Tissue Immunostaining of Candidate Prognostic Proteins in Metastatic and Non-metastatic Prostate Cancer

doi:10.21203/rs.3.rs-1343436/v1

Download PDF

Research Article

Tissue Immunostaining of Candidate Prognostic Proteins in Metastatic and Non-metastatic Prostate Cancer

https://doi.org/10.21203/rs.3.rs-1343436/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Prostate cancer (PCa) lacks specific markers capable of distinguishing aggressive tumors from those with indolent behavior. Therefore, the aim of this study was to evaluate the immunostaining of four candidate proteins (PTEN, AKT, TRPM8, and NKX3.1) through the immunohistochemistry technique (IHC), in tissues from patients with metastatic and non-metastatic PCa. Tissues from 60 patients were divided into three groups categorized according to prognostic parameters: better prognosis (n=21), worse prognosis (n=22), and metastatic (n=17). Immunostaining was analyzed by a pathologist and staining classifications were considered according to signal intensity: (0) no staining, (+) weak, and (++ and +++) intermediate to strong. AKT protein was independently associated with the presence of extraprostatic extension (p=0.012; OR=0.050; 95% CI=0.005-0.524). The immunostaining for TRPM8 (p=0.005) and NKX3.1 proteins (p<0.001) differed between malignant tumor and adjacent tissue as well as for proteins cellular location (nucleus and cytoplasm). NKX3.1 showed positive and predominantly strong immunostaining in all patients, in both tumoral and adjacent tissues. The staining in the prognostic groups showed that all metastatic samples had a positive immunostaining, with strong intensity for NKX3.1 (p=0.035). In the non-metastatic group, this strong protein staining was not observed in any patients. This study confirmed that the NKX3.1 protein is highly specific for prostate tissue and indicated that NKX3.1, AKT and TRPM8 may be prognostic markers for prostate cancer.

Immunohistochemistry

Metastasis

PTEN

AKT

TRPM8

NKX3-1

According to the World Health Organization (WHO), prostate cancer (PCa) is the fourth most incident cancer, with approximately 1.4 million new cases worldwide. In men, it is the second most common type after lung cancer (Global Cancer Observatory 2020; Culp et al. 2020; Sung et al. 2021) and the fifth leading cause of death (Bray et al. 2018; INCA 2019; Sung et al. 2021). For each year of the 2020–2022 triennium, 625,000 new cases of cancer are estimated in Brazil (INCA 2019), with prostate cancer being the second most common type (Global Cancer Observatory 2020; INCA 2019).

Currently, in Brazil, digital rectal examinations and prostate-specific antigen (PSA) measurements are used as screening methodologies for PCa, and patients with abnormalities in the exam and/or PSA dosages above 10ng/mL are referred for a transrectal ultrasound-guided needle biopsy (Sociedade Brasileira de Urologia 2018; Porcaro et al. 2019; Vendrami et al. 2019).

Additionally, it is known that PSA is an excellent marker for identifying prostatic alterations, however, it is not specific and exclusive for malignant alterations (Vendrami et al. 2019; Lomas and Ahmed 2020). In this context, the search for specific biomarkers that could become potential molecular markers for PCa, capable of predicting clinical and pathological complications in the patient, is important and is under study. Immunohistochemistry (IHC) is a widely used tool in clinical routine to confirm diagnoses with tissue markers (Giannico et al. 2017; Orakpoghenor et al. 2018; Comperát 2018).

Some molecules are already being studied by IHC as they are considered potential candidates as markers of PCa, among them those involved in the cell survival pathway, phosphatidylinositol-3-kinase/serine-threonine kinase/mammalian target of the rapamycin complex (PI3K/AKT/mTOR), in addition to transient melastatin 8 (TRPM8) and NK3 homeobox 1 (NKX3.1) stand out, as they play an important role in prostate carcinogenesis.

It is known that the PI3K/AKT/mTOR signaling pathway is one of the pathways that is most dysregulated in cancer (Koundouros and Poulogiannis 2018), and an aberrant expression of this pathway has already been demonstrated in studies in the early and late phases of PCa (Taylor et al. 2010; Sreenivasulu et al. 2018). Studies also show that deletion of the PTEN tumor suppressor gene in PCa is very common, being very present in metastatic and castration-resistant tumors (Robinson et al. 2015; Wozniak et al. 2017; Jamaspishvili et al. 2018).

The TRPM8 channel is a homotetramer formed by subunits, showing 8 putative glycosylation sites and an immunogenic epitope. It is highly expressed in the prostate, as its ion channel functions as a testosterone receptor, suggesting a role in the regulation of androgenic responses (Asuthkar et al. 2015b). Furthermore, evidence demonstrates that it has important role in the development and progression of neoplasms, especially in PCa, being overexpressed in malignant tumor tissue compared to non-malignant tissue. This protein is present in PCa refractory hormone and with a high Gleason score (Yee 2015).

The NKX3.1 gene is a tumor suppressor member of the NK family of homeobox genes, participating in cell specification and organogenesis processes in several species. In humans, this gene is primarily related to normal prostate development. Its loss of expression leads to defects in prostate protein secretion and ductal morphogenesis and contributes to prostate carcinogenesis (Abate-Shen et al. 2008).

Therefore, the current study aimed to evaluate tissue immunostaining of the tumor suppressor proteins PTEN and NKX3.1, of AKT protein, involved in a cell survival pathway, in addition to the testosterone receptor TRPM8, in samples from patients with metastatic and non-metastatic PCa, in the search for candidate markers for malignant prostate cancer.

Study Group and Sample Characterization

In this retrospective longitudinal study, it was evaluated samples of prostatic malignant and adjacent non-tumor tissues embedded in paraffin from 60 male patients with a confirmed diagnosis of PCa after radical prostatectomy, at the Hospital do Cancer de Londrina (HCL). The study was approved by the Research Ethics Committee Involving Human Beings of the State University of Londrina- Brazil, under number 176/2013. Patients participated voluntarily and signed a free and informed consent form and answered a modified personal questionnaire based on Carrano and Natarajan (1988).

Clinical and pathological data were obtained from medical records, which were used, together with the guidelines of the National Comprehensive Cancer Network (NCCN version 4.2019), for classification of patients into three experimental groups: 1) PCa with a better prognosis (n = 21); 2) PCa with a worse prognosis (n = 22); and 3) metastatic PCa (n = 17). Patients with a Gleason score ≤ 7 (3 + 4), staging ≤ T2b, and PSA ≤ 10 ng/mL were considered to have better prognosis PCa. Patients with a Gleason score ≥ 7 (3 + 4), staging ≥ T3a, and PSA ≥ 20 ng/mL were considered to have worse prognosis PCa. Patients with metastasis were classified according to the presence of lymph node invasion and/or distant metastasis.

The sample's protein profiles were compared between the metastatic versus non-metastatic group (with better and worse prognosis), as well as their malignant and adjacent non-tumor tissues. All samples in the present study were from biopsy and radical prostatectomy, without neoadjuvant chemotherapy.

Histopathological Analysis

Tissues obtained from the biopsy were stained with hematoxylin and eosin to confirm the clinical diagnosis of PCa and to verify the presence of tumor and adjacent non-tumor tissue for further analysis and comparison of immunostaining of proteins in the tissues. This step was performed by pathologists from the HCL. The histopathological classification used was based on international standards established by the WHO, such as Gleason score and clinical staging determined by the Tumor/Node/Metastasis (TNM) system, following the recommendations of the AJCC (American Joint Committee on Cancer).

Immunohistochemistry

Experiments were carried out according to a protocol based on Guembarovski et al. (2018) with modifications, regarding antigenic recovery. Formalin-fixed paraffin-embedded tissue samples of metastatic and non-metastatic malignant and adjacent non-tumor tissues were obtained. Cuts were made, 5–6 µm thick, and fixed on silanized StarFrost® slides (Knittel glass, ALE).

Samples were washed in absolute xylol and rehydrated with absolute alcohol and distilled water. Antigen recovery was performed in a Panasonic Junior smart microwave oven (Panasonic da Amazônia S/A, Manaus, AM, BRA) using three cycles of time and power; the first cycle being 4 minutes at 80w, and the second and third cycles of 5 minutes at 60 w each, using a 250mL aqueous medium solution containing citric acid buffers (4.5 mL to 21 g/L) and sodium citrate (20.5 mL to 29.4 g/L), in addition to 130 µL of Tween 20 detergent (Synth, Diadema, SP, BRA). Next, the endogenous peroxidase blocker available in the mouse/rabbit HRP/DAB ABC detection kit (Abcam, Cambridge, MA, USA) was used, one drop per cut in each reaction, for 30 minutes, in the dark, and at room temperature. SuperBlock™ (Thermo Fisher Scientific, Rockford, IL, USA) was also added to minimize binding to secondary structures, using one drop per cut in each reaction, for 1 hour, at room temperature.

Subsequently, overnight incubation of the four primary antibodies was carried out in the refrigerator: 1) rabbit pan-AKT (polyclonal clone EPR9941-2, Abcam, Cambridge, MA, USA) 1:1000 dilution; 2) rabbit PTEN (monoclonal clone, Abcam, Cambridge, MA, USA) dilution 1:50; 3) rabbit TRPM8 (polyclonal clone, Abcam, Cambridge, MA, USA) dilution 1:300; and 4) rabbit NKX3.1 (EPR16653 monoclonal clone, Abcam, Cambridge, MA, USA) dilution 1:500. Antibody dilutions were tested following the manufacturer's instructions on positive control tissues: colon tumor (AKT), breast cancer (PTEN), normal prostate (TRPM8), and benign prostatic hyperplasia (NKX3.1). Negative controls were performed to verify the specificity of the primary antibody in all slide batteries, where it was replaced by phosphate-buffered saline (PBS).

The secondary antibody kit (mouse/rabbit detection kit HRP/DAB ABC, Abcam, Cambridge, MA, USA) was used according to the manufacturer's instructions, and as a chromogen, the Pierce™ DAB substrate kit (Thermo Fisher Scientific, Rockford, IL, USA), using concentrated DAB ([2x]) for NKX3.1 and ([4x]) for the other antibodies (AKT, PTEN, and TRPM8), based on the manufacturer's protocol, for 5 minutes of incubation at room temperature. The slides were then washed with running water and counterstained with Harris hematoxylin, and mounted with Entellan® Novo (Merck, Billerica, MA, USA,).

Immunostaining for protein profiles in experimental groups was analyzed by an experienced pathologist. The classifications were considered according to staining signal strength: (0) no staining, (+) weak, and ( + + and +++) strong, according to Figs. 1 and 2.

Statistical Analysis

The comparison of the mean ages of the experimental groups was performed using the Student's t test. To compare the staining in tumor and adjacent non-tumor tissues for each protein evaluated, the McNemar test for related samples was used.

Kendall's Tau test was used to analyze the correlations between the proteins immunostaining by IHC and clinicopathological parameters, and logistic regression was performed for the variables that showed significance, to verify whether they were independently associated with protein staining. To analyze the interaction between protein tags, the Kendall Tau correlation test was also performed.

All statistical analyses were carried out using IBM® SPSS® software Statistics for Windows, version 20.0 (IBM® Corp., Armonk, N.Y., USA), considering a significance level (α) of 5%.

The mean age of the total sample was 67.6 ± 7.4 years. The mean age according to prognostic group was 64.4 ± 4.6 (better prognosis), 67.6 ± 5.7 (worse prognosis), and 71.4 ± 10.1 years (metastatic), with a significant difference between the better prognosis and metastatic groups (p = 0.008). In addition, a significant difference was also observed between the mean age of the metastatic versus non-metastatic group (better and worse prognosis together) (p = 0.010).

As a general overview, the proteins PTEN, AKT, and TRPM8 were not expressed or presented a weak immunostaining in practically all samples, in both the malignant tissue, PTEN: 28/55 (50.9%); AKT: 30/55 (54.5%); TRPM8: 42/59 (71.9%) and in the adjacent non-tumor tissue, PTEN: 23/54 (42.6%); AKT: 34/54 (63.0%); TRPM8: 38/58 (65.5%) (Online Resource 1–6). The NKX3.1 protein, on the other hand, showed a more prominent immunostaining in both tissues, with strong intensity in malignant tumor tissue 48/57 (84.2%) and in adjacent non-tumor tissue 29/57 (50.9%) (Online Resource 4–6). Representative images of the protein's immunostainings are shown in Figs. 1 (PTEN, AKT, and TRPM8) and 2 (NKX3.1).

Kendall's correlation analysis showed that the prognosis groups (better and worse prognosis and metastatic) were positively correlated with Gleason score (p < 0.001; Tau = 0.437), TNM (p < 0.001; Tau = 0.670), PSA (p < 0.001; Tau = 0.552), seminal vesicle invasion (p < 0.001; Tau = 0.499), extraprostatic extension (p < 0.001; Tau = 0.697), and perineural invasion (p < 0.001; Tau = 0.547).

Photomicrograph of weak intensity immunostaining using the immunohistochemistry technique for PTEN, AKT, and TRPM8 proteins, evaluated in tumor tissue samples and adjacent non-tumor tissue from patients with PCa. Letters represent the evaluated proteins, being a (negative control), b (PTEN), c (AKT), and d (TRPM8). 40x magnification. Source: author himself.

Photomicrograph of strong intensity immunostaining using the protein immunohistochemistry technique for NKX3.1, evaluated in tumor tissue samples and adjacent non-tumor tissue from patients with PCa. Letters represent the evaluated proteins, being a (negative control) and b (NKX3.1). 40x magnification. Source: author himself

PTEN

PTEN protein did not show differences in immunostaining between tumor and adjacent non-tumor tissues (p = 0.513) or in the cellular locations (cytoplasm: p = 0.100) and (nucleus: p = 0.587). PTEN immunostaining did not demonstrate any significative association with the prognostic groups, the prognostic parameters or biochemical recurrence and metastasis (Online Resource 7).

AKT

AKT protein was also not expressed differently in the tissues of the same patients (p = 0.515) or in relation to cellular locations: cytoplasm (p = 0.092) and nucleus (p = 0.264) (Table 1). Furthermore, it was associated with the prognosis groups (better and worse prognosis and metastasis) (p = 0.005) and with the extraprostatic extension parameter (p = 0.011) (Online Resource 8).

Table 1

Comparative proteins immunostaining between tumor and adjacent non-tumor tissues in PCa patients
Protein evaluated	Staining in tumor tissue x adjacent tissue	p value of McNemar
PTEN
	Tumor x Adjacent non-tumor	0.513
	Cytoplasm x Cytoplasm	0.100
	Nucleus x Nucleus	0.587
AKT
	Tumor x Adjacent non-tumor	0.515
	Cytoplasm x Cytoplasm	0.092
	Nucleus x Nucleus	0.264
TRPM8
	Tumor x Adjacent non-tumor	0.005*
	Cytoplasm x Cytoplasm	0.036*
	Nucleus x Nucleus	0.012*
NKX3.1
	Tumor x Adjacent non-tumor	< 0.001*
	Cytoplasm x Cytoplasm	< 0.001*
	Nucleus x Nucleus	< 0.001*
McNemar's Test. * Significance level of p < 0.05.

TRPM8

TRPM8 protein was expressed differently in tumor and adjacent non-tumor tissue (p = 0.005), with higher immunostaining in malignant tumor, even if of low intensity; the same result was observed for cellular locations of the immunostaining: cytoplasm (p = 0.036) and nucleus (p = 0.012), in both tissues (Table 1). In addition, protein immunostaining was associated with the Gleason score parameter (p = 0.035) (Online Resource 9).

NKX3.1

NKX3.1 protein was expressed differently between tumor and adjacent non-tumor tissue (p < 0.001), with higher immunostaining and strong intensity in malignant tumor tissue when compared to the adjacent non-tumor tissue; the same result was observed for cellular locations of the immunostaining: cytoplasm (p < 0.001) and nucleus (p < 0.001), in both tissues (Table 1).

There was no association between NKX3.1 immunostaining with prognostic parameters or biochemical recurrence and metastasis (Table 2). When NKX3.1 tumor immunostaining was associated with the prognostic groups (non-metastatic and metastatic), it was observed that all patients in the metastatic group (17/17, 100%) presented positive and strong immunostaining, while in the non-metastatic group although predominantly strong immunostaining was also verified (p = 0.035; χ² = 0.033), this result was not observed in all patients (31/40, 77.5%). Furthermore, in the Kendall correlation analysis, NKX3.1 immunostaining was positively correlated with metastasis (p = 0.035; Tau = 0.282).

Table 2

Comparison analysis of patient ages and clinicopathological data in relation to NKX3.1 protein immunostaining in tumor tissue of PCa patients
Clinical-pathological data	Absence of staining (%)	Weak staining (%)	Strong staining (%)	p value of χ²	p value of Kendall (value of Tau)
Experimental groups
Non-metastatic	0 (00.0%)	9 (100%)	31 (64.6%)	0.033*	0.035* (0.282)
Metastatic	0 (00.0%)	0 (00.0%)	17 (35.4%)	0.033*	0.035* (0.282)
Experimental groups
Better prognosis	0 (00.0%)	4 (44.4%)	15 (31.2%)	0.100	0.099 (0.208)
Worse prognosis	0 (00.0%)	5 (55.6%)	16 (33.3%)
Metastatic	0 (00.0%)	0 (00.0%)	17 (35.4%)
Age
< 65 years	0 (00.0%)	4 (44.4%)	16 (33.3%)	0.522	0.525 (0.085)
> 65 years	0 (00.0%)	5 (55.6%)	32 (66.7%)	0.522	0.525 (0.085)
Gleason score
6	0 (00.0%)	1 (11.1%)	14 (29.8%)	0.423	0.195 (-0.166)
7	0 (00.0%)	5 (55.6%)	24 (51.1%)
> 8	0 (00.0%)	3 (33.3%)	9 (19.1%)
TNM
T1 to T2a	0 (00.0%)	2 (22.2%)	9 (23.1%)	0.805	0.728 (0.048)
T2b-T2c	0 (00.0%)	3 (33.3%)	9 (23.1%)
>T3	0 (00.0%)	4 (44.4%)	21 (53.8%)
PSA (ng/mL)
< 10	0 (00.0%)	5 (55.6%)	16 (34.0%)	0.468	0.250 (0.146)
10–20	0 (00.0%)	2 (22.2%)	14 (29.8%)
> 20	0 (00.0%)	2 (22.2%)	17 (36.2%)
Seminal vesicle invasion
No	0 (00.0%)	7 (77.8%)	29 (76.3%)	0.926	0.927 (0.014)
Yes	0 (00.0%)	2 (22.2%)	9 (23.7%)	0.926	0.927 (0.014)
Extraprostatic extension
No	0 (00.0%)	5 (55.6%)	19 (51.4%)	0.821	0.823 (0.033)
Yes	0 (00.0%)	4 (44.4%)	18 (48.6%)	0.821	0.823 (0.033)
Perineural invasion
No	0 (00.0%)	7 (87.5%)	26 (74.3%)	0.425	0.430 (0.122)
Yes	0 (00.0%)	1 (12.5%)	9 (25.7%)	0.425	0.430 (0.122)
Relapse
No	0 (00.0%)	4 (66.7%)	13 (41.9%)	0.266	0.272 (0.183)
Yes	0 (00.0%)	2 (33.3%)	18 (58.1%)	0.266	0.272 (0.183)
Kendall's Tau correlation and Chi-square test. *Significance level of p < 0.05. Due to lack of data, some variables did not include the total of 60 patients.

Multinomial Logistic Regression

To verify whether the clinical-pathological variables that showed statistical significance were independently associated with tumor staining of the AKT, TRPM8, and NKX3.1 proteins, a multinomial logistic regression analysis was used.

It was found that the AKT protein was independently associated with the presence of extraprostatic extension (p = 0.012), with weak immunostaining being a protective factor against the presence of extraprostatic extension when compared to strong immunostaining (OR = 0.050; 95%CI = 0.005–0.524). In the prognostic groups (better and worse prognosis and metastatic), AKT immunostaining was associated, but not independently. TRPM8 and NKX3.1 proteins, on the other hand, were associated with the Gleason score parameters and prognostic groups (non-metastatic and metastatic), respectively, but not independently, as shown in Table 3.

Table 3

Association between AKT, TRPM8, and NKX3.1 proteins staining in tumor tissue with clinical-pathological parameters by multinomial logistic regression
Clinical-pathological data	Tumor immunostaining	χ² _Wald	OR (95%CI)	p value
Experimental Groups (Better and Worse Prognosis and Metastatic)	AKT
	Weak	1.026	0.608 (0.232–1.592)	0.311
	Strong	0.001	1.016 (0.357–2.893)	0.976
Extraprostatic extension	AKT
	Weak	6.256	0.050 (0.005–0.524)	0.012*
	Strong	1.984	0.171 (0.015–1.994)	0.159
Gleason score	TRPM8
	Weak	0.734	0.624 (0.212–1.837)	0.392
	Strong	3.382	4.740 (0.903–24.893)	0.066
Experimental Groups (Non-metastatic and Metastatic)	NKX3.1
	Strong	2.405	2.254 (0.807–6.295)	0.121
Age-adjusted multinomial logistic regression analysis, considering the staining of AKT, TRPM8, and NKX3.1 proteins in tumor tissue. *Significance level of p < 0.05.

Protein Interaction

To assess whether the immunostaining results are related to each other, an interaction analysis was performed, considering the staining only in the tumor tissue for all possible protein combinations. Significant interactions were observed between PTEN and AKT (p < 0.001; χ² < 0.001) and PTEN and TRPM8 proteins (p = 0.010; χ² = 0.086) (Table 4).

Table 4

Comparison of the interaction between protein immunostaining in tumor tissue of PCa patients
Protein interaction	p value of χ²	p value of Kendall (value of Tau)
PTEN X AKT	< 0.001*	< 0.001* (0.566)
PTEN X NKX3.1	0.792	0.529 (0.084)
PTEN X TRPM8	0.086	0.010* (0.326)
AKT x NKX3.1	0.434	0.255 (0.152)
AKT x TRPM8	0.126	0.133 (0.189)
NKX3.1 x TRPM8	0.759	0.556 (0.077)
Kendall's Tau correlation and Chi-square test. *Significance level of p < 0.05.

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.

NKX3.1 protein is highly specific for prostate tissue, as it showed positive immunostaining in all samples in the present study, both in tumor and in adjacent non-tumor tissues. Furthermore, the fact that it showed a significant difference in staining between tumor and non-tumor tissue, as well as being expressed in all patients in the metastatic group and with strong intensity, raises the possibility that NKX3.1 can be a candidate biomarker for metastasis and prognosis, besides identification of the primary site in prostate cancer. Also, AKT and TRPM8 may be prognostic markers for prostate cancer, that deserve future research.

WHO: World Health Organization; PCa: Prostate Cancer; PSA: Prostate-Specific Antigen; IHC: Immunohistochemistry; PI3K: Phosphatidylinositol-3-kinase; AKT: Serine/Threonine Kinase; mTOR: Mammalian target of the rapamycin complex; TRPM8: Transient receptor potential for melastatin 8; NKX3.1: NK3 homeobox 1; HCL: Londrina Cancer Hospital; CEP/UEL: Research Ethics Committee Involving Human Beings of the State University of Londrina; NCCN: National Comprehensive Cancer Network; TNM: Tumor/Node/Metastasis; AJCC: American Joint Committee on Cancer; PBS: Phosphate-Buffered Saline; ISUP: International Society of Urological Pathology.

ACKNOWLEDGEMENTS

All authors would like to thank the development the Hospital do Câncer de Londrina and Angela Navarro Gordan for providing the samples for this study.

Funding

This study was supported by Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico do Paraná (Grant 185/2014) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES - Finance Code 001). Pereira, E.R. received scholarship of CAPES and Cólus, I.M.S. received investigator fellowship awards from CNPq (Proc.308231/2017-1).

Competing interests

The authors declare that they have no competing interests.

Author contributions

Érica Romão Pereira participated in study design and acquisition of data, experimental procedures, performed statistical analysis and interpretation, and drafted the manuscript. Amanda Letícia Francelino, Laís Capelasso Lucas Pinheiro participated in the collection of samples and medical records, participated in the study design and immunohistochemical reactions. Carlos Alberto Miqueloto participated in study design and experimental procedures. Alda Fiorina Maria Losi Guembarovski participated the histopathological assays for selection of tumor tissues and adjacent non-tumor tissues and performed the immunohistochemical analysis of the samples. Karen Brajão de Oliveira participated in the statistical analysis and data interpretation. Paulo Emílio Fuganti made the sample collection possible. Ilce Mara de Syllos Cólus participated in the design of the study and reviewed the manuscript for important intellectual content. Roberta Losi Guembarovski participated in the design of the study, interpretation of data and gave final approval of the version to be published. All authors read and approved the final manuscript.

Data Availability

All data generated or analyzed during the current study are included in this published article.

Ethics approval

This study was approved by the Institutional Ethics Committee Involving Humans at State University of Londrina, Londrina – Paraná (PR), Brazil (CEP/UEL 176/2013; CAAE 19769913.0.0000.5231, in accordance with Resolution 466/12 of the National Research Ethics Commission).

Consent to participate

Study purpose and procedures were explained to all patients and written informed consent was obtained from all individual participants included in the study.

Consent to publish

Not applicable.

Abate-Shen C, Shen MM, Gelmann E (2008) Integrating differentiation and cancer: the NKx3.1 homeobox gene in prostate organogenesis and carcinogenesis. Differentiation. https://doi.org/10.1111/j.1432-0436.2008.00292.x
Abouhashem NS, Salah S (2020) Differential expression of NKX3.1 and HOXB13 in bone metastases originating from prostatic carcinoma among the Egyptian males. Pathology - Research and Practice.
Asuthkar S, Demirkhanyan L, Mueting SR, Cohen A, Zakharian E (2017) High-throughput proteome analysis reveals target TRPM8 degradation in prostate cancer. Oncotarget. http://doi.org/10.18632/oncotarget.14178
Asuthkar S, Demirkhanyan L, Sun X, Elustondo PA, Krishnan V, Baskaran P, Velpula KK, Thyagarajan B, Pavlov EV, Zakharian E (2015a) The TRPM8 protein is a testosterone receptor: II. Functional evidence for na ionotropic effect of testosterone on TRPM8. Journal of Biological Chemistry. http://doi.org/10.1074/jbc.M114.610873
Asuthkar S, Elustondo PA, Demirkhanyan L, Sun X, Baskaran P, Velpula KK, Thyagarajan B, Pavlov EV, Zakharian E (2015b) The TRPM8 protein is a testosterone receptor: I. Biochemical evidence for direct TRPM8-testosterone interactions. Journal of Biological Chemistry. https://doi.org/10.1074/jbc.M114.610824
Asuthkar S, Velpula KK, Elustondo PA, Demirkhanyan L, Zakharian E (2015c) TRPM8 channel as a novel molecular target in androgen-regulated prostate cancer cells. Oncotarget. http://doi.org/10.18632/oncotarget.3948
Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A (2018) Glocal cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. https://doi.org/10.3322/caac.21492
Cambruzzi E, Zettler CG, Pegas KL, Teixeira SL (2010) Relação entre escore de Gleason e fatores prognósticos no adenocarcinoma acinar de próstata. J Bras Patol Med Lab. http://doi.org/10.1590/S1676-24442010000100011
Carrano AV, Natarajan AT (1988) Considerations for population monitoring using cytogenetic techniques. Mutation Research/Genetic Toxicology. https://doi.org/10.1016/0165-1218(88)90036-5
Compérat E (2019) New markers in prostate cancer: inmunohistochemical. Archivos Espanoles de Urologia
Culp MB, Soerjomataram I, Efstathiou JA, Bray F, Jemal A (2020) Recent global patterns in prostate cancer incidence and mortality rates. European Urology. https://doi.org/10.1016/j.eururo.2019.08.005
Epstein JI, Egevad L, Amin MB, Delahunt B, Srigley JR, Humphrey PA, Grading Committee (2016) The 2014 International Society of Urological Pathology (ISUP) consensus conference on gleason grading of prostatic carcinoma: definition of grading patterns and proposal for a new grading system. International Society of Urological Pathology. http://doi.org/10.1097/PAS.0000000000000530.
Giannico GA, Arnold SA, Gellert LL, Hameed O (2017) New and emerging diagnostic and prognostic immunohistochemical biomarkers in prostate pathology. Adv Anat Pathol. https://doi.org/10.1097/PAP.0000000000000136
Global Cancer Observatory (2020) Câncer Today. International Agency for Research on Cancer. https://gco.iarc.fr/today/online-analysis-multi-bars?v=2020&mode=cancer&mode_population=countries&population=900&populations=900&key=total&sex=0&cancer=39&type=0&statistic=5&prevalence=0&population_group=0&ages_group%5B%5D=0&ages_group%5B%5D=17&nb_items=10&group_cancer=1&include_nmsc=1&include_nmsc_other=1&type_multiple=%257B%2522inc%2522%253Atrue%252C%2522mort%2522%253Afalse%252C%2522prev%2522%253Afalse%257D&orientation=horizontal&type_sort=0&type_nb_items=%257B%2522top%2522%253Atrue%252C%2522bottom%2522%253Afalse%257D. Accessed 07 Jan 2021
Goncalves MD, Hopkins BD, Cantley LC (2018) Phosphatidylinositol 3-Kinase, growth disorders, and cancer. N Engl J Med. http://doi.org/10.1056/NEJMra1704560
Guembarovski AL, Guembarovski RL, Hirata BKB, Vitiello GAF, Suzuki KM, Enokida MT, Watanabe MAE, Reiche, EMV (2018) CXCL12 chemokine and CXCR4 receptor: association with susceptibility and prognostic markers in triple negative breast cancer. Molecular Biology Reports. https://doi.org/10.1007/s11033-018-4215-7
Gurel B, Ali TZ, Montgomery EA, Begum S, Hicks J, Goggins M, Eberhart CG, Clark DP, Bieberich CJ, Epsteins JI, De Marzo AM (2010) NKX3.1 as a marker of prostatic origin in metastatic tumors. Am J Surg Pathol. http://doi.org/10.1097/PAS.0b013e3181e6cbf3
Hamid AA, Gray KP, Huang Y, Bowden M, Pomerantz M, Loda M, Sweeney CJ (2019) Loss of PTEN expression detected by fluorescence immunohistochemistry predicts lethal prostate cancer in men treated with prostatectomy. European Urology Oncology. https://doi.org/10.1016/j.euo.2018.09.003
https://doi.org/10.1016/j.prp.2020.153221
Huang H, Guma SR, Melamed J, Zhou M, Lee P, Deng FM (2018) NKX3.1 and PSMA are sensitive diagnostic markers for prostatic carcinoma in bone metastasis after decalcification of specimens. Am J Clin Exp Urol
Instituto Nacional de Câncer José Alencar Gomes da Silva (2019) Estimativa 2020: Incidência de Câncer no Brasil. INCA, Rio de Janeiro
Jamaspishvili T, Berman DM, Ross AE, Scher HI, De Marzo AM, Squire JÁ, Lotan TL (2018) Clinical implications of PTEN loss in prostate cancer. Nature Reviews Urology. https://doi.org/10.1038/nrurol.2018.9
Junior FNF, Vasilceac FA, Gabriotti LFB, Spessoto LCF (2016) Câncer de próstata – atualidades. Artmed Panamericana, Porto Alegre
Koundouros N, Poulogiannis G (2018) Phophoinositide 3-Kinase/AKT signaling and redox metabolism in cancer. Front Oncol. https://doi.org/10.3389/fonc.2018.00160
Kristiansen G. (2018) Markers of clinical utility in the differential diagnosis and prognosis of prostate cancer. Modern Pathology. https://doi.org/10.1038/modpathol.2017.168
Kristiansen I, Stephan C, Jung K, Dietel M, Rieger A, Tolkach Y, Kristiansen G (2017) Sensitivity of HOXB13 as a diagnostic immunohistochemical marker of prostatic origin in prostate cancer metastases: comparison to PSA, prostein, androgen receptor, ERG, NKX3.1, PSAP, and PSMA. Int J Mol Sci. https://doi.org/10.3390/ijms18061151
Kurose K, Zhou XP, Araki T, Cannistra SA, Maher ER, Eng C (2001) Frequent loss of PTEN expression is linked to elevated phosphorylated Akt levels, but not associated with p27 and cyclin D1 expression, in primary epithelial Ovarian Carcinomas. American Journal of Pathology. http://doi.org/10.1016/S0002-9440(10)64681-0
Löbler R, Pereira DV, Cóser VM, Neto NBF, Batuira GAC (2012) Avaliação do escore de Gleason como fator prognóstico em pacientes com cancer de próstata em hormonioterapia. Revista Brasileira de Oncologia Clínica
Lomas DJ, Ahmed HU (2020) All change in the prostate cancer diagnostic pathway. Nature Reviews: Clinical Oncology. https://doi.org/10.1038/s41571-020-0332-z
Lotan TL, Gurel B, Sutcliffe S, Esopi D, Liu W, Xu J, Hicks JL, Park BH, Humphreys E, Partin AW, Han M, Netto GJ, Isaacs WB, De Marzo AM (2011) PTEN protein loss by immunostaining: analytic validation and prognostic indicator for a high risk surgical cohort of prostate cancer patients. Clinical Cancer Research. http://doi.org/10.1158/1078-0432.CCR-11-1244
Lotan TL, Heumann A, Rico SD, Hicks J, Lecksell K, Koop C, Sauter G, Schlomm T, Simon R (2017) PTEN loss detection in prostate cancer: comparison of PTEN immunohistochemistry and PTEN FISH in a large retrospective prostatectomy cohort. Oncotarget. http://doi.org/10.18632/oncotarget.19217
NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines^®) (2019) Prostate Cancer. NCCN
Orakpoghenor O, Avazi DO, Markus TP, Olaolu OS (2018) A short review of immunochemistry. Immunogenetics: Open Access
Porcaro AB, Tafuri A, Sebben M, Novella G, Processali T, Pirozzi M, Amigoni N, Rizzetto R, Shakir A, Panunzio A, De Michele M, Brunelli M, Cerruto MA, Migliorini F, Siracusano S, Artibani W (2019) Prostate volume index is able to differentiate between prostatic chronic inflammation and prostate cancer in patients with normal digital rectal examination and prostate-specific antigen values <10 ng/mL: results of 564 biopsy naïve cases. Urol Int. https://doi.org/10.1159/000502659
Robinson D, Allen EMV, Wu YM, et al (2015) Integrative clinical genomics of advanced prostate cancer. Cell. https://doi.org/10.1016/j.cell.2015.05.001
Sociedade Brasileira de Urologia (2018) Nota oficial SBU e SBPC/ML – Rastreio de Câncer de Próstata. https://portaldaurologia.org.br/medicos/noticias/nota-oficial-sbu-e-sbpc-ml-rastreio-de-cancer-de-prostata/. Accessed 07 Jan. 2021
Sreenivasulu K, Nandeesha H, Dorairajan LN, Ganesh RN (2018) Over expression of PI3K-AKT reduces apoptosis and increases prostate size in benign prostatic hyperplasia. The Aging Male. https://doi.org/10.1080/13685538.2018.1519014
STRING CONSORTIUM 2020 (2021) Interation between PTEN and TRPM8. https://www.string-db.org/cgi/network?taskId=bLKI2jQjRS0r&sessionId=bjI64PH3Fa3s. Accessed 28 Jul 2021
Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F (2021) Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. https://doi.org/10.3322/caac.21660
Taylor BS, Schultz N, Hieronymus H, Gopalan A, Xiao Y, Carver BS, Arora AK, Kaushik P, Cerami E, Reva B, Antipin Y, Mitsiades N, Landers T, Dolgalev I, Major JE, Wilson M, Socci ND, Lash AE, Heguy A, Eastham JA, Scher HI, Reuter VE, Scardino PT, Sander C, Sawyers CL, Gerald WL (2010) Integrative genomic profiling od human prostate cancer. Cancer Cell. https://doi.org/10.1016/j.ccr.2010.05.026
THE HUMAN PROTEIN ATLAS (2021a) AKT. https://www.proteinatlas.org/ENSG00000142208-AKT1. Accessed 28 Jul 2021
THE HUMAN PROTEIN ATLAS (2021b) NKX3.1. https://www.proteinatlas.org/ENSG00000167034-NKX3-1. Accessed 28 Jul 2021
THE HUMAN PROTEIN ATLAS (2021c) PTEN. https://www.proteinatlas.org/ENSG00000171862-PTEN. Accessed 28 Jul 2021
Tsaveler L, Shapero MH, Morkowski S, Laus R (2001) Trp-p8, a novel prostate-specific gene, is up-regulated in prostate cancer and other malignancies and shares high homology with transiente receptor potencial calcium channel proteins. Cancer Research
Tse LA, Ho WM, Wang F, He YH, Ng CF (2018) Environmental risk factors of prostate cancer: a case-control study. Hong Kong Med J
Vaidyanathan V, Karunasinghe N, Jabed A, Pallati R, Kao CHJ, Wang A, Marlow G, Ferguson LR (2016) Prostate Cancer: Is It a Battle Lost to Age?. Geriatrics. https://doi.org/10.3390/geriatrics1040027
Vendrami CL, McCarthy RJ, Chatterjee A, Casalino D, Schaeffer EM, Catalona WJ, Miller FH (2019) The utility of prostate specific antigen density, prostate health index, and prostate health index density in predicting positive prostate biopsy outcome is dependent on the prostate biopsy methods. Urology. https://doi.org/10.1016/j.urology.2019.03.018
Wozniak DJ, Kajdacsy-Balla A, Macias V, Ball-Kell S, Zenner ML, Bie W, Tyner AL (2017) PTEN is a protein phosphatase that targets active PTK6 and inhibits PTK6 oncogenic signaling in prostate cancer. Nature Communications. https://doi.org/10.1038/s41467-017-01574-5
Yee NS (2015) Roles of TRPM8 ion channels in cancer: proliferation, survival and invasion. Cancers. https://doi.org/10.3390/cancers7040882
Yu S, Xu Z, Zou C, Wu D, Wang Y, Yao X, Ng CF, Chan FL (2014) Ion channel TRPM8 promotes hypoxic growth of prostate cancer cells via an O2-independent and RACK1-mediated mechanism of HIF-1α stabilization. Journal of Pathology. http://doi.org/10.1002/path.4413

Download PDF

Reviews received at journal
09 Mar, 2022
Reviewers invited by journal
08 Mar, 2022
Editor invited by journal
16 Feb, 2022
Editor assigned by journal
15 Feb, 2022
First submitted to journal
09 Feb, 2022

You are reading this latest preprint version

Tissue Immunostaining of Candidate Prognostic Proteins in Metastatic and Non-metastatic Prostate Cancer

Status:

Version 1

Abstract

Figures

Introduction

Material And Methods

Study Group and Sample Characterization

Histopathological Analysis

Immunohistochemistry

Statistical Analysis

Results

PTEN

AKT

TRPM8

NKX3.1

Multinomial Logistic Regression

Protein Interaction

Discussion

Conclusions

Abbreviations

Declarations

References

Supplementary Files

Status:

Version 1