Concurrent Antitumor and Bone-Protective Effects of Everolimus in Osteotropic Breast Cancer

Andrew J. Browne; Marie L. Kubasch; Andy Göbel; Peyman Hadji; David Chen; Martina Rauner; Friedrich Stölzel; Lorenz C. Hofbauer; and Tilman D. Rachner

Disclosures

Breast Cancer Res. 2017;19(92) 

In This Article

Results

Effects of Everolimus on Cancer Growth In Vitro and In Vivo

In the B16-F10, MDA-MB-231, and MCF-7 cell lines, everolimus exerted a potent negative effect on the growth of all cell lines tested as assessed by the CellTiter-Blue cell viability assay (Figure 1a). In the murine B16-F10 melanoma cell line, concentrations of 10 and 100 nM were effective at significantly impairing cell viability when assessed at 72 h (p < 0.05 and 0.01, respec tively). In the human breast cancer cell line MDA-MB-231, everolimus at a concentration as low as 1 nM was sufficient to induce significant suppression in viability, most notably after 72 h of treatment, when a 45% reduction in viability was observed compared with control treated cells (p < 0.05). Antitumor effects of everolimus were also apparent and equally effective for all three concentrations used in the ER-positive MCF-7 cell line. Effective inhibition of the mTOR pathway was confirmed by Western blot assessment of mTOR phosphorylation and the downstream target of mTOR, p70 S6 kinase (Figure 1b). Increasing everolimus concentrations inhibited the phosphorylation of mTOR in a dose-dependent manner, with 100 nM inducing a significant suppression in all three cell lines investigated (p < 0.01) (Additional file 1: Figure S1). Interestingly, all concentrations were sufficient to significantly suppress the phosphorylation of p70 S6 kinase by ≥50% (p < 0.01) (Additional file 1: Figure S1). In murine models of subcutaneous tumor growth, everolimus at a dose of 1 mg/kg/day was sufficient to significantly inhibit the growth of B16-F10 and MDA-MB-231 cells over a period of 2 and 4 weeks for each respective tumor model. Tumor weight was reduced by 71% (345 ± 66 mg to 103 ± 25 mg, p < 0.01) and 81% (34 ± 5 to 7 ± 1 mg, p < 0.001) in the B16-F10 and MDA-MB-231 models, respectively (Figure 1c).

Figure 1.

Everolimus (EV) inhibits cancer cell growth in vitro and in vivo. a The murine melanoma cell line B16-F10 and the human breast cancer cell lines MCF-7 and MDA-MB-231 were treated with EV in a dose-dependent (0, 1, 10, and 100 nM) and time-dependent (0, 24, 48, and 72 h) manner. Cell viability was assessed with the CellTiter-Blue® assay. b Western blots used to assess the ability of EV concentrations to inhibit the phosphorylation of the mammalian target of rapamycin (mTOR) protein and p70 S6 kinase after 24 h of treatment in the cell lines investigated. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is shown as the housekeeping control. c Female immunocompetent (C57BL/6) and immunocompromised (NMRI nude) mice were inoculated subcutaneously with B16-F10 and MDA-MB-231 cells, respectively. Tumor growth was assessed after daily treatment with 1 mg/kg of EV for 2 and 4 weeks in each respective model. In vitro and in vivo data are shown as mean ± SD of at least three independent experiments or ten mice per group, respectively. Cell viability assays were analyzed for each time point using two-way analysis of variance and in vivo data by Student's t test. Significance between EV treatments and the control condition was apparent only at 72 h and is indicated by asterisks on the graphs. In the B16-F10 graph, EV treatment was significant at inhibiting viability only at 72 h at concentrations of 10 and 100 nM. In the MDA-MB-231 and MCF-7 graphs, all concentrations of EV were significant to the same degree. **p < 0.01, ***p < 0.001. Equal volumes of dimethyl sulfoxide used to prepare and administer EV treatments were used in both in vitro and in vivo control conditions

Effects of Everolimus on Osteoclast Differentiation

RAW 264.7 osteoclastic precursor cells predifferentiated with RANKL for 5 days and exposed to everolimus for 48 h showed a significant reduction of cell viability with incremental decreases of 13%, 21%, and 28% for the increasing concentrations of 1, 10, and 100 nM everolimus, respectively (Additional file 2: Figure S2a). In addition, everolimus exerted potent negative effects on osteoclast formation. The number of TRAP-positive cells developing in the presence of RANKL was reduced by 58% at an everolimus concentration of 1 nM (p < 0.001) (Figure 2a). In accordance with this, markers of osteoclast differentiation, including Trap and Oscar, were significantly reduced by all concentrations of everolimus (p < 0.001) (Figure 2b). There was a trend that the expression of Cstk was also reduced at all everolimus concentrations; however, this observation was not significant. Comparable effects of everolimus were observed in osteoclasts differentiated from murine bone marrow-derived mononuclear cells, where concentrations of 10 nM were sufficient to completely block osteoclastogenesis and expression of osteoclast marker genes (Figure 2c and d). Of note, primary murine cells were more resistant to the lowest concentration of 1 nM everolimus, where no inhibitory effects were observed on osteoclast differentiation. Osteoclasts derived from primary murine cells were also used to assess the effect of everolimus on functional bone resorption by mature osteoclasts in vitro. Here, mononuclear cells were differentiated into osteoclasts on bone slices, and after a treatment period of 5 days with everolimus, it could be clearly observed that concentrations of 10 and 100 nM significantly decreased the levels of the bone resorption marker CTx present in the culture supernatants by 34.2% and 33.4% (p < 0.01), respectively (Additional file 3: Figure S3).

Figure 2.

Effects of everolimus (EV) on osteoclastogenesis of osteoclast progenitor RAW 264.7 cells and murine bone marrow-derived mononuclear cells in vitro. a RAW 264.7 osteoclast precursors were treated with receptor activator of nuclear factor κB ligand (RANKL) in the presence of increasing EV concentrations (0, 1, 10, and 100 nM) for 5 days. Differentiation was assessed by tartrate-resistant acid phosphatase (TRAP) staining and counting. b Osteoclast marker genes Cstk, Oscar, and Trap were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) following differentiation with RANKL and EV for 5 days. c Murine bone marrow-derived mononuclear cells were isolated from the bone marrow of C57BL/6 mice and treated with macrophage colony-stimulating factor (M-CSF) for 2 days prior to further M-CSF plus the addition of RANKL in the presence of increasing EV concentrations (1–100 nM) for 5 days. Differentiation was assessed by TRAP staining and counting. d Osteoclast marker genes Cstk, Oscar, and Trap were also assessed for osteoclasts differentiated from bone marrow-derived mononuclear cells by qRT-PCR following differentiation with RANKL and EV for 5 days. Data are shown as mean ± SD of at least three independent experiments. Data were analyzed using one-way analysis of variance and the Bonferroni posttest, and significance between the control and EV concentrations is denoted (* p < 0.05, **p < 0.01, ***p < 0.001). Equal volumes of dimethyl sulfoxide used to prepare and administer EV concentrations were used in all control conditions. mRNA Messenger RNA

Effects of Everolimus on Osteoblast Differentiation

In human preosteoblasts derived from hMSC, markers of osteoblastogenesis ALP, OPG, RUNX2, and OCN were quantified by qRT-PCR on day 7 following the addition of osteoblastic differentiation medium and increasing everolimus concentrations (Figure 3a). Here, the messenger RNA expression of ALP, a reliable marker of osteoblast activity, was negatively affected only at higher concentrations of everolimus (10 and 100 nM). In fact, the expression of OPG, RUNX2, and OCN significantly increased at 1 nM and remained elevated at 10 nM for RUNX2 and OCN. No inhibitory effect of everolimus on mineralization as assessed by alizarin red staining was observed after 21 days of exposure to everolimus at concentrations of up to 100 nM (Figure 3b). When assessing the same parameters in murine preosteoblasts derived from mMSC, we observed that the expression of osteoblast marker genes following differentiation for 7 days in the presence of everolimus showed increasing reductions in Alp and Ocn from a concentration of 10 nM and only at 100 nM for Opg and Runx2 (Figure 3c). Whereas 1 nM of everolimus maintained the osteoblast expression profile of all the genes assessed, we could not observe any pro-osteoblastic effects as seen in the osteoblastic gene signature of differentiating hMSC. When the mineralizing ability of everolimus-treated murine osteoblasts was assessed at 21 days, a significant decrease of 29% between control treated cells and cells treated with 1 nM of everolimus was observed (p < 0.01). However, increasing concentrations of everolimus did not result in further impairment of mineralization. When investigating whether everolimus has implications on osteoblastic metabolism, we observed that concentrations increasing from 1 nM to 100 nM did not show any effects on the viability of human preosteoblasts (Additional file 2: Figure S2b)

Figure 3.

Effects of everolimus (EV) on human and murine osteoblastogenesis in vitro. Human mesenchymal stem cells were differentiated along the osteoblast lineage in the presence of increasing EV concentrations (0, 1, 10, and 100 nM). a Osteoblast marker genes ALP, OPG, RUNX2, and OCN were assessed by quantitative real-time polymerase chain reaction (RT-PCR) at 7 days of differentiation. b The mineralizing ability of these cells was quantified using alizarin red S staining on day 21. Murine mesenchymal stem cells were isolated from the bone marrow of C57BL/6 mice and differentiated along the osteoblast lineage in the presence of increasing EV concentrations (1–100 nM). c Osteoblast marker genes Alp, Opg, Runx2, and Ocn were assessed by qRT-PCR at 7 days of differentiation. d The mineralizing ability of these cells was quantified using alizarin red S staining on day 21. Data are shown as mean ± SD of at least three independent experiments. Data were analyzed using one-way analysis of variance and the Bonferroni posttest, and significance between the control and EV concentrations is denoted (* p < 0.05, **p < 0.01, ***p < 0.001). Equal volumes of DMSO used to prepare and administer EV concentrations were used in all control conditions. mRNA Messenger RNA

Effect of Everolimus in an OVX Murine Model of Bone Loss

Because the majority of patients with breast cancer are postmenopausal when they present for diagnosis, or undergo hormone depletion therapies over the course of treatment, we wanted to recapitulate the hormone-deprived microenvironment in an animal model. To this end, 9-week-old wild-type C57BL/6 mice underwent OVX to induce an environment of high bone turnover and bone loss. Treatment with everolimus commenced 4 weeks post-OVX, and mice were treated with 1 mg/kg/day. Assessment of bone was performed after 4 weeks of treatment. As expected, there was a decrease in bone mineral density (BMD) in the OVX group compared with the SHAM-operated group by 27.49% (45.00 ± 24.85 vs. 32.63 ± 14.58) at the femur. Treatment with everolimus had a significant effect on BMD, restoring OVX-induced bone loss (Figure 4a). BMD of the everolimus-treated OVX group was 38.44% (53.00 ± 16.57 vs. 32.63 ± 14.58) higher than that of the OVX control group. The average bone volume over total volume (BV/TV) of everolimus-treated OVX mice was also 37% higher than in the control OVX mice (2.62 ± 0.85 vs. 1.67 ± 0.75). These results were echoed by an increase in trabecular number (2.62 ± 0.43 vs. 2.07 ± 0.41, p < 0.01) and a decrease in trabecular separation (0.40 ± 0.07 vs. 0.51 ± 0.11, p < 0.01) in everolimus-treated OVX mice versus control OVX mice (Figure 4a). Analysis of bone histomorphometry demonstrated a 25% reduction in the number of osteoclasts in contact with the bone surface (14.54 ± 5.08 to 10.87 ± 2.89) in the everolimus-treated OVX mice when compared with control OVX mice (Figure 4b), also depicted as whole sections (Additional file 4: Figure S4). Correspondingly, the BFR/BS increased in control OVX mice by 30% (from 0.64 ± 0.26 to 0.91 ± 0.11) when compared with the rate of control SHAM animals, and everolimus was able to reverse this increase by 41.5% (0.91 ± 0.11 to 0.53 ± 0.23, p < 0.01) (Figure 4b). This demonstrates that everolimus prevents the high bone turnover and bone loss that is induced by OVX.

Figure 4.

Everolimus (EV) protects ovariectomized mice from bone loss. Female C57BL/6 mice were divided into sham (SHAM) and ovariectomized (OVX) groups and subdivided into control or 1 mg/kg/day EV treatment groups (eight to ten mice per group). Four weeks post-OVX, treatment with EV commenced for 4 weeks. Bone parameters of the femur were assessed by micro-computed tomography (μCT) (a), and bone parameters of the tibia were assessed by bone histomorphometry (b). Parameters assessed included bone mineral density (BMD), bone volume over total volume (BV/TV), trabecular number (Tb.N), and trabecular separation (Tb.Sp). The number of osteoclasts per unit of bone surface (Oc.N/BS) was assessed by tartrate-resistant acid phosphatase (TRAP) staining (femur), and assessment of the double calcein labels (tibia) was used to determine the bone formation rate per unit of bone surface (BFR/BS). Representative μCT images are shown of the trabecular bone of the femur for the control OVX and EV OVX groups. Representative TRAP staining (with red arrowheads indicating osteoclasts) (original magnification × 40, scale bar 20 μm) and double calcein labels for these two groups are also provided (original magnification × 20, scale bar 100 μm). Data represent mean ± SD. Statistical analysis was performed by two-way analysis of variance for the effect of surgery, treatment, and the interaction of the two (surgery × treatment). Statistical significance of multiple comparisons are denoted (*p < 0.05, **p < 0.01, ***p < 0.001). Equal volumes of dimethyl sulfoxide used to prepare and administer EV concentrations were used in all control conditions. HA Hydroxyapatite

Effects of Everolimus on Growth of Bone Metastases

Having established the bone-protective effects of everolimus at a concentration capable of exerting antitumor potential, the ability of everolimus to inhibit the development of osteolytic breast cancer bone metastases was assessed. Firefly luciferin-labeled MDA-MB-231 breast cancer cells (MDA-MB-231-LucA12) were intracardially injected into 6-week-old NMRI nude mice. Images of all injected mice with the observed bioluminescent signal at sites of tumor burden are shown prior to their being killed at day 36 (Figure 5a). The number of overt lesions per mouse in animals with bioluminescent signals >1 × 107 photons/second/cm2/sr were counted and compared between the groups. Everolimus-treated animals had a significantly reduced number of overt lesions compared with control animals (−70.4%, 7.33 ± 5.32 to 2.17 ± 1.17, p < 0.05) (Figure 5b). Each metastatic signal was also individually quantified, and this was reduced by 45.4% (8.62 ± 8.69 to 4.71 ± 3.86, p < 0.01) in the everolimus group compared with the control group (Figure 5c). Osteolytic lesions corresponded with bioluminescent signals and could be visualized by reconstructing μCT scans of analyzed bones. Representative images of affected and unaffected femurs and tibiae are shown (Figure 5d). Trabecular bone quality in the femurs of these animals was assessed by μCT analysis (Figure 5e). Animals in the everolimus-treated group had an increased BMD and BV/TV of >50% (p < 0.001) compared with the placebo-treated group. Trabecular parameters reflected this observation, with the control group having 24.25% (2.33 ± 0.52 to 3.07 ± 0.75, p < 0.01) less trabeculae and 31.52% (0.46 ± 0.12 to 0.35 ± 0.11, p < 0.01) more separation between the trabeculae than the everolimus group. Interestingly, the total number of osteoclasts in the femurs of everolimus-treated mice were decreased by 42.88% (98.30 ± 39.21 to 58.11 ± 19.68, p < 0.05) compared with control-treated mice (Additional file 5: Figure S5). This experiment confirmed combined antiosteoclastic and antitumor effects of everolimus in the metastatic bone microenvironment.

Figure 5.

Everolimus (EV) inhibits growth of breast cancer bone metastases in vivo. a Female NMRI nude mice received intracardial injections with MDA-MB-231 cells expressing the firefly luciferase gene. Mice received daily treatments of control (nine mice) or 1 mg/kg EV (nine mice), and developing metastases were monitored weekly using the Xenogen IVIS 200 in vivo imaging system until mice were killed on day 36. Of note, one mouse in the control group died early as a result of paralysis on day 34. For this mouse, the measurement on day 28 was included. No animals in the EV treatment group developed paralysis. Representative dorsal-facing images with the observed bioluminescent signal at sites of tumor burden are shown, with animals arranged from left to right according to increasing bioluminescent signals. b The number of lesions per animal with signals ≥1 × 107 photons/s/cm2/sr were counted and compared between the groups, and the results are presented in a box plot. c The average luciferase signal intensity (per second per centimeter squared per steradian) from regions of interest was calculated per metastatic signal focus (EV n = 57 detectable lesions, control n = 90 detectable lesions). d The sites of bioluminescent signal in the knee joint were confirmed by 3D micro-computed tomography (μCT) and corresponded with osteolysis (as indicated by red and white arrowheads). e Bone parameters of the femur where assessed by μCT: bone mineral density (BMD), bone volume over total volume (BV/TV), trabecular number (Tb.N), and trabecular separation (Tb.Sp). Data are shown as mean ± SD and were analyzed using Student's t test (*p < 0.05, **p < 0.01, ***p < 0.001). Equal volumes of dimethyl sulfoxide used to prepare and administer EV concentrations were used in all control conditions. HA Hydroxyapatite

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