OBJETIVOS ESPECÍFICOS

• Avaliar e descrever os dados clínicos das lesões em estudo;

• Analisar a imunorreatividade nuclear e citoplasmática para as proteínas XRCC-1, APE- 1 e XPF em relação às camadas epiteliais dos ceratocistos odontogênicos isolados, ceratocistos odontogênicos sindrômicos e cistos dentígeros;

• Analisar a imunoexpressão nuclear e citoplasmática para as proteínas XRCC-1, APE- 1 e XPF em relação à localização no parênquima dos ameloblastomas;

• Determinar os percentuais de imunoexpressão nuclear e citoplasmática das proteínas XRCC-1, APE-1 e XPF no componente epitelial das lesões e dos folículos dentários e compará-los de acordo com os grupos de estudo (ameloblastomas, ceratocistos odontogênicos isolados, ceratocistos odontogênicos sindrômicos, cistos dentígeros e folículos dentários);

• Verificar se existem diferenças entre a imunorreatividade nuclear e citoplasmática de proteínas de reparo do DNA (XRCC-1, APE-1 e XPF) e as lesões estudadas (ameloblastomas, ceratocistos odontogênicos isolados, ceratocistos odontogênicos sindrômicos, cistos dentígeros) e os folículos dentários;

• Investigar possíveis correlações entre a imunoexpressão das proteínas XRCC-1, APE-1 e XPF nos ameloblastomas, ceratocistos odontogênicos isolados, ceratocistos odontogênicos sindrômicos, cistos dentígeros e folículos dentários.

3 ARTIGO

O artigo será submetido ao periódico Histopathology (ISSN 1365 – 2559, Fator de impacto: 3.267, Qualis Odontologia A1), cujas normas para submissão de trabalhos se encontram no Anexo B.

APE-1, XRCC-1 AND XPF ARE OVEREXPRESSED IN AMELOBLASTOMAS AND SYNDROMIC AND NON-SYNDROMIC ODONTOGENIC KERATOCYSTS

Short title: DNA repair proteins in odontogenic lesions

Hellen Bandeira de Pontes Santos,1 Roseana de Almeida Freitas1

1_{Graduate Program in Oral Pathology, Department of Dentistry, Federal University of Rio}

Grande do Norte, Natal, RN, Brazil

Conflict of interest statement: The authors state that they have no potential conflict of interest that could bias the results obtained in this study.

Word count: 4467 words

*Corresponding author: Roseana de Almeida Freitas

Departamento de Odontologia, Universidade Federal do Rio Grande do Norte. Av. Senador Salgado Filho, 1787, Lagoa Nova, CEP 59056-000 Natal, RN, Brasil. Phone/Fax: +55843215-4138. E-mail: [email protected]

Abstract

Aim: To evaluate the immunoexpression of base excision repair (BER) (APE-1 and XRCC-1) and nucleotide excision repair (NER) (XPF) DNA proteins in benign epithelial odontogenic lesions with different biological behavior.

Methods and results: Thirty solid ameloblastomas (AMEs), 30 non-syndromic odontogenic keratocysts (NSOKCs), 29 syndromic odontogenic keratocysts (SKOCs), 30 dentigerous cysts (DCs) (n = 30) and 20 dental follicles (DFs) were evaluated quantitatively for APE-1, XRCC- 1 and XPF through immunohistochemistry. The results indicated expression of nuclear APE-1 was significantly higher in NSOKCs, SOKCs, and AMEs in comparison to DCs (p < 0.001). Nuclear expression of XRCC-1 was higher in NSOKCs and SOKCs than in DCs (p < 0.05). At the nuclear level, XPF expression was higher in NSOKCs and SOKCs than in DCs and AMEs (p < 0.05). All the odontogenic lesions studied revealed a statistically significant expression of APE-1 (nuclear), XRCC-1 (nuclear), and XPF (nuclear and cytoplasmic) when compared to DFs (p < 0.05). For all lesions, a positive correlation between nuclear expression of APE-1 and XRCC-1 or XPF at the nuclear level was verified (p < 0.05).

Conclusions: Our results suggest a potential involvement of APE-1, XRCC-1 and XPF proteins in the pathogenesis of benign epithelial odontogenic lesions, especially in those with more aggressive biological behavior such as AMEs, NSOKCs, and SOKCs. We also showed that the expression of APE-1 may be synergistic with the expression of nuclear XRCC-1 and XPF, which may suggest an interaction between the BER and NER pathways in all studied odontogenic lesions.

Keywords: Odontogenic tumours; odontogenic cysts; DNA repair; immunohistochemistry; APE-1; XRCC1-1; XPF.

Introduction

Odontogenic lesions represent a heterogeneous group of cysts, tumours and hamartomatous processes with complex clinicopathological features and biological behavior. Ameloblastoma (AME) is the most frequently encountered and clinically significant benign odontogenic tumour of the jaws, characterized by its aggressive biological behavior and high recurrence potential.1-3 The odontogenic keratocyst (OKC) is one of the most frequent odontogenic cysts in gnathic bones, representing a distinct form of odontogenic developmental cyst that deserves special attention, due to its aggressive clinical behavior, high recurrence rate and possible association with Gorlin Syndrome.4-6 _{On the other hand, the dentigerous cyst (DC)} presents an excellent prognosis exhibiting very low recurrence rates when correctly treated.5,7 In turn, the mechanisms involved in the pathogenesis of these lesions and the reasons that justify these different biological behaviors are not yet fully elucidated, which might be attributed to the scarce knowledge concerning their molecular features.

The deoxyribonucleic acid (DNA) repair pathways act on specific types of damage to the genetic material, regulating several cellular processes.8 Among the main DNA repair pathways, the most notable are the base excision repair (BER) and the nucleotide excision repair (NER).9-11 _{Key proteins such as apurinic/apyrimidinic endonuclease 1 (APE-1) and X-ray} repair cross-complementing 1 (XRCC-1) are required for viability and efficient repair of DNA damage in the BER pathway.12 APE-1, also known as redox effector factor 1 (Ref-1), is a multifunctional enzyme that plays an essential role in the BER pathway as an

apurinic/apyrimidinic-site endonuclease and is also involved in the redox regulation of

important transcription factors, such as nuclear factor-κB (NF-κB), p53 and hypoxia inducible

factor-1α (HIF-1α). XRCC-1 recognizes and binds to the DNA breaks and facilitates BER pathway by acting as a scaffold protein to recruit and physically interact with several components of the repair machinery.13.14

As akind of NER protein, xeroderma pigmentosum complementation group F (XPF), also known as the excision repair crosscomplementing group 4 (ERCC4), is one of the most important DNA repair proteins, providing the enzymatic activity of XPF-ERCC1 heterodimer, an endonuclease that incises at the 5’ side of various DNA lesions. Thus, this protein is critically involved in the NER pathway and also has an important role in recombination repair, mismatch repair and possibly immunoglobulin class switching.15,16 Investigations have shown that APE- 1, XRCC-1 and XPF are dysregulated and sometimes highly expressed in some malignancies, contributing to tumour development and progression.12,17-22

In the context of benign epithelial odontogenic lesions, although investigations have demonstrated the participation of growth factors, matrix metalloproteinases, tumor suppressor genes and oncogenes in the biological behavior of these lesions,1,6,23,24 there are only few studies that have evaluated the possible involvement of DNA repair proteins in these lesions. Among the various proteins involved in DNA repair pathways, studies involving benign odontogenic lesions have explored only proteins from the mismatch repair system, such as hMLH, hMSH2, hMSH3 and hMSH6.25-28

Considering the heterogeneous biological behavior of odontogenic lesions and the absence of studies that evaluated the immunoexpression of DNA repair proteins of the BER and NER pathways in benign epithelial odontogenic lesions (Pubmed Database, Scopus, Web of Science, LILACS, SIGLE), we evaluated the immunoexpression of BER proteins APE-1 and XRCC-1 and NER protein XPF in solid AMEs, non-syndromic odontogenic keratocystics (NSOKCs), syndromic odontogenic keratocysts (SOKCs) (related to Gorlin syndrome) and dentigerous cysts (DCs) in order to provide insights for a better understanding of the role of these proteins in relation to differences in the biological behavior between these lesions.

Sample

This cross-sectional study was approved by the Research Ethics Committee of the Federal University of Rio Grande do Norte (UFRN, Natal, RN, Brazil) (Approval number: 2.535.458). A total of 139 formalin-fixed paraffin-embedded (FFPE) tissue samples were retrieved from the files of the Oral Pathology Service at the UFRN, comprising of 30 solid AMEs, 30 NSOKCs, 29 SOKCs, 30 DCs and 20 dental follicles (DFs) obtained from healthy patients submitted to extractions of impacted third molars. DFs were used as a control group, representing healthy adult odontogenic tissues. Microscopic aspects of all lesions were reviewed by two oral pathologists to confirm their diagnoses following current World Health Organization (WHO) guidelines.5 AME samples were comprised of multiple histopathological types. In 18 cases, plexiform type prevailed and in the other 12 cases, the most abundant type was follicular, with 6 cases demonstrating expressive additional areas of acanthomatous or granular pattern. Gorlin’s syndrome patients had been diagnosed according to the criteria proposed by Evans et al.29 and presented multiple odontogenic keratocysts (OKCs). The patients with NSOKCs had single lesions and had been submitted to clinical and radiographical evaluation to exclude the presence of other manifestations of Gorlin syndrome. All odontogenic lesions submitted to the marsupialization technique prior to the biopsy and those that presented, after histopathological analysis, secondary inflammation were not included. Clinical data such as age, gender, and anatomic location of the lesions were collected from medical records and biopsy requisition forms. Clinical characterization of the patients is summarized in Table 1. All procedures were conducted in full accordance with the World Medical Association Declaration of Helsinki.

Histological sections with 3μm were obtained from the FFPE material and mounted on glass slides previously prepared with organosilane (3-aminopropyltriethoxysilane, Sigma Chemical Co., St Louis, MO, USA) as adhesive. The sections were deparaffinized, rehydrated and submitted to antigen retrieval with Trilogy (1:100, Cell-Marque, CA, USA) in a Pascal pressure cooker (Dako, Carpinteria, CA, USA) for 30 minutes. Next, the tissue sections were immersed in 10 volumes of hydrogen peroxide solution to block endogenous peroxidase and then incubated with protein block (Thermo Scientific, Runcorn, UK) for 5 minutes. Subsequently, the tissue sections were incubated with the following monoclonal primary antibodies: anti-APE-1 (clone C4; sc-17774; Santa Cruz Biotechnology, CA, USA; 1:3000; 60 minutes), anti-XRCC-1 (clone 33-2-5; Thermo Scientific, Barrington, IL, USA; 1:1500; overnight) and anti-XPF (clone 219; Thermo Scientific, Barrington, IL, USA; 1:800; overnight). Sections were then washed twice in PBS and incubated in the HiDef visualization system (HiDef Detection™ HRP Polymer System, Cell-Marque, CA, USA) at room temperature. The reactions were revealed with 3.3’-diaminobenzidine (Liquid DAB + Substrate; Dako, Carpinteria, CA, USA), resulting in a brown reaction product. Finally, tissue sections were counterstained with Mayer's hematoxylin and coverslipped. As positive controls, human ovarian carcinoma specimens were used for the APE-1 and XRCC-1 reactions and human tonsil fragments for XPF. Negative controls were performed by omitting the primary antibody in the protocol described above.

Immunostaining assessment

All slides were scanned into high-resolution images using a digital slide scanner system (Pannoramic MIDI II, 3DHISTECH, Budapest, Hungary) and the images were visualized by the Pannoramic Viewer 1.15.2 software (3DHISTECH Kft.29-33, Budapest, Hungary). The tissue sections were analyzed in a blind fashion by one previously trained examiner. APE-1,

XRCC-1 and XPF immunoexpression was analyzed quantitatively in the parenchymal cells of AMEs and in the epithelial lining of OKCs, DCs and DFs. Nuclear and cytoplasmic reactivity were analyzed separately for APE-1 and XPF while only nuclear immunoexpression was evaluated for XRCC-1. Adapting the method from Zhang et al.30_{, the five areas of highest anti-} APE-1, anti-XRCC-1 and anti-XPF immunoreactivity were selected along the epithelial component. These five fields were digitally photographed at a magnification of 400×, each field corresponding to an area of 0.0998mm2 _{and the images were transferred to the ImageJ®} software (Image Processing and Analysis in Java, National Institute of Mental Health, Bethesda, Maryland, USA). Immunostained and negative cells were counted in each photographed field, and the percentage of positive cells in relation to the total number of cells counted was established for each antibody.

Statistical analysis

The results were analyzed using the IBM SPSS Statistics 20.0 program (IBM Corp., Armonk, NY, USA). Descriptive statistics was used for characterization of the sample. Immunopositivity percentages were submitted to distribution analysis by means of the Kolmogorov-Smirnov test, which revealed the absence of normal distribution. Thus, the non- parametric Kruskal-Wallis (KW) and Mann-Whitney (U) tests were performed to compare the median percentages of immunoreactivity between the groups of lesions. Correlations between APE-1, XRCC-1 and XPF immunoexpressions were evaluated by the Spearman’s correlation test. The level of significance for all statistical tests was set at 5% (p ≤ 0.05).

Results

Analysis of the expression of APE-1, XRCC-1 and XPF in AMEs revealed a greater 1immunoexpression of these proteins at the peripheral/ ameloblastic layer and in solid areas (Figure 1A-B). In the epithelial lining of NSOKCs, SOKCs, and DCs, it was observed a varied expression for these proteins with no particular pattern of distribution in the different cell layers (Figure 1D-L). In DFs, a discrete nuclear staining was observed in some cases

Nuclear APE-1 and XRCC-1 are overexpressed in NSOKCs, SOKCs and AMEs

All NSOKCs and SOKCs showed nuclear APE-1 immunoexpression in the epithelial lining while 29 AMEs (96.66%), 24 DCs (80.0%), and 7 DFs (35.0%) exhibited nuclear expression of this protein in the epithelial component (Figure 1A, 1D, 1G, 1J, 1M). Among all groups, only 4 cases of SOKCs (13.79%) and 6 NSOKCs (20.0%) exhibited cytoplasmic immunoreactivity of APE-1 (Figure 1D and 1G). XRCC-1 nuclear immunoexpression was observed in 27 AMEs (90.0%), 28 NSOKCs (93.33%), 28 SOKC (96.55%), 24 DCs (80.0%) and in only 4 DFs (20.0%) (Figure 1B, 1E, 1H, 1K, 1N). Among all groups, no case exhibited cytoplasmic immunoreactivity of XRCC-1.

It was observed a significantly higher nuclear APE-1 expression in NSOKCs compared to DCs (p < 0.0001) and AMEs (p = 0.001). Similarly, SOKCs demonstrated higher nuclear expression of this protein compared to DCs (p < 0.001) and AMEs (p = 0.009). A significantly higher nuclear APE-1 expression was found in AMEs when compared to DCs (p = 0.014). However, no statistically significant differences of APE-1 expression were observed between NSOKCs and SOKCs at the nuclear (p = 0.301) and cytoplasmic (p = 0.366) levels (Figure 2A). Nuclear XRCC-1 expression was significantly higher in NSOKCs when compared to DCs (p = 0.016). Similarly, SOKCs also demonstrated higher nuclear expression of this protein compared to DCs (p < 0.0001). However, no statistically significant differences of nuclear XRCC-1 expression were observed between NSOKCs and SOKCs (p = 0.114) and between

NSKOCs and AMEs (p = 0.667). In addition, although without statistical significance, a greater expression of nuclear XRCC-1 was observed in AMEs in comparison to DCs (p = 0.066) (Figure 2B).

Interestingly, all odontogenic lesions exhibited higher significantly expression of nuclear APE-1 and XRCC-1 when compared to DFs (p < 0.0001) (Figure 2A-B).

Nuclear XPF is overexpressed in NSOKCs, SOKCs and AMEs

Nuclear XPF immunoreactivity was observed in the epithelial cells of 27 AMEs (90.0%), all NSOKCs (100.0%), 28 SOKCs (96.55%), 25 DCs (83.33%) and 4 DFs (20.0%). Cytoplasmic XPF expression was noted in the epithelial component of 15 AMEs (50.0%), 18 NSOKCs (60.0%), 9 SOKCs (31.03%) and 15 DCs (50.0%) (Figure 1C, 1F, 1I, 1L). No case of DF presented XPF cytoplasmic expression (Figure 1O).

The non-parametric Mann-Whitney test demonstrated significantly higher nuclear XPF expression in NSOKCs compared to DCs (p < 0.0001) and AMEs (p = 0.025). Likewise, SOKCs also demonstrated higher nuclear expression of this protein compared to DCs (p = 0.001) and AMEs (p = 0.043). However, no statistically significant differences of nuclear XPF expression were observed between NSOKCs and SOKCs (p = 0.724). Although without statistical significance, nuclear XPF immunoreactivity was higher in AMEs compared to DCs (p = 0.156) (Figure 2C).

Regarding the expression of XPF at the cytoplasmic level in the studied groups, it was found a higher expression of this protein in NSOKCs in comparison to SOKCs (p = 0.039). A higher cytoplasmic expression of XPF was also found in DCs compared to SOKCs (p = 0.04), but no statistical difference of cytoplasmic XPF reactivity was seen between NSOKCs and DCs (p = 0.864). In addition, no significant difference was found between AMEs when compared to NSOKCs (p = 0.072), SOKCs (p = 0.845) and DCs (p = 0.094) (Figure 2D).

Importantly, a greater number of XPF immunopositive cells was noted in all epithelial odontogenic lesions when compared to DFs, at nuclear (p < 0.0001) and cytoplasmic (p < 0.01) levels (Figure 2C-D).

Correlation between APE-1, XRCC-1 and XPF

Possible correlations between APE-1, XRCC-1 and XPF expression were analyzed for the five assessed groups. In AMEs, nuclear APE-1 expression correlated positively with both nuclear XRCC-1 (r = 0.850; p < 0.0001) and nuclear XPF expression (r = 0.600; p < 0.001). Similarly, it was also observed a positive correlation between nuclear XPF and nuclear XRCC- 1 (r = 0.566; p = 0.001). A significant positive correlation was found between nuclear XPF and cytoplasmic XPF (r = 0.597; p < 0.0001) (Table 2).

Concerning NSOKCs, nuclear APE-1 was positively correlated with nuclear XPF (r = 0.571; p = 0.001). Cytoplasmic XPF was positively correlated with both nuclear XPF (r = 0.459; p = 0.011) and nuclear XRCC-1 (r = 0.465; p = 0.01). Furthermore, cytoplasmic APE-1 expression was positively and significantly correlated with nuclear APE (r = 0.388; p = 0.034) and cytoplasmic XPF (r = 0.429; p = 0.018) (Table 2).

In SOKCs, nuclear APE-1 expression was positively and statistically correlated with both nuclear XRCC-1 (r = 0.762; p < 0.0001) and nuclear XPF (r = 0.470; p = 0.01). Likewise, at the nuclear level, XPF was correlated positively with XRCC-1 (r = 0.381; p = 0.041). Cytoplasmic APE-1 and XPF expressions were not significantly related to the expression of the other analyzed proteins in SOKCs (p > 0.05) (Table 2).

Regarding DCs, nuclear APE-1 correlated positively with both nuclear XRCC-1 (r = 0.631; p < 0.0001), nuclear XPF (r = 0.622; p < 0.0001) and cytoplasmic XPF (r = 0.635; p < 0.0001). At cytoplasmic level, XPF expression was positively correlated to expression of nuclear XRCC-1 (r = 0.560; p = 0.001) and nuclear XPF (r = 0.760; p < 0.0001) (Table 2).

In DFs, at nuclear level, APE-1 was positively correlated to XPF (r = 0.498; p < 0.025), but not with XRCC-1 (r = 0.188; p = 0.428). Also, nuclear XPF was not significantly correlated with nuclear XPF (r = -0.042; p = 0.859) (Table 2).

Discussion

Cells have evolutionarily-conserved mechanisms of DNA repair and maintenance that can cope with threats to their genetic material. It is, therefore, unsurprising that deregulation or defects in DNA repair pathways might result in the development and progression of several diseases, including neoplasms.31,32 Little is known about the role of DNA repair proteins in odontogenic cysts and tumours and no previous investigation evaluated the expression of BER proteins APE-1 and XRCC-1 and the NER protein XPF in these lesions. In this study, we found a nuclear overexpression of these proteins in AMEs, NSOKCs and SOKCs, lesions which commonly show locally aggressive behavior and tendency to recur which might suggest their involvement in the regulation of these mechanisms.

The BER is the most used pathway to handle with simple modifications (alkylations and oxidations) of single bases. The BER pathway is also involved in repairing the DNA single- strand breaks (SSB) induced by free radical agents.12 One of the key enzymes of the BER pathway in mammals is APE-1. The APE-1 is a multifunctional protein involved both in the BER pathway of DNA lesions, acting as the major apurinic/apyrimidinic endonuclease, and in transcriptional regulation of gene expression.12,19,35 This effect is obtained as a redox co- activator of different transcription factors such as the early growth response protein-1 (Egr-1), NF-κB, p53, hypoxia inducible factor 1α (HIF-1α), activator protein-1 (AP-1), and paired boxcontaining proteins (Pax) in different cell systems.12,19,35 In malignancies, transcription factors downstream of APE-1 promote growth, migration, and survival in tumour cells as well as angiogenesis and inflammation in the tumour microenvironment.20 In this study, we found a significantly higher expression of nuclear APE-1 in AMEs, NSOKCs and SOKCs when

compared to DCs and DFs. Taken together, these findings suggest that the BER pathway is probably upregulated due to the greater genomic instability and also that APE-1 may trigger a higher transcriptional activity of genes related to proliferation and migration in solid AMEs, NSOKCs and SOKCs, lesions which present a higher proliferation index, a more aggressive behavior, as well as the presence of important molecular genetic changes.1,6,23

Importantly, the compound APX3330 (formerly called E3330), is a specific Ref-1/APE- 1 redox inhibitor. APX3330 has been extensively characterized as a direct, highly selective inhibitor of Ref- 1/APE-1 redox activity that does not affect the protein’s endonuclease activity in tumours. Treatment with APX3330 slows tumour growth and progression, with limited toxicity, in both in vitro and in vivo models20,32-34 and is entering clinical trials in various cancers and other diseases bringing bench discoveries to the clinic.20 Thus, it would be valid to evaluate the possible functional effects of APX3330 on AMEs and OKCs in order to discover future therapeutic targets that could provide a personalized treatment approach for these lesions.

APE-1 subcellular distribution, in different mammalian cell types, is mainly nuclear due to its major DNA repair and co-transcripitional activity, controlling cellular proliferative rate and other activities.20,35 Most cell types exhibit only nuclear, others display only cytoplasmic, while others show both nuclear and cytoplasmic localization.20,35 _{Such a complex distribution} pattern suggests that localization is not random but, on the contrary, is controlled by a strictly regulated process. It has been showed that a shift to greater cytoplasmic localization is present in some aggressive malignant tumours with poor prognoses20,36 and this pattern of expression is very often associated with lesions with high metabolic or proliferative rates, being related to a cell cycle-dependent expression.9,35,36 _{Possible explanatory hypotheses for the cytoplasmic} expression of APE-1 may come from repair activites in mitochondrial DNA9 or by the need to maintain newly synthesized transcription factors in a reduced state during their translocation to the nucleus.9 Interestingly, from all groups herein evaluated, we only found APE-1 cytoplasmic