Low proteolytic processing of histone H3 in cervical tissue positive to hrHPV

During the different stages of cervical carcinogenesis there is an accumulation of epigenetic alterations leading to changes in gene expression. High-risk human papillomavirus (hrHPV) is a primary risk factor for cervical cancer (CC). Impaired proteolytic processing of histone H3 constitutes a potential epigenetic mechanism in CC. However, whether this event occurs in early stages of the HPV infection is unknown. Using human cervical samples with normal pathology but positive and negative to hrHPV, we identied that the H3 cleavage was low in the hrHPV positive cervix compared to the hrHPV negative cervix. These results suggest that low H3 processing previously observed in CC may be a primary effect of hrHPV infection.


Introduction
Worldwide, cervical cancer (CC) is the fourth most common neoplasm and could be accounted for approximately 7.5% of all cancer deaths in women. However, in low-and middle-income countries the CC impact is greater being the second most common cancer [1].
A growing body of evidence suggests that the proteolytic cleavage of the amino terminal end of histone H3 (H3) is a conserved process in Eukaryotes [7][8][9][10][11]. H3 cleaved products may be involved in cell cycle promotion, DNA replication, cell proliferation, apoptosis and migration [8]. Notably, proteolytic cleavage of H3 is reported to occur during viral infections [12].
Proteolytic cleavage of the amino terminal end of H3 has been documented in cancer [13,14]. Speci cally, we reported that the proteolytic cut of H3 is low in CC [13]. However, it is unknown whether reduced H3 processing is product of advanced stages of the cancer or whether it is associated with the HPV infection before the onset of CC. Therefore, we sought to investigate the H3 processing status in the cervix infected or not infected with HPV but negative for CC.

Patients
Cervical samples from 8 patients diagnosed with abnormal uterine bleeding (AUB) or uterine myomatosis (UM) and adnexal tumor (AT) were obtained by hysterectomy procedures. The patients diagnosed with AUB were classi ed according to the International Federation of Gynecology and Obstetrics (FIGO). The information concerning FIGO classi cation is included in Table 1. Additionally, all samples, were negative to pathological analysis to cervical cancer (NCC). Importantly 4 NCC samples (Lane P5 -P8) were positive for hrHPV by PCR, but negative for cervical intrapithelial neoplasia (CIN) by histopathological analysis (Table 1). In addition, the sample of each patient was divided into two parts; the rst one for obtaining DNA and the remaining part was used to obtain total proteins extracts.

Immunoblot Analysis
Immunoblot analysis Immunoblotting assays were performed using total proteins extracts from NCC tissue. All samples were obtained with RSB buffer (10 mM Tris-Cl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 1% NP40) supplemented with complete protease inhibitor cocktail. Immunoblotting assays were performed as described elsewhere [13]. Brie y, samples were resuspended in 2X Laemmli-buffer with β-mercaptoethanol, heated to 95ºC for 8 min, and loaded onto a 15% SDS-polyacrylamide gel. Proteins were transferred to a PVDF membrane by damp blotting at 40 mA for 3 h. The PVDF membrane was blocked with 5% milk in TBS-T (10X TBS-T: 198 mM Tris, 1400 mM NaCl, 0.01% Tween 20, pH 7.6) for 1 h. Then the PVDF membrane was incubated overnight at 4ºC with the primary anti-H3 antibody (E.960.2, Thermo Fisher Scienti c) or HRP conjugated-GAPDH antibody (14C10, Cell Signaling Technology). Anti-H3 antibody was used at a 1:500 dilution and GAPDH (used as loading control) at a 1:1000 dilution. The membrane was subsequently washed three times in TBS-T followed by incubation with HRP-conjugated secondary antibody (Jackson ImmunoResearch) for 1 h at room temperature. Then, the membrane was washed three times in TBS-T. Signals were detected using by chemiluminescence C-Digit (LI-COR).

Results & Discussion
Histone Clipping has been observed in different cell lines [13,14] including HeLa cells [13]. Previously, a report showed that H3 clipping is reduced in samples of CC [13]. However, if the impaired H3 processing occurred as a primary effect of the HPV infection or it is a consequence of cervical carcinogenesis as result of the high number of alterations is unknown. To address this, we analyzed the H3 clipping in samples of positive hrHPV cervical tissue with NCC. In addition, negative-HPV samples diagnosed with NCC were included as observed in Fig. 1. Characteristics of the analyzed samples are showed in the Table 1. Two forms of truncated H3 were detected principally in almost all the samples (Fig. 1). However, the presence of truncated H3 forms was reduced in positive hrHPV samples. These results indicate that H3 clipping is decreased in positive hrHPV NCC samples as compared with negative HPV NCC samples. Thus, this data suggest that aberrant H3 processing is inherently associated with hrHPV infection. Moreover, since low H3 clipping is also observed in samples with CC, it is possible that impaired H3 clipping is preserved during all the process of cervical carcinogenesis. Whether the low H3 processing is required for the development of CC remains to be demonstrated.
Alterations in histone clipping in host cells promoted by infectious microorganisms have been described previously [12,15]. Proteins named adhesion and penetration protein (App) and meningococcal serine protease A (MspA) are secreted by Neisseria meningitidis, a bacterium that can cause meningitis and/or septicemia in children and young adults [16]. These proteins, involved in meningococcal pathogenesis, can bind histones of human cells and generate proteolytic processing of H3 due to their serine endopeptidase activity [15]. H3 clipping also can be generated by the 3C protease of the foot and mouth disease virus (FDMV). The truncated H3 losses 20 amino acids from the N-terminus [12].
Oncogenic viruses can promote aberrant epigenetic changes in the host [17]. HPV oncoprotein speci cally can promote PTM [18]. It is known that E6 protein of HPV can bind cellular serine protease thought the PDZ domain [19] and we recently demonstrated that serine and aspartyl proteases are implicated in the H3 clipping [13].
Even though a limitation is the number of samples, the present work is the rst to suggest that low H3 processing may not be a consequence of the gene expression alteration in CC but a primary step associate to the hrHPV infection. If this event is determinant for viral virulence in the progression to CC requires further investigation but if this is the case, it could offer a potential therapeutic preventive target for the development of the disease.

Declarations
The data analyzed in the study are in the archives of the Hospital Juárez de México and are included in this article.
Ethics approval and consent to participate Ethical approval was obtained from the ethical committee of the Hospital Juarez de Mexico (HJM2231/13-B).

Consent for publication
Participants gave a written consent for participation and for their data to be published in a scienti c journal.