2 German Cancer Research Centre (DKFZ), Germany
Received Date: 01 Nov, 2019 ; Accepted Date: 20 Nov, 2019 ; Published Date: 26 Nov, 2019
Breast cancer is one of the leading cause deaths among women. Although there are many advanced treatments, some types of breast cancer are refractory to therapy. Neoadjuvant systemic therapy has been regarded as an effective way that can be used throughout the cancer treatment process, which including topoisomerase inhibitors such as doxorubicin. Topoisomerase enzymes play a specific role in changing of DNA topology, which are the premise of DNA replication, transcription and repair. Topoisomerase inhibition researches are increasing, because topoisomerase inhibitors have a good targeting effect on rapidly proliferating tumor cells, and are not depend on hormone receptors in breast cancer cells. Since the difference between catalytic inhibition and poisons, the mechanism of topoisomerase inhibitors are not unique, but the path of cell death after DNA damage is roughly the same. Recent years, intensive efforts were devoted to explore the mechanism on resistance to topoisomerase inhibitors. In order to reverse resistance, topoisomerase inhibitors can also exert anti-tumor effects in conjunction with other anti-cancer drugs, such as olaparib (PARP inhibitor). This review presents biology and molecular mechanics of the topoisomerase enzymes, and the effects of their inhibition in breast cancer, are discussed to explain and support the application of topoisomerase inhibitors in breast cancer.
Breast Cancer, Resistance, Topoisomerase, Topoisomerase Inhibition
Breast cancer is one of the most common malignant tumors in women . According to a survey conducted in 2018 , the incidence of breast cancer is the highest, and the mortality rate is the second among women in the United States. Similarly, breast cancer mortality among women in Asia is increasing every year . Collectively, breast cancer still remains a life-threatening malignant disease. The regimens are related to the classification of diseases. The main treatments in breast cancer methods include surgery, chemotherapy, radiotherapy, endocrine therapy and gene-targeted therapy . Treatments for early breast cancer, with cytotoxic drugs, have a better outcomes such as minimized surgical management . DNA topoisomerase inhibitors play a basic role in treatment programs not only for those who did not participate anthracycline or taxane chemotherapy, but in neoadjuvant for many kinds of breast cancer .
With the development of modern pharmacology, we know the relationship between topoisomerase and cancer. The topological configuration of DNA in cells can keep the stability of genetic material, but it is also the biggest obstacle to DNA activity. DNA topoisomerases exist in all biological cells which can break single or double strands of the DNA to release the tension caused by entanglement, so that the cell life activities can continue . Therefore, DNA topoisomerases play important roles in DNA replication, repair, cell proliferation and other cell processes . DNA topoisomerase inhibitors have been used as chemotherapy drugs for malignant tumors with remarkable clinical effect, especially in breast cancer. The studies on DNA topoisomerases have increasingly gained attention. This review summarizes the results of topoisomerase enzymes study and the effect of their inhibitors in breast cancer.
Advancing replication forks cause double-strand DNA strain, leading to gene expression errors, chromatin rupture, mitosis failure, eventually cell death . The main function of DNA topoisomerase is to regulate the topological structure of DNA which can reduce the tension generated by both positive and negative DNA superhelix, and enable the normal process of DNA to replicate, transcript and repair [10,11]. According to the mechanism of DNA topoisomerase and structural characteristics, it can be divided into two main categories: type I topoisomerase and type II topoisomerase .
Type I topoisomerase (Topo I) belongs to the monomer enzyme, including the Topo IA, Topo IB and Topo IC . There are four domains: amino-terminal domain, core domain, linker domain and carboxy-terminal domain . The concentration level of Topo I is stable in mammal cells . Among the multiple active sites in Topo I, Arg488, Arg590, His632 and Tyr723 are the four active sites that are well-defined in the study. Except Tyr723 is located in the carboxy-terminal domain, the other three are located in the core domain of Topo I. In the catalytic process of the enzyme, Tyr723 covalently binds to the 3’ phosphate end of the DNA single-strand break.
Type II topoisomerase (Topo II) is a dimer enzyme ranging from 160-180kDa that causes DNA double-strand broken [10,16]. Topo II can be divided into three domains: N-terminal domain, the central domain of the enzyme and C-terminal domain . The central domain contains the active site, tyrosine 782 residue, which forms the covalent bound to DNA . The dimer consists of two subtypes, Topo IIα and Topo IIβ, whose physiological functions are quite different: Topo IIα can release the tension generated by positively supercoiled DNA. The concentration of Topo IIα is highly expressed in cell growth phase and is associated with cell proliferation; Topo IIβ is similar to Topo I, which can release positive and negative supercoiled DNA; Topo IIβ is expressed stably in cells, but has no direct relationship with cell cycle [19,20]. Moreover, Topo IIβ cannot compensate for the loss of Topo IIα in mammalian cells, which means two isoforms have different roles in cells [21,22].
The Topo I inhibitors used in clinical practice are mostly water-soluble derivatives of camptothecin, and the Topo II inhibitors with better effect are podophyllotoxin derivatives, such as etoposide. The structure and activity of topoisomerase targeting agents are shown in Table 1.
Topo I first recognizes and binds to a specific DNA sequence. Topo I breaks one strand of DNA in the absence of energy . Tyrosine in Topo I attacks DNA phosphodiester bond in one strand of DNA by nucleophilic way, then tyrosine in Topo I binds covalently to the 3’ phosphate of one DNA strand, and the 5’ end forms a hydroxyl group. In this way to form the topo I-DNA covalent complex (Topo I cc). Because the tension of the DNA drives the helix to unwind in the direction of reducing tension, the broken single strand with the end of the 5’ hydroxyl group is rotated around another complete single strand, untying the positive or negative superhelix. After changing the topology of DNA, Topo I is released from DNA . The nickle DNA single strand is rejoined under the action of repair proteins such as poly (ADP-ribose) polymerase (PARP), then the DNA is restored intact and subsequent life activities continued . The specific process is shown in Figure 1.
There are two closely related isoforms of topoisomerase II in mammalian cells, one is Topo IIα (170 kDa form), and the other is Topo IIβ (180 kDa form) . Biochemical and function of Topo II indicate that this enzyme is essential in the segregation of newly replicated pairs of intertwined chromosomes, which highly associated with mitosis . Topo II creates a DNA double-strand broken with the participation of two ATP molecules and divalent metal ions to form the gate of DNA. A complete DNA duplex near the gate passing through this gap for increasing or decreasing the linking numbers of the DNA rings . After the complete DNA double-strand is passed, the dimer undergoes a certain configuration change, and the separated DNA ends 5', 3' are re-ligated. Then, the formed gate of DNA structure is closed and DNA recovery integrity  (Figure 2).
DNA topoisomerase inhibitors are used as chemotherapeutic drugs in the clinical treatment of tumors. In general, their inhibition function referred to two categories: catalytic inhibition mechanism and poison mechanism [26,36].
Catalytic inhibition means that they can directly affect topoisomerase enzymes, for example, interferencing the action of the enzyme or disrupting the enzyme. Topo II catalytic inhibitors can be used in various steps of enzyme-catalyzed reactions. The main reaction mechanisms are as follows:
In order to blocking DNA recovery, poisons can be linked to the DNA-Topo cleavage complex to form ternary complexes. Topo poisons stabilize DNA fragments in cell cycle process, increasing the concentration of the cleavage complex. The fragments of DNA could activate the apoptotic pathway to kill the cells . Drug-DNA-topo ternary complexes are not always to cause cytotoxicity results (cell death) directly. As for Topo II poisons (anthracyclines in particular), they can inhibit the DNA repair way, then active the cell death pathway [39,40]. Allan Chen, et al.  demonstrated that the cytotoxicity of poisons are highly related to DNA synthesis, so that they can arrest cancer cells in S phase and inhibitors can work at low concentration to topoisomerase in S phase. This finding demonstrates that topoisomerase inhibitors working depend on topoisomerase protein concentration.
Studies in Topo I have shown that Topo I agents, DNA and Topo I protein formed a complex by itself is not fatal, but collision with the replication forks or transcription complex will cause cell death . If the cell cycle checkpoint kinase detects DNA nicks before the collision, the cells will be arrested in G2/M phase. However, if not repaired in time, the cells will die . In the molecular model, the Topo I-DNA covalent complex (Topo Icc) is transiently present . Under normal conditions, the complex acts as intermediate in the DNA re-connection process, and the concentration is within a certain range in nucleus . When the Topo I inhibitor is inserted into the base pair of the Topo I cleavage complex, there are certain requirements for the site of the ligation with drugs:
In Camptothecin (CPT), three hydrogen bonds are formed between camptothecin residues 1,17and 20 and amino acid residues Arg364, Asn722, and Asp533 respectively . Noticeably, mutations in other amino acid sites of Topo I do not prevent Topo I agents from attaching to DNA covalent complex . However, if there were changes in connected sites of Topo I protein, these will cause CPT to fail to connect with drug and lead to high resistance to CPT. Topo I from CPT-producing plants have 4 amino acid substitutions in the positions that are proposed to influence CPT binding: Ile-420 to Val, Asn-421 to Lys, Leu-530 to Ile and Asn-722 to Ser. Recently, it was found that Asn722Ser in human tumor cells has changed which caused CPT resistance .
The mechanism of action of Topo II poison is divided into two types: one group of poisons are traditional, interfacial, noncovalent, and redox-independent [17,22,48,49]. These chemicals form noncovalent with Topo II protein interface in the vicinity of the active site tyrosine . The other group of poisons are covalent to Topo II amino acid residues and require redox activity to against Topo II [22,48]. Both of two mechanisms can enhance the DNA cleavage. But the letter group can abrogate the Topo II poisons activity by reducing agents and delay DNA recovery that can not affect compounds in former group [50,51]. Topo II poisons decrease DNA re-ligation rate, increase intracellular DNA broken and chromatin irregular recombination. When these poisons cause permanent DNA breaks in sufficient concentration, they can trigger death pathways [22,52].
Actually, Topo II inhibitors are the most effective anticancer drugs but often cause serious side effects, such as secondary malignancies [53,54]. Some studies found that the drawbacks of targeting Topo IIβ include the induction of cardiotoxicity and the potential development of secondary malignancies, such as doxorubicin [28,55]. Therefore, inhibitors that target Topo IIα will have a better effect in chemotherapy .
According to website, type of breast cancer can be divided into invasive and non-invasive breast cancers . Early studies identified two types of Estrogen Receptor (ER) positive breast cancer subtypes by using cDNA microarray technology: ductal A and B breast cancers, which have highly response to treatment . As for ER-negative patients, they can be divided into three groups: HER-2 high expression type, basal type, and normal type. Compared with ER-positive breast cancer, these three subtypes have poor response to treatment and poor prognosis . Triple-Negative Breast Cancer (TNBC) defined by Estrogen Receptor (ER), Progesterone Receptor (PR), and human epidermal growth factor receptor 2 (HER2) negativity is a group with poor prognosis, refractory to therapy, and become a major problem in the treatment of breast cancer . Topoisomerase inhibitors as cytotoxic drugs plays a certain roles in treatment. On the one hand, Topo I inhibitors can exert anti-tumor effects in combination with other anti-cancer drugs, such as olaparib (PARP inhibitor) . On the other hand, there exist a positive correlation between Topo IIα expression levels and pathological grades of breast cancer. Topo IIα expression is up-regulated in breast cancer cells [61,62]. So Topo II inhibitors could exert anti-tumor effects. Due to expression of Topo IIα, anthracyclines may be effective in treatment of TNBC .
As the mechanism of Topo I inhibitors, they stabilize the Topo I-DNA cleavage complex, and DNA single strand broken in the complex can become DNA double-strand broken near the advancing replication fork, which is the main cause of cell death [63,64]. Topo I inhibitors are not frequently used as a monotherapy in clinical, and they are not interfere with anti-metabolites drugs either . Iben Kümler and colleges have investigated and reviewed the use of Topo I inhibitors in metastatic breast cancer, and they concluded: Irinotecan seem to be effective in some patients previously treated with anthracyclines and taxanes . However, Topo I inhibitors have certain Adverse Events (AE), such as neutropenia, diarrhea and nausea/vomiting . Some studies have suggested that following biomarkers to select which patients are sensitive to Topo I inhibitors can increasing effect of Topo I inhibitors in individualized treatments [60,68]. These biomarkers are mainly involved in DNA repair functions such as PTEN, Chk I, TDP I, PARP, BRCA I, P53, WRN protein, and Topo I gene copy numbers (Table 2).
The peak concentration of Topo II is in G2/M phase, which means that Topo II is connected with proliferation cells . Cells that express high levels of the enzymes are sensitive to Topo II poisons, and the decrease of Topo II inhibitors activity contributes to the drug resistance [17,70]. There are many researches about Topo II and breast cancer [71-73]. Topo IIα is overexpression in Estrogen Receptor (ER), Progesterone Receptor (PR) negative and vimentin positive breast cancer . Vimentin is a protein that has relationship with invasion and metastasis of tumor cells. In addition, high level of Topo IIα is correlated with the high expression of the proliferation markers Ki67, RacGAPI and c-Myc [73,74]. But why is Topo II inhibitors more effective in ER-negative breast cancer, only for levels of Topo II protein? What is the correlation mechanism between Topo II and cancer-promoting genes? As highlighted by Qi, et al.  that Topo II drugs can active p38γ specifically in intrinsically sensitive cells leads to phosphorylation and stabilization of Topo IIα, promoting the action of drugs to inhibit cell proliferation eventually. However, p38γ lose activity in ER-positive breast cancer cells. It has long been recognized that p38γ is belonged to p38/MAPK pathway, and their experiments also indicate that Mitogen-Activated Protein Kinase (MAPK) activity is up-regulated by treatment of breast cancer with Topo II drugs. Besides this, rare signal pathway be found to clarify the regulator mechanism about Topo II drugs and breast cancer types so far. There is an analysis from multiple advanced breast cancer clinical trials established that monotherapy with mitoxantrone achieved an overall response rate of approximately 33% in patients with no prior exposure to chemotherapy [76,77]. Generally, targeted therapies in Triple-Negative Breast Cancer (TNBC) have no effect. If there is high expression of Topo IIα, the anthracycline anticancer drug can be used as a viable chemotherapy proposal. Some studies have shown that mutations in BRCA1 and BRCA2 account for the majority of families with hereditary susceptibility to breast and ovarian cancer . In this population with breast cancer, Topo II mediated Double-Strand Broken (DSB) is more sensitive than other cells, and therefore etoposide has better results . In summary, monotherapy with Topo II inhibitors can get therapeutic effects for breast cancer.
Resistance to topoisomerase inhibitors may result from many processes. We can summarize several reasons as follows. From Topo I inhibitors aspect, resistance maybe from:
As for Topo II inhibitors, resistance involved in Topo II activity which including:
Tumors are sensitive to topoisomerase inhibitors in initial treatment, but acquire resistance eventually. In the research of acquired resistance of mammary tumors to the Topo I inhibitors, Serge Zander AL, et al. illustrated that topotecan resistance can be delayed in Abcg2-/- tumors, and overexpress of ABCG2 is related to camptothecin/topotecan resistance [81,82]. The drug efflux transporters ABCG2 has been reported as a crucial factor in resistance of various tumor types [83,84]. During the therapy of topoisomerase inhibitors, P-glycoprotein (P-gp) is not effective in resistance. P-gp leading to resistance may work with other weak resistance mechanisms .
Nowadays, on the one hand, there are many effective topoisomerase inhibitors under preclinical studies. Indenoisoquinoline agents which belong to non-camptothecin Topo I inhibitors are chemically stable and are not as substrates for the ABCG2 transporters that related to resistance of topotecan [43,85]. As a kind of promise direction, targeting Topo IIα compounds continue to be isolated from etoposide-related nature products . Wang Chen, et al.  demonstrated that design and synthesis podophyllotoxin derivatives have good prospects. Nevertheless, betulinic acid which from plant sources has multiple molecular targets in breast cancer, including function of topoisomerase inhibition . On the other hand, researchers can repress some overexpression factors, such as DFF40 , phosphoglycerate dehydrogenase , substance P . Perhaps we can select another compound as a topoisomerase inhibitor sensitizer to overcome resistance. It is effective in combination of PARP inhibitor and topoisomerase inhibitor . A novel histone deacetylase inhibitor TMU-35435 can enhance etoposide cytotoxicity in triple-negative breast cancer . In the way of sensitization, topoisomerase drugs can work at a lower dose and minimized side effects.
DNA topoisomerases are widely presented in mammalian cells. Topoisomerases in cancer cells can efficiently unwind DNA and make cells proliferation rapidly. Breast cancer is an aggressive disease that affects a large number of patients and families every year. Chemotherapy plays an important role in most types of breast cancer. As for high level of topoisomerase I and α in breast cancer, targeting topoisomerase is recognized as effective regimens and always be used in combination with other basic chemotherapy drugs, even target drugs, such as taxanes, gefitinib. Generally, many patients treated with chemotherapies develop drug resistance, therefore, it is pivotal to find effective sensitization methods and detect new topoisomerase inhibitors. Nowadays, there are many continuing researches in molecular targets promoted the development of breast cancer drugs, which could enhance the power of topoisomerase inhibitors, for example, Epidermal Growth Factor Receptor (EGFR), Vascular Endothelial Growth Factor (VEGF) receptors, protein tyrosine kinases, phosphatases, PI3K/Akt signal pathway, microRNAs (miRs) and long non-coding RNAs (lncRNAs) . However, it is still a problem that how to connect topoisomerases work with those molecular targets. Actually, many topoisomerase inhibitors not only affect protein itself, but cause changes of other genes and protein function. If those changed molecules can enhance the effect of drugs, can we think those kinds of cell lines or cancer are sensitive to topoisomerase inhibitors, or they could become targets to reverse topoisomerase inhibitors resistance, such as p38γ . All much we want are to control the disease and improve the quality of life for patients. This review as a summary of the topoisomerase mechanisms and their inhibitors in breast cancer treatment to provide a reference for future research. It is our hope that these potential biomarkers mechanism works and signal transduction pathways about topoisomerase enzymes could be getting clearer.
Figure 1: Mechanisms of Topo I.
Figure 2: Mechanism of Topo II.
|Name||Function||Type of response||Reference|
|TDP I||A enzyme can recognize Top1cc then remove 3’-phosphoglycolates lesions to restore DNA integrity.||TDP I inhibition|||
|PARP||A DNA binding protein that repair damaged region of DNA.||PARP inhibition||[60,91]|
|ERCC I||A DNA repair protein involved in processes of nucleotide excision repair as well as Homologous Recombination (HR).||Low levels of ERCC1 protein expression|||
|Chk I||Checkpoint kinase I makes cell cycle arrest in G2/M phase and helps stabilize stalled replication forks.||Chk I down-regulation or disruption of ATR/Chk1 pathway||[95,96]|
|BRCA I||A repair protein for error-free DSB by Homologous Recombination (HR).||Spontaneous and transplanted BRCA1-/- mammary tumors|||
|P53||A repair protein for DSB that can stimulate TOP1 catalytic activity.||In breast cancer, MCF-7 p53-/- is hypersensitive to CPT.||[80,81]|
|PTEN||A tumor suppressor gene||Overexpression of PTEN|||
|Mismatch Repair genes (MMR)||MMR proteins involved in DSB repair and induction of apoptosis in response to DNA damage.||MMR-deficient tumors|||
|Topo I gene copy number||-||>4 copies of the TOP1 gene per nuclei|||
|Werner syndrome protein (WRN)||WRN is a RecQ helicase that participates in DNA repair.||WRN degradation|||
Citation: Liao Y, Cheng X, Wang G, Luo K, Wan S, et al. (2019) Topoisomerase Inhibitors in Breast Cancer. J Obstet Gynecol Rep. 1: 001. JOGR-001.000001