Maria Niewolna

A Bone‐Seeking Clone Exhibits Different Biological Properties from the MDA‐MB‐231 Parental Human Breast Cancer Cells and a Brain‐Seeking Clone In Vivo and In Vitro

Breast cancer has a predilection for spreading to bone. The mechanism of preferential metastasis of breast cancer to bone is unknown. We hypothesize that breast cancer cells that develop bone metastases have the capacity to facilitate their colonization in bone. To examine this hypothesis, we established bone‐seeking (MDA‐231BO) and brain‐seeking (MDA‐231BR) clones of the human breast cancer cell line MDA‐MB‐231 by repeated sequential passages in nude mice and in vitro of metastatic cells obtained from bone and brain metastases, respectively. These clones were examined for distinguishing biological characteristics and compared with the MDA‐231 parental cells (MDA‐231P) in vivo and in vitro. Both the MDA‐231BR and the MDA‐231BO showed identical tumorigenicity to MDA‐231P at the orthotopic site. MDA‐231P that was inoculated into the heart developed metastases in bone, brain, ovary, and adrenal glands. On the other hand, MDA‐231BO exclusively metastasized to bone with larger osteolytic lesions than MDA‐231P. MDA‐231BR exclusively disseminated to brain and failed to develop bone metastases. In culture, MDA‐231BO produced greater amounts of parathyroid hormone‐related protein (PTH‐rP) than MDA‐231BR and MDA‐231P in the absence or presence of transforming growth factor β (TGF‐β). Furthermore, the anchorage‐independent growth of MDA‐ 231BO in soft agar was not inhibited by TGF‐β, whereas TGF‐β profoundly inhibited the growth of MDA‐231P and MDA‐231BR. Insulin‐like growth factor I (IGF‐I) markedly promoted the anchorage‐independent growth of MDA‐231BO, whereas marginal or no stimulation was observed in MDA‐231BR or MDA‐231P, respectively. Our data suggest that these phenotypic changes allow breast cancer cells to promote osteoclastic bone resorption, survive, and proliferate in bone, which consequently leads to the establishment of bone metastases.

Actions of bisphosphonate on bone metastasis in animal models of breast carcinoma.

Bone, which abundantly stores a variety of growth factors, provides a fertile soil for cancer cells to develop metastases by supplying these growth factors as a consequence of osteoclastic bone resorption. Accordingly, suppression of osteoclast activity is a primary approach to inhibit bone metastasis, and bisphosphonate (BP), a specific inhibitor of osteoclasts, has been widely used for the treatment of bone metastases in cancer patients. To obtain further insights into the therapeutic usefulness of BP, the authors studied the effects of BP on bone and visceral metastases in animal models of metastasis.

Herbimycin A, a pp60c-src tyrosine kinase inhibitor, inhibits osteoclastic bone resorption in vitro and hypercalcemia in vivo.

Since absence of expression of the c-src gene product in mice indicates that the pp60c-src tyrosine kinase is required and essential for osteoclastic bone resorption, we tested the effects of the antibiotic herbimycin A, which is an inhibitor of pp60c-src on osteoclastic bone resorption in vitro and on hypercalcemia in vivo. We examined the effects of herbimycin A on the formation of bone resorbing osteoclasts in mouse long-term marrow cultures, on isolated rodent osteoclasts and on bone resorption in organ cultures of fetal rat long bones stimulated by parathyroid hormone. We found that herbimycin A in concentrations of 1-100 ng/ml inhibited bone resorption in each of these systems. We determined the effects of herbimycin A (100 ng/ml) on src tyrosine kinase activity in mouse marrow cultures and found that it was decreased. Herbimycin A also decreased elevated blood calcium levels that were induced either by repeated subcutaneous injections of recombinant human interleukin-1 alpha or by a human tumor. There was no evidence for toxicity in any of these culture systems or in mice treated with herbimycin A. A different tyrosine kinase inhibitor that does not inhibit pp60c-src was used as a control and caused none of these effects. These data suggest that pp60c-src tyrosine kinase inhibitors may be useful pharmacologic inhibitors of osteoclastic bone resorption and hypercalcemia.

The Effect of the Bisphosphonate Ibandronate on Breast Cancer Metastasis to Visceral Organs

Bisphosphonate (BPs), specific inhibitors of osteoclastic bone resorption, are widely used therapeutic agents for bone metastases in breast cancer patients. Nevertheless, the effects of BPs on visceral metastases are controversial. Here we specifically studied the effects of the BP ibandronate on visceral metastases of breast cancer using two animal models. In the first set of experiments, we examined the effects of ibandronate on lung metastasis using 4T1 mouse mammary tumor that developed pulmonary and bone metastases following orthotopic inoculation in syngeneic female Balb/c mice. In the second set of experiments, we examined the effects of ibandronate on adrenal metastasis using a clone of the MDA-MB-231 (MDA-231) human breast cancer (MDA-231AD cells) that developed adrenal and bone metastases following intracardiac inoculation in female nude mice. These breast cancer cells were stably transfected with a firefly luciferase cDNA to facilitate quantification of the metastatic tumor burden in visceral organs. Ibandronate (4 µg/day, sc, daily) was given either after metastases were established (therapeutic administration) or at the time of tumor cell inoculation (preventative administration). In both models with each protocol, ibandronate reproducibly reduced bone metastases, establishing that BPs are effective pharmacological agents for the treatment of bone metastases in breast cancer. In the 4T1 model, neither the preventative nor therapeutic administration of ibandronate caused any effects on lung metastases. In the MDA-231 model, the preventative administration of ibandronate significantly increased adrenal metastases. However, no increase in the adrenal metastases was observed when an anti-cancer agent doxorubicin was co-administered. Therapeutic administration of ibandronate showed no effects on the adrenal metastases. Our results suggest that BPs cause no adverse effects on visceral metastases when administered in the manners that breast cancer patients usually receive.