MAZ promotes prostate cancer bone metastasis through transcriptionally activating the KRas-dependent RalGEFs pathway
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Clinically, prostate cancer (PCa) exhibits a high avidity to metastasize to bone. Myc-associated zinc-finger protein (MAZ) is a well-documented oncogene involved in the progression and metastasis of multiple cancer types, even in PCa. However, the clinical significance and biological roles of MAZ in bone metastasis of PCa remain unclear.
MAZ expression was examined in PCa tissues with bone metastasis, PCa tissues without bone metastasis and metastatic bone tissues by real-time PCR and immunohistochemistry (IHC), respectively. Statistical analysis was performed to evaluate the clinical correlation between MAZ expression and clinicopathological features and bone metastasis-free survival in PCa patients. Biological roles of MAZ in bone metastasis of PCa were investigated both in vitro by transwell assay, and in vivo by a mouse model of left cardiac ventricle inoculation. The bioinformatics analysis, western blot, pull-down assays, chromatin immunoprecipitation (ChIP) and luciferase reporter assays were applied to demonstrate and examine the relationship between MAZ and its potential downstream signalling pathway. TaqMan copy number assay was performed to identify the underlying mechanism responsible for MAZ overexpression in PCa tissues.
MAZ expression is elevated in PCa tissues with bone metastasis compared with that in PCa tissues without bone metastasis, and is further increased in metastatic bone tissues. High expression of MAZ positively correlates with poor overall and bone metastasis-free survival in PCa patients. Upregulating MAZ elevates, while silencing MAZ represses the invasion and migration abilities of PCa cells in vitro and bone metastasis ability in vivo. Our results further reveal that MAZ promotes bone metastasis of PCa dependent on KRas signalling, although MAZ transcriptionally upregulates KRas and HRas expression, where the Ral guanine nucleotide exchange factor (RalGEF) signaling is responsible for the different roles of KRas and HRas in mediating the pro-bone metastasis of MAZ in PCa. Finally, our results indicate that recurrent gains contribute to MAZ overexpression in a small portion of PCa tissues.
These results indicate that the MAZ/Kras/ RalGEF signalling axis plays a crucial role in promoting PCa cell bone metastasis, suggesting a potential therapeutic utility of MAZ in bone metastasis of PCa.
KeywordsMAZ Bone metastasis Prostate cancer Ras signalling And RalGEFs
Epidermal growth factor receptor;
Extracellular signal-regulated kinase
Gene set enrichment analysis
Hematoxylin and Eosin Stain
Myc-associated zinc-finger protein
Extracellular-signal regulated kinase kinase
Nuclear factor kappa-light-chain-enhancer of activated B cells
Ral Guanine nucleotide exchange factor
The Cancer Genome Atlas-Prostate adenocarcinoma
Transforming growth factor-β
Novelty and impact
Prostate cancer (PCa) metastasis is the major determinant of cancer-related death and life quality of patients. A hallmark of PCa metastasis is the predominant propensity of bone metastasis. By in vitro and in vivo experiments, our research firstly proves that MAZ/Kras/ RalGEFs signaling axis plays an important role in the bone metastasis of PCa, suggesting a potential therapeutic utility of MAZ in bone metastasis of PCa.
Prostate cancer (PCa) metastasis is the major determinant of cancer-related death and quality of life of patients . A distinguishing feature of PCa metastasis is the predominant susceptibility to bone metastasis . Unlike liver, lung and brain tissues, bone marrow contains less arterial blood flow; thus, the hemodynamics determining metastatic colonization may not be important organ-specific factors contributing to bone metastasis [3, 4]. Therefore, unveiling the specific molecular mechanism underlying bone metastasis in PCa has become an urgent need.
To date, several cellular signalling pathways have been confirmed to be closely related to bone metastases of PCa based on a large amount of literature [5, 6], such as TGF-β , Wnt , NF-kB , EGFR , and Notch . Notably, accumulating evidence has focused great attention on the crucial role of aberrant activation of RAS signaling in bone metastasis of PCa [12, 13]. Ras proteins are prototypical G-proteins that have been shown to play an important role in signal transduction, proliferation, and malignant transformation . The Ras gene family consists of 3 functional genes, H-Ras, K-Ras and N-Ras, which primarily regulate multiple downstream signalling pathways, including the Ras–Raf–mitogen-activated and extracellular-signal regulated kinase kinase (MEK)–extracellular signal-regulated kinase (ERK) cascade, phosphatidylinositol 3-kinase (PI3K), and members of the Ral guanine nucleotide exchange factor (RalGEF) family . All three of these signalling pathways have been proven to be critical for bone metastasis in various types of cancer, including PCa. For example, Ni et al. have informed that PI3K/Akt signaling -mediated stabilization of histone methyltransferase WHSC1 greatly raised bone metastasis in PCa . In addition, Yin et al. demonstrated that activation of the RalGEF/Ral pathway promotes prostate cancer metastasis to bone . Suppression of ERK signaling by XRP44X, an inhibitor of Ras/Erk signalling, effectively inhibits bone metastasis of PCa cells in an intracardiac injection mouse model . Although mutations of activating Ras occur in ~ 30% of human cancers, Ras mutations in prostate cancer are infrequent [18, 19]. Interestingly, several lines of evidence have shown that transcriptional regulation is another primary mechanism accountable for the activation of Ras signalling in several types of cancer [20, 21, 22]. Therefore, it is of paramount importance to identify the underlying transcription factor responsible for constitutive activation of Ras signalling in bone metastasis of PCa.
The Myc-associated zinc-finger protein (MAZ) has been recognized as a transcription factor that binds to a GA box (GGGAGGG) at the ME1a1 site, to the attenuator region of P2 within the first exon of the c-myc gene and to a related sequence that participates in the termination of gene transcription of complement 2 (C2) [23, 24]. The MAZ gene is located on chromosome 16p11.2 and encodes a single, unique gene [24, 25]. Despite being ubiquitous in human tissue, the MAZ expression level varies depending on the organ. The mRNA expression levels of MAZ in the human heart, placenta, pancreas, thymus, prostate, testis, colon, peripheral blood leukocytes, thyroid, and adrenal gland are higher than those in other tissues such as bone marrow . In cancers, MAZ is generally highly expressed in a variety of human tumors, which further promotes the development, progression, and metastasis of cancer by transcriptionally activating multiple downstream target genes [26, 27, 28, 29]. In PCa, a marked increase in MAZ promotes proliferation and metastasis of PCa through reciprocal regulation of androgen receptor , implicating MAZ in the metastatic phenotype of PCa. However, the clinical significance and biological function of MAZ in bone metastasis of PCa remain to be elucidated.
In the present study, the expression levels of MAZ increased steadily from nonbone metastatic PCa tissues and bone metastatic PCa tissues to metastatic bone tissues, and high expression of MAZ was positively correlated with advanced clinicopathological characteristics and poor overall and bone metastasis-free survival in PCa patients. Recurrent gains have been reported to be responsible for MAZ overexpression in a small portion of PCa patients. Furthermore, the upregulation of MAZ promotes while silencing it inhibits invasion and migration of PCa cells in vitro, as well as the bone metastasis ability in vivo. Our results further demonstrated that MAZ activated the RalGEF signalling pathway via transcriptionally activating KRas signaling, which further promoted the bone metastasis of PCa. The clinical correlation revealed that MAZ positively correlated with KRas and RalGEFs signalling activity in PCa and metastatic bone tissues. Collectively, our results unveil a new mechanism responsible for the constitutive activation of the Ras pathway in bone metastasis of PCa, supporting the significance of the transcriptional event in bone metastasis of PCa.
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 .
Plasmid, small interfering RNA and transfection
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.
Patients and tumor tissues
The relationship between MAZ and clinicopathological characteristics in 243 patients with prostate cancer
Number of cases
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).
RNA extraction, reverse transcription, and real-time PCR
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.
TaqMan copy number assay
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 . Bone metastasis lesions were indicated on radiographs in the bone as previously described . 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 . Tumor lesions were monitored by BLI and X-ray.
The MAZ, HRas, and KRas expression levels were detected by immunohistochemistry following the previous description . 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.
Chromatin immunoprecipitation (ChIP)
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.
Affinity pull-down assays for Ral
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.
All values are presented as the mean ± standard deviation (SD). All analyses were performed using GraphPad 7.0 or SPSS 24.0. Student’s t-test was used to determine significant differences between two groups. The chi-square test was used to analyse the relationship between MAZ expression and clinicopathological characteristics. P < 0.05 was considered significant. All experiments were repeated three times.
MAZ is upregulated in PCa tissues with bone metastasis and further enhanced in metastatic bone tissues
Recurrent gains are the potential mechanism responsible for MAZ overexpression in a part of PCa tissues
High expression of MAZ correlates with poor bone metastasis-free survival in PCa patients
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.
MAZ promotes bone metastasis of PCa cells
MAZ transcriptionally activates RAS signalling
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 , 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.
KRas is essential for the pro-bone metastasis role of MAZ in vivo
MAZ promotes bone metastasis of PCa cells in a manner dependent on KRas-mediated RalGEFs signalling
Clinical correlation of MAZ with KRas and activated RalA in human PCa tissues
As a transcription factor, the upregulation of MAZ has been extensively implicated in tumorigenesis, progression, and metastasis in various types of cancer [26, 27, 29]. In PCa, a study from Jiao et al. has shown that overexpression of MAZ contributes to proliferation and metastasis of PCa through reciprocal regulation of androgen receptor , suggesting that MAZ may play a role in the metastatic phenotype of PCa. However, the clinical significance and biological role of MAZ in bone metastasis of PCa have not been reported until now. In this study, we found that MAZ was elevated in bone metastatic PCa tissues compared with nonbone metastatic PCa tissues, and it was further enhanced in metastatic bone tissues, which positively correlated with advanced clinicopathological characteristics and poor overall and bone metastasis-free survival in PCa patients. Interestingly, the expression level of MAZ was relatively lower in normal bone than in several other tissues under physiological conditions . This finding greatly ignites our interest in further exploring the mechanism responsible for the dramatic differential expression of MAZ between physiological and cancerous situations, which was demonstrated to be associated with recurrent gains in the current study. Next, our results revealed that the upregulation of MAZ promoted while its silencing inhibited the invasion and migration of PCa cells in vitro, as well as the bone metastasis ability in vivo. Our results further demonstrate that MAZ promoted bone metastasis of PCa via transcriptionally activating Ras signalling. Thus, these findings indicate that MAZ functions as a pro-bone metastasis gene in PCa. Indeed, several lines of evidence have demonstrated that the metastatic potential of prostate cancer depends on the expression of several metastasis-related genes, or metastasis-promoting genes [45, 46, 47], which further determine the pivotal role of MAZ in bone metastasis of PCa.
As shown in the CHIP experiments Fig. 4e, MAZ bind to thenuclease-hypersensitive element (NHE) of KRas’ promoter which activated the KRas transcription. Theoretically, KRas mutation in this site will block the transcriptional activation of KRas by MAZ and thus affect the occurrence of bone metastasis of prostate cancer. Although Ras signalling was constitutively activated by mutations in approximately 30% of human cancers , Ras mutations were very rare in PCa [19, 48], suggesting that other regulatory mechanisms are involved in aberrant activation of Ras signalling in PCa. It is worth noting that transcriptional regulation is reported to be another primary mechanism responsible for the activation of Ras signalling in several types of cancer [20, 21, 22]. Multiple studies have revealed that MAZ plays an important role in the activation of RAS signalling by varying mechanisms in several tumor types [20, 22, 49]. For example, MAZ promoted angiogenesis through transcriptional activation of the RAS signalling pathway in breast cancer . In addition, HRas is silenced by two neighbouring G-quadruplexes and activated by MAZ in bladder cancer cells . Interestingly, Yu et al reported that MAZ mediated by FOXF2 plays dual roles in basal-like breast cancer: promotion of proliferation and suppression of progression . However, whether these Ras protein members are separately or simultaneously activated by MAZ in bone metastasis of PCa, and whether activation of these Ras signalling members produces subsequent functional roles in mediating the effects of MAZ on bone metastasis of PCa, need to be further elucidated. In the current study, our results demonstrated that MAZ simultaneously transcriptionally activated KRas and HRas signalling in bone metastatic PCa cells. Importantly, our results demonstrated that KRas, but not HRas, was indispensable to the pro-bone metastasis role of MAZ in PCa. Therefore, our results reveal a new mechanism responsible for the sustained activation of Ras signalling in bone metastasis of PCa.
The primary downstream effector pathways of Ras signalling include ERK, PI3K and RalGEFs [15, 18], each of which has been reported to play important roles in bone metastasis of PCa [12, 16, 17]. In this study, we reported that MAZ simultaneously activated ERK, PI3K, and RalGEFs signalling in PCa cells, where ERK and PI3K signalling were under concomitant regulation of KRas and HRas, and RalGEFs were only regulated by KRas. Strikingly, our results showed that KRas silencing markedly abrogated the invasion and migration abilities of MAZ-overexpressing PC-3 cells in vitro and pro-bone metastasis ability in vivo; however, silencing HRas had no significant effect on the bone metastasis ability of MAZ-overexpressing PC-3 cells. These findings support the notion that RalGEFs signalling may be a potential downstream effector signalling contributing to the pro-bone metastasis of MAZ in PCa. In fact, RalGEFs inhibitor RBC8 dramatically suppressed the invasion and migration abilities of MAZ-overexpressing PC-3 cells. However, ERK inhibitor or PI3K inhibitors had no obvious influence on the invasion and migration abilities in MAZ-overexpressing PC-3 cells. More importantly, RBC8 dramatically inhibited the bone metastasis ability of MAZ-overexpressing PCa cells. Therefore, our results reveal that MAZ promotes bone metastasis of PCa cells in a manner that is dependent on KRas-mediated RalGEFs signalling, although ERK and PI3K/AKT signalling are hyperactive in MAZ-overexpressing PCa cells.
Our results unveil that overexpression of MAZ elicited by recurrent gains activates the Kras/ RalGEFs signalling pathway, which further promotes bone metastasis in PCa, providing theoretical evidence that the MAZ/Kras/ RalGEFs signalling axis plays an important role in bone metastasis of PCa. Therefore, a comprehensive understanding of the mechanism responsible for the activation of the Ras signalling pathway will facilitate the development of an effective therapeutic strategy to inhibit bone metastasis of PCa.
Xinsheng Peng and Dong Ren developed ideas and drafted the manuscript. Qing Yang, Chuandong Lang and Zhengquan Wu conducted the experiments and contributed to the analysis of data. Yuhu Dai, Shaofu He, Wei Gu, and Shuai Huang contributed to the analysis of data. Hong Du edited the manuscript. All authors contributed to revising the manuscript and approved the final version for publication.
This study was supported by the Natural Science Foundation of China (No. 81472505).
No conflict of interest was declared.
Ethics approval and consent to participate
In order to use those clinical specimens for research, patients’ consents and approval from the Institutional Research Ethics Committee were acquired, and in accordance with the Declaration of Helsinki. The ethics approval statements for animal work were provided by The Institutional Animal Care and Use Committee of Sun Yat-Sen University Cancer Center. The ethics approval number for animal work was L102012017080Q.
Consent for publication
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