Abstract
Head and neck carcinoma (HNC) comprises a variety of pathological entities. Among them, squamous cell carcinoma (SCC) is histo-pathologically prominent. Specific malignancies, such as nasopharyngeal carcinoma (NPC) arise also from the same anatomical region. In all of them, genomic instability (GI) is implicated not only in the early stages of epithelial malignant transformation, but also in the aggressiveness of the corresponding phenotypes. Among the molecules that are frequently deregulated in solid malignancies including HNCs, topoisomerases (Topo) are of increased significance due to their involvement in DNA topological, structural, and functional stability. The main members are Topo I (20q11), Topo II alpha (17q21) and Topo IIb (3p24). In the current article, we describe the mechanisms of Topo I and Topo IIa deregulation leading to GI in a variety of HNCs. Furthermore, novel data regarding the corresponding targeted therapeutic strategies are presented.
Concerning solid malignancies, cancerous genomes demonstrate two main types of instability: gross chromosomal instability, including polysomy, monosomy or aneuploidy, and specific gene imbalances including all of the numerical or structural changes (mutations, deletions, translocations, and amplifications) detectedon specific chromosomal loci (1-3). These mechanisms of molecular deregulation lead to dysfunctional and aberrant protein production driving epithelial cells to neoplastic and finally malignant transformation (4, 5). Genetic events that affect significantly signal transduction pathways, cell cycle regulation, proliferation/apoptosis mechanisms, DNA stability, and damage response are mediated by a broad spectrum of genes (6-8). Among them, topoisomerases (Topo) are of great importance due to their implications in DNAtopological structural and functional stability (9). In the current article, we describe Topo I and Topo IIa mechanisms of deregulation in a variety of head and neck carcinomas (HNCs) including oral squamous cell carcinoma (OSCC), laryngeal squamous cell carcinoma (LSCC), and specific malignancies such as nasopharyngeal carcinoma (NPC) arising also from the same anatomical region. Furthermore, we present novel data regarding the corresponding targeted therapeutic strategies.
Topoisomerases: Gene & Protein Structure and Function
Topoisomerases act as nucleic acid enzymes that modify DNA topological structure. Topoisomerase I (gene location 20q11), Topoisomerase II alpha (Topo IIa gene location at 17q21) and Topoisomerase IIb (gene location at 3p24) represent the main members of the topoisomerase super-family (10) (Figure 1). Concerning their specific actions, Topo I, Topo IIa, and b isomers cut and rejoin temporarily the DNA double helix, enhancing also DNA double strand winding and unwinding. In fact, this is a critically significant molecular mechanism regulating replication, transcription, and repair of chromosome structure (11). Topo IIa has a molecular weight of 170 kDa. Its highest expression is observed in proliferating cells – especially in late S phase-with a peak in G2-M, promoting normal chromosome condensation (12). Based on cell cycle phase-dependent expression, Topo IIa recognizes more specifically actively proliferating cells than Ki-67. For this reason, it is considered a reliable marker of proliferation in a variety of solid malignancies (13-19). Topo I has a molecular weight of 91 kDa and consists of 765 amino acids. It is involved in the unwinding and uncoiling of supercoiled double helix DNA by transiently cleaving one of the two strands. This mechanism provides rotation of one strand over the other and rejoining of ends of the cleaved strand (20). Furthermore, in contrast to Topo II, Topo I is not an ATP-depended enzyme (21). Additionally, Topo II a and b isoforms participate in DNA’s molecule stereotype domain stability by passing one DNA double-stranded segment in a second one (22-25).
Schematic of chromosomes 17, and 20 containing the Topo IIa & Topo I genes. Topo IIa protein over-expression in a case of laryngeal squamous cell carcinoma (LSCC). Note the prominent dark, black/grey nuclear staining pattern (center) (Immunohistochemistry technique, DAB chromogen, original magnification: 100×).
Regarding topoisomerase inhibition strategies, new agents have been developed and applied to promote cancer cell death such as targeted specific chemotherapy. Regarding also the efficacy of Topo IIa inhibition, many clinical studies have shown that adjuvant chemotherapy strategies, which include anthracyclines (doxorubicin) and podophyllotoxins (etoposide) in conjunction with fluorouracil and cyclophosphamide or carboplatin/paclitaxel are most effective, especially in handling patients with breast cancer and other gynecologic malignancies, such as endometrial or ovarian cancer (26-28). Furthermore, since Topo I down-regulation enhances cell death by leading to DNA damage. It is a target for specific chemotherapy (camptothecin, topotecan, irinotecan) in solid malignancies (29-31).
Topoisomerases in OSCC
Among HNSCCs, OSCC is characterized by a broad spectrum of genomic imbalances, including gross chromosomal alterations, such as polysomy/aneuploidy, and specific gene aberrations. Development and progression of OCSSC is a multistep procedure triggered by a variety of causes, such as chronic tobacco, alcohol, andbetel quid consumption. Furthermore, persistent viral infections - especially high-risk human papilloma virus (HR-HPV) – are also involved in the carcinogenic process (32, 33). HPV positivity is observed in significant proportions of premalignant lesions in the oral mucosa (intraepithelial dysplasias), such as oral atrophic lichen planus (OALP). HPV persistent infection in these cases leads to a progressive transformation of the normal mucosa to malignant tissue. A study group investigated the role of Topo IIa expression and HPV DNA existence in OALP tissues. They reported a strong correlation between HPV-infection and subgroups of atrophic OLP. Topo IIα over-expression was also related to HPV positivity in these lesions (34). Similarly, another study group focused on the role of Topo IIa, Ki-67 and cytokeratin-19 (CK-19) in OALP pre-malignant lesions. The latter molecule represents an intra-cytoplasmic, intermediate filament protein. They showed that Topo IIa should be considered a reliable proliferation and apoptotic marker in OALP lesions indicating also a progressive malignant transformation of the oral mucosa (35). Concerning the influence of Topo IIa on proliferation and apoptosis in oral pre-neoplastic lesions and OSCCs, another protein analysis showed that Topo IIa demonstrated progressively increased expression in hyperplastic, dysplastic, and OSCC tissues (36). Interestingly, Topo IIa expression seems to be involved also in the progression and metastatic expansion of OSCC, especially correlated also to lymph node metastasis (37). Besides Topo IIa, cyclooxygenase (COX)-2 also seems to be critical in oral mucosa carcinogenetic process. A study group co-analyzed cyclooxygenase (COX)-2 and Topo IIa expression in precancerous and cancerous lesions of the oral mucosa. They concluded that patients who demonstrated elevated protein expression levels of both COX-2 and Topo IIa showed poor prognosis (38).
Topoisomerases in LSCC
LSCC represents the most frequent malignancy of this anatomical region (39). Oncogenic high-risk human papillomavirus (HR-HPV) persistent infections, combined or not with chronic alcohol and tobacco consumption, are well established significant pathogenetic factors for LSCC development and progression (40, 41). Because increased cell proliferation is a significant marker for evaluating development and progression rates of carcinogenesis, over-expression of Topo IIa and Ki-67 (cytogenetic band: 10q26.2) in LSCC appears to be a very important factor (42-44). Additionally, it has been shown that Topo IIa deregulation seems to be an early genetic event in LSCC and the Topo IIa-to-Ki-67 ratio could be used as a sensitive proliferation marker (45). Topo IIa gene deregulation mechanisms (amplification) and chromosome 17 status (normal diploid or aneuploid) have been shown to be implicated in aggressive LSCC phenotypes, but only gross chromosome abnormalities in the expression patterns of Topo IIa (46). Another critical observation is that Topo IIa over-expression is associated predominantly with the grade of the examined tumors (47). Concerning the impact of Topo I aberrant expression in LSCC, there are very limited published data. Extensive mRNA–based quantitative RT-PCR analyses suggested that normalized Topo I/G6PDH mRNA ratios were strongly associated with Topo I/porphobilinogen deaminase (PBGD) expression, as has been observed in colorectal tumors but not in pharyngeal/laryngeal tumors (48). In combination with the previous observations, other similar analyseshave shownthat aberrant expression of Topo IIa, but not Topo I, may be a potentially strong indicator of tumor aggressiveness and poor outcome in LSCC, as in esophageal carcinoma (49).
Topoisomerases in NPC
NPC represents a unique, aggressive histo-type entity in the HNC family of malignancies. NPC’s tissue origins are the nasopharyngeal epithelia. The malignancy is characterized by a highly invasive and distal metastatic potential negatively affecting patients’ prognosis. Concerning the pathogenetic factors implicated in its rise and progression, Epstein-Barr virus’s (EBV) persistent infection is prominent. EBV’s oncogenic activity in the target cells results to an aberrant expression of specific critical proteins including LMPs and EBNA1 modified by endogenous EBV micro-RNAs (miRs) (50, 51). Concerning Topo IIa aberrant expression in NPC, immunohistochemistry-based studies reported that its over-expression is significantly associated with tumor aggressiveness (advanced stage) and may modify the response to novel target chemo-therapeutic regimens (52). A multi-gene functional molecular analysis revealed a significant number of genes implicated in NPC development and progression (179 up-regulated/238 down-regulated). Topo IIa deregulation is part of specific gene signatures of the corresponding patients (53). Impact of EBV persistent infection on Topo IIa activation has been also investigated. A study group concluded that the specific viral kinase EBV BGLF4 enhances the decatenation activity of topoisomerase II inducing pre-mature chromosome condensation (54). They also observed and reported a significantly elevated EBV-depended reactivation of condensation of cellular chromatin in host NPC cells was critical for explaining the EBV-mediated reorganization of nuclear architecture.
Anti-topoisomerase Strategies in HNCs
Since the last decade, novel anti-topoisomerase inhibition strategies have been developed. Concerning breast adenocarcinoma, anthracyclines act as critical inhibitors of Topo IIa aberrant expression, especially in gene amplified cases (55). Additionally, Topo IIa gene amplification or deletion modifies the response rates of patients suffering from solid malignancies, such as endometrial or pancreatic cancers and also correlates with poor prognosis (56, 57). Interestingly, a study group analyzing Topo IIa over-expression in patients with LSCC that receive radiotherapy, showed that aberrant expression of Topo IIa-due to its hypermethylation - increases radio-resistance rates (58). For this reason, novel anti-Topo IIa agents including epipodophyllotoxin-based F14512 drug and cisplatin induce radio-sensitivity in sub-groups of LSCC patients (59). The role of anti-Topo IIa drugs combined or not with radiotherapy is under investigation in HPV-depended cases. Recently, a clinical-molecular study analyzed the efficacy of pemetrexed and etoposide combined with cisplatin as therapeutic regimens in HPV positive patients with oral and oropharyngeal cancer. They observed that in Topo IIa and thymidylate synthase (TS) over-expressed cases these regimens offer increased response rates, although extended clinical trials are necessary for evaluating their benefits in patients characterized by specific molecular signatures (60). Concerning Topo I in LSSC and OSCCs, camptothecin, irinotecan, SN-38 etoposide, and teniposide are important inhibitors. Especially, a study group concluded that SN-38 is highly cytotoxic to OSCC cell lines (61). In combination with previously reported data regarding teniposide, another clinical-molecular study showed that the agent could significantly induce apoptotic and Topo IIa & I activity in OSCC, and inhibits cell growth (62).
Critically, mutations in Topo I gene negatively affect the response rates to the targeted therapeutic regimens mentioned above. A study group analyzing the Topo I mutational landscape observed that a single amino acid substitution (E418K) is responsible for increased resistance to camptothecin in NPC cell line cultures (63). Interestingly, in hematological malignancies Topo I affects camptothecin efficiency by inducing cleavage of specific tandem repeats (64). Additionally, mechanisms of anti-Topo I camptothecin-based therapy include decreased expression of O(6)-methylguanine-DNA methyltransferase (MGMT), an important enzyme involved in DNA stability and repair procedure, regulating also cytotoxicity in NPC cell line cultures treated by camptothecin (65). Concerning NPC, targeted therapy with combination of agents seems to provide a better result than monotherapy. In an experimental study, the synergistic activity of topotecan and chronomodulated radiation was analyzed on xenografted NPC tumors. The study group reported a significant enhancement of radiosensitivity in NPC cells (66). In addition to the previous mentioned anti-Topo I & IIa targeted strategies, there is a variety of experimentally analyzed agents, such as voreloxin, mansonone F (MF) derivatives, providing important results for future therapeutic protocols (67, 68). Furthermore, in modern anti-Topo regimens, the combination of two agents, such as docetaxel and topotecan, is experimentally enriched with other inhibitors, including granulocyte-colony-stimulating factor (G-CSF) or dovitinib - an orally bioavailable molecule which triggers a G2/M arrest and interrupts receptor tyrosine kinases mediated signal transduction to nucleus (69, 70).
Conclusion
In conclusion, topoisomerases are significant molecules that regulate DNA structural and functional stability and homeostasis. A variety of HNC histopathological entities including LSCC, OSCC, and NPC demonstrate Topo I and IIa gene deregulation leading to an aberrant over-expression of these molecules. Novel anti-topoisomerase targeted strategies are crucial for preventing their abnormal activity and regulating the corresponding negative genetic events that drive malignancies to more aggressive phenotypes.
Footnotes
This article is freely accessible online.
Authors’ Contributions
ET, EK, VR, VP, AC searched the literature and drafted the article, with ET being the major contributor in writing the article. DP, NM, DS, PP, AN collected the data provided by the corresponding references. All Authors read and approved the final article.
Conflicts of Interest
The Authors declare no conflicts of interest associated with this article.
- Received March 18, 2021.
- Revision received May 6, 2021.
- Accepted May 7, 2021.
- Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.