TOP2A plays a key role in cell survival. It helps unwind an entangled double strand, introduces a break in double stranded DNA to permit another strand to cross the site, and subsequently reseals the nicked end to form a topological structure that favors DNA transcription and replication, thus making it a necessary vital enzyme for cells to maintain life activities. This process requires a conformational change in the structure of TOP2A for it to complete catalytic reactions. Enhanced expression of TOP2A protein in rapidly proliferating cells makes it an important therapeutic target for cancer therapy. The optimal defined cut-off value for TOP2A expression in various types of tumors according to previous studies is 13-30% in breast cancer (An et al. 2018; Nikolényi et al. 2012; O’Malley et al. 2011), 18% in epithelial ovarian cancer (Ghisoni et al. 2019), 15% in neuroblastoma, 15% in luminal fluid cytology (Kimura et al. 2010), and 75% in NSCLC (Sakurai et al.2020). However, such high expression levels were not detected in current samples, probably because of the differences related to patient selection, tissue processing, and fixation methods. Therefore, a median cut-off value of 50% was used in the present study.
Previous studies believed that the increase of protein expression mainly depended on gene amplification and the increase of transcription (Bagci et al. 2015). In the present study, aneuploidy was consistent with elevated TOP2A expression as detected by kappa consistency test. Thus, in the current study, gene transcription is proposed as the major regulatory process associated with the enhanced expression of TOP2A protein. An earlier published data indicated translation regulation of TOP2A in Hela cells (Subramanya et al. 2011). To increase the expression of TOP2A protein, HuR promotes the translation of TOP2A by enhancing the binding and not by altering the level of TOP2A mRNA (Hitti et al. 2016). The results of IHC and FISH obtained in the present study are inconsistent with the above-published in vitro studies. In addition, the mRNA of zinc finger protein 148 (ZFN148) and TOP2A regulate each other through ceRNA in colorectal cancer cells, with their target miRNAs such as miR101, miR144, miR335, and miR365 negatively regulating TOP2A mRNA (Gao et al. 2017), suggesting a novel regulatory mechanism of TOP2A. Thus, the mechanisms regulating the expression of TOP2A protein are diverse and needs to be elucidated.
In the present study, although TOP2A amplification was not significant in the analyzed 244 NSCLC samples, patients with enhanced expression of TOP2A protein showed varied degree of heteroploidy in tumor cells as determined by IHC and FISH. The presence of heteroploid karyotype is related to the physiological mechanism associated with the action of TOP2A (cell cycle progression and chromosome segregation in mammalian cells cultured in the presence of the topoisomerase II inhibitors ICRF-187 [(+)-1,2-bis(3,5-dioxopiperazinyl-1-yl) propane; ADR-529] and ICRF-159 (razoxane); Daniloski et al. 1994). The primary action of TOP2A during meiosis is when the DNA structure fails to successfully deconvolve, leading to abnormal cell division and chromosomal segregation, resulting in a heteroploid karyotype (Lee and Berger 2019). The mechanism associated with increasing the aneuploidy has been partially elucidated in recent years. A previous study identified the role of osteoprotegerin (OPG) in inducing an increase in the TOP2A copy number (Goswami 2015). In relapsed diffuse large B cell lymphoma (DLBCL) patients, an increase in polyploidy 17 reflected the genetic compensation of tumor cells in response to TOP2 inhibition (Pedersen et al. 2015). Several studies have suggested the involvement of aneuploidy in tumor development and drug resistance (Replogle et al. 2020) Aneuploidy predicted shorter overall survival (OS) and progression free survival (PFS) in patients with DLBCL (Chen et al. 2012) and response to polyethylene glycol liposomal adriamycin in epithelial ovarian cancer (Erriquez et al. 2015). The significant correlation between heteroploidy and poor patient prognosis warrants the need for alternate novel methods to replace FISH in detecting chromosomal concordance. Several studies have shown the applicability of quantitative polymerase chain reaction (qPCR) in screening patients with aneuploid tumors. The method was used to detect expression markers related to chromosomal instability, such as AURKA, FOXM1, TOP2A, and TPX2, to classify patients with grade II breast tumors into two groups with good and poor prognosis(Szászet al 2013).Compared with immunohistochemistry and FISH to assess the karyotype of patients, qPCR is undoubtedly inexpensive and rapid, however screening for tumor-associated markers associated with chromosomal instability-dependent expression, needs to be further explored. As a retrospective study, we were unable to collect blood specimens from patients for screening biochemical markers, which limits the expansion of the research direction. Future research could aim at analyzing blood specimens for TOP2A mRNA-related data, to not only elucidate TOP2A protein regulatory mechanisms, but also to decipher the ability of TOP2A to screen NSCLC patients with aneuploid chromosomes using qPCR.
In the present study, the 16th case was a patient with squamous lung cancer with haploid tumor cells exhibiting a total deletion of chromosome 17 and a single signal each for TOP2A and gsp17, and with normal diploid precancerous cells in the surroundings. Tumor cells of diverse haplogroups have different mutations to resist the action of targeted drugs (Real and Marsiglia 2020). Accordingly, haploinsufficiency in acute lymphoblastic leukemia was found to be associated with poor patient prognosis (Safavi et al. 2013; Safavi and Paulsson 2017). The present study was limited in analyzing the role of haplogroups in NSCLC because of the small sample size.
Currently two major types of antitumor drugs targeting TOP2A are available. The first type converts the topoisomerase into a toxin that induces DNA double-strand breaks to inhibit DNA replication, resulting in the death of tumor cells expressing high levels of TOP2A. However, its use is severely limited by the dose, as it tends to cause cardiotoxicity and secondary malignancies. The second type are less commonly used in clinical practice and comprises of a catalytic inhibitor of TOP2A, which uses an enzymatic reaction to inhibit the ATPase activity to block the conformational conversion of TOP2A enzyme (Delgado et al. 2018; Hasan et al. 2008). Anthracyclines are a type of TOP2A toxins, whose cumulative dose-induced side effect s include cardiotoxicity, myelosuppression, and gastrointestinal reactions (Qiu et al. 2021). The exact mechanism of anthracycline-induced cardiotoxicity is unknown, however, several theories are proposed to explain the same, such as oxygen radical damage theory, iron ion metabolism disorder theory, calcium overload theory, and anthracycline-induced apoptosis (Hurvitz, 2021). Assessment of the risk of the toxic effects of anthracyclines remains limited to real-time monitoring of the patient's physical status during administration using ECG scanners, cardiac enzyme profiles, echocardiography, and calcium-regulated tetraphosphate tests (Alexandre et al. 2020). In addition, secondary tumors and drug resistance related to the use of anthracyclines remains inevitable. The issue of adverse effects and safety of drug therapy remains a challenge in systemic oncology treatment. In drug therapy, control of drug safety together with ensuring effectiveness remains an issue to be explored. Since current medical research does not support "de-anthracycline,” adjuvant combination of anthracyclines with targeted drugs has become a routine treatment for patients with advanced cancer. Accordingly, the use of anthracyclines is continued in oncology treatment regimens because of their "cost-effectiveness.” Anthracyclines are widely used in the treatment of lung cancer. However, the clinical evaluation of anthracyclines is not routinely based on the IHC results of TOP2A. Patients with enhanced expression of TOP2A show higher sensitivity to anthracyclines. Cell lines resistant to anthracyclines exhibit low expression of TOP2A. However, the occurrence of this phenotype pre- or post-drug resistance remains uncertain. The ability of IHC to predict sensitivity to anthracyclines remains to be studied (Burgess et al. 2008).
The results of the present study indicate the association of aneuploidy with high expression of TOP2A protein. The enhanced expression of TOP2A protein was primarily based on the increase in the number of the transcripts. In addition, the patient's aneuploid karyotype indicated an increased risk of death. Thus, TOP2A protein could be used as a potential biochemical marker for poor prognosis in patients with NSCLC.