New Achievements for the Treatment of Triple-Negative Breast Cancer

Abstract

Triple-negative breast cancer (TNBC) constitutes a heterogeneous group of malignancies that are often aggressive and associated with a poor prognosis. The development of new TNBC treatment strategies has become an urgent clinical need. Diagnosis and subtyping of TNBC are essential to establish alternative treatments and targeted therapies for every TNBC patient. Chemotherapy, particularly with anthracycline and taxanes, remains the backbone for medical management for both early and metastatic TNBC. More recently, immune checkpoint inhibitors and targeted therapy have revolutionized cancer treatment. Included in the different strategies studied for TNBC treatment is drug repurposing. Despite the numerous medications available, numerous studies in medicinal chemistry are still aimed at the synthesis of new compounds in order to find new antiproliferative agents capable of treating TNBC. Additionally, some supplemental micronutrients, nutraceuticals and functional foods can potentially reduce the risk of developing cancer or can retard the rate of growth and metastases of established malignant diseases. Finally, nanotechnology in medicine, termed nanomedicines, introduces nanoparticles of variable chemistry and architecture for cancer treatment. This review highlights the most recent studies in search of new therapies for the treatment of TNBC, along with nutraceuticals and repositioning of drugs.

1. Introduction

Breast cancer (BC) is one of the most frequent types of cancer among women. The global cancer project (GLOBOCAN 2012) reports that breast cancer is considered the second most common disease in the world [1]. It is the second leading cause of death among women worldwide and receives increasing attention nowadays [2]. In high income countries, BC mortality is decreasing, largely owing to improved laboratory, epidemiological and clinical research, but incidence has been steadily increasing [3,4]. Therefore, it is urgent to develop new treatment regimens and targets [5], and for this reason numerous research studies are directed towards new strategies for the treatment of BC [6,7,8,9].

Among BCs, triple-negative breast cancer (TNBC), which does not express estrogen receptor (ER) or progesterone receptor (PR) and does not overexpress the human epidermal growth factor receptor 2 (HER2), is one of the most aggressive subtypes. TNBC includes the more common invasive ductal and lobular carcinomas plus rarer subtypes of BC [10]. The latest classification divides TNBC into four different subtypes: basal-like 1 (BL1), basal-like 2 (BL2), mesenchymal stem cell-like (MSL) and luminal androgen receptor (LAR) [11]. TNBC is a heterogeneous disease, with subtypes characterized by distinct pathologic, genetic, and clinical features [12]. Its clinical features include high invasiveness, high metastatic potential, proneness to relapse and poor prognosis. It accounts for approximately 15% to 20% of all BCs [13]. Cancer stem cells (CSCs), also called tumor initiating cells (TICs), seem to contribute to aggressive phenotypes of TNBC [14]. Brain metastasis (BM) is a serious, life-threatening issue that occurs more frequently in TNBC patients, and TNBC has a worse prognosis than other BCs [15,16]. The identification of TNBCs with a high level of accuracy compared with other tumor sub-types is due to magnetic resonance imaging (MRI) [17]. As TNBCs lack ER and PR expression and do not overexpress HER2, they are not sensitive to endocrine therapy or HER2 treatment, and standardized TNBC treatment regimens are still lacking.

A large number of novel therapeutic agents have been evaluated for their efficacy in TNBC [18]. TNBC has traditionally been treated with systemic chemotherapy (e.g., paclitaxel, capecitabine, eribulin, ixabepilone) [19]. However, standard chemotherapy is associated with low response rates and short progression-free survival, especially among patients with pretreated metastatic triple-negative breast cancer (mTNBC). Indeed, the predominant systemic therapy for most mTNBC remains chemotherapy, but responses are often short-lived, and patients have a median overall survival (OS) of 12 to 18 months [20]. Moreover, various sensitivities of cancer sufferers critically limit chemotherapy’s clinical results due to infection, antibiotic resistance [21], diverse pathogens, individual differences and chemotherapy resistance [22]. Recent data suggest that matrix alterations in triple-negative cancer cells are epigenetically regulated, and that matrix-associated events increase tumor cell survival and resistance to therapy [23]. Recently, ferroptosis-inducing agents have also been suggested as interesting antitumor candidates [24,25].

Despite recent treatments, most mTNBC patients have disease progression, and the 5-year OS is estimated at only 11%. TNBC greatly benefits from precision oncology and therapeutic targeting [26,27]. Targeted agents for TNBC include inhibitors of poly(ADP-ribose) polymerase (PARP), epidermal growth factor receptor (EGFR), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), angiogenesis, microtubules, proto-oncogene tyrosine-protein kinase Src (Src kinase), AKT, checkpoint kinase 1 (Chk1) and mammalian target of rapamycin (mTOR) [28]. Anti-programmed death receptor-1/programmed death ligand-1 (PD-1/PD-L1) antibodies, androgen receptor blockers, tumor necrosis factor-related apoptosis-inducing ligand receptor agonists and transforming growth factor-β antagonists represent also targeted agents for TNBC [28]. Moreover, signal transducer and activator of transcription 3 (STAT3) [29], EGFR [30], gamma-butyrobetaine hydroxylase (BBOX1) [31], cyclin-dependent kinases CDK12/CDK13 [32], mammalian transcription factor BACH1 [33], cMET oncogene [34] and integrin subunit alpha-V gene (ITGAV) [35] have been described as potential therapeutic targets for TNBC in the literature [36]. It is also known that other pathways, such as the insulin-like growth factor-1 (IGF-1)/insulin-like growth factor receptor-type 1 (IGF-1R)—focal adhesion kinase (FAK)—yes-associated protein (YAP) transduction pathway, are able to trigger the growth of TNBC cells [37,38]. Dysfunctions of micro RNAs (miRs), a class of ~22 bp noncoding regulatory RNAs, may play an important role in the initiation and progression of cancer, and recent studies on the possible involvement of miR-331-3p [39], miR-490-3p [40], miR-424-5p [41] and miR-335-3p [42] in the tumor microenvironment have been carried out in TNBC. Neoadjuvant chemotherapy (NACT) is an important form of therapy for TNBC patients, though with limited effectiveness, narrow response durations and considerably toxic profiles [43]. Platinum-based NACT has been recently reviewed for the treatment of TNBC [44]. In the last years, great effort has been spent to identify new predictive biomarkers and relative therapies [45,46]. Moreover, novel drug combinations and nanotechnology may represent promising strategies for the treatment of TNBC [47,48]. The combination of anti-angiogenic therapy and immunotherapy has been used in the treatment of advanced metaplastic breast carcinoma (MBC) [49]. In recent studies, it has been suggested as an interesting strategy to combat TNBC [50,51].

It is noteworthy that there are still no FDA-approved targeted therapies for patients with TNBC. The use of an old drug for a new disease indication, named drug repositioning or repurposing, is a very promising strategy used in different fields of drug discovery [52]. Recently, it has also been proposed for targeted therapy in TNBC, as for the anti-leprotic drug clofazimine, which efficiently suppresses tumor growth and is correlated with in vivo inhibition of the Wnt pathway [53]. Adoptive cell therapy, particularly chimeric antigen receptor (CAR)-modified T cell therapy, has gained much attention in the last years [54]. CAR T cell-based immunotherapy redirects the patient’s immune system to directly recognize and eradicate tumor cells expressing tumor-associated antigens (TAAs) [55]. However, unlike the remarkable clinical success of CAR-T cell therapies in hematologic cancer, resulting in FDA approval of Kymriah and Yescarta, the development of this type of therapy for solid tumors has been much slower. Recent studies show that the combination of CAR-T cells with other therapies seems to improve the clinical outcome in patients with TNBC [56].

Finally, the Coronavirus disease (COVID-19) pandemic [57] induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 completely reprioritized healthcare resources [58,59]. The neglect of breast cancer (particularly TNBC) patients during the outbreak could have negatively impacted their overall survival, as delays in treatment and medical consulting are essential for tumor progression and metastasis. Moreover, it should be considered that SARS-CoV-2 is a respiratory virus, with systemic consequences in the management of TNBC patients with metastatic versus localized disease. This review is focused on the novel therapies used for the treatment of TNBC, including nutraceuticals, and studies regarding recent updates obtained in medicinal chemistry in this field.

2. Therapeutic Treatments

Several therapeutic strategies have been proposed for early and advanced TNBC and are summarized in Figure 1 [60]. This disease differs in several characteristics from other BC subtypes, and it is more likely than other BC subtypes to benefit from immune checkpoint blockade [61]. Immunotherapy is a useful strategy for the treatment of TNBC [62,63], and immune checkpoint inhibitors (ICIs) can block immunosuppressive receptors to improve the cytotoxicity and proliferative capacity of tumor-infiltrating lymphocytes (TILs) [64,65]. ICIs include monoclonal antibodies against PD-1 (e.g., pembrolizumab, nivolumab, cemiplimab) [66,67], PD-L1 (e.g., atezolizumab, durvalumab, avelumab) [68], and CTLA-4 (e.g., ipilimumab and tremelimumab) [69,70]. TNBC has more TILs and thus better responds to ICIs; moreover, high levels of TILs in TNBC are associated with improved prognosis in early-stage TNBC. Futher, TNBC has higher levels of PD-L1 expression on both tumor and immune cells, thus providing direct targets for ICIs. However, immune checkpoint inhibitors are not able to hamper the ex-novo expression of oncoproteins such as PD-L1 in tumoral cells. Instead, microRNA-based gene therapy leads to the restoration of specific microRNAs, which can downregulate tumoral PD-L1 expression, inhibiting TNBC development. This approach suppresses tumor proliferation and migration and regulates different oncogenic signaling pathways in TNBC cells [71].

Taxane-based chemotherapy is still the mainstay of treatment in early stages of TNBC. Among taxanes, paclitaxel protein-bound is a well-known microtubule inhibitor used for TNBC treatment, even if there are some limitations, including the barriers to effective drug delivery of highly lipophilic agents. In order to overcome the latter limitation, a next–generation taxane, nab-paclitaxel, was specifically designed and showed a therapeutic index higher than paclitaxel, as demonstrated in Phase III trials [72]. Docetaxel (Taxotere^®), discovered in the 1980s, has been one of the most significant chemotherapeutic medicines used to treat cancer [73], but it may present side effects on the normal microbial flora of the body, leading to antibiotic resistance. Since the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway is frequently activated in TNBC, Capivasertib (AZD5363) [74], a potent selective oral inhibitor of all three isoforms of the serine/threonine kinase AKT, demonstrated preclinical activity in TNBC models, and drug sensitivity has been associated with activation of PI3K or AKT and/or deletions of PTEN [75]. Vinblastine, a Vinca alkaloid [76], shows various biochemical effects, such as microtubule disorganization, inhibition of protein and nucleic acid synthesis, increased oxidized glutathione, alteration of lipid metabolism and the lipid content of membranes, elevation of cyclic adenosine monophosphate (cAMP) and inhibition of calcium–calmodulin regulated cAMP phosphodiesterase. Particularly, it induces the assembly of tubulin into non-microtubule polymers such as para-crystals or spiral proto-filamentous structures [77]. Finally, dasatinib, an approved second-generation inhibitor of multiple tyrosine kinases (Src, BCR-ABL, c-KIT, PDGFR-α, PDGFR-β and ephrin receptor kinase), was suggested in preclinical studies as a potential targeted treatment for TNBC. However, in Phase II clinical trials it showed limited single-agent activity in patients with locally advanced or metastatic TNBC, thus further studies are needed.

3. Novel Approaches to Therapy in TNBC

Since the drugs used to treat TNBC often exhibit their action without any selectivity between cancer and normal cells, new effective treatments are needed that are capable of killing cancer cells without negatively interfering with normal cells [78]. Therefore, the design and synthesis of new targeted agents for TNBC is required due to the high heterogeneity of this disease in order to find new bioactive and safe molecules. In this regard, several strategies may be addressed. In addition, drug repositioning is one of the easiest and cheapest strategies [79], particularly in oncology, where finding new safe and effective therapeutics is becoming more and more difficult and expensive. There is enhanced interest regarding the use of previously approved drugs as possible anticancer treatments [80]. Finally, nanoparticles have also been recently studied since they may improve bioavailability and solubility of anticancer compounds, minimizing their side effects. Further, nutraceuticals, which are able to interfere selectively with cancer cells without being toxic on healthy cells, have gained interest. All these therapeutic approaches are described below and summarized in Figure 2.

3.1. Drug Repurposing for TNBC

Drug repurposing or drug repositioning is a strategy that significantly reduces the time and the cost of drug development [81]. Indeed, it is known that the discovery and development of a new drug requires much time and countless investments. In addition, almost 90% of newly discovered drugs are later withdrawn due to side effects, thus drug repurposing accelerates clinical trials in light of their previous safety and toxicological studies [82]. This is particularly relevant for TNBC, which currently has the worst prognosis and for which there are few viable treatment options (

Table 1. Structures of repurposed drugs studied for the treatment of TNBC.

Structure	Name	Original Indication	Ref
	Propranolol	Hypertension	[85]
	ICI-118,551	Hypertension	[85]
	Atenolol	Hypertension	[86]
	Osthole	Osteoporosis	[87]
	Risedronate sodium	Osteoporosis	[88]
	Zoledronic acid	Osteoporosis	[88]
	1	Thalidomide analogues	[90]
	2	Thalidomide analogues	[90]
	Flubendazole	Anthelmintic	[91]
	Niclosamide	Anthelmintic	[92]

) [83]. Several data demonstrated that β-adrenergic signaling in BC activates genes involved in inflammation, ETM processes and angiogenesis [84]. Literature studies demonstrated the ability of β-adrenergic blocker drugs to interfere with TNBC cell proliferation and migration. Xie et al. (2019) [85] assessed that propranolol and the β₂-adrenoceptor antagonist ICI 118,551 caused a significant reduction in MDA-MB-231 cell viability, inducing a G0/G1 and S phase cell cycle arrest and apoptosis. This activity is probably linked to the decrease of the phosphorylation levels of extracellular-signal-regulated kinase (ERK)1/2 and the expression levels of cyclo-oxygenase 2 (COX-2) induced by the β-blockers. Talarico et al. (2016) [86] also demonstrated that the selective β1-blocker atenolol inhibited in vitro cell proliferation in MDA-MB-435 cells and enhanced the ability of metformin to reduce in vivo angiogenesis and metastasis in TNBC cells. Dai et al. (2018) [87] investigated the effect of osthole, a coumarin derivative mainly used in osteoporosis, in TNBC treatment in vitro and in a xenograft model. The authors found that osthole reduced the growth of a TNBC cell panel, inducing apoptosis in vitro and in vivo. This activity is due to the capability of osthole to inhibit STAT3 phosphorylation and nuclear translocation. Stella Sravanthi et al. (2019) [88] identified two other oral bisphosphonates useful to treat osteoporosis, risedronate sodium and zoledronic acid, as STAT3 inhibitors by using in silico studies, and they also proved their toxicity to TNBC cells in vitro. Thalidomide, another repurposed cancer drug, was first used for relief of morning sickness in pregnant women and then withdrawn from the market because of its dramatic teratogenic effects [89]. However, it has recently been successfully used to treat different diseases, including cancer. Iacopetta et al. (2017) [90] reported that some thalidomide-correlated compounds have shown interesting anticancer activity, higher than thalidomide’s, against MDA-MB-231 cancer cell lines. Two of them (compounds 1 and 2) were able to drastically reduce the migration of BC cells by regulation of the two major proteins involved in epithelial-to-mesenchymal transition (EMT): vimentin and E-cadherin. Finally, recent literature studies indicated good anticancer activity in several cancer types by flubendazole and niclosamide, two widely used anthelmintic agents. In particular, Oh et al. (2018) [91] reported that flubendazole treatment produced a significant induction of apoptosis together with G2/M phase accumulation, caspase-3/-7 activation and dysregulation of STAT3 activation in TNBC cells. Flubendazole also increased the activity of two drugs used for TNBC treatment, fluorouracil and doxorubicin, reducing tumor resistance and side effects. The FDA-approved anthelmintic agent niclosamide was found to inhibit TNBC cell growth in vitro and in vivo, reversing EMT and inhibiting the stem-like phenotype in cancer cells [92]. Moreover, in a recent work, repositioning natural products for BC has been proposed [93].

3.2. Synthetic Compounds with Antiproliferative Activity in Preclinical Studies

Several recent studies on new synthetic compounds are listed below, subdivided on the basis of their chemical structure, and for some of them the mechanism for their antiproliferative activity has been described.

3.2.1. Carbazoles Derivatives or Bioisosters

Recent advances in the scientific literature regarding TNBC antitumor activity of molecules with a carbazolic core have been extensively carried out [94]. Structures are reported in

Table 2. Structure of carbazole derivatives or bioisosters studied for the treatment of TNBC.

Structure	Compound	Ref
	3	[95]
	Ellipticine	[96]
	4	[96]
	5	[96]
	6	[97]
	7	[97]
	8	[100]
	9	[100]
	10	[100]
	11	[100]
	12	[101]
	13	[102]

. Listing them, N,N,N-trimethylethanammonium iodide alkylcarbazole 3 showed activity against MDA-MB-231 cell lines, and this activity was demonstrated to be strictly connected to the observed inhibition of human topoisomerase II (hTopo II) [95]. Moreover, the same research group studied the anticancer activity of four 1,4-dimethylcarbazole derivatives, analogues of a known antitumoral compound, ellipticine [96]. Particularly, compounds 4 and 5 possessed good anticancer activity on different BC cells, including triple-negative MDA-MB-231. This activity seems to be linked to their ability to inhibit DNA topoisomerase II. A study on 3-(alkyl(dialkyl)amino)benzothieno[2,3-f]quinazolin-1(2H) showed that compounds 6 and 7 exhibited good antitumor activity against MDA-MB-231 cell lines without cytotoxicity against normal cells. The compounds were suggested as multi-target tools due to their dual inhibition of different cellular proteins, e.g., tubulin and human topoisomerase I, involved in relevant cellular processes [97]. Interestingly, a nitrocarbazole acting against triple-negative MDA-MB-231 cell lines was recently proposed as a new microtubule-targeting agent in BC treatment [98,99]. Other research groups studied carbazole derivatives as anti-cancer and anti-migratory agents. In the study by Vlaar et al. (2018) [100], compounds 8 and 9 inhibited the migration of metastatic cell line MDA-MB-231 by 32% and 34%, respectively, at 10 µM, comparable with parent compound EHop-016, which exhibited anti-migratory activity of 33% at 10 μM (and 17% at 5 µM), but they did not inhibit the growth of the cell lines tested at concentrations ≤ 50 μM. On the other hand, compounds 10 and 11 inhibited the growth at ~15 µM but did not significantly inhibit migration. Hou et al. (2014) [101] demonstrated that compound 12, a carbazole derived with fluorophore, strongly induced apoptosis in vitro in a wide number of TNBC cancer cell lines. Moreover, it reduced the growth of human TNBC xenograft tumors (SUM149) in vivo without inducing severe toxicity. Both in vitro and in vivo studies showed that phospho-STAT3 inhibition was due to protein-tyrosine phosphatase PTPN6 upregulation. Xiao et al. (2018) [102] examined new derivatives of racemosin B, a natural indolo[3,2-a]carbazole isolated from the green alga Caulerpa racemose. Among several alkylamide derivatives, compound 13 exhibited good growth inhibition against human BC cell lines, inducing G2/M cell cycle arrest and apoptosis in MDA-MB-231 cancer cell lines.

3.2.2. Indole Derivatives

Recent literature data reviewed the encouraging anti-breast cancer potential of indole derivatives, which have multiple mechanisms of action, such as aromatase and microtubule inhibition, DNA-binding mechanism, induction of apoptosis or inhibition of PI3K/AkT/NFkB/mTOR (

Table 3. Structure of indole derivatives studied for the treatment of TNBC.

Structure	Compound	Ref
	14	[104]
	15 and 16	[105]
	17–20	[106]
	21–24	[107]

) [103]. In a recent study by Qin et al. [104], benzopyran and indole pharmacophores were combined using molecular hybridization, obtaining a new library of 1,3,4,9-tetrahydropyrano[3,4-b]indoles. Among them, compound 14 exerted the most effective activity against MDA-MB-231 cells, with an IC₅₀ value of 2.29 μM. This compound reduced cell growth, blocking the cell cycle at the G0/G1 phase. It disrupted the mitochondrial membrane potential (MMP), caused accumulation of reactive oxygen species (ROS) and reduced glutathione, triggering cell apoptosis through caspase pathway activation. This cell death mechanism is linked to the ability of compound 14 to inhibit the regulators of PI3K/AKT/mTOR pathway in MDA-MB-231 cells. Kwon and colleagues in 2021 designed 2-(substituted arylmethyl)-1-oxo-1,2,3,4-tetrahydropyrazino[1,2-a]indole-3-carboxamide analogues. Some of them showed potent anticancer activity against two BC cell lines together with selectivity against the MDA-MB-468 cell line. In some cases, this activity was higher than the anticancer effect of gefitinib, an EGFR- tyrosine kinase inhibitor. Particularly, compounds 15 and 16 inhibited Akt T308 phosphorylation, selectively inhibiting a kinase involved in its phosphorylation, phosphoinositide 3-kinase β (PI3Kβ) [105]. In another recent work, two libraries of indole derivatives were designed and synthesized. Four of the derivatives (17–20) were extensively evaluated for their pharmacological effect on BC [106]. These four compounds showed anti TNBC activity comparable or better than that of paclitaxel, causing autophagy in MDA-MB-231 cells and triggering cell cycle arrest and apoptosis by ROS generation, mitochondrial membrane disruption and caspase activation. Moreover, they stabilized microtubules, similar to paclitaxel, and some of them were safe and well-tolerated up to a 300 mg/kg oral dose in Swiss albino mice. Finally, Guerra and co-authors [107] described good anticancer activity against MDA-MB-231 by four new carbohydrate-based benzo[f]indole-4,9-dione derivatives (21–24). These compounds increased ROS, producing DNA damage and G2/M cell cycle arrest. Additionally, the four derivatives cleaved caspases 3 and 9 plus activate the Bax/Bcl-2 pathway in vitro, thereby triggering the intrinsic apoptotic pathway.

3.2.3. Metal Complexes

Recently, great interest has been focused on complexes with metals for their anticancer activity [108,109,110,111]. A series of complexes with scandium, yttrium, and neodymium metallocene and half-metallocene were studied. It was found that all the compounds with yttrium, metallocene and half-metallocene show a good inhibition effect on MDA-MB-231 cell lines, whereas the scandium-based compounds did not show a significant effect, except for one compound and the neodymium-based compounds showed a slight effect [112]. Complexes with ruthenium are also widely studied as anticancers [113,114]. Moreover, gold, silver and copper complexes targeting human topoisomerases have been recently reviewed [115]. Li et al. (2018) [116] demonstrated that five new phosphine–gold complexes exhibited good anticancer activity against TNBC MDA-MB-231 breast cancer cells (

Table 4. Structure of metal complexes studied for the treatment of TNBC.

Structure	Compound	Ref
	(SS)-25	[116]
	26	[117]
	27	[119]
	28	[119]
	29	[121]
	30–33	[122]
	34–38	[123]
	39,40	[124]

). The most interesting compound was complex 25. Methylester functional groups in the carbon skeleton of the phosphine ligand, as found in complex 25, dramatically increased their activity and their selectivity toward cancerous cells with low toxicity for normal cells. The authors also found that cytotoxicity of normal cells also depends on chirality, as (SS)-25 had less impact on healthy cells than its (RR)-25 enantiomer. In 2020, Ortega et al. [117] evaluated the anticancer activity of an erlotinib triphenylphosphane gold(I) conjugate (1). It was 68-fold more cytotoxic than Erlotinib against MDA-MB-231 cell lines. The gold conjugate 26 also caused DNA damage, ROS increase and triggered apoptosis, inducing cell cycle arrest in S and G2/M phases. Recently, N-heterocyclic carbene-gold (I) and silver (I) have been studied for their ability to interfere with the growth of different cancer cell lines, including TNBC cells [118,119,120,121]. Among them, 27 demonstrated interesting antitumor activity against MDA-MB-231 cells, possessing the ability to interfere with at least four important intracellular targets: human topoisomerases I and II, tubulin and actin. Moreover, the silver complex 28 also exhibited good anticancer activity on this cell line [119,121]. Regarding silver complexes, Silva et al. (2020) [121] demonstrated that compound 29 [AgCl(PPh3)2(L)] (PPh3 = triphenylphosphine; L = VTSC = 3-methoxy-4-hydroxybenzaldehyde thiosemicarbazone) exerted interesting anticancer activity against MDA-MB-231 cells, inducing the typical morphological alterations of cell death, an increase of cells at the sub-G1 phase, apoptosis and mitochondrial membrane depolarization. Finally, allyl substituted N-heterocyclic carbene silver (I) complexes (30–33) [122], silver(I) complexes with mixed-ligands of thiosemicarbazones and diphenyl(p-tolyl)phosphine (34–38) [123] and benzimidazole-based N-heterocyclic carbene silver(I) complexes (39,40) [124] exhibited high activity against TNBC cells without showing cytotoxicity to normal cells.

3.3. Nanoformulations and Nanobiosensors

The current drugs used for BC treatment are associated with several limitations, such as poor oral bioavailability, non-selectivity and inadequate pharmacodynamics properties, which are critical factors to determine the efficacy of a drug. Nanoparticles (NPs) and nanomedicines may improve their bioavailability and specificity for cancer cells and diminish side effects. NPs are widely used in all areas of medicine and drug development, including oncology [125,126]. A review of targeted NPs for image-guided treatment of TNBC discussed subtypes, biomarkers and potential surface targets [127]. However, the therapeutic efficacy of nanoparticulate drugs is suppressed by a series of biological barriers. Nano-liposomes, which are lipid-based vesicles widely used for cancer diagnostics and targeted drug and gene delivery, have been studied for the treatment of TNBC, demonstrating that lipid composition, as well as concentration of nanoscale drug delivery systems, can be adjusted to modulate their cellular uptake [128]. An interesting study reported the synthesis of albumin-coated silver nanoparticles (ASNPs) and their anti-cancer effects against MDA-MB 231, suggesting them as good candidates as a chemotherapeutic drug [129]. Gold NPs have also been demonstrated to induce cell death in TNBC. Indeed, a recent work by Surapaneni et al. (2018) [130] describes the role of AuNPs in promoting oxidative stress in MDA-MB-231 and MDA-MB-468 cells, together with anticancer effects depending on their different surface charges. Remarkably, AuNP treatment also makes MDA-MB-231 cells more responsive to 5-fluorouracil (5-FU). Recently, dasatinib was encapsulated in poly(styrene-co-maleic acid) (SMA) micelles, obtaining SMA–dasatinib NPs, which protects it from fast pharmacokinetic degradation in order to prolong its activity. It showed pronounced in vivo activity against TNBC cell lines compared to a hormone-sensitive cell line [131]. Among the nanoformulations, nanoemulsions (NEs) represent another effective therapeutic strategy for the encapsulation of anticancer drugs [132]. Recently, adelfosine, an alkyl-lysophospholipid, nanoemulsions, formulated with Miglyol 812 and phosphatidylcholine as excipients, reduced highly aggressive and invasive TNBC cell proliferation in vitro and in vivo [133]. Considering the essential role of ROS as oncogene activators in TNBC, Shashni et al. (2018) [134] formulated ROS scavenging nitroxide radical-containing nanoparticles with interesting anticancer activity against MDA-MB-231 cells. In particular, these NPs downregulated NF-κB and MMP-2 expression, reducing ROS-mediated MDA-MB-231 migration both in vitro and in vivo. Fan et al. (2017) reported a novel cationic liposome nanocomplex for gemcitabine and docetaxel co-delivery. The new formulation exerted a synergistic induction of cytotoxicity, apoptosis and inhibition of wound healing, without showing systemic toxicity [135]. Finally, nanobiosensors are biosensors (simple, robust, sensitive, cost-effective) combined with nanomaterials [136] that are applicable for TNBC diagnosis [137]. Specific biomarkers, such as miRs and long noncoding RNAs (lncRNAs), are used for cancer detection. For instance, miR-199a-5p has been suggested as a suitable candidate for the diagnosis of TNBC in patients in the early stages of the disease. An electrochemical nanobiosensor to detect serum miR-199a-5p has been studied [138].

3.4. Nutraceuticals in the Prevention and Treatment of TNBC

Numerous studies have demonstrated that high consumption of fruit and vegetables guarantees a reduced onset of cancer because of the presence of several natural bioactive molecules with anticancer properties [139]. One advantage of natural anticancer compounds is that they are able to selectively interfere with transformed or cancer cells without being toxic to healthy cells. Nutraceuticals, ranging from active phytochemicals, minerals and vitamins all the way to whole functional foods, are extensively studied for the treatment of TNBC [140]. Some recent examples of nutraceuticals used for their antitumor properties are listed with their structures in Table 5.

One of the most studied nutraceuticals is curcumin, a bioactive component obtained from the rhizome of turmeric (Curcuma longa) with a wide spectrum of pharmacological properties [141]. It has been shown to inhibit triclocarban cytotoxicity in BC cells [142] Indeed, human breast cell carcinogenesis may also be induced by endocrine disruptors, such as triclocarban, an antimicrobial agent widely used in soaps, deodorants, shampoos, toothpastes and cosmetics [143,144]. Moreover, Guan et al. (2016) [145] showed that exposure of MDA-MB-231 cells to curcumin reduced Akt protein expression in a dose- and time-dependent manner, together with activation of autophagy and suppression of ubiquitin-proteasome system (UPS) function.

Generally, cell lines derived from aggressive tumor subtypes, such as TNBC, express low levels of vitamin D receptor (VDR) and are less sensitive to 1α,25-dihydroxy vitamin D₃ (1,25D) than those derived from more-differentiated tumor types. The hyaluronic acid synthase 2 (HAS2) gene is overexpressed in a high percentage of breast tumors, especially in basal-like TNBC. It has been reported that 1,25D inhibits HAS2 expression and hyaluronic acid (HA) synthesis in murine TNBC cells. Recently, it was demonstrated that Hs578T cells retain VDR and sensitivity to 1,25D, and that 1,25D suppresses proliferation, HAS2 gene expression and HA production in cells selected for high HA production [146]. The antitumor properties of vitamin D have been recently reviewed [147].

Piperine, a major bioactive constituent of black pepper (Piper nigrum) [148], has shown activity against TNBC cells. Particularly, piperine decreased the percentage of TNBC cells in the G2 phase, decreasing G1- and G2-associated protein expression and increasing p21(Waf1/Cip1) expression. Piperine also inhibited survival-promoting Akt activation in TNBC cells and induced the caspase-dependent mitochondrial apoptosis pathway [149]. Luteolin is a natural flavonoid found in citrus fruits, leafy greens, cruciferous vegetables and spices that is very effective against TNBC [150]. Literature data demonstrated that luteolin suppressed in vivo metastasis of human MDA-MB-435 and MDA-MB-231 (4175) LM2 TNBC cells to the lungs. It also inhibited in vitro cell migration and the viability of MDA-MB-435 and MDA-MB-231 (4175) LM2 cells, inducing apoptosis [151]. In addition, Lin et al. (2017) [152] reported that luteolin effectively suppressed metastases of TNBC by reversing epithelial–mesenchymal transition (EMT), which may be mediated by downregulation of β-catenin, in vitro and in vivo.

It has been known for many years that resveratrol (RSV), whose major dietary sources include grapes, wine, peanuts and soy, possesses anticancer properties against several types, including BC [153,154]. Recent studies confirmed the potential use of RSV for the treatment of TNBC. For instance, Liang et al. [155] revealed that RSV may induce TNBC cell apoptosis by decreasing the expression of the DNA polymerase delta catalytic subunit (POLD1), implicated in the activation of the apoptotic pathway. Furthermore, RSV treatment decreased the anti-apoptotic markers of MDA-MB-231 cells, such as proliferating cell nuclear antigen (PCNA) and BCL-2, and increased apoptosis markers, such as caspase-3. It has been also recently demonstrated that RSV enhanced the in vitro anticancer activity of FL118, a novel camptothecin analogue, in TNBC. Consecutive administration of RSV and FL118 inhibited cell viability in both TNBC MDA-MB-436 and MDA-MB-468 cell lines, also reducing the migratory and invasive capabilities and increasing the number of apoptotic cells [156].

Another natural compound, widely present in onions, apples, grapes, berries, broccoli and other fruits and vegetables, for which anticancer effects have been known for many years, is quercetin [157]; it is also known for its multiple biological activities [158]. Recently, Umar et al. [159] proved the capacity of quercetin to inhibit cytoplasmic HuR protein, an RNA binding protein critical for many disease conditions, and for which elevated levels are associated with TNBC progression and degeneration. Quercetin treatment significantly inhibited cytoplasmic HuR in MDA-MB-231 and MDA-MB-468 TNBC cell lines, decreasing adhesion and migration through the HuR-β-catenin axis and CD44. Chen et al. [160] also demonstrated quercetin’s in vitro inhibition of IGF1R activation and its downstream kinases Akt and Erk1/2 in a dose-dependent manner in human MDA-MB-231 cells, suppressing the metastatic phenotype and reducing the expression of EMT transcription factors. Further, in xenograft mouse models, quercetin blocked the growth of MDA-MB-231 tumor xenografts and their lung metastasis, downregulating the expression of specific markers related to invasion (Snail, Slug, fibronectin and vimentin).

Olive oil, the consumption of which is connected to a low occurrence of BC, is rich in hydroxytyrosol (HT) and oleuropein (OL). Nevertheless, the effects and mechanisms of action of HT and OL in BC cells are still uncertain. A recent study on MDA-MB-231 cell lines showed that treatment with HT or OL reduced MDA-MB-231 cell viability in a dose-dependent manner, and MDA-MB-231 cells were more sensitive to HT than OL treatment. These data suggested that HT and OL may inhibit migration and invasion of TNBC cells by activating autophagy [161]. Another study on OL demonstrated a difference between African American (AA, MDA-MB-468) and Caucasian American (CA, MDA-MB-231) cell lines. OL effectively inhibited cell growth in both cell lines, together with S-phase cell cycle arrest-mediated apoptosis. However, MDA-MB-468 cells were two-fold more sensitive to OL’s antiproliferative effect than MDA-MB-231 cells [162].

Fisetin is an important phytoflavonoid present in vegetables (onions and cucumbers), fruits (persimmon, apples, strawberries), wine and nuts, and possesses antioxidant and anticancer activities. Its effect against MDA-MB-231 cells has been demonstrated [163]. Bitter melon or bitter gourd (Momordica charantia) was shown to suppress triple-negative breast cancer cell growth [164], and recently, mango (Mangifera indica) extracts were suggested for the treatment of TNBC. M. indica bark and seed extracts significantly halted the growth of MDA-MB-231 cells with IC₅₀ values of 108 μg/mL and 33 μg/mL, respectively [165].

Pomegranate (Punica granatum L. Punicaceae) has shown antiproliferative, anti-invasive, and pro-apoptotic activity against MDA-MB-231 cell lines [166]. Moreover, pomegranate peels, the major byproducts of pomegranate juice processing, particularly the Akko variety, showed antiproliferative activity towards human BC cell lines [167]. A sea-cucumber (Holothuria scabra) extract suppressed the viability of MDA-MB-231 in a dose- and time-dependent manner by enhancing apoptosis, indicated by a marked increase of proapoptotic Bax and pro-caspase three expressions, and decreased expression of anti-apoptotic Bcl-2 [168]. Frankincense, an oleogum resin from trees of the genus Boswellia Roxb. ex Colebr., belonging to the Burseraceae family, has been studied for its antitumor properties. Its extracts, with total contents of boswellic and lupeolic acids > 30%, showed high activity against MDA-MB-231 cells [169].

Coffee and tea consumption is generally considered as protective against cancer [170,171,172,173]; however, several studies suggest that there is not an association between coffee and tea intake and cancer risk [174,175,176]. However, more epidemiologic research is required before solid, science-based recommendations can be made with regard to coffee consumption [177]. The antitumor, pro-apoptotic and antioxidant effects of Salvia fruticosa subsp. Thomasii was recently reported. The methanol-soluble fraction inhibited cancer growth in MDA-MB-231 breast cancer cell lines, leading to death by apoptosis [178]. In addition, a methanol extract of Anchusa azurea Mill. (Boraginaceae) aerial parts, a wild plant native to Europe with wide application in folk medicine, showed activity against four tumor cell lines, including MDA-MB-231 [179]. Finally, plant-derived compounds in combination with classical chemotherapeutic agents may be more efficient in TNBC treatment, possibly with lessened side effects and overcoming of resistance phenomena [180].

Table 5. Structure of nutraceuticals studied for the treatment of TNBC.

Structure	Name	Ref
	Curcumin	[142,145]
	1,25D	[146]
	Piperine	[148,149]
	Luteolin	[150,151]
	Resveratrol (RSV)	[155,156]
	Quercetin	[159,160]
	Hydroxytyrosol (HT)	[160]
	Oleuropein (OL)	[160]
	Fisetin	[163]

Table 5. Structure of nutraceuticals studied for the treatment of TNBC.

Structure	Name	Ref
	Curcumin	[142,145]
	1,25D	[146]
	Piperine	[148,149]
	Luteolin	[150,151]
	Resveratrol (RSV)	[155,156]
	Quercetin	[159,160]
	Hydroxytyrosol (HT)	[160]
	Oleuropein (OL)	[160]
	Fisetin	[163]

4. Discussion and Conclusions

TNBC, distinct by the lack of ER and PR expression and HER2 overexpression, remains the most challenging BC subtype to treat. It accounts for 15% to 20% of all BCs and typically shows aggressive behavior, including earlier recurrence and metastasis. New and more effective therapies for the treatment of BC are needed. TNBC was formerly considered a disease unapproachable with molecular therapy; however, new targeted therapies have recently been proposed due to intrinsic molecular TNBC subtyping and accurate classification and prediction of prognosis progress. In this review, repurposing studies in TNBC are described, along with the most recent medicinal chemistry studies carried out on triple-negative MDA-MB-231 cell lines. Moreover, the importance of nutraceuticals in cancer prevention and treatment of TNBC has been underlined. Novel drug combinations and predictive biomarkers may also represent promising strategies for the treatment of TNBC. The combination of anti-angiogenic therapy and immunotherapy may represent a new and interesting strategy to combat TNBC. Interestingly, nanoparticles may represent new promising strategies for TNBC therapy, and nanobiosensors can be utilized as future TNBC diagnostics. Further in-depth studies could ensure monitoring of the efficacy of current treatments and discover new therapeutic approaches to treat TNBC, a disease characterized by a poor prognosis. To date, no valid therapeutic regimens have been established and standardized.