The bone is the most common site of metastases in patients with breast carcinoma. Patients with ER-positive bone metastases usually have a prognosis better than that of patients with ER-negative bone metastases [17
], suggesting a protective role of ER in tumor progression associated with bone metastasis. ER-positive tumors in breast cancer patients are more differentiated and have lower metastatic potential, in comparison to ER-negative tumors [18
]. In this study, we examined whether stable reintroduction of functional human ERα in ERα-negative human breast cancer cells could alter their bone-metastatic potential in an experimental bone metastasis model. We used two bone-metastatic ERα-negative human breast cancer cell lines for the stable transfection of ERα, MDA-MB-231 cells (which are highly osteolytic) [19
], and MDA-MB-435-F-L cells (which are highly invasive and capable of widespread skeletal metastasis) [11
] in animal models. We isolated and characterized the ERα clones of each cell line after stable transfection with an expression vector for wild-type human ERα. The expression of ERα was detected by Western blot analysis in MDA-MB-231 ERα clones 11 and 15 and in MDA-MB-435-F-L ERα clones 3 and 6, even though it was lower than that in ERα-positive breast cancer MCF-7 cells (). ERα was undetectable in control vector-transfected cells (). The estrogen sensitivity of the ERα clones of both cell lines was confirmed with E2
-mediated induction of an estrogen-responsive promoter activity reported by luciferase activity and compared also with the ERα-positive human breast cancer MCF-7 cell line. Both ERα-expressing clones of MDA-MB-231 cells () and MDA-MB-435-F-L cells () showed high levels of promoter activity, which was further stimulated after treatment with E2
, suggesting that ectopic ERα is functional.
Figure 1 Ectopic expression of functional ERα in human breast cancer cells. (A) MDA-MB-231 and MDA-MB-435-F-L cells were transfected with either a retroviral ERα expression vector or an empty vector, which carried a puromycin-resistant gene. Puromycin-resistant (more ...)
Although the expression of ERα greatly increased estrogen-responsive promoter activity, it showed a limited effect on the growth of both cell lines on plastic (). Previous studies have shown that the expression of ectopic ERα in ERα-negative human mammary epithelial cells led to growth inhibition through treatment with E2
at concentrations between 10-10
]. However, we only observed a moderate growth inhibition of some ERα-transfected clones through treatment with E2
within the same concentration range (data not shown). This discrepancy is likely due, in part, to the fact that ectopic ERα levels in our clones appear to be lower than those in published studies [20–22
] when the ERα level in MCF-7 cell was used as standard.
To investigate the bone-metastatic potential of ERα-expressing clones compared to that of parental cells in both cell lines, we used an intracardiac injection model of experimental metastasis. Female nude mice were inoculated with MDA-MB-231 and MDA-MB-435-F-L control cells or their respective ERα clones through the left ventricle of the heart. Bone metastasis induced by MDA-MB-435-F-L control cells and ERα clones (clones 3 and 6) was monitored by whole-mouse EGFP imaging, as described earlier [14
]. Because MDA-MB-231 transfectants did not retain EGFP expression and the cell line was known to induce osteolytic bone metastasis, whole-mouse X-ray imaging was used to detect osteolytic bone metastasis induced by MDA-MB-231 control and ERα clones. Whole-mouse EGFP imaging on week 4 after inoculation revealed that the expression of ERα markedly reduced the widespread skeletal metastasis incidence induced by MDA-MB-435-F-L cells (). The incidence of metastasis in the femur/tibia, spine, and mandible of control cell-inoculated mice was 75%, whereas in ERα clone-inoculated mice, it ranged between 0% and 33%. The difference in metastasis incidence between the femur/tibia and the mandible was statistically significant by Fisher's exact probability test. Similarly, the incidence of osteolytic bone metastasis as detected by X-ray imaging in the legs and arms of mice inoculated with ERα clones of MDA-MB-231 cells for 3 weeks was significantly reduced in comparison to that of control mice (P
< .05) ( and ). It was 80% and 40%, respectively, in the legs and arms of control cell-inoculated mice, whereas in ERα clone-inoculated mice, it ranged between 0% and 10%. We also examined the presence of tumor cells in stained histologic sections of the femur and tibia of mice inoculated with both control cell lines or their ERα-expressing clones. Representative bone histology pictures are shown in , B
. The incidences of bone metastasis in the femora and tibiae obtained by histology examination were identical to those obtained by EGFP imaging or X-ray imaging, as shown in . In these stained bone sections, it is evident that the growth of metastatic tumors was associated with osteolysis, as reflected by the loss of trabecular bones in control cell-inoculated mice (, B
). Because the majority of mice inoculated with ERα clones did not have tumor cells in the tibiae and femora, it is not clear whether ERα expression in the two breast cancer cell lines can also inhibit osteolysis. Thus, our studies indicate that the inhibition of skeletal metastasis by the expression of ERα in ERα-negative breast cancer cells appears due to the inhibition of cancer cell homing in the bone marrow. At present, it is not clear whether ERα cells failed to invade the bone marrow or whether they did not proliferate in the bone marrow. Ectopic expression of ERα in MDA-MB-231 cells has been previously shown to inhibit its invasive potential in vitro
and to reduce the number and burden of lung metastases when the cells were inoculated through the tail vein of nude mice [22
]. Thus, it is likely that the attenuated invasive potential after ERα expression contributed significantly to the loss of bone-metastatic potential in MDA-MB-231 and MDA-MB-435-F-L cells.
Inhibition of Bone Metastasis in Breast Cancer Cells By the Ectopic Expression of ERα.
Figure 2 Inhibition of bone invasion and osteolytic metastasis by the ectopic expression of ERα. Control and ERα cells of MDA-MB-231 and MDA-MB-435-F-L were inoculated through the left cardiac ventricle of female nude mice at 0.1 x 106 cells/mouse. (more ...)
Integrins, which are cell surface receptors involved in cell adhesion, migration, invasion, and survival, have been implicated in the malignant progression of cancer [23
]. Several studies indicated a role for integrin αv
in the promotion of both spontaneous and experimental metastases to the bone in animal models [14,24–26
]. In a clinical study, integrin αv
was found to be overexpressed in metastatic breast cancer cells within the bone, compared to primary breast adenocarcinoma [27
]. The level of integrin αv
was found to be significantly lower in tumors initiated from an ERα-transfected human endometrial cancer cell line than in tumors originated from an ERα-negative parental cell [28
]. Because systemic treatment with an antagonist of integrin αv
has been shown to inhibit bone metastasis induced by MDA-MB-435 parental cells [24
], we examined whether integrin αv
was associated with ERα-induced inhibition of bone metastasis by MDA-MB-435-F-L cells. As shown in , the protein levels of both integrin αv
subunits were markedly lower in the two ERα clones of MDA-MB-435-F-L cells than in control cells, suggesting that the inhibition of integrin αv
may be one of the mechanisms by which ERα inhibits bone metastasis. The expression of integrin αv
in MDA-MB-231 cells was undetectable with Western blot analysis. Previous studies have shown that ectopic expression of integrin αv
stimulated the bone-metastatic potential of MDA-MB-231 cells [26
]. Thus, ERα-induced inhibition of the bone-metastatic potential of MDA-MB-231 is not likely due to the inhibition of integrin αv
Figure 3 Inhibition of integrin αvβ3 and RANK protein expression in ERα-transfected clones. Western blot analyses were performed with cell lysates from the MDA-MB-435-F-L cells and their respective ERα clones for integrin α (more ...)
Another cell surface receptor that has been shown to promote the homing of cancer cells to the bone is RANK. It has been shown that cytokine RANK ligand, which is expressed by stromal cells and osteoblasts, can trigger the migration and bone metastasis of human epithelial cancer cells expressing RANK [29
]. We found that both MDA-MB-231 and MDA-MB-435-F-L cells express RANK protein, which was noticeably reduced in their respective ERα clones (, A
). Thus, reduction of RANK expression in ERα-expressing cells may be another mechanism for the impairment of bone-metastatic potential by ERα. Future studies will be necessary for the elucidation of whether the dramatic blockade of bone-metastatic potential in the two breast cancer cell lines by the expression of ERα is mainly due to the attenuation of the integrin αv
and RANK pathways.
In summary, our study demonstrates that restoration of functional ERα expression in ERα-negative human breast cancer cells can block their aggressive bone-metastatic potential, supporting the notion that ERα confers a less aggressive phenotype of breast cancer. Although the mechanism by which ERα suppresses metastatic potential is currently not clear, our data suggest a potential involvement of the integrin αvβ3 and/or the RANK pathway in mediating the metastasis-suppressive action of ERα signaling.