Yuping Tang

Inhibition of Pulmonary and Skeletal Metastasis by a Transforming Growth Factor-β Type I Receptor Kinase Inhibitor

Transforming growth factor-beta (TGF-beta) signaling has been shown to promote invasion and metastasis in various models of human cancers. In this study, we investigated the efficacy of a TGF-beta type I receptor kinase inhibitor (TbetaRI-I) to limit early systemic metastases in an orthotopic xenograft model of lung metastasis and in an intracardiac injection model of experimental bone and lung metastasis using human breast carcinoma MDA-MB-435-F-L cells, a highly metastatic variant of human breast cancer MDA-MB-435 cells, expressing the enhanced green fluorescent protein (EGFP). Treatment of the cells with the TbetaRI-I had no effect on their growth but blocked TGF-beta-stimulated expression of integrin alpha(v)beta(3) and cell migration in vitro. Systemic administration of the TbetaRI-I via i.p. injection effectively reduced the number and size of the lung metastasis in both orthotopic xenograft and experimental metastasis models with no effects on primary tumor growth rate compared with controls. TbetaRI-I treatment also reduced the incidence of widespread early skeletal metastases in the femur, tibia, mandible, and spine detected by whole-body EGFP fluorescence imaging. Tumor burden in femora and tibiae was also reduced after TbetaRI-I treatment as detected by histomorphometry analysis compared with the placebo controls. Our results indicate for the first time that abrogation of TGF-beta signaling by systemic administration of the TbetaRI-I can inhibit both early lung and bone metastasis in animal model systems and suggest antimetastatic therapeutic potential of the TbetaRI-I.

Doxorubicin in Combination with a Small TGFβ Inhibitor: A Potential Novel Therapy for Metastatic Breast Cancer in Mouse Models

Background Recent studies suggested that induction of epithelial-mesenchymal transition (EMT) might confer both metastatic and self-renewal properties to breast tumor cells resulting in drug resistance and tumor recurrence. TGFβ is a potent inducer of EMT and has been shown to promote tumor progression in various breast cancer cell and animal models. Principal Findings We report that chemotherapeutic drug doxorubicin activates TGFβ signaling in human and murine breast cancer cells. Doxorubicin induced EMT, promoted invasion and enhanced generation of cells with stem cell phenotype in murine 4T1 breast cancer cells in vitro, which were significantly inhibited by a TGFβ type I receptor kinase inhibitor (TβRI-KI). We investigated the potential synergistic anti-tumor activity of TβR1-KI in combination with doxorubicin in animal models of metastatic breast cancer. Combination of Doxorubicin and TβRI-KI enhanced the efficacy of doxorubicin in reducing tumor growth and lung metastasis in the 4T1 orthotopic xenograft model in comparison to single treatments. Doxorubicin treatment alone enhanced metastasis to lung in the human breast cancer MDA-MB-231 orthotopic xenograft model and metastasis to bone in the 4T1 orthotopic xenograft model, which was significantly blocked when TβR1-KI was administered in combination with doxorubicin. Conclusions These observations suggest that the adverse activation of TGFβ pathway by chemotherapeutics in the cancer cells together with elevated TGFβ levels in tumor microenvironment may lead to EMT and generation of cancer stem cells resulting in the resistance to the chemotherapy. Our results indicate that the combination treatment of doxorubicin with a TGFβ inhibitor has the potential to reduce the dose and consequently the toxic side-effects of doxorubicin, and improve its efficacy in the inhibition of breast cancer growth and metastasis.

Blockade of transforming growth factor-beta (TGFβ) signaling inhibits osteoblastic tumorigenesis by a novel human prostate cancer cell line

The skeleton is the most common site of prostate cancer metastasis, which often results in osteoblastic lesions. The role of transforming growth factor‐beta (TGFβ) signaling in prostate cancer‐induced osteoblastic metastasis is not clear. We investigated the role of TGFβ signaling in prostate cancer‐induced bone metastasis using a novel human prostate cancer cell line, PacMetUT1.

Characterization of PacMetUT1, a recently isolated human prostate cancer cell line.

Existing prostate cancer cell lines have limitations.

Expression, purification and characterization of BGERII: a novel pan-TGFβ inhibitor

Transforming growth factor beta (TGFbeta) isoforms are known to be upregulated during the progression of some diseases. They have been shown to stimulate invasion and metastasis during carcinogenesis and promote many pathological fibrotic diseases when overstimulated. This involvement in late-stage carcinoma and pathological fibrosis makes TGFbeta isoforms prime targets for therapeutic intervention. Although soluble ectodomains of TGFbeta type II (RII) and betaglycan (BG) have been utilized as TGFbeta inhibitors, their antagonistic potency against different TGFbeta isoforms varies considerably because RII does not appreciably bind to TGFbeta2 whereas BG binds weakly to TGFbeta1 and TGFbeta3. In this study, we have successfully constructed and expressed a recombinant fusion protein containing the endoglin domain of BG (BG(E)) and the extracellular domain of RII. The fusion protein (named BG(E)RII) was purified from bacterial inclusion bodies by immobilized metal ion chromatography, refolded and characterized. It bound with higher affinity to TGFbeta1 and TGFbeta3 than a commercially available soluble RII and to TGFbeta2 than a commercially available soluble BG. More significantly, whereas BG(E) or RII alone showed no antagonistic activity towards TGFbeta2, BG(E)RII inhibited the signaling of both TGFbeta1 and TGFbeta2 in cell-based assays including TGFbeta-induced phosphorylation of Smad2 and Smad3, and transcription from a TGFbeta-responsive promoter more effectively than equimolar concentrations of either RII or BG. After further purification by gel filtration chromatography, BG(E)RII was found to have greater activity than other potent TGFbeta inhibitors in blocking the signaling of TGFbeta1 and TGFbeta3. Thus, BG(E)RII is a potent pan-TGFbeta inhibitor in vitro and has potential for blocking TGFbeta-induced pathogenesis in vivo.

Expression, purification and characterization of BG(E)RII: a novel pan-TGFbeta inhibitor.

Transforming growth factor beta (TGFbeta) isoforms are known to be upregulated during the progression of some diseases. They have been shown to stimulate invasion and metastasis during carcinogenesis and promote many pathological fibrotic diseases when overstimulated. This involvement in late-stage carcinoma and pathological fibrosis makes TGFbeta isoforms prime targets for therapeutic intervention. Although soluble ectodomains of TGFbeta type II (RII) and betaglycan (BG) have been utilized as TGFbeta inhibitors, their antagonistic potency against different TGFbeta isoforms varies considerably because RII does not appreciably bind to TGFbeta2 whereas BG binds weakly to TGFbeta1 and TGFbeta3. In this study, we have successfully constructed and expressed a recombinant fusion protein containing the endoglin domain of BG (BG(E)) and the extracellular domain of RII. The fusion protein (named BG(E)RII) was purified from bacterial inclusion bodies by immobilized metal ion chromatography, refolded and characterized. It bound with higher affinity to TGFbeta1 and TGFbeta3 than a commercially available soluble RII and to TGFbeta2 than a commercially available soluble BG. More significantly, whereas BG(E) or RII alone showed no antagonistic activity towards TGFbeta2, BG(E)RII inhibited the signaling of both TGFbeta1 and TGFbeta2 in cell-based assays including TGFbeta-induced phosphorylation of Smad2 and Smad3, and transcription from a TGFbeta-responsive promoter more effectively than equimolar concentrations of either RII or BG. After further purification by gel filtration chromatography, BG(E)RII was found to have greater activity than other potent TGFbeta inhibitors in blocking the signaling of TGFbeta1 and TGFbeta3. Thus, BG(E)RII is a potent pan-TGFbeta inhibitor in vitro and has potential for blocking TGFbeta-induced pathogenesis in vivo.

Abstract B108: Inhibition of osteoblastic tumorigenesis of a novel human prostate cancer cell line by the blockade of TGF‐β signaling

The skeleton is the most common site of prostate cancer metastasis, which often results in osteoblastic lesions. The associated fractures and pain are major causes of morbidity and mortality in prostate cancer patients. Transforming growth factor‐beta (TGFβ) signaling has been shown to developmentally regulate bone mass and bone matrix properties. It has also been shown to promote osteolytic metastasis induced by breast cancer through its regulation of some secreted proteins that are involved in bone resorption. However, its role in prostate cancer‐induced osteoblastic metastasis is much less clear. This is in part due to a lack of prostate cancer cell lines that can efficiently induce bone metastasis and osteoblastic lesions in animal models. We found that a newly established human prostate cancer PacMetUT1 cell line produces copious amounts of active TGFβ1 and induces extensive bone metastases in calvaria, rib, femur, and tibia when inoculated in the left cardiac ventricle of male nude mice. Histology revealed extensive bone formation in the metastatic lesions of the calvaria and femur with some areas showing bone resorption. Stable knockdown of TGFβ1 with a shRNA in the PacMetUT1 cells resulted in significantly reduced TGFβ1 secretion and decreased tumor incidence when the cells were directly injected into the tibiae of male nude mice. Systemic administration of either a small inhibitor of TGFβ type I receptor kinase or a pan TGFβ binding protein also decreased osteoblastic intratibial tumor incidence and growth. PacMetUT1 is a novel prostate cancer cell line for the investigation of mechanisms that regulate formation of metastatic osteoblastic bone lesions. Future studies will reveal whether targeting the TGFβ signaling pathway is feasible for the prevention and treatment of prostate cancer‐induced bone metastasis. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B108.

Expression, purification and characterization of BG E RII

Transforming growth factor beta (TGFbeta) isoforms are known to be upregulated during the progression of some diseases. They have been shown to stimulate invasion and metastasis during carcinogenesis and promote many pathological fibrotic diseases when overstimulated. This involvement in late-stage carcinoma and pathological fibrosis makes TGFbeta isoforms prime targets for therapeutic intervention. Although soluble ectodomains of TGFbeta type II (RII) and betaglycan (BG) have been utilized as TGFbeta inhibitors, their antagonistic potency against different TGFbeta isoforms varies considerably because RII does not appreciably bind to TGFbeta2 whereas BG binds weakly to TGFbeta1 and TGFbeta3. In this study, we have successfully constructed and expressed a recombinant fusion protein containing the endoglin domain of BG (BG(E)) and the extracellular domain of RII. The fusion protein (named BG(E)RII) was purified from bacterial inclusion bodies by immobilized metal ion chromatography, refolded and characterized. It bound with higher affinity to TGFbeta1 and TGFbeta3 than a commercially available soluble RII and to TGFbeta2 than a commercially available soluble BG. More significantly, whereas BG(E) or RII alone showed no antagonistic activity towards TGFbeta2, BG(E)RII inhibited the signaling of both TGFbeta1 and TGFbeta2 in cell-based assays including TGFbeta-induced phosphorylation of Smad2 and Smad3, and transcription from a TGFbeta-responsive promoter more effectively than equimolar concentrations of either RII or BG. After further purification by gel filtration chromatography, BG(E)RII was found to have greater activity than other potent TGFbeta inhibitors in blocking the signaling of TGFbeta1 and TGFbeta3. Thus, BG(E)RII is a potent pan-TGFbeta inhibitor in vitro and has potential for blocking TGFbeta-induced pathogenesis in vivo.