Abstract
Background
The scarcity of effective therapeutic approaches for prostate cancer (PCa) has encouraged steadily growing interest for the identification of novel antigenic targets. Placenta-specific 1 (PLAC1) is a novel cancer–testis antigen with reported ectopic expression in a variety of tumors and cancer cell lines. The purpose of the present study was to investigate for the first time the differential expression of PLAC1 in PCa tissues.
Methods
We investigated the differential expression of PLAC1 in PCa, high-grade prostatic intraepithelial neoplasia (HPIN), benign prostatic hyperplasia (BPH), and nonneoplastic/nonhyperplastic prostate tissues using microarray-based immunohistochemistry (n = 227). The correlation of PLAC1 expression with certain clinicopathological parameters and expression of prostate-specific antigen (PSA), as a prostate epithelial cell differentiation marker, were investigated.
Results
Placenta-specific 1 (PLAC1) expression was increased in a stepwise manner from BPH to PCa, which expressed highest levels of this molecule, while in a majority of normal tissues, PLAC1 expression was not detected. Moreover, PLAC1 expression was positively associated with Gleason score (p ≤ 0.001). Interestingly, there was a negative correlation between PLAC1 and PSA expression in patients with PCa and HPIN (p ≤ 0.01). Increment of PLAC1 expression increased the odds of PCa and HPIN diagnosis (OR 49.45, 95 % CI for OR 16.17–151.25).
Conclusion
Our findings on differential expression of PLAC1 in PCa plus its positive association with Gleason score and negative correlation with PSA expression highlight the potential usefulness of PLAC1 for targeted PC therapy especially for patients with advanced disease.
Electronic supplementary material
The online version of this article (doi:10.1007/s00262-014-1594-z) contains supplementary material, which is available to authorized users.
Keywords: Immunohistochemistry, Gleason, PLAC1, Prostate cancer, PSA
Introduction
Due to the existence of few effective therapeutic strategies and the associated morbidity, new therapeutic approaches such as immunotherapy for the treatment for prostate cancer (PCa) are highly desired for several reasons. First, several overexpressed and/or altered antigens exist in PC, which could be potential candidates for immunotherapy [1, 2]. Second, it takes a long time for localized PCa to metastasize to other tissues providing enough time for an immunotherapeutic approach to take effect [1, 3]. Third, biomarkers such as prostate-specific antigen (PSA) [1, 3] and prostate acid phosphatase (PAP) [3] can be used for monitoring tumor progression.
The aim of the new therapeutic approaches was to target tumor-initiating rather than more differentiated cells [4]. CD133+/α2β1 integrinhigh/CD44+ phenotype has been proposed for selecting cancer-initiating cells in human prostate tumors [5]. Similarly, in prostate xenografts and cell lines, tumorigenic cells with coexpression of CD44/α2β1 integrin or CD44 expression alone have been isolated [6, 7]. In this regard, the value of markers such as CD133 in partitioning stem cell-like cells from PCa cell lines has been questioned by recent investigation [8]. Collectively, the prognostic value of these markers in prostate tumors is still a matter of debate. Moreover, many normal cell types also express substantial levels of these markers reflecting the ambiguity regarding their usefulness as an immunotherapy target. Several studies have demonstrated a positive correlation between the PSA protein expression and overall degree of differentiation in PCa [9–11]. Interestingly, it has been recently shown that PCa cells that do not express or express low levels of PSA (PSA−/lo), which have high clonogenic potential, are refractory to androgen deprivation and reveal a long-term tumor-propagating capacity. In contrast, PSA+ PCa cells possess more limited tumor-propagating capacity [12].
Although, based on the aforementioned features, the PSA−/lo cell population could be regarded as an appropriate target of immunotherapy, its targeting needs a specific marker, which is overexpressed instead of being downregulated. Some critical factors for the selection of a candidate biomarker suitable for cancer immunotherapy include lack of expression in normal tissues, a tumor-promoting function, and expression in most cancerous tissues of the same type [13]. Placenta-specific 1 (PLAC1) is a novel X-linked gene [14] and a new member of the cancer–testis antigens [15, 16] with 212 amino acids [14]. PLAC1 expression is restricted to placenta [15, 17, 18]. Much lower levels of this molecule are detected in testis [15, 16], while other normal human tissues have no detectable expression of PLAC1 [16]. Predicted topology shows PLAC1 is a transmembrane protein, suggesting that it is localized to a membranous compartment [15, 19]. The physiological role of PLAC1 remains to be elucidated. In a murine model, it has been shown that PLAC1 is essential for normal placental development [20]. Recently, ectopic expression of PLAC1 transcript, and in few instances PLAC1 protein, has been reported in a wide variety of tumor types such as lung [19], gastric [21], colorectal, hepatocellular [22], breast [19], and epithelial ovarian cancers [23] as well as in numerous cancer cell lines [16, 19, 22]. It has been shown that knocking down PLAC1 by siRNA in MCF-7 and BT-549 breast cancer cell lines induces G1-S cell cycle arrest with nearly complete abrogation of cell proliferation and impaired motility, migration, and invasion [19].
The purpose of the present study was to investigate for the first time the differential expression of PLAC1 in a series of prostate tissues including PCa, high-grade prostatic intraepithelial neoplasia (HPIN), benign prostatic hyperplasia (BPH), and nonneoplastic/nonhyperplastic prostate tissue, to correlate PLAC1 expression with clinicopathological parameters in PCa, and to examine a potential link between PLAC1 and PSA expression.
Materials and methods
Production of anti-PLAC1-specific polyclonal antibody
A New Zealand White Rabbit was immunized against KLH-conjugated PLAC1-specific epitope corresponding to the published amino acid sequence (CVFSEEEHTQVP) [24]. Specific polyclonal antibodies were purified using peptide-coupled affinity column. The purity and reactivity of purified antibodies were assessed by SDS–PAGE and ELISA, respectively.
Patients and tissue samples
Paraffin-embedded prostate tissue blocks were collected from Hasheminejad Kidney Center hospital, a major university-based referral urology hospital in Tehran, Iran. All patients were diagnosed between 2006 and 2011 with either prostate adenocarcinoma (PCa), high-grade prostatic intraepithelial neoplasia (HPIN), or benign prostate hyperplasia (BPH). Prostate tissues were collected for pathological examination after radical prostatectomy or performing transrectal ultrasound (TRUS)-guided needle biopsy. The patients had no history of preoperation systemic treatment. Pathologic parameters were extracted from pathology report such as Gleason grade, perineurial and vascular invasion, and adjacent tissue involvement including seminal vesicles and bladder neck, lymph node involvement, and pathologic tumor stage. Medical records of all patients were also reviewed to obtain patients’ age and serum PSA level. Gleason scoring was performed based on the guidelines released in 2005 by the International Society of Urological Pathology [25]. Tumor stage was determined based on the AJCC/UICC TNM staging system [26]. As the negative control group, nonneoplastic/nonhyperplastic prostate tissues adjacent to the tumor site were collected and processed similarly. Specimens from patients with benign prostate hyperplasia (BPH) were collected upon simple prostatectomy. Patient demographic data were collected from medical reports. A total of 273 prostate samples were collected at the initial step, and after excluding 46 samples due to technical problems in tissue processing, 227 samples comprising 154 PCa (103 radical prostatectomy and 51 needle biopsy), 23 HPIN, 27 BPH, and 23 nonneoplastic/nonhyperplastic prostate tissues were studied. This research was approved by the ethical committees of Avicenna Research Institute and Iran University of Medical Sciences, and all patients provided written consent. Patient data are fully anonymous.
Tissue microarray (TMA) preparation
Hematoxylin and Eosin (H&E)-stained slides for each case were reviewed by a pathologist to find the best area of benign and cancerous tissue and also foci of high-grade PIN for preparing TMA. Tissue microarray blocks were constructed as published previously [27, 28]. In brief, three cores of 0.6 mm diameter were punched from the cancerous regions of the donor blocks and precisely arrayed into a new recipient paraffin block using Tissue Arrayer Minicore (ALPHELYS, Plaisir, France). Each TMA had nonneoplastic/nonhyperplastic tissue specimens for comparing the IHC signal intensity. Tissue microarray blocks were constructed in three copies for each specimen, and the mean scoring of three cores was then calculated as the final score. Needle biopsies were not included in TMA slides, but stained simultaneously with TMA sections.
Immunohistochemistry (IHC)
Immunohistochemistry was performed on 3 micron paraffin sections of TMA as we published elsewhere with some modifications [29]. Human term placenta tissue and normal human endometrium served as positive and negative control tissues for PLAC1, respectively. Briefly, deparaffinized sections were subjected to heat-activated antigen retrieval in citrate buffer (10 mM, pH 6) at 95 °C for 30 min in water bath.
Endogenous peroxidase activity and nonspecific binding sites were blocked by 1 % H2O2 and 5 % normal sheep serum diluted in protein block (Dako, CA, USA), respectively.
Anti-PLAC1 (10 µg/ml) or Rabbit anti-PSA (Dako, CA, USA) antibody was added to the slides followed by Envision secondary antibody (Dako, Denmark) (for PLAC1) or peroxidase-conjugated sheep anti-rabbit Ig (for PSA)(Avicenna Research Institute, Tehran, Iran). Signals were visualized by 3,3′-Diaminobenzidine (DAB) (Roche, USA). In negative reagent control slides, primary antibodies were blocked with saturating concentrations of immunizing peptide or protein (1:100 molar ratio) prior to being applied. Slides were counterstained with Harris hematoxylin, dehydrated, and mounted. Digital images were captured by a BX51 microscope and a DP70 CCD camera (Olympus, Japan).
Immunostaining evaluation and scoring
The immunostained tissue arrays were observed on a multi-headed microscope simultaneously by three expert observers in a blind manner without having previous knowledge of pathological diagnosis. Each sample was scored independently by observers, and a consensus was achieved based on the similarity of the scores. All IHC signals were scored according to the semiquantitative scoring system [30]. Scoring was classified as 0 (no expression), 1+ (weak), 2+ (moderate), and 3+ (strong). After 2 months, re-scoring was blindly repeated to confirm the reproducibility of our initial scoring system.
Statistical analysis
Regarding PLAC1 and PSA expression score categories, Kruskal–Wallis and Pearson’s χ 2 tests were used to compare continuous and categorical variables, respectively. In the case of unbalanced values in table cells, exact tests were replaced. Spearman’s correlation test analysis was performed to find the correlation between categorical variables. To investigate whether or not Gleason grades 3 + 4 and 4 + 3 show different patterns of PLAC1 or PSA expression, χ 2 test was used. Binary logistic regression analysis was performed to estimate the odds ratio (OR) and 95 % confidence intervals (CI) of PCa and HPIN versus BPH and nonneoplastic/nonhyperplastic diagnosis, using PLAC1 and PSA expressions as predictors. To exclude the effect of empty cells in regression analysis, PLAC1 and PSA expressions were also converted to dichotomous variables “no to weak expression” vs. “moderate to strong expression”. p < 0.05 was considered to be statistically significant.
Results
Study population
Overall mean age of the study population was 66.83 years (ranged 39–90). Mean age of different groups was as follows: BPH 69.96 (SD 9.04), HPIN 65.70 (SD 7.08), needle biopsy adenocarcinoma 69.43 (SD 9.08), and radical prostatectomy adenocarcinoma 65.58 (SD 7.83). PSA levels (range 0.6–89, mean 17.19 ng/ml) were grouped as <4, 4–10, and >10 ng/ml [31]. Of 99 cases for whom the PSA data were available, 7 (7.0 %) had a PSA of <4 ng/ml, 46 (46.5 %) had PSA of 4–10, and 46 (46.5 %) had PSA >10 ng/ml. The Gleason score of patients was categorized as 6–7 and ≥8. Of 154 PCa patients, 96 (62.3 %) had Gleason score of 6–7 and 58 (37.7 %) had Gleason score of ≥8. Clinicopathological data of adenocarcinoma patients are summarized in Table 1.
Table 1.
Clinicopathological prognostic factors in prostate adenocarcinoma patients (n = 154)
| Clinicopathological parameters† | Value [n (%)] | Clinicopathological parameters† | Value [n (%)] |
|---|---|---|---|
| PSA (n = 89) | Laterality (n = 95) ‡ | ||
| <4 ng/mL | 3 (3.4) | Unilateral | 19 (20.0) |
| 4–10 ng/mL | 41 (46.1) | Bilateral | 76 (80.0) |
| >10 ng/mL | 45 (50.6) | ||
| Gleason score (n = 154) | Involvement of margins (n = 88) ‡ | ||
| 6–7 | 96 (62.3) | Not identified | 54 (61.4) |
| ≥8 | 58 (37.7) | Present | 34 (38.6) |
| Extraprostatic extension (n = 143) | Involvement of bladder neck (n = 53) ‡ | ||
| Not identified | 95 (66.4) | Not identified | 46 (86.8) |
| present | 48 (33.6) | Present | 7 (13.2) |
| Perineural invasion (n = 143) | |||
| Not identified | 11 (7.7) | ||
| Present | 132 (92.3) | ||
| Involvement of regional lymph nodes (n = 92) ‡ | |||
| Free | 87 (94.6) | ||
| Tumor stage (n = 97) ‡ | Involved | 5 (5.4) | |
| T2 | 2 (2.1) | Involvement of seminal vesicle (n = 93) ‡ | |
| T2a | 6 (6.2) | Free | 73 (78.5) |
| T2b | 10 (10.3) | Involved | 20 (21.5) |
| T2c | 42 (43.3) | Vascular invasion (n = 79) ‡ | |
| T3a | 15 (15.5) | Not identified | 75 (94.9) |
| T3b | 22 (22.6) | Present | 4 (5.1) |
PSA prostate-specific antigen
† Number of cases with the available data for each parameter is presented in parenthesis
‡ These variables were present only for radical prostatectomy PCa patients
Validation of anti-PLAC1 antibody
To determine the specificity of anti-PLAC1 antibody, IHC was carried out on human term placenta and normal human endometrium as negative tissue control. The results clearly showed the specific pattern of immunoreactivity on human term placenta as shown in Supplementary Figure 1. The antibody recognized PLAC1 in differentiated trophoblasts, and localization was restricted mostly to the cytoplasmic compartment and to some extent to microvillus plasma membrane of syncytiotrophoblasts. Moreover, very few cytotrophoblasts exhibited specific immunoreactivity. Endometrium, the tissue adjacent to placenta, was assessed for its reactivity with anti-PLAC1 and shown to be always negative (Supplementary Figure 1). No signal was detected in negative reagent control sections in which primary antibody was pre-adsorbed or substituted by pre-immune-purified rabbit IgG.
Expression of PLAC1 in PCa, HPIN, BPH, and nonneoplastic/nonhyperplastic prostate tissues
Figure 1 depicts PLAC1 expression in nonneoplastic/nonhyperplastic, BPH, HPIN, and PCa tissues. PLAC1 was expressed with varying intensities in nearly all examined tissues except for the nonneoplastic/nonhyperplastic ones. PLAC1 was localized to both cytoplasmic compartment and plasma membrane. In most cases, PLAC1 expression was localized predominantly to the apical surface of epithelial cells. Immunoreactivity was restricted to the epithelial cells of prostate tissues, and no expression was observed in nonepithelial cells including stromal and endothelial cells. Interestingly, expression of PLAC1 was increased in a stepwise manner from the nonneoplastic/nonhyperplastic group with the lowest PLAC1 expression to HPIN and PCa groups with the highest expression levels of this molecule (Fig. 2) (χ 2 tests, p < 0.001). The results of Spearman’s correlation test also showed a significant positive correlation between PLAC1 expression and diagnosis groups (r = 0.650, p < 0.001). Notably, none of the PCa and HPIN samples showed “no expression” of PLAC1, while 69.6 % of HPIN and 86.4 % of PCa samples expressed moderate or high levels of PLAC1. Moreover, no moderate or strong staining was observed in the nonneoplastic/nonhyperplastic group. On average, 65 % of nonneoplastic/nonhyperplastic samples failed to express PLAC1 and 34.8 % of them showed weak immunoreactivity. In BPH group, all staining patterns were observed except strong staining with 22.2, 63.0, and 14.8 % of samples showing no expression (0), weak (1 +), and moderate (2 +) expression of PLAC1, respectively (Fig. 2).
Fig. 1.
PLAC1 and PSA expression pattern in nonneoplastic/nonhyperplastic prostate, BPH, HPIN, and PCa. A series of 227 samples including PCa (n = 154), HPIN (n = 23), BPH (n = 27), and nonneoplastic/nonhyperplastic prostate tissues (n = 23) were collected and assessed for the expression of PLAC1 and PSA by immunohistochemistry using specific antibodies. In each panel (PLAC1 and PSA), photographs of low- and high-power fields are represented. In PCa, two series of photographs representing PLAC1 and PSA staining of samples with Gleason scores of 6 (m-p) and 9 (q-t) are shown
Fig. 2.
Distribution of PLAC1 expression score in nonneoplastic/nonhyperplastic, BPH, HPIN, and PCa groups. n = 227, χ 2 test: p < 0.001
Placenta-specific 1 (PLAC1) expression in relation to clinicopathological features of prostate cancer
The association between clinicopathological prognostic parameters and PLAC1 expression was also assessed in prostate adenocarcinoma patients (Table 2). There is a strong positive correlation between Gleason scores and PLAC1 expression in PCa patients (χ 2 test: p < 0.001) (Fig. 3a). No significant correlation was observed between PLAC1 expression and other clinicopathological parameters. There was no significant difference in PLAC1 expression between Gleason scores 3 + 4 and 4 + 3, but PCa tissues with 4 + 3 Gleason score showed significantly lower percent of moderate and high PSA expression compared to those with 3 + 4 Gleason score (p < 0.05).
Table 2.
Association of clinicopathological prognostic factors with PLAC1 expression score in prostate adenocarcinoma patients (n = 154)
| Clinicopathological parameters | PLAC1 expression‡ | p value | ||
|---|---|---|---|---|
| Weak (n = 21) | Moderate (n = 69) | Strong (n = 64) | ||
| Age† (n = 154) | 63.05 (9.29) | 67.55 (7.76) | 67.36 (8.65) | 0.251 |
| Serum PSA (n = 89) | ||||
| <4 ng/mL | 0 (0.0 %) | 3 (7.0 %) | 0 (0.0 %) | 0.394 |
| 4–10 ng/mL | 7 (46.7 %) | 21 (48.8 %) | 13 (41.9 %) | |
| >10 ng/mL | 8 (53.3 %) | 19 (42.2 %) | 18 (40.0 %) | |
| Gleason score (n = 154) | ||||
| 6–7 | 20 (91 %) | 49 (72.1 %) | 27 (42.2 %) | <0.001* |
| ≥8 | 2 (9 %) | 19 (27.9 %) | 37 (57.8 %) | |
| Extraprostatic extension (n = 143) | ||||
| Not identified | 11 (52.4 %) | 44 (69.8 %) | 40 (67.8 %) | 0.340 |
| Present | 10 (47.6 %) | 19 (30.2 %) | 19 (32.2 %) | |
| Perineural invasion (n = 143) | ||||
| Not identified | 0 (0.0 %) | 5 (7.8 %) | 6 (10.2 %) | 0.364 |
| Present | 20 (100 %) | 59 (92.2 %) | 53 (89.8 %) | |
| Laterality (n = 95)§ | ||||
| Unilateral | 4 (20.0 %) | 9 (18.8 %) | 6 (22.2 %) | 0.937 |
| Bilateral | 16 (80.0 %) | 39 (81.3 %) | 21 (77.8 %) | |
| Tumor stage (n = 97) ‡ | ||||
| T2 | 0 (0.0 %) | 2 (4.1 %) | 0 (0.0 %) | |
| T2a | 0 (0.0 %) | 3 (6.1 %) | 3 (10.7 %) | |
| T2b | 2 (10 %) | 5 (10.2 %) | 3 (10.7 %) | |
| T2c | 6 (30 %) | 22 (44.9 %) | 14 (50 %) | 0.423 |
| T3a | 6 (30 %) | 5 (10.2 %) | 4 (14.3 %) | |
| T3b | 6 (30 %) | 12 (24.5 %) | 4 (14.3 %) | |
| Margins involvement (n = 88)§ | 7 (36.8 %) | 18 (40.0 %) | 9 (37.5 %) | 0.964 |
| Bladder neck involvement (n = 53)§ | 4 (28.6 %) | 1 (4.2 %) | 2 (13.3 %) | 0.089 |
| Lymph node metastasis (n = 92)§ | 1 (5.0 %) | 4 (8.5 %) | 0 (0.0 %) | 0.355 |
| Seminal vesicle involvement (n = 93)§ | 6 (30.0 %) | 10 (20.8 %) | 4 (16.0 %) | 0.518 |
| Vascular invasion (n = 79)§ | 2 (12.5 %) | 2 (5.0 %) | 0 (0.0 %) | 0.240 |
* Statistically significant
† Represented values show mean (standard deviation)
‡ Categorical variable values are presented as number (%)
§ These variables were present only for radical prostatectomy specimens
Fig. 3.
Association of Gleason score with PLAC1 and PSA expression in PCa patients. Gleason score and PLAC1 a; n = 152, Gleason score and PSA b; n = 147, χ 2 test: p < 0.001 for both analyses
PSA expression and its correlation with clinicopathologic parameters and PLAC1 expression
The expression of PSA in serial sections of all tissues was determined and scored. Results showed that the majority of nonneoplastic/nonhyperplastic (76.2 %) and BPH (92.6 %) tissues exhibited intense staining (3+) for PSA. In HPIN group, the percentage of specimens with moderate and weak staining was increased, and this pattern of reactivity was further fortified in the cancer group in which most specimens (64.5 %) showed weak or moderate PSA expression.
Interestingly, in patients with higher Gleason scores, lower PSA expression was more frequent (p < 0.001) (Fig. 3b). Evaluation of the association between PSA expression and clinicopathological prognostic parameters showed that there was a significant negative association between PSA expression and extra-prostatic extension (p < 0.01). The correlation of PSA expression with PLAC1 in PCa and HPIN groups was also analyzed. As shown in Fig. 4, PSA expression showed a significant negative correlation with PLAC1 expression (p < 0.001). By performing binary logistic regression, we found a significant predictive value of PLAC1 and PSA expression in patients’ diagnosis (PCa and HPIN vs. nonneoplastic/nonhyperplastic and BPH). The analysis showed that increment of PLAC1 expression increased the odds of PCa and HPIN diagnosis (OR 49.45, 95 % CI for OR 16.17–151.25), whereas increment of PSA expression decreased the mentioned odds (OR 0.08, 95 % CI for OR 0.01–0.70).
Fig. 4.
Association of PLAC1 and PSA expression in HPIN and PCa groups. n = 171, Spearman correlation test; r = −0.218, p < 0.01
Discussion
One major drawback of PCa immunotherapy is the paucity of specific markers that could be potentially used for targeting. More importantly, most of the proposed markers for PC immunotherapy including PSA have not been shown so far to have a known role in cancer development and progression. Indeed, as we showed here, there was an inverse correlation between PSA expression and tumor aggressiveness and prognosis as judged by Gleason scoring. The elegant work done by Qin et al. [12] clearly demonstrated that PSA−/lo rather than PSA+ prostate cells are highly clonogenic and possess long-term tumor-propagating capacity. This implies that novel immunotherapeutic strategies for PCa should focus on PSA−/lo cells as a more suitable target. These observations raise the fundamental question of how PSA−/lo PCa cells could be targeted?
In this study, we investigated for the first time the differential expression profile of PLAC1 and PSA expression in a wide variety of prostate tissues with different diagnosis including nonneoplastic/nonhyperplastic, BPH, HPIN, and PCa.
Immunohistochemistry (IHC) results displayed a discriminating profile of PLAC1 expression in carcinoma tissues relative to the nonneoplastic/nonhyperplastic controls (p < 0.001). PLAC1 was mostly absent or weakly expressed in nonneoplastic/nonhyperplastic tissues, while its expression level was increased in a stepwise manner in BPH, HPIN, and PCa, suggesting PLAC1 expression as an early event in prostate carcinogenesis. These findings strongly suggest PLAC1 as a potential target for PCa immunotherapy. Expression of PLAC1 in healthy individuals was reported to be restricted to placenta and testis with no detectable expression in other normal tissues [15–18]. Thus, one may not expect nonneoplastic/nonhyperplastic prostate tissues to express PLAC1; nonetheless, in our study, about 35 % of nonneoplastic/nonhyperplastic tissues expressed marginal levels (1+) of this antigen. We obtained nonneoplastic/nonhyperplastic tissues from areas adjacent to cancerous tissues; therefore, it is possible that these areas would be affected by molecular events responsible for carcinogenesis.
Placenta-specific 1 (PLAC1) may play a role in cancer cell proliferation [19]. Our results on relatively increased level of PLAC1 expression in BPH specimens compared to nonneoplastic/nonhyperplastic tissues are in line with this assumption. Nonetheless, the exact function of this cancer–testis antigen in prostate tumor biology remains to be elucidated. Expression of PLAC1 transcript in PCa cell line has also been reported recently [16]. Nonetheless, no functional activity of PLAC1 in these cells has been surveyed.
In this study, we did not find statistical associations between PLAC1 expression and most of the examined clinicopathological parameters except Gleason score for which we observed a positive correlation. Prostate cancer is a heterogeneous mixture of differentiated and undifferentiated tumor cells with different proliferation potentials. Whether PLAC1 expression is a function of the differentiation state of PCa cells is not clear; however, our finding on the positive correlation between PLAC1 expression and Gleason score suggests PLAC1 as a marker of poorly differentiated PC cells. In line with this assumption, we found that tumors with higher expression of PLAC1 showed lower PSA expression. According to a recent report, PSA expression positively correlates with the degree of differentiation, and differentiated cells expressed high levels of PSA, while PSA−/lo cells exhibited high clonogenic and long-term tumor-propagating capacity [12].
Interestingly, lower tissue PSA transcript in PCa correlates with worse clinical outcomes, including high tumor grade, lymph node positivity, metastasis, recurrence, and reduced patient survival [12]. Given that high-grade PCas are usually unresponsive to conventional therapeutic regimens, PLAC1 may be considered as a proper candidate molecule to be targeted in such cases. Considering the inverse correlation between PSA and PLAC1 expression, this molecule may also be regarded as a potential therapeutic target in metastatic PCas. Importantly, recent work by Chen et al. [32] showed that Tp53 mutations in cancer cells trigger the derepression of a PLAC1 promoter. Interestingly, p53 mutation is reported in advanced stages of PCa, as well as in the recurrent and metastatic disease [33]. Our finding on the positive correlation between PLAC1 expression and Gleason score support these data.
Conclusion
Here, we showed for the first time the differential expression of PLAC1 in PCas and nonneoplastic/nonhyperplastic prostate tissues. The positive association between PLAC1 expression and Gleason score may highlight the potential usefulness of PLAC1 in targeted PC therapy especially for patients with advanced disease. Whether other epithelial solid tumors take advantage of PLAC1 expression is our ongoing research plan.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Acknowledgments
This study was funded by grants from Iran University of Medical Sciences (Grant No: 92-02-13-22395), and Tehran University of Medical Sciences (Grant No: 90-03-13-13530). The authors would like to thank Dr M. Bozorgmehr for carefully proofreading the manuscript, Mr. E. Mirzadegan for technical assistance and preparation of figures, and Mrs. J. Taeb for arranging clinicopathological data.
Conflict of interest
All authors declare that there is no conflict of interest.
Abbreviations
- BPH
Benign prostatic hyperplasia
- CI
Confidence interval
- DAB
Diaminobenzidine
- HPIN
High-grade prostatic intraepithelial neoplasia
- IHC
Immunohistochemistry
- OR
Odds ratio
- PAP
Prostate acid phosphatase
- PCa
Prostate cancer
- PLAC1
Placenta-specific 1
- PSA
Prostate-specific antigen
- TMA
Tissue microarray
- TRUS
Transrectal ultrasound
Contributor Information
Mohammad-Hossein Ghahremani, Phone: +98 2188991118, Email: mhghahremani@tums.ac.ir.
Amir-Hassan Zarnani, Phone: +98 2122432020, Email: zarnani@avicenna.ac.ir, Email: zarnania@gmail.com.
References
- 1.Slovin SR. Toward maximizing immunotherapy in metastatic castration-resistant prostate cancer–rationale for combinatorial approaches using chemotherapy. Front Oncol. 2012;2:43. doi: 10.3389/fonc.2012.00043. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Agarwal N, Sonpavde G, Sternberg CN. Novel molecular targets for the therapy of castration-resistant prostate cancer. Eur Urol. 2012;61:950–960. doi: 10.1016/j.eururo.2011.12.028. [DOI] [PubMed] [Google Scholar]
- 3.Di Lorenzo G, Buonerba C, Kantoff PW. Immunotherapy for the treatment of prostate cancer. Nat Rev Clin Oncol. 2011;8:551–561. doi: 10.1038/nrclinonc.2011.72. [DOI] [PubMed] [Google Scholar]
- 4.Lang SH, Frame FM, Collins AT. Prostate cancer stem cells. J Pathol. 2009;217:299–306. doi: 10.1002/path.2478. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Collins AT, Berry PA, Hyde C, Stower MJ, Maitland NJ. Prospective identification of tumorigenic prostate cancer stem cells. Cancer Res. 2005;65:10946–10951. doi: 10.1158/0008-5472.CAN-05-2018. [DOI] [PubMed] [Google Scholar]
- 6.Patrawala L, Calhoun T, Schneider-Broussard R, Li H, Bhatia B, Tang S, Reilly JG, Chandra D, Zhou J, Claypool K, Coghlan L, Tang DG. Highly purified CD44 + prostate cancer cells from xenograft human tumors are enriched in tumorigenic and metastatic progenitor cells. Oncogene. 2006;25:1696–1708. doi: 10.1038/sj.onc.1209327. [DOI] [PubMed] [Google Scholar]
- 7.Patrawala L, Calhoun-Davis T, Schneider-Broussard R, Tang DG. Hierarchical organization of prostate cancer cells in xenograft tumors: the CD44 + alpha2beta1 + cell population is enriched in tumor-initiating cells. Cancer Res. 2007;67:6796–6805. doi: 10.1158/0008-5472.CAN-07-0490. [DOI] [PubMed] [Google Scholar]
- 8.Pfeiffer MJ, Schalken JA. Stem cell characteristics in prostate cancer cell lines. Eur Urol. 2010;57:246–254. doi: 10.1016/j.eururo.2009.01.015. [DOI] [PubMed] [Google Scholar]
- 9.Abrahamsson PA, Lilja H, Falkmer S, Wadstrom LB. Immunohistochemical distribution of the three predominant secretory proteins in the parenchyma of hyperplastic and neoplastic prostate glands. Prostate. 1988;12:39–46. doi: 10.1002/pros.2990120106. [DOI] [PubMed] [Google Scholar]
- 10.Feiner HD, Gonzalez R. Carcinoma of the prostate with atypical immunohistological features: clinical and histologic correlates. Am J Surg Pathol. 1986;10:765–770. doi: 10.1097/00000478-198611000-00003. [DOI] [PubMed] [Google Scholar]
- 11.Gallee MP, Visser-de Jong E, van der Korput JA, van der Kwast TH, ten Kate FJ, Schroeder FH, Trapman J. Variation of prostate-specific antigen expression in different tumour growth patterns present in prostatectomy specimens. Urol Res. 1990;18:181–187. doi: 10.1007/BF00295844. [DOI] [PubMed] [Google Scholar]
- 12.Qin J, Liu X, Laffin B, Chen X, Choy G, Jeter CR, Calhoun-Davis T, Li H, Palapattu GS, Pang S, Lin K, Huang J, Ivanov I, Li W, Suraneni MV, Tang DG. The PSA(-/lo) prostate cancer cell population harbors self-renewing long-term tumor-propagating cells that resist castration. Cell Stem Cell. 2012;10:556–569. doi: 10.1016/j.stem.2012.03.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Madu CO, Lu Y. Novel diagnostic biomarkers for prostate cancer. J Cancer. 2010;1:150–177. doi: 10.7150/jca.1.150. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Cocchia M, Huber R, Pantano S, Chen EY, Ma P, Forabosco A, Ko MS, Schlessinger D. PLAC1, an Xq26 gene with placenta-specific expression. Genomics. 2000;68:305–312. doi: 10.1006/geno.2000.6302. [DOI] [PubMed] [Google Scholar]
- 15.Fant M, Farina A, Nagaraja R, Schlessinger D. PLAC1 (Placenta-specific 1): a novel, X-linked gene with roles in reproductive and cancer biology. Prenat Diagn. 2010;30:497–502. doi: 10.1002/pd.2506. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Silva WA, Jr, Gnjatic S, Ritter E, Chua R, Cohen T, Hsu M, Jungbluth AA, Altorki NK, Chen YT, Old LJ, Simpson AJ, Caballero OL. PLAC1, a trophoblast-specific cell surface protein, is expressed in a range of human tumors and elicits spontaneous antibody responses. Cancer Immun. 2007;7:18. [PMC free article] [PubMed] [Google Scholar]
- 17.Fant M, Weisoly DL, Cocchia M, Huber R, Khan S, Lunt T, Schlessinger D. PLAC1, a trophoblast-specific gene, is expressed throughout pregnancy in the human placenta and modulated by keratinocyte growth factor. Mol Reprod Dev. 2002;63:430–436. doi: 10.1002/mrd.10200. [DOI] [PubMed] [Google Scholar]
- 18.Massabbal E, Parveen S, Weisoly DL, Nelson DM, Smith SD, Fant M. PLAC1 expression increases during trophoblast differentiation: evidence for regulatory interactions with the fibroblast growth factor-7 (FGF-7) axis. Mol Reprod Dev. 2005;71:299–304. doi: 10.1002/mrd.20272. [DOI] [PubMed] [Google Scholar]
- 19.Koslowski M, Sahin U, Mitnacht-Kraus R, Seitz G, Huber C, Tureci O. A placenta-specific gene ectopically activated in many human cancers is essentially involved in malignant cell processes. Cancer Res. 2007;67:9528–9534. doi: 10.1158/0008-5472.CAN-07-1350. [DOI] [PubMed] [Google Scholar]
- 20.Jackman SM, Kong X, Fant ME. Plac1 (placenta-specific 1) is essential for normal placental and embryonic development. Mol Reprod Dev. 2012;79:564–572. doi: 10.1002/mrd.22062. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Chen J, Pang XW, Liu FF, Dong XY, Wang HC, Wang S, Zhang Y, Chen WF. PLAC1/CP1 gene expression and autologous humoral immunity in gastric cancer patients. Beijing Da Xue Xue Bao. 2006;38:124–127. [PubMed] [Google Scholar]
- 22.Dong XY, Peng JR, Ye YJ, Chen HS, Zhang LJ, Pang XW, Li Y, Zhang Y, Wang S, Fant ME, Yin YH, Chen WF. Plac1 is a tumor-specific antigen capable of eliciting spontaneous antibody responses in human cancer patients. Int J Cancer. 2008;122:2038–2043. doi: 10.1002/ijc.23341. [DOI] [PubMed] [Google Scholar]
- 23.Tchabo NE, Mhawech-Fauceglia P, Caballero OL, Villella J, Beck AF, Miliotto AJ, Liao J, Andrews C, Lele S, Old LJ, Odunsi K. Expression and serum immunoreactivity of developmentally restricted differentiation antigens in epithelial ovarian cancer. Cancer Immun. 2009;9:6. [PMC free article] [PubMed] [Google Scholar]
- 24.Ghods R, Ghahremani MH, Darzi M, Mahmoudi AR, Yeganeh O, Bayat AA, Pasalar P, Jeddi-Tehrani M, Zarnani AH. Immunohistochemical characterization of novel murine monoclonal antibodies against human placenta-specific 1. Biotechnol Appl Biochem. 2014;61:363–369. doi: 10.1002/bab.1177. [DOI] [PubMed] [Google Scholar]
- 25.Epstein JI, Allsbrook WC, Amin MB, Egevad LL. The 2005 International Society of Urological Pathology (ISUP) consensus conference on Gleason grading of prostatic carcinoma. Am J Surg Pathol. 2005;29:1228–1242. doi: 10.1097/01.pas.0000173646.99337.b1. [DOI] [PubMed] [Google Scholar]
- 26.Cheng L, Montironi R, Bostwick DG, Lopez-Beltran A, Berney DM. Staging of prostate cancer. Histopathology. 2012;60:87–117. doi: 10.1111/j.1365-2559.2011.04025.x. [DOI] [PubMed] [Google Scholar]
- 27.Kononen J, Bubendorf L, Kallioniemi A, Barlund M, Schraml P, Leighton S, Torhorst J, Mihatsch MJ, Sauter G, Kallioniemi OP. Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat Med. 1998;4:844–847. doi: 10.1038/nm0798-844. [DOI] [PubMed] [Google Scholar]
- 28.Mohsenzadegan M, Madjd Z, Asgari M, Abolhasani M, Shekarabi M, Taeb J, Shariftabrizi A. Reduced expression of NGEP is associated with high-grade prostate cancers: a tissue microarray analysis. Cancer Immunol Immunother. 2013;62:1609–1618. doi: 10.1007/s00262-013-1463-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Mahmoudi AR, Zarnani AH, Jeddi-Tehrani M, Katouzian L, Tavakoli M, Soltanghoraei H, Mirzadegan E. Distribution of vitamin D receptor and 1alpha-hydroxylase in male mouse reproductive tract. Reprod Sci. 2013;20:426–436. doi: 10.1177/1933719112459235. [DOI] [PubMed] [Google Scholar]
- 30.Zarnani AH, Shahbazi M, Salek-Moghaddam A, Zareie M, Tavakoli M, Ghasemi J, Rezania S, Moravej A, Torkabadi E, Rabbani H, Jeddi-Tehrani M. Vitamin D3 receptor is expressed in the endometrium of cycling mice throughout the estrous cycle. Fertil Steril. 2010;93:2738–2743. doi: 10.1016/j.fertnstert.2009.09.045. [DOI] [PubMed] [Google Scholar]
- 31.Barry MJ. Clinical practice. Prostate-specific-antigen testing for early diagnosis of prostate cancer. N Engl J Med. 2001;344:1373–1377. doi: 10.1056/NEJM200105033441806. [DOI] [PubMed] [Google Scholar]
- 32.Chen Y, Schlessinger D, Nagaraja R. T antigen transformation reveals Tp53/RB-dependent route to PLAC1 transcription activation in primary fibroblasts. Oncogenesis. 2013;2:e67. doi: 10.1038/oncsis.2013.31. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Eastham JA, Stapleton AM, Gousse AE, Timme TL, Yang G, Slawin KM, Wheeler TM, Scardino PT, Thompson TC. Association of p53 mutations with metastatic prostate cancer. Clin Cancer Res. 1995;1:1111–1118. [PubMed] [Google Scholar]
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