Introduction
The National Cancer Institute of the United States estimated that approximately 175,000 new prostate cancer cases will be identified in 2020, while there will be a projected 32,000 cancer-related deaths among American males. Approximately 14% of men will acquire the disease throughout their lifetimes (1). To date, radiation therapy, chemotherapy and hormone therapy are the most common treatments for PC. Radiation therapy is not ideal as it destroys surrounding healthy cells and tissues, leading to a number of side-effects. Hormone treatment implies clinical castration and the usage of anti-androgens, that can have adverse effects and can affect the lifestyle of the recipient (2). Moreover, PC treatments involve the use of chemotherapeutic agents, such as docetaxel. Docetaxel only increases the survival time of the patient by approximately 100 days, primarily due to the emergence of resistance (3).
In previous studies by the authors, protein kinase C-ι (PKC-ι) was recognized as a major driving factor of prostate and melanoma carcinogenesis, and was therefore proposed as a potential and novel therapeutic target (4-10). PKC is a part of the protein kinase enzyme class that post-translationally modifies certain other proteins and participates in a variety of cellular signaling cascades. A total of 15 PKC isoforms are recognized in humans; these are further grouped as classical, novel and atypical PKCs (aPKCs). aPKCs comprise two structurally and functionally distinctive isoforms; PKC-ζ and PKC-ι (11-13). Other than PC and melanoma, PKC-ι has been found to function as an oncogene in several other cancer types, such as neuroblastoma, ovarian cancer and glioma, where the upregulation of PKC-ι expression has been shown to be associated with a low survival rate (10,11,14). In previous studies by the authors, it was also reported that higher levels of PKC-ι were observed in DU-145 and PC-3 cells compared to undetectable levels in normal tissues and the normal prostate epithelial cell line, RWPE-1 (7,10). In addition, it was demonstrated that PKC-ι phosphorylates to activate IκB kinase (IKKα/β). This p-IKKα/β activation triggers the dissociation of nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor (IκB) from the nuclear factor NF-κB complex, which leads to the ubiquitination of IκB. IκB releasing from NF-κB activates the translocation of NF-κB to the nucleus. Previous studies by the authors suggested that PKC-ι specific leads to the suppression of NF-κB nuclei translocation, thereby causing a downregulation of NF-κB activity (9,10). These findings indicated that the inhibition of PKC-ι not only impaired pathways regulated by PKC-ι, but also downgraded its protein expression (7-10). There are data to suggest that PKC-ι maintains a self-propagative mechanism, as observed in certain other cancer-related cycles, such as the transformation of the growth factor (TGF)-β and CD147(15). Since transcription factors play a pivotal role in gene expression, the aim of the present study was to determine which transcription factors were key PKC-ι regulators, as well as which pathways were integral components for these transcription factors.
In the present study, the outcomes of the knockdown of the expression of c-Jun, FOXO1, PKC-ι and NF-κB are demonstrated, giving emphasis to pathways associated with PKC-ι. The findings suggested that c-Jun is a crucial transcriptional activator, while FOXO1 functions as a transcriptional suppressor of PRKCI expression. The roles performed by these transcription factors were determined in an inflammatory process that promotes PKC-ι and is dependent on PKC-ι for the continuation of the process. Moreover, the pathway through which cytokines stimulate PKC-ι expression to release activated NF-κB is demonstrated, which leads to the production of additional cytokines; this process is used in certain types of cancer as part of a loop through which to grow and propagate. In addition, IL-8 promotes c-Jun and NF-κB signaling to enhance PKC-ι production. On the other hand, PKC-ι inhibition induces the production of ICAM-1, which promotes FOXO1 to reduce PKC-ι production. In general, these findings indicate that with a dynamic and closely controlled expression profile, PKC-ι plays a central role in the development of prostate cancer. The specific inhibition of PKC-ι may interact with its own regulatory process, contributing to a distortion of its oncogenic function in prostate cancer.
Discussion
In our previous study, the selective binding of ICA-1T to an allosteric site located in the C-lobe of PKC-ι kinase domain was recognized. This binding led to the inhibition of PKC-ι activity (9). This consequentially leads to a reduction in cellular differentiation, proliferation, migration and invasion, whilst simultaneously driving the apoptosis of prostate cancer cells via the diminution of the NF-κB pathway in vitro. Subsequently, PKC-ι was established as a key factor in the induction of cell growth, differentiation and survival (8,9,11). It was also recognized that PKC-ι undergoes self-regulation as a consequence of its inhibition and a decrease in its expression in the PC-3 and DU-145 cell lines. Thus, the aim of the present study was the identification of the underlying processes of PKC-ι regulation in the aforementioned cell lines in vitro.
In order to investigate PKC-ι regulation and expression, the roles of transcription factors which interacted with the PRKC1 promoter region were investigated. The gene which codes of PKC-ι is the PRKCI gene, which is positioned on chromosome 3 (3q26.2), which is an amplicon known to undergo replication events (20). In order to deduce key TFs in PRKCI regulation, a sequence encompassing the PRKCI promotor with a motif feature was selected, as well as a promoter flank and an enhancer. This was selected as it provides the ideal platform in which the TFs can bind to regulate transcription. Two systems, PROMO and Genomatix Matinspector, were utilized to predict probable transcription factor bindings. This led to the identification of 5 TFs two of which were FOXO1 and c-Jun. Subsequently, these TFs were silenced in order to analyze the downstream effect they would have on PKC-ι expression.
c-Jun was the first transcription factor found to be associated with numerous types of cancer, including metastatic breast cancer and non-small lung cancer (21). It functions through the formation of an early response complex containing AP-1 and c-Fos (22). The activation of c-Jun occurs via phosphorylation events by c-Jun N-terminal kinases (JNKs) on S63 and S73, and is regulated via multiple extracellular stimuli, i.e., cytokines (23). Upon phosphorylation at S63 and S73, not only is c-Jun activated, but it also leads to an increase in the transcription of c-Jun-targeted genes. Extracellular signal-regulated kinase (ERK) is also upregulated by activated c-Jun (24-26). c-Jun is also known to promote the oncogenic transformation of ‘ras’ and ‘fos’ in several cancer types (27,28). FOXO1 is known to play a role in regulating various metabolic pathways, such as gluconeogenesis, adipogenesis and insulin signaling. Similar to c-Jun, phosphorylation plays a crucial role in FOXO1 function (29,30). FOXO1 is deactivated by AKT through phosphorylation on T24, leading to the induction of nuclear exclusion, which leads to ubiquitylation (31,32). Therefore, it is important to note that the phosphorylation of FOXO1 indicates its inactivation and the downregulation of FOXO1 signaling. In relation to cancer, FOXO1 is a well-established tumor suppressor (33-35). As such, there is a known association between FOXO dysregulation and cancer progression, as it is also plays a role in both intrinsic and extrinsic pathways of apoptosis (36,37). Experiments in vitro and in vivo have confirmed that the overexpression of FOXO1 causes a reduction in cell migration, proliferation and tumorigenesis in cancer cells (38). Furthermore, ERK1/2, PKC-ι and AKT can downregulate FOXO1(35). Thus, in the present study, it was demonstrated that through the specific inhibition of PKC-ι, the expression of active PKC-ι decreases, which renders it ineffective in its role to deactivate FOXO1 through phosphorylation events. This is a crucial indication of PKC-ι involvement in the regulation of its own expression, as PKC-ι inhibition leads to the continuous upregulation of FOXO1.
At the same time, previous data have demonstrated that the inhibition of PKC-ι causes the significant downregulation of the PI3K/AKT pathway and in particular, downregulates the activation of AKT (10). In the present study, as shown in Figs. 3 and 6, NF-κB downregulation led to elevated levels of active c-Jun (phospho c-Jun), which upregulated PKC-ι expression. These results validate our previous observation that PKC-ι inhibition, by which the phosphorylation of IKKα/β is reduced, inhibits NF-κB activation and translocation to the nucleus (10). Subsequently, NF-κB depletion induces an increase in c-Jun expression, which then attempts to increase the production of PKC-ι, which then needs to phosphorylate IKKα/β to restore NF-κB signaling. The tight regulation of PKC-ι expression through c-Jun may explain these results as it enhances PRKCI transcription. There was also no significant alteration in the levels of FOXO1 and phosphorylated FOXO1 resulting from NF-κB siRNA knockdown. This suggests that the downregulation of NF-κB does not disrupt PKC-ι expression through FOXO1, but rather that c-Jun provides cancer cells with resistance to apoptosis through interplay with NF-κB upon cytokine stimulation (21). In our previous study, it was demonstrated that in melanoma, TNF-α upregulates NF-κB, phosphor-AKT and PKC-ι expression (9). However, the results of the present study demonstrate that c-Jun ‘switches on’ PKC-ι expression and FOXO1 ‘switches off’.
Apart from identifying c-Jun and FOXO1 out of 5 TFs which could bind to the PKC-ι gene promoter region, other key molecular factors were also identified. Through the conduction of ELISA using IPAD assay and a cytokine array, crosstalks between multiple pathways were examined. The data indicated links between PKC-ι expression with cytokines IL-8 and ICAM-1, along with some other key cellular signaling points.
As shown in Fig. 4, the IPAD ELISA data revealed that there was a significant increase in the expression levels of p-STAT3 (Y705), p-JNK (T183) and p-mTOR, whilst displaying a significant decrease in p-AKT (S473), p-β-catenin and CD44 levels. Moreover, it has been demonstrated that irregular STAT3/5 is associated with the progression of various cancer types (39-44). Cell survival in multiple cancers has been shown to be induced by upregulated STAT signaling, which is often stimulated by the cytokines, IL-6 and IL-8 (39,40,45). STAT3 signaling enhances the production of c-Jun, thereby inducing c-Jun-targeted transcription (39,46). The IPAD data of the present study strongly suggested that STAT3 was upregulated due to PKC-ι inhibition, suggesting that the deprivation of PKC-ι tries to accelerate the production of c-Jun through the upregulation of STAT3, JNK and mTOR. Connections between the JNK pathway and FOXO1 have been explored in few studies (35,47,48). Hornsveld et al summarizes the tumor-suppressing features of FOXO1 resulting in a decreased JNK activity (47). Whilst JNK activates c-Jun, by contrast, PKC-ι inhibition renders it ineffective at increasing c-Jun or phospho-c-Jun levels, as can be seen in Fig. 3. Instead, the FOXO1 levels were increased, while the phosphor-FOXO1 levels along with the levels of phosphor-AKT and STAT3 were reduced in both cell lines. This demonstrates that the activation of FOXO1 leads to a reduction in c-Jun levels by blocking the activity of phosphor-JNK. Therefore, it was deduced that FOXO1 plays a major role in c-Jun regulation only upon PKC-ι inhibition. This process likely employs multiple mechanisms, such as JNK signaling inhibition, causing the further retardation of PKC-ι expression, which will eventually lead to cell cycle arrest. This is further corroborated by FOXO1 being established as being able to induce cell cycle arrest. It accomplishes this through the promotion of the transcription of cell cycle kinase inhibitors or cyclin-dependent kinase inhibitor (CKI). p21 and p27 are two of the most well-known FOXO-induced downstream CKIs (35,47). FOXO1 has also been shown to be associated with the induction of anoikis (apoptosis that occurs when cells detach from the extracellular matrix) (47). Once again, this displays another downstream effect of PKC-ι involvement, as the inhibition of its expression augments FOXO1 antitumor activity.
As shown in Fig. 7, it is summarized that the expression of PRKCI is negatively affected by FOXO1, whilst being positively affected by c-Jun. The inhibition of PKC-ι leads to the following downstream effects. The downregulation of NF-κB activity through the lack of phosphor-IKKα/β, decreases the levels of phosphor-AKT (S473), thereby diminishing AKT activity. Subsequently the low activity of AKT, along with PKC-ι, lead to the decreased phosphorylation of FOXO1, also leading to elevated levels of active unphosphorylated FOXO1. These elevated levels of activated FOXO1 lead to the further suppression of PRKCI gene expression. This acts as a ‘switch off’ effect on PRKCI expression. PKC-ι downregulation also leads to decreased STAT3, mTOR and JNK signaling. As a consequence, this reduces c-Jun activity, leading to the cancellation of the positive effects of c-Jun towards PKC-ι expression. Furthermore, STAT3 and STAT5 upregulate NF-κB transcription in addition to c-Jun (46,49). Due to this, it was deduced that PKC-ι inhibition causes the downregulation of NF-κB and STAT3, leading to a decrease in both the transcription and activation of c-Jun. Therefore, these data suggest that the PKC-ι levels were decreased when c-Jun expression was silenced by siRNA (Figs. 1 and 6B).
In the present study, further in vitro experiments (Figs. 5 and 6A) demonstrated the deviations in cytokine expression (IL-8 and ICAM-1) in the PC-3 and DU-145 cells upon PKC-ι knockdown. In both cell lines, the protein levels of IL-8 and ICAM-1 (as well as their mRNA levels) were shown to undergo a significant intensification following PKC-ι knockdown by siRNA, as proven by western blot and RT-qPCR analyses. These data suggest that PKC-ι self-regulation is involved in autocrine signaling. Tumor cellular environments, with prostate cancer in particular, are constantly exposed to a variety of immune cells and inflammatory factors. The effects of which function to either promote chronic inflammation or engage in antitumor activity (50). Examples of these inflammatory factors are cytokines; they play a crucial role in controlling the tumor microenvironments (51). To achieve their functions, cytokines utilize multiple signaling pathways. They can either act to promote or downregulate tumor progression and metastasis. Examples of tumor promoting cytokines are; as CXCL-1, CXCL-12, IL-18, CXCL-10, IL-6 and IL-8. CXCL1, also known as melanoma growth-stimulatory activity/growth-regulated protein α, functions in processes of wound healing, angiogenesis and inflammation after being secreted by cancer cells. It has also been linked to tumor formation (52). Metastatic regulation has also been linked to high levels of CXCL10/CXCR3, with CXCL10 playing an important role in the promotion of tumor growth and metastasis (52). Metastatic regulation has also been linked to high levels of CXCL10/CXCR3, with CXCL10 playing an important role in the promotion of tumor growth and metastasis (52,53). CXCL12 (stromal-derived factor-1) utilizes the receptors CXCR4 and CXCR7 and it has been linked to playing a role in the regulation of tumor metastasis. However, CXCL-10, CXCL-12 and IL-18 were not observed as being significantly altered as result of PKC-ι inhibition.
IL-6 has been linked to the stimulation of the degradation of IκB-α, which in turn results in the upregulation of NF-κB translocation. As previously demonstrated, PKC-ι stimulates NF-κB translocation through IκB-α degradation (9). Upon translocation to the nucleus, NF-κB induces cell survival via the transcription of multiple survival factors and cytokines (39,45,53), with IL-8 being one such cytokine. It plays a key role in the regulation of polymorphonuclear neutrophil mobilization. It is also associated with the extravasation in the steps of cancer metastasis. IL-8 has been shown through studies to be regulated by NF-κB in prostate cancer cells. As such, an increased IL-8 expression has been connected to the promotion of a favorable microenvironment for metastasis (54,55). Notably, the findings of the present study demonstrated that upon transfection with PKC-ι siRNA, the IL-8 expression levels increased. This may be a result of a backup mechanism in order to upregulate IL-8. IL-8 also plays an essential role in upregulating c-Jun through JNKs. As the inhibition of PKC-ι downregulates c-Jun, the cells may be attempting to reinstate these downregulated pathways by a higher IL-8 production. As shown in Fig. 7, the expression of IL-8 is regulated through both NF-κB and STATs. These results indicate that IL-8 is important in upregulating PKC-ι expression, activating c-Jun, while deactivating FOXO1. Though it would appear that due to the high activity of FOXO1, the effect of IL-8 are canceled out.
Through utilizing an immune response, some cytokines promote antitumor activity. One such cytokine is ICAM-1, which plays a role in the immune response, including antigen recognition and lymphocyte activation (56,57). As such, ICAM-1 has beeb linked to the inhibition of tumor progression via the inhibition of the PI3K/AKT pathway. In its role in inhibiting this pathway, ICAM-1 exposes tumor cells to attack and death through cytotoxic T-lymphocytes (57). ICAM-1 expression inhibition has also been shown in clinical research to be associated with an increased risk of metastasis within the first 5 years of ovarian cancer diagnosis (57). Of note, the results of the present study demonstrated that upon the silencing of PKC-ι by siRNA, the ICAM-1 levels increased. This confirmed that upon the knockdown of oncogenic PKC-ι, antitumor/pro-apoptotic signaling was upregulated through an autocrine manner via ICAM-1. Furthermore, these results demonstrate that ICAM-1 plays an important downregulatory role in the regulation of PKC-ι expression along with FOXO1, opposite to c-Jun and IL-8.
To conclude, the results of the present study illustrate that PKC-ι plays an imperative role in its own expression via an intricate signaling grid that involves the transcriptional activation/deactivation of c-Jun and FOXO1. The inhibition of PKC-ι activity, based on its specific inhibition, downregulates the NF-κB pathway along with the transcriptional activity of STAT3 and IL-8. The results in a decrease in AKT activity that leads to FOXO1 upregulation. FOXO1 was identified to be the most important transcription factor when it comes to regulating PKC-ι, along with ICAM-1 stimulation. FOXO1 negatively regulates PKC-ι expression, diminishing JNK activity and further suppressing the activation of c-Jun. The consequence of this process is that it leads to the downregulation of NF-κB and c-Jun, and further upregulates FOXO1. This continues to deplete the PKC-ι expression, subsequently leading to a decrease in the total PKC-ι levels in prostate cancer cells. The regulation of PKC-ι is complex, and PKC-ι itself plays a key role in that process. As such, when inhibited, it leads to a decrease in PKC-ι production, prompting multiple antitumor/pro-apoptotic signaling. PKC-ι is therefore a key factor to target when attempting to treat prostate cancer in vitro. Finally, the results of the present study demonstrate that PKC-ι is not only a novel biomarker to target for personal therapeutics for prostate cancer, but also that ICA-1T shows promise as one such therapy in relation to the proposed mechanism.