Abstract
Prostatic diseases such as hyperplasia and cancer are a consequence of glandular aging due to the loss of homeostasis. Glandular homeostasis is guaranteed by the delicate balance between production and cell death. Both cell renewal and apoptosis are part of this delicate balance. We will explore the predictive capacity for biochemical progression, following prostatectomy, of some members of the Bcl-2 family and of proteins involved in cell cycle inhibition in conjunction with established classical markers. The expression of Bcl-2, Bcl-xL, Mcl-1, Bax, Bim, Bad, PUMA, Noxa, p21, p27, Rb and p53 were analyzed by immunochemistry in 86 samples of radical prostatectomy and correlated with each of the markers established clinicopathological tests using statistical tests such as Sperman, Kaplan–Meier curves, unifactorial Cox, and multifactorial. The most relevant results are: (1) Positive correlation between: p27 with clinical T stage; and PUMA with pathological T stage; (2) Negative correlation between: Bcl-2 with clinical T stage, Bcl-xL with survival, Noxa and pRb with Gleason score.
Our results suggest that the expression of Bcl-2, Bcl-xL, PUMA, Noxa, p27, and Rb were related to some of the classic markers established to predict biochemical progression after prostatectomy.
Background
Prostate cancer is the second most prevalent cancer in men worldwide. Likewise, most men will develop benign prostatic hyperplasia (BPH) around the age of 60. Both pathologies develop in a different anatomical area. BPH appears in the transition zone of the gland while prostate cancer develops from epithelial cells located in the peripheral zone of the prostate gland along with a small percentage that arises from those cells that are in the transition zone. However, in 20% of the cases, BPH and cancer coexist in the same prostatic area [Citation1]. We can define the BPH as a noncancerous increase in size of the prostate, perhaps due to the influence of cytokines [Citation2–4]. To alleviate the most significant symptoms, different treatments have been used, including the use of alpha-blocking inhibitors of 5α-reductase, such as finasteride or dutasteride, which have different efficacy [Citation5]. In the last time, nucleotide polymorphisms are significantly associated with the efficacy of BPH treatment [Citation6]. Prostate cancer is related to many factors. Among them, there would be factors as different as cytokines [Citation7], human papillomavirus [Citation8], or even nutrition. In this sense, some recent works relate the importance of a good diet may lower the risk of prostate cancer [Citation9–11].
In the last decade, BPH and cancer are related as glandular aging due to the loss of homeostasis. Glandular homeostasis is guaranteed by the delicate balance between proliferation and cell death. Through the cell cycle, the tissues and organs guarantee the cellular renewal produced by losses due to normal wear and tear [Citation12]. If the balance mitosis vs. apoptosis is altered, the individual is exposed to immune disorders, degenerative diseases, and cancer [Citation13].
One of the hallmarks of cancer cells is to prevent apoptosis. Several signaling pathways may modulate apoptosis involving Bcl-2 proteins, especially the intrinsic mitochondrial cell death pathway [Citation12]. The members of the Bcl-2 family are subdivided according to their function in anti-apoptotic (Bcl-2, Bcl-xL or Mcl-1) and pro-apoptotic proteins [Citation14]. A high expression of anti-apoptotic proteins has been demonstrated in various types of cancer [Citation15]. At the same time, pro-apoptotic family are subdivided into two groups. The first group are formed by Bax and Bak that participate directly in the formation of pores in the mitochondrial outer membrane (MOMP), which leads to cell death at the point of no return [Citation16]. The second group of members of the pro-apoptotic Bcl-2 family is composed of proteins that share only the BH3 domain such as Bad, Bim, Noxa, or PUMA. Basically, BH3 proteins only promote apoptosis in a direct way through the binding and oligomerization of Bax and Bak or indirectly by the neutralization of anti-apoptotic family members [Citation17].
Another key process that ensures the maintenance of homeostasis is the cell cycle with regulatory proteins that accurately guarantee that no aberrant DNA or damaged or altered cells are synthesized. In the last years, cell cycle regulators are considered attractive targets in cancer therapies [Citation18]. In the cell cycle control are implicated by several members such as the retinoblastoma tumor suppressor protein (Rb), p53, p27, or p21. All of them have been previously described in cancer [Citation19,Citation20]. Some of them (Rb, p53, and p21) have even been described by our group in prostate cancer [Citation21,Citation22].
Rb regulates the transition between G1 and S phases in the cell cycle. The cyclin D-CDK4/6 and cyclin E-CDK2 complexes are responsible for phosphorylation of pRb [Citation23]. Nevertheless, the hypophosporylated forms of Rb block entry into S phase because inhibit the E2F transcriptional action [Citation24].
The p53 protein is another critical contributor at the control points, being the product of the TP53 gene. P53 is known as a tumor suppressor and pleotropic transcription factor that receives multiple signals of stress and cellular damage by activating genes that function variably in the arrest cell cycle. If the cell fails to repair DNA damage, p53 induces apoptosis. Apoptosis induced by p53 is mediated through the Bcl-2 family [Citation25].
P21 is induced in response to DNA damage through p53 tumor suppressor protein activity and mediates cell cycle arrest in the G1 and G2 phase [Citation26]. p21 binds to the cyclin D-CDK4/6 and E-CDK2 complexes, inhibiting pRb phosphorylation in the G1 phase [Citation27]. In phase S, p21 can inhibit the cyclin A-CDK1 complex and halt the G2M transition [Citation28]. The p21 protein acts as the main effector of anti-proliferative processes independent of the classical p53 pathway [Citation29]. P21 expression is associated with prostate cancer aggressiveness, while its decreased levels may in fact confer protection against prostate tumorigenesis [Citation29].
The p27 protein is a member of the CIP/KIP family. P27 negatively regulates the progression of the G1 cell to the S phase of the cell cycle by binding and inhibition of cyclin-dependent kinases (CDKs) [Citation30]. P27 facilitates the nuclear import of cyclin D-CDK4/6 complexes and inactivates cyclin A and cyclin E-CDK2 complexes [Citation31]. In addition to its role as a cell cycle inhibitor, p27 also regulates apoptosis. P27 could have tumor suppressor activity, and the decrease in p27 expression is associated with aggressive tumor behavior in several human malignant tumors. Genetic alterations of p27 are rare, in which case the mechanisms associated with transcription are poorly regulated [Citation32].
Since cell cycle regulation and apoptosis are closely linked cellular processes, we propose to study some of the proteins involved in homeostasis and their involvement in prostate cancer, as well as their possible involvement in biochemical progression to find some factor that together with the PSA allows us to know early on both the appearance of prostate cancer and possible biochemical recurrences.
Methods
Study patients
All the procedures followed were examined and approved by the University of Alcalá and Principe de Asturias Hospital Ethics Committees (reference number 2013/003/20130214) and were in accordance with the ethical standards of the Committee for Human Experimentation, with the Helsinki Declaration of 1975 (revised in Tokyo 2004) and the Committee on Publication Ethics (COPE) guidelines. This study was made with the consent of the patients’ relatives or their family in autopsy cases.
Prostates were obtained from: (a) histologically normal prostates (NP) obtained at autopsy (8–10 h after death) from 20 men (aged between 20 and 38 years) without histories of reproductive, endocrine, or related diseases; (b) radical prostatectomies from 86 men (aged between 52 and 74 years) with prostate cancer. Prostate cancer was detected by serum PSA (prostate specific antigen) screening and rectal examination, and diagnostic was confirmed by histopathological examination of needle biopsy cores. The median age (range) at the time of surgery was 66 (52–74). Patients were generally scheduled to have a serum PSA measure every 3 months for the first year and every 6 months thereafter. Median follow-up (range) time of the cohort was 75.9 (15.6–159.2) months, being defined as the time between the surgery and the endpoint of the study or the last record. Clinic pathological features of patients are shown in .
Table 1. Clinic pathological feacture of patients.
Reagents
Total serum PSA was measured by the AxSYM system (Abbott, Chicago, IL). All primary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The primary antibodies used were: rabbit anti-human Mcl-1, Bim, p27; mouse anti-human Bcl-2, Bcl-xL, Bax, PUMA, p21, Rb, pRb, mp53and goat anti-human Bad, Noxa. For immunohistochemistry analysis, antibodies were diluted at PUMA and pRb (used at 1:100), Bcl-2 and p21 (1:75), Bcl-xL, Bax, Bim, Noxa and Rb (1:50), Mcl-1, Bad and p21 (1:25), mp53 (1:1) in TBS. Biotin-conjugated antibodies (DAKO, Barcelona, Spain) and vectastain ABC (avidin–biotin complex) kit (Vector Labs, Barcelona, Spain) were at a 1:500 dilution.
Immunohistochemical analysis and scoring
Immediately after surgery prostate tissues were fixed, dehydrated, and embedded in paraffin, and 5 µm thick sections were processed following the avidin–biotin–peroxidase complex (ABC) method as described previously [Citation33].
Specificity controls for immunohistochemistry were performed as previously published [Citation7]. Briefly, for negative controls tissues were incubated with blocking peptides or preimmune serum (Santa Cruz Biotechnology). As external positive controls, histologic sections of human lymph nodes, skin, or thymus were incubated with the same antibodies.
Immunostaining was assessed by two independent pathologists (Pilar Martínez Onsurbe and Gabriel Olmedilla), blinded for the endpoint, in five randomly selected fields per section and six sections per patient. First, patients were stratified as positive (those showing staining in more than 5% of the surface of the corresponding compartment) or negative. Moreover, a score combining both intensity and percentage of immunostained surface was assigned to each sample, and accordingly patients were stratified as positive (those showing staining in more than 5% of the surface of the corresponding compartment) or negative. Moreover, a score combining both intensity and percentage of immunostained surface was assigned to each sample, and accordingly patients were stratified as having ‘‘negative’’, ‘‘low’’, ‘‘intermediate’’, or ‘‘high’’ immunostaining [Citation34].
Statistical analysis
The endpoint of the study was time to biochemical progression, defined as the time between definitive therapy to the first of at least two consecutive elevations in the total serum PSA level above 0.2 ng/ml. Established prognostic variables included in the study were preoperative serum PSA, pathological, and clinical T stages (2010 AJCC/UICC TNM classification [Citation35], postoperative Gleason score (2014 International Society of Urological Pathology (ISUP) [Citation36]), lymph node involvement and surgical margins status. To evaluate the association between clinicopathological (clinical T stage, pathological T stage, preoperative serum PSA, and Gleason score) and immunohistochemical variables Spearman test was performed. Log-rank test and Kaplan–Meier analyses were used for survival comparisons. To explore the correlation of the studied immunohistochemical parameters and established prognostic variables with biochemical progression, univariate, and multivariate Cox proportional hazard regression analyses were performed. All statistical analyses were performed using the SPSS 22.0 software (SPSS Inc. Chicago, IL, USA). p Values <.05 were considered as significant.
Results
Comparative analysis of expression of the studied proteins in NP, BPH, and PC
In previous manuscripts, we have studied by immunohistochemistry the expression of Bcl-2, Bax, p21, Rb, pRb, mcl-1, or p53 in normal, BPH and prostate cancer [Citation21,Citation22,Citation37]. The results obtained in these manuscripts were the same as those presented now, but with minor variations due to the increased number of patients present in this study ().
Table 2. (A) Percentages of patients showing positive immunohistochemical reactions to all the antibodies studied in normal prostate (NP), (B) benign prostatic hyperplasia (BHP), (C) prostatic carcinoma (PC); and average optical densities (O.D.) of immunostainings in positive patients, (D) statistical frequence of patients positive for antibodies under study.
In NP immunoreaction was found for Bcl-2, Bcl-xL, Mcl-1, Bax, Bim, Bad, PUMA y Noxa. 100% of the samples were positive for Bcl-xL and Noxa. Immunostaining was weak for Bcl-2, Bax, Bim, Bad, and Noxa. No immunoreaction was found to p21, p27, Rb, pRb, y mtp53 ( and ).
Figure 1. Expression of Bcl-2 (A–D), Bcl-xL (E,F), Mcl-1 (G,H), Bax (I,J), Bim (K,L), Bad (M,N) y PUMA (O,P) in normal prostate (A,H), BPH (B,E,G,K,M,O) and prostate cancer (C,D,F,H,J,L,N,P). Scale bars: 30 μm (O,P), 25 μm (A,B,G,I,J,K) and 20 μm (C,E,F,H,L,M,N), 15 μm (D).
Figure 2. Expression of Noxa (A,B), p27 (C,D), pRb (E,F), mp53 (G–I) in normal prostate (G), BPH (B,D,F,I) and prostate cancer (C–F). Scale bars: 25 μm (A,D,G,H,I) and 20 μm (B,C,E,F).
In BPH, the expression increased significantly in Bim and p21, although there were no expression changes for Bcl-xL, Mcl-1, Bax, Bad, and PUMA with respect to normal prostates. 100% of the samples were positive for Bcl-xL, PUMA, Noxa, p21, and mp53 ( and ).
In PC, Bim, Bad, p21, Rb, and pRb increased their expression. However, there were no changes in the expression of Bcl-2, Bcl-xL, Bim, PUMA, and Noxa. The number of positive samples also increased for Bcl-2, Mcl-1, Bim, Bad, and pRb ( and ).
Correlation between immunohistochemical variables and clinicopathological features
When the correlation between the study proteins and the classical markers was studied, a significant positive correlation was found between expression of p27 and clinical T stage. With respect to the intensity of the expression Mcl-1, Bax, Bim, Bad, p21, pRb, and p53 mut did not show any correlation with established classical markers. In addition, preoperative serum PSA, perineural invasion, node involvement, and biochemical progression were not correlated with any of the study proteins ().
Table 3. Correlation expression of studied proteins and clinic pathological feature.
In our cohort, there was a negative correlation between Bcl-2 and clinical T stage; between Bcl-xL with survival; between Noxa and Rb with Gleason score. The correlation was direct between PUMA and p27 with clinical T stage ().
Table 4. Correlation between immunohistochemical score of studied proteins and clinic pathological feature.
Correlation of immunohistochemical variables with time to biochemical progression
We used the Kaplan–Meier method combined with the log-rad test to evaluate the correlation between the expression of these protein and time to biochemical progression. The proteins used in this cohort when doing the Kaplan Meier survival analysis with the long-term test, none of them had a significant relationship with survival. When we performed the unifactorial Cox analysis, we confirmed the results of the survival curves.
After evaluating Kaplan–Meier survival curves for our proteins, we proceeded with the analysis of survival curves for the classical markers. The classical markers that had the most influence on the biochemical progression, according to the statistical method used were: pathological T stage (p = .017), Gleason score (p = .000), and node involvement (p = .001) [Citation34]. Factors such as preoperative serum PSA (p = .146), perineural invasion (p = .077) and positive surgical margins (p = .404) did not present any biochemical recurrence-related orientation in this cohort. These results were confirmed when the unifactorial Cox analysis was performed. In previous studies of this research group, we obtained similar results [Citation38,Citation39].
Discussion
Defective apoptosis comprises the main reason for tumor aggressiveness and tolerance to chemotherapy in solid neoplasms [Citation5,Citation40,Citation41]. In the same way that nucleotide polymorphisms have been related with the clinical characteristics BPH treatment [Citation6], recent research describes that a diet higher in fruits, vegetables, legumes, and fish (rich in omega-3 fatty acids) may lower the risk of prostate cancer [Citation9,Citation10,Citation11]. On the other hand, it is not unknown that BcL-2 family proteins contribute to tumor development, maintenance, and resistance to therapy, through the up-regulation of anti-apoptotic members and mutations or defects in pro-apoptotic genes [Citation42]. The prognostic value of Bcl-2 is contradictory. Previous papers in our group related to the increased expression of Bcl-2 and Bax in cancer of the prostate with the higher apoptosis index reported in this disease [Citation21]. At the same time, we found a significant relationship between Bcl-2 expressions in the stroma with pathological T stage but were also related to Bcl-2 intensity with clinical T stage [Citation39]. In this study, we only found that the Bcl-2 intensity in the tumor was related to the clinical T stage. This comparative evaluation between stromal and/or tumor has not been performed previously, whereas relating Bcl-2 overexpression with malignant phenotype [Citation43], other investigators found that the expression of Bcl-2 had no relationship to the prognosis of prostate cancer [Citation44–46].
In previous studies of our group [Citation39], we found a significant inverse relationship between Bcl-xL intensity in the tumor and survival. In this study, we have obtained the same results. Overexpression of Bcl-xL is also observed in many types of cancer because Bcl-xL also promotes metastasis [Citation47–48]. The reduction of Bcl-xL prevents migration, whereas overexpression promotes its migration in colorectal cancer cell lines [Citation49]. In xenograft mouse model, Bcl-xL promotes cell migration and metastasis, independently of its anti-apoptotic activity, but related with the Bcl-xL extranuclear expression [Citation49].
Although previous studies associate the anti-apoptotic function of Mcl-1 with poor prognosis, in this manuscript we observed that expression of Mcl-1 was higher in prostate cancer than in normal prostate, but no significant relationship with the classical markers evaluated was found [Citation50].
In our cohort of patients, the expression of Bax increase in prostate cancer patients samples respect normal or BPH patients. However, any significant relation between our data and classic markers was found. In colorectal cancer, patients undergoing surgery with low levels or absence of Bax expression had mortality 5.33 times greater than those with Bax overexpression [Citation51]. Previous work has shown that Bax acts as tumor suppressor, since its inactivation in many types of cancer is related to tumor progression by escaping apoptotic death [Citation52–53].
We did not find any relation between the expression of Bim or Bad. We did not expect these results since Bim is a hallmark for tumorigenesis [Citation54] and Bad has been shown to promote prostate tumor growth in a mouse model [Citation55]. The efficacy of many anticancer drugs depends on Bim, and insufficient induction of Bim or Bim function is often an underlying cause of therapeutic failure [Citation54]. Bad phosphorylated form is associated with treatment resistance and survival failure in several types of cancer [Citation56]. Other authors associate the high expression of Bad to greater global disease-free survival [Citation57] or Bad expression as an indicator of good prognosis in gastric, hepatocellular, and colon carcinomas [Citation58,Citation59].
We find a direct and significant relationship between PUMA intensity and pathological T stage. The intensity of Noxa presented an inverse relation with the Gleason score. Previously, we found a significant relationship between Noxa overexpression and biochemical recurrence in prostate cancer using tissue microarrays, however, they found no association with biochemical progression [Citation60]. Overexpression of PUMA is associated with increased apoptosis in several types of cancer cells [Citation61]. The tyrosine kinase inhibitor Pazopanib induce the PUMA expression, but also could activate Bax in colon cancer cells [Citation62]. Noxa was initially identified as a p53-responsive gene but has also been considered as a positive regulator of cell cycle progression in ER-positive breast cancer cells [Citation63]. In addition, several cancer cell lines have been reported to effectively resist chemotherapy by modifying the stability of Noxa [Citation64,Citation65].
In this manuscript and in this cohort, expression of p21 had no association with classical markers. The expression of p21 has been extensively studied, however, the results are discordant. In previous studies using the same antibody in our laboratory related that p21 had no role in the prediction of biochemical relapse [Citation66]. P21 deletion reduces prostate tumorigenesis and that p21 overexpression is associated with aggressive prostate cancer, possibly mediated by the ability of p21 to inhibit apoptosis [Citation67]. The overexpression of p21 has been associated with higher Gleason Score, poor survival and increase of recurrent prostate cancer in a transgenic mouse model [Citation29].
The cytoplasmic location of p27 has been detected in many human cancers, including melanoma, ovarian carcinoma, renal cell carcinoma, osteosarcoma, acute myelogenous leukemia, and breast cancer [Citation68]. It is generally associated with poor prognosis, high-tumor grade, and metastasis [Citation69,Citation70]. The tumorigenic effect of cytoplasmic p27 could be mediated by suppression of apoptosis, which may cause increased resistance to anti-tumor therapies that induce cancer cell death in breast cancer [Citation69]. The cytoplasmic localization of p27 suggests a possible application as a clinical prognostic marker [Citation68]. Prostate cancer showed expression of less than 50% p27 together with Gleason score, sufficient to predict clinically significant disease [Citation71]. Similarly, in our results, we found both expression and intensity of p27 had a direct relationship with clinical T stage. P27 is a tumor suppressor, and partial loss of the p27 function has been observed to be more oncogenic than complete loss, although the mechanisms of this loss remain unclear [Citation68].
Previously, we published that Rb (both the phosphorylated and dephosphorylated forms), are markedly increased in prostates with proliferative alterations [Citation21]. In this cohort of patients, Rb is associated with Gleason score but pRb has no relation to classical markers. Similar results were obtained in colorectal cancer [Citation72]. In contrast, expression of Rb protein in metastatic lymph nodes represents an independent prognostic indicator in patients with unknown primary tumor cervical metastases [Citation73]. Rb phosphorylation allowed inhibition of the mesenchymal epithelial transition (EMT) by decreasing invasiveness, in cultures of highly invasive breast cancer cells [Citation74].
In this study, the expression of mtp53 was not related to any prognostic factor, similar results have been described by other authors [Citation75,Citation76] and suggest that mutations in the p53 tumor suppressor gene do not influence the degree of differentiation or the clinical stage of prostate cancer [Citation76]. Discordant results have been found in the literature, argue that the expression of p53 is significantly related to several prognostic factors [Citation77]. The increased expression of 53 was related to shorter biochemical progression times [Citation78]. The abnormal p53 expression in prostate cancer is associated with an increased risk of disease-specific death, as well as the development of distant metastases at 5 years [Citation79]. The p53 mutation promotes cancer progression through up-regulation of the transcription factor Egr-1 in prostate cell lines [Citation79]. Erg-1 is an early response transcription factor induced by a wide range of growth factors and stress signals.
Tumor suppressor p53 and factor Bcl-2 are actively involved in the regulation of cell growth and apoptosis. PSA-based recurrence was associated with increased expression of p53 and Bcl-2 in patients who had undergone radical prostatectomy [Citation80].
Conclusions
In summary, we found that expression of Bcl-2, Bcl-xL, PUMA, Noxa, p27, and Rb as prognostic markers correlates with clinicopathological feature of prostate cancer. The objective of future studies could be directed to know the different mechanisms that these proteins involve with the development of prostate cancer and its use as therapeutic targets.
Ethical approval
All the procedures were examined and approved by the Joint committee of the University of Alcalá and Principe de Asturias Hospital Ethics Committees (PI13/1801; 2013/003/20130214) and were in accordance with the ethical standards of the Committee for Human Experimentation, with the Helsinki Declaration of 1975 (revised in Tokyo 2004) and the Committee on Publication Ethics guidelines. This study was performed with the written consent of the patients or their relatives. All pathological, clinical or personal data were anonymized and separated from any personal identifiers.
Disclosure statement
The authors have no conflict of interest to declare.
Data availability
All data generated or analyzed during this study are included in this published article.
Additional information
Funding
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