Anoikis Resistance and Development in Prostate Cancer: The Possible Role of HPV/EBV Coinfection

Javid Sadri Nahand Iran university of medical sciences Khadijeh Khanaliha Iran university of medical sciences Hamed Mirzaei Kashan University of Medical Sciences Mohsen Moghoofei Kermanshah University of Medical Sciences Hossein Bannazadeh Baghi Tabriz University of Medical Sciences Maryam Esghaei Iran university of medical sciences AliReza Khatami Iran university of medical sciences Maryam Fatemipour Iran university of medical sciences Farah Bokharaei-Salim (  bokharaeifarah@gmail.com ) Iran university of medical sciences

induction of tumor cell proliferation, higher invasion of tumor, and increased metastasis [9]. Prostate tissue can be impacted by many pathogens, including viruses [2], such as Epstein-Barr virus (EBV), human herpes simplex virus type 2 (HSV2), herpesviruses including cytomegalovirus (CMV) and human papillomavirus (HPV) [10]. However, there is no substantiated evidence to prove a direct correlation between infection frequency and the development of the in ammatory response with prostate carcinogenesis [3,10].
The EBV and HPV are well-established oncoviruses capable of initiating and progressing various human carcinomas [11][12][13]. In the high-risk HPV types, E5, E6, and E7 proteins interference with the tumor suppressor proteins in the cell. In addition, the oncoproteins of HPV E6 and E7 can modify the tumor milieu by regulating the certain pro-in ammatory chemokines and cytokines, which can impact the immune reaction e cacy [14]. On the other hand, the oncogenic proteins of LMP1 and EBNA1 in EBV prevent the apoptosis, stimulate the cell motility and proliferation, induce the angiogenesis and cell motility, which all prove the function of EBV in the carcinogenesis [15,16]. The EBV and HPV co-infection in some carcinomas have been reported by numerous studies [17][18][19][20][21][22]. Besides, the presence of both HPV and EBV sequences in healthy, malignant, and benign prostate samples [23][24][25][26] is of great importance because many experimental ndings showed that the HPV and EBV maybe act a role in the development of PCa [27]. In this regard, the function of EBV/HR-HPV co-infection is unclear. The current study aimed to evaluate the association of EBV/HPV co-infection with the PCa development by deregulating the cellular events linked to in ammation (RNS, ROS, TGF-β, VEGF, IL-17, IL-11, IL-8, IL-6, IL-1, TNF-α and NF-kB) and tumor progression (N-cad, Rb, P53, Survivin, Bcl-2, CD44, TWIST, SLUG, PTPN13 and E-cad). Also, we investigate the level of viral gene expression (E7, E6, E2, LMP-1, LMP-2 EBER 1 and EBER2) and the associations among viral genes and cellular factors involved in PCa development.

The collection of samples
The present multicenter case-control research was conducted during December 2018 to April 2020. First, 67 prostate tumor samples (n = 67) were obtained considering the inclusion and exclusion criteria during the study period in Tehran, Iran. Moreover, healthy tissues (n = 40) dissected from the peripheral area of adenoma removed by surgical procedure were collected as control (in terms of age). At least 24 hours prior to surgery, serum samples were prepared from blood by venipuncture and stored at -80 °C.
Additionally, the tumor stage was detected based on the TNM system in accordance with the consultation of a team of experts in cancer including a cancer surgeon, a radiologist and an oncologist. The ethical considerations were in accordance with the Helsinki Declaration, and both verbal and written informed consents were achieved from all research units. Snap-frozen tissue samples in liquid nitrogen were stored at − 80 °C. The clinical pro les of patients included tumor stage, tumor type and age (Table 1).

Detection of HPV and EBV by PCR
Total DNA was extracted from the tissue samples by the QIAamp DNA Mini Kit based on the kit protocol from the frozen samples of PCa. The quality of extracted DNA was analyzed using a 268-bp fragment ampli cation of the b-globin gene with the aid of HotStarTaq DNA polymerase (Qiagen, Dusseldorf, Germany) in the presence of G074 (5′-CAACTTCATCCACGTTCACC-3′) and G073 (5′-GAAGAGCCAAGGACAGGTAC-3′) primers. The cycling program was 95 C for 9 min, and then 35 cycles at 95C for 30 seconds, 55 C for 30 seconds, 72 C for 1 minute, and a nal extension at 72 C for 10 minutes [28].

Cellular gene (SLUG)
The SLUG gene was ampli ed by the qRT-PCR in the presence of speci c primers. The real time PCR device (Rotor-Gene® Q; Qiagen, Hilden, Germany) was utilized exploiting the Power SYBR Green PCR Master Mix (TaKaRa Bio; Kusatsu; Japan). The normalization of the relative expression level for the gene was carried out by a GAPDH as housekeeping gene. The sequences of F and R primers to amplify the SLUG gene were as follows: the forward primer sequence was as: 5'-GCCTCCAAAAAGCCAAACTACA-3', the reverse primer sequence was as: 5'-GAGGATCTCTGGTTGTGGTATGACA-3' [36].
In addition, the expression level was measured by the equation of 2 (−ΔΔCt) exploiting the online data analysis tool of QIAGEN (Gene Globe; http://www.qiagen.com/us/ shop/genes-and-pathways/dataanalysis-center-overview-page/). All reactions were repeated three times, and the internal control was considered to be GAPDH for the normalization of gene expression level (E7, E6, and E2) for various specimens. Kolmogorov-Smirnov test was used to assesse the normality of data distribution. Data were reported as mean ± standard deviation (SD) of parametric variables, and the median and the interquartile range for nonparametric data. In the comparison of central tendency parameters, including the mean for the normal variables and the median for the non-normal variables, two-independent samples t-test or Mann-Whitney non-parametric tests were applied between two groups, as well as one-way ANOVA or kruskalwallis tests were applied between more than two groups. Fisher's exact test or Chi-square was utilized to evaluate the associations between the categorical data. Spearman's rank correlation was measured for obtaining the extent of coexpression between the cellular factors and the viral genes. The statistically sini cance level was considered to be P < 0.05, adjusted by Bonferroni's correction test for ANCOVA analysis of data. Benjamini-Hochberg method was applied to correct the false discovery rate for multiple comparisons. Heat maps were plotted by one matrix CIMminer using Ward cluster algorithm and Euclidean distance approachs.

The pro les of research units
The demographic pro les of the study participants (n = 107) are shown in  and E7 oncogenes, thereby resulting in cell transformation [38]. It should be noted that the association of integration with contributing to prostate malignancy has not been the reported. A fully integrated type was seen in 42.8% of PCa group and 16.6% of control group. In 9.5% of PCa and 16.6% control groups, HPV genome was present in a purely episomal form. Also, mixed forms (episomal and integrated genomes) were found in 47.6% and 66.66% of the PCa and control specimens, respectively (Table 2).
Furthermore, the frequency of the fully HPV genome integrated type was signi cantly higher in the PCa group co-infected with the HPV/EBV when comparing with the mono-HPV infected PCa group (P: 0.0009 and 0.002, respectively). It is noteworthy that all integrated form of HPV DNA was found in the HPV/EBV co-infection samples. The  (Table 2).  Table 3 shows the expression level calculated for the EBV genes (EBER-1, EBER-2, LMP-1 and LMP-2) and HPV genes (E2, E6 and E7) in both stages and types of PCa. The maximum expression level of EBV genes examined was EBER 2 in stage IIA samples (mean ± SD:22.4 ± 9.7) as well as the lowest expression level of EBV genes was that of LMP 2 in stage IIIC samples (mean ± SD: 7.1 ± 8.3).The lowest and highest expression of LMP-1 gene were observed in stage IIIB (mean ± SD: 7.5 ± 10.2) and IVB (mean ± SD: 15 ± 11.9) samples, respectively. The expression of both LMP-2 and EBER-1 had the maximum mean levels in stage I specimens, and EBER 2, has the lowest expression in stage IIIB samples. Strati cation of the specimens in terms of the types of cancer demonstrats the EBER 1 level in SCC (mean ± SD: 18.6 ± 7.5) and EBER 2 in ductal adenocarcinoma (mean ± SD: 8.7 ± 9.5) samples were respectively the maximum and the minimum. The LMP-1 and LMP-2 gens had the highest expression level in acinar adenocarcinoma and SCC samples, respectively (Table 3). Figure 1B shows that a signi cant difference was in the EBV gene expression between mono EBV-positive PCa samples, whereas, except for EBER-2 gene, no signi cant difference was found between the mono EBV-positive PCa and HPV/EBV-coinfection positive PCa samples. The lowest and highest levels were found for both E6 and E7 genes in stage IIB (mean ± SD: 6.6 ± 2.7 and 7.3 ± 1.5, respectively) and IVB (mean ± SD: 15.5 ± 2.7 and 17.2 ± 2.5, respectively) samples, respectively ( Table 3). The minimum HPV gene expression level was related to E2 that was observed in the stage IIIC (mean ± SD: 3 ± 0.54) samples. Concerning the cancer type, the E7 gene expression level were the highest in the SCC (mean ± SD: 19.17 ± 4.3), as well as, the highest E6 gene level was observed in the SCC. According to Fig. 1, the expression level of HPV genes between the mono HPV-positive PCa cases and the mono HPV-positive controls as well as between the mono HPV positive PCa cases and the HPV/EBV coinfected PCa cases was not signi cantly different. Nonetheless, the expression level of E7 gene was signi cantly higher in the HPV/EBV coinfected PCa group when comparing with the HPV/EBV coinfected controls.

Comparison of expression pattern of in ammatory-related factors among study groups
We examined the expression levels of different in ammatory mediators between the PCa samples with the control samples and between EBV/HPV-positive samples with EBV/HPV-negative samples. The result is shown in Tables 4, 5, and 6. According to the results obtained, the level of in ammation-related factors including NF-κB, TNF-α, IL-17, IL-11, IL-8, IL-7, IL-6, IL-1, VEGF, TGF-β, ROS, and RNS in the PCa group higher than in the control group. For detection of whether HPV and/or EBV infection or/and HPV/EBVcoinfection are implicating in regulating in ammation in PCa, we analyzed the expression level of these factors among EBV infected, HPV infected, and HPV/EBV coinfected samples. All in ammatory factors mentioned above was higher signi cantly in the groups positive for HPV compared to the groups negative for HPV (Table 5). Furthermore, as shown in the Table 5, the maximum levels of expression were seen for the ROS (P < 0.0001, fold change: 7.5), RNS (P < 0.0001, fold change: 2.6) and IL-6 (P < 0.0001, fold change: 2.51) in the PCa group positive for HPV compared with the PCa group negative for HPV. The correlation results in Table 7 showed signi cantly positive correlation between the expression level of the viral proteins (E2, E6 and E7) and the in ammatory agents (P < 0.001). The strongest positive correlation was between ROS and E6/E7 (r = 0.763, P = 0.0001), and then between Survivin and E6/E7 (r = 0.75, r = 0.76, respectively, p value = 0.0001 for both).     Compared with the EBV-negative group, EBV-negative PCa group and EBV-negative control group, the expression level of VEGF, IL-6, IL-1, NF-κB and TNF-α was higher in EBV-positive control, EBV-positive PCa and EBV-positive groups, respectively ( Table 4). As shown in Table 7, there was a strongest correlation between LMP-1 and IL-6 (R = 0.66, P = 0.001). Table 7 presents more details. Since some of the samples studied were both infected with the EBV and infected with the HPV (HPV/EBV coinfection), the question may now be whether the increased expression of in ammatory factors was the result of the effect of EBV-infection or the presence of HPV-infection and/or effect of HPV/EBV-co-infection? Therefore, as can be seen in Table 6, the level of in ammatory factors was compared between the co-infected and mono-  Table 4). As well, a signi cant positive correlation was observed between the EBV-LMP1 gene expression levels and the expression levels of CD44 (r = 0.8, P < 0.0001) and Survivin (r = 0.76, P < 0.0001) ( Table 7).
As seen in Table 5, there was a signi cant reduction and elevation in the concentration of tumor suppressor proteins (p53 and Rb) and the anti-apoptotic proteins (survivin and Bcl-2), respectively, in the HPV-positive groups in comparison with the HPV-negative groups. Also, the concentration of E-cad and PTPN13 proteins was signi cantly lower and the concentration of N-cad, SLUG, TWIST was signi cantly higher in the HPV-positive groups when comparing with the HPV-negative groups. In addition, no statistically signi cant difference was found in the expression pattern of CD44 between HPV-positive groups when comparing with the HPV-negative groups (P > 0.05). As shown in Table 7, there was a signi cantly positive correlation between the E7 and E6 level and Survivin (r:0.75, and 0.76, respectively, P < 0.001), Bcl-2 (r:0.74, and 0.74, respectively, P < 0.001), TWIST (r:0.83, and 0.86, respectively, P < 0.0001) and N-cad (r:0.85, and 0.87, respectively, P < 0.0001). Beside, there was a signi cantly negative correlation between E7 and E6 level and Rb (r:-0.75, and − 0.75, respectively, P < 0.0001), p53 (r:-0.71, and − 0.71, respectively, P < 0.0001), E-cad (r:-0.71, and − 0.71, respectively, P < 0.0001).
Based on correlation results in Table 6, signi cantly lower and higher expression levels were reported for the tumor suppressor proteins (p53 and Rb) and Bcl-2 in the HPV/EBV co-infection PCa group, respectively in compared to the mono EBV positive PCa group, and the non-HPV/non-EBV PCa group.As well, the mean expression level of these factors between HPV/EBV co-infected PCa and mono HPVinfected PCa groups are different which it probably due to the presence of the EBV infection and it can contribute to the progression of prostate cancer by modulation of cellular factors.In comparing Survivin between HPV/EBV co-infection PCa group with mono HPV-and mono-EBV-infected PCa group, it was observed that the mean expression level of survivin had no signi cant difference between the these groups (P > 0.05). Therefore, it can be concluded which high concentration of Survivin in coinfected PCa group maybe can due to the simultaneous presence of HPV and EBV infections.
Expression level of CD44 in PCa and EBV-positive groups were signi cantly higher than control and EBVnegative groups, respectively ( Table 4). As shown in Table 6, the mean CD44 expression level was signi cantly different only between the two groups (HPV/EBV coinfection PCa group versus mono HPVpositive PCa group), but not between coinfection PCa group and mono EBV-positive PCa group. Thus, the increase in mean expression level of CD44 in the coinfection group is most likely due to the presence of EBV virus.
According to the results of Table 4, among anoikis-related factors, the expression level of N-cad and PTPN13 in the PCa group is signi cantly different from the control. Also, no signi cant difference was seen in the mean expression level of anoikis-related factors between EBV-positive and EBV-negative samples (Table 4). Conversely, a signi cant difference was reported between the mean expression levels of all of these factors in HPV-positive compared to HPV-negative samples ( Table 5). The mean expression level of TWIST, N-cad and SLUG in HPV/EBV co-infected PCa group was higher compared to EBV/HPV negative PCa, mono EBV-infected PCa, non HPV/non EBV PCa groups. As shown in Table 7, a signi cant negative correlation was seen between the expression level of E-cad with E7 and E6 (r=-0/71, P < 0.0001), and also between E7 and E6 levels with the TWIST (r = 0/83, r = 0/86, respectively, P < 0/0001) and with the N-cad (r = 0/85, r = 0/87, respectively, P < 0/0001). More information is given in Table 7.
In compared HPV/EBV coinfected PCa group with coinfection negative PCa, mono EBV-positive PCa and non-HPV/non-EBV PCa groups, the E-cad level and the TWIST and N-cad levels had signi cantly decreased and increased expression, respectively (Table 6). However, no signi cant difference was found in the mean expression level of Anoikis-related factors between the mono HPV positive PCa group and the co-infection positive PCa group. In conclusion, the HPV infection is probably the cause of these changes and HPV plays important role in contribute to anokis resistance than the EBV virus.

Discussion
The The viral infections globally accounts for about 12% of cancers, predominantly (> 85%) in developing countries [11,12]. The HPV and EBV are reportedly associated with 38% of all cancers linked with viruses [39]. Evidence has proved an associstion between the HPV infection and the cervical cancer prognosis and carcinogenesis. There is also reportedly a strong association between the HPV infection and other cancers as an etiologic factor [40]; as well, studies reported the presence of EBV in the cervical cancer [41] and the prostate cancer [26]. In addition, the EBV/HPV co-infection has also been detected in the breast cancer [42,43]. In our study, the HPV and EBV were isolated from in 31.3% and 49.3% of the PCa group, as well as in 15% and 40% of the control group, respectively. The EBV/HPV co-infection was present in 14.9% of PCa patients, 7.5% of control samples. Moreover, the high-risk strains (HPV 16 and 18) were responsible for 50% and 30% of HPV/EBV co-infected PCa samples, respectively. Also, the most common EBV genotype in the study was genotype 2. The EBV infection has been reported in the prostate tissues samples. The presence of the EBV was reported in 8.8% of malignant and benign prostate tissue samples (31 of 352) in Sweden [24], and in 8% of malignant, benign and normal prostate tissues (16 of 200) in the United States [25]. A study reported the EBV infection in about 37% of prostate cancer patients (n = 19) [23]. HPV-18 and EBV (EBNA1) gene sequences were detected in high proportions and almost equal to normal, benign and prostate cancer samples [26]. The EBV/HPV co-infection was signi cantly more prevalent in prostate cancer (55%) when comparing with benign samples (15%) and normal prostate samples (30%) [26]. Moreover, the EBV and HPV act simultaneously to enhance the cultured cervical cell proliferation, indicating that the same may happens to prostate epithelial cells [26,44], in line with the present experiments about the high-risk HPVs and about the EBV in PCa.
The HPV genome integration is the key genetic phenomenon in the cervical carcinogenesis. Some studies revealed that the EBV infection accelerates the HPV genome integration into cervical cell genome in the cervix, which increases their genomic instability [18,45] and develops lymphoepithelioma-like cervical carcinoma in some cases [41]. In line with this hypothesis, our results showed the maximum HPV genome integration rate was detected in the HPV/EBV co-infected PCa group (8/10, 80%). Also, the purely integrated HPV (47.4%) was signi cantly higher than the purely episomal HPV (5.2%) in the tumor tissues positive for the HPV. The purely integrated HPV was signi cantly higher in HPV/EBV co-infected PCa samples compared to the mono HPV-infected PCa samples (P: 0.0009).
Since no studies have been performed on the effect of the HPV/EBV coinfection on PCa development, we compared inevitably our ndings with the results obtained from other cancers. The EBV acts probably as a co-factor for the HPV to induce the pathology of uterine cervix, veri ed by Szkaradkiewicz et al. [46] who reported a possible sexually EBV transmission route. Also, It has been found that sexually transmissible infections have been associated with increased risks of PCa [47]. and NF-κB signaling [48]. Similarly, as mentioned in result 3.5 section, mean expression level of p53 and Rb in co-infecction group were lower than mono HPV-and mono EBV-infected groups but was not signi cantly different between co-infected group with mono HPV-infected group, while, there was a signi cant between coinfected group with mono EBV-infected group. Besides, there was no signi cant difference between EBV-positive samples with EBV-negative specimens. Additionally, a signi cant reverse correlation was found between the HPV E7 and E6 mRNA expression levels with those of p53 and Rb, respectively (Table 7). Therefore, it can be concluded which lower concentration of Rb and p53 in coinfected PCa group probably more due to the simultaneous presence of HPV and EBV infections but maybe HPV infection effect was more than EBV infection.
One of the important defense strategies of innate immunity against pathogens is in ammation that helps to heal damaged tissue [49]. However, in ammation during pathogenesis can play a dual role. Prolonged chronic in ammation promotes tissue damage and autoimmune disorders, in addition to can lead to the accumulation of damage to DNA and give rise to cancers [50,51]. Chronic or recurrent in ammation can be developed following the persistent viral infection. The pivotal pathogenic mechanism in the development of cancer caused by EBV and HPV is to induce the cytokine impact and the chronic in ammation [14] but the function of HPV/EBV co-infection in PCa is unknown. In addition, the NF-κB has a key role in coordinating the expression of genes related to the chronic in ammatory diseases [52]. A wide variety of factors such as viral infection, oxidative stresses, cytokines (TNF-α, IL-1β), and tumor promoters can activate NF-κB. Also, NF-κB modulates expression of the various genes including cytokines (e.g. IL-1, 6, 8, TNF) and cell cycle regulatory molecules [49,52]. A crosstalk between in ammation and cancer mediates by NF-kB thoroughly accepted [49]. It has been observed that an elevated EBER1 level in cervical cells may play a role in the transition from the in ammation to the oncogenesis of cervical cancer caused by HPV through the modulation of innate immunity [53]. The results of the current study showed that all in ammatory factors had signi cantly higher expression level in HPV-positive specimens when comparing with the HPV-negative ones ( Table 5, result 3.4 section), while the concentration of VEGF, IL-11, IL-6, IL-1 and NF-kB was signi cantly elevated in the EBV-positive specimens in comparison with the EBV-negative ones ( Table 6). There was also a signi cant association among the expression levels of LMP-1, E7 and E6 with NF-kB as well as between IL-6 and LMP-1 (Table 7). These results indicate that a possible association between in ammation with HPV and EBV in PCa. The level of NF-κB, TNF-α, IL-6, IL-17, VEGF, ROS and RNS in HPV/EBV co-infected PCa samples was higher than non-coinfected PCa samples. Besides, the mean expression level of TNF-α, IL-8, RNS, ROS, TGF-β, VEGF and IL-17 in co-infection positive PCa samples was signi cantly higher than mono EBV positive samples. Also, the in ammatory factors had greater mean expression level in the HPV/EBV coinfection group when comparing with mono HPV and mono EBV-infected groups but no signi cant difference was reported between coinfection and mono HPV groups. Therefore, by comparing the mean expression level of in ammatory factors between co-infection and mono-infection (Table 6, result 3.4 section) it can be concluded that HPV/EBV coinfection can increase the mean expression of in ammatory factors compared to mono HPV or mono EBV infection.
In a study by Grace et al., a signi cantly positive correlation was observed between the HPV-induced SCC and the expression levels of Bcl2/p53 proteins [54], con rming that the HPV could develop the cervical lesions by deregulating the induction of apoptosis [54]. In fact, it can be said that the high-risk HPV-E6 oncoprotein is able to enhance the Bcl-2 protein expression in the cervical cancer through the elimination of inhibitory activity of p53 over the Bcl-2 [55]. Survivin and Bcl-2 (as anti-apoptotic proteins), which widely expressed in most tumor tissues, are critical factors to regulate the progression of cell cycle and to impede the apoptosis [56]. In a study by Guo et al., the p53-induced survivin upregulation is promoted by LMP1 through an elevation activity of survivin promoter and activity of p53-survivin DNA binding, which means that the complexity of p53 regulation in Survivin occurs via the viral oncoprotein of LMP1 in NPC.
Their model of p53-induced G1/S cell cycle progression could upregulate the LMP1-mediated expression of survivin in the pathogenesis of nasopharyngeal carcinoma [57]. Moreover, an LMP1-induced upregulation of Bcl-2 has been reported in B cells [58], while LMP1 has no effect on Bcl-2 levels in the leukemic T cell line Jurkat [59]. In a study of Muzio  The evidence suggested that the expression level of survivin may be in uenced directly or indirectly by the HPV [60]. In this study, it was observed that the Survivin and Bcl-2 expression level in the HPVinfected, EBV-infected and HPV/EBV-coinfected samples were signi cantly higher than in HPV-negative, EBV-negative and non-HPV/EBV-coinfected samples, respectively. Additionally, in the current study, there was a direct association between the E6 and E7 expression level and the Survivin and Bcl-2 expression level as well as between the expression levels of LMP-1 and Survivin. The EBV/HPV co-infection may initiate a neoplastic transformation of carcinogenesis [22]. Moreover, an in vivo interaction occurs between HPV and EBV as well as an interaction is there between EBV and HPV oncoproteins [44].
According to result of this study, the Survivin and Bcl-2 expression level signi cantly was higher in HPV/EBV coinfected PCa group than in non coinfected PCa group as well as there was not signi cantly different between mean expression level of Survivin in HPV/EBV co-infected PC with mono HPV-and mono EBV-infected PCa samples. In general, these results suggest that the co-presence of HPV and EBV infection may lead to the resistance of cancer prostate cells to apoptosis, although probably the effect of the HPV infection is greater than that of EBV.
Metastasis refers to the removal of tumor cells from their original location and then their dissemination to and proliferation in distant locations [61]. To this end, epithelial cancer cells must achieve a exible migratory phenotype. In this regard, the epithelial-mesenchymal transition acts as a developmental pathway for tumor cells to reach this phenotype [62]. One of the physiological barriers for the tumor metastasis is anoikis, but most tumor cells become resistant to the anoikis [63].
According to the ndings, the Slug and Twist (EMT-inducing transcription factors) are responsible for the anoikis resistance, in contrast, E-cadherin signi cantly inhibits anoikis resistance [63]. The CD44 as a cell membrane receptor with multifunctional potential is highly expressed in many cancers such as PCa and accounts for cell-cell adhesion and tumor metastasis and invasion [64]. The tumor aggression may be inhibited by the PTPN13 phosphatase. The invasion of different cancer cells is suppressed by the PTPN13 [65]. In a study by Castilla et al., the PTPN13 gene silencing elevated the expression level of invasion-related genes in the PCa cells [66]. The HPV-E6, by losing the PTPN13, triggers the anchorageindependent growth in the human epithelial cells [67,68]. Accordingly, the present study aimed to analyze the expression levels of SLUG, Twist, N-cadherin, E-cadherin and PTPN13 proteins were in prostate tissue lysates. As mentioned in result 3.5, the mean expression level of N-cad, SLUG and TWIST were signi cantly higher in the cases positive for HPV compared to that in the groups negative for HPV, while the PTPN13 and E-cad level in the HPV-positive PCa group was signi cantly downregulated compared with HPV-negative PCa group (Table 5). Based on the ndings, the E6 and E7 expression levels had a signi cant negative and positive correlation with E-cadherin and N-cadherin/TWIST/SLUG, respectively (Table 7). It should be noted that no signi cant difference in the concentrations of PTPN13, TWIST, SLUG, N-cad and E-cad was found between EBV-positive and EBV-negative groups (Table 4). However, the mean CD44 expression level was signi cantly greater in EBV-positive samples than in EBV-negative ones and there was a positive correlation between the LMP-1 and LPM-2 expression level and the CD44 expression level (Tables 4 and 7) but no signi cant difference was seen between the HPV-positive and HPV-negative groups. It has been reported that the LMP1 down-regulates the E-cadherin expression and up-regulates TWIST and other transcription factors linked to the cell motility [69,70]. The EBV-protein, LMP-1, leads to anoikis resistance by inducing the expression of anti-apoptosis proteins survivin, CD44, the inhibitor of DNA binding 1 (Id1), Bim and ROS [63,71]. Moreover, the co-expression of E6 and LMP1 triggers some processes, including tumor-formation ability, anchorage-independent growth, resistance to apoptosis and cell proliferation, in nude mice when comparing with the expression of E6 or EBNA1 alone [72]. The LMP1 and HR-HPV E6 co-expression is associated with more aggressive malignant tumors, including cervical SCC and breast adenocarcinoma [73,74]. In current study, the level of TWIST, and N-cad in HPV/EBVcoinfected PCa group compared to non-coinfected PCa group were increased, while the E-cad had signi cantly decreased expression level. However, no signi cant difference was reported in the mean expression level of anoikis-related factors between the PCa group positive for co-infection and the PCa group positive for mono HPV. As well as, the mean level of CD44 expression was not signi cant differences between HPV/EBV coinfection PCa group vs mono EBV-positive PCa group. Thus, the increase in mean expression level of CD44 in the coinfection group is most likely due to the presence of EBV virus. In conclusion, the existence of the HPV and EBV is probably the cause of these changes and viral infection plays important role in contribute to anoikis resistance and PCa development.
One of the limitations of this study was we had to sample a peripheral area of surgically dissected benign prostatic hyperplasia because of lacking access to normal or healthy prostate samples.

Conclusion
According to the results from the present study, the HPV/EBV coinfection was present in 14.9% of PCa samples and that the high-risk strains (HPV 16 and 18) were responsible for 50% and 30% of 10 PCa samples co-infected with HPV/EBV, respectively. The maximum percentage of HPV genome integration was found in HPV/EBV coinfected PCa group (8/10, 80%). In current study, although there were not signi cant association between the HPV/EBV co-infection with PCa, the expression pattern of some cellular factors involved in in ammation, tumor progression and metastasis were different between PCa infected with virus and between HPV/EBV co-infected PCa samples with mono EBV or mono HPV infection. These differences may mean that the simultaneous presence of these viruses has altered the pattern of expression of cellular factors compared to mono infection (Fig. 2), suggesting the HPV/EBV coinfection as a contributing factor in the development of PCa, in addition to the fact that the EBV has a role in the HPV genome integration. Availability of data and materials

List Of Abbreviations
The datasets used and/or analyzed during the current study could become available through the corresponding author on reasonable request.