Parathyroid hormone related-protein (PTHrP) in tissues with poor prognosis in prostate cancer patients
Parathyroid hormone-related protein (PTHrP) serves multiple developmental and physiological roles, and it is recognized for its involvement in the advancement of several cancer types. Elevated PTHrP levels are prevalent in lung, breast, colon, and prostate cancers, while nonneoplastic or healthy tissues typically exhibit minimal PTHrP expression.  Previous studies indicated that PTHrP could stimulate prostate cancer cell invasion, growth, and metastasis.  These demonstrate that PTHrP likely plays a pivotal role in the development of prostate cancer. Prostate-specific antigen (PSA) in combination with prostate biopsy is the most commonly employed method for prostate cancer screening. Despite the increased early-stage diagnosis rate, approximately 25% to 50% of patients experience disease recurrence after surgery, leading to a significant mortality rate.  Hence, novel biomarkers of prostate cancer are urgently needed for prognostication. A recent study showed that increased expression of PTHrP is associated with poor survival for patients with pancreatic cancer.  Furthermore, it was the first to report that serum PTHrP levels can differentiate between indolent and aggressive forms of prostate cancer in patients.  These findings suggest the potential utility of PTHrP as a biomarker for certain malignancies, including prostate cancer. However, the clinical implications of PTHrP in prostate cancer patients and the underlying mechanisms remain unexplored at present. Epithelial-to-mesenchymal transition (EMT) can be characterized by the fact that epithelial cells undergo morphological and cytoskeletal changes to obtain a mesenchymal phenotype and are critically involved in tumor invasion and metastasis. Decreased expression of epithelial markers including E-cadherin and acquisition of mesenchymal proteins such as vimentin are commonly involved in the EMT process.  It has been indicated that PTHrP can promote prostate cancer cell invasion and growth via EMT.  Hence, combined with the previous content, we speculate that a pathway of PTHrP-mediated EMT may take effect in the disease progression of prostate cancer patients. To explore the clinical and survival significance of PTHrP in patients with prostate cancer and the potential mechanisms involved, the expression of PTHrP, E-cadherin, and vimentin were measured in prostate cancer tissues in the current study. Then we analyzed the correlation between PTHrP and E-cadherin, vimentin, or clinicopathologic parameters and evaluated the association between PTHrP and the outcomes of patients with prostate cancer.   2.1. Patients and tissue samples The study involved 88 patients diagnosed with prostate cancer who underwent transrectal ultrasound-guided biopsies or radical prostatectomy at the First Affiliated Hospital of Soochow University between November 2017 and January 2020. All patients received a confirmed diagnosis of prostate cancer through pathological examination. None of the patients had undergone antiandrogen treatment, radiotherapy, or chemotherapy prior to surgery. Additionally, no adjuvant therapy was administered before observing biochemical or clinical progression. The staging procedures included transrectal ultrasonography, digital rectal examination, serum PSA assay, MRI, CT, and bone scan. The median duration of follow-up was 28 months (range of 3–36 months). The main pathological and clinical features are recorded in Table . Patients were followed by periodic measurement of the serum PSA value. In the context of castrate serum testosterone levels, biochemical progression was defined as a sequence of 3 consecutive PSA increases, each occurring at least 1 week apart, leading to a cumulative rise of at least 50% from the lowest recorded level. The onset of new symptoms due to local progression, or systemic or lymphatic metastases indicated a clinical progression. Progression-free survival (PFS) was defined as the time interval from the initiation of treatment to the confirmed first time of biochemical or clinical progression. The 3-year overall survival (OS) data were also collected. The surface of the specimen was inked and fixed, and whole-mount step sections were cut at 3 mm intervals transversely. The current study was performed according to the Declaration of Helsinki and approved by the Ethics Committees of the First Affiliated Hospital of Soochow University and Xuzhou Cancer Hospital (FASU20120107x and XCH20110012x). Written informed consent was obtained from each participant. 2.2. Immunohistochemical analysis and evaluation Tissue sections were stained by immunohistochemistry using specific antibodies for PTHrP, E-cadherin, and vimentin. Briefly, sections from formaldehyde-fixed, paraffin-embedded tissues were deparaffinized by xylene and rehydrated in ethanol. After blocking peroxidase with hydrogen peroxidase, the sections were boiled in 0.01 M citrate buffer for 10 minutes and then incubated with 5% blocking serum for 20 minutes. After that, the sections were incubated with the following antihuman antibodies: PTHrP rabbit polyclonal antibody (Proteintech, Rosemont, IL), E-cadherin mouse monoclonal antibody (Dako, Carpinteria, CA) and vimentin mouse monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA). After incubation with biotinylated goat anti-rabbit or mouse IgG (Vector Laboratories, Burlingame, CA), the samples were incubated in an avidin–biotin peroxidase complex for 30 minutes. The samples were then exposed to diaminobenzidine tetrahydrochloride solution and counterstained with methyl green. Two unbiased assessors, who were unaware of the clinicopathological information, evaluated the staining results. As mentioned in previous studies,  the extent of staining was graded on a scale of 0 (0–5%), 1 (5–25%), 2 (26–75%), or 3 (75–100%), and staining intensity was rated on a scale of 0 (no staining), 1 (weak), 2 (moderate), or 3 (strong). The extent and intensity scores were combined to calculate a composite score, with a score > 3 indicating strong expression and a score ≤ 3 denoting weak expression. We defined high PTHrP expression as strong expression and low PTHrP expression as weak expression. 2.3. Statistical analysis Values were expressed as mean ± SD. Continuous variables were analyzed by the approach of independent-samples Student t test. And chi-square test or Fisher exact test was used for categorical variables. Kaplan–Meier survival analysis and log-rank test were used for survival analysis. Univariate and multivariate Cox proportional hazards model analysis was used to evaluate the prognostic value. The statistical analysis was carried out using SPSS 21.0 software. A P -value < .05 was considered statistically significant.   The study involved 88 patients diagnosed with prostate cancer who underwent transrectal ultrasound-guided biopsies or radical prostatectomy at the First Affiliated Hospital of Soochow University between November 2017 and January 2020. All patients received a confirmed diagnosis of prostate cancer through pathological examination. None of the patients had undergone antiandrogen treatment, radiotherapy, or chemotherapy prior to surgery. Additionally, no adjuvant therapy was administered before observing biochemical or clinical progression. The staging procedures included transrectal ultrasonography, digital rectal examination, serum PSA assay, MRI, CT, and bone scan. The median duration of follow-up was 28 months (range of 3–36 months). The main pathological and clinical features are recorded in Table . Patients were followed by periodic measurement of the serum PSA value. In the context of castrate serum testosterone levels, biochemical progression was defined as a sequence of 3 consecutive PSA increases, each occurring at least 1 week apart, leading to a cumulative rise of at least 50% from the lowest recorded level. The onset of new symptoms due to local progression, or systemic or lymphatic metastases indicated a clinical progression. Progression-free survival (PFS) was defined as the time interval from the initiation of treatment to the confirmed first time of biochemical or clinical progression. The 3-year overall survival (OS) data were also collected. The surface of the specimen was inked and fixed, and whole-mount step sections were cut at 3 mm intervals transversely. The current study was performed according to the Declaration of Helsinki and approved by the Ethics Committees of the First Affiliated Hospital of Soochow University and Xuzhou Cancer Hospital (FASU20120107x and XCH20110012x). Written informed consent was obtained from each participant.   Tissue sections were stained by immunohistochemistry using specific antibodies for PTHrP, E-cadherin, and vimentin. Briefly, sections from formaldehyde-fixed, paraffin-embedded tissues were deparaffinized by xylene and rehydrated in ethanol. After blocking peroxidase with hydrogen peroxidase, the sections were boiled in 0.01 M citrate buffer for 10 minutes and then incubated with 5% blocking serum for 20 minutes. After that, the sections were incubated with the following antihuman antibodies: PTHrP rabbit polyclonal antibody (Proteintech, Rosemont, IL), E-cadherin mouse monoclonal antibody (Dako, Carpinteria, CA) and vimentin mouse monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA). After incubation with biotinylated goat anti-rabbit or mouse IgG (Vector Laboratories, Burlingame, CA), the samples were incubated in an avidin–biotin peroxidase complex for 30 minutes. The samples were then exposed to diaminobenzidine tetrahydrochloride solution and counterstained with methyl green. Two unbiased assessors, who were unaware of the clinicopathological information, evaluated the staining results. As mentioned in previous studies,  the extent of staining was graded on a scale of 0 (0–5%), 1 (5–25%), 2 (26–75%), or 3 (75–100%), and staining intensity was rated on a scale of 0 (no staining), 1 (weak), 2 (moderate), or 3 (strong). The extent and intensity scores were combined to calculate a composite score, with a score > 3 indicating strong expression and a score ≤ 3 denoting weak expression. We defined high PTHrP expression as strong expression and low PTHrP expression as weak expression.   Values were expressed as mean ± SD. Continuous variables were analyzed by the approach of independent-samples Student t test. And chi-square test or Fisher exact test was used for categorical variables. Kaplan–Meier survival analysis and log-rank test were used for survival analysis. Univariate and multivariate Cox proportional hazards model analysis was used to evaluate the prognostic value. The statistical analysis was carried out using SPSS 21.0 software. A P -value < .05 was considered statistically significant.   3.1. PTHrP, E-cadherin, and vimentin in tumor tissues of prostate cancer patients Immunohistochemical analysis showed that the expression rates of PTHrP, E-cadherin, and vimentin in prostate cancer tissues were 95.5%, 88.6%, and 84.1% respectively. Representative immunohistochemical results are shown in Figure A. Patients with a high level of PTHrP had a decreased expression of E-cadherin (2.42 ± 2.22 vs 3.53 ± 1.31, P = .013) and an increased expression of vimentin (3.99 ± 1.47 vs 2.97 ± 1.83, P = .010) compared with patients with a low level of PTHrP (Fig. B). 3.2. Correlations between PTHrP and clinicopathological features The primary objective of this study was to assess the clinical relevance of PTHrP in prostate cancer tissues. Subsequently, we examined the correlation between PTHrP levels and clinicopathological characteristics. The results showed that PTHrP in tumor tissues was significantly correlated with a higher level of initial PSA ( P = .026), positive lymph node metastasis ( P = .010), osseous metastasis ( P = .004), and Gleason score ( P = .026). These findings are shown in Table . 3.3. The prognostic value of PTHrP in tissues of prostate cancer patients The main challenge in clinical practice for prostate cancer is to predict the outcomes accurately. For this reason, the relationship between PTHrP in tumor tissues and patient outcomes was evaluated. The Kaplan–Meier survival analysis and log-rank test were conducted, and the results indicated that patients with a high level of PTHrP had a shorter PFS than those with a low level of PTHrP ( P = .002, as shown in Fig. ). To further determine the predictive value of PTHrP in prostate cancer tissues, the Univariate and Multivariate Cox proportional hazard model analysis was performed then. The parameters that were risk factors in univariate analysis were further entered into multivariate analysis. The results indicated that PTHrP is an independent prognostic factor for PFS (HR: 0.584, 95% CI: 0.347–0.984, P = .043) of prostate cancer patients. These findings are presented in Table . However, for the 3-year OS, we did not observe a significant difference between the 2 groups ( P = .118, as shown in Fig. ).   Immunohistochemical analysis showed that the expression rates of PTHrP, E-cadherin, and vimentin in prostate cancer tissues were 95.5%, 88.6%, and 84.1% respectively. Representative immunohistochemical results are shown in Figure A. Patients with a high level of PTHrP had a decreased expression of E-cadherin (2.42 ± 2.22 vs 3.53 ± 1.31, P = .013) and an increased expression of vimentin (3.99 ± 1.47 vs 2.97 ± 1.83, P = .010) compared with patients with a low level of PTHrP (Fig. B).   The primary objective of this study was to assess the clinical relevance of PTHrP in prostate cancer tissues. Subsequently, we examined the correlation between PTHrP levels and clinicopathological characteristics. The results showed that PTHrP in tumor tissues was significantly correlated with a higher level of initial PSA ( P = .026), positive lymph node metastasis ( P = .010), osseous metastasis ( P = .004), and Gleason score ( P = .026). These findings are shown in Table .   The main challenge in clinical practice for prostate cancer is to predict the outcomes accurately. For this reason, the relationship between PTHrP in tumor tissues and patient outcomes was evaluated. The Kaplan–Meier survival analysis and log-rank test were conducted, and the results indicated that patients with a high level of PTHrP had a shorter PFS than those with a low level of PTHrP ( P = .002, as shown in Fig. ). To further determine the predictive value of PTHrP in prostate cancer tissues, the Univariate and Multivariate Cox proportional hazard model analysis was performed then. The parameters that were risk factors in univariate analysis were further entered into multivariate analysis. The results indicated that PTHrP is an independent prognostic factor for PFS (HR: 0.584, 95% CI: 0.347–0.984, P = .043) of prostate cancer patients. These findings are presented in Table . However, for the 3-year OS, we did not observe a significant difference between the 2 groups ( P = .118, as shown in Fig. ).   Prostate cancer is a leading cause of cancer-related deaths, primarily affecting middle-aged men in Western countries.  Approximately 25% to 50% of prostate cancer patients experience postsurgical recurrence, leading to a significant number of fatalities.  Currently, clinical and pathological parameters such as serum PSA, Gleason score, and clinical stage are the most commonly employed prognostic factors in prostate cancer. While these established tools are valuable, there remains a demand for enhancements or the discovery of new markers to improve the accuracy of prostate cancer prognosis and refine treatment strategies.  Our previous research has shown that serum protocadherin-8 methylation could serve as a potential prognostic marker for patients with low Gleason scores, highlighting the presence of novel markers in addition to the traditional ones in prostate cancer.  In the present study, we focused on PTHrP as well as relevant proteins in tissues and its clinical significance to discover a new potential marker for prostate cancer. PTHrP is known to participate in various cellular processes, including proliferation, hypertrophy, differentiation, and the regulation of intracellular calcium.  In the context of prostate cancer, PTHrP functions to stimulate the growth of prostate cancer cells and plays a crucial role in tumor development.  In clinical prostate cancer patients, Bryden et al  showed that high-frequency expression of PTHrP was detected in primary tumor tissues. Moreover, previous studies also indicated that PTHrP could improve the process of prostate cancer bone metastasis in animal models.  In our present study, our results revealed a frequent expression of PTHrP in tumor tissues of prostate cancer patients, providing additional evidence of a potential association between PTHrP and the progression of prostate cancer. In clinical practice, the primary challenge in treating prostate cancer is differentiating between aggressive and indolent forms of the disease. Previous studies have defined Gleason scores of ≥8 as indicative of aggressive forms, while scores of ≤6 were associated with indolent cancer.  Dhanapala et al  demonstrated that PTHrP can be detected in serum, and its expression correlates with the Gleason score in prostate cancer patients. Moreover, their findings indicated that PTHrP levels could differentiate between indolent and aggressive prostate cancers with specificity ranging from 78% to 96% and sensitivity from 83% to 91%. These results suggest that PTHrP may be a suitable candidate for predicting the malignancy level of prostate cancer. In our current study, we assessed PTHrP expression in prostate cancer tissues and investigated its relationship with the Gleason score. Our findings revealed a distinct disparity in PTHrP expression between tumors with Gleason scores of ≤6 and those with Gleason scores of ≥8, affirming the utility of PTHrP in distinguishing between indolent and aggressive forms of prostate cancer. Osseous metastasis represents a significant milestone in the progression of prostate cancer, often leading to a poor prognosis and diminished quality of life for affected patients.  In our study, we also examined the relationship between PTHrP and the M stage, and our results revealed an elevation in PTHrP expression when comparing M1b and M1c phases to M0 and M1a phases. This suggests that PTHrP may play a role in the process of osseous metastasis in prostate cancer patients. Furthermore, we explored the connections between PTHrP and various clinicopathological features. We observed a significant association between high PTHrP expression in prostate cancer tissues and higher initial PSA levels, as well as the presence of positive lymph node metastasis. Prior studies have suggested a correlation between elevated levels of PTHrP expression and unfavorable survival outcomes in individuals diagnosed with pancreatic cancer, implying that PTHrP could serve as a potential biomarker for malignancy.  At present, there is no report that has investigated the correlation between PTHrP in tumor tissues and clinical survival significance in prostate cancer. In the current study, we explored this and our results indicated that patients with high expression of PTHrP in tumor tissues had shorter PFS than patients with a low level of PTHrP. Moreover, multivariate Cox proportional hazards model analysis indicated that high expression of PTHrP in tumor tissues was an independent prognostic factor for worse PFS in patients with prostate cancer. These data imply that PTHrP in tissues may be used as a potential biomarker for predicting prostate cancer prognosis. However, in this study, we did not observe a significant difference in 3-year OS between the high PTHrP expression group and the low expression group. This may be attributed to the relatively short follow-up duration. Additionally, the number of patients in the present study was relatively small, emphasizing the need for further studies with larger sample sizes and longer-term follow-up research to corroborate these findings and enhance the protocol and strategy for this purpose. EMT contributes to tumor progression and is characterized by losing epithelial markers including E-cadherin and the acquisition of mesenchymal proteins such as Vimentin.  Studies indicated that PTHrP interacts with different cytokines to modulate EMT.  In prostate cancer cells, the mechanism of PTHrP-mediated EMT takes effect in the process of cell invasion.  In the current study, we checked the expression of vimentin and E-cadherin in tumor tissues besides PTHrP. We found that patients with a high level of PTHrP had a decreased expression of E-cadherin and an increased expression of vimentin compared with patients with a low level of PTHrP. These findings hint that a pathway of PTHrP-mediated EMT may take part in tumor progression in patients with prostate cancer, which should be confirmed by further studies.   In conclusion, our study firstly indicates that PTHrP in tumor tissues is associated with malignant clinicopathological characteristics and poor prognosis in prostate cancer patients. Besides, decreased expression of E-cadherin and increased expression of vimentin were found in prostate cancer tissues with high expression PTHrP, implying that a mechanism of PTHrP-mediated EMT may take effect in the progression of prostate cancer. Further research is needed to confirm our findings.   Conceptualization: Yan Zhao, Qian Wang, Jian-Hua Long. Data curation: Yan Zhao, Sheng-Ming Lu, Bing Zhong, Gong-Cheng Wang. Formal analysis: Yan Zhao, Sheng-Ming Lu, Bing Zhong, Gong-Cheng Wang, Rui-Peng Jia. Project administration: Qian Wang, Jian-Hua Long. Writing – original draft: Yan Zhao. Writing – review & editing: Sheng-Ming Lu, Bing Zhong, Gong-Cheng Wang.   