Stimulation of Host Bone Marrow Stromal Cells by Sympathetic Nerves Promotes Breast Cancer Bone Metastasis in Mice

<div><p>Bone and lung metastases are responsible for the majority of deaths in patients with breast cancer. Following treatment of the primary cancer, emotional and psychosocial factors within this population precipitate time to recurrence and death, however the underlying mechanism(s) remain unclear. Using a mouse model of bone metastasis, we provide experimental evidence that activation of the sympathetic nervous system, which is one of many pathophysiological consequences of severe stress and depression, promotes MDA-231 breast cancer cell colonization of bone via a neurohormonal effect on the host bone marrow stroma. We demonstrate that induction of RANKL expression in bone marrow osteoblasts, following β2AR stimulation, increases the migration of metastatic MDA-231 cells in vitro, independently of SDF1-CXCR4 signaling. We also show that the stimulatory effect of endogenous (chronic stress) or pharmacologic sympathetic activation on breast cancer bone metastasis in vivo can be blocked with the β-blocker propranolol, and by knockdown of RANK expression in MDA-231 cells. These findings indicate that RANKL promotes breast cancer cell metastasis to bone via its pro-migratory effect on breast cancer cells, independently of its effect on bone turnover. The emerging clinical implication, supported by recent epidemiological studies, is that βAR-blockers and drugs interfering with RANKL signaling, such as Denosumab, could increase patient survival if used as adjuvant therapy to inhibit both the early colonization of bone by metastatic breast cancer cells and the initiation of the “vicious cycle” of bone destruction induced by these cells.</p> </div

Propranolol inhibits the increase in MDA-231 metastastic bone colonization induced by endogenous sympathetic activation.

<p>(A) Mice were given propranolol (Pro) in the presence or absence of 2 h daily chronic immobilization stress (CIS). (B) Bone lesion number and (C) lesion area in the femurs, tibiae, and humeri of nude mice, measured by Faxitron analysis (<i>n</i> = 10). (D) Representative Faxitron images of hind limbs 28 d after tumor inoculation, showing osteolytic lesions (white arrows). (E) Number of osteolytic lesions and (F) total osteolytic lesion area/mouse following control versus Isoproterenol (ISO) treatment by Faxitron analysis (<i>n</i> = 8). Data are plotted as means ± SEM ; * <i>p</i><.05.</p

Osteoblast-derived RANKL promotes the migration of MDA-231 cells in response to ISO treatment.

<p>(A) Whole bone (tibia and femur, including marrow, excluding growth plate) <i>Rankl</i> expression in response to ISO treatment (3 mg/kg) in vivo compared to other soft tissues measured by qPCR (2 h treatment, control PBS versus ISO, <i>n</i> = 4). (B) <i>Rankl</i> expression in ISO-treated BMSCs measured by qPCR. (C) <i>Rankl</i> expression time course in long bones from mice subjected to daily restraint stress for 2 wk (CIS) (<i>n</i> = 3). (D) Schematic of the osteoblast-MDA231 co-culture transwell migration assay. (E) Transwell migration assays of MDA-231 cells toward BMSCs (<i>n</i> = 3), in the presence of 10 uM ISO (<i>n</i> = 3) or Pro (10 uM) treatment (<i>n</i> = 2). (F) Transwell migration assays of MDA-231 cells toward BMSCs in the presence of ISO or OPG (1 ug/mL) treatment (<i>n</i> = 2). (G) Transwell migration assays of MDA-231 toward BMSCs in the presence of ISO or OPG or AMD 3100 (10 ng/mL) treatment (<i>n</i> = 3). (H) MDA-231 transwell migration assay in response to soluble rRANKL (<i>n</i> = 3). All in vitro assays repeated at least 2 times. Data are plotted as means ± SEM; *** <i>p</i><0.001.</p

ß2 Adrenoreceptor signaling in bone increases tumor colonization.

<p>(A) Athymic nude mice were treated daily with ISO (3 mg/kg) for 2 wk prior to or for 4 wk after intracardiac injection of MDA-231 cells. (B) Number of osteolytic lesions per mouse as quantified from Faxitron radiographs at day 28 in humeri, femurs, and tibiae (<i>n</i> = 8). (C) Total osteolytic lesion areas visible per mouse, as assessed by Faxitron (<i>n</i> = 8). (D) Representative Faxitron images of hind limbs at day 28. (E) Tumor number counted in femurs and tibiae by histology (<i>n</i> = 8). (F) Total bone tumor burden in femurs and tibiae by histology (<i>n</i> = 8). (G) MDA-231 tumors appear as pink masses in the metaphyses (modified H&E). Data are plotted as means ± SEM; * <i>p</i><.05; ** <i>p</i><.005.</p

ß2AR stimulation decreases tumor cell growth in vitro

<p><b>and in vivo.</b> (A) ß2AR mRNA expression in two bone metastatic mammary carcinoma lines MDA-231 and 4T1-592 assessed by RT-PCR. (B) In vitro effects of ISO (1 µM) on MDA-231 cell number over 4 d (<i>n</i> = 3). (C) ISO treatment dose-response on MDA-231 cell number versus control (<i>n</i> = 3). (D) Subcutaneous MDA-231 cell growth quantification (tumor volume) following daily PBS (control) or ISO injections (<i>n</i> = 6). (E) 4T1-592 cell number in vitro in response to ISO versus control (<i>n</i> = 3). (F) ISO treatment dose-response on 4T1-592 cells (<i>n</i> = 3). (G) Mammary fat pad tumor mass of 4T1-592 cells in BalbC mice as measured by end point tumor mass after 4 wk of ISO treatment (<i>n</i> = 9; <i>ns</i>, not significant, <i>p</i> = 0.22). Data are plotted as means ± SEM; ** <i>p</i><.005.</p