The second most frequent cancer for men, on a worldwide scale, is prostate cancer (Sung et al., 2021). When diagnosed at early stages the disease is relatively manageable however, at tumor progression, patients may gain resistance to treatment and create metastatic lesions (Sharma et al., 2018). Prostate cancer patients with distant metastasis, have aggressive disease with poor survival (Sharma et al., 2018; Zhang et al., 2022), justifying prostate cancer as the second leading cause of cancer mortality for men in the western world (Zhou et al., 2017). Thus, it is crucial to identify new targets for treatment to overcome acquired resistance to therapy and metastasis.

CTCs are the major contributors to the disease progression and the initiation of metastatic dissemination, as they can intravasate blood vessels, travel through the bloodstream, and extravasate from the vessels to colonize other tissues and organs (van Zijl et al., 2011). Most CTCs entering the bloodstream, are apoptotic, however, a subset of them can create metastasis (Kallergi et al., 2013). The detection of CTCs is a rather difficult process (Lianidou et al., 2014; Heidrich et al., 2021; Pantazaka et al., 2021), because tumor cells to detach from the primary tumor and migrate into the bloodstream must lose their epithelial characteristics and acquire mesenchymal features, through a process called epithelial-mesenchymal transition (EMT) (Kallergi et al., 2011; Nieto et al., 2016; Satelli et al., 2017). Therefore, there is an urgent need to identify these aggressive subclones of CTCs, that are responsible for generating metastases, through their expression of distinct biomarkers (Kallergi et al., 2013).

Specific intracellular signal transduction pathways promote Ca²⁺ to be released from the endoplasmic reticulum (ER) stores. For normal cells to retain their homeostasis, store-operated calcium entry (SOCE) is used for Ca²⁺ intracellular control (Xu et al., 2015; Zhang et al., 2017). SOCE is an ion channel consisting of two important components, stromal-interacting molecule 1 (STIM1) and calcium release-activated calcium channel protein 1 (ORAI1). When Ca²⁺ levels decrease, STIM1 as a calcium sensor, aggregates and moves to membrane areas interacting with pores formed from ORAI1. STIM1 mediates ORAI1 channel activation to promote Ca²⁺ influx through SOCE (Zhang et al., 2017). SOCE is the major influx of Ca²⁺ in non-excitable cells (Zhou et al., 2017), however, high expression of STIM1, ORAI1, and activated SOCE were found to be involved in cancer progression, migration, and metastasis for cervical cancer (Chen et al., 2011; Chen et al., 2013), breast cancer (Yang et al., 2009) and hepatocarcinoma (Yang et al., 2013).

According to previous publications from our group and other research teams, STIM1 and ORAI1 are implicated in metastasis of prostate cancer, as it has been shown to promote invasion, migration, and EMT in prostate cancer cells (Xu et al., 2015; Stagno et al., 2017; Zhou et al., 2017). We have recently shown that ORAI1 and STIM1 expression was controlled by KDM2B which is a histone demethylase belonging to the F-box protein family, regulating phosphorylation-dependent ubiquitination. KDM2B is a novel oncogene, and it is upregulated in a wide range of cancers. It is implicated in SOCE calcium signaling, actin cytoskeleton rearrangement, and cell migration (Zacharopoulou et al., 2018a; Zacharopoulou et al., 2018b).

Since CTCs are the major players in metastasis in the current study, we investigated for the first time the expression of STIM1, ORAI1, and KDM2B simultaneously in Circulating Tumor Cells from the same blood draw of metastatic prostate cancer patients. We also correlated our results with clinical data to identify potential aggressive CTCs’ subclones and evaluate the clinical relevance of the aforementioned biomarkers.

CTCs have a fundamental role in metastatic dissemination by infiltrating the bloodstream and migrating to establish distant lesions (Chambers, Groom, and MacDonald 2002). They are considered the seeds of metastasis since it has been recently proved that they create cancer and promote metastasis in mice xenograft models (Baccelli et al., 2013; Hodgkinson et al., 2014). It is also widely accepted that most of the CTCs harbor both epithelial and mesenchymal properties (Kallergi et al., 2011; Nieto et al., 2016; Satelli et al., 2017; Kallergi et al., 2018; Ntzifa et al., 2021). However, the mesenchymal nature of CTCs poses a challenge to their isolation (Kallergi et al., 2011; Satelli et al., 2017; Zavridou et al., 2020). Many current isolation methods rely on epithelial markers, such as CellSearch, which is the golden standard for CTC detection particularly in metastatic breast (Cristofanilli et al., 2004), colorectal (Gazzaniga et al., 2013), and prostate cancer (Olmos et al., 2009). However, these methods fail to detect CTCs under EMT process, due to the absence of EpCAM and other epithelial antigens (Adams et al., 2015; Hyun et al., 2016; Messaritakis et al., 2017; Satelli et al., 2017; Zavridou et al., 2020; Ntzifa et al., 2021). In an older study, 34 human breast cancer cell lines were tested. Normal-like breast cancer cell lines were not identified from the CellSearch system (Sieuwerts et al., 2009). Another study in prostate cancer patients showed that identification of CTCs with an antibody against cell-surface vimentin had higher sensitivity and specificity, while in castration-resistant patients, more CTCs were identified compared to CellSearch (Satelli et al., 2017). A previous study of our group, using Ficoll density gradient and immunofluorescent staining, in patients with small cell lung cancer, showed that CK⁺/Vim⁺ CTCs (EMT CTCs), could be detected in patients whereas in the same samples, CellSearch had negative results. Furthermore, this phenotype was correlated with poor survival (Messaritakis et al., 2017). Another study examined the isolation of CTCs from 30 patients with breast and prostate cancer, through a microfiltration system (CellSieve) and the CellSearch system; showed that the CellSieve isolated more CTCs as opposed to the CellSearch, and they could be divided into five morphologically distinct subpopulations, characteristic of CTC heterogeneity (Adams et al., 2015). Overall the heterogeneous nature of CTCs is a well-described observation in terms of their morphological characteristics, protein expression patterns, and molecular profile (Polzer et al., 2014; Adams et al., 2015; Hyun et al., 2016). Therefore, new biomarkers are necessary to improve the identification rate of CTCs and to provide potential new therapeutic targets.

CTCs can persist either individually or as clusters, remaining dormant for years until they give rise to new lesions, underscoring their resistance to conventional therapies (Vishnoi et al., 2015; Matikas et al., 2022). The reason for this resistance is mainly their non-proliferating physiology making them unable to be destroyed by drugs targeting mitosis or cell proliferation. However, in castration, resistant prostate cancer patients’ chemotherapy is the main therapeutic strategy. Thus, it is extremely important the identification of new therapeutic targets in CTCs for improving survival of prostate cancer patients. It has also been proved that the enumeration of CTCs is not as important as finding the specific subpopulations which are the most aggressive subclones, related to patients’ metastasis (Kallergi et al., 2013; Satelli et al., 2016). Interestingly, recent studies showed that CTCs can be destroyed by targeted therapy, and this approach is related to patients’ survival (Georgoulias et al., 2012; Fehm et al., 2021).

On the other hand, store-operated calcium entry (SOCE), a primary mechanism for cellular calcium influx, is controlled by a store-operated calcium channel composed of ORAI1 and STIM1 proteins. The oncogenic potential of ORAI1/STIM1 has been proved, as their inhibition has been shown to suppress proliferation, migration, and invasion. Previous research from our group has demonstrated that lithium significantly enhances ORAI1 and STIM1 transcript and protein levels, along with increasing SOCE in the neurodegenerative disease Chorea-Acanthocytosis. These effects were reversed upon treatment with wogonin, an NFκB inhibitor (Sukkar et al., 2018), suggesting that this pathway holds promise for future exploration. We have also recently shown that overexpression of KDM2B in prostate cancer cells resulted in a significant upregulation of ORAI1 and Stim1 transcription. Additionally, KDM2B upregulation increased the mRNA expression of Nhe1, a Na+/H+ exchanger. Notably, ORAI1, STIM1, and Nhe1 have all been reported to be upregulated by SGK1. Previous studies have underscored the significance of SGK1 in upregulating SOCE and influencing migration and cell survival (Lang et al., 2010; Lang and Stournaras, 2013; Lang et al., 2019). In the same study, we explored the expression of KDM2B in CTCs of a small cohort of prostate cancer patients. It was interesting that 80% of the CTC-positive patients harbored KDM2B-positive CTCs. Based on the previous data the current study investigated at protein level the expression of ORAI1, STIM1, and KDM2B in CTCs from prostate cancer patients. This study confirmed the increased expression of KDM2B in CTCs from prostate cancer patients revealing that 85% of the patients harbored KDM2B-positive CTCs, which is line with the results from the smaller group of patients, implying that this expression is a common phenomenon for prostate cancer.

The method of ORAI1 and STIM1 staining was developed using PC-3 and DU-145 spiked in PBMCs cells. Interestingly as it is shown in Figure 1 the ORAI1 and STIM1 were exclusively expressed in cancer cells and not in the nearby PBMCs making them interesting biomarkers for identifying CTCs in the bloodstream. We also investigated this expression in prostate cancer patients’ CTCs, isolated with Ficoll density gradient. We found that the vast majority of prostate cancer CTCs had simultaneously both STIM1 and ORAI1 overexpression (94%). Only one patient had one CTC with STIM1 and not ORAI1 (6%) expression. None of the patients had CTCs with ORAI1 overexpression alone and none of the patients had CTCs in with absence of both STIM1 and ORAI1. In addition, all 100% of the CK-positive patients harbored CTCs with STIM1 and 94% with ORAI1 expression. The above indications are very interesting for two reasons: a) These biomarkers have extremely high expression in CTCs, higher than most of the other identified biomarkers so far, making them important potential targets for the corresponding therapeutic approaches, b) The expression of ORAI1 and STIM1 in PBMCs is very low implying that they can be interesting biomarkers for the identification of these cells in the bloodstream. These results are in line with previous publications, referring that STIM1 and ORAI1 are implicated in metastasis in prostate cancer (Xu et al., 2015; Stagno et al., 2017; Zhou et al., 2017). STIM1 overexpression has been shown to promote cell migration and EMT through TGF-β, Snail and Wnt/β-Catenin signaling in prostate cancer cell lines of PC-3 and DU-145 (Xu et al., 2015). STIM1 knockdown inhibited migration and invasion of PC-3 and DU-145 cells through the inactivation of PI3K/Akt signaling pathway while it was also proposed as a potential target against prostate cancer metastasis (Zhou et al., 2017). In a previous work of our group, a tested drug called istaroxime caused a strong inhibition of cell migration and motility in treated DU-145 cells, through the reduction of Stim1, ORAI1 transcripts, ORAI1 protein, and activated SOCE and FAK. The inhibition of cancer cell migration from istaroxime was further enhanced by blocking ORAI1 and FAK with the corresponding inhibitors (Stagno et al., 2017).

This study also revealed that the expression of the double positive phenotype (CK+STIM1+/ORAI1+) is related to bone metastasis and the (CK+STIM1+/ORAI1–) phenotype to disease recurrence. This is also in line with a previous study showing that STIM1 mediates cell invasion and bone metastasis in prostate cancer patients (Zhou et al., 2023). In the current study, the correlation of this phenotype with bone metastasis underlies the important role of these cells and the necessity of their enumeration and characterization in every patient in the precision medicine approach.