Selective expression of the Runx2 transcription factor in metastatic prostate cancer cells and functional activities
We compared four widely studied prostate cancer cell lines with distinct metastatic and tumor growth potential to characterize Runx2 expression in relation to their phenotypic properties. These included highly metastatic PC3 cells isolated from bone metastases, non-metastatic LNCaP cells that do not grow in bone and C4-2B cells that are derived from LNCaP cells and form osteoblastic lesions in the bone (Thalmann et al., 1994
). PC3 cells express the highest level of Runx2 compared to LNCaP, C4-2B and RWPE by both qRT-PCR and western blot analysis (). Because prostate cell lines exhibit genomic instability and centrosome defects that lead to gene alterations (Glinsky et al., 2006
), we further analyzed Runx2 among metastatic PC3 sublines, maintained in distinct media (see methods). PC3 cells were designated PC3-H (high), PC3-M (moderate), and PC3-L (low) according to Runx2 mRNA expression levels, determined by qRT-PCR (, top panel). Runx2 mRNA levels in PC3-H cells are 2 fold higher than PC3-M and 15 fold greater in PC3-L cells. Because protein levels in whole cell lysates are low in PC3-M and PC3-L cells, nuclear extracts were examined demonstrating highest levels in the PC3-H line. Because Runx factors recognize the same regulatory sequence (Blyth et al., 2005
), we compared Runx2 expression to hematopoietic Runx1 and nerve related Runx3 factors in all sublines (Supplementary figure 2
). Runx1 protein was very low in all sublines, while Runx3 mRNA and protein levels were significantly higher in PC3-L cells. These results show that the three PC3 sublines can be examined for further studies for relative levels of Runx2 influencing bone metastatic properties of prostate cancer cells.
Runx2 expression in prostate cancer cell lines regulates metastatic properties
To determine if Runx2 target genes are functionally linked to metastatic properties of PC3-H cells, we selectively inhibited Runx2 expression by RNA interference. Runx2 protein was completely inhibited () and RNA reduced by 80% compared to controls (). Metastatic genes established to be regulated by Runx2 (e.g., VEGF, MMP9) and bone related genes (e.g., OC) were reduced by 80-90% compared to the non-silencing oligo control (). Runx2 siRNA treated PC3-H cells exhibited a 30% increased adhesion to fibronectin and significantly decreased migration and invasion through Matrigel (). Thus, increased adhesion is consistent with decreasing invasive properties of PC3-H cells by deletion of Runx2. Zaidi et al (32) reported a correlation between Runx2 expression and either cell survival or tumorigenesis depending on the cellular context. We addressed if Runx2 contributed to survival of PC3-H cells and show that the cell survival marker survivin is highly expressed in PC3-H cells. A 50% decrease occurred in response to Runx2 knockdown (). These results suggest that Runx2 regulates cellular activities associated with tumor cell invasion and survival of prostate cancer cells.
Runx2 mediates prostate tumor-induced osteolytic bone disease
To identify the functional activities of Runx2 in the PC3 sublines in-vivo, cells were inoculated into the intramedullary cavity of SCID mice (). Osteolysis was first evident in PC3-H and PC3-M tumors by radiography at 3 weeks (data not shown) and by 4 weeks osteolysis of the bone cortex was evident, along with tumor cell invasion in muscle by 6 weeks (). PC3-L cells however, produced a mixed lesion phenotype of mild osteolysis at 3 and 4 weeks, followed by later osteoblastic disease observed more prominently by 6 weeks in tumor tissue (). Histologically, active osteoclasts were found at the tumor-bone interface resorbing bone by the PC3-H () and PC3-M cells (not shown). PC3-L tumors showed unorganized woven bone, especially at the interface of the cortical bone and tumor growing in the muscle (). This is consistent with the irregular cortical bone surface in the PC3-L radiographs showing woven bone, in contrast to radiographs of the eroded cortical surface of PC3-H and PC3 M tumors (, 6 week). Immunohistochemistry (IHC) of PC3-H tumor showed robust Runx2 expression in the majority of cells positive for human cytokeratin (), which was used to distinguish between implanted human prostate cancer cells and mouse marrow cells. In addition, Runx2 expression in PC3 sublines was analyzed post implantation verifying that differences were maintained (). In summary, PC3-H cells resulted in the most aggressive osteolysis, PC3-M cell-mediated osteolytic disease occurred at a slower rate, while PC3-L derived tumors exhibited limited osteolysis (that is, smaller lesions at 3 and 4 weeks), concurrent with osteoblastic disease. These findings indicate a positive correlation of Runx2 mRNA with severity of osteolytic bone disease induced by PC3 tumor cells.
Runx2 expression in PC3 sublines promotes osteolytic bone disease in vivo and osteoclast activation in vitro
To provide further support that Runx2 promotes osteolytic disease, we analyzed expression of IL8 and PTHrP (), signals known to activate osteoclast activity (Bendre et al., 2005
; Liao and McCauley, 2006
). Robust expression of PTHrP in PC3-H and IL8 in PC3-M cells was found, consistent with their aggressive osteolysis. PC3-L cells exhibited low expression of both bone resorbing factors. Analysis of expression levels of Runx2 and these factors post implantation, revealed that the PC3 sublines maintained their unique characteristics in vivo (supplementary figure 3
) Thus, expression of Runx2 in PC3 cells appears to promote activity of secreted osteolytic factors.
PC3-H cells contribute to proliferation of osteoclast precursors and osteoclastogenesis
To delineate the mechanism(s) for tumor-related resorption, PC3-H cells were co-cultured with osteoclast precursors (RAW 264.7). PC3-H cells were cultured for 7 days in the upper compartment of a Boyden chamber, while RAW 264.7 cells were seeded in the bottom chamber to allow for the exchange of secreted factors that generate osteoclasts. Three groups were compared as indicated in . A 3-7 fold increased expression of genes associated with activation of osteoclasts (TRAP, Cathepsin K, MMP9, RANK) and cell growth (histone H4) was observed when RAW 264.7 cells were co-cultured with PC3-H cells, compared to control groups (RAW 264.7 and RAW 264.7 with RANKL). These results indicate that the aggressive stimulation of osteolytic disease is due in part to proliferation of osteoclast precursors that differentiate to active osteoclasts under the influence of secreted factors PTHrP and IL8 as shown in . This finding is consistent with Runx2 positive PC3-H and PC3-M cells promoting osteolytic bone disease, but not PC3-L cells which lack expression of these factors.
Figure 3 (A) Gene expression of markers for osteoclast differentiation detected by qRT-PCR after co-culturing PC3-H cells with RAW 264.7 cells. (B) Histochemical staining of MC3T3 cells for alkaline phosphatase shows inhibited osteoblast differentiation when medium (more ...)
Given the osteomimetic properties of prostate cancer cells (Huang et al., 2005
; Guise et al., 2006
), we next addressed if PC3-H cells influenced the activity of osteoblasts. CM (10% and 20%) from PC3-H cells was used to supplement MC3T3 osteoprogenitor cell cultures during osteoblast differentiation. We find reduced alkaline phosphatase staining on days 14 and 21 reflecting an inhibition of the activity of osteoblasts and competency further to differentiate to mature osteoblasts (). In summary, prostate tumor cells expressing high levels of Runx2 influence both osteoclast and osteoblast activities at the tumor-bone interface (in vivo studies) by modulating target genes.
Exogenous expression of Runx2 promotes activation of metastatic target genes in prostate cancer
Runx2 increases expression of numerous genes essential for cell growth, angiogenesis, and metastasis in breast cancer cells. In order to address if Runx2 can alter the metastatic properties of PCa cells, we first evaluated basal expression of metastatic Runx2 target genes in the cell lines (). We found each PC3 subline exhibited a distinct profile of metastasis related genes. PC3-H cells preferentially expressed MMP9, PC3-M cells expressed MMP13 and PC3-L cells had high MMP2 levels. LNCaP cells did not express MMPs at significant levels, but showed high levels of VEGF. Notably, the bone matrix proteins osteocalcin and osteopontin were expressed at much lower levels than the MMPs in all cell lines.
Forced expression of Runx2 leads to activation of metastatic markers
From these results, we selected PC3-M, PC3-L and LNCaP cells to address mechanisms directly related to Runx2 activities in PCa cells by adenoviral delivery of Runx2 (). Our results reveal that only those MMP genes which are differentially expressed at low endogenous levels () become induced by Runx2 (). For example in PC3-M cells, MMP9, but not MMP13, was induced because basal MMP13 levels are very high and not further stimulated by Runx2. Likewise PC3-L cells, which only express endogenous MMP2, and LNCaP cells, which do not express MMP2, both showed robust induction of MMP9 and MMP13 by Runx2. In summary, these studies reveal that prostate cells differentially express genes that are Runx2 targets and that Runx2 can activate metastatic genes in PCa cells that are expressed at low levels, suggesting Runx2 can contribute to metastatic properties of PCa cells. Our findings provide an insight to the specific genes that are regulated by Runx2 in the various prostate cancer cells.
Knockdown of Runx2 expression in PC3 cells blocks tumor growth and metastatic lesions formed by PC3-H cells in vivo
To determine the direct roles of Runx2 in formation of bone metastatic lesions, parental PC3-H cells were infected with scrambled or Runx2 shRNA for intra-tibial injections. Radiography at 4 weeks showed that mice (n=12; 3 representative mice) with PC3-H cells expressing scrambled shRNA formed aggressive osteolysis, similar to mice tibiae with parental PC3-H cells ( top and center panels). In contrast, PC3-H cells infected with Runx2 shRNA ( lower panel) showed no evidence of osteolytic disease in 2 of 3 mice, while one mouse exhibited the onset of mild bone lysis (arrows). Knockdown of Runx2 by shRNA was confirmed by western blot and qRT-PCR revealing an almost complete knockdown of bone resorbing factors IL8 and PTHrP (). These studies indicate that Runx2 expression is essential for tumor survival in the bone microenvironment and formation of osteolytic lesions. summarizes the activities of Runx2 regulation and contributions to prostate cancer metastasis and formation of bone lesions.
Runx2 knockdown reduces metastatic bone disease
Runx2 expression in human prostate cancer tissue is associated with tumor metastasis
To address the clinical significance of our in vivo findings, we analyzed a prostate cancer tissue microarray to determine if Runx2 expression correlates with prostate cancer progression by immunohistochemistry. The tumor tissue was classified as either negative or positive for Runx2 (). The majority of non-neoplastic tissues and PIN lesions were negative for Runx2, while 48% and 46% of the primary tumors and metastases were Runx2 positive. The staining intensity determined by immunohistological scoring (see methods and supplementary figure 1
) was evaluated in . We find that the majority (92-93%) of non-neoplastic and PIN lesions had a staining intensity of zero, while half (almost 48%) of the advanced primary tumors had a staining intensity of 1 and 2, and metastatic lesions (45.8%) showed intensities of 1 to 3. Thus Runx2 expression is detected in human prostate tumors at advanced stages of the disease.
Runx2 expression in primary and metastatic prostate tumor tissue microarrays