provided expertise to develop 18 F nutrient uptake assays. F.X. and M.N.T injected and handled mice for 18 F nutrient uptake assays, and performed and provided expertise for PET imaging and autoradiography. T.H. and W.D.M. performed and provided expertise for intrarenal Renca experiments. R.W.J. and V.T.M generated and provided expertise for PyMT GEMM tumors. R.E.B and C.S.W. generated and provided expertise for AOM/DSS CRC tumors. B.I.R. R.T.O. and M.H.W. generated the pTZeo-EL-thy1.1 transposon construct and engineered MC38 cells using this transposon system. B.I.R, M.Z.M, and A.S. performed in vivo 2NBDG studies. J.E.B. provided expertise in characterizing TAM. A.R.P provided expertise in flow sorting for mRNA transcript analysis. B.I.R. and M.Z.M performed extracellular flux and mRNA transcript experiments. F.M.M. and E.F.M performed and provided expertise in cell staining for light microscopy. E.F.M performed light microscopy and pathologic examination of MC38 tumors. A.A (VU) conducted transcriptomic analysis. B.I.R and M.Z.M. analyzed all data generated in this study. J.C.R. and W.K.R. obtained funding for this study.Data Availability Statement (DAS) All data will be made available upon reasonable request to JCR/WKR. Tumor mRNA transcript data that support the findings of this study have been deposited in Gene Expression Omnibus (GEO) under accession GSE165223. These data are also found in Supplementary Information Table 4. Code Availability Statement (CAS)The code used to support tumor mRNA transcript analysis has been previously published (see methods references) and will be made available upon request to JCR/WKR.

Breast cancer cells frequently home to the bone marrow, where they may enter a dormant state before forming a bone metastasis. Several members of the interleukin-6 (IL-6) cytokine family are implicated in breast cancer bone colonization, but the role for the IL-6 cytokine leukemia inhibitory factor (LIF) in this process is unknown. We tested the hypothesis that LIF provides a pro-dormancy signal to breast cancer cells in the bone. In breast cancer patients, LIF receptor (LIFR) levels are lower with bone metastases and are significantly and inversely correlated with patient outcome and hypoxia gene activity. Hypoxia also reduces the LIFR:STAT3:SOCS3 signaling pathway in breast cancer cells. Loss of the LIFR or STAT3 enables otherwise dormant breast cancer cells to down-regulate dormancy, quiescence, and cancer stem cell-associated genes, and to proliferate in and specifically colonize the bone, suggesting LIFR:STAT3 signaling confers a dormancy phenotype in breast cancer cells disseminated to bone.

Breast cancer frequently metastasizes to bone, in which tumor cells receive signals from the bone marrow microenvironment. One relevant factor is TGF-b, which upregulates expression of the Hedgehog (Hh) signaling molecule, Gli2, which in turn increases secretion of important osteolytic factors such as parathyroid hormonerelated 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-b-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-b propagation of a vicious osteolytic cycle in this MDA-MB-231 model of bone metastasis. Accordingly, in the absence of TGF-b 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-b to stimulate PTHrP expression and that blocking Hh-independent Gli2 activity will inhibit tumor-induced bone destruction. Cancer Res; 71(3); 822-31. Ó2010 AACR.