Introduction
Prostate cancer (PCa), the second most frequent cancer among men, is a heterogeneous disease and the fifth cause of death in men (D’Elia et al.
2022; Torre et al.
2016). Approximately 1,414,259 people were diagnosed with PCa in 2020, and 375,304 of them died. This number will almost double to 2.3 million cases and 740,000 deaths in 2040 worldwide (Chung et al.
2019; Culp et al.
2020; Ferlay et al.
2019; Sung et al.
2021). While prostate-specific antigen (PSA)-based screening for PCa has had a beneficial effect in reducing PCa mortality, it has also resulted in the problem of overdiagnosis, overtreatment, and a substantial number of unnecessary biopsies (Srivastava et al.
2019). More precise markers can identify individuals with aggressive forms of the disease, enabling tailored treatment approaches. Novel markers may also reveal new therapeutic targets, leading to targeted treatments that disrupt cancer cell pathways or enhance immune response (Nandana and Chung
2014). Overall, the search for new markers aims to enhance diagnostic accuracy, refine prognostic assessments, and develop more effective and personalized therapeutic strategies. Tumor markers, which are substances produced in excessive amounts by the body or cancer cells as cancer develops and progresses, offer valuable information for the diagnosis, prognosis, and prediction of different cancers (Saoud et al.
2020; Yang et al.
2023).
CTAs have emerged as a large family of tumor‐associated antigens expressed in human tumors of different histological origin, that warrant further investigation, as they have not been thoroughly explored (Kulkarni et al.
2012; Wei et al.
2020). They are a family of proteins by their importance in development and in cancer immunotherapy and typically expressed in male testicles, though they involve in many other cancer cell types (Yang et al.
2015). Based on the available information in the CT database and GeneBank, more than 200 CTAs are found which are classified into 40 groups (Chong et al.
2022; Nin and Deng
2023). Because of their tumor-restricted expression, low level of MHC class I, and low immunogenicity, CTAs are considered as ideal targets for tumor specific immunotherapeutic approaches and prompted the development of several clinical trials of CTAs-based vaccine therapy (Salmaninejad et al.
2016). Previous studies have shown its expression in various malignancies is heterogeneous and often correlates with progression of tumor, invasion, and metastasis (Gjerstorff et al.
2016; Shang et al.
2014).
TPTE is a gene belonging to members of the CTA family expressed only in the testis of healthy individuals but absent in healthy tissues (Atanackovic et al.
2006). It encodes 551 amino acids with an approximate weight of 64 kDa and possesses a minimum of two transmembrane domains, a phosphatase domain, and a domain homologous to tensin (Atanackovic et al.
2006; Dong et al.
2003). It exhibits a genetic sequence resemblance to the extensively expressed suppressor, phosphatase and tensin homolog (PTEN). Notably, TPTE distinguishes itself through an N-terminal extension featuring three transmembrane regions, leading to its alternate designation as PTEN2 (Leslie et al.
2007). The human genome contain many copies of TPTE located on acrocentric chromosomes 13, 15, 21, and 22 and the Y chromosome; and, only one copy on chromosome 21 can be expressed (Chen et al.
1999). The function of TPTE remains incompletely elucidated, yet it is presumed to contribute to signal transmission through tensin and interaction with the cytoskeleton (Singh et al.
2008). The presence of TPTE protein has been detected in diverse cancer types, such as hepatocellular carcinoma (Dong et al.
2003), lung cancer (Kuemmel et al.
2015), and ovarian cancer (Adepiti and Odunsi
2022). Earlier investigations have provided evidence that TPTE exhibits elevated expression in androgen-independent PCa cell lines, as opposed to hormone-sensitive cells. Differences in TPTE protein expression show promise as a valuable tool for diagnosing and predicting both localized and metastasized cancer (Singh et al.
2008). Previous research has highlighted the presence of TPTE on the cellular membrane (Walker et al.
2001), designating it as a viable candidate for targeted therapy in treating PCa. This study represents the inaugural investigation assessing TPTE expression patterns in a series of PCa samples, employing newly generated anti-TPTE antibodies for immunohistochemistry (IHC) analysis. Examining the association between TPTE expression and various clinicopathological features in patients with PCa, our study aimed to identify a potential protein marker for the assessment and management of the disease.
Material and methods
To generate antibodies against the TPTE protein, a sequence corresponding to the TPTE was obtained from the Uniport database (ID: P56180). The extracellular regions of the protein sequence were subjected to computational modeling using the Swiss-model program, which employs homology-based modeling techniques to generate a three-dimensional structure. The IEDB website algorithms were employed to assess both linear and non-linear epitopes linked to B-cells, aligning with the extracellular regions of the protein sequence. Peptide fragments, spanning 15–20 amino acids, were extracted, and subjected to analysis for physicochemical properties, antigenicity, and allergenicity. Ultimately, two sequences TPTE-p1 (N-IYSIPRYVRDLKIQIEMEK-C) andTPTE-p2 (N-ELDNLHKQKARRIYPSDF-C) were selected and synthesized (Pepmic CO, China) for this study.
Polyclonal Ab production
The peptide sequences of TPTE were linked to bovine serum albumin (BSA) from Sigma-Aldrich, USA, to enhance immunogenicity. To fulfill this purpose, a sulfo-SMCC (Sulfo-succinimidyl-4-(N-Maleimidomethyl) Cyclohexane-1-Carboxylate) linker from Sigma-Aldrich, USA, was utilized. In summary, sulfo-SMCC was dissolved in distilled water and combined with BSA in a pH 7.2 phosphate buffer solution. The resulting mixture underwent incubation at room temperature (RT) for one hour. Subsequently, the product obtained underwent purification through a desalting column filled with Superdex 200 Prep Grade from Sigma-Aldrich, USA. The maleimide activated protein was then combined with peptide solutions with a molar ratio of 1:30 (BSA to peptide), in a phosphate buffer solution (pH 7.2). After incubating at RT for two hours, the reaction underwent dialysis at 4°C in phosphate-buffered saline (PBS) for 12 h. Subsequently, the analysis of the conjugated products was conducted using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (Mohsenzadegan et al.
2013). The modified proteins were subsequently used to immunize animals to produce antibodies. To produce polyclonal antibodies, four adult female New Zealand white rabbits, sourced from the Pasteur Institute in Iran, were utilized. The conjugated protein was thoroughly dissolved in complete Freund’s adjuvant from Sigma-Aldrich, USA. This solution was then injected into various areas of the rabbits’ backs during the immunization stage. Booster injections, consisting of a conjugated peptide solution in Freund’s incomplete adjuvant, were administered one month after the initial injection to attain the desired antibody titer. Blood samples were collected before the first injection and 10 days after each booster injection, from which serum samples were prepared (Mohsenzadegan et al.
2015).
Immunoreactivity analysis of the rabbit sera by ELISA
The ELISA technique was employed to assess the immunoreactivity of rabbit sera. Antibody concentrations were measured through an indirect ELISA method. Initially, 96-well flat-bottom microtiter plates were loaded with TPTE-p1 and p2 peptides (1μg/well) and incubated for one hour at 37 °C to quantify antibody levels. The wells were blocked using 5% skim milk in PBS (pH 7.4) and incubated for 1 h at RT. Plates were washed three times with wash solution (PBS plus 5% Tween 20), incubated with 100 μl of immunized rabbit serum at serial dilutions (1/400, 1/800, 1/1600, 1/3200, 1/6400, and 1/12800), and then incubated for 30 min at 37 °C. Subsequently, we introduced a secondary antibody, goat anti-rabbit IgG, conjugated with HRP from Sigma-Aldrich, USA, at a 1:10,000 dilution. This was followed by a 30-min incubation at 37 °C. After each step, the plates underwent a washing process with a wash solution. Subsequently, 100 μl of the 3,3′,5,5′-tetramethylbenzidine (TMB) substrate reagent from Dako, Denmark, was added to all wells. The color development was halted by the addition of 0.2 M H
2SO
4 (100 μl). The outcomes were analyzed by ELISA reader using optical density of 450 nm (Dana Tajhiz, Iran). TPTE-p1 and p2 immobilized on NHS-Activated Agarose beads (Pierce Chemicals Co., USA) and specific anti-peptide antibodies were purified from rabbit sera using the suggested protocol (Mohsenzadegan et al.
2015).
Sample preparation
A total of 150 paraffin-embedded tissue samples, comprising both PCa and BPH as the control group, were procured from patients at Hasheminejad Urology-Nephrology Hospital in Iran, spanning the years 2016 to 2017. The sample set encompassed 102 PCa tissues and 48 BPH tissues. Notably, none of the patients underwent preoperative treatments, such as hormonal therapy or radiotherapy. The PCa samples were categorized based on the Gleason grading system. Comprehensive analysis involved the examination of medical records and slides stained with hematoxylin and eosin (H&E) to extract details on specific pathological and clinical features, including patient age, serum PSA levels, and Gleason score. Survival data for patients, including disease-specific survival (DSS), overall survival (OS), and biochemical recurrence (BCR), were meticulously documented until the end of 2022. Whereas OS is characterized as the duration from the surgical procedure to the individual's demise (Rygalski et al.
2021). DSS is delineated as the timeframe commencing with the surgical intervention and extending until either the patient's cancer-induced death or the most recent follow-up (Shoup et al.
2003). And BCR is defined as the period from the primary treatment or radical prostatectomy to the point when serum PSA levels exhibit an increase beyond 0.2 ng/ml (Matsumoto et al.
2018).
Immunohistochemical analysis
To examine TPTE expression in tissue samples, immunohistochemical staining was conducted on paraffin-embedded sections of PCa. After deparaffinization and rehydration of the tissues, endogenous peroxidase activity was quenched by immersing the tissue in methanol containing 0.3% hydrogen peroxide for 15 min. Heat-induced epitope retrieval was performed using a pressure cooker and a solution comprising 10 mM sodium citrate and 0.05% Tween 20 at pH 6.0. Following cooling at RT, the sections underwent two PBS washes. Incubation with the primary antibody (anti-TPTE-p2 Ab) occurred at serial dilutions of 1:20, 1:40, and 1:80 in a humid chamber at 4 °C, with the optimal dilution determined as 1:20. Subsequently, incubation with poly-HRP-goat anti-mouse/rabbit IgG (DPVB110HRP, BrightVision, Netherlands) was carried out at RT for 30 min. The samples were visualized using 3,3ˊ-diaminobenzidine (DAB, Dako, Denmark), counterstained with hematoxylin (Dako, Denmark), and observed under an optical microscope (Nikon, Japan) (Shafiei et al.
2019). Testis tissue served as a positive control for validating the anti-TPTE-p2 antibody, while the absence of the primary antibody in PCa tissue acted as the negative control. To validate the antibody’s specificity in immunohistochemical staining, the TPTE-p2 peptide underwent a pre-incubation of 2 h with the anti-TPTE-p2 antibody at RT before application to the sample. The assay utilized peptide concentrations ranging from 1 to 20 μg/ml, combined with the TPTE-p2 antibody at an optimal dilution of 1:20.
Scoring system
An uninformed professional pathologist assessed the immunohistochemical staining of tissue slides through a semi-quantitative scoring system. Staining intensity was categorized as negative, weak, moderate, or strong. The percentage of TPTE-positive cells received grades: 0 (no staining), 1 (< 50% positive cells), 2 (50–80% positive cells), and 3 (> 80% positive cells). The total score was determined using the histochemical score (H-score) for each case, calculated by multiplying the staining intensity by the percentage of positive cells, resulting in an ultimate score ranging from 0 to 300. The mean H-scores, namely 188 for cytoplasmic, 134.88 for membrane, and 191.67 for nuclear expression, were employed as the threshold values to categorize samples into low or high TPTE expression.
Cell culture
The MCF-7 (human breast cancer) and PC-3 (human PCa) cell lines procured from the National Cell Bank of Pasteur Institute, Iran, were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with penicillin and streptomycin (Gibco, UK) and 10% fetal bovine serum (FBS) (Gibco, UK). The culturing process took place at 37 °C in a humidified atmosphere containing 5% CO2 and 95% air.
Flow cytometry
To assess the percentage of cells expressing TPTE protein, indirect flow cytometry was performed on MCF-7 and PC-3 cells using an anti-TPTE antibody (LSBio, USA). The test involved isolating adherent cells using 0.25% trypsin–EDTA (Gibco, UK) at 37 °C for 1 min. The cells were washed with FACS buffer (3% BSA/PBS), followed by incubation with an anti-TPTE antibody at 10 µg/ml on ice for 30 min. After two additional washes with FACS buffer, a fluorescently labeled secondary antibody (PE-conjugated goat anti-rabbit IgG; Biolegend, USA) was added and incubated on ice for 30 min. Subsequently, the cells underwent analysis for TPTE expression using FlowJo software (Bagherian et al.
2022). Two control groups were implemented: one with Ig G fraction as primary antibody (isotype control), and the other with PBS as the primary antibody instead of the anti-TPTE antibody (unstained). These controls served the purpose of distinguishing between non-specific background fluorescence and authentic TPTE protein expression in the cells.
Cell viability
The cell viability was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit from Bio Idea Co., Iran. MCF-7 and PC-3 cells were seeded in 96-well plates at a density of 5 × 10
3 cells per well in 200 μl of DMEM and incubated overnight at 37 °C. Subsequently, the cells were treated with varying concentrations of the anti-TPTE-p2 antibody (2.5, 5, and 10 µg/ml) or PBS buffer (control) in 200 μl of fresh medium for 24 or 48 h. Following treatment, MTT solution (20 μl) was added to all wells, and incubation continued for an additional 4 h. The culture medium was then removed, and each well was exposed to dimethyl sulfoxide (DMSO) (100 μl) to dissolve the frozen crystals. The absorbance of the samples was measured at 570 nm with a reference filter of 630 nm. This experimental procedure was repeated three times, and cell viability was calculated using the formula: (mean OD sample/ mean OD control) × 100 (Kamiloglu et al.
2020).
Statistical analysis
Graphs were generated using Prism 8.3.0 software, and data analysis was conducted with SPSS 26. A two-way analysis of variance (ANOVA) was employed for comparing ELISA data. The correlation between clinicopathological parameters and TPTE expression underwent analysis through Pearson’s χ2 and Pearson’s R tests. Group comparisons were executed using Kruskal–Wallis and Mann–Whitney U tests. Survival analysis utilized the Kaplan–Meier method, and resulting curves for each group were compared using the log-rank test. Flow cytometry and MTT assay experiments were performed in triplicates. All data were presented as mean ± standard deviation (SD), and statistical significance was determined by a P-value < 0.05.
Discussion
CTAs hold promise as targeted molecules for personalized immunotherapy and as prognostic or diagnostic biomarkers for PCa. Among these antigens, the TPTE family are particularly attractive targets due to their robust immunogenic properties and limited expression in normal tissues. Nonetheless, the role of TPTE is still poorly understood where CTAs are emerging as potential tumor markers for various malignant tumors, showcasing the continuous exploration of novel diagnostic and therapeutic avenues. TPTE is implicated in cell signaling modulation through interactions with membrane-bound molecules, exerting its influence via de-phosphorylation processes (Atanackovic et al.
2006). This multifaceted role extends to the association between elevated TPTE expression and enhanced survival outcomes, a phenomenon observed across various cancer types. The cancer/testis antigen TPTE is aberrantly expressed in various tumors, including lung cancer, implicating its involvement in the immune responses against cancer cells (Dong et al.
2003; Bansal et al.
2015). TPTE is considered a member of the PTENs family, which shares lineage with the well-known PTEN. Situated on chromosome 10, PTEN plays a pivotal role in regulating the cell cycle and proliferation. Importantly, the common association of PTEN loss with tumor development underscores its significance in the tumorigenesis process (Gao et al. 2021).
Understanding the intricate TPTE signaling pathways involved in PCa provides potential diagnostic tumor marker to intervene in disease progression. Hence, in our investigation, we identified specific extracellular regions of TPTE that demonstrate the highest effectiveness in initiating an immune response in B cells, as determined by IEDB algorithms. Peptide fragments covering amino acid sequences 421–436 and 514–531 were pinpointed as crucial regions. These specific segments displayed robust efficacy in activating B cell immune responses. ELISA tests validated the significant immunogenicity of both TPTE-p1 and p2 by generating elevated antibody levels.
In this research, a novel discovery emerged as we identified a correlation between the cytoplasmic expression level of TPTE and crucial prognostic indicators in PCa, including the Gleason score and PSA level. This finding shed light on the potential significance of TPTE in understanding and predicting the prognosis of PCa patients. Aligned with prior investigations (Adepiti and Odunsi
2022) our study reveals a significant increase of TPTE expression in PCa samples compared to BPH samples. The pivotal discovery of our research lies in the substantial correlation we identified—higher TPTE expression is significantly associated with escalated disease severity, as evidenced by elevated Gleason scores. Furthermore, a significant association was observed between the severity of the disease and PSA level. Elevated PSA levels were consistently linked to heightened protein expression and H-score. We also found a significant positive correlation between TPTE membrane expression and the pathological index of PNI, suggesting that this protein may play a role in the progression of PCa. Adepiti et al. conducted a study employing RT-PCR to assess TPTE expression in ovarian tumors. Similar to our findings, they identified TPTE expression in a significant proportion of tumors (45.1%). However, their study did not establish significant correlations between TPTE expression and variables such as patient age, tumor stage, grade, histology, or response to therapy (Adepiti and Odunsi
2022). Nonetheless, these findings suggest that TPTE could be a potential marker for tracking the progression of PCa.
In a computational investigation, researchers explored the genes associated with clinical features and prognosis in oral squamous cell carcinoma (OSCC) using RNA-sequencing data. Through comprehensive analyses, they identified 17 Gene Ontology terms and 4 KEGG pathways using functional enrichment and protein–protein interaction network analysis. Notably, six specific genes—DCAF4L2, OPRPN, R3HDML, TPTE, ACTL8, and PCDHA11—emerged as having a substantial impact on patient survival. Remarkably, their results underscore a positive correlation between the presence of TPTE and an enhanced likelihood of increased patient survival (Wu et al.
2019). Additionally, in the Adepiti et al. study, a pattern emerged suggesting a connection between TPTE expression and slightly prolonged progression-free survival (PFS) and OS. Notably, it is crucial to emphasize that this correlation did not achieve statistical significance (Adepiti and Odunsi
2022). In our research, we observed that patients with elevated H-scores of TPTE expression exhibited diminished survival rates; however, this discrepancy lacked statistical significance. The PTEN gene holds a pivotal function in overseeing cell cycle regulation and the proliferation of cells. Importantly, the noteworthy association between the diminished expression of PTEN and the onset of tumor development underscores its substantial role in the intricate process of tumorigenesis (Gao et al.
2021). Consequently, there is a belief that TPTE, akin to PTEN, could exert a noteworthy influence on the progression of cancer. To achieve a thorough comprehension of the specific role played by the TPTE protein, additional in-depth studies are deemed necessary.
To check cell viability, in addition to prostate cell line, we also used breast cell line to study the different effect of the anti-TPTE-p2 antibody on these two cell lines. We conducted experiments exposing PC-3 and MCF-7 cells to the anti-TPTE-p2 antibody to demonstrate its efficacy against cancer. The results revealed that varied doses of the anti-TPTE-p2 antibody effectively restrained the proliferation of both PC-3 and MCF-7 cell lines. Notably, the inhibitory effect demonstrated a direct correlation with the antibody dosage, indicating more pronounced suppression at higher doses. These findings underscore the potential of the anti-TPTE-p2 antibody as a promising inhibitor for curtailing the growth of PCa and breast cancer cells, offering valuable insights for innovative treatment development. Additionally, Sahin and collaborators (
2020) conducted phase I trials testing the melanoma liposomal RNA vaccine (BNT111) against four non-mutated antigens, including TPTE, Tyrosinase, melanoma-associated antigen A3 (MAGE-A3), and New York Esophageal Squamous Cell Carcinoma 1 (NY-ESO-1). The study revealed that the combination of these antigens elicited a robust immune T-cell response, with heightened CD4 + T-cell responses against MAGE-A3, TPTE, and NY-ESO-1, and CD8 + T-cell responses against NY-ESO-1 and MAGE-A3. Moreover, the vaccine, either independently or combined with checkpoint inhibitors, successfully induced persistent immune responses in individuals diagnosed with advanced stage 3 and 4 melanoma cancer (Sahin et al.
2020). These cumulative findings underscore the significance of TPTE as a pivotal factor in cancer diagnosis, prognosis, and therapy, laying a robust groundwork for ongoing research and the advancement of groundbreaking management of PCa patients.
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