SOX9 elevation in Pca is positively correlated with cancer aggressiveness
In order to verify the clinical significance of SOX9 in human Pca, we firstly examined SOX9 expression in specimens of radical prostatectomy from a cohort of 108 consecutive Pca patients by IHC staining. For pathological analysis, Pca with higher Gleason scores had higher expression of SOX9 (Fig. 1A, B). Particularly, specimens with Gleason scores no less than 8 showed significantly higher expression of SOX9 than 3+3, 3+4 and 4+3 tissues (Fig. 1B). Moreover, Pca with advanced tumor stage (III and IV) also showed higher SOX9 expression (Fig. 1C). Spearman correlation analysis indicated that SOX9 expression was significantly associated with the percentage of Ki67-positive tumor cells (Spearman r =0.301, p =0.0017, n =108) (Fig. 1D). In addition, high SOX9 IHC staining in Pca patients was positively associated with Gleason scores (p =0.004), seminal vesicle invasion (p =0.040) and nerve invasion (p =0.019) (Table S1). These results validated previous reports [7, 8, 10] that SOX9 levels correlated with Pca progression.
To explore the possible roles of SOX9 in Pca tumorigenesis and progression, we subsequently assessed the effects of SOX9 on proliferation, migration, invasion and stemness in Pca cells. DU145 cells transiently transfected with pcDNA3.1(+)-SOX9 (Fig. S1A, B) showed a remarkably enhanced growth rate compared with the negative control group (Fig. S1C). Transwell assay revealed that forced expression of SOX9 notably accelerated cell migration and invasion of DU145 cells (Fig. S1D, E). Spheroid formation assay indicated that over-expression of SOX9 significantly increased both the size and number of cell spheres (Fig. S1F, G). Consistently, immunoblotting analysis showed that SOX9 overexpression led to the increase of MMP1 (metastasis-associated protein) and CD44, CD133, c-Myc, Oct4, ALDH1A1 (tumor stemness-associated markers) (Fig. S1H, I). Then, another Pca cell line PC3, which had a higher endogenous expression of SOX9, was knocked down by siRNA (siSOX9-1 and siSOX9-2) (Fig. S2A, B). Compared with negative control siRNA (siNC), in vitro assays revealed that SOX9 interfering markedly inhibited cell growth and viability (Fig. S2C, D), as well as cell migration, invasion (Fig. S2E, F) and cancer stemness (Fig. S2G, H). In addition, decreased protein levels and mRNA expression of CD44, CD133 and c-Myc were also detected in the SOX9 knockdown cells (Fig. S2I, J).
To investigate whether forced expression of SOX9 promote tumor growth of Pca cells in vivo, DU145 cells stably overexpressing SOX9 or vector control (Fig. 1E, F) were injected subcutaneously in nude mice. Compared with the control group, xenografts of SOX9 over-expressing cells had a greater tumor volume and growth rate (Fig. 1G, H). Moreover, IHC staining showed that over-expression of SOX9 promoted the expression of Ki67 and CD44, CD133 in DU145 xenograft tumors (Fig. 1I).
SOX9 in Pca is upregulated by CAFs and essential for CAF-induced tumor-promoting effects
To assess whether SOX9 in Pca cells could be regulated by paracrine factors from CAFs, we isolated CAFs and corresponding normal fibroblasts (NFs) from fresh prostate biopsy specimens (Fig. S3A). Immunofluorescence staining showed that in the biopsy tissues of both normal prostate and prostate cancer, epithelial cells expressed E-cadherin prominently, while stromal cells expressed Fibronectin markedly (Fig. S3B). As for CAF-specific biomarker, α-SMA, was barely detected in the stromal area of normal tissues, while strongly stained in prostate cancer stromal, nearly co-localized with Fibronectin (Fig. S3B). Cell immunofluorescence (Fig. S3C), western blotting and qRT-PCR (Fig. S4A, B) were also performed to verify the purity of cultured fibroblasts. Altogether, the fibroblast identity of NFs and CAFs has been confirmed, and found to be sustaining after several passages (data not shown).
Then, we verified the tumor-promoting effects of CAFs by using an in vitro model of paracrine interaction between primary CAFs and Pca cells. Wound healing assay, transwell assay and spheroid formation assay revealed that treatment with CAF-CM remarkably increased the abilities of cell migration, invasion and cancer stemness (Fig. S5A-F), in comparison with both control medium and conditional medium from NFs (NF-CM). Consistently, western blotting results showed that MMP1 and MMP3, and markers of cancer stemness (CD44, CD133, c-Myc) were elevated in the CAF-CM treated cells (Fig. S5G, H).
To investigate the paracrine effects of CAFs on SOX9 expression in Pca cells, CAF-CM and NF-CM were added to DU145 and 22Rv1 cells for 12 and 24 h. Western blotting and qRT-PCR revealed that both CAF-CM and NF-CM increased SOX9 protein expression and mRNA level, but the effect of CAF-CM was more apparent (Fig. 2A, B). Immunocytochemical staining also demonstrated that since 3 hours after CAF-CM stimulation, SOX9 expression started increasing (Fig. S8J). These results suggested that paracrine factors from stromal fibroblasts upregulated SOX9 expression in Pca cells.
Since CAFs promoted both the aggressiveness of Pca and the expression of SOX9, we wondered whether SOX9 played the role as a bridge in the process of CAF-mediated tumor promotion. RNA interference involving three siRNA targeting different regions was employed to knock down SOX9 expression, then transwell invasion assay and spheroid formation assay showed that depletion of SOX9 significantly reduced the capabilities of cell invasion and sphere formation potentiated by CAF-CM (Fig. 2C-F). Concomitantly, Western blotting analysis further showed that protein expressions of CD44 and CD133 were reduced in SOX9 knockdown groups compared with siNC cells (Fig. 2G). These data implied that SOX9 played an essential role in the CAF-mediated Pca progression.
HGF is a key paracrine factor secreted by CAFs to mediate SOX9 upregulation in Pca cells
In order to identify the key paracrine factors secreted by CAFs involved in the activation of SOX9, a home-made qRT-PCR array consisting of 52 TME-related secretory factors was applied to screen in DU145 cells and three pairs of NFs and CAFs for the possibly responsible factors. Hierarchical clustering and heatmap indicated that in the aggregate that genes expressed lower in DU145 cells but highly in fibroblasts, HGF was the most upregulated in CAFs compared with NFs (Fig. 3A). Considering HGF and its tyrosine kinase receptor c-Met are known involved in promoting Pca progression [32, 33], we focused our attention on the role of HGF in CAF-mediated SOX9 upregulation and tumor-promoting effects. Firstly, qRT-PCR results verified that mRNA level of HGF in CAFs was about 13 folds higher than in NFs, while barely detected in DU145 cells (Fig. 3B). ELISA data showed that no or negligible levels of HGF were produced by Pca cells (22Rv1, PC3, DU145), while all CAFs secreted more abundant HGF compared with their paired NFs, suggesting that HGF was preferentially secreted by CAFs in TME (Fig. 3C). Consistently, western blotting analysis confirmed the strikingly increase of c-Met phosphorylation (Tyr1234/1235) in DU145 and 22Rv1 cells following the treatment with CAF-CM compared to NF-CM, while c-Met remained nearly unphosphorylated in control medium (Fig. 3D), which suggested that tyrosine residues 1234 and 1235 of c-Met in Pca cells would not auto-phosphorylated without stimulation of stromal fibroblasts. The correlation between the expression of HGF and ACTA2 (which encodes α-SMA) was evaluated by Pearson correlation analysis using mRNA expression profiles of Pca from TCGA, and it turned out that HGF expression was significantly correlated with ACTA2 (Pearson r =0.406, p <0.0001, n =499) (Fig. 3E).
Previous studies have demonstrated that HGF/c-Met played an important role in tumorigenesis and tumor progression of Pca [32, 33]. Herein, we treated DU145 and 22Rv1 cells with recombinant human HGF (Peprotech, 100-39H, Rocky Hill, NJ, USA) in vitro, and the abilities of cell migration, invasion and sphere formation were detected. As shown in Fig. S6, 10 ng/ml HGF was sufficient to significantly increase the number of cells migrated or invaded to the down surface of transwell membrane (Fig. S6A-D), and enhance the number and size of formed spheroids (Fig. S6E, F). The key role of HGF in the paracrine effects of CAFs was further validated by an ATP-competitive c-Met inhibitor, capmatinib. In vitro assays showed that the inhibition of HGF/c-Met pathway in DU145 and 22Rv1 cells with pre-treatment of 10 nM capmatinib, remarkably abrogated the effects of CAF-CM on the cell migration, invasion and tumor initiating abilities (Fig. S7A-F).
Since HGF has been identified as a key secretory factor in CAF-CM mediating tumor promotion, and SOX9 was also verified essential for Pca progression induced by CAFs, we wonder whether there was a connection between HGF and SOX9 expression. Western blotting analysis demonstrated that treatment with 30 ng/ml recombinant human HGF obviously upregulated SOX9 expression since 1 hour upon stimulation in both DU145 and 22Rv1 cells (Fig. 3F). 10 ng/ml HGF was already sufficient to induce the increase of SOX9 expression, as well as 30 ng/ml HGF (Fig. 3G). When capmatinib was combined in the HGF or CAF-CM treatment, the upregulation of SOX9 caused by HGF or CAF-CM was markedly abrogated (Fig. 3G, Fig. S8A, B). In addition, in vivo experiments were performed to further investigate the comprehensive role of HGF/c-Met signaling in the regulation of tumor growth and SOX9 expression. DU145 cells alone or companied with equivalent CAFs were injected subcutaneously into nude mice, and three weeks later, vehicle or capmatinib were given to the tumor-bearing mice twice a day. Compared with the DU145 alone group, tumors of DU145 with CAFs had a more rapid growth rate and greater tumor size, while capmatinib treatment significantly abrogated the CAF-mediated acceleration of tumor growth (Fig. 3H, I). IHC staining demonstrated that SOX9 expression in Pca cells was upregulated by CAFs in vivo, and could be restored by capmatinib which strongly inhibited the phosphorylation of c-Met in Pca cells (Fig. 3J). Correspondingly, Ki67 and CD44 showed a similar pattern to SOX9 (Fig. 3J). Taken together, HGF plays a central role in upregulating SOX9 in Pca cells and mediating the tumor-promoting effects of CAFs.
HGF upregulates SOX9 expression via MEK1/2-ERK1/2 pathway
Previous studies have reported that HGF induced dimerization and phosphorylation of c-Met, resulting in the activation of several downstream oncogenic signaling pathways including RAS/MAPK, PI3K/AKT and STAT3 . To specify which pathway was involved in the regulation of SOX9 expression by HGF, we tested the total expression and phosphorylation level of representative proteins of RAS/MAPK, PI3K/AKT and STAT3 pathways in DU145 cells treated with 30 ng/ml HGF, by western blotting. It turned out that the phosphorylation of ERK1/2 (Thr202/Tyr204) was most enhanced in cells treated with HGF, while phosphorylation of JNK (Thr183/Tyr185) was increased slightly and others including p38, Akt, Stat3 and NF-κB remained unaffected (Fig. 4A). In a time course of HGF treatment, phosphorylation of ERK1/2 in both DU145 and 22Rv1 cells started rising as early as 10 mins upon stimulation and lasted for at least 2 hours (Fig. 4B). Further analysis revealed that phosphorylation of ERK1/2 by HGF was dose-dependent, and 30 ng/ml HGF induced a strikingly increased phosphorylation of ERK1/2, which could be abolished by capmatinib pre-treatment (Fig. 4C). As expected, CAF-CM also caused a remarkable enhancement of phosphorylation of ERK1/2 compared with the control and NF-CM treatment, which could be abolished by capmatinib (Fig. S8C,D). Then, MEK1/2 inhibitor U0126 (Selleck Chemicals, S1102, Boston, MA, USA) and ERK1/2 inhibitor SCH772984 (Selleck Chemicals, S7101) were adopted to block MEK1/2-ERK1/2 pathway, and we found that the upregulation of SOX9 induced by HGF or CAF-CM was obviously abrogated (Fig. 4D, Fig. S8E). In addition, when MEK1/2-ERK1/2 pathway was inhibited, it repressed the reinforced abilities of cell invasion and sphere formation induced by HGF (Fig. 4E-H). These results implied that MEK1/2-ERK1/2 pathway was responsible for the upregulation of SOX9 and the tumor-promoting effects by HGF secreted from CAFs.
FRA1 is the downstream effector of MEK1/2-ERK1/2 signaling and transcriptionally upregulates SOX9
Previous reports showed that HGF treatment of hepatocellular carcinoma cells resulted in enhancement of the phosphorylation and total expression levels of AP-1 proteins which then mediated HGF-induced effects [35-37]. Multiple heterodimers of the FOS and JUN family bind to the TPA-responsive element (TRE, 5'-TGAG/CTCA-3') sequence and transcriptionally upregulate target genes. Since previous research has demonstrated that putative AP-1 binding site was found in the promoter region of mouse Sox9 gene , we deduced that AP-1 transcription factors may be the downstream mediator of activated MEK1/2-ERK1/2 to upregulate SOX9 expression. In order to identify which AP1 proteins were specifically activated by HGF, we analyzed the protein expression of all the members of AP-1 family, including c-Jun, JunB, JunD, c-Fos, FosB, Delta FosB, FRA1 and FRA2, in DU145 cells stimulated with 30 ng/ml HGF. The results revealed an obviously increase of FRA1 protein level, while the other members remained nearly unaffected (Fig. 5A). Further investigation showed that HGF treatment induced remarkable phosphorylation (Ser265) and increase of total expression of FRA1, as early as 10 mins in DU145 cells and 30 mins in 22Rv1 cells after HGF exposure, indicating that the inducement of phosphorylation and upregulation of FRA1 protein was quite rapid (Fig. 5B). Similar to the patterns of phosphorylation of ERK1/2, phosphorylation and upregulation of FRA1 by HGF was also in a dose-dependent fashion, and the markedly phosphorylation and upregulated expression of FRA1 induced by 30 ng/ml HGF could be restored by capmatinib to that observed in the control treatment (Fig. 5C). Correspondingly, CAF-CM induced higher upregulation of FRA1 protein expression as well as its phosphorylation level compared to NF-CM (Fig. S8F). When capmatinib was added along with CAF-CM, the levels of phos-FRA1 and total FRA1 expression were restored to levels in the control treatment (Fig. S8G). U0126 and SCH772984 were used to inhibit MEK1/2-ERK1/2 pathway, then both the phosphorylation and upregulation of FRA1 stimulated by HGF or CAF-CM were abolished obviously (Fig. 5D, Fig. S8H). These data suggested that FRA1 was a downstream transcription factor activated by CAF-secreted HGF through MEK1/2-ERK1/2 pathway.
To evaluate the hypothesis that FRA1 transcriptionally upregulate SOX9 expression, we transiently overexpressed FRA1 as well as FOS and JUN in DU145 cells (AR negative). Forced expression of FRA1, FOS and JUN all increased protein level of SOX9, but FRA1 had the most obvious upregulating effect (Fig. 5E, left). qRT-PCR also showed a similar outcome that FRA1 increased SOX9 mRNA level most efficiently (Fig. 5E, right). In another two AR positive Pca cell lines, 22Rv1 and LNCaP, and an immortalized prostate epithelial cell, RWPE-1, the expression of SOX9 also increased upon FRA1 overexpression (Fig. 5F, Fig. S10A), indicating that upregulation of SOX9 by FRA1 was AR-independent. When FRA1 was depleted by specific siRNA, the upregulation of SOX9 induced by HGF or CAF-CM was obviously abrogated (Fig. 5G, Fig. S8I). Interestingly, FRA1 knockdown also visibly decreased the phosphorylation level of c-Met (Tyr1234/1235), suggesting that a positive feedback loop may exist between the HGF/c-Met pathway and FRA1 expression (Fig. 5G, Fig. S8I). To investigate the transcriptional regulation of SOX9 by FRA1, we analyzed the sequence of human SOX9 promoter and one TRE sequence (-551 ~ -545) was found (Fig. 5H). ChIP assay was performed using specific FRA1 and phos-FRA1 (Ser265) antibodies in DU145 cells stably overexpressing FRA1, and the precipitated DNA was amplified by two sets of primers across TRE sequence. The results showed both FRA1 and phos-FRA1 (Ser265) could bind to the target segment more than normal rabbit IgG (Fig. 5I, J), which indicated FRA1 could transcriptionally upregulate SOX9 expression. Further investigation showed that in DU145 cells treated with HGF (Fig. 5K) or CAF-CM (Fig. S8J), co-localization of FRA1 and SOX9 within the nuclei was quite apparent. In addition, Pca cells with FRA1 overexpressed formed larger xenograft tumors in mice, compared with the control group (Fig. S9). These data collectively indicated that FRA1 was the downstream effector of MEK1/2-ERK1/2 activated by HGF and transcriptionally mediated SOX9 upregulation.
HGF/c-Met-FRA1-SOX9 axis is validated in mouse Pca cells and TCGA database
On the one hand, mouse Hgf shares remarkable homology with human HGF at both mRNA (percent identity: about 88%) and protein (percent identity: higher than 90%) levels, and human and murine HGF/Hgf are cross-reactive. On the other hand, TRE sequences were found in the promoter region of both human and mouse SOX9/Sox9 gene (Fig. 5H, Fig. 6E). In addition, binding site B (-600 bp ~ --593 bp) in the mouse Sox9 promoter was found conserved with that found in the human SOX9 promoter (-551 ~ -545). Therefore, we assumed that Hgf/c-Met-Erk1/2-Fra1 signaling was also responsible for the upregulation of Sox9 in mouse. Western blotting was carried out in a mouse Pca cell line, RM-1, and the results showed that recombinant murine Hgf (Peprotech, 315-23) also promoted Sox9 expression through c-Met-Erk1/2-Fra1 axis (Fig. 6A-D). ChIP assay with antibody against phos-Fra1 (Ser265) was performed in RM-1 cells treated with 30 ng/ml Hgf for 6 hours, and it turned out that the activated Fra1 could bind to all the three TRE sequence located in the promoter of Sox9 gene (Fig. 6E-G). We also exploited TCGA database and found that Pearson correlation analysis showed significant positive correlation between MET and SOX9 (Pearson r =0.268, p <0.0001, n =499, Fig.6H), MET and FRA1 (Pearson r =0.268, p <0.0001, n =499, Fig.6I), FRA1 and SOX9 (Pearson r =0.312, p <0.0001, n =499, Fig.6J). These results strongly suggested that SOX9 expression regulated by HGF/c-Met-ERK1/2-FRA1 signaling was a relatively common mechanism in Pca cells.