An insight into the diagnostic, prognostic, and taxanes resistance of double zinc finger and homeodomain factor’s expression in naïve prostate cancer

Abstract

Currently, clinical biomarkers are urgently needed to improve patient management to guide personal therapy for cancer. In this study, we investigate the deregulation of Zeb-1 in prostate cancer (PC) Tunisian patients. Expression patterns of the Zeb-1 were investigated in prostate adenocarcinoma and benign prostate biopsies using quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR) and 2-ΔΔCt method. Statistical analysis was used to identify differences across groups depending on gene expression level. Furthermore, we exploited a follow-up over 15 years to correlate Zeb-1 deregulation and clinical outcomes in PC patients. Based on ROC curve analyses, the AUC was found in discriminating PC patients from controls (AUC = 0.757; p < 0.001). In addition, the higher expression level was significantly associated with PSA, Digital Rectal Examination, Gleason score, tumor stage, and distant lymph node metastases. Moreover, Zeb-1 overexpression was correlated with shorter overall survival (OS) (p = 0.042), poor progression-free survival (PFS) (p = 0.007), and with resistance to taxanes (p = 0.012). Our data provide the aberrant expression of Zeb-1 in PC patients suggesting its potential diagnostic, prognostic, and theranostic role. Further functional studies are mandatory to strengthen these results and to uncover the molecular mechanism of this neoplasm.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-024-03941-8.

Introduction

Prostate cancer (PC) is a complex and heterogeneous tumor, characterized by interesting clinical variability and unpredictable disease outcome. Although the majority of PC cases are indolent with a slow rate of progression, a proportion of tumors behave aggressively and could threaten patient lives despite the availability of several treatment options (Grozescu and Popa 2017). Following initial treatment, patients may suffer from progression contributing to metastasis, which allows malignant tumors to spread beyond the prostate gland to other parts of the body and represent one of the most deaths causes of PC, associated with an average survival rate of 3–5 years (Ritch and Cookson 2016). According to the recent database of the Global Cancer Observatory (GCO), this neoplasm ranks the third leading cause of cancer-related death in European men (Bray et al. 2018; Rawla 2019; Siegel et al. 2017). In Tunisia, the death percentage is lower compared to lung cancer (Bray et al. 2018), but the mortality-to-incidence ratio (MIR) is still important with a value close to half (Bray et al. 2018). Although PC has been studied for decades, prediction of clinical severity and aggressiveness remains one of the greatest challenges for this entity, which may affect patient care decisions. Efforts are being made and several studies are being conducted to identify molecular biomarkers that could predict tumor prognosis, stratify patient risk to guide treatment options, and improve disease management, but this remains insufficient. Recently, new potential biomarkers that complement and improve the diagnostic/screening potential and accuracy of prostate-specific antigen (PSA) have been identified (Siegel et al. 2017). Yet PSA has been proven as a diagnostic test to be controversial because of its limitations (Prensner et al. 2012). Thus, there is an urgent need for further research in the area of candidate biomarker identification. Epithelial–mesenchymal transition (EMT) plays a critical role in cancer progression and metastasis, which is the leading cause of death from PC (Vasaikar et al. 2021). EMT induces migration, invasion, and dissemination of epithelial cancer cells that facilitate metastasis, whereas the reverse process mesenchymal–epithelial transition (MET) promotes metastatic colonization of cancer cells that migrate from the tumor site of origin to distant sites (Vasaikar et al. 2021). Recently, it has been suggested that targeting and antagonizing the EMT process might represent a promising potential therapeutic approach for PC (Di Gregorio et al. 2020). In addition, EMT-associated biomarkers, which are represented by pleiotropic transcription factors (TF) that could inhibit epithelial genes and/or up-regulate mesenchymal genes, have been revealed in PC cell lines and even rarely also been shown in PC patients' tissues. Among these markers, double zinc finger and homeodomain factor (Zeb-1) have been described as master and "curious" regulator of EMT (Madany et al. 2018; Shah et al. 2015; Zhang et al. 2019). It has been shown that Zeb-1 is associated with aggressive behavior, treatment recurrence following radical prostatectomy, and metastasis in several cancers through the repression of epithelial genes (Karihtala et al. 2013; Krebs et al. 2017; Liu et al. 2014; Spaderna et al. 2008; Wellner et al. 2009). In PC cell lines and mouse models, Zeb-1 is generally associated with metastasis and resistance to hormonotherapy and/or chemotherapy (Pérez et al. 2021). Nevertheless, the expression of formalin-fixed paraffin-embedded (FFPE) from primary endoscopic resection with detailed specificities of PC tissues and its correlation to clinical pathology is not reported.

In this present study, we aimed to investigate the expression level of the transcription factor Zeb-1 in primary prostate adenocarcinoma compared to benign prostate biopsies, and explored its clinical value.

Materials and methods

Human tissue and specimens

This study was approved by the Charles Nicolle Ethics committee (17–03–2016), according to the ethical guidelines of the Helsinki Declaration in 1983, and a written informed consent form was obtained from the included volunteers and their families. We conducted a retrospective study on formalin-fixed paraffin-embedded biospecimens. Samples were collected from the pathology department of Charles Nicolle Hospital (Tunis, Tunisia) from 2002 to 2019.

Cohort selection and clinicopathologic evaluation

Based on the histological evaluation, confirmed by two pathologists after Hematoxylin–Eosin (H&E) staining of the slides, we analyzed 111 FFPE specimens from PC patients recruited following the endoscopic resection of prostate adenocarcinoma without any history of treatment, including 36 tumors of early-stage (T1–T2) and 75 of locally advanced (T3a) and advanced form (T3b–T4) of PC with representative malignant tumor areas containing no less than 85% tumor cells.

A histological examination of twenty specimens from benign biopsies was assessed to confirm the absence of any evidence of malignancy (used as controls) and a PSA test was done and showed a value of less than 2 ng/ml.

The clinical, anatomopathological, and epidemiological data for the samples were obtained from the medical record at the Urology Department, Charles Nicolle Hospital—Tunis, Tunisia, and summarized in Table S1 (Supplementary material).

Molecular analysis and gene expression study

Total RNA was extracted from tissue specimens using the High Pure RNA Paraffin Kit—Roche Applied Science (Mannheim 68,298, Germany), following the manufacturer's instructions. The concentration and purity of the total isolated RNA were assessed using Nanodrop^™ 2000/2000c spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA).

Total RNA was subjected to reverse transcription (RT) using High-capacity cDNA Reverse Transcription Kits (Applied Biosystem) using 1 ug of RNA for all samples. The cDNA synthesis was performed in a total volume of 20ul. The mixture was incubated in a thermocycler (Applied Biosystem, USA) at 25 °C for 10 min, 37 °C for 120 min, 85 °C for 5 min, and 4 °C for 10 min.

To analyze Zeb-1 gene expression, quantitative real-time PCR (q-PCR) was performed with SsoAdvanced^TM Universal Supermix according to the manufacturer's protocol (BIO-RAD), using specific Primers PCR Assay^TM (BIO-RAD) for Zeb-1 (Unique Assay ID: qHsaCED0045418), and GAPDH (Unique Assay ID: qHsaCEP0041396) genes. The relative expression level of was normalized according to the endogenous gene expression on the Applied Biosystem 7500 sequence Detection system. Relative fold change (FC) in Zeb-1 expression was calculated using the comparative cycle thresholds (Ct) method 2−ΔΔCt (Livak and Schmittgen 2001). Experiments were done in triplicate for Zeb-1 and GAPDH.

Survival analysis

The outcome measures used in this study were overall survival (OS) and progression-free survival (PFS). The overall survival was defined as the time from the first date of initial treatment until cancer-specific death. However, progression-free survival was recorded as the time from treatment initiation until the PC progression. Patients lost to follow-up were censored.

Statistical analysis

Data were performed by descriptive statistics using the Statistical Package for the Social Sciences, version 21.0 (SPSS Inc., Chicago, IL, USA). Differences across groups were inspected using the Kruskal–Wallis, Mann–Whitney U, Chi-squared, Spearman, and Pearson exact tests. AUC–ROC (Area under the Curve–Receiver Operating Characteristic) was used to distinguish between controls and PC patients. An excellent model for a good separation has AUC near one, which means it has a good measure of separation. OS and PFS were assessed by the Kaplan–Meier method, a log-rank test, and Cox regression analysis. A multivariate analysis was used to determine whether it is an independent prognostic indicator for progression after accounting into account the grade and the stage. A p value < 0.05 for all statistical tests was considered statistically significant.

Results

Clinical and epidemiological features of PC patients

In our cohort, the median age was 71 years old, ranging from 60 to 80 years old. Age at diagnosis, symptomatology, rectal touch (RT), PSA and lymph node metastasis (NM), tumor stage, Gleason score, International Society of Urological Pathology grade (ISUP), type of therapeutic care (curative or palliative) are recorded in Table S1. Epidemiological parameters such as coronary vascular disease (CVD), diabetes, and arterial hypertension (PAH) are summarized in Table S1 (Supplementary material). The patient’s follow-up was ranked between 1 day and 15.25 years (183 months). Analysis criteria of the follow-up including the presence or absence of progression after primary resection and cancer-specific death are described in Table S2.

Up-regulation of Zeb-1 in primary prostate cancer

We analyzed Zeb-1 gene expression level by RT-qPCR between 111 naïve neoplasm and 20 control samples. Our data indicate that Zeb-1 was significantly up-regulated in patients compared to control, with a median fold change of 3.519 vs. FC = 1.028, respectively (p values for Student’s t test = 0.016) (Fig. 1a). Indeed, ROC curve analysis showed that Zeb-1 expression level could discriminate between tumor/non-tumor state, with an AUC = 0.757 (95% confidence interval [CI] = [0.678–0.837], p < 0.001) (Fig. 1b).

Fig. 1.

Diagnosis utility of Zeb-1: a Box plot presentation of Zeb-1 folds changes in different groups. The tissues from patients (PC in diagnosis) and controls were analyzed for Zeb-1 expression by real-time RT-qPCR. The method 2-ΔΔCt was used for Fold change determination. The use of a boxplot aims to compare the distribution of Zeb-onefold changes in controls, and PC based on the five-number summaries: minimum (lower whisker), first quartile, median, third quartile, and maximum (upper whisker). The median (middle quartile) marks the mid-point of the fold change marked by the line that divides the box into two parts. Following the norms of relative gene expression, the expression of controls is close to 1, and the patient's expression is up regulated (Fold Change > 2). b ROC curve for Zeb-1 overexpression to distinguish PC patients from controls (p < 0.001; AUC = 0.757). The p value is the probability that the observed sample area is under the ROC curve (AUC), and is significant when the true area under the ROC curve is 0.5 (null hypothesis: area = 0.5). In this analysis, the p value < 0.001. We concluded that the area under the ROC curve is significantly different

Association of Zeb-1 expression with clinical, anatomopathological factors, and outcome

We aimed to explore the association between Zeb-1 expression level and clinicopathological parameters of PC patients, including Digital Rectal Examination (DRE), PSA levels, Gleason score, ISUP, tumor stage, lymph node metastasis, and postoperative care. Our data demonstrated that the high expression level of this gene was associated with these parameters (p < 0.05) (Table 1). We found that Zeb-1 expression level correlates with clinical screening/diagnostic factors, including Digital Rectal Examination which might discriminate between PC and controls, and with a higher PSA level (> 100 ng/ml) (p < 0.05). Moreover, our analysis revealed an association between Zeb-1 up-regulation with the higher Gleason score (≥ 8 vs. < 8) (p < 0.001), the higher ISUP grades (3, 4–5 vs. the lowest grades 1, 2) (p < 0.05), locally advanced and metastasis stages vs. local stage (p < 0.001) and with distant and lymph node metastasis (NxM1 vs. N0M0) (p < 0.001) (Table 1).

Table 1.

Correlation between Zeb-1’s expression levels and clinical parameters of PC patients recruited from surgery

Clinical parameters of PC patients in the diagnosis	The expression level of Zeb-1 p value for Kruskal–Wallis test	The expression level of Zeb-1 p value for U de Mann–Whitney
RT (RT1; RT2; RT3; RT4)	0.000*	[RT1–RT2] (p < 0.05)** [RT1–RT3] (p < 0.05)** [RT1–RT4] (p < 0.05)**
PSA level groups ([0–20]; [20–100]; ≥ 100 ng/ml)	0.011*	[0–20]; ≥ 100 (p = 0.01)** [0–20]; [20–100] (p > 0.05) [20–100]; ≥ 100 (p > 0.05)
Gleason score groups (< 8; ≥ 8)	–	0.000**
ISUP 1.2.3.4.5	0.001*	[1–2]; [1–3]; [1–4]; [1–5] (p < 0.05) [2–5] (p = 0.002)**; [3–5] (p = 0.001)**; [3–4] (p = 0.06) ; [4–5] (p > 0.05)**
Tumor stages (localized; locally advanced; lymph node; distant metastatic)	0.001*	[Localized–Metastatic] (p = 0.001)** [Localized–Locally advanced] (p = 0.001)** [Locally advanced–Lymph node metastatic] (p = 0.002)**
NM statues (N0M0; NxM1)	–	0.001**
Therapeutic care (Curative or palliative)	–	0.001**
Postoperative care (W.w; neoadjuvant treatment)	–	0.001**
Tumor evolution (controlled disease, progression and not available information)	–	0.001**
Cancer-specific death (Yes; No)	–	0.007**

RT rectal touch, PSA prostate-specific antigen, ISUP International Society of Urological Pathology grade, NM lymph node metastasis

*, **The p values for Kruskal–Wallis or U de Mann–Whitney

Furthermore, we aimed to investigate the clinical utility of Zeb-1 in predicting surgical outcomes between patients who received a postoperative treatment compared to those who underwent only resection. We found that the overexpression of Zeb-1 in resected patients who developed progression later, according to increased PSA, which is used as a tool to estimate the risk of adverse pathology after treatment, compared with those with stable status (p = 0.001) (Table 1).

We achieved a multivariate logistic regression analysis including age range, PSA classification, stage, Zeb-onefold change, and Gleason score. We found a significant correlation between progression and advanced age, higher grade of Gleason score, metastatic lymph node stage, higher PSA level, and Zeb-1’s overexpression (p < 0.001) (Table 2).

Table 2.

Logistic regression: effect of PSA, tumor stage, node metastasis, and expression of Zeb-1 on progression parameter (Gleason score)

Estimations des paramètres
Progressiona		B	Std. error	Wald	ddl	Sig	Exp (B)	95% Confidence interval for Exp (B)
								Lower bound	Upper bound
1.00	Constant	2.128	0.981	4.708	1	0.030
	[PSA class = 1]	– 1.162	0.727	2.557	1	0.110	0.313	0.075	1.300
	[PSA class = 2]	0.496	0.757	0.430	1	0.512	1.642	0.373	7.234
	[PSA class = 3]	0b			0
	[NM class = 1]	14.377	0.766	352.123	1	0.000	1,753,973.631	390,702.429	7,874,083.373
	[NM class = 2]	− 0.941	1.390	0.459	1	0.498	0.390	0.026	5.945
	[NM class = 3]	0b			0
	[Tumor stage = 0]	− 17.058	0.811	442.372	1	0.000	3.908E-008	7.974E-009	1.916E-007
	[Tumor stage = 1]	− 14.943	0.000		1		3.239E-007	3.239E-007	3.239E-007
	[Zeb-1 expression =]	1.383	1.554	0.792	1	0.373	3.988	0.190	83.934
	[Zeb-1 expression = A]	0.323	0.841	0.148	1	0.701	1.381	0.266	7.173
	[Zeb-1 expression = B]	0.225	0.745	0.091	1	0.763	1.252	0.291	5.388
	[Zeb-1 expression = C]	0b			0
	[Gleason score class = 1]	– 0.619	0.736	0.706	1	0.401	0.539	0.127	2.280
	[Gleason score class = 2]	0b			0
	[Age class = 1.00]	0.226	0.572	0.156	1	0.693	1.253	0.408	3.847
	[Age class = 2.00]	0b			0

PSA classification = 1 Prostate-specific antigen value (1–20 ng/ul), PSA classification = 2 Prostate-specific antigen value (20–100 ng/ul), PSA classification = 3 Prostate-specific antigen value > 100 ng/ul, NM classification = 1 Absence of nodes metastasis, NM classification = 2 Presence of node and absence of metastasis, NM classification = 3 the presence of node and metastasis, Tumor stage = 0 localized tumor, Tumor stage = 1 Locally advanced, Zeb-1 expression = A No expression, Zeb-1 expression = B Over expression, Zeb-1 expression = C Under expression

^aThe reference category is PC with no progression

^bThis parameter is set to zero because it is redundant

In addition, the expression level of Zeb-1 was correlated with epidemiological parameters such as coronary vascular disease (CVD), diabetes, and arterial hypertension (PAH). A positive correlation was revealed between CVD and Zeb-1 (0.001; r² = 0.353) in a univariate regression model. In contrast, the relationship between the deregulation of Zeb-1 and age was irrelevant (p = 0.111).

Zeb-1 and OS and PFS

Kaplan–Meier method and a log-rank test showed that higher expression of Zeb-1 was associated with poorer survival (p = 0.042) (Fig. 2a) and shorter progression-free survival (Fig. 2b) (p = 0.007). Furthermore, to delve deeper into the matter we examined how the presence of Zeb-1 relates to survival rates in each Gleason grade. Our findings indicate that, in low grade cases (Gleason score < 8) the expression of Zeb1 does not have an effect, on either overall survival (Log rank p = 0.287) (Fig. 3a) or progression-free survival (Log rank p =0.695) (Fig. 3c). However in high-grade cases (Gleason score ≥ 8), an elevated level of this gene is linked to poorer survival (Fig. 3b) (p < 0.001) and progression (Fig. 3d) (p = 0.006) outcomes.

Fig. 2.

Kaplan–Meier analysis of Zeb-1: a Overall survival (OS) of PC patients according to the expression levels of Zeb-1 PC patients (p = 0.042); b Progression-free survival (PFS) of PC patients relative to the expression levels of Zeb-1 PC patients (p = 0.007). The classification of gene expression is according to the standards of relative gene expression: the Fold Change is lower than 0.5, the gene expression is under-expressed; it is not-expressed if it is between [0.5–2], the gene expression, and it is over-expressed because the FC is higher than 2

Fig. 3.

Survival rates of Zeb-1 within Gleason score Stratification (GSS): a Overall survival of PC patients according to the expression levels of Zeb-1 PC patients within [Gleason score stratification = 1 (GSS1) < 8] (Log rank p = 0.287); b Overall survival of PC patients according to the expression levels of Zeb-1 PC patients within [Gleason score stratification = 2 (GSS2) ≥ 8] (Log rank p < 0.001); c Progression Free survival of PC patients according to the expression levels of Zeb-1 PC patients within [Gleason score stratification = 1 (GSS1) < 8] (Log rank p = 0.695), d Progression Free survival of PC patients according to the expression levels of Zeb-1 PC patients within [Gleason score stratification = 2 (GSS2) ≥ 8] (Log rank p = 0.006). The classification of gene expression is according to the standards of relative gene expression: the Fold Change is lower than 0.5, the gene expression is under-expressed; it is not-expressed if it is between [0.5–2], the gene expression, and it is over-expressed because the FC is higher than 2

Zeb-1 expression and taxanes resistances

During follow-up, we are interested in resistance to the taxanes drug (docetaxel, paclitaxel). Using a Pearson correlation, we have found a positive correlation between Zeb-1 overexpression for patients with primary prostate cancer and resistance to taxanes drugs, later, prescribed for patients who required taxanes as neoadjuvant therapy (p = 0.012; r² = 0.247).

Discussion

Zeb-1 is a major key promoter of the EMT process in cancer progression. Concerning aberrant expression of Zeb-1, previous evidence has demonstrated the implication of this gene on several carcinomas with aggressive disease, poor clinical prognosis, and metastasis development (Graham et al. 2008; Okugawa et al. 2012; Shen et al. 2012; Singh et al. 2008). In the present retrospective study, we aimed to explore the clinical value of Zeb-1 in PC patients. Our genetic interest in Zeb-1 is based on the existence of biological differences in the pathogenesis of PC between racially disparate populations as recently discovered (Rebello et al. 2021), and on the deficiency of data for Zeb-1 expression, especially about patient’s follow-up, resistance to treatment, and correlation with epidemiological parameters. For this purpose, the expression level of Zeb-1 was investigated in malignant tumor tissues compared to benign biopsies used as healthy controls, by RT-qPCR as a result of overexpression in PC samples. As expected, the up-regulation of Zeb-1 was with previous data concerning several malignancy types (Farfán et al. 2018; Orellana-Serradell et al. 2018; Pérez et al. 2021; Zhang et al. 2019). Indeed, the overexpression of Zeb-1 has also been reported in PC cell lines and human prostate cancer cells (Pérez et al. 2021). This transcription factor has no expression in normal prostate tissue obtained from patients treated with cystoprostatectomy for bladder carcinoma (Figiel et al. 2017), matched non-neoplastic prostatic tissue (NPT) (Cha et al. 2016), and benign biopsies as demonstrated in our study. This explains that despite the difference in control tissues the activation of this transcription factor occurs only in prostate cancer cells. Moreover, our analysis highlights the diagnostic utility of Zeb-1 in primary PC patients, which is being investigated for the first time in patients, to our knowledge. Meanwhile, this factor has only been designated as a diagnostic biomarker in a recent functional study using primary cell culture of PC cell lines transfected with this gene (Farfán et al. 2018). Overall, we think that the regulation of Zeb-1 at diagnosis could be modulated by transcriptional and post-transcriptional regulations. This might explain both its molecular function as RNA-binding protein (RBPs), as transcription regulator activity, and its implication on the development process revealed by the Panther database. On this basis, it will be interesting to further investigate the diagnostic value of this gene, by analyzing precancerous PC lesions compared to healthy tissues to reveal its usefulness in early PC diagnosis. In addition, in vitro studies from PC cell lines from patient’s tissues are warranted to decipher the involvement of Zeb-1 in PC initiation besides cancer progression. In addition, our findings are in agreement with several studies, which outlined Zeb-1 as a prognostic biomarker in PC, based on the prognostic utility of the Gleason score which is defined as a prognostic parameter in PC (Figiel et al. 2017; Marín-Aguilera et al. 2014). In line with the finding of Figiel and his collaborators (Figiel et al. 2017), our study demonstrates that Zeb-1 expression level is associated with Tumor stage, Gleason grade, ISUP grading, and distant and lymph node metastases in multivariate analysis. In resume, we support the evidence that higher levels of Zeb-1 expression are associated with a poor prognosis, for individuals diagnosed with PC.

Indeed, our results demonstrated that Zeb-1 up-regulation might predict a shorter overall survival and poor progression-free survival especially with a high grade of prostate cancer (Gleason score ≥ 8), which highlight that it could be an independent prognostic indicator for survival rates outcome (OS and PFS). The similar observation relates to the OS, in naive prostate cancer patients. However, our study takes an approach by focusing on clinical progression rather than biochemical progression unlike the emphasis given by the Figiel and his collaborators (Figiel et al. 2017). Understanding the interaction, between aspects is vital when dealing with PC since their importance may differ from patient to patient. It is crucial to highlight the significance of progression as it goes beyond just relying on biochemical markers offering a more holistic comprehension of the overall advancement of the disease.

Today, discovering the molecular mechanism of chemoresistance in PC is one of the current challenges. In this study, we confirm the utility of Zeb-1 as a potential marker of chemotherapy resistance. We have found a positive link between this transcription factor and resistance to taxanes drugs (docetaxel and paclitaxel) prescribed for patients from this cohort. Our result was consistent with previous new data concerning the role of Zeb-1 on chemoresistance in radical prostatectomy specimens after systemic therapy and in prostate cancer cell lines such as PC3 (Orellana-Serradell et al. 2018), DU 145 (Orellana-Serradell et al. 2019), but not in primary prostate cancer specimens from resection. However, we propose a descriptive study on serum samples from large sample sizes at diagnosis and patients treated or not with chemotherapy to confirm these findings.

In addition to the clinical interest in Zeb-1, our data showed a positive correlation between Zeb-1 expression and epidemiological features, including coronary vascular disease, which fits with previous studies revealed that death event CVD is commonly detected in patients with cancer, such as prostate cancer (Finke et al. 2021), and that EMT factors, including Zeb-1, are involved in CVD, which could explain its role in biological regulation. It has also been suggested that EMT factors represent promising therapeutic targets for coronary vascular disease (Duffy 2020; Kovacic et al. 2019; Li et al. 2021). In contrast, we did not find any correlation between age and mRNA Zeb-1 in our population unlike in the Polish population (Jędroszka et al. 2017).

A potential limitation of this study is the relatively small sample of individuals, due in part to the low incidence of the disease in Tunisia. Further analysis of functional studies may help to better understand the importance of Zeb-1 gene deregulation in the pathobiology of PC and its contribution to the aggressiveness of this neoplasm.

Conclusion

In conclusion, we have shown that mRNA Zeb-1 expression was expressed in clinical prostate cancer specimens and correlated with poor prognosis (higher Gleason score and metastasis). Our findings also suggest that this transcriptional factor could be a potential diagnostic and therapeutic target in chemotherapy resistance, especially after the development of a novel mRNA vaccine for targeted prostate cancer therapy.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

RS, JHL, and SO conceived and designed the study. RS, AH, and SR provided clinical samples. RS, JHL, RLH, and TM provided technical support. RS, SK, RJ, and AD collected clinical parameters. RS performed experiments. RS analyzed data. RS wrote the manuscript. RS, JHL, and SO revised the manuscript. All authors approved the final manuscript submitted for publication.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability

Data sharing does not apply to this article as no datasets were generated or analyzed during the current study.

Declarations

Conflict of interest

The authors have no conflicts of interest to declare that are relevant to the content of this article.

Ethical approval

This project was approved by a Charles Nicolle ethical committee, Tunis, Tunisia. Relevant protocols followed in our retrospective study were performed following guidelines and regulations of The Code of Ethics of the World Medical Association (Declaration of Helsinki).

Consent to participate

Informed consent was obtained from all individuals according to the ethical standards of the Ethic committee of Charles Nicolle Hospital, Tunis, Tunisia and the 1964 Helsinki Declaration. All patients included in any research, experiment, or clinical trial described in this paper have given written consent to the inclusion of material about themselves, they acknowledge that they cannot be identified via the paper, and we have fully anonymized them.