ABSTRACT
Breast cancer frequently metastasizes to bone, in which tumor cells receive signals from the bone marrow microenvironment. One relevant factor is TGF-ß, which upregulates expression of the Hedgehog (Hh) signaling molecule, Gli2, which in turn increases secretion of important osteolytic factors such as parathyroid hormone-related protein (PTHrP). PTHrP inhibition can prevent tumor-induced bone destruction, whereas Gli2 overexpression in tumor cells can promote osteolysis. In this study, we tested the hypothesis that Hh inhibition in bone metastatic breast cancer would decrease PTHrP expression and therefore osteolytic bone destruction. However, when mice engrafted with human MDA-MB-231 breast cancer cells were treated with the Hh receptor antagonist cyclopamine, we observed no effect on tumor burden or bone destruction. In vitro analyses revealed that osteolytic tumor cells lack expression of the Hh receptor, Smoothened, suggesting an Hh-independent mechanism of Gli2 regulation. Blocking Gli signaling in metastatic breast cancer cells with a Gli2-repressor gene (Gli2-rep) reduced endogenous and TGF-ß-stimulated PTHrP mRNA expression, but did not alter tumor cell proliferation. Furthermore, mice inoculated with Gli2-Rep-expressing cells exhibited a decrease in osteolysis, suggesting that Gli2 inhibition may block TGF-ß propagation of a vicious osteolytic cycle in this MDA-MB-231 model of bone metastasis. Accordingly, in the absence of TGF-ß signaling, Gli2 expression was downregulated in cells, whereas enforced overexpression of Gli2 restored PTHrP activity. Taken together, our findings suggest that Gli2 is required for TGF-ß to stimulate PTHrP expression and that blocking Hh-independent Gli2 activity will inhibit tumor-induced bone destruction.
Subject(s)
Bone Neoplasms/metabolism , Bone Neoplasms/secondary , Breast Neoplasms/metabolism , Hedgehog Proteins/metabolism , Kruppel-Like Transcription Factors/metabolism , Nuclear Proteins/metabolism , Parathyroid Hormone-Related Protein/biosynthesis , Transforming Growth Factor beta/pharmacology , Animals , Bone Neoplasms/drug therapy , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Female , Hedgehog Proteins/antagonists & inhibitors , Humans , Kruppel-Like Transcription Factors/antagonists & inhibitors , Mice , Mice, Nude , Nuclear Proteins/antagonists & inhibitors , Recombinant Proteins/pharmacology , Signal Transduction , Veratrum Alkaloids/pharmacology , Zinc Finger Protein Gli2ABSTRACT
Transforming growth factor (TGF)-beta promotes breast cancer metastasis to bone. To determine whether the osteolytic factor parathyroid hormone-related protein (PTHrP) is the primary mediator of the tumor response to TGF-beta, mice were inoculated with MDA-MB-231 breast cancer cells expressing a constitutively active TGF-beta type I receptor. Treatment of the mice with a PTHrP-neutralizing antibody greatly decreased osteolytic bone metastases. There were fewer osteoclasts and significantly decreased tumor area in the antibody-treated mice. TGF-beta can signal through both Smad and mitogen-activated protein (MAP) kinase pathways. Stable transfection of wild-type Smad2, Smad3, or Smad4 increased TGF-beta-stimulated PTHrP secretion, whereas dominant-negative Smad2, Smad3, or Smad4 only partially reduced TGF-beta-stimulated PTHrP secretion. When the cells were treated with a variety of protein kinases inhibitors, only specific inhibitors of the p38 MAP kinase pathway significantly reduced both basal and TGF-beta-stimulated PTHrP production. The combination of Smad dominant-negative blockade and p38 MAP kinase inhibition resulted in complete inhibition of TGF-beta-stimulated PTHrP production. Furthermore, TGF-beta treatment of MDA-MB-231 cells resulted in a rapid phosphorylation of p38 MAP kinase. Thus, the p38 MAP kinase pathway appears to be a major component of Smad-independent signaling by TGF-beta and may provide a new molecular target for anti-osteolytic therapy.
