Estrogen receptors β1 and β2 have opposing roles in regulating proliferation and bone metastasis genes in the prostate cancer cell line PC3

Abstract

The estrogen receptor (ER)β1 is successively lost during cancer progression, whereas its splice variant, ERβ2, is expressed in advanced prostate cancer. The latter form of cancer often metastasizes to bone, and we wanted to investigate whether the loss of ERβ1 and/or the expression of ERβ2 affect such signaling pathways in prostate cancer. Using PC3 and 22Rv1 prostate cancer cell lines that stably express ERβ1 or ERβ2, we found that the ERβ variants differentially regulate genes known to affect tumor behavior. We found that ERβ1 repressed the expression of the bone metastasis regulator Runx2 in PC3 cells. By contrast, RUNX2 expression was up-regulated at the mRNA level by ERβ2 in PC3 cells, whereas Slug was up-regulated by ERβ2 in both PC3 and 22Rv1 cells. In addition, the expression of Twist1, a factor whose expression strongly correlates with high Gleason grade prostate carcinoma, was increased by ERβ2. In agreement with the increased Twist1 expression, we found increased expression of Dickkopf homolog 1; Dickkopf homolog 1 is a factor that has been shown to increase the RANK ligand/osteoprotegerin ratio and enhance osteoclastogenesis, indicating that the expression of ERβ2 can cause osteolytic cancer. Furthermore, we found that only ERβ1 inhibited proliferation, whereas ERβ2 increased proliferation. The expression of the proliferation markers Cyclin E, c-Myc, and p45(Skp2) was differentially affected by ERβ1 and ERβ2 expression. In addition, nuclear β-catenin protein and its mRNA levels were reduced by ERβ1 expression. In conclusion, we found that ERβ1 inhibited proliferation and factors known to be involved in bone metastasis, whereas ERβ2 increased proliferation and up-regulated factors involved in bone metastasis. Thus, in prostate cancer cells, ERβ2 has oncogenic abilities that are in strong contrast to the tumor-suppressing effects of ERβ1.

Fig. 1.

Stable expression of ERβ1 and ERβ2. A and B, qPCR analysis of ERβ1 and ERβ2 mRNA in stably transfected PC3 cells. Control +Dox (Doxycycline) (bar 1), control −Dox (bar 2), ERβ1/β2 +Dox (bar 3), and ERβ1/β2 −Dox (bar 4). C, The protein expression of ERβ1 and ERβ2 was verified by Western blotting using the ERβ antibody 14C8 (Abcam). Lane 1 represents the control cells, lane 2 ERβ1-expressing cells, and lane 3 ERβ2-expressing cells. The FLAG-ERβ2 band is slightly lower than the ERβ1 band. β-Actin was used as a loading control. D and E, qPCR analysis of ERβ1 and ERβ2 mRNA in stably transfected 22Rv1 cells. All the experiments were performed n = 3.

Fig. 2.

The effects of ERβ1 and ERβ2 on cell proliferation. A, The BrdU assay of the cells expressing ERβ1 showed decreased uptake of BrdU, whereas the cells expressing ERβ2 showed an increased incorporation of BrdU compared with the control. B, Quantification of BrdU incorporation as calculated by counting the number of BrdU-positive cells in three fields per DAPI-positive cells. The ERβ1-expressing cells showed lower BrdU incorporation compared with control cells. Data are mean values ± se, calculated using Student's t test, P < 0.003. ERβ2-expressing cells showed larger BrdU incorporation compared with control cells. Data are mean values ± se, calculated using Student's t test, P < 0.008. C, 25,000 PC3 cells were seeded in a 12-well plate with 0 or 10% FBS. After 48 h, the cells were counted with a Neubauer chamber. ERβ1 significantly decreased cell proliferation (0% FBS, P < 0.0004 and 10% FBS, P < 0.0003), whereas ERβ2 significantly increased cell proliferation both at 0 and 10% FBS, respectively (0% FBS, P < 0.0007 and 10% FBS, P < 0.017). D, 25,000 22Rv1 cells were seeded in a 12-well plate with 0 or 10% FBS. After 48 h, the cells were counted with a Neubauer chamber. ERβ1 significantly decreased cell proliferation (0% FBS, P < 0.037 and 10% FBS, P < 0.0003), whereas ERβ2 significantly increased cell proliferation at 10% FBS (P < 0.0053) and no significant change at 0% FBS. E, 10,000 cells/well were seeded in 96-well cell culture plates. After 48 h, an MTS assay kit was used to determine the rate of cell proliferation. ERβ1 decreases cellular proliferation (P < 0.0001), whereas ERβ2 enhances proliferation (P < 0.05) compared with the control. Data are mean values ± se, calculated using Student's t test. Data are mean values ± se, calculated using Student's t test. All the experiments were performed n = 3. ns, Nonsignificant.

Fig. 3.

The effects of ERβ1 and ERβ2 on the expression of proproliferation genes. A, The qPCR analysis of p45^Skp2 in PC3 cells showed less expression in the ERβ1-expressing cells than in the control cells (P < 0.0006). Data are mean values ± se, calculated using Student's t test. B, The qPCR analysis of p45^Skp2 in 22Rv1 cells showed less expression in the ERβ1-expressing cells than in the control cells (P < 0.022). Data are mean values ± se, calculated using Student's t test. C, Western blot analysis showed a decrease in p45^Skp2 in ERβ1-expressing PC3 cells, whereas an increase was shown in cells expressing ERβ2. D, The qPCR analysis of c-Myc in PC3 cells showed increased expression in the ERβ2-expressing cells (P < 0.01) but no significant difference for ERβ1 cells compared with the control. Data are mean values ± se, calculated using Student's t test. E, The qPCR analysis of c-Myc in 22Rv1 cells showed increased expression in the ERβ2-expressing cells (P < 0.08) but no significant difference for ERβ1 cells compared with the control. Data are mean values ± se, calculated using Student's t test. F, Western blot analysis showed an increase in c-Myc in PC3 cells expressing ERβ2 and a sharp decrease in cells expressing ERβ1 compared with the control. G, Western blot analysis showed an increase in c-Myc in 22Rv1 cells expressing ERβ2 and a decrease in cells expressing ERβ1 compared with the control. H, Western blot analysis of p27^Kip1 in PC3 cells showed an increase in expression, both in ERβ1- and ERβ2-expressing cells as demonstrated in the top panel. The middle panel shows phosphorylation of p27^Kip1 at T198. There is a significant increase in the phosphorylation of p27^Kip1 (T198) only in cells expressing ERβ2. β-Actin was used as a loading control. I, Western blotting of p21^WAF1/CIP1 expression in PC3 cells showed an increase in p21^WAF1/CIP1 in the cells expressing ERβ1 and a significant decrease in the cells expressing ERβ2 compared with the control. All the experiments were performed n = 3. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; ns, nonsignificant.

Fig. 4.

The effects of ERβ1 and ERβ2 on the cell cycle. A, Immunocytochemical staining of Cyclin E in ERβ1- and ERβ2-expressing PC3 cell pellets. The left panel shows control cells, the middle panel ERβ1 cells, and the right panel ERβ2 cells. The expression of Cyclin E decreased in the cells expressing ERβ1 but remained unchanged in ERβ2-expressing cells. B, Western blot analysis showed a decrease in Cyclin E in PC3 cells expressing ERβ1 compared with control and ERβ2-expressing cells. C, The qPCR analysis of ERβ1 and ERβ2 significantly increased Cyclin D1 expression in PC3 cells (P < 0.0001). Data are mean values ± se, calculated using Student's t test. D, Western blot analysis showed an increase in Cyclin D1 expression in ERβ1 cells compared with control cells and no change in ERβ2 cells. E, Western blot analysis of Rb proteins. The top panel shows the total Rb protein. The second panel shows phosphorylation at Ser780, and the third panel shows phosphorylation at Ser795. The fourth panel is the β-actin loading control. The total Rb and pRb780 increased, whereas pRb795 decreased by ERβ1. In the ERβ2-expressing cells, total Rb and pRb795 were unchanged, and there was a small increase in pRb780. All the experiments were performed n = 3. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase.

Fig. 5.

The effects of ERβ1 and ERβ2 on the expression of genes involved in bone metastasis. A, qPCR analysis of ERβ1 cells showed a decrease in Runx2 expression (P < 0.01), whereas the expression of Runx2 increased in ERβ2 cells (P < 0.001). Data are mean values ± se, calculated using Student's t test. B, Western blot analysis of the Runx2 protein (50 μg of nuclear extract were loaded in each lane). β-Actin was used as a loading control. Runx2 expression decreased in the ERβ1 cells, but there was no reduction in the ERβ2 cells. C, qPCR analysis of the ERβ2 cells showed an increase in Slug expression (P < 0.03), whereas there was no significant change in ERβ1 cells. D, qPCR analysis of the 22Rv1 cells expressing ERβ2 also showed an increase in Slug expression (P < 0.034), whereas there was no significant change in ERβ1 cells. Data are mean values ± se, calculated using Student's t test. E, Western blot analysis of PC3 cells showed a small increase in Slug protein in ERβ2-expressing cells but no significant change in cells expressing ERβ1. F, Quantification of the Slug protein expression by ImageJ software (National Institutes of Health, Bethesda, MD). All the experiments were performed n = 3. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; ns, nonsignificant.

Fig. 6.

The effects of ERβ1 and ERβ2 on the expression of DKK1. A, qPCR analysis showed increased Twist1 mRNA in ERβ2-expressing cells (P < 0.0033) but no significant change in ERβ1-expressing cells. Data are mean values ± se, calculated using Student's t test. B, Western blot analysis showed an increase in the Twist1 protein level in ERβ2-expressing cells. C, qPCR analysis of the cells expressing ERβ2 showed an increase in DKK1 transcripts (P < 0.09) but no significant change in ERβ1-expressing cells. D, Western blot analysis showed an increase in the DKK1 protein in ERβ2-expressing cells, whereas no significant change in DKK1 was observed in ERβ1-expressing cells. All experiments were performed n = 3. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; ns, nonsignificant.

Fig. 7.

The effects of ERβ1 and ERβ2 on the expression of β-catenin. A, qPCR analysis of the ERβ1 cells showed a decrease in β-catenin expression (P < 0.00001). Data are mean values ± se, calculated using Student's t test. B, Western blot analysis showed a small increase in the β-catenin protein in ERβ2-expressing cells, whereas there was a decrease in ERβ1-expressing cells. C, The luciferase (TCF/LEF) reporter assay for β-catenin showed a 17β-estradiol(E2)-independent decrease in activity for vehicle-treated ERβ1-expressing cells compared with the mock-transfected cells, which was further repressed by 17β-estradiol compared with the vehicle-treated ERβ1-transfected cells (P < 0.0001). Data are mean values ± se, calculated using Student's t test. D, The luciferase (TCF/LEF) reporter assay for β-catenin showed an ERβ2-concentration-dependent increase in activity (P < 0.001 for 200 ng) compared with the empty vector transfection. Data are mean values ± se, calculated using Student's t test. All the experiments were performed n = 3. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase.

Fig. 8.

Xenograft transplantation of control PC3 cells and PC3 cells expressing ERβ1 and ERβ2. Nude (nu/nu) mice transplanted with PC3 cells expressing ERβ1 (n = 3), ERβ2 (n = 3), or with empty vector control (n = 3). The cells (5 × 10⁵) were mixed with 50% Matrigel and implanted sc into the flank region. The tumor was monitored regularly and after 40 d, the mice were killed and photographed. A, PC3-control, PC3-ERβ1, and PC3-ERβ2 cells transplanted mice, with arrows pointing at the injection site and the picture of the excised xenografts at the end point. Scale in centimeters. B, Tumor weight at the end point. C, Tumor size at the end point. D, Representative pictures of Ki67 immunohistochemically stained slides from PC3-control, PC3-ERβ1, and PC3-ERβ2 xenografts. E, Number of Ki67-positive cells in xenograft slides per visual field (×20 objective) identified by immunohistochemistry. Bar 1, PC3-control; bar 2, PC3-ERβ1; and bar 3, PC3-ERβ2 xenografts. Number of positive cells was counted in three independent areas of each xenograft slide. Columns, average number; bars, sd. PC3-control vs. PC3-ERβ1, P < 0.01 and PC3-control vs. PC3-ERβ1, P < 0.001.

Fig. 9.

The regulation of proliferation and of bone-metastasis genes by ERβ1 and ERβ2. This figure incorporates data of the genes that were found to be regulated by the expression of ERβ1 or ERβ2, together with data of their relative relationships from the proprietary software database. A, Model of the antiproliferative circuit promoted by ERβ1. The down-regulation of c-Myc, Cyclin E, and p45^SKP2 and the up-regulation of Cyclin D1 result in an overall inhibition of cell growth. Also shown is the ERβ2-mediated up-regulation of c-Myc and p45^SKP2, which causes ERβ2 cells to proliferate faster than the control cells. B, Pathway diagram showing how ERβ2 regulates factors that control osteoblastic and osteolytic activity by up-regulating RUNX2, Twist1, and β-catenin, resulting in increased DKK1, which controls the RANK ligand/osteoprotegerin (OPG) ratio. In addition, other genes that are either up-regulated or down-regulated by ERβ2 may have possible net effects that contribute to ERβ2-mediated effects.