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

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 [ ].

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 [ ].

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 [ ].