPK activation, metabolic stress generation, inhibition of cell proliferation, DNA replication apoptosis through the BAX/BCL-2 apoptotic pathway and AMPK/mTOR/p70S6 growth pathway, cytotoxicity, and apoptosis via toll-like receptor (TLR) signaling
DSF FDA approved 1951 as Antabuse in alcohol deaddiction Inhibition of alcohol dehydrogenase Inhibition of proteasome (E3 ubiquitin ligase), generation of ROS, inhibition of SOD-1 activity, activation of MAPK pathway, inhibition of P-gp, inhibition of NF-kB, inactivation of Cu/Zn SOD, inactivation MMP, inhibition of DNA topoisomerase, inhibition of DNMT, inhibition of GSTP1, inhibition of MGMT, upregulation of RECK, reduction of CDK1 expression levels, causing G2 arrest, reduction in expression of PLK1 protein and mRNA
Propranolol FDA approved in 1967 for the management of hypertension. It is used for other cardiovascular conditions cardiac arrhythmias, postmyocardial infarction to reduce mortality. Other noncardiovascular uses are for prophylaxis of migraine, essential tremors, anxiety, portal hypertension, hyperthyroidism, and pheochromocytoma Nonselective beta-adrenergic receptor blockage—blocks the action of catecholamines (adrenaline and noradrenaline) at both beta-1 and beta-2 adrenergic receptors Reduced expression of hexokinase-2
Agonism at MT1 and MT2 melatonergic receptors
Antivirals Ombitasvir, dasabuvir, and paritaprevir were FDA approved in July 2016 for the treatment of hepatitis C
Ritonavir was FDA approved in 1999 for the treatment of HIV AIDS
Ombitasvir—HCV NS5A inhibition
Dasabuvir—HCV NS5B inhibition
Paritaprevir—HCV NS3/4A inhibition
Ritonavir—cytochrome P3A4 inhibition causing protease inhibition
Inhibition of P-gp, inhibition of breast cancer–resistance protein (BCRP), inhibition of transcriptional and translational factors, suppression of telomerase activity, and activation of human DNA polymerase, increase plasma exposure in drugs, binding to heat shock protein 90 (HSP90), and partial inhibition of its chaperone
Antipsychotics Risperidone, 9-hydroxy-risperidone (paliperidone), olanzapine, quetiapine, clozapine, haloperidol, and chlorpromazine Exact mechanisms are not known, but drugs of this class are believed to inhibit mainly dopaminergic D2 and serotonergic 5HT2A receptors and sometimes alpha-1 adrenergic, H-1 histaminic, and muscarinic receptors Inhibition of cellular uptake of mitoxantrone, inhibition of RANKL-mediated MAPK and NF-kB signaling pathways, inhibition of breast cancer–resistance protein (BCRP)
Thalidomide It was previously used in pregnant women to relieve morning sickness. However, its use was restricted because of its teratogenic effect on the developing fetus. Over the last decades, thalidomide showed a cytotoxic effect on different cancer cell lines, including breast cancer The CD147 and MCT1 protein complex is stabilized and developed by the binding of the cereblon protein and this effect on the complex stimulates cell growth and facilitates the excretion of metabolic products like lactate. It is because of this that an increased abundance of this protein complex enables tumor cells to spread rapidly in diseases such as multiple myeloma. If such a cancer is treated with IMiDs, the complex is displaced from its binding to cereblon and therefore the protein complex of CD147 and MCT1 can no longer be activated, which in turn causes tumor cells to die. The growth and progression of breast cancer cells including MCF-7 and MDA-MB-231 were inhibited by a small series of thalidomide-correlated compounds, which are very effective to induce cancer cell death via triggering TNFα-mediated apoptosis
Artemisinin Artemisinin (ART) is a chemical that was isolated from the sweet wormwood by Youyou Tu at the Chinese Academy of Traditional Chinese Medicine in 1972 . Originally used for the treatment of malaria, during the past two decades, studies revealed the anticancer activity of artemisinin and its derivatives, which indicates the effectiveness of these compounds as cancer therapeutic drugs Artemisinin and its semisynthetic derivatives were originally formulated to be used against malaria. These drugs contain endoperoxide bridges, which are needed for antimalarial activity. The mechanism of action for this original use was believed to be a two-step mechanism. First, ART is activated by intraparasitic heme iron which catalyzes the cleavage of the endoperoxide bridges which then causes a free radical intermediate to be formed and kill the parasite by alkylating and poisoning one or more essential malarial proteins ARS and DHA were reported to inhibit TGF-β signaling that inactivates cancer-associated fibroblasts (CAFs) which play an important role in tumor growth and metastasis, such as stimulating angiogenesis, cell proliferation, migration, and invasion
Mebendazole It was approved by the US FDA in 1974 for the treatment of nematode infections. Also, MBZ revealed efficacy against different types of solid tumors in vitro and in vivo, such as lung cancer, melanoma, colon cancer, glioblastoma multiforme, medulloblastoma, and head and neck squamous cell carcinoma Mebendazole binds to the colchicine-sensitive site of tubulin and therefore causes degenerative alterations in the tegument and intestinal cells of the parasite. The parasite then has its glycogen stores depleted because this loss of cytoplasmic microtubules eventually leads to disruption in the glucose uptake by its larval and adult stages. This causes the parasites immobilization and eventual death It was reported that MBZ was able to induce cell cycle arrest in the radiosensitive G2/M phase of the cell cycle in TNBC cells. This cell cycle arrest was followed by significant induction of apoptotic cell death in a dose- and time-dependent manner. Different studies revealed induction of apoptosis as the primary mode of cell death by MBZ in other tumor types, such as melanoma, lung cancer, and medulloblastoma. Also, tubulin depolymerization was reported to be the major target for benzimidazoles, including MBZ. MBZ exhibited induction of depolymerization of tubulin and inhibition of normal spindle formation in different cancer cell lines, resulting in mitotic arrest and apoptosis
Flunarizine Not FDA approved. Not prescribed in the United StatesIn other countries, include prophylaxis of migraine, peripheral vascular disease, and vertigo Selective calcium entry block with calmodulin-binding properties and histamine H1–blocking activity N-Ras degradation, inhibition of colony formation through TG101348 and FLN combination, inhibition of growth and transforming activity of BLBC cells, induction of autophagy-like activity

Metformin—mechanism of action of the original use

MTF is an orally bioavailable drug metabolized in the liver. It is transported into hepatocytes by organic cation transporter-1 (OCT1). It then accumulates in the mitochondrial inner membrane, inhibits complex 1 of the electron transport chain, and reduces ATP production. The increase in AMP and ADP causes AMPK activation. Activated AMPK inhibits fat synthesis and promotes fat oxidation instead, thus reducing hepatic lipid stores and enhancing hepatic insulin sensitivity. The increase in AMP:ATP ratio also inhibits fructose-1,6-bisphosphatase, resulting in the acute inhibition of gluconeogenesis. MTF also acts via other pathways to inhibit gluconeogenesis, glycogenolysis, and release of glucose in the blood ( Table 5.1 ).

Metformin—preclinical studies

Metformin's anticancer effects

In vitro and in vivo data of MTF treatment revealed its ability to inhibit the growth of ovarian cancer stem cells (CSCs) [ ], glioma-initiating cells [ ], breast cancer cells [ ], endometrial cancer cells [ ], and non-small cell lung cancer cells [ ].

Anticancer properties of MTF have been shown either through its direct effect on the cancer cells by activating AMPK/inhibiting mammalian target of rapamycin (mTOR) pathway [ ] or via its indirect effect on the host by decreasing the blood glucose level in addition to its antiinflammatory effects [ , ] ( Table 5.1 ). The inhibition of mTOR in tumor cells was reported as one of the potential vital mechanisms of the anticancer properties of MTF [ ]. AMPK could inhibit mTORC1 via phosphorylation of mTOR-binding raptor [ ]. In addition, HER2 expression in human breast cancer cells treated with MTF was declined via direct inhibition of p70S6K1, which is a downstream effector of mTOR [ ]. Several studies also suggested that MTF causes many biological and molecular effects on breast cancer cells in addition to its ability to reduce cellular response to the tumorigenesis critical factors as insulin, interleukin 6 (IL-6), and epidermal growth factor through downregulation of IGF-1R, p-Stat-3, and EGFR [ ].

In MCF-7 breast cancer cells ( Table 5.2 ), MTF reduced phosphorylation of S6 kinase, ribosomal protein S6, and eIF4E-binding protein, inhibited mTOR, and reduced translation initiation via AMPK activation [ ].

Table 5.2
Inhibition of various subtypes of breast cancer cells by repurposed drugs.
Drug Cancer cell line ER PR HER2 BRCA1 mutation Subtype Source
Metformin BT474 + + + WT LB L
MCF7, T47D + + WT LA L
ZR7530 + + WT LB L
MDAMB453, SKBR3 + WT H L
MDAMB231, MDAMB157, MDAMB468, HCC70, BT20, BT549 WT TNB L
HCC38, HCC1143, HCC1187, HCC1806 ND TNB L
HCC1937, MDAMB436 MU TNB L
DSF BT474 + + + WT LB L
MCF7, T47D + + WT LA L
SKBR3, SUM190 + WT H L
MDAMB157, MDAMB231, BT549 WT TNB L
SUM149 MU TNB L
HCC70, BT20 WT TNA L
MDAMB436 MU TNA L
Propranolol MCF7 + + WT LA L
MDAMB231 WT TNB L
Antiviral drugs MCF7, T47D + + WT LA L
MDAMB231 WT TNB L
MDAMB436 MU TNB L
Antipsychotic drugs MCF7 + + WT LA L
MDAMB231 WT TNB L
Antimalarial drugs,
Artemisinin (ART)
MCF7 + + WT LA L
MDAMB231 WT TNB L
Mebendazole MCF7, T47D + + WT LA L
MDAMB231, SUM159PT WT TNB L
Thalidomide MCF7 + + WT LA L
MDAMB231 WT TNB L
Flunarizine SUM102PT WT TNB L
SUM149PT MU TNB L
H , HER2 positive; LA , luminal A; LB , luminal B; MU , mutant; ND , not decided; TNA , triple-negative A; TNB , triple-negative B; WT , wild type.

Zordoky et al. reported that excess glucose amount in triple-negative MDA-MB-231 cells ( Table 5.2 ) blocked the MTF-induced cell death, which suggested that high glucose levels resulted in the production of enough energy for cell proliferation via aerobic glycolysis [ ]. In addition, Wahdan-Alaswad et al. reported that glucose increases the aggression of breast cancer cells and reduces the efficacy of MTF. Their preclinical studies on triple-negative breast cancer (TNBC) showed that MTF blocked several key enzymes necessary to glucose metabolism, but its effect was glucose-dependent. Thus, glucose monitoring for patients with breast cancer is suggested to be a crucial factor [ ]. In vivo studies also revealed the antiproliferative effect of MTF in breast cancer [ , ].

Metformin enhances the anticancer activity of other drugs

In vitro and in vivo preclinical studies of MTF in combination with other drugs also revealed a synergistic effect to reduce cell proliferation in both breast cancer cell culture and animal models [ ].

Metformin in combination with natural compounds

Curcumin with MTF exhibited dose-dependent cytotoxicity and antiproliferative activity, as shown by a declined hTERT expression, reduced vascular endothelial growth factor (VEGF) expression, induced transformation related protein 53–independent apoptosis, and triggered Th2 immune response [ ]. In mice models, Falah et al. reported that the combination of MTF and curcumin in breast cancer caused angiogenesis inhibition, immune system modulation, and induction of p53-independent apoptosis [ ].

A combination of ursolic acid with MTF at low concentrations significantly inhibited invasion and migration of transforming growth factor-β (TGF-β)–induced breast cancer in MDA-MB-231 and MCF-7 cells. Combination of ursolic acid and MTF also downregulated expression of CXCR4, uPA, vimentin, E-cadherin, N-cadherin, and MMP-2/9 proteins in breast cancer cells [ ].

Vitamin D3 in combination with MTF increased levels of cleaved caspase-3, BAX, and AMPK and inhibited that of BCL-2, c-Myc, IGF-IR, mTOR, P70S6K, and S6, causing cell cycle arrest in breast cancer cells [ ].

Silibinin in combination with MTF synergistically downregulated expression levels of hTERT and cyclin D1 and enhanced inhibition of breast cancer cell growth [ ].

Chrysin with MTF synergistically caused breast cancer cell death by reducing cyclin D1 and hTERT gene [ ]. Flavone with MTF decreased MDMX protein expression, regulated p53 downstream target gene Bcl-2, and cleaved caspase-3 [ ].

Melatonin with MTF reduced tumor growth and size by increasing the expression of Bax and caspase-3 and inhibiting DMBA-induced breast cancer tumor growth [ ].

Lily polysaccharide-1 with MTF downregulated Bcl-2 expression and upregulated Bax, causing enhanced antiproliferation and apoptosis in breast cancer cells [ ].

Metformin in combination with other drugs used as anticancer agents

Recent studies revealed the ability of MTF to enhance the efficacy of chemotherapeutics in combination therapy [ ]. It was reported that efficacies of several chemotherapeutics increased dramatically when given in combination with MTF [ ].

A combination of MTF with doxorubicin inhibited NF-κB activity in breast tumor cells, which further decreased TNF-α and IL-6 expressions in breast tumor cells, suppressed tumor cell proliferation, and enhanced apoptosis. Moreover, the therapy enhanced nuclear doxorubicin accumulation and overcame drug resistance by downregulating P-gp (P-glycoprotein) and intracellular ATP content in a reduced dose [ , ]. Combination therapy of doxorubicin + 2-deoxyglucose (2-DG) with MTF showed substantial metabolic stress at low dose via inhibition of glucose uptake and suppression of lactate, fatty acid, and ATP production. This combination therapy was associated with decreased cell viability, increased the intracellular oxidation, induced apoptosis and autophagy, and completely inhibited colony formation by activation of AMPK [ ]. Another combination experiment of MTF with doxorubicin and 2FDG increased phospho-AMPK but decreased phospho-Akt and phospho-ERK expressions [ ].

Sahra et al. reported that the combination of MTF with 2-DG revealed inhibition of mitochondrial respiration and glycolysis via p53-dependent apoptosis through AMP pathway. Furthermore, MTF showed a synergistic effect when combined with paclitaxel (PAC), carboplatin, or doxorubicin in xenograft mouse models of breast, lung, and prostate cancer [ ].

A combination of MTF with PAC exhibited significant inhibition of cell viability and induction of the G 2 /M phase arrest [ ]. In vitro studies revealed that MTF and PAC had a synergistic effect, and codelivery of the micelles induced higher cytotoxicity and apoptosis in 4T1 breast cancer cells than each free drug [ ].

Tamoxifen with MTF synergistically inhibited cell proliferation and DNA replication and triggered apoptosis at reduced doses, through regulating both Bax/Bcl-2 and AMPK/mTOR/p70S6 pathways [ ].

MTF in combination with 5-fluorouracil, epirubicin, and cyclophosphamide accelerated glucose consumption and lactate production in breast CSCs and significantly hampered intracellular ATP leading to a severe energy crisis and thus inducing DNA damage in breast cancer cells [ ].

Combination of everolimus with MTF inhibited cell survival, clonogenicity, mTOR signaling activity, mitochondrial respiration, and abrogated S6 and 4E-BP1 phosphorylation in breast cancer cells [ , ]. In addition, the combination of MTF with erlotinib in breast cancer cells enhanced the reduction of EGFR, AKT, S6, and 4EBP1 phosphorylation, associated with inhibition of mammosphere outgrowth [ ].

Aspirin and MTF combination enhanced breast cancer 4T1 cell apoptosis by inducing secretion of TGF-β1. The effect of this combination partly relied on cyclooxygenase-2 upregulation, without production of lipoxins [ ]. In both immune-deficient and immune-competent breast cancer preclinical models, atenolol increased MTF activity against angiogenesis, local and metastatic growth of HER2+, and triple-negative BC. Aspirin increased the activity of MTF only in immune-competent HER2+ BC models. Both aspirin and atenolol, when added to MTF, significantly reduced the endothelial cell component of tumor vessels, whereas pericytes were reduced by the addition of atenolol but not aspirin. These data indicate that the combination of aspirin or atenolol with MTF might be beneficial for BC treatment and that this anti-BC activity is likely due to the inhibitory effects on both BC and microenvironment cells [ ].

A combination of propranolol and MTF inhibited glucose metabolism via downregulation of ADRB2-dependent hexokinase-2, and posttranscription and activation of AMPK, along with the antioxidation activity [ ].

Dichloroacetate and MTF combination revealed synergistic induction of caspase-dependent apoptosis via oxidative damage through PDK1 inhibition and decreased lactate production in breast cancer cells. Expression of glycolytic enzymes, including HK2, LDHA, and ENO1, were downregulated, associated with induction of cell death [ , ].

Topotecan with MTF was shown to activate AMPK, downregulate excision repair cross-complementation group 1, and suppress DNA replication by inhibiting nuclear enzyme topoisomerase I. This combination also depolarized mitochondrial membrane and induced cell cycle arrest in breast cancer cells [ ]. MTF was also reported to prevent BRCA1 haploinsufficiency-driven RANKL gene overexpression and disrupt the autoregulatory feedback control of RANKL-addicted CSCs. The synergistic effect of MTF and denosumab decreased breast cancer–initiating cell (BCIC) population and self-renewal capacity [ ].

Metformin—clinical trials

There are a total 45 registered clinical trials at different stages of development that have studied the efficacy of MTF as monotherapy or in combination with chemotherapy and/or radiotherapy exclusively for the management of breast cancer ( Table 5.3 ). The studies also focus on the process of establishing the effects of MTF on markers of cellular proliferation, pathological response rate, progression-free survival, tolerated safe dose, and recurrence-free survival for breast cancer.

Table 5.3
Clinical trials of repurposed drugs in breast cancer.
Clinical trial Condition N1 N2 Design Primary outcome Status
1 NCT00897884 Breast cancer 40 39 Metformin 500 mg tablet, TID X 2–3 weeks prior to surgery Reduction in cell proliferation rates in tumor tissue Completed
2 NCT01266486 Breast cancer 40 41 Extended release metformin 1500 mg OD X 14–21 days Measure metformin induced effects in phosphorylation of S6K, 4e-BP-1 and AMPK Completed
3 NCT02882581 Breast cancer 10 7 400MBq of 11C-metformin is injected in the cubital vein, followed by PET scan Metformin uptake in breast cancer Completed
4 NCT01310231 Metastatic breast cancer 78 40 Metformin 850 mg BID + standard chemotherapy (anthracyclines, platinum, taxanes, or capecitabine; first or second line)
Placebo + standard chemotherapy
Progression-free survival Completed
5 NCT01650506 Breast cancer 20 8 Standard 3 + 3 dose escalation
Metformin 850 mg BID to TID
Erlotinib 150 mg daily
Maximum tolerated dose of metformin in combination with a fixed dose of 150 mg erlotinib daily Completed
6 NCT01340300 Colorectal cancer
Breast cancer
200 139 1. Exercise training
2. Exercise training + oral metformin QD X 2 weeks, then BID
3. Oral metformin QD X 2 weeks, then BID
Control—educational information
Change in fasting insulin level Completed
7 NCT01589367 Hormone receptor–positive malignant neoplasm of breast 208 208 Arm 1: Metformin experimental
Arm 2: Letrozole alone/placebo
Clinical response rate comparing with RECIST 1.1 from baseline Completed
8 NCT01793948 Breast cancer
Obesity
24 24 Experimental: Arm I: Metformin hydrochloride PO OD on days 1–30 in course 1 and BID on days 1–30 thereafter X 12 courses
Arm II: Placebo
Changes in the phosphorylation of proteins after metformin exposure Completed
9 NCT01885013 Human epidermal growth factor 2 negative carcinoma of breast 112 126 Arm A: Metformin (day 1–day 3, 1000 mg OD; day 4 to day 13, 1000 mg BID) + myocet 60 mg/m 2 , IV on day 1/21 days + cyclophosphamide 600 mg/m 2 IV on day 1/21 days
Arm B: Myocet + cyclophosphamide
Progression-free survival (PFS) Completed
10 NCT00909506 Breast cancer 105 105 Placebo comparator: Placebo
Active comparator: Metformin 500 mg/day OD X 1–2 weeks
Active comparator: Metformin 1000 mg/day (dose-eascalate)
Weight loss Completed
11 NCT00933309 Breast cancer 24 25 Group 1: Exemestane 25 mg PO OD
Group 1: Exemestane 25 mg PO OD + avandamet (rosiglitazone 2 mg + metformin 500 mg PO OD)
Dose-limiting toxicity (DLT) Completed
12 NCT00659568 Breast cancer
Endometrial cancer
Kidney cancer
Lung cancer
Lymphoma
Unspecified adult solid tumor, protocol specific
28 28 Metformin hydrochloride OD/BID/TID on d1–d28 + temsirolimus IV d1, 8, 15, 22/28 days Maximum tolerated dose and recommended Phase II dose of metformin hydrochloride when administered with temsirolimus Completed
13 NCT02145559 Breast neoplasms
Lung neoplasms
Cancer of liver
Lymphoma
Cancer of kidney
64 24 Experimental: Sirolimus (3 mg daily) d1–7 + metformin XR (500 mg PO OD d8–d28; from d15 1000 mg PO OD)
Experimental: Sirolimus (3 mg daily) d1–7, + delayed metformin XR (500 mg PO OD from d22; after 1 week 1000 mg PO OD)
Pharmacodynamic biomarker p70S6K Completed
14 NCT02431676 Breast cancer
Prostate cancer
Lung cancer
Colon cancer
Melanoma of skin
Endometrial cancer
Liver cancer
Pancreatic cancer
Rectal cancer
Kidney cancer
Other solid malignant tumors
120 121 Active comparator: Self-directed behavioral self-control weight loss
Experimental: Coach-directed behavioral weight loss
Experimental: Metformin up to 2000 mg daily X 12 months
IGF-1 levels
IGF-1 levels: IGFBP3 levels (ratio)
Completed
15 NCT02028221 Breast cancer 150 151 Metformin 850 mg PO OD X 4 weeks followed by 850 mg PO BID.
Placebo
Change from baseline in breast density at 6 and 12 months Active, not recruiting
16 NCT02488564 HER2-positive breast cancer 46 49 Day 1: Liposome-encapsulated doxorubicin, 50 mg/m 2 IV 1 h
Day 2 and 9: Docetaxel, 30 mg/m 2 IV 1 h
Day 2, 9, and 16: Trastuzumab 4 mg/kg loading dose, 2 mg/kg/week for subsequent injections
Day 13–11: Metformin 1000 mg OD;
Day 10–0: Metformin 1000 mg BID until end of the study treatment.
Pathologic complete response rate (pCR) Active, not recruiting
17 NCT01101438 Breast cancer 3582 3649 Experimental Arm I: Oral metformin HCL BID (OD in weeks 1–4) continue up to 5 years in ER+, PR+
Placebo Arm II: Placebo BID (OD in weeks 1–4) continue up to 5 years in ER+, PR+
Invasive disease-free survival in hormone receptor negative and positive subgroups Active, not recruiting
18 NCT02278965 Stage 0—III breast carcinoma
Breast neoplasms
20 19 Metformin 850 mg, oral, twice a day for 12 months
Omega-3 fatty acids 2 capsules (560 mg each) oral, twice a day for 12 months
Assess the safety and feasibility of a 1-year intervention of metformin and omega-3 fatty acids in early stage breast cancer patients who completed adjuvant treatment Active, not recruiting
19 NCT04143282 Metastatic breast cancer (nondiabetic) 50 50 Metformin 850 mg to 1 gm, BID Disease progression through tumor size assessed by CT scan (chest–abdomen–pelvis), bone scan, and MRI Recruiting
20 NCT04170465 Breast cancer female 60 60 1. Oral metformin HCl 850 mg BID X 6 months + AC-T chemotherapy regimen ([doxorubicin 60 mg/m 2 IV + cyclophosphamide 600 mg/m 2 IV[ X 4 cycles, every 3 weeks + [paclitaxel 80 mg/m 2 IV] every week X 12 weeks)
2. AC-T chemotherapy regimen alone
Evaluation of the effect on tumor proliferation as measured by Ki-67 immunohistochemical (IHC) assessment (%)
Tissue level of Ki-67 expression in the excised tumor
Evaluation of the effect on tumor apoptosis as measured by caspase-3
Chemotherapy toxicities
Recruiting
21 NCT03238495 HER2-positive breast cancer 100 100 1. Active comparator: Chemotherapy only—taxotere, carboplatin, herceptin + pertuzumab (TCH + P)
1. Experimental: Metformin 850 mg OD during the first cycle, then 850 mg twice daily for the remaining 5 cycles + (TCH + P)
Pathologic complete response Recruiting
22 NCT01980823 Breast cancer
Breast tumors
40 40 Metformin 1500 mg per day: Divided 500 mg in the morning and 1000 mg in the evening + atorvastatin 80 mg OD Change in tissue levels of the proliferation marker Ki-67 Recruiting
23 NCT02506777 Breast cancer 96 96 1. Experimental: Metformin 850 mg BID + FDC (fluoruracil 500 mg/m 2 , doxorubicin 50 mg/m 2 , cyclophosphamide 500 mg/m 2 ) once every 21 days
2. Experimental: melatonin 3 mg before sleeping daily + FDC
3. Active comparator: FDC
Response rate evaluated by RECIST criteria
Pathomorphological response assessed after surgery by miller and payne sscale
Recruiting
24 NCT02506790 Breast cancer 96 96 Experimental: Toremifene 60 mg daily + metformin 850 mg BID
Experimental: Toremifene 60 mg daily with melatonin 3 mg before sleep daily
Active comparator: Toremifene
Response rate evaluated by RECIST criteria
Pathomorphological response assessed after surgery by miller and payne scale
Recruiting
25 NCT01929811 Breast cancer 200 Experimental: Metformin 500 mg PO TID (500 mg PO OD for 1st cycle) + docetaxel + epirubicin + cyclophsophomide
TEC: Docetaxel 75mg/m2 IV d1 q3w∗6 + epirubicin 75mg/m2 IV d1 q3w∗6 + cyclophsophomide 500mg/m2 IV d1 q3w∗6
Pathologic complete response rate (pCR) Recruiting
26 NCT01905046 Atypical ductal breast hyperplasia
BRCA1, BRCA2
DCIS, LCIS
400 128 Arm I: Metformin hydrochloride PO (OD/BID X 24months; 850 mg BID 13–24months)
Arm II: Placebo OD/BID X 12 months; patients may cross over to Arm I for months 13–24
Cytological atypia in unilateral or bilateral RPFNA aspirates Recruiting
27 NCT03006172 Breast cancer
Solid tumor
156 104 Experimental Stage I Arm A: GDC-0077 6 mg (escalating doses) d1, OD from d8/28 days cycle (35 days cycle 1)
Experimental Stage I Arm B: GDC-0077 3 mg (escalating doses) OD, d1–d28 + palbociclib d1–d21 + letrozole d1–d28/28 days cycle
Experimental Stage I Arm C: GDC-0077 (escalating doses) OD, d1–d28 + letrozole d1–d28/28 days cycle
Experimental Stage II Arm B: GDC-0077 OD, d1-–28 + palbociclib d1–d21 + letrozole d1–d28/28 days cycle
Experimental Stage II Arm C: GDC-0077 OD, d1–d28 + letrozole d1–d28/28days cycle
Experimental Stage II Arm D: GDC-0077 d1–d28 + fulvestrant d1, d15/cycle 1, d1 from cycle 2/28 days
Experimental Stage II Arm E: GDC-0077, d1–d28 + palbociclib d1–d21 + fulvestrant d1, d15/cycle 1, d1 from cycle 2/28 days
Experimental Stage II Arm F: GDC-0077, d1–d28 + palbociclib d1–d21 + fulvestrant d1, d15/Cycle 1, d1 from cycle 2+ metformin d1–d28/28 days
Dose-limiting toxicities
Recommended Phase II dose of GDC-0077
Adverse events and serious adverse events
Recruiting
28 NCT03168880 Triple-negative breast cancer A: Paclitaxel 100 mg/m2/week X 8 weeks + AC/EC (60/600 or 90/600)/3 weekly Disease-free survival (DFS)
Overall survival (OS)
Recruiting
29 NCT04001725 Brain metastases
Melanoma
Lung cancer
Breast cancer
110 A: Dexamethasone 8 mg(minimum) PO/IM/IV OD/BID
B: Dexamethasone + metformin (850 mg/day, progressively increased to 1700 mg/day on d4 and 2550 mg/day on d7, if well tolerated)
Prevention of precocious (14 days) dexamethasone-induced diabetes Recruiting
30 NCT02874430 Breast carcinoma
Endometrial clear cell adenocarcinoma
Endometrial serous adenocarcinoma
Uterine corpus cancer
Uterine corpus carcinosarcoma
74 46 Metformin hydrochloride PO OD d1–3, BID from d4 + doxycycline PO BID/7 days Change in the percent of stromal cells expressing caveolin-1 (CAV1) at an intensity of 1+ or greater assessed by immunohistochemistry analyzed using the wilcoxon signed-rank test Recruiting
31 NCT01042379 Breast neoplasms
Breast cancer
Breast tumors
800 1920 1. Active comparator: Paclitaxel, herceptin followed by doxorubicin, cyclophosphomide
2. AMG 386 with or without trastuzumab
3. AMG 479 (ganitumab) plus metformin
4. MK-2206 with or without trastuzumab
5. T-DM1 and pertuzumab
6. Pertuzumab and trastuzumab
7. Ganetespib
8. ABT-888
9. Neratinib
10. PLX3397
11. Pembrolizumab—4 cycles
12. Talazoparib plus irinotecan
13. Patritumab and trastuzumab
14. Pembrolizumab—8 cycles
15. SGN-LIV1A
16. Durvalumab plus olaparib
17. SD-101 + pembrolizumab
18. Tucatinib
Pathologic complete response (pCR) for experimental + standard neoadjuvant chemotherapy versus standard neoadjuvant chemotherapy Recruiting
32 NCT02695121 Breast cancer
Bladder cancer
NA Dapagliflozin, insulin, metformin, sulfonylureas Incidence of breast cancer and bladder cancer Recruiting
33 NCT02201381 Cancer 2000 207 Experimental: Metabolic treatment
Atorvastatin PO up to 80 mg OD + metformin up to 1000 mg PO OD, increased to BID after 2 weeks + Doxycycline 100 mg PO OD +mg PO OD.
Overall survival (OS) Recruiting
34 NCT01302002 Breast cancer 30 0 Metformin 500 mg tablet, BID X 3 weeks Determine the in situ effects of metformin on proliferation (Ki67) and apoptosis (TUNEL), phosphorylate AKT, CD1a CD83, CD68, F40/80, arginase iNOS and T cells CD4(+), CD45RA(+), CD 45RO, CD4, CD8, and FOXP3(+) Withdrawn
35 NCT00984490 Breast cancer 30 5 Metformin 850 mg PO BID X 7–21 days Change in Ki67 levels before and after treatment Terminated (poor accrual)
36 NCT01627067 Breast cancer 40 23 Exemestane 25 mg OD + everolimus 10 mg OD + metformin 500 mg OD every 3 days—increase to 1000 mg BID if no toxicity Progression-free survival (PFS) Terminated (komen foundation funding terminated)
37 NCT01477060 Metastatic breast cancer 168 32 ARM A: Hormonal therapy + lapatinib (1250mg/die)
ARM B: Hormonal therapy + metformin (1500 mg/die)
ARM C: Hormonal therapy + lapatinib + metformin
Rate of patients free from disease progression Terminated (poor accrual)
38 NCT02472353 Breast cancer
Breast tumors
44 30 1. Active comparator—doxorubicin
Experimental: Metformin + doxorubicin
Decrease in the incidence of change in left ventricle ejection fraction (LVEF) Terminated (poor accrual)
39 NCT02360059 Breast cancer 42 1 12 days prior to start of paclitaxel
1. Experimental arm: Metformin 500 mg OD X 5 days, followed by 500 mg BID X 5 days, followed by 1000 mg BID X 2 days
Questionnaires; sensory and fine -motor tests
2. Placebo
Mean change in neuropathy Terminated (poor accrual)
40 NCT00930579 Breast cancer 15 35 Metformin 1500 mg: divided 500 mg in the morning and 1000 mg in the evening, for at least 2 weeks prior to surgery Effects of metformin on AMPK/mTOR signaling pathway Unknown
41 NCT03192293 Breast cancer 28 28 Experimental: 7 days lead-in period:
Metformin 850 mg OD + simvastatin 20 mg OD
If well-tolerated, fulvestrant at standard doses:
Cycle 1: 500 mg at day 1 and day 15/28 days cycle
Cycle 2 and beyond: 500 mg at day 1/28 days cycle
Clinical benefit rate (CBR) Unknown
42 NCT01666171 Breast cancer 458 Metformin hydrochloride clinical observation, diagnostic laboratory biomarker analysis, imaging biomarker analysis, medical chart review; procedure: Digital mammography Change in percent mammographic breast density in contralateral (unaffected) breast from baseline to 1 year using two-sample t-test or wilcoxon rank-sum test Unknown
43 NCT01566799 Locally advanced malignant neoplasm 60 Paclitaxel X 12 weeks followed by 4 cycles of FAC combined with metformin 500 mg PO OD X 24 weeks Pathologic complete response (pCR) Unknown
44 NCT01286233 Breast cancer
Depression
Fatigue
Sleep disorders
454 394 Group 1: Metformin 850 mg PO BID X 5 years
Group 2: Placebo PO BID X 5 years
Questionnaire scores about fatigue, stress, sleep, depression, general quality of life, and behavioral risks; biological correlates of fatigue
DNA polymorphisms
Changes in RNA gene expression
Unknown
45 NCT01302379 Breast neoplasms 340 333 1. Metformin: (week 1 - 500 mg PO OD, week 2–4–1000 mg PO OD, weeks 5+–500 mg PO daily) + lifestyle intervention
2. Placebo + lifestyle intervention
3. Metformin + standard dietary guidelines
4. Placebo + standard dietary guidelines
Biological markers associated with breast cancer survival Unknown
46 NCT03323346 Breast neoplasm
Metastatic breast cancer
150 150 Disulfiram 400 mg PO daily + copper (2 mg elemental cu) PO daily Clinical response rate (RR)
Clinical benefit rate (CBR)
Recruiting
47 NCT01847001 Locally advanced malignant neoplasm
Breast cancer
20 10 Propranolol (20 mg BID up to 40 mg BID) + neoadjuvant chemotherapy (regimen I: paclitaxel; Regimen II: doxorubicin and cyclophosphamide; if tumor is HER2 positive add trastuzumab and pertuzumab) Percentage of patients compliant with taking > 80% take the drug while on chemotherapy Active, not recruiting
48 NCT02596867 Breast cancer 30 2 Propranolol 1.5 mg/kg/day, BID X 3 weeks Reduction the tumor proliferative index using Ki-67 Terminated (poor accrual)
49 NCT00502684 Primary operable breast cancer 460 32 Experimental: propranolol 10 mg QID, starting on d-3 pre-op, X 6 days, till POD 2 + etodolac 400 mg BID, starting on d-3 pre-op, for 6 days, till POD 2
Placebo
Number and cytotoxic activity of NK cells, levels of NKT cells, lymphocytes, monocytes, and granulocytes; cytokine levels; in vitro cytokine secretion; levels of cortisol and VEGF. Cancer recurrence in 5 years Unknown
50 NCT02013492 Male breast cancer, recurrent melanoma, Stage IV breast cancer, Stage IV melanoma, Stage IV ovarian epithelial cancer, Stage IV ovarian Germ cell tumor, unspecified adult solid tumor, protocol specific hepatocellular carcinoma 35 35 Propranolol PO BID X 4 months Incidence of toxicity graded according to common criteria for adverse events (CTCAEs) V. 4.0
Change in vascular endothelial growth factor (VEGF)
Effect of beta-adrenergic blockade on the tumor microenvironment and on the host immune system
Recruiting
51 NCT02649101 Metastatic breast cancer 60 60 Thalidomide tablet 100 mg QN, PO Progression-free survival (PFS) Unknown
52 NCT00193102 Breast cancer 40 40 Thalidomide Overall response rate
Time to disease progression
Terminated
53 NCT00049296 Cancer 26 26 Thalidomide PO, BID
Docetaxel IV over 30 min once weekly
Determine maximum tolerated dose of docetaxel when administered with thalidomide Completed
54 NCT00764036 Metastatic breast cancer
Locally advanced breast cancer
18 23 Artesunate PO QD add-on therapy of 100, 150 or 200 mg Dose-limiting adverse events with possible, probable, or definite relation with the respective dose level of the add-on therapy
BID , twice a day; FDC , fixed dose combination; HCL , hydrochloride; IGF , insulin-like growth factor; IM , intramuscular; IV , intravenous; N 1, Original estimated enrollment; N 2, Estimated enrollment; OD , once daily; PO ,per oral; QD , once daily; QN , every night; TID , three times a day; XR , extended release.

In vitro studies showed that MTF at 5–10 μM was able to activate AMPK [ ]. However, clinically such low concentrations of MTF were not sufficient to cause AMPK activation, although metabolic alteration could be evoked [ ]. Therefore, scientists have focused on using pharmacological combinations that can aid in producing anticancer effects at an effective and safe dose profile of MTF [ ].

Other pharmacological agents may be used together with MTF for enhancing the therapeutic efficacy of the drug. Still, the effective and safe dose for breast cancer is a great challenge. A Phase I study was conducted using radioactive 400MBq 11C-MTF, injected in cubital veins of 10 males and females age >50 years with breast cancer, and PET images were taken after 120 min. Quantitative polymerase chain reaction confirmed the drug transportation pathway through OCT, specifically OCT1-3, MATE 1 and 2, and PMAT. PET imaging also confirmed the drug uptake by breast tissue [ ]. In postmenopausal women with breast cancer, MTF therapy changed the levels of protein phosphorylation. In obese patients with the ordinal level of breast density, MTF decreased the risk of breast cancer [ ].

MTF therapy prior to surgery was shown to reduce cancer cell proliferation and inhibit tumor growth in early stage breast cancer patients [ ]. MTF was also shown to improve general body condition including weight loss in overweight or prediabetes mellitus–operable breast cancer patients [ ]. In Stage I to III colorectal or breast cancer patients, MTF was able to decrease fasting insulin levels and change other insulin-related biomarkers like C-peptide, IGFR, IGFBP-3, adiponectin, and lectin [ ].

In another trial, MTF was found to be able to inhibit cell proliferation and tumor growth in patients with early stage breast cancer before surgery. MTF before surgery in operable breast cancer patients improved general body condition. In a subset of breast cancer patients with tumor size >3 cm, 1500 mg extended-release MTF was shown to cause an alteration of S6K, 4E-BP-1, and AMPK via histochemical analysis, thus effectively altering breast cancer metabolism [ ].

Phase I trial of MTF with exemestane and rosiglitazone was well tolerated in obese postmenopausal ER+ and PR+ breast cancer patients [ ]. Another Phase I trial was conducted using MTF and temsirolimus to determine the maximum tolerated dose (MTD) for the combination in breast cancer patients [ ]. A combination study of MTF with standard first-, second-, third- or fourth-line chemotherapy like anthracycline, taxane, platinum, capecitabine, or vinorelbine-based regimens showed improved survival and tumor response in women with metastatic breast cancer [ ]. Another combination therapy of MTF and erlotinib showed good tolerance in TNBC patients [ ]. MTF acted as a biomarker for tracing the mechanism of cancer cell resistance. MTF, along with myocet and cyclophosphamide in HER2-negative breast cancer patients, led to an increase in the progression-free survival as well as characterization of sensitivity in insulin levels [ ].

In invasive breast cancer patients, when MTF was used before PAC therapy, there was a reduction in the occurrence of some adverse effects, specifically peripheral neuropathy [ ]. In another subset of breast cancer patients, combination therapy of MTF and doxorubicin reduced cardiotoxicity by decreasing the tendency of significant change in left ventricle ejection fraction [ ].

An ongoing trial of MTF in postmenopausal obese women is studying the effect of MTF in the obesity-related breast cancer and breast density along with serum IGF-1 to IGFBP-3 ratio [ ]. Another trial of MTF in women who have had a prior breast biopsy demonstrating atypical hyperplasia is studying the efficacy of MTF for prevention and alteration of RPFNA or blood biomarkers of atypical hyperplasia in unilateral or bilateral RPFNA aspirates of breast cancer [ ].

The combined effect of MTF with 5-fluorouracil, doxorubicin, and cyclophosphamide is being studied in women with locally advanced breast cancer [ ]. The pathomorphological and toxicological effect of MTF and toremifene is being studied in women above the age of 18 [ ]. A presurgical trial of combination therapy of MTF and atorvastatin in women with operable DCIS breast cancer is studying the benefits of the combination as well as a change in the proliferation marker Ki-67 levels in the breast tissue of these patients [ ]. In patients with ER+ breast cancer, the efficacy of neoadjuvant therapy of a combination of MTF and letrozole QD is currently being studied in a clinical trial. This trial also investigates the response rate of breast-conserving surgery and changes in Ki67 level after treatment with the combination [ ].

An ongoing clinical trial with repurposed MTF along with taxotere, carboplatin, herceptin, and pertuzumab in patients with HER2+ breast cancer with cT1c-cT4a-d node without metastasis is studying the effect of the combination as adjuvant therapy [ ].

An ongoing trial of MTF, doxorubicin, docetaxel, and trastuzumab focuses on the efficacy of the combination for operable and locally advanced HER2+ breast cancer. This trial evaluates pCR and systemic tolerance, with precise consideration to cardiac toxicity [ ]. The clinical benefit rate and toxicity profile of combination therapy of MTF and simvastatin are currently being studied in a trial for ER+ breast cancer patients [ ]. The pCR after combination therapy of MTF with docetaxel, epirubicin, and cyclophosphamide is also being studied in breast cancer patients with a life expectancy of >12 months [ ].

An ongoing trial of a combination of MTF and doxycycline (DOX) is being studied in women with localized breast or uterine cancer. The main purpose of the study is to analyze the change in stromal cells expressing caveolin-1 at an intensity of 1+ by immunohistochemistry [ ].

Metformin—summary

MTF has been off-patent protection since 2002 and can be easily synthesized with dimethylamine hydrochloride and 2-cyanoguanidine. It is known to have well-tolerated side effects making its compliance better than other anticancer medications. By reducing insulin, MTF reduces the anabolic capability of the body, which in turn reduces tumor growth. In the tumor cells, MTF alters the high energy metabolism, reducing their ability to survive and proliferate. The hydrophilic characteristics of the drug facilitate its direct transport through the cell membrane and its action via OCTs, plasma monoamine transporter, and multidrug and toxin extrusion protein.

Disulfiram

Disulfiram (DSF, tetraethylthiuram disulfide, or Antabuse; Fig. 5.1 ) is one of the most popular repurposed drugs. In the beginning, it was used in the rubber industry as an industrial catalyst [ ], then it was introduced into medicine as scabicide and vermicide [ ]. It was accidentally found to be a bad combination with alcohol when it was used in a study to treat stomach ailments. The US FDA-approved DSF in 1951 as Antabuse in alcohol deaddiction ( Table 5.1 ). For about 50 years, DSF was used as an antialcoholism drug and now is being repurposed in cancer treatment [ , ] ( Table 5.1 ). DSF repurposition as a potential anticancer drug is based on its unique properties in the human body, e.g., low cost, fewer side effects, high selectivity against different cancers, and synergistic activity with other drugs [ ]. Moreover, DSF showed proteasome inhibition and suppressed different cancer-associated pathways [ , , ].

Disulfiram—mechanism of action of the original use

DSF is the best-known aldehyde dehydrogenase (ALDH) irreversible inhibitor [ ]. Inhibition of ALDH by DSF is through the formation of an intramolecular disulfide bond by two mechanisms: (i) between DSF and an active site thiol in the enzyme or (ii) between the active site thiol and thiol of another cysteine residue via unstable mixed disulfide adduct [ ]. When alcohol enters the body, it is converted into acetaldehyde by alcohol dehydrogenase and then rapidly metabolized into acetic acid by the action of ALDH in the liver. DSF was reported to block the action of ALDH that causes an increased serum acetaldehyde concentration, which is toxic and results in an unpleasant DSF-ethanol reaction to the individual ( Table 5.1 ).

Disulfiram—preclinical studies

Since the 1970s, scientists have noticed the tumor-suppressive effect of DSF [ ]. Many anticancer properties of DSF have been demonstrated in different preclinical models of breast, prostate, myeloma, leukemia, lung cancer, cervical adenocarcinoma, melanoma, neuroblastoma, and colorectal cancer [ ] ( Table 5.1 ).

Disulfiram complexes with metals

DSF, as a thiuram disulfide of dithiocarbamate, could form complexes with metals. The interaction of DSF with copper (II) chloride in solution reveals a rapid formation of the bis (N, N-diethyldithiocarbamate) copper (II) complex in situ [ ]. The anticancer activity of DSF is copper-dependent [ ]. Up to 10 μM, DSF alone did not exhibit cytotoxic effect in cancer cells [ ], while DSF/Cu at lower concentrations exerted anticancer activity [ ] and suppressed cell proliferation, tumor growth, invasion, and migration [ , ], in addition to eliminating tumor-initiating cells and inhibiting colony formation and tumor formation in different animal models [ ]. The antiangiogenic effect was also seen following DSF/Cu treatment via reducing microvessel density and VEGF expression [ ]. DSF with exogenous copper had better antitumor effect when compared to DSF alone in a breast cancer xenograft model [ ].

DSF–Cu complex in breast cancer stem cells

CSCs are a subpopulation of cells that have the ability of self-renewal and differentiation. Levels of CSCs correlate with tumorigenesis, metastasis, radio-/chemoresistance, and cancer recurrence clinically [ , ]. DSF exhibited a highly cytotoxic effect on breast cancer stem cells (BCSCs), which are considered a significant cause of chemoresistance that leads to the failure of breast cancer chemotherapy.

DSF–Cu complex exhibited proteasome inhibition and induction of apoptosis in breast cancer cell cultures. It suppressed the growth of breast cancer xenografts, which is independent of the status of PIK3CA, the gene encoding α form of class IA PI3Ks, that heightened and overexpressed in a variety of human cancers, including breast cancer. Treatment of a DSF–Cu mixture decreased the expression of PTEN protein in a time- and dose-dependent manner and activated AKT in cell lines regardless of the presence of PIK3CA mutations, which are thought to lead to the activation of the AKT signaling pathway. Therefore, DSF appears to trigger two conflicting signaling pathways in breast cancer cells. First, DSF–Cu inhibits the proteasome, thus bringing about the death pathway and inducing apoptosis. Second, DSF–Cu activates the PI3K/PTEN/AKT survival signaling pathway [ ].

HER2 is an important determinant of survival for BCSCs that are associated with a high risk of tumor recurrence [ , ]. HER2 overexpression [ ] is able to alter the apoptotic and molecular signaling pathways [ ]. DSF/Cu combination was reported to inhibit HER2/Akt signaling and eliminated BCSCs, suggesting the potential effectiveness of DSF for HER2-positive breast cancer treatment [ ].

DSF–Cu complex for TNBC therapy

TNBC represents an aggressive subtype, for which radiation and chemotherapy are the only options [ ]. Acquired chemoresistance remains the primary cause of therapeutic failure of TNBC [ ]. In the clinic, the relapsed TNBC is commonly pan-resistant to various drugs with entirely different resistance mechanisms. Investigation of the resistance mechanisms and development of new drugs to target pan-chemoresistance will potentially improve the therapeutic outcomes of TNBC patients [ ].

The MDA-MB-231 PAC10 cell line is made up of a high population of cells expressing stem cell markers that may play a vital role in the pan-resistance. These cells express high ALDH activity and a panel of embryonic stem cell–related proteins (e.g., Oct4, Sox2, Nanog) and nuclearization of HIF2a and NF-kBp65. These cells are highly cross-resistant to PAC, cisplatin (CDDP), docetaxel, and doxorubicin. DSF was reported to abolish CSC characters and completely reverse PAC and CDDP resistance in MDA-MB-231 PAC10 cells ( Table 5.2 ). In addition, DSF/Cu exposure for 4 h leads to inhibition of both ALDH activity and expression of Sox2 and Nanog in the resistant cells. In combination with DSF/Cu, the cytotoxicity of PAC and CDDP in MDA-MB-231 PAC10PAC10 cells was significantly higher than PAC, CDDP, or DSF/Cu single-drug exposure. The cytotoxicity of DSF/Cu plus PAC was synergistic in a wide range of concentrations [ ].

DSF/Cu combination induced the expression of Bax while inhibited the expression of Bcl2 in MDA-MB-231 PAC10 cells, which lead to a significant increase in Bax/Bcl2 ratio in the resistant cell line. Expression of p21 and p53 proteins was also enhanced by DSF/Cu combination, which did not effect on that of CDK2 and cyclin D1 and E [ ].

Analysis of the impact of various treatments on CSCs population in MDA-MB-231 and T47D cell lines revealed that formation of mammosphere from both cell lines was blocked entirely by exposure to the combination of DSF (1 μM)/Cu (1 μM) plus PAC (40 nM) for 48 h but not affected by PAC, DSF, or Cu alone. In addition, DSF/Cu, but not DSF or Cu, treatment significantly inhibited the ALDH-positive population in mammospheres [ ].

DSF–Cu complex for the treatment of inflammatory breast cancer

Although there have been advances in multimodality treatment, inflammatory breast cancer (IBC) is a discrete, advanced BC subtype characterized by high rates of residual disease and recurrence [ , ]. Metagene analysis of patient samples revealed significantly higher oxidative stress response (OSR) scores in IBC tumor samples when compared to normal or non-IBC tissues. These higher OSR scores were responsible for the inadequate response of IBC tumors to standard treatment strategies. DSF–Cu antagonized NF-kB signaling [ ], ALDH activity, and antioxidant levels [ ], causing the induction of oxidative stress–mediated apoptosis in multiple IBC cellular models [ ].

Under in vivo condition, DSF–Cu caused apoptosis and inhibited growth exclusively in tumor cells without significant toxicity. It has been suggested that strong redox adaption of IBC tumors may contribute to their resistance to ROS-inducing therapies. DSF, through redox modulation, may be advantageous in the manner of enhancing chemo- and/or radiosensitivity for extreme BC subtypes that showed therapeutic contention. DSF's potency was significantly enhanced by the addition of 10 μM Cu (DSF–Cu), with a decrease of roughly 100-fold in IC 50 values in SUM149 and rSUM149 ( Table 5.2 ). Remarkably, DSF-Cu caused cell death in redox-adapted rSUM149 cells at levels comparable to parental redox-sensitive SUM149 cells [ ].

DSF–Cu complex is nontoxic, at up to 20 µM , to normal, immortalized breast cells (MCF10A). Similar to SUM149 cells, DSF-induced, Cu-dependent cell death was observed in other IBC cell lines tested, which included MDA-IBC-3 (HER2-overexpressing), SUM190 (HERer2-overexpressing, ROS sensitive), and rSUM190 (an isogenic derivative of SUM190 with therapeutic resistance, redox adaptation) ( Table 5.2 ). SUM190 cells, even at extremely reduced concentrations of DSF, were exceedingly susceptible to DSF–Cu treatment. In the presence of DSF–Cu, the lessening of IBC cell viability corresponded with a drop off in levels of XIAP, the most potent mammalian caspase inhibitor and antiapoptotic protein. This is coherent with the induction of oxidative stress triggering the intrinsic apoptotic pathway. XIAP overexpression in IBC cells was identified to be correlated with resistance to therapeutic apoptosis. DSF–Cu-mediated cell death was also related to the decreased expression of eIF4G1 (a disease pathogenesis factor identified in IBC tumors) and increased PARP cleavage. These findings suggested the induction of apoptotic cell death being a fundamental mode of action of DSF–Cu [ ].

Addition of bathocuproine disulfonate or tetrathiomolybdate (TTM), two high affinity chelators to sequester free Cu, hindered DSF–Cu-induced cell death altogether in SUM149 and SUM149 cells, thus showing the importance of Cu binding in the intensification of DSF's cytotoxic effects. Although TTM is able to induce cancer cell death through sequestering Cu, the TTM–Cu mixture showed no effect in these models of IBC. These data confirmed that the activity of DSF is not related to Cu sequestration, but rather a gain of function that results in increased ROS production [ ].

Han et al. reported that without copper supplementation, exogenous SOD potentiated subtoxic DSF toxicity antagonized by subtoxic TTM or by the antioxidant N-acetylcysteine. Exogenous glucose oxidase, another H 2 O 2 generator, paralleled exogenous SOD in potentiating subtoxic DSF. It was confirmed that potentiation of sublethal DSF toxicity by extracellular H 2 O 2 against the human tumor cell lines only needed basal Cu and heightened ROS production. This is critical as these findings accentuate the importance of extracellular H 2 O 2 as a novel mechanism to improve the anticancer effects of DSF while minimizing copper toxicity [ ]. The authors also documented that DSF could significantly suppress the TGF-β–induced upregulation of vimentin and N-cadherin as well as downregulation of E-cadherin in a dose-dependent manner. These findings suggested that DSF can inhibit TGF-β–induced EMT by modifying the expression of EMT-related proteins.

DSF–Cu complex as a BC proteasome inhibitor

DSF was able to affect 20S proteasome activity at low micromolar concentrations, but through a mechanism different from that of the authentic proteasome inhibitors as bortezomib or MG132 that block the active site of the proteasome. DSF acts at some other sites on the proteasome as a slow-binding partial noncompetitive inhibitor [ ]. However, DSF and its combination with copper exhibited a strong inhibitory effect on 26S proteasomes [ ], and the inhibition was copper-dependent [ ].

MDA-MB-231 BC cells with high levels of cellular copper showed proteasome inhibition in the presence of DSF alone, which suggested that copper acts as an endogenous element in the cell, which can form a complex with DSF and induce cell death. Similar findings were observed in mice bearing MDA-MB-231 tumor xenografts treated with DSF that inhibited tumor growth and proteasome activity and induced apoptosis in vivo [ ] ( Table 5.2 ).

DSF–cadmium complex

A study reported that breast cancer MCF-10 DCIS cells were more sensitive to DSF–Cd-induced 20S proteasome inhibition and apoptosis induction than nontumorigenic human breast MCF-10A cells [ , ]. Thus, the DSF–Cd complex could selectively induce proteasome inhibition and apoptosis in human breast tumor cells [ ].

DSF and zinc availability

The availability of extracellular zinc was suggested to influence DSF efficacy significantly. Live cell confocal microscopy using fluorescent endocytic probes and the zinc dye FluoZin-3 showed that DSF elevated zinc levels selectively and speedily in endolysosomes. DSF was reported to cause the spatial disorganization of late endosomes and lysosomes, suggesting that they could be used as novel targets for DSF. Moreover, DSF was shown to increase the intracellular zinc levels in breast cancer cells specifically. It was reported that 10–100 μM DSF greatly heightened intracellular zinc levels in both MCF-7 and MDA-MB-231 cell lines ( Table 5.2 ), while zinc levels in the noncancerous MCF-10A cells remained unaffected by the same treatment. In serum-free media containing low zinc and copper, neither sodium pyrithione nor DSF induced a statistically significant increase in intracellular zinc in MCF-7 cells. Supplementation of serum-free media with 20 μM zinc was enough to restore completely, and exaggerate, the ionophore ability of both DSF and sodium pyrithione, demonstrating that this ionophore activity is dependent on extracellular zinc levels [ ].

Disulfiram combination with other drugs

DSF combination with doxorubicin, daunorubicin, mitoxantrone, colchicine, or paclitaxel

Robinson et al. reported that DSF, as well as the related compound thiram, were identified as the most potent growth inhibitors against multiple TNBC cell lines. Combination treatment with four different drugs commonly used to treat TNBC revealed that DSF synergizes most effectively with doxorubicin to inhibit cell growth of TNBC cells. DSF and doxorubicin cooperated to induce cell death as well as cellular senescence and targeted the ESA+/CD24−/low/CD44+ CSC population. The results suggested that DSF may be repurposed to treat TNBC in combination with doxorubicin [ ].

Studies performed on HCC70, MDA-MB-231, MDA-MB-436, and Bt549 cell lines revealed that DSF was more effective against each cell line than doxorubicin, daunorubicin, mitoxantrone, colchicine, or PAC. Notably, MDA-MB-436 cells were resistant to the mitotic inhibitors, colchicine, and PAC but highly susceptible to DSF. In a panel of 13 human-derived TNBC lines, both DSF and thiram effectively suppressed the growth of TNBC cells, with an average IC 50 across all lines of 300 and 360 nM, respectively ( Table 5.2 ). Similar effect was seen when these drugs were used in basal-A or basal-B TNBC cell lines. These and other results all indicate that DSF/Cu is cytotoxic and nonspecific to breast cancer cells and can kill all subtypes of breast cancer.

There was a strong synergistic effect between DSF/Cu and PAC over a wide range of concentrations (IC 50 – IC 90 ). DSF/Cu significantly enhanced (3.7- to 15.5-fold) the cytotoxicity of PAC in BC cell lines. Contrary to the slight induction of apoptosis at low concentration of PAC alone (1 nM), the proportion of apoptotic cells was greatly elevated by DSF/Cu (DSF 100–150 nM/Cu 1 μM) and PAC in combination. DSF/Cu-induced cytotoxicity in BC cell lines was caused by ROS activation and was reversed by the addition of NAC, a ROS inhibitor, in the culture [ ].

DSF/Cu combination was also seen to inhibit NF-kB activity in BC cell lines. The NF-kB is a ROS-induced transcription factor with substantial antiapoptotic activity, which in turn dampens the proapoptotic effect of ROS [ ]. Therefore, the blocking of NF-kB activation enhances ROS-induced cytotoxicity. Both PAC and DSF/Cu inhibited NF-kB DNA-binding activity in BC cell lines. The most potent inhibition was observed in the BC cells treated with PAC/DSF/Cu in combination.

The clonogenicity of BC cell lines was significantly inhibited by DSF/Cu and eradicated by exposure to PAC plus DSF/Cu [ ]. Furthermore, inhibition of ALDH activity by DSF has been suggested to contribute to reduce CSCs and overcome drug resistance [ ].

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