Abstract
Prostate cancer is the second most common neoplasm among men, with a high mortality rate in advanced stages. Poly (ADP-ribose) polymerase (PARP) plays an important role in repair to DNA damage, being associated with resistance to tumor cell death. Conversely, Caspase-3 is a crucial mediator of programmed cell death, being highly expressed in apoptotic cells. The aim of the present study was to characterize the expression of PARP and Caspase-3 by immunohistochemistry in patients with advanced prostate cancer. PARP and Caspase-3 were independently correlated to patients’ evolution, in accordance with the classification of prognostic groups. The increase in PARP expression was positively correlated with tumor patients with poor prognosis (P < 0.0001). In contrast, a decrease in Caspase-3 expression was identified in patients with poor prognosis, when compared with prostate cancer patients with good prognosis (P = 0.0007). Numerically, 92.3% of patients previously classified with poor prognosis showed higher PARP expression, while 93.75% of patients previously classified with good prognosis showed higher levels of Caspase-3. We conclude that PARP and Caspase-3 are potential prognostic markers for prostate cancer patients with different prognosis.
Prostate cancer (PCa) is the second most common cancer among men,1 with an estimated occurrence of 65,840 new cases in Brazil over the next 2 years (2020-2022).2 PCa is a heterogeneous disease with multiple outcomes, and to date, the treatment options for advanced disease stage remain scarce.3
Androgen deprivation therapy (ADT) has been the primary treatment option for male patients with advanced symptomatic PCa for seven decades. This treatment strategy often results in decreased prostate specific antigen (PSA) levels. However, following a median of 18-24 months of endocrine therapy, most patients progress to a poor prognosis stage castration resistant prostate cancer (CRPC), which is known as hormone-refractory PCa.4
Despite the scientific progress in PCa treatment, which includes surgery, radiotherapy, hormone blocking, and chemotherapy, patients with advanced PCa still face severe outcomes. The early-stage detection is of great value for PCa management due to a significant number of subclinical cases in patients over 50 years-of-age.5 Therefore, the identification of new predictive and prognostic biomarkers may be a determining factor in PCa treatment.
The prostate-specific antigen (PSA) is the only widely used biomarker for PCa diagnosis and prognosis. However, PSA is unable to differentiate between indolent and aggressive forms of PCa. Also, despite some individuals presenting with initially low serum levels of PSA, many men may harbor aggressive PCa.6
The Gleason grading system is commonly used to differentiate prostate biopsy samples and help evaluate the prognosis of PCa patients. Recently, the simplified PCa grading system using five stages has shown more accurate stratification, compared with the current Gleason grading system. Along with other parameters, it is incorporated into a strategy of PCa staging that predicts prognosis and helps to guide treatment.7,8
There are recent studies evaluating the role of poly (ADP-ribose) polymerase (PARP) and Caspase-3 as prognostic markers. PARP proteins are a family of 17 enzymes involved in the regulation of transcription, DNA damage response, genome stability, cell cycle, energy metabolism, cell death, and tumorigenesis.9 PARP-1 is the main member of the PARP family, and it induces cell survival through DNA repair. During apoptosis, PARP-1 is cleaved into two fragments by caspases, resulting in protein inactivation. The caspase-mediated PARP-1 inactivation suggests that PARP-1 activity blockade is essential for the proper functioning of the apoptotic machinery via DNA fragmentation.10
PARP-1 was previously demonstrated to be overexpressed in several malignant tumors and is associated with invasiveness and poor prognosis.9 Because some types of PCa may activate DNA repair signaling, the development of anticancer targets that affect this system may be promising for PCa diagnosis and treatment.11
The caspase protein family includes protease enzymes that are programmed cell death regulators. Caspase-3 (cysteine protease protein 32) is responsible for the proteolytic cleavage of various proteins, being the ultimate executioner caspase essential for the apoptosis.12,13 Caspase expression and activation represent an important step in apoptotic cell death.14 Loss of apoptotic control in association with cell proliferation are key events responsible for PCa initiation and progression.15
Predictive biomarkers are important for the advancement of targeted therapies. This allows more effective triage of patients to determine beneficial clinical interventions.16 Therefore, to better establish the clinical evolution of PCa patients, we investigated the prognostic value of PARP and Caspase-3 and correlated them with clinicopathological parameters of PCa patients.
Material and Methods
Sample Characterization
In the present experimental protocol, paraffin blocks containing tumor fragments were selected from 29 men diagnosed with PCa in the Oncology Section of São José do Rio Preto Medicine School – FAMERP, between the years of 2011–2012. The information on clinical parameters included pathological findings (phenotypic classification of the tumor, clinical aspects, and pathological staging) (Table 1). All patients had adequate follow-up time and overall survival for patients diagnosed with worse prognosis was, on average, 2 years. All data about treatment and clinical outcomes were tracked by the electronic medical system records of the Base Hospital of São José do Rio Preto, and each patient’s samples were collected and analyzed by pathologists. Treatment modalities followed the protocol of the oncology staff and were categorized among patients with worse and good prognosis. The patients with good prognosis received the protocol including hormone therapy or radical prostatectomy, either isolated or combined with adjuvant radiotherapy, while patients with worse prognosis received hormone therapy without adjuvant therapy, except for cases in which patient status was not eligible for any treatment due to critical clinical conditions. In both groups, therapeutic strategies were determined in agreement with the latest National Comprehensive Cancer Network (NCCN) clinical practice guidelines in oncology, combined with the Brazilian Society of Clinical Oncology (SBOC) terms. For localized disease, treatment varied between radical prostatectomy and radiotherapy, with or without the association of hormonal blockage; in cases of systemic disease, the main option was hormone therapy. Two experimental groups were included in this study. The study was approved by the Ethics and Research Committee/FAMERP (registration number #15716819.2.0000.5415 and protocol #073430/2019).
Description of patients’ clinicopathological data included in this study
Prognosis Determination
In this retrospective study, patient´s pathological prognostic staging was determined by analyzing characteristics such as the anatomical profile of the tumor (TNM staging, regarding the International Union against Cancer [UICC], TNM classification recommended in 1989), histological grade, Gleason score at the time of biopsy, being fundamentally based on individual patient’s clinical evolution. Eventually, patients who developed aggressive PCa and died were considered as poor prognosis, while patients with indolent PCa and alive were considered as good prognosis (with or without the disease).
PARP and Caspase-3 Analysis by Immunohistochemistry
The immunoexpression of PARP and caspase-3 was analyzed by immunohistochemical assay. For this purpose, the paraffin blocks were cut at 4 µm thickness, and histological sections were then fixed to previously silanized slides. Subsequently, antigen retrieval was performed in a steam cooker (ARNO, Sao Paulo, SP, Brazil) with citrate buffer (pH = 6.0). Endogenous peroxidase was blocked by incubating the slides with 3% hydrogen peroxide for 15 minutes, followed by protein unspecific blocking for 10 minutes. The slides were incubated with the primary antibodies, following standard dilutions according to the concentrations provided by manufacturers; anti-PARP (1:200 dilution; Thermo Fisher Scientific Inc – Invitrogen), and anti-caspase-3 (1:500 dilution; Abcam plc.) for 18 h at 4°C in a humid and dark chamber. After the incubation period, the slides were washed with phosphate buffered saline (PBS), incubated with secondary antibody, and then with a horseradish peroxidase conjugate. Immunoreactions were revealed using the chromogenic substrate DAB (Bioscience, Pleasanton, CA). Section staining was counterstained with Harris Hematoxylin for 40 seconds, followed by mounting in Erv-Mount (Erviegas, Sao Paulo, SP, Brazil). All immunoreactions were accompanied by a positive control for the tested antibody (human spleen tissue for anti-PARP and human tonsil for anti-caspase-3) and a negative control (absent primary antibody).
Immunohistochemical Quantification
The evaluation of the immunoreactive areas for PARP and caspase-3 was performed by examining different fields in each slide based on different staining levels. Slides were observed under a ZEISS–AXIOSKOP 2 microscope (Leica Nikkon Eclipse E200; Nikon Instruments, Melville, NY, USA) using 40 X objective. For each patient, three fields of tumor tissue were photographed and 20 random spots from each photographed field were selected using Image J software (NIH, Bethesda, MD, USA). The immunohistochemical staining was then quantified by means of optical densitometry (M.O.D.). Overall, 60 different spots of every patient sample were analyzed, resulting in an average of the correspondent immunostaining intensity, and the correlation with PARP and caspase-3 expression was determined using arbitrary units (a.u.).
Statistical Analysis
Results were initially subjected to descriptive analysis to determine normal distribution. The analysis of each PARP and caspase-3 expression in predefined prognostic groups (expression in patients with good prognosis compared with expression in patients with poor prognosis) was performed by applying the unpaired Student t test. Subsequently, the groups were stratified into six further risk classification criteria according to clinicopathological parameters, and Pearson’s chi-square test was applied to evaluate high or low variation according to each parameter. The cut-off points for the expression of both PARP and caspase-3 were established through the receiver operating characteristic (ROC) curve using the values of expression obtained from the patients in the present study. From the ROC curve, cut-off points were then fixed according to their best sensibility and specificity simultaneously. Data were presented as mean ± standard error (SEM). P values < 0.05 were considered statistically significant, and all analyses were performed with GraphPad Prism 5 software (GraphPad Software, Inc., San Diego, CA, USA).
Results
The immunoexpression evaluation revealed increased levels of PARP in tumor tissues of patients previously classified as worse prognosis compared with tumor expression levels of patients classified as good prognosis (P < 0.0001) (Figure 1). In accordance with these results, a strong PARP immunostaining was observed in patients with worse prognosis (Figure 2). The validation of the results was performed by analyzing the immunohistochemical cytoplasmic staining of caspase-3 in the selected patients, followed by its comparison between the prognostic groups. Patients previously classified as poor prognosis revealed lower levels of caspase-3 expression, when compared with the good prognosis group (P = 0.0007) (Figure 3). A strong caspase-3 immunolabeling was found in prostate tumors of patients with good prognosis, while it was nearly absent in PCa patients classified as poor prognosis (Figure 4). Thereafter, all selected patients were stratified according to six other parameters, to compare the simultaneous influence of PARP and caspase-3 with clinicopathological factors and risk classification. This statistical analysis confirmed a significant positive correlation between the older age group (> 70 years) and PARP expression (P = 0.0001). Based on the Brazilian Manual of Clinical Oncology (MOC) criteria, a significant correlation was also detected between the unfavorable risk group and higher levels of PARP expression (P = 0.033). No additional significant correlations were determined when comparing other parameters to immunoexpression of PARP and caspase-3 (Table 2).
Statistical analysis of poly (ADP-ribose) polymerase protein levels in patients with good and poor prognosis. * P < 0.0001. Student’s t-test.
Immunohistochemical evaluation of poly (ADP-ribose) polymerase (PARP) protein. (A) Low nuclear expression of PARP in tumors from patients classified as good prognosis. (B) High nuclear expression of PARP in tumors from patients classified as poor prognosis. (C) Absence of PARP expression in negative control (absent primary PARP antibody). (D) Positive control for PARP antibody. 40 X magnification. Scale bar = 20 μm.
Statistical analysis of caspase-3 protein level in patients with good and poor prognosis. *P < 0.0007. Student t-test.
Immunohistochemical evaluation of caspase-3 protein. (A) High cytoplasmic expression of Caspase-3 in tumors from patients classified as good prognosis. (B) Low cytoplasmic expression of Caspase-3 in tumors from patients classified as poor prognosis. (C) Absence of Caspase-3 expression in negative control (absent primary Caspase antibody). (D) Positive control for caspase-3 antibody. 40 X magnification. Scale bar = 20 μm.
Distribution of patients considering different clinicopathological parameters and average protein expression of PARP and Caspase-3*
The PARP sensitivity was 92.31% (CI = 63.97-99.81%) and specificity was 81.25% (CI = 54.35 – 95.95%), while the area under the ROC curve was 92.31% (P = 0.0001). Caspase-3 sensitivity was 69.23% (CI 38.57-90.91%) and specificity was 93.75% (CI = 69.77-99.84%), while the area of ROC curve was 84.13% (P = 0.0018). Considering the ROC curves, they were established and tested to show the diagnostic or the prognostic prediction ability of both biomarkers. The positive predictive value for high PARP expression was 80% (that is, the probability of a patient with high PARP expression presenting a poor prognostic evolution), while the negative predictive value was 92.85%. For low caspase-3 expression, the positive predictive value was 83.3% (that is, the probability of a patient with low caspase-3 expression presenting with poor prognosis), while the negative predictive value was 65.22%.
To correlate prognostic groups with protein levels, patients were divided into high or low PARP expression according to the cutoff value established on the ROC curve. Patients with expression higher than the cut-off value of M.O.D. = 172.8 a.u. (92.31% sensitivity and 81.25% specificity) were considered to be the high expression group, while patients with expression below this value were considered to be the low expression group. Similarly, patients were also divided between low and high expression groups for caspase-3, considering the cut-off value of M.O.D. = 127.6 a.u. according to the ROC curve (69.23% sensitivity and 93.75% specificity).
Two distinct prognosis groups were generated, taking into consideration the specific clinical evolution of the disease, by comparing the expression of PARP and caspase-3 proteins with better or worse prognosis. The prognostic value of these biomarkers was also examined. Out of a total of 29 men, 13 (44.82%) were classified as poor prognosis, and 12 of them (92.30%) showed higher PARP expression (values above the established cut-off). Furthermore, from the remaining 16 patients who were previously classified as good prognosis group (55.17% of total samples), 15 of them (93.75%) showed higher caspase-3 expression.
Discussion
As the main member of the PARP family, PARP-1 induces cell survival through DNA repair, and it was previously demonstrated to be overexpressed in a variety of malignant tumors. It is also associated with invasiveness and poor prognosis.9 This corroborates our results, where PARP did show a significant increase in the prostate cancer group of patients with worse prognosis when compared to the group with better prognosis.
PARP plays a key role in DNA damage repair and has been systematically studied in multiple cancer types, especially in targeted therapy. PARP inhibitors (PARPi) act in the DNA during replication, forming a complex with the PARP-1 and PARP-2 enzymes. This complex leads to unrepaired DNA and programmed cell death.18-20
Other researchers have also investigated the therapeutic applications of PARPi in prostate cancer and have observed that when given concomitantly with androgen receptor (AR) signaling inhibitors, a greater sensitivity to the therapeutic response was reached. The role of AR inhibitors was related to a lower expression of repair genes (DDR), leading to a higher rate of DNA damage, and consequently increased PARP activation, which promoted higher sensitivity to PARPi.21 The role of PARP as an important component in a deficient prostate cancer DNA repair system—also attested by other previously studies—led to the assumption of the potential for this enzyme as a prognostic marker for prostate cancer. Since some types of PCas lead to activation of signaling pathways associated with DNA damage, new agents targeting DNA repair could become prognostic and potentially predictive markers for prostate cancer.11
Apoptosis relies on different cell signaling mechanisms, and cancer cells may use distinct strategies to evade apoptosis. Consequently, this may lead to disease progression by promoting cell survival and resistance to antineoplastic drugs.22 The cysteine protease protein 32, known as caspase-3, has been studied in different modalities of PCa.12 This protein family plays a central role in the final execution of cell death, being expressed in prostate epithelial tissues under normal conditions.
Caspase-3 is the ultimate executioner enzyme responsible for the nuclear changes associated with apoptosis, including chromatin condensation.13 Caspase expression and activation represent an important cell marker of apoptosis,14 since loss of apoptotic control in association with cell proliferation is responsible for the initiation and progression of PCa.15 Altered caspase expression may represent an additional component related to the resistance to cell death.14
A recent study suggested that suppression of caspase-3 expression profoundly augments the ability of prostate cancer cells to survive, thus contributing to disease progression.23 In addition, other studies demonstrate caspase activation as a way of apoptosis induction, also observed in antitumor properties of natural compounds.24
To determine whether caspase-3 activity had prognostic value in colorectal cancer, the enzyme activity was matched with clinical parameters. A high caspase-3 activity was found in tumors significantly correlated with higher risk of recurrence, but other clinical parameters such as age, sex, tumor stage, presence of metastases, overall survival, or all-cause mortality, did not correlate with caspase-3 activity.25
Motta et al26 reported that caspase-3 and CD-34 showed high expression regarding label index and presented low expression in 34 histopathological PCa patient samples. There was no statistical significance among expressions and tumor severity according to Gleason´s score, and no significant correlation was set between the biomarkers.26
To identify the significance of caspases in prostate cancer progression, Winter et al13 examined the expression of three key caspases (caspase-1, caspase-3, and caspase-9) in normal and malignant human prostates. Immunohistochemical analysis revealed the pattern of immunostaining and distribution was homogeneous in the normal prostate, and the epithelial cells exhibited a diffuse cytoplasmic staining for caspase-3. Caspase-3 expression was also reduced in moderately and poorly differentiated prostate tumors compared with well-differentiated prostate adenocarcinomas and the normal prostate (P < 0.05). No significant correlation was observed between the apoptotic index or Gleason grade and the caspase expression. This study suggested that caspase-3 expression in prostatic tumors may have prognostic significance over disease progression. Under benign conditions, prostate samples showed homogeneous and uniform caspase-1 and caspase-3 expression levels, whereas in prostate carcinomas, these levels were significantly reduced.13
Our results revealed strong caspase-3 expression in the prostate cancer group previously classified as good prognosis compared to the poor prognosis group (P = 0.0007), as well as high probability of presenting poor prognosis when caspase-3 expression was reduced. By comparing the expression of PARP and caspase-3 in patients’ prognostic groups, a high percentage of patients with poor prognosis revealed high expression of PARP. Once patients presented high PARP expression, there was an increased probability of developing poor prognosis, thereby indicating PARP as a potential prognostic biomarker in PCa.
According to a previous study, PARP inactivation prevents substrate and ATP depletion, a necessary condition for the occurrence of apoptotic events.27 These data strengthen our results by comparing high expression of PARP and low expression of caspase-3 and their relationship to the worst clinical outcome PCa patients, leading to their potential as prognostic markers in prostate cancer. In this context, it is speculated that PARP and caspase-3 expression, when analyzed together, can provide more reliable information about tumor behavior and evolution, due to the antagonist feature of these proteins in terms of disease progression.
Conclusion
Current research in PCa treatment demands identification of reliable biomarkers, so as to guide proper therapeutic decisions in challenging clinical scenarios.28 Our results suggest that the expression of PARP and caspase-3 may be of significant prognostic value for patients with PCa and may provide predictive data on the behavior of prostate cancer and patient’s clinical outcome. Nevertheless, further studies are necessary to confirm the present findings.
Note in Proof
The authors wish to acknowledge Luiz Gustavo de Almeida Chuffa for statistical analysis of the study results. We are grateful for his work and invaluable contribution to the study.
Footnotes
Financial Support: This work was supported by FAPESP – Brazil (Research Support Foundation of the State of Sao Paulo). Process No. 2019/11381-1.
Disclosures: The authors report no conflicts of interest.
↵† Deceased
- Received July 28, 2020.
- Revision received February 27, 2021.
- Revision received July 2, 2021.
- Accepted July 20, 2021.
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