Background
Prostate cancer (PCa) is the most common malignant cancer and the second leading cause of cancer-related death in men worldwide [
1]. The primary issue derived from PCa is its propensity to metastasize to bone, which occurred in up to 90% of patients with advanced PCa [
2]. Despite great advances in systemic and individualized treatments of PCa in the last decades, distant bone metastasis remains a principal issue, which severely affects the quality of life and survival time of PCa patients [
3]. Thus, it is of great importance to better understand the underlying mechanisms contributing to bone metastasis of PCa, which will facilitate the development of novel anti-bone metastasis therapeutic avenues in PCa.
Epithelial-mesenchymal transition (EMT) is an imperative phenotypic conversion that occurs during several processes, including embryonic development, tissue remodelling and metastasis, where epithelial cells obtain mesenchymal-like properties in combination with reduced intercellular adhesion and enhanced motility [
4,
5]. EMT is a transient and dynamic process that primarily emerges at the onset of invasion and is tightly controlled by several cellular signaling pathways, such as ErbB, Wnt, NF-kB and TGF-β pathways [
6‐
8]. Among these, transforming growth factor (TGF)-β is identified as the most important inducer of EMT process due to stimulation of the expression of EMT-inducing transcription factors, including Snail1, Twist1 and ZEB1/2 [
9‐
11]. Furthermore, accumulating studies have demonstrated that NF-kB signaling pathway is essential for the induction and maintenance of EMT in a large number of cancers [
7,
12,
13]. The NF-κB pathway was discovered nearly three decades ago [
14], and the critical roles of the NF-κB pathway in physiologic processes, such as immunity and inflammation, have been well documented [
15,
16]. NF-κB signaling has been reported to be constitutively activated in a number of human cancers, which contributed to the initiation and progression of a large array of malignancies [
15,
17]. Furthermore, accumulating literatures reported that NF-κB signaling plays a crucial role in the bone metastasis of various types of cancers [
18,
19]. Park and colleagues reported that constitutive NF-κB activity in breast cancer cells was crucial for the bone resorption characteristic of the osteolytic bone metastasis via transcriptionally regulating granulocyte macrophage-colony stimulating factor (GM-CSF) that mediated osteolytic bone metastasis of breast cancer by stimulating osteoclast development [
20]. Furthermore, several lines of evidence have implied that NF-κB activation was also associated with the metastatic phenotype of PCa progression [
19,
21]. Chen et al. reported that NF-κB activation was crucial for the development of PCa bone metastasis [
19]. However, the underlying mechanism responsible for constitutive activation of NF-κB signaling in the bone metastasis of PCa remains largely unknown.
MicroRNAs (miRNAs) are small endogenous non-coding RNAs that are responsible for post-transcriptional regulation of target genes by binding with specific sequences in the 3′ untranslated region (3’UTR) of downstream target genes, leading to mRNA degradation and/or translational inhibition [
22]. miRNAs play important roles in many cellular and biological processes such as proliferation, apoptosis, differentiation, metabolism, cardiogenesis, development and function of the nervous and immune systems [
22,
23]. The dysregulation of miRNAs in cancers is widely documented, and several studies have revealed a correlation of miRNAs expression levels and metastatic tumors [
24,
25]. Furthermore, several miRNAs have been reported as mediators in the bone metastasis of PCa [
26,
27]. Our previous studies demonstrated that loss of wild-type P53 induced downregulation of miR-145 promoted bone metastasis of PCa via regulating several positive regulators of EMT [
28‐
30]. These studies indicate that aberrant expression of miRNAs elicited by unknown mechanism plays a crucial role in the bone metastasis of PCa.
In this study, we report that miR-210-3p expression is elevated in PCa tissues compared with the adjacent prostate epithelial tissues (ANT). Interestingly, the expression levels of miR-210-3p increases steadily from non-bone metastatic PCa tissues, bone metastatic PCa tissues to metastatic bone tissues and high expression of miR-210-3p positively correlates with the clinicopathological characteristics and bone metastasis status of PCa patients. Furthermore, upregulating miR-210-3p enhances, while silencing miR-210-3p suppresses the EMT, invasion and migration of PCa cells in vitro. Importantly, silencing miR-210-3p significantly inhibits bone metastasis of PC-3 cells in vivo. Furthermore, our results demonstrate that miR-210-3p promotes EMT, invasion and migration of PCa cells via targeting negative regulators of NF-κB signaling (TNF-α Induced Protein 3 Interacting Protein 1) TNIP1 and (Suppressor Of Cytokine Signaling 1) SOCS1, resulting in constitutive activation of NF-κB signaling pathway. Our results further indicate that recurrent gains are responsible for miR-210-3p overexpression in a small number of PCa patients. The analysis of clinical correlation reveals that miR-210-3p inversely correlates with SOCS1 and TNIP1, but positively correlates with NF-κB signaling activity in human PCa and metastatic bone tissues. Taken together, these findings uncover a plausible mechanism responsible for constitutive activation of NF-κB signaling in bone metastasis of PCa, suggesting that miR-210-3p may serve as a novel target for clinical intervention in PCa.
Discussion
The key findings of the current study present novel insights into the critical role of miR-210-3p in the sustained activation of NF-κB signaling, which further promotes bone metastasis of PCa. Here, we reported that miR-210-3p expression was elevated in bone metastatic PCa tissues, which was caused by recurrent gains, and high expression of miR-210-3p correlated with PSA levels, Gleason grade and bone metastasis status in PCa patients. Our results further indicate that miR-210-3p activates NF-κB signaling in PCa cells via directly targeting SOCS1 and TNIP1, resulting in the development of PCa bone metastasis. Therefore, our results uncover a novel mechanism by which miR-210-3p sustains constitutive activation of NF-κB signaling, elucidating the oncogenic function of miR-210-3p in bone metastasis of PCa.
Extensive research efforts have shown that NF-κB signaling was constitutively activated in several types of human cancer, which was significantly associated with the tumor progression and metastasis [
15,
17]. For example, in glioma, activation of NF-κB signaling was crucial for the promotion of glioma cell invasion and migration [
45,
46]; in addition, a study by Helbig and colleagues has noted that expression of chemokine receptor CXCR4 was induced by activation of NF-κB signaling, which promoted the migration and metastasis of breast cancer cells [
47]. Emerging literatures have shown that NF-κB signaling plays an important role in the bone metastasis of cancers [
18,
19]. Park and colleagues reported that constitutive NF-κB activity in breast cancer cells was crucial for the bone resorption characteristic of osteolytic bone metastasis. The identified gene mediated osteolytic bone metastasis of breast cancer was a key target of NF-κB signaling: granulocyte macrophage-colony stimulating factor (GM-CSF) promoted osteolytic bone metastasis of breast cancer cells by stimulating osteoclast development [
20]. Importantly, Chen et al. reported that NF-κB activation also played a pivotal role in the development of PCa bone metastasis [
19]. However, the underlying mechanism responsible for constitutive activation of NF-κB signaling in the bone metastasis of PCa remains largely unknown. Here, we report that miR-210-3p activated NF-κB signaling through directly targeting SOCS1 and TNIP1 in PCa cells, which promoted the development of bone metastasis of PCa. Furthermore, NF-κB signaling activity repressed by the specific inhibitors attenuated the stimulatory role of upregulating miR-210-3p on invasion and migration of PCa cells. Taken together, our results indicate that high expression of miR-210-3p constitutively activates NF-κB signaling, which is essential for bone metastasis of PCa.
Numerous lines of evidence have indicated that deficiencies or downregulation of negative regulators of the NF-κB signaling pathway could lead to constitutive activation of NF-κB signaling, which further promoted tumor progression and metastasis [
48‐
50]. Multiple well-known negative regulators of NF-κB signaling, such as CYLD, TNIPs and A20, have been reported to restrict the activity of NF-κB signaling via different negative feedback mechanisms. TNIPs, which were found to exert functions by linking A20 to NEMO and accelerate A20-mediated NF-κB signaling activity inhibition through deubiquitination of NEMO, have been reported to participate in the inhibition of NF-κB signaling activity [
51]. On the other hand, extensive crosstalk between inhibitors or negative regulators of other signaling pathways, such as JAK/STAT signaling, and NF-κB signaling activity were broadly reported. For example, PIAS4, a member of the PIAS (protein inhibitor of activated STAT) protein family, which was originally identified as inhibitors of the STAT proteins, has been reported to be an important repressor of NF-κB signaling activation via regulating TRIF-induced NF-κB signaling activation [
52,
53]. Moreover, STAT3 signaling inhibitor suppressor of cytokine signaling (SOCS1) has been reported to promote the degradation of the DNA-bound p65 protein, leading to the suppression of NF-κB activity [
54‐
57]. However, how cancer cells simultaneously take priority over these feedback loops in PCa remains obscure. In this study, our results demonstrated that high expression of miR-210-3p constitutively activated NF-κB signaling via simultaneously suppressing negative regulators of NF-κB signaling TNIP1 and SOCS1, resulting in the bone metastasis of PCa. Therefore, our finding uncover a novel mechanism by which miR-210-3p disrupts the negative feedback loops of NF-κB signaling in PCa cells, which results in constitutive activation of NF-κB signaling, supporting the notion that NF-κB signaling contributes to the bone metastasis of PCa.
Hypoxia has been identified as a critical contributor to the tumor development, progression and metastasis, where the hypoxic environment exerts its functions via inducing the production of hypoxia inducible factor (HIF), which then transcriptionally activates a wide array of downstream molecules for adaptation to the hypoxic condition [
58,
59]. The bone marrow microenvironment possesses extensive hypoxic regions [
42,
43] that are characterized by abundant HIF-1α staining and HIF target proteins including MCT4 and Glut1 [
44]. It’s notable that the hypoxic microenvironment of the bone marrow is conductive to subsequent bone colonization of cancer cells, and therapies targeting HIF/HIF targets has potential value in the prevention of bone colonization [
60‐
62]. Furthermore, accumulating studies revealed that miRNAs are emerging as a novel class targets of hypoxia-responsive molecules [
63,
64]. It’s worth noting that miR-210-3p has been broadly demonstrated to be a direct target of HIF-1α in a variety of tumor cells [
40,
41]. Therefore, it’s conceivable that miR-210-3p expression in bone tissues will be elevated compared with primary PCa tissues due to the inducible effects of abundant HIF within the hypoxic bone marrow microenvironment. Indeed, our results revealed that miR-210-3p expression in metastatic bone tissues was upregulated compared with primary PCa tissues. Furthermore, several lines of evidence reported that activation of NF-κB signaling promoted the attachment and growth of cancer cells in bone via upregulating multiple osteoclastogenesis-associated genes, including RANKL, PTHrP and GM-CSF, resulting in osteolytic bone metastasis of cancer [
20,
65]. In this study, our results demonstrated that overexpression of miR-210-3p augmented the NF-κB signaling activity via targeting TNIP1 and SOCS1 in PCa cells. Therefore, these findings suggest that a hypoxic bone microenvironment promotes bone colonization of cancer cells to bone via activation of miR-210-3p/ NF-κB signaling axis, which contributes to the development of bone metastatic disease in PCa.
Several studies have indicated that miR-210-3p was upregulated in multiple human cancers and that high expression of miR-210-3p promoted cancer cell invasion and metastasis via different mechanisms and predicted poor survival [
40,
41,
66‐
68]. Furthermore, recent literatures have identified miR-210-3p as a serum marker in many types of cancer, which will facilitate the early detection of metastatic tumors [
68,
69]. Notably, Tewari and the colleagues reported that miR-210-3p was dramatically elevated in the serum of metastatic castration resistant prostate cancer patients compared with healthy controls, indicating that miR-210-3p was involved in the metastasis of PCa [
41]. Moreover, a study by Taddei showed that hypoxia-induced miR-210 in fibroblasts enhanced the senescence-associated features, which promoted PCa aggressiveness by inducing EMT and by secreting energy-rich compounds to support PCa cell growth [
70]. However, the biological roles and clinical significance of miR-210-3p in bone metastasis of PCa remains largely unknown. In this study, our results revealed that miR-210-3p was elevated in human bone metastatic PCa tissues and cells. High expression of miR-210-3p correlated with serum PSA level, Gleason grade and distant bone metastasis status in PCa patients. Moreover, our results revealed that miR-210-3p activated NF-κB signaling via targeting TNIP1 and SOCS1, which further promoted the EMT, invasion, migration and bone metastasis of PCa cells in vitro and in vivo. Furthermore, our finding demonstrated that recurrent gains are the underlying mechanism contributing to miR-210-3p overexpression in bone metastatic PCa tissues. Collectively, our findings indicate that miR-210-3p plays an important role in the bone metastasis of PCa.