The evaluation of four proteins immunostaining by IHC (PTEN, AKT, NKX3.1, and TRPM8) in malignant tumor and adjacent non-tumor tissues of patients with prostate cancer indicated that TRPM8 protein was differentially expressed, with higher immunostaining in malignant tumor tissue. However, our most prominent result was the fact that NKX3.1 immunostaining was observed in all samples in the present study, both in tumor tissue and in the adjacent tissue non-tumor, indicating high specificity for prostate tissue. In addition, there was a strong tumor immunostaining for NKX3.1 in all patients of the metastatic group.
Certain results obtained from biopsies may be inconclusive, as the biological material collected is insufficient, with few atypical glands for analysis, and a repeat examination may be necessary. Therefore, the use of complementary techniques and the use of new biomarkers are extremely important. A widely used and highly relevant tool is the IHC technique (Kristiansen 2018; Orakpoghenor et al. 2018), which allows identification of the presence or absence of certain proteins in specific tissues, and the staining intensity is generally used as the gold standard (Jamaspishvili 2018).
It is known that age is one of the main risk factors linked to PCa (Vaidyanathan et al. 2016; Junior et al. 2016; Tse et al. 2018; INCA 2019), therefore, aging increases the risk of developing the disease and its possible aggravation. The significative results obtained in this study comparing the mean ages between patients in the better prognosis and metastatic groups (p = 0.008), in addition to the result of the comparison between the metastatic versus non-metastatic groups (better and worse prognosis) (p = 0.010), confirm that PCa is a disease of advanced age and that late diagnosis may be associated with a more severe condition of the disease, such as the development of metastases.
The genomic deletion of PTEN is very common in PCa, as it is the most lost tumor suppressor in the early stages of the disease (Lotan et al. 2011; 2017; Jamaspishvili et al. 2018; Hamid et al. 2019). Consequently, an increase in AKT expression would possibly occur, as these proteins participate in the same signaling pathway (Kurose et al. 2021). Low or absent expression of PTEN was expected in the worse prognosis and metastatic groups; on the other hand, the AKT protein should be more expressed in tumor tissue in these groups of patients. However, we verified that the immunostaining intensities of PTEN and AKT were similar, showing a similar pattern of immunostaining in many of the samples, in the tumor tissue PTEN: 28/55 (50.9%) and AKT: 30/55 (54.5%) and in the adjacent non-tumor tissue PTEN: 23/54 (42.6%) and AKT: 34/54 (63.0%). Our data are not distinct from the literature, as according to the database The Human Protein Atlas (2021c), the PTEN protein has predominantly cytoplasmic immunostaining, and the prostate tissue has low expression of this protein (The Human Protein Atlas 2021a). The AKT protein, on the other hand, presents nuclear immunostaining and in normal prostate tissue, its expression is median (The Human Protein Atlas 2021a).
Tumor immunostaining of PTEN protein did not show any significant association with the prognostic groups, prognostic parameters, or recurrence and metastasis. AKT protein immunostaining, on the other hand, was associated with groups of prognosis (better and worse prognosis and metastatic) and extraprostatic extension, being independently associated with the latter mentioned parameter. AKT is an oncogenic protein, so the presence of immunostaining may favor tumor growth. Extraprostatic extension is a parameter indicative of tumor aggressiveness, which can lead to invasion of adjacent tissues and, consequently, to metastases.
Literature data demonstrate that PTEN and AKT are part of the same cell survival pathway, PI3K/AKT/mTOR, where PI3K acts as an agonist of the pathway converting PIP2 into PIP3, favoring the binding of AKT to PIP3, which it will mediate, through activation of proteins, cell growth, proliferation, survival, and migration (Gonçalves et al. 2018). PTEN protein, on the other hand, acts as a direct antagonist of the pathway, converting PIP3 into PIP2, therefore having a role as a lipid phosphatase (Gonçalves et al 2018; Jamaspishvili et al. 2018). This role of antagonist (PTEN) and agonist (AKT) of the cell survival pathway was confirmed in our protein interaction analysis, in which it was observed that strong AKT intensity immunostaining was present when absent or weak intensity immunostaining of PTEN was observed.
Asuthkar et al. (2015a, b, c, 2017) suggested that TRPM8 is a key element in the testosterone-induced response pathway, and that its activity can significantly contribute as an anti-tumor defense mechanism, serving as a new therapeutic target. The immunostaining of the TRPM8 protein, in general, was quite weak in the samples of the present study, in the tumor tissue: 42/59 (71.9%) and in the adjacent non-tumor tissue TRPM8: 38/58 (65.5%). In addition, it was more present in tumor tissue compared to adjacent non-tumor tissue and a significant difference was observed in immunostaining of this protein in the cellular location. According to literature data, this protein is highly expressed in the prostate, both in tumor-free individuals and in patients with PCa (Asuthkar 2017). Our results do not support this study, but the fact that this protein was more marked in the tumor tissue than in the adjacent tissue non-tumor, suggests the need for future studies with new sample groups, to verify whether it may have any correlation with the malignant change.
Asuthkar et al. (2015b) found that TRPM8 protein and testosterone are directly involved in localized interactions in the plasma membrane of cells in the periphery of the prostate and in the plasma membrane of the endoplasmic reticulum of cells in the lumen. Furthermore, the authors suggested that testosterone-induced TRPM8 might be an important regulator of Ca2 homeostasis and the cell cycle in prostate cells. Although TRPM8 mRNA levels increase during prostate tumor progression (Tsaveler et al. 2001), protein levels are not proportionally equal. Asuthkar et al. (2015c) found that TRPM8 is redirected to degradation in PCa, while protein recovery effectively suppresses tumor cell growth. This fact could explain the low expression of this protein in the samples of the present study.
One of the main prognostic factors described in the literature is the histological grade, with the Gleason score being the most used (Cambruzzi et al. 2010). This is a very important prognostic parameter in the assessment of tumor progression and aggressiveness (Löbler et al. 2012). TRPM8 was not associated with the prognostic groups or biochemical recurrence and metastasis, however, it was associated with the Gleason score parameter, but not independently. Yu et al. (2014) and Yee et al. (2015) demonstrated an association of TRPM8 with Gleason score, in which TRPM8 immunostaining was correlated with a high Gleason score.
Another interesting result was the interaction observed between PTEN and TRPM8 proteins, in which it was verified that the presence of one protein is associated with the presence of the other. However, when analyzing the interaction through the String Database (2021), we found no direct interaction between these proteins, but that TRPM8 can interact with other peripheral proteins of the PI3K/AKT/mTOR and PTEN signaling pathway, such as PPP1CA, PPP1CB, and PPP1CC, for example, which are catalytic serine/threonine-protein phosphatase subunits, and associate with several regulatory proteins to form highly specific holoenzymes, aiming to dephosphorylate hundreds of biological targets.
NKX3.1 protein presented the most intense immunostaining in the tissues of the patients with PCa in the present study, with a significant difference being observed in the tumor tissue (48/57; 84,2%) versus adjacent non-tumor tissue (29/57; 50.9%). Differences in immunostaining regarding cellular locations (nucleus and cytoplasm) were also observed. These results corroborate the study of Gurel et al. (2010), that clearly showed the nuclear immunostaining of NKX3.1 present in practically all analyzed samples, presenting a high pattern of nuclear staining and a high rate of positivity in metastases. According to the database The Human Protein Atlas (2021b), NKX3.1 presents immunostaining both in the nucleus and in the cytoplasm, with nuclear staining being the most evident. In addition, this protein has a high expression rate in the prostate tissue.
It's already well established that clinicopathological parameters used when studying neoplasms are extremely important, as the data help to confirm and classify patients, and can help guide more effective therapy. The prognosis of PCa is fundamentally related to some histopathological data, such as topography/laterality, tumor volume/size, histological type, degree of differentiation, presence of capsular to extraprostatic neoplastic invasion, state of the surgical margins and the presence of metastases in regional or distant lymph nodes (Cambruzzi et al. 2010). Within this context, PTEN, AKT, and TRPM8 proteins were not significantly correlated with prognostic parameters or biochemical recidive and metastases evaluated in our samples.
NKX3.1 immunostaining was positively correlated and significantly associated, but not independently, with the metastatic group, where all patients had intense immunostaining. Previous studies found that NKX3.1 expression was strongly present in metastatic PCa samples, showing high sensitivity for the prostate, and even when associated with PSA (Kristiansen 2017), PSMA (Huang et al. 2018), or HOXB13 (Abouhashem and Salah 2020), they were considered good markers for detecting metastases of prostatic origin. In addition to these studies, the International Society of Urological Pathology (ISUP) (Epstein et al. 2016) has already indicated the NKX3.1 protein as an excellent biomarker of prostate origin in PCa metastases, being highly specific for this tissue. Our results confirm that NKX3.1 is a specific marker of prostate tissue and indicate that it can be used to identify a primary site of metastasis, but also that it could be an early predictor of this phenomenon, given its immunostaining profile in metastatic patients.