Therapeutic Potential of Metal-Based and PARP Inhibitor Chemotherapy for BRCA1-Associated Triple-Negative Breast Cancer

Abstract

Triple-negative breast cancer (TNBC) accounts for about 10–15% of all breast cancers and is an aggressive disease with a poor prognosis. There is currently no standard treatment regimen for TNBC patients; thus, chemotherapy remains the main treatment. Anthracycline- and taxane-based regimens are the most widely used in a clinical setting, either alone or in combination with other chemotherapeutic agents, including poly (ADP-ribose) polymerase (PARP) inhibitors and platinum drugs. Platinum drugs have been used particularly in patients with BRCA1-mutated TNBC. Preclinical and clinical trials revealed that the response to PARP inhibition was directly correlated to the sensitivity to platinum chemotherapies. Inhibition of PARP enzymes has been shown to specifically target BRCA1 dysfunctional cells. Therefore, targeting breast cancer cells that possess genetic alterations that are absent in normal cells could be attained by the exploitation of synthetic lethality for the discovery of other candidate metals, i.e., ruthenium-derived compounds, as next-generation drugs for the treatment of TNBC. This prospective approach provides new insight into alternative treatments for breast cancers with BRCA1-associated TNBC.

1. Introduction

Breast cancer is a diverse histologic disease according to standardized clinicopathologic criteria. In clinical and medical situations, breast cancer has been classified into several biological subtypes with distinct histopathological, genetic, and epigenetic characteristics, clinical outcomes, and dramatically different responses to systemic therapy [1,2,3]. According to molecular subtypes, breast cancer can be categorized as luminal A, luminal B, human epidermal growth factor receptor 2 (HER2) expression, basal-like (BL), and normal breast-like [4]. The BL breast cancer subtype has distinct intrinsic characteristics, including chemotherapeutic responses as well as clinical outcomes [5]. Treatment decisions as well as options of breast cancer are made by the patient and the physician after careful consideration of the suitable treatment available based on subtype, anatomic cancer stage/grade, and biological characteristics of the cancer; patient preferences; and the risks and benefits associated with each treatment protocol. Over the past decades, treatment of breast cancer was considerably uniform, depending on standard protocols such as surgery, radiotherapy, and chemotherapy, with suboptimal outcomes. Surgery is the primary goal to remove the cancer from the breast and to assess the stage of the disease; it is used to treat breast cancer, including lumpectomy, mastectomy, sentinel node biopsy, and axillary lymph node dissection [6]. Radiation therapy using X-rays is recommended to destroy cancer cells remaining in the breast after breast surgery or mastectomy; moreover, it can prevent cancers that have spread to the lymph nodes. Some breast cancers are treated with external or internal radiation (brachytherapy) or both types of therapy [7]. Surgery is often combined with other treatments, including radiation therapy, hormone therapy, chemotherapy, and targeted therapy [8,9,10,11,12]. Hormone therapy is often used to treat breast cancers that are sensitive to hormones. Hormone therapy can be used after surgery or other treatments to decrease the chance of cancer relapse. Chemotherapy refers to the use of anticancer drugs to kill breast cancer cells, and is the most effective when the full dose and cycle of drugs is completed in a timely manner. Several studies support the use of a variety of anticancer agents or regimens. Some of the most common chemotherapy drugs used to treat breast cancer include paclitaxel, docetaxel, doxorubicin, cyclophosphamide, methotrexate, 5-fluorouracil, vincristine, vinblastine, gemcitabine, epirubicin, and platinum drugs [13]. Different chemotherapy drugs exert their actions in different ways and interfere with cancer cells at different phases of their growth and progression. Often called molecular targets, targeted breast cancer therapies are drugs or promising substances that can inhibit the growth and spread of breast cancer cells by interfering with specific molecules involved in cell proliferation.

Triple-negative breast cancer (TNBC) represents the BL subtype overlapping with breast cancer that lacks estrogen receptor (ER), progesterone receptor (PR), and HER2. It is frequently detected in African American or dark-skinned women (15–20%) [14,15,16]. However, not all basal-like cancers are determined via the absence of ER, PR, and HER2, and conversely, not all TNBCs show a basal-like cancer [17]. It has been shown that only two-thirds of triple-negative breast cancers are of the basal-like subtype. The TNBC subtype occurs in 10–15% of all breast cancer cases and is associated with high histological grade, large tumor size, high rates of proliferation with poor prognosis, and high rates of recurrence and metastases within 5 years of the initial diagnosis. TNBC patients have a poorer overall survival than patients with other subtypes of breast cancers, i.e., TNBC patients have a higher risk of the local recurrence rate (LRR) and shorter progression-free survival (PFS) and overall survival (OS) than non-TNBC patients [18,19]. Several lines of evidence have indicated that no expression of ER, PR, and HER2 in conjunction with the expression of high molecular weight basal cytokeratins (CK5/6, 14, and 17), epidermal growth factor receptor (EGFR), proliferating gene marker Ki-67, androgen receptor (AR), cyclin E, and low expression levels of cyclin D1 are commonly found in patients with TNBC [15,20].

TNBC is a highly aggressive subtype of breast cancer with limited targeted treatment options. TNBC has a specific biological profile with many potential molecular targets, including the overexpression of vascular endothelial growth factors (VEGFs) and EGFR, and high rates of BRCA mutation or deficiency in BRCA function (BRCAness). Bevacizumab is the most widely used drug as an anti-VEGF inhibitor with an improvement in PFS for randomized trials of first-line bevacizumab and chemotherapy treatment [21]. Currently, there is emerging evidence on the use of VEGF, EGFR, PARP, and mammalian target of rapamycin (mTOR) inhibitors for the treatment of TNBC. Available prospective randomized data support the use of such targeted agents in TNBC. However, the search for more specific and reliable molecular biomarkers to identify TNBC patients who are more likely to benefit from treatment is ongoing and being evaluated. Several promising therapeutic agents are currently being evaluated for their effectiveness and safety in TNBC. However, the evaluation of therapeutic agents still poses a clinical challenge due to the lack of known targeted agents, guidelines, recognized standards for treatments, and selection of TNBC patients from early prediction of pathologic complete response (pCR) to neoadjuvant chemotherapy. A recent study developed an MRI-based model for predicting complete pathologic response to neoadjuvant chemoimmunotherapy in patients with TNBC [19,22]. Moreover, blood circulating tumor DNA (ctDNA) has been introduced to assess response to treatment and recurrence prediction in patients with early-stage TNBC [15,23,24]. There is currently no single recommended first-line chemotherapy or preferred standard regimen for TNBC treatment. The first-line treatment normally includes a combination of surgery, radiation, and neoadjuvant/adjuvant chemotherapy [25]. TNBC patients have shown high response rates to neoadjuvant chemotherapy. Anthracycline/taxane/platinum chemotherapy-based regimens have been routinely used in the neoadjuvant setting for TNBC; however, anthracycline treatment is associated with undesired cardiotoxicity [26]. Homologous recombination deficiency (HRD) score has been introduced to predict the response to platinum-based neoadjuvant chemotherapy in TNBC patients [27,28]. Retrospective analysis reveals that the combination of paclitaxel with anthracycline may be more advantageous for the treatment of ER-negative and HER2-negative breast cancers. However, the addition of available targeted treatments, including PARP inhibitor (iniparib) and EGFR inhibitor (cetuximab), both in the adjuvant and metastatic settings, unfortunately failed to provide favorable outcomes [29].

Collected data have demonstrated that 20–30% of TNBC patients harbor germline BRCA1 mutations and correlates with decreased BRCA1 mRNA and protein expression [30,31]. In addition, 15–60% of TNBC has been associated with CpG island hypermethylation of the BRCA1 promoter region that shares similar histological characteristics and clinical outcomes with BRCA1 mutation carriers. It has been reported that BRCA1 promoter methylation occurs in 32% of TNBC patients and 21% of non-TNBC patients [32,33,34]. In vitro studies showed that a sporadic breast cancer cell, UACC3199, carrying a hypermethylated BRCA1 promoter, conferred a similar degree of sensitivity to the poly(ADP-ribose) polymerase (PARP) inhibitor, olaparib, as did the mutated BRCA1 breast cancer cells, MDA-MB-436 and HCC1937, and a combination treatment with carboplatin was more effective than either drug alone [35]. In a similar way, BRCA1 promoter methylation conferred sensitivity to platinum compounds [36,37]. In recent clinical studies of TNBC patients, BRCA1 promoter methylation has increased disease-free survival (DFS) and disease-specific survival (DSS) compared with non-TNBC patients who received adjuvant chemotherapy [32].

2. Role of BRCA1 in BRCA1-Associated Triple-Negative Breast Cancer

Since the discovery of the breast cancer susceptibility gene, BRCA1, which is located on chromosome 17q, significant advances have been made in understanding the structure and function of the BRCA1 protein. The BRCA1 protein accounts for its ability to maintain genomic integrity and suppress tumor formation through several cellular processes, such as the DNA damage repair pathway, transcription regulation, cell cycle progression, apoptosis, and protein ubiquitination [14,38]. Several lines of evidence have supported an important role of the BRCA1 protein in repairing DNA double-stranded breaks (DSBs) via the homologous recombination (HR) pathway. HR depends on the presence of sister chromatids formed during DNA synthesis as a template for exchanging a homologous sequence with a single-stranded sequence. Thus, this pathway is only available during the S/G2 phases of the cell cycle. Such a repair process requires the recruitment of many proteins to make up a complex that acts as a concert in repairing DNA damage. The underlying molecular mechanism of the HR repair pathway is schematically proposed in Figure 1. A DSB, after being exposed to chemotherapeutics, ionizing radiation, or DNA-damaging agents, activates the ataxia telangiectasia mutated (ATM) kinase, which subsequently catalyzes the MRE11/RAD50/NBS1 (MRN) complex. The MRN complex then recognizes the site of the DNA damage and uses its 5′ ⟶ 3′ exonuclease activity (depicted by scissors) to create single-stranded 3′ ends. Participating proteins such as BRCA1, BRCA2, RAD51, and RAD52 are simultaneously recruited to the DNA-damaged sites [39,40]. BRCA1 interacts with BRCA2 to facilitate the nuclear transport of RAD51. RAD52 aids in RAD51 binding to the damaged ends to form a nucleoprotein filament. RAD51 exchanges a homologous sequence from a single strand within a double-stranded molecule (the sister chromatid as a template) with a single-stranded sequence. Resolvases then restore the junctions (termed Holliday junctions) as a result of homologous recombination. Finally, two copies of an error-free DNA molecule are produced. However, in the absence or impairment of BRCA1, the alternative pathway of non-homologous end joining repair (NHEJ) may be required. The NHEJ pathway is less accurate than the HR pathway because it does not use the sister chromatid as a template [41,42]. This may lead to defective repair and enhance the toxicity of the DNA damage. In addition, BRCA1 has been shown to be involved in nucleotide excision repair (NER), demonstrating a close association between the transcription-coupled repair (TCR) pathway [43,44] and the increased cisplatin sensitivity in BRCA1-deficient cells [45,46].

There is a substantial relationship between dysfunctional BRCA1 and triple-negative status. Breast cancer patients with a BRCA1 mutation are also frequently triple-negative and basal-like [47]. BRCA1-associated breast cancers are well categorized as “BRCAness”, which lacks ER/PR/HER2, high-grade, and basal phenotypes. BRCA1 plays an important role in the repair of double-strand breaks (DSBs) through several mechanisms. It is a vital mediator in the HR pathway to repair DSBs (Figure 1). When BRCA1 is inactivated, breast cancer cells become hypersensitive to DNA damage [25]. If not properly repaired, cancerous cells develop genetic instability and simultaneously enter programmed cell death or apoptosis. Therefore, BRCA1-dysfunctional breast cancer may be particularly sensitive to certain classes of DNA-damaging drugs, such as platinum-based drugs as well as their derivatives, and relatively resistant to mitotic spindle poisons, such as taxanes and vinca alkaloids [38]. A decrease in BRCA1 expression was observed in BRCA1-deficient TNBC patients who subsequently became resistant to taxane-based chemotherapy. TNBC patients with non-BRCAness had significantly higher pathological complete responses (pCRs) and a shorter progression-free survival than those with BRCAness tumors. The results indicated that BRCAness breast cancers had a significantly poorer response to taxane regimens than non-BRCAness breast cancers [48]. The neoadjuvant use of cisplatin and carboplatin results in high rates of pCRs in TNBC patients with a defective BRCA1 function [49] (Figure 2).

3. Platinum-Based Chemotherapy for BRCA1-Associated Triple-Negative Breast Cancer

The discovery of the first platinum complex cis-diamminedichloroplatinum(II) as an effective anticancer drug, known as cisplatin, was one of the great success stories in the field of medicinal inorganic chemistry and heralded a new area of anticancer research based on metallopharmaceuticals. The cytotoxicity of cisplatin to cancer cells is directly related to how much drug enters the cell and its cellular accumulation. It is generally accepted that the cytotoxicity of cisplatin results from the interaction of the drug with DNA through the formation of a covalent bond between an activated platinum(II) complex and guanine or adenine in double-stranded DNA. The interaction is apparently preceded by an electrostatic attraction between the positively charged platinum(II) complex and the negatively charged phosphodeoxyribose DNA backbone, and facilitated by a bidirectional diffusion along the DNA backbone [50], followed by the replacement of the remaining chloro ligands before the formation of DNA adducts. The DNA adducts interfere with transcription and translation and lead to apoptosis (Figure 3).

Over the past decades, cisplatin has been widely used in clinical practice for the treatment of a variety of cancers, including testicular, ovarian, head and neck, and lung [51]. Historically, platinum-based chemotherapy has not figured prominently in the treatment of breast cancer. Treatment standards for metastatic breast cancer have included taxane and the use of single-agent capecitabine or vinorelbine for those who relapse shortly after completion of adjuvant taxane treatment. Currently, cisplatin and its derivative carboplatin are among the most effective chemotherapeutic agents in a clinical setting, and are frequently used in combination with other anticancer agents [52] (

Table 1. Platinum drugs/PARP inhibitors as single and combination therapy for the treatment of TNBC.

Chemotherapy	TNBC (n)	Setting	Outcomes	References
Cisplatin	86	Metastatic	RR 37%	[55]
Olaparib	27	Metastatic	pCR 41%	[56]
Cisplatin/veliparib	162	Neoadjuvant	pCR 74%	[57]
Cisplatin/docetaxel	27	Metastatic	ORR 59%	[58]
Carboplatin/docetaxel	28	Neoadjuvant	pCR 86%	[59]
Carboplatin/veliparib	634	Neoadjuvant	pCR 47%	[60]
Carboplatin/veliparib	107	Neoadjuvant	pCR 61%	[61]
Gemcitabine/carboplatin/iniparib	258	Metastatic	ORR 34%	[62]
Paclitaxel/carboplatin	24	Neoadjuvant	pCR 33%	[63]
Paclitaxel/doxorubicin/cyclophosphamide/carboplatin	60	Neoadjuvant	pCR 51%	[64]
Eribulin/carboplatin	22	Neoadjuvant	pCR 40%	[26]
Paclitaxel/carboplatin/olaparib	559	Neoadjuvant	pCR 51%	[65]
Mitomycin C/vinblastine/cisplatin	34	Metastatic	ORR 41%	[66]
Gemcitabine /carboplatin/iniparib	80	Neoadjuvant	ORR 36%	[67]

pCR, pathological complete response; ORR, overall response rate; RR, response rate; and CR, complete response.

). Platinum drugs have been used as a combination regimen of docetaxel and trastuzumab, which is an alternative to anthracycline, taxane, and trastuzumab-based treatments of HER2-positive early breast cancer patients [53]. More experimental neoadjuvant regimens, including platinum drugs combined with taxane, have been shown to have high pCR rates in TNBC patients. Moreover, the role of platinum-based chemotherapy as an alternative therapy to anthracyclines and taxane-resistant breast cancer had an overall response rate of 31%, and a median overall survival of 3–4 months [54]. Therefore, it is necessary to acquire a better understanding of biology and etiology of TNBC, therapeutic targets or molecular biomarkers, as well as patient preferences for the successful treatment of each TNBC subtype.

Despite increasing evidence indicating that BRCA1-mutated breast cancer and TNBC confer sensitivity to platinum-derived compounds (Figure 2), randomized data comparing such platinum-based agents versus standard regimens for breast cancer patients in terms of efficacy and safety are still lacking and remain a subject of argument [49,68]. Clinical trials show that carboplatin has a clinical advantage over docetaxel in patients with BRCA1-associated TNBC, but not in other TNBC patients. In contrast, recent findings have demonstrated that the application of carboplatin in conjunction with paclitaxel, doxorubicin, and an antiangiogenic drug (bevacizumab) did not improve pCR rates in patients harboring HER2-positive or BRCA1-associated TNBC [68]. The association between BRCA1 deleterious somatic mutation status and response to a taxane/carboplatin neoadjuvant chemotherapy in women with TNBC has been investigated, with the results showing a higher pCR rate than non-carriers (60% vs. 30%, p = 0.32). Therefore, breast cancer patients with BRCA1 somatic mutations are more likely to respond to the platinum drug carboplatin [27,69]. Nonetheless, a controversial finding showed that there is no advantage of neoadjuvant cisplatin (pCR rate of 18%) over an anthracycline (doxorubicin) and cyclophosphamide (AC-based regimen, pCR rate of 26%), after surgery, in patients with HER2-negative, BRCA1-associated breast cancer, whether HER-positive, HER-negative, or TNBC [68]. Consequently, the decision on chemotherapeutic treatment should be carefully considered to avoid any other adverse drug reactions (ADRs) or events. To meet criteria, the application of poly(ADP-ribose) polymerase (PARP) inhibitors alone or in combination with chemotherapeutic drugs/agents seems to be an attractive neoadjuvant option for patients carrying these breast cancer subtypes. The results of a Phase II randomized trial of cisplatin in the presence/absence of veliparib in metastatic TNBC and/or germline BRCA-associated breast cancer (Swog S1416) demonstrated superior progression-free survival (PFS) with PARP inhibitor monotherapy compared with chemotherapy for gBRCA mutation [57].

4. PARP Inhibitors for BRCA1-Associated Triple-Negative Breast Cancer

One of the bottlenecks in cancer-related drug discovery is the identification of tumor-selective characteristics. Normally, cancer cells often have DNA repair defects. This leads to the conceptual framework that a combinative inhibition within DNA repair pathways in cancer cells may result in synthetic lethality. Therefore, several investigators have exploited this idea to gain a better understanding of the concept of loss-of-function mutations found in DNA repair genes. In eukaryotic cells, there are more than 100 genes that repair DNA damage [70]. A large body of evidence has demonstrated that HR-defective cancers are highly sensitive to DNA-damaging agents, cytotoxic drugs, and PARP inhibitors [71,72]. In addition, the DSB repair defect in BRCA1-mutated cells has been exploited in a synthetically lethal approach with PARP inhibition [52,73]. Moreover, BRCA1-mutated breast cancers have been shown to be more immunogenic than HR-proficient cancers.

PARP is a highly abundant DNA-binding protein that plays an important role in estrogen-related transcription, chromatin remodeling, and the DNA repair pathway, particularly in the base excision repair of single-stranded DNA breaks (SSBs) [3,74]. Inhibition of PARP leads to the accumulation of SSBs that finally cause the formation of DSBs (Figure 4) after interfering with DNA replication processes. These DSBs cannot be accurately repaired in cancer cells with HR deficiency [75]. Therefore, the inhibition of PARP using synthetic killing agents could be advanced as a novel targeted therapy for breast cancer patients with dysfunctional BRCA1 (Figure 5) [76].

Several PARP inhibitors (PARPis) have been developed with different antitumor activity [77,78,79]. It is well known that they exhibit synthetic lethal effects on tumors defective in the BRCA1 gene, which encodes the protein required for efficient HR repair [80]. Cells harboring BRCA1 mutations are much more sensitive to PARP inhibitors than wild-type cells [73]. As previously mentioned, triple-negative breast cancer patients are closely associated with defective BRCA1. PARP inhibitors impair BER through PARP inhibition by trapping the PARP complex to DNA, subsequently causing DSBs [81]. BRCA mutations have been validated as biomarkers for patient selection for PARP inhibitors in clinical settings. PARPis have been developed as single-agent treatments for BRCA1/2-deficient breast, ovarian, and prostate cancers [82,83]. Knockout of the PARP genes in experimental cell models impaired normal embryonic development and led to high levels of SSBs, eventually resulting in the accumulation of DSBs, cell cycle arrest, and/or cell death [84]. Many PARPis have progressed to clinical trials, including olaparib, talazoparib, veliparib, rucaparib, niraparib, mefuparib hydrochloride, pamiparib, and suraparib [17,85,86,87]. FDA approved the PARP inhibitors olaparib and talazoparib in 2018/2019 for clinical treatment of refractory metastatic breast cancer with deleterious germline BRCA1/2 mutations, BRCA-mutated HER2-negative metastatic breast cancer, gBRCAm metastatic pancreatic cancer, and maintenance of BRCA-mutated (gBRCAm or sBRCAm) advanced epithelial ovarian cancers [88,89]. In addition, the trial demonstrated a positive correlation between higher HRD scores and better response to talazoparib, particularly in patients with PALB2 mutations [89]. Nonetheless, in germline BRCA1/2 (gBRCA1/2)-mutated HER2-negative advanced breast cancer, talazoparib did not significantly improve overall survival over chemotherapy [90]. Compared with other PARP inhibitors, pamiparib showed improved penetration across the blood–brain barrier in mice [86,91]. Recently, a potent PARP inhibitor, senaparib, has been developed and shown to be highly potent in cell viability tests against tumor cells with BRCA1/2 mutations [76]. However, acquired resistance to PARP inhibitors limits their clinical efficacy [92]. Novel PARP inhibitors with increased PARP trapping capacity, such as veliparib derivatives, have been developed as promising candidates [93]. In addition, it has been reported that the emergence of acquired resistance caused by secondary genetic or epigenetic modification that restores HR repair frequently occurs [94,95,96].

5. Cellular Resistance to Platinum Drugs

Presently, there is concern about the clinical limitations of the anticancer platinum drugs, cisplatin and its derivatives, that are widely used as chemotherapeutic drugs for the treatment of TNBC. The clinical disadvantages of cisplatin include severe toxicity, such as nephrotoxicity and neurotoxicity; its limited applicability to a narrow range of tumors; and limited efficacy for cancer cells that have developed platinum resistance [97,98]. Clinical data have shown that breast cancer patients at first responded well to platinum drugs; however, patients became drug-resistant after prolonged treatment. Cellular resistance to cisplatin is caused by a wide variety of mechanisms, including decreased drug accumulation, increased levels of the intracellular thiols, and increased DNA repair [99] as depicted in Figure 6.

5.1. Decreased Drug Accumulation in Cisplatin Resistance

Decreased drug accumulation could be attributed to decreased influx, decreased intracellular binding, or increased efflux. The mechanism of cisplatin uptake is believed to occur through a combination of passive diffusion and carrier-mediated transport processes [100]. Several copper transport proteins are found to play significant roles in drug transport and drug resistance [101,102]. The high-affinity copper transporter CTR1 has been implicated in cisplatin transport in non-renal cells. Evidence derived from cell models has indicated that CTR1 regulates cisplatin cytotoxicity by interfering with drug uptake. The copper exporters ATP7A and ATP7B have been found to be involved in cisplatin resistance by enhancing efflux [103,104]. A recent study showed that Cox17, a copper chaperone that delivers cuprous ions to mitochondria for the activation of cytochrome c oxidase, can transport platinum to mitochondria. An increased Cox17 level potentiates platinum accumulation in mitochondria and enhances the cytotoxicity of cisplatin. Moreover, dysfunctional Cox17 was found to reduce cisplatin sensitivity of the cell [105,106].

5.2. Increased Binding to Intracellular Thiol Molecules

Cisplatin resistance has resulted from an increase in the intracellular level of glutathione (GSH) [107]. GSH is a tripeptide thiol, γ-glutamylcysteinylglycine, and the major cellular non-protein thiol with high concentrations (0.5–10 mM) in cells. It plays an important role in a variety of physiological functions in cellular defense and metabolism, including the protection of cells from oxidative stress and the detoxification of electrophilic compounds [108]. Intracellular GSH can regulate the amount of platinum being transported. An increase in GSH can lead to a decrease in copper concentration, which enhances the expression of CTR1 [103]. The binding of cisplatin to GSH is usually considered to be associated with drug resistance due to the reduced DNA platination [109]. Elevated GSH levels are observed in some cisplatin-resistant cancer cells. The GSH–platinum complex is eliminated from cancer cells using an ATP-dependent glutathione S-conjugate export pump [108], which results in a reduction in the intracellular accumulation of the platinum complex. In addition, overexpression of metallothionein (MT) has been consistently observed in cisplatin-resistant tumor cell lines. These findings indicate that cisplatin resistance may be prevented or reduced through the regulation of metallothionein synthesis.

5.3. Increased DNA Repair

There are several DNA repair pathways involved in the repair of DNA damage caused by various anticancer drugs, including the platinum drug cisplatin [110]. Each DNA repair pathway can increase repair activity in cancer cells, enabling them to survive DNA damage that is induced by chemotherapeutic treatments, which results in resistance to chemotherapeutic drugs [111]. Although many patients initially respond favorably to cisplatin-based chemotherapy. Such resistance is often driven by enhanced DNA repair capacity, as evidenced by decreased platinum adduct accumulation, increased repair synthesis, and reactivation of cisplatin-damaged plasmids.

Cisplatin–DNA adducts can be excised and repaired before replication or DNA synthesis via base excision repair (BER) or nucleotide excision repair (NER). BER excises a single damaged DNA base or a short strand harboring the damaged base [112], while NER excises a single-stranded DNA molecule (24–32 base pairs) containing the DNA lesions [113]. Preclinical-derived data have shown that the NER pathway is required for the majority of intrastrand cisplatin–DNA adducts [114]. In addition, the excision repair cross-complementing 1 (ERCC1) gene, a key factor in NER, has been shown to undergo transcriptional upregulation in certain patients following cisplatin treatment. Collectively, these results underscore the importance of gene-level DNA repair mechanisms as critical determinants of cisplatin resistance.

The overexpression of breast cancer suppressor protein 1 (BRCA1)-mediated HR was observed in cisplatin-resistant breast and ovarian carcinoma cell lines [115,116] as well as the occurrence of secondary BRCA1 mutations in breast cancers previously treated with platinum drugs [117]. The tumor suppressor gene p53 has also been linked to DNA repair capacity. Disruption of p53 in human breast cancer cells increases cisplatin sensitivity, likely due to impaired DNA repair ability. Moreover, inhibition of the BRCA/Fanconi anemia pathway sensitizes tumor cells to cisplatin, whereas deficiencies in this pathway confer cisplatin resistance. Furthermore, activation of proto-oncogenes c-fos and c-myc enhances downstream gene expression and stimulates repair protein activity, thereby further strengthening cellular DNA repair capacity [118].

5.4. Epigenetics in Resistance to Cisplatin

Epigenetics, including DNA methylation, histone modifications, and microRNA silencing, play a pivotal role in the development of cisplatin resistance. Aberrant hypermethylation of CpG islands within tumor suppressor gene promoters disrupts key cellular processes such as cell cycle, DNA repair, apoptosis, and carcinogen metabolism, thereby facilitating cancer progression [119]. Recent studies have reported that TNBC patients with germline BRCA1 mutation and BRCA1 promoter methylation become resistant to platinum-based therapy [120,121]. In addition, epigenetic remodeling mechanisms are responsible for DNA damage response (DDR) dysregulation and hence drug resistance. The emergence of acquired resistance caused by secondary genetic or epigenetic modification that restores HR repair frequently occurs [122].

However, no clear-cut dominant mechanism of resistance to cisplatin has been identified in human cancer cells. Cisplatin resistance of a particular cell may be due to several possible mechanisms. Moreover, a given cell may use more than one mechanism or can simultaneously develop several mechanisms of resistance. Other mechanisms may be involved in cellular resistance to cisplatin, such as altered folate metabolism [123], changes in oncogene expression [118], modulation by protein kinases [124], and loss of DNA mismatch repair [125]. In addition, an increased tolerance to DNA damage can be another explanation for increased resistance to cisplatin. Cells can achieve damage tolerance by bypassing DNA lesions during replication or transcription [126].

6. Cellular Resistance to PARP Inhibitors

Cellular resistance to PARP inhibitors is caused by a wide variety of mechanisms, including reverse mutation, restoration of replication fork stability, dysregulation within molecular signaling pathways, and enhanced drug efflux, as depicted in Figure 7.

6.1. Reverse Mutation

PARP inhibitor resistance is closely associated with the reversion of BRCA1/2 mutations, which restore the open reading frame (ORF) and are, therefore, capable of the synthesis of native proteins. This reversion mutation reduces the synthetic lethality induced by PARP inhibition, ultimately leading to PARP inhibitor resistance [127]. Moreover, HR deficiency has been shown to activate the non-homologous end joining (NHEJ) pathway. NHEJ is a major pathway in the G1 phase of the cell cycle that directly connects both ends of truncated DNA double-strands and is, therefore, error-prone. In contrast, proliferating cells most likely employ HR, an error-free pathway that utilizes the sister chromatid as a template and is particularly activated during the late S and G2 phases of the cell cycle. While HR is active during the S and G2 phases of the cell cycle, NHEJ functions during interphase. In HR-defective cells, treatment with PARP inhibitors induces synthetic lethality. When NHEJ remains functional, cells still undergo cell death upon PARP inhibition. In contrast, when NHEJ is blocked, HR-defective cells can escape synthetic lethality, thereby contributing to the development of resistance to PARP inhibitors [128].

6.2. Restoration of Replication Fork Stability

BRCAness cancer cells exhibit HR deficiency and undergo replication fork collapse mediated by MRE11, which underlies their strong cytotoxic response to PARP inhibitors [129]. Replication fork remodelers such as SMARCAL1, ZRANB3, and HLTF can partially counteract this vulnerability by promoting fork reversal, recruiting protective factors, stabilizing forks, preventing excessive DNA damage accumulation, and allowing cancer cell survival despite PARP inhibitor treatment. This fork remodeling represents a critical mechanism of acquired PARP inhibitor resistance in BRCA1 mutant cancers, playing a role irrespective of HR restoration [130].

6.3. Dysregulation Within Molecular Signaling Pathways

Poly-ADP-ribosylation (PARylation) is a post-translational modification characterized by the addition of ADP-ribose units to substrate proteins. This process is catalyzed by the poly(ADP-ribose) polymerase (PARP) family of ADP-ribosyl transferases and exerts multiple functions in cells, including proliferation, differentiation, and DNA repair [131]. PARylation requires the binding of PARP1 or PARP2 to DNA. If this binding is disrupted, the cytotoxic effects of PARP inhibitors are reduced, leading to PARP inhibitor resistance and the failure to achieve synthetic lethality [132]. It has been implicated that the PTEN protein (phosphatase and tensin homolog) and the PI3K/AKT signaling pathways are involved in PARP inhibitor resistance [133]. Loss of PTEN activity enhances PI3K/AKT signaling, interferes with DSB repair, and promotes the growth of BRCA1-deficient cancer cells by upregulating BRCA1 expression [134]. PTEN-deficient cancer cells have been closely associated with decreased RAD51 expression, one of the key proteins involved in HR [135], thereby rendering cancer cells sensitive to PARP inhibitors. In contrast, PTEN-proficient cancer cells can contribute to PARP inhibitor resistance [136].

6.4. Enhanced Drug Efflux

Cellular resistance through enhanced drug efflux is mediated primarily by ATP-binding cassette (ABC) transporters. Overexpression of Multidrug-Resistance Protein 1 (MDR1)/P-glycoprotein (PgP, encoded by Abcb1a/b) has been observed in BRCA-mutated breast and ovarian cancer cells, leading to a reduced intracellular concentration of PARP inhibitors, and consequently, PARP inhibitor resistance [137,138]. Inhibition of MDR1/PgP with agents such as tariquidar, verapamil, or elacridar has been shown to reverse resistance to PARP inhibitors [139,140].

In addition, it has been reported that cellular resistance to platinum drugs and PARP inhibitors in BRCA1-defective cancer cells is caused by overexpression of a microRNA(miR-622)-mediated regulation of NHEJ [141]. Moreover, the really interesting gene (RING) domain-deficient BRCA1 proteins (Rdd-BRCA1) are capable of contributing to PARP inhibitor and platinum resistance [142]. Therefore, to overcome resistance to platinum drugs and PARP inhibitors, prospective possibilities including the discovery of next-generation Pt/PARP selective inhibitors, novel drug delivery carriers, as well as an antibody–drug conjugation system (ADC) could be alternative approaches to address the drawbacks of chemotherapy and meet medical requirements [84,94,143,144,145,146].

7. Ruthenium-Based Chemotherapy for BRCA1-Associated Triple-Negative Breast Cancer

A search for non-platinum metal-based chemotherapeutics has been extensively investigated with recent advances in the development of new anticancer agents [147,148,149,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,169,170,171,172,173,174,175,176]. Among non-platinum-based compounds, a number of ruthenium-centered complexes have received much attention as promising alternatives to conventional anticancer platinum-based drugs. These complexes can form DNA adducts and induce crosslinking, such as effective anticancer platinum compounds, but often with significantly lower general toxicity. The lower toxicity of ruthenium complexes is partly due to a function of the ligands attached to the metal center, and it is believed to be a function of the metal itself. Ruthenium can adopt different oxidation states under physiological conditions [147,148], and the more active Ru(II) oxidation state is promoted in the hypoxic environment of cancer cells, whereas the more inert Ru(III) state is promoted in healthy tissues. Moreover, some ruthenium complexes can mimic iron (Fe) in binding to important carrier proteins such as transferrin [149,150,151,152,153], which is postulated to be a specific delivery mechanism to rapidly dividing cells, including tumor cells, because of their higher iron requirement. Ruthenium complexes appear to exhibit different mechanisms of action compared with platinum compounds (Figure 8). They can interact with DNA and disrupt replication and transcription processes. They also interfere with telomerase activity by binding to G-quadruplex structures of telomeric DNA and inhibit topoisomerases, ultimately inducing programmed cell death. These properties of ruthenium complexes give rise to less toxicity than the approved cisplatin or any other anticancer platinum(II) complexes, and may be able to overcome drug resistance that develops in the current platinum-based treatments. Moreover, they may also target cancer types that are not sensitive to existing drugs. A number of ruthenium complexes with a variety of ligands have been shown to display promising anticancer properties [154,155,156,157,171,172,173,174,175,176,177,178,179,180,181,182,183,184,185]. Several ruthenium complexes have already been shown to exhibit excellent in vivo antitumor activity, and some complexes have even entered clinical trials [150,151,177,178,179,180] (Figure 9,

Table 2. Preclinical/clinical studies and relevant anticancer activity of ruthenium complexes.

Ruthenium Complexes	Phase/ Status	Mechanism of Action/Clinical Challenges	Ref.
NAMI-A	Phase II	- Inhibits the formation of new blood vessels - Inhibits adhesion, migration, and α5β1 integrin - Strong inhibition of tumor malignancy and metastasis; however, clinical development was limited by patient side effects	[177,178]
KP-1019	Phase I	- Modulates intracellular ROS levels - Induces apoptosis through mitochondria or the MAPK/P38 pathway, and blocks the cell cycle in the G2/M phase - Enters Phase I for colorectal tumors, but clinical development was limited due to low solubility	[177,178,179]
KP-1339	Phase I	- Modulates intracellular ROS levels - Induction of apoptosis through mitochondria or the MAPK/P38 pathway, and blocks the cell cycle in the G2/M phase - Early clinical trials	[177,178]
TLD1433	Phase Ib	- Light activation, generating cytotoxic singlet oxygen and radical oxygen species, leading to cell death - Currently in phase trials for the treatment of non-muscle invasive bladder cancer	[177,178,180]
BOLD-100	Phase I	- Currently in Phase II trials for advanced gastrointestinal cancer	[177,178,180]
RM175	Preclinical	- Successful in vitro and in vivo assessments with IC50 values similar to cisplatin in vitro - Being studied in advanced clinical trials	[177]
RAED-C	Preclinical	- Targets the DNA of chromatin - Activity is similar to cisplatin (DNA-targeting proclivity and apoptosis profile) - Cytotoxicity against various cancer cell lines, including cisplatin-resistant cells - Highly active in the primary tumor - Being studied in advanced clinical trials	[177,181,182,183]
RAPTA-C	Preclinical	- Mono-aquated form is the most abundant species - Multitargeted anticancer activity with antimetastatic properties and induced pH-dependent DNA damage - Exhibits broad acting antitumor efficacy with intrinsic angiostatic activity - Suppresses cathepsin B and thioredoxin reductase activities - Modifies proteins and histone–DNA interactions - The steric bulk of the phosphaadamantane ligand is the primary factor that distinguishes histone/DNA site selectivity - Being studied in advanced clinical trials	[177,181,182,183,184]

).

Dyson et al. developed the ruthenium-based complexes, called the RAPTA family or Ru(η⁶-arene)(PTA)Cl₂ (PTA = 1,3,5-triaza-7-phosphaadamantane) [155,171,175]. A monodentate ligand, PTA, exhibits remarkable water solubility and thermal stability and forms stable metal complexes. Due to its unique properties, PTA-containing ruthenium complexes have been shown to be promising antimetastatic agents [154,172,182,183,184]. The biochemical mode of action of the RAPTA derivatives is different from that of anticancer platinum-based drugs on cancer cells [149,152,155,171]. It is generally accepted that cellular DNA is the main target for classical platinum drugs, but intracellular proteins, including thiol-containing proteins, are evidenced for the RAPTA compounds [181,182,183]. This class of ruthenium compounds shows high affinity to the thiol moiety of cysteine residues of proteins that play important roles in DNA regulation, histone modification, signal transduction, cell growth, and epigenetic pathways [181,182,183]. Despite the lower affinity for DNA, several RAPTA complexes display excellent in vivo activity, reducing the number and weight of solid metastases, but not affecting the primary tumor. The prototype compound, [Ru((η⁶-p-cymene)Cl₂(pta)], termed RAPTA-C (Figure 5), remains the best characterized compound of the series, and the underlying molecular mechanism has been demonstrated to involve mitochondrial-induced apoptosis [157]. RAPTA-C exhibited its IC₅₀ value of >300 μM for both normal and cancer cell lines. As such, it would not be considered a cytotoxic agent. In addition, RAPTA-C exhibited broad-acting antitumor efficacy with intrinsic angiostatic activity. The drug combination between RAPTA-C and the epidermal growth factor receptor (EGFR) inhibitor, erlotinib, resulted in strong synergistic inhibition of cell viability in human endothelial and human ovarian carcinoma cells [184,185]. Moreover, RAPTA-C accumulated on cellular chromatin, potentially forming adducts at specific histone sites on the nucleosome core [182]. This implied that RAPTA-C primarily targets proteins that are becoming increasingly implicated as relevant to the mode of action of RAPTA compounds.

Previous studies have demonstrated that RAPTA complexes regulate the marker genes involved in the apoptotic pathway, cell cycle progression, and the expression of BRCA1 [154,156,173,186,187]. RAPTA-EA1 (a RAPTA compound with ethacrynic acid (EA) tethered to the arene ring) and Ru(η⁶-toluene)(pta)Cl₂, termed RAPTA-T, significantly decreased BRCA1 replication in BRCA1-defective HCC1937 cells compared with BRCA1-proficient MCF-7 cells [188]. Conversely, BRCA1 mRNA expression was apparently upregulated in HCC1937 cells in the presence of the ruthenium compound, whereas it was downregulated in MCF-7 cells. However, such a ruthenium compound caused a reduction in BRCA1 expression in the tested breast cancer cells. In this case, BRCA1 mRNA expression was inversely proportional to its protein expression, as it was in HCC1937 cells. This negative correlation may derive from diverse subtypes of breast cancer [4]. It was implied that downregulation of BRCA1 mRNA expression in sporadic BRCA1-proficient MCF-7 cells might be due to ruthenation of the BRCA1 promoter region that may interfere with the transcription factors required for BRCA1 transcription activity and finally lead to decreased mRNA expression [154]. In contrast, in HCC1937 cells, whose BRCA1 mRNA expression was increased while having a significantly reduced replication, this might result from more favorable ruthenation within the BRCA1 structural gene, meanwhile removing the Ru-BRCA1 adducts. The resulting data have revealed a differential cellular response for BRCA1-deficient and BRCA1-proficient breast cancer cells to the compound. Moreover, RAPTA compounds have been found to interact with the N-terminal region of the BRCA1 RING domain proteins, both wild-type and clinically relevant variant proteins (D67E and D67Y) [189]. The binding of the compounds to BRCA1 proteins led to the release of Zn²⁺ ions in a dose- and time-dependent manner; moreover, changes in thermal events eventually resulted in the impaired function of the RING heterodimer BRCA1/BARD1-mediated E3 ubiquitin ligase activity that played an important role in DNA damage response [45,46]. The D67Y variant exhibited a decrease in ubiquitination function and was more susceptible to RAPTA treatment than the other variants tested. Moreover, treatment of the BRCA1 protein with the ruthenium compound in combination with the PARP inhibitor olaparib resulted in 5-fold inhibition of the E3 ligase activity, indicating a synergism [189]. Likewise, metallo-intercalator ruthenium(II) polypyridyl complexes caused the inactivation of BRCA1-mediated E3 ubiquitin ligase activity [47]. These findings indicate that the zinc finger motif of dysfunctional BRCA1 proteins could be a molecular target for ruthenium-based agents in the treatment of breast cancer.

8. Synergistic Effects of Olaparib in Combination with Platinum/Ruthenium-Based Anticancer Agents in BRCA1-Associated Triple-Negative Breast Cancers

The rational combination of the metal-based agents with PARP inhibitors has long been hypothesized and developed to achieve synergistic activity [65,190,191,192,193,194,195,196,197,198,199,200]. A combination treatment between platinum-containing drugs and a PARP inhibitor is one of the therapeutic strategies to improve their efficacy in BRCA1/2-deficient breast cancer cells [26,64,65,66,67]. Preclinical and clinical trials revealed that the response to PARP inhibition was directly correlated to sensitivity to platinum chemotherapies [49,52,55,58,72,73]. The assessment for olaparib in combination with chemotherapy in patients with advanced ovarian, breast, and other solid tumors has shown encouraging efficacy [60,61,62]. The excellent sensitivity of these cancers to olaparib, either single or combined treatment with the platinum-containing drugs, has provided strong support for using olaparib in combination with promising metal-based drugs as novel targeted therapeutics against BRCA-deficient cancers. With the positive outcome on progression-free survival, olaparib has been further evaluated in Phase III clinical trials in combination with cisplatin/carboplatin with gemcitabine in BRCA1-associated and triple-negative breast cancers [65]. However, it has been demonstrated that neoadjuvant olaparib did not improve any pCR rates, PFS, or OS when added to carboplatin–paclitaxel and anthracycline-based chemotherapy in TNBC patients with wild-type BRCA1/2 [65]. This lethality is a possible explanation because the cancer cells with defects in the BRCA1/2 gene are defective in HR repair. Nonetheless, resistance to PARP inhibitors or platinum chemotherapy in BRCA1 mutant metastatic breast cancer has been found [65,196]. Additionally, a recent study revealed that olaparib combined with cisplatin may exert its synergistic action on breast cancer through platinum drug resistance and the longevity regulating pathway, and downregulate the expression of targeted genes involved in apoptotic and DNA metabolic pathways [197,198]. RAPTA derivatives have been shown to be significantly more effective against BRCA1-deficient breast cancer cells than cisplatin [52,187,188]. Moreover, the combination treatment of RAPTA with olaparib exhibited a synergistic effect in BRCA1-deficient HCC1937 (5382insC mutation) cell line, in a dose-dependent manner [52]. Drug sensitivity in BRCA1-mutated cells might likely be related to a defective BRCA1 that is incapable of repairing DNA damage induced by the ruthenium compound or olaparib treatment, ultimately leading to cell death. Moreover, olaparib can be exploited for other types of cancer, regardless of BRCA1 status. Synergism has been observed for triple-negative BRCA1-proficient breast cancer cells treated with anticancer organometallic compounds and other promising inhibitors [199]. Recently, the combination of DNA-binding ruthenium(II) polypyridyl complexes and olaparib showed synergy in triple-negative BRCA1-proficient breast cancer cells [192,199]. However, a ruthenium-derived glutathione transferase (GSTP-1) inhibitor, combined with olaparib, dramatically reduced the expression of the BRCA1 protein as well as the inhibition of BRCA1 replication in triple-negative BRCA1 wild-type MDA-MB-231 breast cancer cells [52]. It appears that drug susceptibility in triple-negative BRCA1-proficient breast cancer cells is linked to the reduced expression of the BRCA1 protein in repairing DSBs after ruthenium exposure; meanwhile, olaparib targets the PARP enzyme, which leads to cancer cell death via synthetic lethality.

Although preclinical and clinical trials have shown that PARP inhibitors are effective for BRCA1-associated cancers, some limitations to the synthetic lethality approach still exist, such as evidence for therapeutic efficacy, side effects, and resistance to PARP inhibitors. Not all patients carrying BRCA1-deficient breast cancer responded equally well; some patients experienced relevant side effects from PARP inhibitors, including myelosuppression, gastrointestinal toxicity, hypercholesterolemia, fatigue, and teratogenicity [200,201]. Moreover, side effects from platinum drugs included nephrotoxicity, neurotoxicity, ototoxicity, myelosuppression, and damage to normal tissue for prolonged treatment [202] (

Table 3. Mechanism, toxicity, and efficacy of platinum complexes, PARP inhibitors, and ruthenium complexes on TNBC.

Class	Primary Mechanism	Toxicity	Efficacy
Platinum complexes	Platinum complexes exert cytotoxicity by forming DNA crosslinks that disrupt replication and transcription, leading to the accumulation of unrepaired DNA lesions, cell cycle arrest, and apoptosis in TNBC [203]	- Dose-limiting - Nephrotoxicity - Neurotoxicity - Ototoxicity - Myelosuppression - Damage to normal tissues if used for a long time [202]	- Increase the pathologic complete response (pCR) rate in TNBC when combined with chemotherapy in the neoadjuvant setting [204]
PARP inhibitors	PARP inhibitors indicate antitumor effects in TNBC by blocking the repair of single-strand DNA breaks (SSBs), leading to the accumulation of double-strand breaks (DSBs) during replication. In BRCA1/2 mutant or HR-deficient TNBC, these lesions cannot be effectively repaired, resulting in synthetic lethality [205]	- Myelosuppression - Gastrointestinal toxicities - Fatigue - Hypercholesterolemia - Teratogenicity [201]	- Strong efficacy in BRCA-mutated TNBC [205]
Ruthenium complexes	Ru(III) complexes are involved in TNBC by accumulating in mitochondria, causing mitochondrial dysfunction, ROS generation, and membrane depolarization, which leads to DNA damage and cell death. Additionally, ruthenium inhibits the protein expression of macrophage colony-stimulating factor (M-CSF), which is relevant to the PI3K/AKT/mTOR pathway, thereby reducing migration, invasion, and angiogenesis of cancer cells [181]	- Ru(II) complexes induce toxicity in TNBC through multiple mechanisms depending on the ligands - Several Ru(II) complexes exhibit low IC50 values in TNBC, which typically shows poor response to current drugs [206]	- Ru(II) complexes can be designed with various ligands, including traditional drugs or bioactive ligands, to achieve more effective treatment of TNBC [206]

and

Table 4. Comparison of the mechanism, advantages, limitations, and clinical status of platinum drugs, PARP inhibitors, and ruthenium complexes.

Class	Mechanism	Advantages	Limitations	Clinical Status
Platinum drugs	The cytotoxicity of platinum complexes arises from the covalent binding of platinum atoms to the N7 position of purine bases in DNA, forming platinum adducts that generate intrastrand and interstrand crosslinks. This blocks replication and transcription, inhibits DNA synthesis, induces cell cycle arrest, and ultimately triggers apoptosis in cancer cells [207]	Widely used for the treatment of cancer - Ovarian cancer - Lung cancer - Gastrointestinal cancer - Germ-cell cancer and various other malignancies [208]	High toxicity - Nephrotoxicity - Ototoxicity - Neurotoxicity Drug resistant [207]	- Cisplatin FDA approved (1978) - Carboplatin FDA approved (1989) - Oxaliplatin FDA approved (2002) - Nedaplatin Not FDA approved; approved in Japan (1995) - Heptaplatin Not FDA approved; approved in South Korea (1999) - Lobaplatin Not FDA approved; approved in China (2010) - Satraplatin Not FDA approved [208,209]
PARP inhibitors	PARP inhibitors induce synthetic lethality by inhibiting PARP1/2 catalytic activity, preventing the repair of single-strand DNA breaks (SSBs) and leading to their accumulation. This results in the formation of double-strand breaks (DSBs). In HR-deficient tumors (e.g., BRCA1/2 mutants), DSBs cannot be efficiently repaired, ultimately leading to cell death (apoptosis) [95]	- Extended progression-free survival (PFS) in ovarian and breast cancer [210] - Highly effective in HR-deficient tumors and BRCA1/2 mutation [207] - Exhibited promising clinical activity against various solid tumors [211]	- Drug resistance caused by HRR restoration (e.g., BRCA1/2 reversion mutations, loss of PARP-1 binding, or PARP-1 mutations) [212] - Limited efficacy in tumors without BRCA1/2 mutations [211] - Development of therapy-related myeloid neoplasms (t-MNs) [213]	- Olaparib FDA approved (2014) - Rucaparib FDA approved (2016) - Niraparib FDA approved (2017) - Talazoparib FDA approved (2018) [214]
Ruthenium complexes	Ru(II) complexes inhibit tumor growth and metastasis by entering the nucleus, binding to DNA, inducing DNA damage, and causing cell cycle arrest. Additionally, ruthenium can localize to mitochondria, leading to mitochondrial dysfunction, increased ROS generation, and apoptosis of cancer cells [215]	- High redox potential, allowing Ru(II) complexes to selectively damage target tumor cells [177] - Administered via multiple routes (e.g., oral, intravenous, and intraperitoneal) [177] - Exhibited tumor cell selectivity with minor effects on healthy cells [177] - Mimicked the iron-binding properties of serum albumin and transferrin [177] - Enters cells via transferrin receptors, inducing apoptosis [176] - Several oxidation states (e.g., Ru(II), Ru(III), and Ru(IV)) under physiological conditions [176] - Pro-drug potential: the inactive Ru3+ state in circulation can be reduced to the active Ru2+ state within target cells [178] - Slow ligand exchange kinetics [178] - Less toxic than platinum-based drugs [216] and capable of overcoming platinum-drug resistance [178]	- The anticancer mechanism of Ru(II) complexes remains poorly understood [179] - Clinical challenges persist; for example, NAMI-A remains in Phase I trials due to patient adverse effects [177]	Ru(II) complexes are not FDA-approved and are still under clinical evaluation in humans - KP-1019 (Phase I) - KP-1339 (Phase I) - NAMI-A (Phase II) - TLD1433 (Phase Ib) - BOLD-100 (Phase I) - RM175, RAED-C, and RAPTA-C remain in preclinical studies [177,178]

). In addition, it has been shown that tumors develop resistance to PARP inhibitors by a variety of mechanisms, such as upregulation of the proteins involved in drug efflux pumps, hypomorphic activity of mutant BRCA1 alleles, and rewiring of the DNA damage response [196]. Moreover, cancer cells can survive or share some characteristics by using a salvage DNA repair pathway because DNA repair pathways can overlap, although this is not well defined, to overcome a defect in a single pathway.

9. Future Perspectives

A major drawback of anticancer agents is their lack of selectivity for tumor tissues, including breast cancer. This causes systemic toxicity, severe side effects, and gives rise to poor response rates. Accordingly, increasing the therapeutic index of the drug while minimizing systemic toxicity would be the best strategy to improve breast cancer therapy. It is well established that the characteristics of breast cancerous tissue are quite different from normal tissues; for instance, breast cancerous tissue is characterized by a lack of a functional lymphatic drainage system and more permeable and leakier blood vessels. However, differential responses to chemotherapy among patients with breast cancer become relevant, as well as the development of drug resistance. Hence, it is crucial to assess the responses of early breast cancer patients to a given chemotherapeutic agent without the presence of adverse drug effects. Combining PARP inhibitors with ruthenium-based agents to induce BRCAness or inactivate the BRCA1 protein via its HR deficiency not only represents a potential approach for TNBC treatment but also a challenging strategy that remains largely unexplored [217]. In addition, other molecularly targeted biomarker proteins beyond BRCA mutations, including HR-related (ATM, BARD1, CHEK2, MRE11, NBS51, RAD50, RAD51, PALB2, FANCA, 53PB1, CDC25), cell cycle/apoptosis signaling pathways (CHKs, CDKs, PD-L1), replication fork stability (ATR/WEE1), hypoxia-induced signaling (HIF-1), cellular stresses/oxidative stresses (p38 MAPK, NRF2, NAD⁺/NADH-SIRT3 axis), and therapeutic approaches for TNBC without germline BRCA mutations are encouraging to achieve preclinical and clinical outcome assessments [218,219,220,221,222,223,224]. To the best of our knowledge, the responsiveness of TNBC to targeted therapy is significantly influenced by the expression of molecularly predictive biomarkers and hormone receptor status. Therefore, precision medicine in combination with suitable drug delivery as a tool for tailoring therapeutic strategies is required to improve both preclinical and clinical outcomes. In addition, a better understanding of the precise mechanisms of action of the anticancer metal-based candidates and their potential synergy with existing therapeutic drugs could pave the way for the development of novel metal-centered drugs that overcome treatment resistance and improve efficacy and selectivity.