MAZ promotes prostate cancer bone metastasis through transcriptionally activating the KRas-dependent RalGEFs pathway

The human PCa cell lines 22RV1, LNCaP, DU145, PC-3, VCaP, and normal.

prostate epithelial cells RWPE-1 were purchased from the Shanghai Chinese Academy of Sciences Cell Bank (China). RWPE-1 cells were cultured in Keratinocyte-SFM (1×) (Invitrogen, USA). PC-3, 22Rv1, and LNCaP cells were grown in RPMI-1640 medium (Life Technologies, USA) supplemented with penicillin G (100 U ml^− 1), streptomycin (100 mg ml^− 1) and 10% fetal bovine serum (FBS, Life Technologies, USA). The C4-2B cell line was purchased from the MD Anderson Cancer Center and cultured in T-medium (Invitrogen) supplemented with 10% FBS. DU145 and VCaP cells were grown in Dulbecco’s Modified Eagle’s Medium (Invitrogen, USA) supplemented with 10% FBS [7].

Human MAZ cDNA was PCR-amplified and cloned into the pMSCV-puro-retro vector (Clontech). Two shRNAs against MAZ,KRas and HRas in the pSuper-puro vector were obtained from Sigma-Aldrich. Transfection of plasmids was performed using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, California, USA) based on the manufacturer’s instructions. Cells (2 × 10⁵) were cultured and infected using a retrovirus produced by pMSCV-puro-MAZ, pSuper-puro-MAZ-shRNA pSuper-puro-KRas-shRNA or pSuper-puro-HRas-shRNA for 3 days. Stable cell lines expressing MAZ, MAZ-shRNAs, MAZ with HRas-shRNAs or MAZ with KRas-shRNAs were selected with 0.5 μg/mL puromycin for 7 days. The human KRas and HRas gene cDNA were obtained from Vigene Biosciences (Shandong, China) and cloned into the pSin-EF2 plasmid (Cambridge, MA, USA). Small interfering RNA (siRNA) for MAZ (siRNA1#: CCUCAACAGUCACGUCAGATT; si RNA2#: AGGUUUUAACGAUUUGUUUTT); KRas (siRNA1#: GCCUUGACGAUACAGCUAATT; siRNA2#: CUAUGGUCCUAGUAGGAAATT) and HRas (siRNA1#: ACACCAAGUCUUUUGAGGATT; siRNA2#: AUGGGAUCACAGUAAAUUATT) consisting of the knockdown and respective scramble RNAs were synthesized and purified by RiboBio. Transfection of plasmids was performed using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, California, USA) in accordance with the manufacturer’s protocol.

The 104 archived fresh specimens, including 53 non-bone metastatic PCa tissues and 36 bone metastatic PCa tissues, and 15 metastatic bone tissues were acquired from The First People’s Hospital of Guangzhou City (Guangzhou, China) between January 2008 and March 2018.To use those clinical specimens for research, patients’ consent and approval from the Institutional Research Ethics Committee were acquired, and in accordance with the Declaration of Helsinki. The 267 paraffin-embedded PCa tissues, including 158 PCa tissues with bone metastasis, 85 PCa tissues without bone metastasis and 24 metastatic bone tissues were also obtained from the First People’s Hospital of Guangzhou City (Guangzhou, China) between 2008 and 2018. The clinicopathological features of the patients are listed in Table 1. The median MAZ expression in PCa tissues was used to distribute the samples by high and low expression of MAZ.

Western blotting was carried out in accordance with a standard method, as previously described. Antibodies against H-Ras (clone-A485), K-Ras (clone-3B10-2F2), N-Ras (clone-2A3), p-ERK1/2(clone-AW39R), ERK (clone-NP2), p-AKT(S473)- polyclonal, p-AKT(T308)-(clone-NL50), AKT (clone-SKB1) and alpha-tubulin (clone-B-5-1-2) were purchased from Sigma-Aldrich (USA).

Cells (4 × 10⁴) were seeded in 24-well plates and cultured for 24 h. Then, Cells were transfected with 250 ng pNFκB-luc, p (CAGAC)12-luc, TOP-Flash, pRaf-luc, EGFR-shc-luc, pGA981–6-luc luciferase reporter plasmid or control plasmids reporter luciferase plasmid, in addition to 5 ng pRL-TK Renilla plasmid (Promega) using Lipofectamine 3000 (Invitrogen) following the manufacturer’s instructions. Both Luciferase and Renilla signals were calculated 36 h after transfection employing a Dual-Luciferase Reporter Assay Kit (Promega) through the manufacturer’s recommendations.

The invasion and migration experiments were conducted following a standard method, as described previously. After culturing for 24–48 h, the cells penetrated the coated membrane to the lower surface, where they were fixed with 4% paraformaldehyde and stained with haematoxylin. The cell number was counted under a microscope (× 100).

Total RNA from cells or tissues was extracted using Trizol reagent (Invitrogen) following the manufacturer’s protocol. The extracted RNA.

was pretreated with RNase-free DNase, and 2 g of RNA from each sample was used for cDNA synthesis primed with random primer. Complementary DNA (cDNA) was amplified and quantified on a CFX96 real-time system (BIO-RAD, USA) with iQ SYBR Green (BIO-RAD, USA). The primers are listed in Additional file 6: Table. Primers for MAZ, KRas and HRas were purchased from by RiboBio (Guangzhou, China). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was conducted as a control. The relative fold expression was calculated using the comparative threshold cycle (2^-ΔΔCt) method.

A TaqMan CNV assay for MAZ (Hs01274263_cn, Applied Biosystems [ABI]) was conducted through the manufacturer’s instructions. TaqMan CNV reactions were carried out in triplicate with the FAM-dye-labelled assay for MAZ and VIC-dye-labelled RNaseP assay as a reference gene. The relative quantity analysis to calculate copy number was conducted by CopyCaller Software V2.0 (ABI). To confirm the results, all samples were run twice through an independent experiment.

All animal experiments were authorized by The Institutional Animal Care and Use Committee of Sun Yat-sen University. The approval number is L102012017080Q. The laboratory animal welfare was based on the state Standard animal-guideline of the People’s Republic of China. For the animal model, after the anesthetic, BALB/c-nu mice (5–6 weeks old, 18–20 g) were inoculated with 1 × 10⁵ PC-3 cells in 100 μl of PBS through left cardiac ventricle injection. Bone metastases were detected through bioluminescent imaging (BLI) following the previous description [9]. Bone metastasis lesions were indicated on radiographs in the bone as previously described [7]. For survival detection, mice were monitored every day for any indications of discomfort and were either euthanized all at one time or individually when they presented signs of distress, such as 10% loss of body weight, head tilting or paralysis. For the intra-tibia injection animal experiment, BALB/c-nu mice (5–6 weeks old, 18–20 g) were anesthetized using isoflurane, and 2 × 10⁶ VCaP cells in 20 μL were injected into the proximal end of the tibia through a 25-G needle attached to a 100-μL Hamilton syringe [30]. Tumor lesions were monitored by BLI and X-ray.

The MAZ, HRas, and KRas expression levels were detected by immunohistochemistry following the previous description [7]. The rating criteria of the specimen are as follows: 0, full negative; 1, < 10% positive; 2, 10–35% positive; 3, 35–75% positive; and 4, > 75% positive. Staining intensity was graded as follows: 0, full negative; 1, weak staining (light yellow); 2, moderate staining (yellow-brown); 3, strong staining (brown). The staining index (SI) was equal to the product of the staining intensity score and the proportion of positive cells. The results of the staining were assessed and scored by two independent pathologists, based on both the proportion of positively stained tumor cell and the intensity of staining. Interobserver reliability was used to examine the consistency of two independent analyses by SPSS statistics 24 software. High and low expression of MAZ were distinguished as follows: SI ≤ 4 defined as low expression of MAZ and SI > 6, defined as high expression of MAZ.

Cells (1 × 10⁷) were cultured in a 10-cm dish,and 1% formaldehyde solution in order was added to cross-link proteins to DNA. Then, the cells were harvested in 1 ml of SDS lysis buffer supplemented with 5 μl of Protease Inhibitor mixture. Cell lysates were Sonicated cells to shear DNA to fragments of 200 to 1000 bp. An equal aliquot of chromatin supernatants was incubated with anti-MAZ antibody (Sigma, #HPA048315), or an anti-IgG antibody (Millipore, Billerica, MA, USA) overnight at 4 °C with rotation. Immunoprecipitation of the DNA–protein complexes were conducted with 60 μl of Protein G Agarose for 1 h at 4 °C. After reversing the cross-linking of protein–DNA complexes to liberate the DNA, the human KRas or HRas promoter was amplified by real-time PCR. The primer sequences are listed in Additional file 6: Table S1.

Cells were cultured in 10-cm tissue dishes and lysed at 75–80% confluency; for tissues, 50–100 mg of fresh tissues were cut into small pieces. Ral activation assays were conducted following the manufacturer’s (Thermo, USA, #88314) protocol.

As previously reported, the expression level of MAZ in normal bone was relatively lower than that in several other tissues under physiological conditions [25]. Strikingly, we found that MAZ expression was significantly upregulated in metastatic bone tissues derived from PCa compared with primary prostate and other common metastatic sites, such as liver and lung, by analysing the publicly available RNA sequencing dataset of PCa from GSE74685 [31](Fig. 1a). This dramatic differential expression of MAZ between physiological and cancerous situations stimulated our interests to speculate that MAZ may correlate with bone metastasis of PCa. To confirm this hypothesis, 89 fresh PCa tissues, including 53 PCa tissues without bone metastasis (PCa/nBM) and 36 PCa tissues with bone metastasis (PCa/BM), as well as 15 metastatic bone tissues were collected. We further inspected the expression of MAZ in these tissues and found that it was upregulated in PCa/BM compared with PCa/nBM and was further increased in metastatic bone tissues (Fig. 1b). Consistently, Western blot analysis revealed a similar protein expression pattern to the mRNA expression pattern of PCa/nBM, PCa/BM and metastatic bone tissues (Fig. 1c). Immunohistochemical (IHC) staining was performed to validate the expression levels in a large number of PCa and bone samples. As shown in Fig. 1d, MAZ expression was detected in the cell nucleus, and its staining intensity was clearly upregulated in PCa/BM and further increased in metastatic bone tissues. The mRNA and protein levels of MAZ in PCa cell lines were further detected. Compared with the normal epithelial prostate cell line RWPE-1, we found that MAZ expression was significantly upregulated in the non-metastatic PCa cell line 22RV1 from a xenograft of CWR22R cells, the lymph node metastatic PCa cell line LNCaP and the bone metastatic PCa cell line VCaP and C4-2B but not in the brain metastatic PCa cell line DU145 and the bone metastatic PCa cell line PC-3 (Fig. 1e and f). These results implicate that the high expression of MAZ is correlated with bone metastasis of PCa.

To investigate the mechanism responsible for MAZ overexpression in PCa tissues, we further analysed the PCa dataset from TCGA-PRAD [32] and GSE74685 and found that the frequencies of recurrent gains (amplification) in TCGA-PRAD and GSE74685 were 6.91 and 28.2% respectively (Fig. 2a and b). In both datasets, the expression level of MAZ in cases with gains was robustly elevated compared with those with the diploid (Fig. 2c and d). Furthermore, we measured the gains levels in our own PCa cases using a TaqMan copy number assay [33] and found that gains were found in 24/104 PCa cases (approximately 23%) (Fig. 2e), and the expression level of MAZ was considerably higher in cases with gains than in those with diploid (Fig. 2f). Importantly, gains occurred in 10/15 PCa metastatic bone tissues (approximately 67%) and in 10/36 PCa tissues with bone metastasis (approximately 28%), but only in 4 out of 53 PCa tissues without bone metastasis (approximately 7.5%) (Fig. 2g). These results show that recurrent gains are responsible for MAZ overexpression in PCa tissues.

To understand in-depth the clinical significance of MAZ in PCa patients, the clinical correlation between MAZ expression and clinicopathological characteristics in PCa patients was first analysed. As shown in Table 1, MAZ overexpression positively correlated with serum PSA levels, Gleason grade and bone metastasis status in PCa patients. Kaplan-Meier survival analysis showed that PCa patients with high MAZ expression presented a shorter overall survival (OS) compared with those with low MAZ expression, as well as poor bone metastasis-free survival (Fig. 2h and i). Furthermore, high expression of MAZ predicted a poor OS and progression-free survival in PCa patients based on analysis of the PCa dataset from TCGA-PRAD (Additional file 1: Figure S1). These findings reveal that overexpression of MAZ correlates with poor prognosis as well as bone metastasis and progression status in PCa patients.

To investigate the role of MAZ in the bone metastasis of PCa, we first constructed MAZ stably expressing cell lines by ectopically overexpressing MAZ and endogenously silencing MAZ in PC-3 by retrovirus infection. (Additional file 2: Figure S2A, C). We also generated MAZ-silencing constructs in VCaP by sh-MAZ plasmid transfection. (Additional file 2: Figure S2B, C). Invasion and migration assays showed that upregulation of MAZ escalated, while silencing MAZ reduced the invasion and migration ability of PCa cells (Additional file 2: Figure S2D-G). However, overexpression of MAZ in VCaP cells has no significant effect on their invasion and migration abilities (data not shown). A mouse model of bone metastasis was used (n = 10 mice per group), in which the luciferase-labelled vector, MAZ-overexpressing PC-3 cells, scramble, and MAZ-silencing PC-3 cells were inoculated into the left cardiac ventricle of male nude mice to monitor the development of distant bone metastasis loci by BLI and X-ray. As shown in Fig. 3a and b, the MAZ-overexpressing PC-3 cells group suffered more bone metastases compared with the vector group, while the MAZ-silencing PC-3 cells group displayed fewer bone metastases compared with the scramble group by BLI and X-ray. H&E staining of tumor sections from the tibias of inoculated mice showed that upregulation of MAZ significantly aggravated while downregulation of MAZ clearly reduced the tumor burden in bone (Fig. 3c). Furthermore, MAZ-overexpressing PC-3 cells exhibited more metastatic foci and severe osteolytic areas of metastatic tumors, as well as a shorter survival and bone metastasis-free survival compared with the vector group; conversely, the mouse group injected with the MAZ-silenced PC-3 cells yielded the opposite results (Fig.3d-g). To explore the effect of MAZ on AR-positive PCa cells in vivo, an intra-tibial injection model (n = 10 mice per group) was used to observe the effect of silencing MAZ on the tumorigenic ability of VCaP cells. As shown in Additional file 2: Figure S2 H-I, the mice injected with MAZ-silenced VCaP cells displayed less bone tumor lesion compared with the control group by BLI and X-ray. H&E staining of the tumors section from the tibias of inoculated mice showed that downregulating MAZ obviously remitted the tumor burden in bone (Additional file 2: Figure S2 J). At the same time, MAZ-silencing VCaP cells exhibited less osteoblastic area of bone tumors, as well as a longer bone tumor burden-free survival time compared with the scramble group (Additional file 2: Figure S2 K-L). These results demonstrate that MAZ promotes bone metastasis of PCa cells in vivo and enhances invasion and migration abilities of PCa cells in vitro.

To determine the underlying specific signalling involved in the pro-bone metastasis role of MAZ in PCa, we further examined the effects of MAZ on multiple well-documented bone metastasis-related signalling pathways, including the TGF-β [7], Wnt [8], NF-kB [9], EGFR [10], and Notch [11] signalling, using a luciferase reporter assay. As shown in Fig. 4a, the luciferase reporter activity of Ras signalling was upregulated by overexpression of MAZ in PC-3 cells and consistently repressed by silencing of MAZ in two bone metastatic PCa cells. The three Ras genes, Hras, Kras and Nras, have been reported to be the most common oncogenes in human cancer [34, 35]. Thus, we further investigated the effects of MAZ on these three Ras protein family members in PCa cells by qPCR and Western blot analysis. We found that MAZ upregulation dramatically increased while silencing remarkably inhibited the KRas and HRas expression in PCa cells, but had no effect on NRas expression in PCa cells (Fig. 4b and Additional file 3: Figure S3A-B). Furthermore, we explored the expression of MAZ, KRAS, and HRAS in each group of bone metastasis animal model in Fig. 3. The analysis of IHC staining showed that upregulating MAZ enhanced, while silencing MAZ repressed HRAS and KRAS expression in the bone tumor tissues from tibia in different mice groups, confirming the positive correlation of MAZ with HRAS and KRAS in these PCa cells (Additional file 3: Figure S3C-F). Three critical downstream effector signalling pathways of Ras activity, namely-ERK, PI3K/AKT, and RalGEFs, were equally examined. Phospho-ERK1/2 and phosphor-AKT were considered as indicators of ERK and PI3K/AKT signalling activation in PCa cells, respectively. The RalGEF function was measured by determining the level of its enzymatic product, RalA-GTP, using a pull-down assay [36]. As shown in Fig. 4b, overexpression of MAZ raised the levels of p-Erk, p-AKT at S473 and T308 and activated RalA in PCa cells, whereas MAZ silencing had the opposite effect.

To unveil the specific mechanism by which MAZ activates RAS signalling in PCa cells, we generated three luciferase reporter constructs containing an approximately 1- kb region of the KRas and HRas promoters, respectively. As shown in Fig. 4c-d, KRas and HRas promoter activity were enhanced by co-transfection with the MAZ-overexpressing plasmid in PC-3 cells and decreased in MAZ-silenced PCa cells compared with the respective empty vector-transfected control cells. Additionally, we performed chromatin immunoprecipitation (ChIP) assays to identify the regions of the KRas and HRas promoter that might be bound by MAZ. Previous studies have indicated that the promoter region of the KRas contains a GC box and nuclease-hypersensitive element (NHE) region [37], and the promoter region of the HRAS also contains a GC box and two G-rich elements (hras-1 and hras-2) [38, 39], all of which are easily bound by the transcription factor. Therefore, according to the characteristics of its promoter region, 4 and 6 pairs of primers were designed for the promoter regions of KRas and HRas, respectively. The results showed that region 2 and 4 of the KRas promoter had the highest affinity for MAZ protein (Fig. 4e), while regions 5 and 6 of the HRas promoter had the highest affinity (Fig. 4f), implying that MAZ upregulates KRas and HRas at the transcriptional level. Taken together, our results indicate that MAZ transcriptionally activates RAS signalling in PCa cells.

As demonstrated above, MAZ transcriptionally activates KRas and HRas in PCa cells. Next, we considered whether one or both mediated the pro-metastatic role of MAZ overexpression in vitro and in vivo. A gene set enrichment analysis (GSEA) of MAZ based on mRNA expression data from the TCGA-PRAD revealed that the MAZ expression level was strongly and positively correlated with the KRas pathway (Additional file 3: Figure S3G, H). And then we constructed MAZ-overexpression with sh-HRas and MAZ-overexpression with sh-KRas stably expressing cell lines by endogenously silencing HRas or KRas in MAZ-overexpression PC-3 by retrovirus infection (Additional file 4: Figure S4A-D). The mice were divided into three groups: MAZ-overexpressing group, MAZ-overexpressing with KRas-silencing group and MAZ-overexpressing with HRas-silencing group. As shown in Fig. 5a-g, we found that KRas silencing markedly abrogated the pro-bone metastasis ability in MAZ-overexpressing PC-3 cells, including a decrease in the bone metastatic score and osteolytic area of tumors and a longer bone metastasis-free survival and OS compared with the MAZ-overexpressing group by BLI, X-ray, H&E staining and survival analysis. However, silencing of HRas had no significant effect on the bone metastasis ability of MAZ-overexpressing PC-3 cells in vivo (Fig. 5a-g). Consistently, invasion and migration assays showed that silencing KRas rescued the invasion and migration abilities of MAZ-overexpressing PC-3 cells (Additional file 4: Figure S4E, F). Thus, these results indicate that MAZ promotes bone metastasis of PCa by transcriptionally activating KRas.

Previous studies have demonstrated that different Ras isoforms exhibited quantitative and qualitative differences in their ability to activate particular downstream effector signalling [15, 40]. To discern the underlying signalling responsible for the different roles of KRas and HRas in mediating the pro-bone metastasis of MAZ overexpression in PCa, the effects of KRas and HRas on the expression levels of p-Erk, p-AKT and activated RalA were further investigated by constructing KRas or HRas expressing PCa cell lines by ectopically overexpressing KRas or HRas in PC-3 cells and endogenously knocking down KRas or HRas by retrovirus infection in PC-3 and VCaP cell lines (Additional file 5: Figure S5A-C). Western blot analysis revealed that the upregulation or silencing of KRas simultaneously increased or reduced the expression levels of p-Erk, p-AKT and activated RalA in PCa cells (Fig. 6a). However, the upregulation or silencing of HRas enhanced or inhibited the expression levels of p-Erk and p-AKT but had no influence on activated RalA (Fig. 6b). Moreover, KRas, but not HRas, had a significant effect on the invasion and migration abilities of PCa cells. (Additional file 5: Figure S5A-G). Our aforementioned results demonstrated that MAZ transcriptionally activated KRas and HRas, where only silencing KRas can rescue the pro-bone metastasis effects of MAZ overexpression in PCa (Figs. 4 and 5). Thus, these findings suggest that RalGEFs signalling may be a potential effector signalling contributing to the different roles of KRas and HRas in mediating the pro-bone metastasis of MAZ overexpression in PCa. To verify this hypothesis, three inhibitors of the activity of the three major effector pathways downstream of Ras (ERK, AKT, and RalGEFs pathway), including RBC8 for RalGEFs [41], FR180204 for ERK [42], BMK120 [43] for PI3K and API2 for AKT [44], were applied to MAZ-overexpressing PC-3 cells. Predictably, only RBC8 effectively attenuated the stimulatory effects of MAZ overexpression on the invasion and migration ability of PC-3 cells (Fig. 6c). Importantly, RBC8 dramatically repressed the bone metastasis ability in MAZ-overexpressing cells (Fig. 6d-j). Collectively, our results indicate that MAZ promotes bone metastasis of PCa via KRas/RalGEFs signalling.

To investigate the clinical significance of MAZ-induced KRas and HRas, IHC staining was performed in the PCa tissues. As shown in Fig. 7a-c, MAZ expression in PCa tissues with bone metastasis was elevated compared with that in PCa tissues without bone metastasis and further increased in metastatic bone tissues; consistently, the expression of HRas and KRas exhibited the same pattern. Since activated RAL cannot be observed by IHC, it is not presented here. Meanwhile, the protein expression levels of MAZ, KRas and activated RalA were examined in PCa tissues and metastatic bone tissues by western blotting. As shown in Fig. 7d, MAZ expression was elevated in PCa tissues with bone metastasis (T4–6) compared with expression in PCa tissues without bone metastasis and was further increased in metastatic bone tissues (T7–9); consistently, protein expression of KRas and activated RalA exhibited the same pattern (Fig. 7d). Taken together, our results indicate that overexpression of MAZ activates KRas and RalGEFS, resulting in the bone metastasis of PCa (Fig. 7e).